Publications
2024
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(2024) International Journal of Molecular Sciences. 25, 1, 578. Abstract
Presenilin 1 (PS1) is a transmembrane proteolytic subunit of γ-secretase that cleaves amyloid precursor proteins. Mutations in PS1 (mPS1) are associated with early-onset familial Alzheimer\u2019s disease (AD). The link between mutated PS1, mitochondrial calcium regulation, and AD has been studied extensively in different test systems. Despite the wide-ranging role of mPS1 in AD, there is a paucity of information on the link between PS1 and neuronal cell death, a hallmark of AD. In the present study, we employed the selective mitochondrial uncoupler carbonyl cyanide chlorophenylhydrazone (CCCP) and compared the reactivity of mPS1-transfected cultured rat hippocampal neurons with PS1 and control neurons in a situation of impaired mitochondrial functions. CCCP causes a slow rise in cytosolic and mitochondrial calcium in all three groups of neurons, with the mPS1 neurons demonstrating a faster rise. Consequently, mPS1 neurons were depolarized by CCCP and measured with TMRM, a mitochondrial voltage indicator, more than the other two groups. Morphologically, CCCP produced more filopodia in mPS1 neurons than in the other two groups, which were similarly affected by the drug. Finally, mPS1 transfected neurons tended to die from prolonged exposure to CCCP sooner than the other groups, indicating an increase in vulnerability associated with a lower ability to regulate excess cytosolic calcium.
2023
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(2023) International Journal of Molecular Sciences. 24, 16, 12940. Abstract
A major route for the influx of calcium ions into neurons uses the STIM-Orai1 voltage-independent channel. Once cytosolic calcium ([Ca2+]i) elevates, it activates mitochondrial and endoplasmic calcium stores to affect downstream molecular pathways. In the present study, we employed a novel drug, carbonyl cyanide chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, to explore the role of mitochondria in cultured neuronal morphology. CCCP caused a sustained elevation of [Ca2+]i and, quite surprisingly, a massive increase in the density of dendritic filopodia and spines in the affected neurons. This morphological change can be prevented in cultures exposed to a calcium-free medium, Orai1 antagonist 2APB, or cells transfected with a mutant Orai1 plasmid. It is suggested that CCCP activates mitochondria through the influx of calcium to cause rapid growth of dendritic processes.
2022
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(2022) International Journal of Molecular Sciences. 23, 20, 12321. Abstract
While neuronal mitochondria have been studied extensively in their role in health and disease, the rules that govern calcium regulation in mitochondria remain somewhat vague. In the present study using cultured rat hippocampal neurons transfected with the mtRCaMP mitochondrial calcium sensor, we investigated the effects of cytosolic calcium surges on the dynamics of mitochondrial calcium ([Ca2+]m). Cytosolic calcium ([Ca2+]c) was measured using the high affinity sensor Fluo-2. We recorded two types of calcium events: local and global ones. Local events were limited to a small, 2\u20135 µm section of the dendrite, presumably caused by local synaptic activity, while global events were associated with network bursts and extended throughout the imaged dendrite. In both cases, cytosolic surges were followed by a delayed rise in [Ca2+]m. In global events, the rise lasted longer and was observed in all mitochondrial clusters. At the end of the descending part of the global event, [Ca2+]m was still high. Global events were accompanied by short and rather high [Ca2+]m surges which we called spikelets, and were present until the complete decay of the cytosolic event. In the case of local events, selective short-term responses were limited to the part of the mitochondrial cluster that was located directly in the center of [Ca2+]c activity, and faded quickly, while responses in the neighboring regions were rarely observed. Caffeine (which recruits ryanodine receptors to supply calcium to the mitochondria), and carbonyl cyanide m-chlorophenyl hydrazine (CCCP, a mitochondrial uncoupler) could affect [Ca2+]m in both global and local events. We constructed a computational model to simulate the fundamental role of mitochondria in restricting calcium signals within a narrow range under synapses, preventing diffusion into adjacent regions of the dendrite. Our results indicate that local cytoplasmic and mitochondrial calcium concentrations are highly correlated. This reflects a key role of signaling pathways that connect the postsynaptic membrane to local mitochondrial clusters.
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(2022) International Journal of Molecular Sciences. 23, 4, 2048. Abstract
Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is caused by a developmentally regulated silencing of the FMR1 gene, but its effect on human neuronal network development and function is not fully understood. Here, we isolated isogenic human embryonic stem cell (hESC) subclones\u2014one with a full FX mutation and one that is free of the mutation (control) but shares the same genetic background\u2014differentiated them into induced neurons (iNs) by forced expression of NEUROG-1, and compared the functional properties of the derived neuronal networks. High-throughput image analysis demonstrates that FX-iNs have significantly smaller cell bodies and reduced arborizations than the control. Both FX-and control-neurons can discharge repetitive action potentials, and FX neuronal networks are also able to generate spontaneous excitatory synaptic currents with slight differences from the control, demonstrating that iNs generate more mature neuronal networks than the previously used protocols. MEA analysis demonstrated that FX networks are hyperexcitable with significantly higher spontaneous burst-firing activity compared to the control. Most importantly, cross-correlation analysis enabled quantification of network connectivity to demonstrate that the FX neuronal networks are significantly less synchronous than the control, which can explain the origin of the development of intellectual dysfunction associated with FXS.
2021
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(2021) Brain Structure and Function. 226, p. 2459-2466 Abstract
Synaptopodin (SP) is localized within the spine apparatus, an enigmatic structure located in the neck of spines of central excitatory neurons. It serves as a link between the spine head, where the synapse is located, and the endoplasmic reticulum (ER) in the parent dendrite. SP is also located in the axon initial segment, in association with the cisternal organelle, another structure related to the endoplasmic reticulum. Extensive research using SP knockout (SPKO) mice suggest that SP has a pivotal role in structural and functional plasticity. Consequently, young adult SPKO mice were shown to be deficient in cognitive functions, and in ability to undergo long-term potentiation of reactivity to afferent stimulation. However, although SP expresses differently during maturation, its role in synaptic and intrinsic neuronal mechanisms in adult SPKO mice is still unclear. To address this knowledge gap we analyzed hippocampus bulk mRNA in SPKO mice, and we recorded the activity of CA1 neurons in the mouse hippocampus slice, with both extracellular and patch recording methods. Electrophysiologically, SPKO cells in CA1 region of the dorsal hippocampus were more excitable than wild type (wt) ones. In addition, exposure of mice to a complex environment caused a higher proportion of arc-expressing cells in SPKO than in wt mice hippocampus. These experiments indicate that higher excitability and higher expression of arc staining may reflect SP deficiency in the hippocampus of adult SPKO mice.
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(2021) Proceedings of the National Academy of Sciences - PNAS. 118, 12, e201845911. Abstract
The interplay between excitation and inhibition is crucial for neuronal circuitry in the brain. Inhibitory cell fractions in the neocortex and hippocampus are typically maintained at 15 to 30%, which is assumed to be important for stable dynamics. We have studied systematically the role of precisely controlled excitatory/inhibitory (E/I) cellular ratios on network activity using mice hippocampal cultures. Surprisingly, networks with varying E/I ratios maintain stable bursting dynamics. Interburst intervals remain constant for most ratios, except in the extremes of 0 to 10% and 90 to 100% inhibitory cells. Single-cell recordings and modeling suggest that networks adapt to chronic alterations of E/I compositions by balancing E/I connectivity. Gradual blockade of inhibition substantiates the agreement between the model and experiment and defines its limits. Combining measurements of population and single-cell activity with theoretical modeling, we provide a clearer picture of how E/I balance is preserved and where it fails in living neuronal networks.
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(2021) Frontiers in Drug Design and Discovery. p. 92-167 Abstract
Alcohol dependence is one of the top priority public health problems on a global scale. The costs of medical treatments of patients with alcohol dependence, a decrease in labor productivity, an increased risk of developing somatic and mental disorders, and early mortality are all consequences of acute and chronic alcohol abuse. The brain is one of the main targets of alcohol intoxication. Extensive neurobiological studies have revealed a number of synaptic and extra-synaptic mechanisms, affected by alcohol. A primary target of it is GABAergic transmission. Nevertheless, the exciting and disinhibiting actions of alcohol at the system and cellular levels have not been satisfactorily elucidated. It remains unclear whether effects of ethanol are highly complex, manifested only at the level of entire brain or concerns also individual cells, their subcellular structures, organelles, ion channels and receptors. With this approach, small, cultured neural networks that are isolated from the rest of the brain are of particular interest. A serious problem of modern pharmaceuticals is the lack of drugs that have a therapeutic effect on alcohol toxicity of the brain and nervous system, despite the abundance of so-called “traditional medicines”. Substances obtained from some herbs containing a mixture of biologically active substances that exhibit a wide range of properties are of particular interest. Among them - flavonoids, which are polyphenols of plant origin and often reveal a sign of sedative, neuroprotective, antidepressant properties, and may improve cognitive function. The aims of our study is to reveal the mechanisms of various concentrations of ethanol, as well as its chronic effects on the functional properties of neurons in small neural networks such as the primary neuronal culture of the rat hippocampus. We have also performed a complex neuropharmacology screening and the study of flavonoids, extracted from Scrophulariaceae plant family, which is known in the traditional medicine for its antialcohol properties.
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Calcium Sensors STIM1 and STIM2 Regulate Different Calcium Functions in Cultured Hippocampal Neurons(2021) Frontiers in Synaptic Neuroscience. 12, 1, 573714. Abstract
There are growing indications for the involvement of calcium stores in the plastic properties of neurons and particularly in dendritic spines of central neurons. The store-operated calcium entry (SOCE) channels are assumed to be activated by the calcium sensor stromal interaction molecule (STIM)which leads to activation of its associated Orai channel. There are two STIM species, and the differential role of the two in SOCE is not entirely clear. In the present study, we were able to distinguish between transfected STIM1, which is more mobile primarily in young neurons, and STIM2 which is less mobile and more prominent in older neurons in culture. STIM1 mobility is associated with spontaneous calcium sparks, local transient rise in cytosolic [Ca2+]i, and in the formation and elongation of dendritic filopodia/spines. In contrast, STIM2 is associated with older neurons, where it is mobile and moves into dendritic spines primarily when cytosolic [Ca2+]i levels are reduced, apparently to activate resident Orai channels. These results highlight a role for STIM1 in the regulation of [Ca2+]i fluctuations associated with the formation of dendritic spines or filopodia in the developing neuron, whereas STIM2 is associated with the maintenance of calcium entry into stores in the adult neuron.
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(2021) European Journal of Neuroscience. Abstract
Early life stress is an important vulnerability factor for the development of anxiety disorders, depression and late-onset cognitive decline. Recently, we demonstrated that juvenile stress (JS) lastingly enhanced long-term potentiation via reduction of steady-state glutamine synthetase mRNA expression and the associated dysregulation of the astrocytic glutamate-glutamine cycle in the rat ventral CA1. We now investigated the regulation of steady-state mRNA expression of neuronal gene products that determine GABAergic and glutamatergic neurotransmission in layers of the ventral and dorsal CA1 after JS. We further studied their interaction with stress in young adult age (AS) to address their putative role in psychopathology development. Strikingly, mRNA levels of the glutamic acid decarboxylase (GAD) isoforms GAD65 and of the GABA-A receptor α2 (Gabra2) were increased after single JS or AS, but not after combined JS/AS stress experience. In fact, JS/AS resulted in layer-specific reduction of Gabra2 and also of Gabra1 mRNA levels in the ventral CA1. Furthermore, GAD65 and Gabra2 mRNAs were correlated with glutamatergic AMPA and NMDA receptor subunit mRNAs after single JS and AS, but not after combined JS/AS. Together, these data indicate a loss of allostatic regulation of steady-state mRNA levels of key GABAergic components that may result in a dysregulation of excitation/ inhibition balance in the ventral CA1 upon dual stress exposure. Finally, individual differences in local glucocorticoid receptor mRNA expression may contribute to this regulation.
2020
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(2020) Neuroscience and Biobehavioral Reviews. 117, p. 281-296 Abstract
This review focuses on the inter- and transgenerational effects of stress experience prior to and during gestation. We provide an overview of findings from studies in humans as well as in animal models on brain structural and physiological functions and on the development of cognitive and executive functions. We also discuss the concept of stress-induced (re-)programming in more detail by highlighting epigenetic mechanisms and particularly those affecting the development of monoaminergic transmitter systems, which constitute the braińs reward system. As the majority of studies have focused on male individuals we will emphasize sex-specific differences in stress vulnerability and resilience. Finally, we offer some perspectives on the development of protective and therapeutic interventions in cognitive and emotional disturbances resulting from pre-conception and prenatal stress.
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(2020) Frontiers in Behavioral Neuroscience. 14, 144. Abstract
Injection of corticosterone (CORT) in the dorsal hippocampus (DH) can mimic post-traumatic stress disorder (PTSD)\u2014related memory in mice: both maladaptive hypermnesia for a salient but irrelevant simple cue and amnesia for the traumatic context. However, accumulated evidence indicates a functional dissociation within the hippocampus such that contextual learning is primarily associated with the DH whereas emotional processes are more linked to the ventral hippocampus (VH). This suggests that CORT might have different effects on fear memories as a function of the hippocampal sector preferentially targeted and the type of fear learning (contextual vs. cued) considered. We tested this hypothesis in mice using CORT infusion into the DH or VH after fear conditioning, during which a tone was either paired (predicting-tone) or unpaired (predicting-context) with the shock. We first replicate our previous results showing that intra-DH CORT infusion impairs contextual fear conditioning while inducing fear responses to the not predictive tone. Second, we show that, in contrast, intra-VH CORT infusion has opposite effects on fear memories: in the predicting-tone situation, it blocks tone fear conditioning while enhancing the fear responses to the context. In both situations, a false fear memory is formed based on an erroneous selection of the predictor of the threat. Third, these opposite effects of CORT on fear memory are both mediated by glucocorticoid receptor (GR) activation, and reproduced by post-conditioning stress or systemic CORT injection. These findings demonstrate that false opposing fear memories can be produced depending on the hippocampal sector in which the GRs are activated.
2019
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(2019) Cell Reports. 29, 8, p. 2144-2153.e7 Abstract
Patients with germline mutations in the urea-cycle enzyme argininosuccinate lyase (ASL) are at risk for developing neurobehavioral and cognitive deficits. We find that ASL is prominently expressed in the nucleus locus coeruleus (LC), the central source of norepinephrine. Using natural history data, we show that individuals with ASL deficiency are at risk for developing attention deficits. By generating LC-ASL-conditional knockout (cKO) mice, we further demonstrate altered response to stressful stimuli with increased seizure reactivity in LC-ASL-cKO mice. Depletion of ASL in LC neurons leads to reduced amount and activity of tyrosine hydroxylase (TH) and to decreased catecholamines synthesis, due to decreased nitric oxide (NO) signaling. NO donors normalize catecholamine levels in the LC, seizure sensitivity, and the stress response in LC-ASL-cKO mice. Our data emphasize ASL importance for the metabolic regulation of LC function with translational relevance for ASL deficiency (ASLD) patients as well as for LC-related pathologies.
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(2019) Basic Neurobiology Techniques. p. 261-284 Abstract
The enhanced ability to visualize small neuronal compartments in live tissue, such as individual dendritic spine, is accompanied in recent years by a need for a precise, high temporal and spatial resolution ability to activate or suppress electrophysiological as well as biochemical properties within such compartments. Parallel rapid progress in molecular, cellular, and physics methodologies enabled the recruitment of novel technologies to the analysis of a wide spectrum of issues, from long lasting imaging of subcellular compartments in vitro as well as in vivo, to simultaneous recording of activities of networks of hundreds of neurons in behaving animals. In the present review, we will focus on a fast UV flash (4 ns) photolysis of caged molecules in a small sphere (
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(2019) Oxidative Medicine and Cellular Longevity. 2019, 7284967. Abstract
Mutations in the presenilin 1 (PS1) gene are a major trigger of familial Alzheimer's disease (AD), yet the mechanisms affected by mutated PS1 causing cognitive decline are not yet elucidated. In the present study, we compared rat hippocampal neurons in culture, transfected with PS1 or with mutant (M146V) PS1 (mPS1) plasmids in several neuronal functions. Initially, we confirmed earlier observations that mPS1-expressing neurons are endowed with fewer mature "mushroom" spines and more filopodial immature protrusions. The correlation between calcium changes in the cytosol, mitochondria, and endoplasmic reticulum (ER) is mitigated in the mPS1 neurons, tested by the response to an abrupt increase in ambient [Ca2+]o; cytosolic [Ca2+]i is higher in the mPS1 neurons but mitochondrial [Ca2+] is lower than in control neurons. Strikingly, mPS1-transfected neurons express higher excitability and eventual lower survival rate when exposed to the oxidative stressor, paraquat. These results highlight an impaired calcium regulation in mPS1 neurons, resulting in a reduced ability to handle oxidative stress, which may lead to cell death and AD.
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(2019) Journal of Neuroscience. 39, 20, p. 3983-3992 Abstract
Disruption in calcium homeostasis is linked to several pathologies and is suggested to play a pivotal role in the cascade of events leading to Alzheimer's disease (AD). Synaptopodin (SP) residing in dendritic spines has been associated with ryanodine receptor (RyR), such that spines lacking SP release less calcium from stores. In this work, we mated SPKO with 3xTg mice (3xTg/ SPKO) to test the effect of SP deficiency in the AD mouse. We found that 6-month-old male 3xTg/ SPKO mice restored normal spatial learning in the Barns maze, LTP in hippocampal slices, and expression levels of RyR in the hippocampus that were altered in the 3xTg mice. In addition, there was a marked reduction in 3xTg-associated phosphorylated tau, amyloid beta plaques, and activated microglia in 3xTg/ SPKO male and female mice. These experiments indicate that a reduction in the expression of SP ameliorates AD-associated phenotype in 3xTg mice.
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(2019) Frontiers in Cellular Neuroscience. 13, 92. Abstract
Palmitoyl-protein thioesterase 1 (PPT1) is a depalmitoylation enzyme that is mutated in cases of neuronal ceroid lipofuscinosis (NCL). The hallmarks of the disease include progressive neurodegeneration and blindness, as well as seizures. In the current study, we identified 62 high-confident PPT1-binding proteins. These proteins included a self-interaction of PPT1, two V-type ATPases, calcium voltage-gated channels, cytoskeletal proteins and others. Pathway analysis suggested their involvement in seizures and neuronal morphology. We then proceeded to demonstrate that hippocampal neurons from Ppt1-/- mice exhibit structural deficits, and further investigated electrophysiology parameters in the hippocampi of mutant mice, both in brain slices and dissociated postnatal primary cultures. Our studies reveal new mechanistic features involved in the pathophysiology of this devastating neurodegenerative disease.
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(2019) Journal of Ethnopharmacology. 229, p. 22-28 Abstract
Ethnopharmacological relevance: A major concern in modern society involves the lasting detrimental behavioral effects of exposure to alcoholic beverages. Consequently, hundreds of folk remedies for hangover have been suggested, most of them without a scientific basis, for lack of proper test systems. Over centuries, yellow toadflax (Linaria vulgaris Mill., Lv) tincture has been used in Russian traditional medicine to treat the spectrum of hangover symptoms such as vertigo, headache, drunken behaviors, and as a sedative.Materials and methods: Here we use in-vitro cultured hippocampal neurons to examine the effect of the Lv extract as well as the flavonoid acetylpectolinarin (ACP) exclusively found in Lv extract, on spontaneous network activity of the cultured neurons exposed to low, physiological concentrations of ethanol.Results: As in previous studies, low (0.25-0.5%) ethanol causes an increase in network activity, which was converted to suppression, with high concentrations of ethanol. Lv extract and ACP, at low concentrations, had no appreciable effect on spontaneous activity, but they blocked the facilitating action of low ethanol. This action of ACP was also seen when the culture was exposed to 1-EBIO, a SK potassium channel opener, and was blocked by apamin, an SK channel antagonist. In contrast, ACP or Lv extracts did not reverse the suppressive effects of higher ethanol.Conclusions: Our results suggest that ACP acts by interacting with the SK channel, to block the facilitatory effect of low concentration of ethanol, on network activity in hippocampal cultures.
2018
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(2018) Neuroscience. 394, p. 156-163 Abstract
Mitochondrial Carrier Homolog 2 (MTCH2) acts as a receptor for the BH3 interacting-domain death agonist (BID) in the mitochondrial outer membrane. Loss of MTCH2 affects mitochondria energy metabolism and function. MTCH2 forebrain conditional KO (MTCH2 BKO) display a deficit in hippocampus-dependent cognitive functions. Here we study age-related MTCH2 BKO behavioral and electrophysiological aspects of hippocampal functions. MTCH2 BKO exhibit impaired spatial but not motor learning and an impairment in long-term potentiation (LTP) in hippocampal slices. Moreover, MTCH2 BKO express an increase in activated microglia, in addition to a reduction in neuron density in the hippocampus, but do not express amyloid-beta plaques or neurofibrillary tangles. These results highlight the role of mitochondria in the normal hippocampus-dependent memory formation. (C) 2018 IBRO. Published by Elsevier Ltd. All rights reserved.
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(2018) Journal of Neurophysiology. 120, 5, p. 2694-2705 Abstract
Extracellular calcium ions support synaptic activity but also reduce excitability of central neurons. In the present study. the effect of calcium on excitability was explored in cultured hippocampal neurons. CaCl2 injected by pressure in the vicinity of a neuron that is bathed only in MgCl2 as the main divalent cation caused a depolarizing shift in action potential threshold and a reduction in excitability. This effect was not seen if the intracellular milieu consisted of Cs+ instead of K-gluconate as the main cation or when it contained ruthenium red, which blocks release of calcium from stores. The suppression of excitability by calcium was mimicked by caffeine, and calcium store antagonists cyclopiazonic acid or thapsigargin blocked this action. Neurons taken from synaptopodin-knockout mice show significantly reduced efficacy of calcium modulation of action potential threshold. Likewise. in Orail knockdown cells, calcium is less effective in modulating excitability of neurons. Activation of small-conductance K (SK) channels increased action potential threshold akin to that produced by calcium ions, whereas blockade of SK channels but not big K channels reduced the threshold for action potential discharge. These results indicate that calcium released from stores may suppress excitability of central neurons.NEW & NOTEWORTHY Extracellular calcium reduces excitability of cultured hippocampal neurons. This effect is mediated by calcium-gated potassium currents, possibly small-conductance K channels. Release of calcium from internal stores mimics the effect of extracellular calcium. It is proposed that calcium stores modulate excitability of central neurons.
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(2018) GLIA. 66, 5, p. 1098-1117 Abstract
To elucidate mechanisms contributing to cortical pathology in multiple sclerosis (MS), we investigated neurovascular aberrations, in particular the association of astrocytes with cortical neurons and blood vessels, in mice induced with experimental autoimmune encephalomyelitis (EAE). Blood-brain barrier (BBB) dysfunction was evident by leakage of the tracer sodium fluorescein, along with reduced expression of claudin-5 by endothelial cells and desmin by pericytes. Immunohistological and ultrastructural analyses revealed detachment of the astroglial cell bodies from the blood vessels and loss of their connections with both the blood vessels and the neuronal synapses. Furthermore, examination of individual astrocytic processes at cortical layer IV, where well-defined neuronal columns (barrels) are linked to functional properties, revealed loss of astrocytic confinement to the functional neuronal boundaries. Thus, in contrast to the highly modulated patches of astrocyte processes in naive mice overlapping the barrel cores, in EAE-mice process distribution was uniform ignoring the barrel boundaries. These aberrations are attributed to the surrounding inflammation, indicated by T-cells presence in the cortex as well as in the subcortical white matter and the meninges. Immunomodulatory treatment with glatiramer acetate partially abrogated the neurovascular damage. These combined findings indicate that under inflammatory conditions, activated perivascular astrocytes fail in neuro-hemodynamic coupling, resulting in obstructed cross-talk between the blood vessels and the neurons. We propose that loss of cortical astrocytic regulation and fine-tuning between the blood supply and the neuronal needs contributes to the neurological impairment and cognitive decline occurring in EAE/MS as well as to the disease progression.
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(2018) Frontiers in Molecular Neuroscience. 11, 32. Abstract
Epilepsy is a devastating disease, with cognitive and emotional consequences that are not curable. In recent years, it became apparent that cannabinoids help patients to cope with epilepsy. We have studied the effects of cannabidiol (CBD) on the ability to produce long term potentiation (LTP) in stratum radiatum of CA1 region of the mouse hippocampus. Exposure to seizure-producing pilocarpine reduced the ability to generate LTP in the slice. Pre-exposure to CBD prevented this effect of pilocarpine. Furthermore, CBD caused a marked increase in ability to generate LTP, an effect that was blocked by calcium store antagonists as well as by a reduction in serotonin tone. Serotonin, possibly acting at a 5HT1A receptor, or fenfluramine (FFA), which causes release of serotonin from its native terminals, mimicked the effect of CBD. It is proposed that CBD enhances non-NMDA LTP in the slice by facilitating release of serotonin from terminals, consequently ameliorating the detrimental effects of pilocarpine.
2017
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(2017) Neuron. 96, 4, p. 730-735 Abstract
Science is ideally suited to connect people from different cultures and thereby foster mutual understanding. To promote international life science collaboration, we have launched “The Science Bridge” initiative. Our current project focuses on partnership between Western and Middle Eastern neuroscience communities. Science is ideally suited to connect people from different cultures and thereby foster mutual understanding. To promote international life science collaboration, Lissek et al. have launched “The Science Bridge” initiative. Theirs current project focuses on partnership between Western and Middle Eastern neuroscience communities.
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(2017) Hippocampus. 27, 8, p. 860-870 Abstract
Stress has a profound effect on ability to express neuronal plasticity, learning, and memory. Likewise, epileptic seizures lead to massive changes in brain connectivity, and in ability to undergo long term changes in reactivity to afferent stimulation. In this study, we analyzed possible long lasting interactions between a stressful experience and reactivity to pilocarpine, on the ability to produce long term potentiation (LTP) in a mouse hippocampus. Pilocarpine lowers paired pulse potentiation as well as LTP in CA1 region of the mouse hippocampal slice. When stress experience precedes exposure to pilocarpine, it protects the brain from the lasting effect of pilocarpine. When stress follows pilocarpine, it exacerbates the effect of the drug, to produce a long lasting reduction in LTP. These changes are accompanied by a parallel change in blood corticosterone level. A single exposure to selective mineralo- or gluco-corticosterone (MR and GR, respectively) agonists and antagonists can mimic the stress effects, indicating that GR's underlie the lasting detrimental effects of stress whereas MRs are instrumental in counteracting the effects of stress. These studies open a new avenue of understanding of the interactive effects of stress and epileptic seizures on brain plasticity.
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(2017) Neurobiology of Learning and Memory. 140, p. 1-10 Abstract
It is well established that neurons are plastic and can change the strength of their connections with other neurons depending on their recent history. While many molecular entities involved in plastic processes were already described, the role of a store-operated calcium channel RAU in neuronal plasticity is not known as yet. Using dominant negative form of ORM, we were able to show that RAH. is needed for formation of new dendritic spines following chemical induction of long term potentiation (cLTP), and that this is due to the release of Ca+2 from ryanodine receptor-associated endoplasmic reticulum stores. We propose that when RAU is deficient, there is less Ca+2 in the stores, less releasable Ca+2 and consequently less LTP and spine formation. (C) 2017 Elsevier Inc. All rights reserved.
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(2017) Scientific Reports. 7, 44401. Abstract[All authors]
Mitochondrial Carrier Homolog 2 (MTCH2) is a novel regulator of mitochondria metabolism, which was recently associated with Alzheimer's disease. Here we demonstrate that deletion of forebrain MTCH2 increases mitochondria and whole-body energy metabolism, increases locomotor activity, but impairs motor coordination and balance. Importantly, mice deficient in forebrain MTCH2 display a deficit in hippocampus-dependent cognitive functions, including spatial memory, long term potentiation (LTP) and rates of spontaneous excitatory synaptic currents. Moreover, MTCH2-deficient hippocampal neurons display a deficit in mitochondria motility and calcium handling. Thus, MTCH2 is a critical player in neuronal cell biology, controlling mitochondria metabolism, motility and calcium buffering to regulate hippocampal-dependent cognitive functions.
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(2017) Neuroscience and Biobehavioral Reviews. 74, p. 21-43 Abstract[All authors]
ALBRECHT, A., MULLER, L, ARDI, Z, CALISKAN, G., GRUBER, D., IVENS, S., SEGAL, M., BEHR, J., HEINEMANN, U., STORK, O., and RICHTER-LEVIN, G. Neurobiological consequences of juvenile stress: A GABAergic perspective on risk and resilience. NEUROSCI BIOBEHAV REV XXX-XXX, 2016. - Childhood adversity is among the most potent risk factors for developing mood and anxiety disorders later in life. Therefore, understanding how stress during childhood shapes and rewires the brain may optimize preventive and therapeutic strategies for these disorders.To this end, animal models of stress exposure in rodents during their post-weaning and pre-pubertal life phase have been developed. Such 'juvenile stress' has a long-lasting impact on mood and anxiety like behavior and on stress coping in adulthood, accompanied by alterations of the GABAergic system within core regions for the stress processing such as the amygdala, prefrontal cortex and hippocampus. While many regionally diverse molecular and electrophysiological changes are observed, not all of them correlate with juvenile stress-induced behavioral disturbances. It rather seems that certain juvenile stress-induced alterations reflect the system's attempts to maintain homeostasis and thus promote stress resilience. Analysis tools such as individual behavioral profiling may allow the association of behavioral and neurobiological alterations more clearly and the dissection of alterations related to the pathology from those related to resilience. (C) 2017 Elsevier Ltd. All rights reserved.
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(2017) Neurobiology of Learning and Memory. 138, p. 3-9 Abstract
The introduction of novel technologies, including high resolution time lapse imaging in behaving animals, molecular modification of the genome and optogenetic control of neuronal excitability have revolutionized the ability to detect subcellular changes in the brain, associated with learning and memory. The sequence of molecular cascades leading to formation, longevity and erasure of memories are being addressed in growing number of studies. Still, major issues concerning the relationship between the morphology and physiology of dendritic spines and memory mechanisms and the functional, neuronal network relevance of such parameters remain unsettled. The present review will summarize recent studies related to the immediate and long lasting changes in density, morphology and function of dendritic spines and their parent neurons following exposure to plasticity-producing stimulation in vivo and in vitro. Standing issues such as the relations between volume/shape and longevity, with respect to different classes of memories in different brain regions will be addressed. These studies indicate that the rules governing the structure/function relations of dendritic spines and memory in the brain are still not conclusive. (C) 2016 Elsevier Inc. All rights reserved.
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(2017) Journal Of Physiology-London. 595, 1, p. 125-140 Abstract
The possible role of store operated calcium entry (SOCE) through the Orai1 channel in the formation and functions of dendritic spines was studied in cultured hippocampal neurons. In calcium store-depleted neurons, a transient elevation of extracellular calcium concentration ([Ca2+](o)) caused a rise in [Ca2+](i) that was mediated by activation of the SOCE. The store depletion resulted in an increase in stromal interacting molecule 2 (an endoplasmic calcium sensor) association with Orai1 in dendritic spines. The response to the rise in [Ca2+](o) was larger in spines endowed with a cluster of Orai1 molecules than in spines devoid of Orai1. Transfection of neurons with a dominant negative Orai1 resulted in retarded maturation of dendritic spines, a reduction in synaptic connectivity with afferent neurons and a reduction in the ability to undergo morphological changes following induction of chemical long-term potentiation. Similarly, small interfering RNA (siRNA)-treated neurons had fewer mature dendritic spines, and lower rates of mEPSCs compared to scrambled control siRNA-treated neurons. Thus, influx of calcium through Orai1 channels facilitates the maturation of dendritic spines and the formation of functional synapses in central neurons.
2016
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(2016) Neuroscientist. 22, 5, p. 477-485 Abstract
Calcium stores in the endoplasmic reticulum play important roles in a variety of mammalian cellular functions. However, the multitude of calcium-handling machineries in neurons, including voltage- and ligand-gated channels, calcium-binding proteins, pumps, and transporters, as well as the rapid mobility of calcium ions among different cellular compartments hampered the singling out of calcium stores as a pivotal player in synaptic plasticity. Despite these methodological obstacles, novel molecular and imaging tools afforded a rapid progress in deciphering the role of specific calcium stores in neuronal functions. In the present review, we will address several key issues related to the involvement of ryanodine receptors and the calcium entry channel Orai1 in dendritic spine development and plasticity as well as their derailing in neurodegenerative diseases.
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(2016) Brain Structure and Function. 221, 4, p. 2393-2397 Abstract
In previous studies we and others have found that activation of ryanodine receptors (RyRs) facilitate expression of long-term potentiation (LTP) of reactivity to afferent stimulation in hippocampal slices, with a more pronounced action in the ventral hippocampus. We have also been able to link the involvement of synaptopodin (SP), an actin-binding protein, with neuronal plasticity via its interaction with RyRs. To test this link more directly, we have now compared the ability of ryanodine to convert short-term to LTP in hippocampal slices taken from normal and SP-knockout (SPKO) mice. Indeed, SPKO hippocampus expresses lower concentrations of RyRs and in slices of these mice ryanodine is unable to facilitate conversion of short-term to LTP. These observations link functionally SP with calcium stores.
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(2016) FRONTIERS IN CELLULAR NEUROSCIENCE. 10, MAY, 121. Abstract
Fragile X Syndrome (FXS) is the most common form of inherited cognitive disability. However, functional deficiencies in FX neurons have been described so far almost exclusively in animal models. In a recent study we found several functional deficits in FX neurons differentiated in-vitro from human embryonic stem cells (hESCs), including their inability to fire repetitive action potentials, and their lack of synaptic activity. Here, we investigated the responses of such neurons to pulse application of the neurotransmitter GABA. We found two distinct types of responses to GABA and sensitivity to the GABA-A receptor antagonist bicuculline; type 1 (mature) characterized by non-desensitized responses to GABA as well as a high sensitivity to bicuculline, and type 2 (immature) which are desensitized to GABA and insensitive to bicuculline. Type 1 responses were age-dependent and dominant in mature WT neurons. In contrast, FX neurons expressed primarily type 2 phenotype. Expression analysis of GABA-A receptor subunits demonstrated that this bias in human FX neurons was associated with a significant alteration in the expression pattern of the GABA-A receptor subunits α2 and β2. Our results indicate that FMRP may play a role in the development of the GABAergic synapse during neurogenesis. This is the first demonstration of the lack of a mature response to GABA in human FX neurons and may explain the inappropriate synaptic functions in FXS.
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(2016) Scientific Reports. 6, 24950. Abstract
Unexpectedly, a post-translational modification of DNA-binding proteins, initiating the cell response to single-strand DNA damage, was also required for long-term memory acquisition in a variety of learning paradigms. Our findings disclose a molecular mechanism based on PARP1-Erk synergism, which may underlie this phenomenon. A stimulation induced PARP1 binding to phosphorylated Erk2 in the chromatin of cerebral neurons caused Erk-induced PARP1 activation, rendering transcription factors and promoters of immediate early genes (IEG) accessible to PARP1-bound phosphorylated Erk2. Thus, Erk-induced PARP1 activation mediated IEG expression implicated in long-term memory. PARP1 inhibition, silencing, or genetic deletion abrogated stimulation-induced Erk-recruitment to IEG promoters, gene expression and LTP generation in hippocampal CA3-CA1-connections. Moreover, a predominant binding of PARP1 to single-strand DNA breaks, occluding its Erk binding sites, suppressed IEG expression and prevented the generation of LTP. These findings outline a PARP1-dependent mechanism required for LTP generation, which may be implicated in long-term memory acquisition and in its deterioration in senescence.
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(2016) Proceedings of the National Academy of Sciences of the United States of America. 113, 12, p. 3341-3346 Abstract
Oscillatory activity is widespread in dynamic neuronal networks. The main paradigm for the origin of periodicity consists of specialized pacemaking elements that synchronize and drive the rest of the network; however, other models exist. Here, we studied the spontaneous emergence of synchronized periodic bursting in a network of cultured dissociated neurons from rat hippocampus and cortex. Surprisingly, about 60% of all active neurons were self-sustained oscillators when disconnected, each with its own natural frequency. The individual neuron's tendency to oscillate and the corresponding oscillation frequency are controlled by its excitability. The single neuron intrinsic oscillations were blocked by riluzole, and are thus dependent on persistent sodium leak currents. Upon a gradual retrieval of connectivity, the synchrony evolves: Loose synchrony appears already at weak connectivity, with the oscillators converging to one common oscillation frequency, yet shifted in phase across the population. Further strengthening of the connectivity causes a reduction in the mean phase shifts until zerolag is achieved, manifested by synchronous periodic network bursts. Interestingly, the frequency of network bursting matches the average of the intrinsic frequencies. Overall, the network behaves like other universal systems, where order emerges spontaneously by entrainment of independent rhythmic units. Although simplified with respect to circuitry in the brain, our results attribute a basic functional role for intrinsic single neuron excitability mechanisms in driving the network's activity and dynamics, contributing to our understanding of developing neural circuits.
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(2016) Neural Plasticity. 2016, 2540462. Abstract
Early life adversaries have a profound impact on the developing brain structure and functions that persist long after the original traumatic experience has vanished. One of the extensively studied brain structures in relation to early life stress has been the hippocampus because of its unique association with cognitive processes of the brain. While the entire hippocampus shares the same intrinsic organization, it assumes different functions in its dorsal and ventral sectors (DH and VH, resp.), based on different connectivity with other brain structures. In the present review, we summarize the differences between DH and VH and discuss functional and structural effects of prenatal stress in the two sectors, with the realization that much is yet to be explored in understanding the opposite reactivity of the DH and VH to stressful stimulation.
2015
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(2015) Journal of Neuroscience. 35, 46, p. 15295-15306 Abstract
Fragile X syndrome (FXS), the most common form of inherited mental retardation, is a neurodevelopmental disorder caused by silencing of the FMR1 gene, which in FXS becomes inactivated during human embryonic development. We have shown recently that this process is recapitulated by in vitro neural differentiation of FX human embryonic stem cells (FX-hESCs), derived from FXS blastocysts. In the present study, we analyzed morphological and functional properties of neurons generated from FX-hESCs. Human FX neurons can fire single action potentials (APs) to depolarizing current commands, but are unable to discharge trains of APs. Their APs are of a reduced amplitudes and longer durations than controls. These are reflected in reduced inward Na+ and outward K+ currents. In addition, human FX neurons contain fewer synaptic vesicles and lack spontaneous synaptic activity. Notably, synaptic activity in these neurons can be restored by coculturing them with normal rat hippocampal neurons, demonstrating a critical role for synaptic mechanisms in FXS pathology. This is the first extensive functional analysis of human FX neurons derived in vitro from hESCs that provides a convenient tool for studying molecular mechanisms underlying the impaired neuronal functions in FXS.
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(2015) Hippocampus. 25, 11, p. 1465-1471 Abstract
A transient ischemic episode causes a reduction in evoked EPSPs in hippocampal slices, followed by an NMDA dependent LTP. We explored the relations between ischemic LTP (iLTP) and the more conventional tetanic LTP (tLTP) in CA1 region of slices along the dorsal/ventral axis of the hippocampus. Dorsal hippocampal (DH) slices produced a much larger iLTP than their ventral hippocampal (VH) counterparts. In both regions, iLTP and tLTP shared the same NMDA mediated potentiation, such that one LTP saturated the ability of the other treatment to generate LTP. The smaller LTP in VH was correlated with a lower NMDA-mediated EPSP, and a parallel lower density of NMDA receptors. Calcium permeable AMPA receptors did not contribute to the DH/VH disparity. We conclude that a differential distribution of NMDA receptor subunits along the septotemporal axis of the hippocampus controls the diverse ability to evoke iLTP and tLTP in the two regions and may underlie their characteristic behavioral outputs as well as their differential sensitivity to ischemia.
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(2015) EBioMedicine. 2, 9, p. 1048-1062 Abstract
Down syndrome (DS) mouse models exhibit cognitive deficits, and are used for studying the neuronal basis of DS pathology. To understand the differences in the physiology of DS model neurons, we used dissociated neuronal cultures from the hippocampi of Ts65Dn and Tc1 DS mice. Imaging of [Ca2+]i and whole cell patch clamp recordings were used to analyze network activity and single neuron properties, respectively. We found a decrease of ~30% in both fast (A-type) and slow (delayed rectifier) outward potassium currents. Depolarization of Ts65Dn and Tc1 cells produced fewer spikes than diploid cells. Their network bursts were smaller and slower than diploids, displaying a 40% reduction in δf/f0 of the calcium signals, and a 30% reduction in propagation velocity. Additionally, Ts65Dn and Tc1 neurons exhibited changes in the action potential shape compared to diploid neurons, with an increase in the amplitude of the action potential, a lower threshold for spiking, and a sharp decrease of about 65% in the after-hyperpolarization amplitude.Numerical simulations reproduced the DS measured phenotype by variations in the conductance of the delayed rectifier and A-type, but necessitated also changes in inward rectifying and M-type potassium channels and in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We therefore conducted whole cell patch clamp measurements of M-type potassium currents, which showed a ~90% decrease in Ts65Dn neurons, while HCN measurements displayed an increase of ~65% in Ts65Dn cells. Quantitative real-time PCR analysis indicates overexpression of 40% of KCNJ15, an inward rectifying potassium channel, contributing to the increased inhibition. We thus find that changes in several types of potassium channels dominate the observed DS model phenotype.
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(2015) Biological Psychiatry. 78, 5, p. 315-326 12490. Abstract
Abstract Animal studies confirm earlier anecdotal observations in humans to indicate that early life experience has a profound impact on adult behavior, years after the original experience has vanished. These studies also highlight the role of early life adversaries in the shaping of a disordered brain. Evidence is accumulating to indicate that the epigenome, through which the environment regulates gene expression, is responsible for long-lasting effects of stress during pregnancy on brain and behavior. A possible differential effect of the environment on the epigenome may underlie the observation that only a small fraction of a population with similar genetic background deteriorates into mental disorders. Considerable progress has been made in the untangling of the epigenetic mechanisms that regulate emotional brain development. The present review focuses on the lasting effects of prenatal stress on brain plasticity and cognitive functions in human and rodent models. Although human studies stress the significance of early life experience in functional maturation, they lack the rigor inherent in controlled animal experiments. Furthermore, the analysis of molecular and cellular mechanisms affected by prenatal stress is possible only in experimental animals. The present review attempts to link human and animal studies while proposing molecular mechanisms that interfere with functional brain development.
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(2015) Journal of Neuroscience. 35, 36, p. 12404-12411 Abstract
The ζ-inhibitory peptide (ZIP) is considered a candidate inhibitor of the atypical protein kinase Mζ(PKMζ). ZIP has been shown to reverse established LTP and disrupt several forms of long-term memory. However, recent studies have challenged the specificity of ZIP, as it was reported to exert its effect also in PKMζknock-out mice. These results raise the question of what are the targets of ZIP that may underlie its effect on LTP and memory. Here we report that ZIP as well as its inactive analog, scrambled ZIP, induced a dose-dependent increase in spontaneous activity of neurons in dissociated cultures of rat hippocampus. This was followed by a sustained elevation of intracellular calcium concentration ([Ca2+]i) which could not be blocked by conventional channel blockers. Furthermore, ZIP caused an increase in frequency of mEPSCs followed by an increase in membrane noise in patch-clamped neurons both in culture and in acute brain slices. Finally, at 5–10 μM, ZIP-induced excitotoxic death of the cultured neurons. Together, our results suggest that the potential contribution of cellular toxicity should be taken into account in interpretation of ZIP’s effects on neuronal and behavioral plasticity.
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(2015) PLoS ONE. 10, 5, e0124694. Abstract
Neuronal networks can generate complex patterns of activity that depend on membrane properties of individual neurons as well as on functional synapses. To decipher the impact of synaptic properties and connectivity on neuronal network behavior, we investigate the responses of neuronal ensembles from small (5-30 cells in a restricted sphere) and large (acute hippocampal slice) networks to single electrical stimulation: in both cases, a single stimulus generated a synchronous long-lasting bursting activity. While an initial spike triggered a reverberating network activity that lasted 2-5 seconds for small networks, we found here that it lasted only up to 300 milliseconds in slices. To explain this phenomena present at different scales, we generalize the depression-facilitation model and extracted the network time constants. The model predicts that the reverberation time has a bell shaped relation with the synaptic density, revealing that the bursting time cannot exceed a maximum value. Furthermore, before reaching its maximum, the reverberation time increases sub-linearly with the synaptic density of the network. We conclude that synaptic dynamics and connectivity shape the mean burst duration, a property present at various scales of the networks. Thus bursting reverberation is a property of sufficiently connected neural networks, and can be generated by collective depression and facilitation of underlying functional synapses.
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(2015) Journal of Ethnopharmacology. 163, p. 220-228 Abstract
Ethnopharmacological relevance The aqueous extract of the plant Malmpyrum pratense (Mp), is widely used in traditional medicine as a sedative, yet the biological basis of its action is not known. Aim of the study The effects of Mp on network activity and intrinsic and synaptic properties were studied in cultured hippocampal neurons in an attempt to analyze its mode of action. Materials and methods Dissociated cultures of rat hippocampal neurons were used. Spontaneous network activity was assessed by variations in intracellular [Ca2+] concentrations, reflecting action potential discharges. Individual neuronal synaptic activity was measured by patch clamp recordings from similar neurons. The effect of exposure to different concentrations of Mp and some of its main ingredients was measured. Results Mp produced complex, dose dependent, reversible effects on network activity, increasing it with low concentrations, and decreasing it at high concentrations. Individual flavonoids contained in Mp mimicked the effects of the extract, both for the facilitating and suppressing effects of the extract. Electrophysiologically, Mp caused a reduction in spontaneous activity, but did not affect membrane properties of individual patch clamped neurons, nor did it affect mEPSCs recorded from these neurons. However, a transient increase in reactivity to pulse application of GABA was evident. Conclusions These results suggest that a main sedative effect of Mp is on GABAergic neurotransmission in cultured hippocampal neurons.
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(2015) NeuroToxicology. 47, p. 62-71 Abstract
The effects of chronic exposure to moderate concentrations of ethanol were studied in cultured hippocampal neurons. Network activity, assessed by imaging of [Ca2+]i variations, was markedly suppressed following 5 days of exposure to 0.25-1% ethanol. The reduced activity was sustained following extensive washout of ethanol, but the activity recovered by blockade of inhibition with bicuculline. This reduction of network activity was associated with a reduction in rates of mEPSCs, but not in a change in inhibitory synaptic activity. Chronic exposure to ethanol caused a significant reduction in the density of mature dendritic spines, without an effect on dendritic length or arborization. These results indicate that chronic exposure to ethanol causes a reduction in excitatory network drive in hippocampal neurons adding another dimension to the chronic effects of alcohol abuse.
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(2015) Behavioural Brain Research. 278, p. 559-562 Abstract
Childhood adversity is a prominent risk factor for developing stress-related disorders in adulthood. It can be modeled in rodents, where altered stress responses in adulthood have been observed. The ventral hippocampus is thought to be involved in emotional responses and displays a unique modulation of synaptic plasticity following exposure to stress. Here, we investigated the long-term effect of juvenile stress (at postnatal age of 27-29 days) on synaptic plasticity in the ventral and dorsal hippocampus of adult, 3 month old rats. The rats that had experienced juvenile stress expressed impaired LTP in the dorsal hippocampus (DH), while ventral hippocampus (VH) LTP was facilitated. Furthermore, juvenile stress caused reduced sensitivity to the beta-adrenergic agonist isoproterenol (Iso; 1. μM) in the adult DH, while it enhanced its action in VH slices. Further, juvenile stress resulted in an increase in the expression of beta1-adrenergic receptors in the VH but not in the DH, as revealed by western blot. Taken together, the ventral hippocampus expresses a lasting sensitivity to adrenergic modulation, thus likely to affect the emotional response to challenging situations in adulthood.
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(2015) Stress. 18, 3, p. 319-327 Abstract
Depending on its severity, timing and context, stress has been shown to have a differential regional effect on hippocampal synaptic plasticity. While the focus of attention in most recent studies is on excitatory synapses and generation, modifications of inhibitory synapses and local interneurons cannot be ignored. We have now examined the effects of corticosterone (CORT) on extrinsic afferent and local circuit plasticity of the perforant path on the dentate gyrus (DG) and the ventral hippocampal commissure on CA1. Local circuit activity was measured by responses to paired-pulse stimulation. Control rats expressed afferent long-term potentiation (LTP) and local circuit plasticity in both the DG and CA1. Administration of a high dosage of CORT-reduced paired-pulse inhibition and increased facilitation in DG but not in CA1, whereas administration of a moderate CORT dosage had no effect. Moderate CORT doses caused enhancement of LTP in the DG but not in CA1, while high CORT doses converted LTP to long-term depression in the CA1 but had no effect in the DG. CORT blocked theta burst stimulation-induced local circuit plasticity otherwise found in control DG. These findings suggest that elevation of the level of CORT results in a regionally differentiated physiological response. In addition, the results indicate that CORT affects aspects of local circuit activity and plasticity in the DG but less so in the CA1. It is possible that these differentiated alterations underlie some of the behavioral consequences and memory processes under stressful conditions.
2014
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Pathology of Down Syndrome manifested in network and single neuron properties of hippocampal neuronal cultures(2014) Abstract
Background: Down Syndrome (DS) is the most common chromosomal abnormality in humans, with many phenotypic characteristics-mental retardation is probably the most prominent one. Embryonic hippocampal neuronal cultures from two mouse models (TC1 and Ts65Dn) for DS were compared to control littermate cultures to detect possible cellular phenotypes of DS. Network behaviour was assessed using calcium imaging, and whole cell patch clamp technique was used to measure differences in single cell properties. Results: Calcium imaging, measured with fluo4, revealed smaller amplitude, shorter duration network bursts, with no change in basal calcium level, which was measured using Fura-2. Baclofen, a GABA-B agonist, suppressed network bursts with lower concentration in DS networks than in control networks. In patch clamped neurons, the threshold for excitation, after-hyperpolarization (AHP) amplitude and input resistance were reduced, while spike height was increased in DS cells compared to controls. Following blockage of sodium currents we found that potassium currents (slow and fast) are reduced in DS cells. Network activity was different with electrophysiological tools as well: bursts were shorter, the total amount of time the network spent bursting was shorter, and network bursts were less synchronized. Most of the differences appeared in both mouse models. Conclusions: DS cells were different both in the basic cell properties and in the assembly into a network. The changes in spike properties, along with reduced potassium currents all point to changes in potassium channels. We speculate that this may be due to possible overexpression of two potassium channel regulators, KCNE1 and KCNE2, whose genes reside on chromosome 21. The altered network activity along with baclofen experiments may be caused by increased inhibition. Two types of inwardly rectifying potassium channels genes reside on chromosome 21: KCNJ6, KCNJ15, and may be the cause of this increased network inhibition.
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(2014) Journal of Neuroscience. 34, 35, p. 11641-11651 Abstract
The role of synaptopodin (SP), an actin-binding protein residing in dendritic spines, in synaptic plasticity was studied in dissociated cultures of hippocampus taken from control and SP knock-out (SPKO) mice. Unlike controls, SPKO cultures were unable to express changes in network activity or morphological plasticity after intense activation of their NMDA receptors. SPKO neurons were transfected with SP-GFP, such that the only SP resident in these neurons is the fluorescent species. The localization and intensity of the transfected SP were similar to that of the native one. Because less than half of the spines in the transfected neurons contained SP, comparisons were made between SP-containing (SP(+)) and SP lacking (SP(-)) spines in the same dendritic segments. Synaptic plasticity was induced either in the entire network by facilitation of the activation of the NMDA receptor, or specifically by local flash photolysis of caged glutamate. After activation, spines that were endowed with SP puncta were much more likely to expand than SP(-) spines. The spine expansion was suppressed by thapsigargin, which disables calcium stores. The mechanism through which SP may promote plasticity is indicated by the observations that STIM-1, the sensor of calcium concentration in stores, and Orai-1, the calcium-induced calcium entry channel, are colocalized with SP, in the same dendritic spines. The structural basis of SP is likely to be the spine apparatus, found in control but not in SPKO cells. These results indicate that SP has an essential, calcium store-related role in regulating synaptic plasticity in cultured hippocampal neurons.
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(2014) Frontiers in Neuroanatomy. 8, 64. Abstract
Despite decades of research, the role of calcium stores in dendritic spines structure, function and plasticity is still debated. The reasons for this may have to do with the multitude of overlapping calcium handling machineries in the neuron, including stores, voltage and ligand gated channels, pumps and transporters. Also, different cells in the brain are endowed with calcium stores that are activated by different receptor types, and their differential compartmentalization in dendrites, spines and presynaptic terminals complicates their analysis. In the present review we address several key issues, including the role of calcium stores in synaptic plasticity, their role during development, in stress and in neurodegenerative diseases. Apparently, there is increasing evidence for a crucial role of calcium stores, especially of the ryanodine species, in synaptic plasticity and neuronal survival.
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(2014) Neurodegenerative Diseases. 13, 2-3, p. 135-138 Abstract
Background: A possible contributing factor to the development of cognitive deficits in Alzheimer's disease (AD) patients involves the exposure to early life stress. Objective: We explored the impact of stress on synaptic plasticity (long-term potentiation, LTP) of 6-month-old triple-transgenic mice (3×Tg-AD). Methods: 3×Tg-AD and control (NonTg) mice were exposed to three stressors at the age of 2 and 4 months. Excitatory postsynaptic potentials were recorded in the stratum radiatum of the CA1 region of hippocampal slices, in a two-pathway paradigm. Results: Slices taken from 3×Tg-AD mice exhibited significant deficits in LTP compared with NonTg slices. Early stress led to a further decrease in LTP in these mice, while it did not affect NonTg mice. LTP in 3×Tg-AD and stressed 3×Tg-AD mice was rescued by pre-exposure to 0.2 μM ryanodine. In an attempt to find a molecular correlate for the effects of stress in the 3×Tg-AD mice, we found that stressed mice have an altered ratio of Aβ42/40 both in the cortex and hippocampus. Conclusions: Stress experiences in young adults may accelerate the cognitive loss in AD mice, adding another dimension to the plethora of factors that lead to AD.
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(2014) Physiological Reviews. 94, 1, p. 141-188 Abstract
The introduction of high-resolution time lapse imaging and molecular biological tools has changed dramatically the rate of progress towards the understanding of the complex structurefunction relations in synapses of central spiny neurons. Standing issues, including the sequence of molecular and structural processes leading to formation, morphological change, and longevity of dendritic spines, as well as the functions of dendritic spines in neurological/psychiatric diseases are being addressed in a growing number of recent studies. There are still unsettled issues with respect to spine formation and plasticity: Are spines formed first, followed by synapse formation, or are synapses formed first, followed by emergence of a spine? What are the immediate and long-lasting changes in spine properties following exposure to plasticity-producing stimulation? Is spine volume/shape indicative of its function? These and other issues are addressed in this review, which highlights the complexity of molecular pathways involved in regulation of spine structure and function, and which contributes to the understanding of central synaptic interactions in health and disease.
2013
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(2013) PLoS ONE. 8, 11, e75988. Abstract
The effects of ethanol on neuronal network activity were studied in dissociated cultures of rat hippocampus. Exposure to low (0.25-0.5%) ethanol concentrations caused an increase in synchronized network spikes, and a decrease in the duration of individual spikes. Ethanol also caused an increase in rate of miniature spontaneous excitatory postsynaptic currents. Higher concentrations of ethanol eliminated network spikes. These effects were reversible upon wash. The effects of the high, but not the low ethanol were blocked by the GABA antagonist bicuculline. The enhancing action of low ethanol was blocked by apamin, an SK potassium channel antagonist, and mimicked by 1-EBIO, an SK channel opener. It is proposed that in cultured hippocampal networks low concentration of ethanol is associated with SK channel activity, rather than the GABAergic receptor.
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(2013) Journal of Neurophysiology. 110, 2, p. 279-285 Abstract
Long-term effects of stress during pregnancy on brain and behavior have been analyzed extensively in recent years. These effects include changes in emotional behavior, a reduction in learning capacity, and ability to generate long-term potentiation (LTP) in the offspring. In earlier studies, we and others have described a difference in ability to express LTP in dorsal and ventral sectors of the hippocampus (DH and VH, respectively) and its modification by prior stress. We now found that norepinephrine (NE) facilitated conversion of short-term potentiation to LTP in the normal DH but not in VH. Prenatal stress (PS) switched the locus of the facilitating action of NE from the DH to the VH. The effects of NE are likely to be mediated by activation of calcium stores. PS also facilitated (S)-3,5-dihydroxyphenylglycine hydrate (DHPG)-induced LTD in the VH, assumed to be mediated by release of calcium from stores. These observations have important implications for the role of the hippocampus in cognitive and emotional memories.
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(2013) Biological Psychiatry. 73, 11, p. 1095-1102 Abstract
Background: Long-term effects of stress during pregnancy on brain and behavior have been analyzed extensively in recent years. One major problem with these studies is the inability to separate between the net effects of the prenatal stress (PS) and the effects of the stressed mother and siblings on the newborn animals. Methods: To address these issues, we studied morphological and electrophysiological properties of neurons in dissociated cultures of the hippocampus taken from newborn PS rats. We complemented these studies with experiments on behaving rats and recordings from slices taken from PS rats and their control rats. Results: While the density of cultured neurons was not different between PS and control rats, there were fewer glutamic acid decarboxylase-positive neurons in the former cultures. Additionally, cells taken from PS pups developed more extensive dendrites than control animals. These differences were correlated with a higher rate of synchronous activity in the PS cultures and a lower rate of spontaneous miniature inhibitory postsynaptic current activity. There were no differences in the excitatory synaptic currents or the passive and active properties of the recorded neurons in the two groups. Young PS rats were more motile in open field and elevated plus maze than control rats, and they learned faster to navigate in a water maze. Slices taken from hippocampus of PS rats expressed less paired-pulse inhibition than slices from control rats. Conclusions: These results indicate that PS affects network properties of hippocampal neurons, by reducing gamma-aminobutyric acidergic inhibition.
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(2013) Developmental Biology. 374, 1, p. 32-45 Abstract
Fragile X Syndrome (FXS) is the most common form of inherited intellectual disability, caused by developmentally regulated inactivation of FMR1, leading to the absence of its encoded protein FMRP. We have previously shown that undifferentiated Fragile X human Embryonic Stem Cells (FX-hESCs) express FMRP, despite the presence of the full FMR1 mutation (>200 CGG repeats). We describe here, for the first time, in-vitro differentiation of FX-hESCs into neurons progressively inactivating FMR1. Abnormal neurogenesis and aberrant gene expression were found already during early stages of differentiation, leading to poor neuronal maturation and high gliogenic development. Human FX neurons fired action potentials but displayed poor spontaneous synaptic activity and lacked reactivity to glutamate. Our dynamic FX-hESCs model can contribute to the understanding of the sequence of developmental events taking place during neurogenesis and how they are altered in FXS individuals, leading to intellectual disability. Furthermore, it may shed light over the striking phenotypic features characterizing FXS in human.
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(2013) FRONTIERS IN CELLULAR NEUROSCIENCE. FEB, Abstract
Thrombin is a serine protease playing an essential role in the blood coagulation cascade. Recent work, however, has identified a novel role for thrombin-mediated signaling pathways in the central nervous system. Binding of thrombin to protease-activated receptors (PARs) in the brain appears to have multiple actions affecting both health and disease. Specifically, thrombin has been shown to lead to the onset of seizures via PAR-1 activation. In this perspective article, we review the putative mechanisms by which thrombin causes seizures and epilepsy. We propose a potential role of PAR-1 antagonists and novel thrombin inhibitors as new, possible antiepileptic drugs.
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(2013) Neurobiology of Disease. 50, 1, p. 171-178 Abstract
Seizures are a common outcome of cerebrovascular events as well as of traumatic brain injuries. Thrombin, a protease-activated receptor (PAR) agonist, has been implicated in the onset of seizures in these settings, yet its mode of action is not entirely clear. In this study, the effect of thrombin and a PAR-1 agonist on neuronal excitability and synaptic currents was assessed by whole cell-patch recordings of pyramidal neurons in rat hippocampal slices. In addition, PAR-1 distribution in different hippocampal regions was assessed using immunohistochemistry. We found that thrombin caused an increase in spontaneous action potential discharges of CA3 but not of CA1 pyramidal neurons. When excitatory synaptic activity was blocked, thrombin caused a marked reduction in spontaneous IPSCs in CA3 neurons and a marked increase in the frequency of IPSCs in CA1 neurons. These effects are likely to be local, as they were reproduced in TTX-treated slices. In parallel, thrombin increased both the frequency and the amplitude of mEPSCs only in CA3 neurons. These effects were blocked by a selective PAR-1 antagonist. The higher expression of PAR-1 in stratum lucidum of CA3 is correlated with the effects of thrombin in this region. These results suggest that thrombin triggers the generation of epileptic seizures by reducing the inhibitory and increasing the excitatory tone in CA3 neurons, providing a novel insight to the pathophysiology of seizures following cerebrovascular events and present new avenues for therapeutic intervention.
2012
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(2012) Cerebral Cortex. 22, 11, p. 2519-2528 Abstract
Protein kinase M zeta (PKMζ), an atypical isoform of protein kinase C (PKC), has been implicated in long-term maintenance of neuronal plasticity and memory. However, the cellular machinery involved in these functions has yet to be elucidated. Here, we investigated the effects of PKMζ overexpression on the morphology and function of cortical neurons in primary cultures. Transfection with a plasmid construct expressing the PKMζ gene modified the distribution of spine morphologies and reduced spine length, while leaving total spine density and dendritic branching unchanged. A significant increase in magnitude but not frequency of miniature excitatory post synaptic currents was detected in the PKMζ overexpressing cells. These results suggest that PKMζ is involved in regulation of dendritic spine structure and function, which may underlie its role in long-term synaptic and behavioral plasticity.
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(2012) Hippocampus. 22, 7, p. 1635-1644 Abstract
The effects of low concentrations of caffeine and ryanodine on field excitatory postsynaptic potentials (EPSPs) and long-term potentiation (LTP) were studied in CA1 region of slices of dorsal and ventral hippocampus (DH and VH, respectively). There was a striking difference between the two regions in the magnitude of effect of both drugs, as well as the ability to interact with a tetanic stimulation to produce LTP. Low concentration of caffeine (1 mM) produced a postsynaptic increase in the slope of population EPSPs in VH, and facilitated LTP in this region. Low concentration of ryanodine (0.2 μM) was able to convert short-term potentiation (STP) to LTP in VH only. These effects are postsynaptic and they reflect a higher concentration of ryanodine receptors (RyRs) in the VH compared to the DH.
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(2012) Experimental Neurology. 234, 1, p. 200-207 Abstract
The role of stress hormones in the initiation of epileptic seizures has been studied extensively in the past decade, with conflicting observations, from suppression to exacerbation of spontaneous seizures. We have now studied the effects of an acute stress on reactivity of juvenile rats to kainic acid (KA), which produces epileptic seizures. With a short (30. s) stress-KA delay, stress exacerbated epilepsy via activation of mineralocorticosterone receptors (MR). With a long (60. min) stress-KA delay, seizures were suppressed through activation of a glucocorticosterone receptor (GR). In a parallel study with CA1 pyramidal neurons in acute hippocampal slices, activation of MRs reduced the frequency of mIPSCs, whereas activation of GRs produced a slow onset, 2.5 fold increase in amplitudes of mIPSCs. GR effects were not mediated by protein synthesis, but did require activation of some protein kinases. These experiments suggest that stress can either facilitate or suppress seizures, in a time and receptor dependent manner.
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(2012) FRONTIERS IN CELLULAR NEUROSCIENCE. MARCH, 12. Abstract
Several new observations have shifted the view of the hippocampus from a structure in charge of cognitive processes to a brain area that participates in the formation of emotional memories, in addition to its role in cognition. Specifically, while the dorsal hippocampus is involved in the processing of cognitive memories; the ventral sector is mainly associated with the control of behavioral inhibition, stress, and emotional memory. Stress is likely to cause this switch in control of hippocampal functions by modulating synaptic plasticity in the dorsal and ventral sectors of the hippocampus through the differential activation of mineralocorticosteroid or glucocorticosteroid receptors. Herein, we will review the effects of stress hormones on synaptic plasticity in the hippocampus and outline the outcomes on stress-related global functions of this structure. We propose that steroid hormones act as molecular switches: by changing the strength of synaptic connectivity in the hippocampus following stress, they regulate the routes by which the hippocampus is functionally linked to the rest of the brain. This hypothesis has profound implications for the pathophysiology of psychiatric disorders.
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(2012) Hippocampus. 22, 2, p. 267-275 Abstract
The ventral hippocampus (VH) was recently shown to express lower magnitude LTP compared to the dorsal hippocampus (DH). Exposure to acute stress reversed this difference, and VH slices from stressed rats expressed larger LTP than that produced in the DH, which was reduced by stress. In an attempt to uncover the mechanisms responsible for this differential action, we found that activation of mineralocorticosteroid receptors (MR) by aldosterone mimics the effects of stress in the VH, to facilitate LTP. We also found that aldosterone reduces GABAergic inhibition in both the DH and VH. We now examined if the reduction in inhibition caused by MRs can underlie the altered LTP in the VH. Rat hippocampal slices were recorded before and after exposure to the GABA antagonist bicuculline and to aldosterone. As expected, blockade of GABA with bicuculline enhanced LTP in both DH and VH. However, its effect did not occlude that of aldosterone in the VH, indicating that the latter drug does not operate by blockade of inhibition. Furthermore, the NMDA receptor antagonist APV blocked LTP induced in the presence of bicuculline, but did not block LTP facilitation by aldosterone, indicating that the effect of aldosterone is not mediated by the conventional NMDA-dependent LTP generating mechanism. Furthermore, rapid effects of aldosterone on LTP were blocked by the L-type calcium channel antagonist nifedipine, indicating that aldosterone facilitates calcium influx via nifedipine-sensitive channels, to enhance LTP in the VH. The locus of effect of aldosterone may be the presynaptic terminal, as it caused a marked facilitation of paired pulse potentiation in the VH but not in the DH. These experiments confirm and extend previous suggestions for the effects of MRs on neuronal plasticity in the hippocampus.
2011
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(2011) Journal Of Physiology-London. 589, 24, p. 5987-5995 Abstract
The presence of calcium stores and their function in dendritic spines is still unsettled. We have now studied the kinetics of calcium released inside dendritic spines of cultured rat hippocampal neurons by flash photolysis of caged calcium. Photolysis of calcium produced a fast rise in [Ca 2+] i, followed by a variable decay. We were able to correlate the decay of elevated [Ca 2+] i with the presence of synaptopodin (SP), an actin-binding protein, in the spines; spines containing SP generated the same initial [Ca 2+] i transient, but their decay time was significantly slower and more complex than that of SP-negative ones. The altered decay kinetics of the flash-elevated [Ca 2+] i transient was blocked by thapsigargin or cyclopiazonic acid (CPA), indicating that this kinetic change is due to compartmentalized release of calcium from intracellular stores. Thus, SP plays a pivotal role in the calcium store-associated ability of spines to locally tune calcium kinetics.
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(2011) Journal of Neurophysiology. 106, 5, p. 2314-2321 Abstract
Synchronized network activity is an essential attribute of the brain. Yet the cellular mechanisms that determine the duration of network bursts are not fully understood. In the present study, synchronized network bursts were evoked by triggering an action potential in a single neuron in otherwise silent microcultures consisting of 4-30 hippocampal neurons. The evoked burst duration, ~ 2 s, depended on the recovery time after a previous burst. While interburst intervals of 35 s enabled full-length bursts, they were shortened by half at 5-s intervals. This reduction in burst duration could not be attributed to postsynaptic parameters such as glutamate receptor desensitization, accumulating afterhyperpolarization, inhibitory tone, or sodium channel inactivation. Reducing extracellular Ca 2+ concentration ([Ca 2+] o) relieved the effect of short intervals on burst duration, while depletion of synaptic vesicles with α-latrotoxin gradually eliminated network bursts. Finally, a transient exposure to high [K +] o slowed down the recovery time following a burst discharge. We conclude that the limiting factor regulating burst duration is most likely the depletion of presynaptic resources.
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(2011) PLoS ONE. 6, 10, e25919. Abstract
Spontaneous activity is an essential attribute of neuronal networks and plays a critical role in their development and maintenance. Upon blockade of activity with tetrodotoxin (TTX), neurons degenerate slowly and die in a manner resembling neurodegenerative diseases-induced neuronal cell death. The molecular cascade leading to this type of slow cell death is not entirely clear. Primary post-natal cortical neurons were exposed to TTX for up to two weeks, followed by molecular, biochemical and immunefluorescence analysis. The expression of the neuronal marker, neuron specific enolase (NSE), was down-regulated, as expected, but surprisingly, there was a concomitant and striking elevation in expression of tissue-type plasminogen activator (tPA). Immunofluorescence analysis indicated that tPA was highly elevated inside affected neurons. Transfection of an endogenous tPA inhibitor, plasminogen activator inhibitor-1 (PAI-1), protected the TTX-exposed neurons from dying. These results indicate that tPA is a pivotal player in slowly progressing activity deprivation-induced neurodegeneration.
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(2011) Brain Behavior And Immunity. 25, 5, p. 1008-1016 Abstract
Recent studies indicate that astrocytes play an integral role in neural and synaptic functioning. To examine the implications of these findings for neurobehavioral plasticity we investigated the involvement of astrocytes in memory and long-term potentiation (LTP), using a mouse model of impaired learning and synaptic plasticity caused by genetic deletion of the interleukin-1 receptor type I (IL-1RI). Neural precursor cells (NPCs), derived from either wild type (WT) or IL-1 receptor knockout (IL-1rKO) neonatal mice, were labeled with bromodeoxyuridine (BrdU) and transplanted into the hippocampus of either IL-1rKO or WT adult host mice. Transplanted NPCs survived and differentiated into astrocytes (expressing GFAP and S100β), but not to neurons or oligodendrocytes. The NPCs-derived astrocytes from WT but not IL-1rKO mice displayed co-localization of GFAP with the IL-1RI. Four to twelve weeks post-transplantation, memory functioning was examined in the fear-conditioning and the water maze paradigms and LTP of perforant path-dentate gyrus synapses was assessed in anesthetized mice. As expected, IL-1rKO mice transplanted with IL-1rKO cells or sham operated displayed severe memory disturbances in both paradigms as well as a marked impairment in LTP. In contrast, IL-1rKO mice transplanted with WT NPCs displayed a complete rescue of the impaired memory functioning as well as partial restoration of LTP. These findings indicate that astrocytes play a critical role in memory functioning and LTP, and specifically implicate astrocytic IL-1 signaling in these processes. The results suggest novel conceptualization and therapeutic targets for neuropsychiatric disorders characterized by impaired astrocytic functioning concomitantly with disturbed memory and synaptic plasticity.
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(2011) Biological Psychiatry. 69, 8, p. 748-753 Abstract
Background: The ventral hippocampus (VH) was recently shown to express lower magnitude long-term potentiation (LTP) compared with the dorsal hippocampus (DH). Exposure to acute stress reversed this difference, and VH slices from stressed rats expressed larger LTP than that produced in the DH, which was reduced by stress. Stressful experience in adolescence has been shown to produce long-lasting effects on animal behavior and on ability to express LTP/long-term depression (LTD) of reactivity to afferent stimulation in the adult. We are interested in possible interactions between juvenile and adult stress in their effects of adult plasticity. Methods: We studied the effects of a composite juvenile (2830 days) stress, followed by a reminder stressful experience in the young adult (60 days) rat, on the ability to produce LTP and LTD in CA1 region of slices of the VH and DH. Results: Juvenile or adult stress produced a transient decrease in ability to express LTP in DH and a parallel increase in LTP in VH. Stress in the young adult after juvenile stress produced a striking prolongation of the DH/VH disparity with respect to the ability to express both LTP and LTD into the adulthood of the rat. Conclusions: These results have important implications for the impact of juvenile stress on adult neuronal plasticity and on the understanding the functions of the different sectors of the hippocampus.
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(2011) Proceedings of the National Academy of Sciences of the United States of America. 108, 12, p. 5081-5086 Abstract
Neurons, astrocytes, and blood vessels are organized in functional "neurovascular units" in which the vasculature can impact neuronal activity and, in turn, dynamically adjust to its change. Here we explored different mechanisms by which VEGF, a pleiotropic factor known to possess multiple activities vis-à-vis blood vessels and neurons, may affect adult neurogenesis and cognition. Conditional transgenic systems were used to reversibly overexpress VEGF or block endogenous VEGF in the hippocampus of adult mice. Importantly, this was done in settings that allowed the uncoupling of VEGF-promoted angiogenesis, neurogenesis, and memory. VEGF overexpression was found to augment all three processes, whereas VEGF blockade impaired memory without reducing hippocampal perfusion or neurogenesis. Pertinent to the general debate regarding the relative contribution of adult neurogenesis to memory, we found that memory gain by VEGF overexpression and memory impairment by VEGF blockade were already evident at early time points at which newly added neurons could not yet have become functional. Surprisingly, VEGF induction markedly increased in vivo long-term potentiation (LTP) responses in the dentate gyrus, and VEGF blockade completely abrogated LTP. Switching off ectopic VEGF production resulted in a return to a normal memory and LTP, indicating that ongoing VEGF is required to maintain increased plasticity. In summary, the study not only uncovered a surprising role for VEGF in neuronal plasticity, but also suggests that improved memory by VEGF is primarily a result of increasing plasticity of mature neurons rather than the contribution of newly added hippocampal neurons.
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(2011) Acta Neurovegetativa. 118, 3, p. 445-451 Abstract
Spiny striatal GABAergic neurons receive most of their excitatory input from the neocortex. In culture, striatal neurons form inhibitory connections, but the lack of intrinsic excitatory afferents prevents the development of spontaneous network activity. Addition of cortical neurons to the striatal culture provides the necessary excitatory input to the striatal neurons, and in the presence of these neurons, striatal cultures do express spontaneous network activity. We have confirmed that cortical neurons provide excitatory drive to striatal neurons in culture using paired recording from cortical and striatal neurons. In the presence of tetrodotoxin (TTX), which blocks action potential discharges, the connections between cortical and striatal neurons are still formed, and in fact synaptic currents generated between them when TTX is removed are far larger than in control, undrugged cultures. Interestingly, the continuous presence of TTX in the co-culture caused striatal cell death. These observations indicate that the mere presence of cortical neurons is not sufficient to preserve striatal neurons in culture, but their synchronous activity, triggered by cortical excitatory synapses, is critical for the maintenance of viability of striatal neurons. These results have important implications for understanding the role of activity in neurodegenerative diseases of the striatum.
2010
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(2010) Hippocampus. 20, 12, p. 1332-1338 Abstract
Recent observations have caused a drastic shift in the conception of the hippocampus as a homogeneous structure that subserves cognitive functions, either spatial maps or short term episodic memory, to a structure that is associated with both cognitive and emotional functions. In fact, the assignment of cognitive functions to the hippocampus is restricted to its dorsal sector. In contrast, the ventral hippocampus (VH) appears to be associated with control of behavioral inhibition, stress and emotional memory, but not with strictly cognitive functions. Curiously, the VH but not the dorsal hippocampus (DH) is associated with the development of affective disorders. In line with these collective observations, we and others have found that the ability to evoke a sustained long term potentiation (LTP), a cellular correlate of learning and memory, is much lower in the VH compared to the DH. Strikingly, acute stress as well as direct exposure to corticosterone affect DH and VH in an opposite manner; causing facilitation of LTP in the VH and its suppression in the DH. This double dissociative action results from activation of different steroid receptor species in the DH and VH. Since the DH and VH differ in efferent connectivity, and since the strength of LTP can be considered as an indicator of strength of synaptic connectivity, these results suggest that stress regulates the routes by which the hippocampus is functionally linked to the rest of the brain such that under stress, the ventral route to the amygdala is enabled while the dorsal route to the neocortex is suppressed. This selective routing may underlie the complex outcome of stress on hippocampal and amygdala physiology and behavior.
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(2010) Journal of Neurophysiology. 104, 2, p. 1052-1060 Abstract
Ivenshitz M, Segal M. Neuronal density determines network connectivity and spontaneous activity in cultured hippocampus. J Neurophysiol 104: 1052-1060, 2010. First published June 16, 2010; doi: 10.1152/jn.00914.2009. The effects of neuronal density on morphological and functional attributes of the evolving networks were studied in cultured dissociated hippocampal neurons. Plating at different densities affected connectivity among the neurons, such that sparse networks exhibited stronger synaptic connections between pairs of recorded neurons. This was associated with different patterns of spontaneous network activity with enhanced burst size but reduced burst frequency in the sparse cultures. Neuronal density also affected the morphology of the dendrites and spines of these neurons, such that sparse neurons had a simpler dendritic tree and fewer dendritic spines. Additionally, analysis of neurons transfected with PSD95 revealed that in sparse cultures the synapses are formed on the dendritic shaft, whereas in dense cultures the synapses are formed primarily on spine heads. These experiments provide important clues on the role of neuronal density in population activity and should yield new insights into the rules governing neuronal network connectivity.
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(2010) European Journal of Neuroscience. 31, 12, p. 2178-2184 Abstract
An emerging view of structure-function relations of synapses in central spiny neurons asserts that larger spines produce large synaptic currents and that these large spines are persistent ('memory') compared to small spines which are transient. Furthermore, 'learning' involves enlargement of small spine heads and their conversion to being large and stable. It is also assumed that the number of spines, hence the number of synapses, is reflected in the frequency of miniature excitatory postsynaptic currents (mEPSCs). Consequently, there is an assumption that the size and number of mEPSCs are closely correlated with, respectively, the physical size of synapses and number of spines. However, several recent observations do not conform to these generalizations, necessitating a reassessment of the model: spine dimension and synaptic responses are not always correlated. It is proposed that spines are formed and shaped by ongoing network activity, not necessarily by a 'learning' event, to the extent that, in the absence of such activity, new spines are not formed and existing ones disappear or convert into thin filopodia. In the absence of spines, neurons can still maintain synapses with afferent fibers, which can now terminate on its dendritic shaft. Shaft synapses are likely to produce larger synaptic currents than spine synapses. Following loss of their spines, neurons are less able to cope with the large synaptic inputs impinging on their dendritic shafts, and these inputs may lead to their eventual death. Thus, dendritic spines protect neurons from synaptic activity-induced rises in intracellular calcium concentrations.
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(2010) International Journal of Developmental Neuroscience. 28, 2, p. 195-205 Abstract
Status epilepticus occurring in early postnatal development protects CA1 hippocampal neurons, the region most sensitive to seizure-induced injury in the developing brain. Here, we developed a "two hit" model in dissociated cultures of the rat hippocampus to test whether pre-exposure of immature neurons to high concentrations of glutamate, N-methyl-d-aspartic acid (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) during a relatively resistant period prevents neurons from dying following a second exposure to the same chemicals after neurons mature and become highly vulnerable to excitatory amino acids (EAAs). Cultures were exposed to varied doses of glutamate, NMDA, or AMPA for 48 h at 5 DIV and again at 14 DIV for 5, 15, or 30 min. NeuN immunohistochemistry showed early exposure to glutamate (500 μM) killed approximately half of the neurons (52 ± 8.6%) compared to the marked depletion that occurs after one exposure at 14 DIV (98 ± 0.79%). When cultures were first challenged with moderate doses of glutamate (200 μM) followed by the high dose 7 days later, a significant population of neurons was spared (35.3 ± 1.2%). Similarly, pre-exposure to maximal doses of NMDA (100 μM) increased the proportion of surviving cells following the second challenge. In contrast, AMPA (100 μM) was equally toxic after early or late applications and did not protect from the second exposure. GluR1 subunit expression was markedly decreased at 48 h after one or two exposures to 200 μM glutamate (by 44.57 ± 3.6%, 45.07 ± 3.69%) whereas GluR2 subunit expression was reduced by a lesser amount (25.7 57 ± 3.8%). Confocal microscopy showed that one or two exposures to NMDA caused GluR2 protein to downregulate even further whereas parvalbumin (PV) was dramatically increased in the same neurons by over four-fold. On the other hand, calbindin (CB) immunoreactivity was nearly absent after the first exposure to 500 μM glutamate. These data indicate that early, transient exposure to certain EAAs at high doses can induce long-lasting neuroprotection. Alterations in the GluR1/GluR2 ratio as well as differential expression of specific calcium binding proteins may contribute to this neuroprotection.
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(2010) Neuroscientist. 16, 2, p. 125-131 Abstract
The spine apparatus (SA) is an essential component of mature dendritic spines of cortical and hippocannpal neurons, yet its functions are still enigmatic. Synaptopodin (SP), an actin-binding protein, colocalizes with the SA. Hippocampal neurons in SP-knockout mice lack SA, and they express lower LTP. SP probably plays a role in synaptic plasticity, but only recently it is being linked mechanistically to synaptic functions. These authors and others have studied endogenous and transfected SP in dendritic spines of cultured hippocampal neurons. They found that spines containing SP generate twice as large responses to flash photolysis of caged glutamate than SP-negative ones. An N-methyl-D-aspartate receptor mediated chemical LTP caused accumulation of GFP-GIuR I in spine heads of control but not of shRNA transfected, SP-deficient neurons. SP is linked to calcium stores, because their pharmacological blockade eliminated SP-related enhancement of glutamate responses. Furthermore, release of calcium from stores produces an SP-dependent delivery of GIuR I into spines. Thus, SP plays a crucial role in the calcium store-associated ability of neurons to undergo long-term plasticity.
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(2010) Thescientificworldjournal. 10, p. 462-469 Abstract
Stress, via release of steroid hormones, has been shown to affect several cellular functions in the brain, including synaptic receptors and ion channels. As such, corticosteroids were reported to modulate plasticity, expressed as long-term changes in reactivity to afferent stimulation. The classical view of the effects of stress on synaptic plasticity and cognitive functions assumes an inverted U-shape curve, such that a low stress level facilitates and a high stress level (i.e., corticosterone levels) impairs cognitive functions. This universal view has been challenged recently in a series of studies that show that stress and corticosterone have immediate and opposite effects on the ability to express long-term potentiation (LTP) in the dorsal and ventral sectors of the hippocampus. This differential role of stress may be related to the different functions associated with these sectors of the hippocampus. Herein, we review the known effects of stress hormones on cellular functions and outline the role of molecular mechanisms in stress-related global functions of the hippocampus.
2009
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(2009) Journal of Neural Transmission. 116, 11, p. 1363-1369 Abstract
Central neurons express persistent spontaneous electrical network activity both in the developing brain in vivo as well as in dissociated cultures. This electrical activity is important for the formation of connections among neurons, and for their survival. Prolonged suppression of the spontaneous activity using the sodium channel blocker tetrodotoxin (TTX) causes the death of the cultured neurons. In the present study, we investigated molecular mechanisms that may underlie the activity-suppressed slow degeneration of cortical neurons in culture. Already after 6-7 days of exposure to TTX, neurons begin to express apoptotic vacuoles and shrunken dendrites. Eventually, neurons activate p53, caspase-3 and BAX, hallmarks of neuronal apoptosis, before they die. This death is restricted to neurons, and no parallel process is seen in glial cells that co-exist in the culture. These experiments may lead to a better understanding of slow neuronal death, akin to that found in neurodegenerative diseases of the brain.
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(2009) European Journal of Neuroscience. 30, 6, p. 998-1010 Abstract
The neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been shown to promote excitatory and inhibitory synapse development. However, a quantitative analysis of their influence on connectivity has proven in general difficult to achieve. In this work we use a novel experimental approach based on percolation concepts that provides a quantification of the average number of connections per neuron. In combination with electrophysiological measurements, we characterize the changes in network connectivity induced by BDNF and NT-3 in rat hippocampal cultures. We show that, on the one hand, BDNF and NT-3 accelerate the maturation of connectivity in the network by about 17 h. On the other hand, BDNF and NT-3 increase the number of excitatory input connections by a factor of about two, but without modifying the number of inhibitory input connections. This scenario of a dominant effect on the excitation is supported by the analysis of spontaneous population bursts in cultures treated with either BDNF or NT-3, which show burst amplitudes that are insensitive to the blockade of inhibition. A leaky integrate-and-fire model reproduces the experimental results well.
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(2009) Journal of Neurochemistry. 110, 4, p. 1203-1213 Abstract
Networks of neurons express persistent spontaneous network activity when maintained in dissociated cultures. Prolonged blockade of the spontaneous activity with tetrodotoxin (TTX) causes the eventual death of the neurons. In this study, we investigated some molecular mechanisms that may underlie the activity-suppressed slow degeneration of cortical neurons in culture. Already after 3-4 days of exposure to TTX, well before the neurons die, they began to express markers that lead to their eventual death, 7-10 days later. There was a reduction in glutamate receptor (GluR2) expression, a persistent increase in intracellular calcium concentration, activation of calpain, and an increase in spectrin breakdown products. At this point, blockade of GluR2-lacking GluR1 or calpain (either with a selective antagonist or through the natural regulator of calpain, calpastatin), protected cells from the toxic action of TTX. Subsequently, mitochondria lost their normal elongated shape as well as their membrane potential. Eventually, neurons activated caspase 3 and PUMA (p53 up-regulated modulator of apoptosis), hallmarks of neuronal apoptosis, and died. These experiments will lead to a better understanding of slow neuronal death, typical of neurodegenerative diseases.
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(2009) Journal of Neuroscience. 29, 27, p. 8633-8638 Abstract
The ventral hippocampus (VH) was recently shown to express lower-magnitude long-term potentiation (LTP) than the dorsal hippocampus (DH). An exposure to acute stress reversed this difference, and VH slices from stressed rats expressed larger LTP than controls, whereas LTP in the DH was suppressed by stress. We have now used long-term depression (LTD)-generating trains of stimulation to examine whether this differential LTP reflects a genuine difference in synaptic modifiability between the two sectors of the hippocampus. Surprisingly, slices of DH and VH express similar magnitudes of LTD. However, while prior stress enhanced LTD in the DH, it actually converted LTD to slow-onset, robust LTP in the VH. These two effects of stress on LTD were blocked by glucocorticosterone receptor (GR) and mineralocorticosterone receptor (MR) antagonists, respectively. Acute exposure of slices to a GR agonist dexamethasone facilitated LTD in slices of both DH and VH, while activation of MRs by aldosterone converted LTD to LTP in both regions. Thus, differential activation of the two species of corticosterone receptors determines the ability of the two sectors of the hippocampus to undergo plastic changes in response to LTD-inducing stimulation.
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(2009) Journal of Neurophysiology. 101, 4, p. 2077-2088 Abstract
Spontaneous synchronized bursts of activity play an essential role in the maturation and plasticity of neuronal networks. To investigate the cellular properties that enable spontaneous network activity, we used dissociated cultures of hippocampal neurons that express prolonged network activity bursts. Acute exposure to a low concentration of N-methyl-D-aspartate (NMDA) caused an increase in spontaneous firing rates and intracellular calcium concentration ([Ca2+]i). However, in the course of a chronic (>1 day) exposure to NMDA, [Ca2+]i recovered back to normal baseline levels, and only sporadic asynchronous calcium transients were detected. Spontaneous network bursts were still absent 1 h after the removal of NMDA, indicating a persistent downregulation of network activity, which did recover eventually 2 days later. This effect of NMDA was activity-dependent as it was blocked by co-application of tetrodotoxin (TTX). The chronic NMDA-treated neurons expressed normal morphology and active membrane properties as well as spontaneous miniature excitatory postsynaptic currents and postsynaptic reactivity to glutamate. However, in response to trains of afferent stimulation in paired recordings, the treated neurons expressed synaptic depression as opposed to synaptic potentiation seen in control cells. Also, treated neurons did not respond to low-intensity electrical field stimulation as did control cells. Finally, Western blot analysis revealed that chronic exposure to NMDA altered presynaptic but not postsynaptic protein expression patterns, suggesting a presynaptic locus of effect. Thus a long-lasting increase in activity downregulates neurotransmitter release to prevent over-excitation of the network and, consequently, blocks the generation of network bursts.
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(2009) Journal of Neuroscience. 29, 11, p. 3395-3403 Abstract
Environmental enrichment (EE) was found to facilitate memory functioning and neural plasticity in normal and neurologically impaired animals. However, the ability of this manipulation to rescue memory and its biological substrate in animals with specific genetically based deficits in these functions has not been extensively studied. In the present study, we investigated the effects of EE in two mouse models of impaired memory functioning and plasticity. Previous research demonstrated that mice with a deletion of the receptor for the cytokine interleukin-1 (IL-1rKO), and mice with CNS-specific transgenic over-expression of the IL-1 receptor antagonist (IL-1raTG) display impaired hippocampal memory and long-term potentiation (LTP). We report here a corrective effect of EE on spatial and contextual memory in IL-1 rKO and IL-1 raTG mice and reveal two mechanisms for this beneficial effect: Concomitantly with their disturbed memory functioning, LTP in IL-1rKO mice that were raised in a regular environment is impaired, and their dendritic spine size is reduced. Both of these impairments were corrected by environmental enrichment. No deficiencies in neurogenesis or hippocampal BDNF and vascular endothelial growth factor secretion were found in IL-1rKO mice that were raised in a regular environment, and both of these variables were increased to a similar degree in enriched IL-1rKO and wild-type mice. These findings suggest that exposure to an enriched environment may be beneficial for individuals with impaired learning and memory related to genetic impairments of IL-1 signaling (and possibly other genetic causes), by reversing impairments in dentate gyrus LTP and spine size and by promoting neurogenesis and trophic factors secretion.
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(2009) Journal of Neuroscience. 29, 9, p. 2857-2866 Abstract
Corticosterone has been known to mediate the effects of stress on cognitive functions associated with the hippocampus. Acting at mineralocorticosteroid receptors (MRs) and glucocorticosteroid receptors (GRs), corticosterone exerts several effects in the hippocampus and elsewhere. Assuming that there are major functional differences between the dorsal hippocampus (DH) and ventral hippocampus (VH), and that these may be regulated by local interneurons, we analyzed the action of corticosterone on inhibitory synaptic currents in patch-clamped pyramidal neurons, recorded in acute slices of DH and VH. Corticosterone, through activation of MRs, reduced the frequency of spontaneous IPSCs in VH but not in DH neurons, and markedly suppressed paired-pulse facilitation of evoked inhibitory synaptic currents. These effects were mimicked by aldosterone, an MR agonist, and were blocked by an MR antagonist. In contrast, corticosterone caused an increase in the magnitude of IPSCs in both the DH and VH via its activation of GRs. This effect was mimicked by a GR agonist, dexamethasone, which produced a slow-onset, large potentiation reaching a peak within 45-60 min after onset of perfusion, and was blocked by a GR antagonist. The amplitude of mIPSCs was markedly increased by the GR agonist, indicating a synaptic locus of effect. These results indicate that corticosterone has a dual action, which may underlie the differential functional effects of stress hormones in the DH and VH.
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(2009) Journal of Neuroscience. 29, 4, p. 1017-1033 Abstract
The spine apparatus is an essential component of dendritic spines of cortical and hippocampal neurons, yet its functions are still enigmatic. Synaptopodin (SP), an actin-binding protein, is tightly associated with the spine apparatus and it may play a role in synaptic plasticity, but it has not yet been linked mechanistically to synaptic functions. We studied endogenous and transfected SP in dendritic spines of cultured hippocampal neurons and found that spines containing SP generate larger responses to flash photolysis of caged glutamate than SP-negative ones. An NMDA-receptor-mediated chemical long-term potentiation caused the accumulation of GFP-GluR1 in spine heads of control but not of shRNA-transfected, SP-deficient neurons. SP is linked to calcium stores, because their pharmacological blockade eliminated SP-related enhancement of glutamate responses, and release of calcium from stores produced an SP-dependent increase of GluR1 in spines. Thus, SP plays a crucial role in the calcium store-associated ability of neurons to undergo long-term plasticity.
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Determinants of Neuronal Network Activity in Cultured Hippocampus(2009) Encyclopedia Of Basic Epilepsy Research, Vols 1-3. p. 819-825 Abstract
2008
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(2008) Brain Research. 1235, p. 21-30 Abstract
The brain generates extensive spontaneous network activity patterns, even in the absence of extrinsic afferents. While the cognitive correlates of these complex activities are being unraveled, the rules that govern the generation, synchronization and spread of different patterns of intrinsic network activity in the brain are still enigmatic. Using hippocampal neurons grown in dissociated cultures, we are able to study these rules. Network activity emerges at 3-7 days in-vitro (DIV) independent of either ongoing excitatory or inhibitory synaptic activity. Network activity matures over the following several weeks in culture, when it becomes sensitive to chronic drug treatment. The size of the network determines its properties, such that dense networks have higher rates of less synchronized activity than that of sparse networks, which are more synchronized but rhythm at lower rates. Large networks cannot be triggered to fire by activating a single neuron. Small networks, on the other hand, do not burst spontaneously, but can be made to discharge a network burst by stimulating a single neuron. Thus, the strength of connectivity is inversely correlated with spontaneous activity and synchronicity. In the absence of confirmed 'leader' neurons, synchronous bursting network activity appears to be triggered by at least several local subthreshold synaptic events. We conclude that networks of neurons in culture can produce spontaneous synchronized activity and serve as a viable model system for the analysis of the rules that govern network activity in the brain.
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(2008) Experimental Neurology. 212, 1, p. 71-84 Abstract
The neurofibrillary-tangles (NTFs), characterisric of tauopathies including Alzheimer's-disease (AD), are the pathological features which correlate best with dementia. The objective of our study was to generate an authentic transgenic (tg) animal model for NFT-pathology in tauopathy/AD. Previous NFT-tg mice were driven by non-related/non-homologous promoters. Our strategy was to use the natural tau promoter for expressing the human-tau (htau) gene with two mutations K257T/P301S (double mutant, DM) associated with severe phenotypes of frontotemporal-dementia in humans. Cellular, biochemical, behavioral and electrophysiological studies were subsequently conducted. The tg mice showed a tolerated physiological level of the DM-htau protein, mostly in cortex and hippocampus. The mice demonstrated tauopathy-like characteristics, which increased with age, that included NFT-related pathology, astrogliosis, argyrophilic plaque-like (amyloid-free) structures in brain, with memory deficits and signs of anxiety. Moreover, the tg mice showed a robust synaptic plasticity deficit selectively expressed in a severe impairment in their ability to maintain hippocampal long-term-potentiation (LTP) in response to stimulation of the perforant path, providing evidence that "tau-pathology only" is sufficient to cause this memory and learning-associated deficit. This is a unique mutant-htau-tg model which presents a wide spectrum of features characteristic of tauopathy/AD, which does not show unrelated motor deficits described in other models of tauopathy. In addition, expressing the DM-htau in a neuronal cell model resulted in tau-aggregation, as well as impaired microtubule arrangement. Both animal and cell models, which were regulated under the natural tau promoter (of rat origin), provide authentic and reliable models for tauopathy, and offer valuable tools for understanding the molecular events underlying tauopathies including AD. (C) 2008 Elsevier Inc. All rights reserved.
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(2008) Developmental Neurobiology. 68, 7, p. 870-876 Abstract
Dendritic spines are assumed to constitute the locus of neuronal plasticity, and considerable effort has been focused on attempts to demonstrate that new memories are associated with the formation of new spines. However, few studies that have documented the appearance of spines after exposure to plasticity-producing paradigms could demonstrate that a new spine is touched by a bona fida presynaptic terminal. Thus, the functional significance of plastic dendritic spine changes is not clearly understood. We have used quantitative time lapse confocal imaging of cultured hippocampal neurons before and after their exposure to a conditioning medium which activates synaptic NMDA receptors. Following the experiment the cultures were prepared for 3D electron microscopic reconstruction of visually identified dendritic spines. We found that a majority of new, 1- to 2-h-old spines was touched by presynaptic terminals. Furthermore, when spines disappeared, the parent dendrites were sometime touched by a presynaptic bouton at the site where the previously identified spine had been located. We conclude that new spines are most likely to be functional and that pruned spines can be transformed into shaft synapses and thus maintain their functionality within the neuronal network.
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(2008) Hippocampus. 18, 1, p. 1-4 Abstract
Synaptopodin (SP), an actin binding protein associated with the spine apparatus, has been proposed to affect the ability of neurons to undergo synaptic plasticity. In the present study, we compared the density of SP puncta in stratum radiatum of the dorsal hippocampus (DH) with that of the ventral hippocampus (VH), a region that expresses only small and short lived LTP. Surprisingly, the VH contains significantly higher density of SP puncta than the DH. Following exposure to an acute stress, LTP was enhanced in the VH and suppressed in the DH. While SP density did change following stress, no similar trends were seen in the two sectors of the hippocampus. Thus, our results indicate a lack of clear interrelation between the density of SP and the ability to express LTP in the hippocampus,
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(2008) Journal of Neuroscience. 28, 3, p. 732-736 Abstract
The effects of thrombin, a blood coagulation serine protease, were studied in rat hippocampal slices, in an attempt to comprehend its devastating effects when released into the brain after stroke and head trauma. Thrombin acting through its receptor, protease-activated receptor 1 (PAR1), produced a long-lasting enhancement of the reactivity of CA1 neurons to afferent stimulation, an effect that saturated the ability of the tissue to undergo tetanus-induced long-term potentiation. This effect was mediated by activation of a PAR1 receptor, because it was shared by a PAR1 agonist, and was blocked by its selective antagonist. An independent effect of thrombin involved the lowering of the threshold for generating epileptic seizures in CA3 region of the hippocampus. Thus, the experiments in a slice mimicked epileptic and cognitive dysfunction induced by thrombin in the brain, and suggest that these effects are mediated by activation of the PAR1 receptor.
2007
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(2007) Developmental Neuroscience. 30, 1-3, p. 187-199 Abstract
Doublecortin (DCX) is a microtubule-associated protein necessary for neuronal migration. In spite of its ubiquitous distribution in dendrites, its possible role in dendrite development has not yet been documented. The present study examined the effects of different expression levels of DCX on the arborization of dendrites in cultured hippocampal neurons. Reduced expression of DCX following RNAi transfection resulted in reduced branch points, total length and complexity of the dendrites. Overexpression of DCX resulted in an increase in branch points and complexity of the dendrites. In contrast to control green fluorescent protein cells, DCX-overexpressing cells maintained highly branched and complex dendritic trees when subjected to reduced neuronal activity by blockade of immature GABAA receptors. These results suggest that DCX supports developing dendrites, in addition to its role in neuronal migration.
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(2007) Cell Calcium. 42, 1, p. 41-57 Abstract
Glutamate receptor trafficking into dendritic spines is a pivotal step in synaptic plasticity, yet the relevance of plasticity-producing rise of [Ca2+]i and of spine morphology to subsequent delivery of glutamate receptors into dendritic spine heads are still not well understood. Following chemical induction of LTP, an increase in eGFP-GluR1 fluorescence in short but not long dendritic spines of cultured hippocampal neurons was found. Repeated flash photolysis of caged calcium, which produced a transient rise of [Ca2+]i inside spine heads caused a selective, actin and protein synthesis dependent increase of eGFP-GluR1 in these spines. Strikingly, GluR1 increase was correlated with the ability of a calcium transient generated in the spine head to diffuse into the parent dendrite, and inversely correlated with the length of the spine: short spines were more likely to raise GluR1 than long ones. These observations link, for the first time, calcium transients in dendritic spines with spine morphology and its ability to undergo synaptic plasticity.
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(2007) Cerebral Cortex. 17, 6, p. 1292-1306 Abstract
When deprived of spontaneous ongoing network activity by chronic exposure to tetrodotoxin (TTX), cultured cortical neurons retract their dendrites, lose dendritic spines, and degenerate over a period of 1-2 weeks. Electrophysiological properties of these slowly degenerating neurons prior to their death are normal, but they express very large miniature excitatory postsynaptic currents (mEPSCs). Chronic blockade of these mEPSCs by the alpha-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist 6,7-Dinitroquinoxaline-2,3-dione (DNQX) had no effect of its own on cell survival, yet, paradoxically, it protected the TTX-silenced neurons from degenerating. TTX-treated neurons also exhibited deficient Ca2+ clearance mechanisms. Thus, upscaled mEPSCs are sufficient to trigger apoptotic processes in otherwise chronically silenced neurons.
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(2007) Journal of Neuroscience. 27, 21, p. 5757-5765 Abstract
The ability to express long-term potentiation (LTP) of reactivity to afferent stimulation along the septotemporal axis was explored in transverse rat hippocampal slices. The ventral pole of the hippocampus (VH) was found to be much impaired in ability to express LTP compared with the rest of the hippocampus. An exposure to acute stress before the rat was killed reversed this trend, and slices from VH now expressed a large LTP, whereas in the rest of the hippocampus, it was much suppressed. The enhanced LTP in VH was mediated by activation of a mineralocorticoid receptor (MR), whereas the suppressed LTP was mediated by activation of a glucocorticoid receptor, and indeed selective agonists of the respective steroid receptors mimicked the effects of stress, whereas selective antagonists blocked them. The MR-enhanced LTP in VH was not mediated by activation of the NMDA receptor but by enhancement of voltage-gated calcium channels. Because the VH has an unique efferent system to the hypothalamus, these results indicate that stress may activate this system while suppressing the ability of the rest of the hippocampus to express plastic properties under stressful conditions.
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(2007) Journal of Neurophysiology. 97, 4, p. 2937-2948 Abstract
Spontaneous activity is typical of in vitro neural networks, often in the form of large population bursts. The origins of this activity are attributed to intrinsically bursting neurons and to noisy backgrounds as well as to recurrent network connections. Spontaneous activity is often observed to emanate from localized sources or initiation zones, propagating from there to excite large populations of neurons. In this study, we use unidimensional cultures to overcome experimental difficulties in identifying initiation zones in vivo and in dissociated two-dimensional cultures. We found that spontaneous activity in these cultures is initiated exclusively in localized zones that are characterized by high neuronal density but also by recurrent and inhibitory network connections. We demonstrate that initiation zones compete in driving network activity in a winner-takes-most scenario.
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(2007) Antioxidants & Redox Signaling. 9, 2, p. 181-189 Abstract
The recent finding that hippocampal slices from aged mice overexpressing the gene for superoxide dismutase (SOD) exhibit long-term potentiation (LTP) of reactivity to afferent stimulation that is significantly larger than that produced in aged wild-type (wt) mice has encouraged the exploration of the effects of reactive oxygen species (ROS) on learning in aged mice. In addition, young-adult and aged wt and SOD transgenic mice were used in an attempt to correlate adult neurogenesis with spatial learning. Aged wt and SOD mice exhibited a 90% reduction in doublecortin-labeled new dentate gyrus neurons as compared to young mice, with no significant difference between genotypes. In addition, aged SOD mice exhibited better performance than wt controls in both reference and working memory tasks in a water maze. These findings provide a behavioral measure to demonstrate that excessive production of H 2O2 is beneficial in aged mice.
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(2007) Hippocampus. 17, 1, p. 10-25 Abstract
Morphological electrophysiological evidence has been accumulated in recent years to indicate that a functional gradient exists along the septo-temporal axis of the hippocampus such that spatial memory is associated primarily with the dorsal (septal) sector while the functions of the ventral sector are not yet clearly defined. Also, the ventral hippocampus (VH) is reported to express a much smaller long term potentiation of responses to afferent stimulation than the dorsal sector. In the present study, we first confirmed that CAI region of VH slices expresses significantly smaller LTP than the dorsal hippocampus. Strikingly, much larger LTP was obtained in VH slices following low frequency priming stimulation applied prior to the tetanic stimulation. DHPG ((S)-3,5-Dihydroxyphenylglycine hydrate) a metabotropic glutamate receptor agonist, produced a similar potentiating effect on LTP as that produced by the priming stimulation. In both cases, the spectral analysis of spontaneous electrical activity recorded from the same location in the slice revealed an increase in peak amplitude around 30 Hz. MCPG, a metabotropic glutamate receptor antagonist, and both thapsigargin and cyclopiazonic acid, inhibitors of Ca2+ release from stores, blocked the potentiating action of both DHPG and the priming stimulation. These results indicate that the ventral hippocampus possesses different network properties compared to the dorsal hippocampus and that its ability to undergo plastics changes is controlled by a metabotropic glutamate receptor.
2006
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(2006) Learning & Memory. 13, 6, p. 719-727 Abstract
A common denominator for the induction of morphological and functional plasticity in cultured hippocampal neurons involves the activation of excitatory synapses. We now demonstrate massive morphological plasticity in mature cultured hippocampal neurons caused by a brief exposure to glutamate. This plasticity involves a slow, 70%-80% increase in spine cross-section area associated with a significant reduction in the width of dendrites. These changes are age dependent and expressed only in cells >18 d in vitro (DIV). Activation of both NMDARs and AMPARs as well as a sustained rise of internal calcium levels are necessary for induction of this plasticity. On the other hand, blockade of network activity or mGluRs does not abolish the observed morphological plasticity. Electrophysiologically, a brief exposure to glutamate induces an increase in the magnitude of EPSCs evoked between pairs of neurons, as well as in mEPSC frequency and amplitude, in mature but not young cultures. These results demonstrate an age-dependent, rapid and robust morphological and functional change in cultured central neurons that may contribute to their ability to express long term synaptic plasticity.
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(2006) Journal of Neuroscience. 26, 36, p. 9130-9134 Abstract
Elevated levels of corticosteroid hormones, presumably occupying both mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs), have been reported to impair synaptic plasticity in the hippocampus as well as the acquisition of hippocampus-dependent memories. In contrast, recent evidence suggests that activation of MRs enhance cognitive functions. To clarify the roles of different steroid receptors in hippocampal plasticity, young adult rats were injected with the GR antagonist RU38486 (mifepristone) or the MR antagonist Spironolactone before the exposure to an acute swim stress. Hippocampal responses to perforant path stimulation were then recorded in anesthetized rats. Stress combined with RU38486 produced a striking facilitation of LTP. Spironolactone enabled only short-term potentiation that reversed to long-term depression (LTD) in the stressed animals. Finally, the blockade of both MRs and GRs led to impairment of long-term potentiation. These findings indicate that MRs and GRs assume opposite roles in regulation of synaptic plasticity after acute exposure to stressors.
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(2006) Journal of Neurochemistry. 97, 5, p. 1379-1392 Abstract
We studied the lysophosphatidic acid receptor-1 (LPA1) gene, which we found to be expressed endogenously in cultured hippocampal neurons, and in vivo in young (1-week-old) rat brain slices. Overexpressed green fluorescent protein (GFP)-tagged, membrane-associated LPA1 accumulated in a punctate manner over the entire dendritic tree and caused an increase in dendritic spine density. About half of the dendritic spines in the LPA 1-transfected neurons displayed distinct fluorescent puncta, and this subset of spines was also substantially larger than puncta-free, LPA 1-transfected or control GFP spines. This phenotype could also be seen in cells transfected with a ligand-binding, defective mutant and is therefore not dependent on interaction with an ambient ligand. While spontaneous miniature excitatory synaptic currents were of the same amplitudes, they decayed slower in LPA1-transfected neurons compared with GFP controls. We propose that LPA1 may play a role in the formation and modulation of the dendritic spine synapse.
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(2006) Journal of Neuroscience. 26, 4, p. 1199-1210 Abstract
A fundamental issue in understanding activity-dependent long-term plasticity of neuronal networks is the interplay between excitatory and inhibitory synaptic drives in the network. Using dual whole-cell recordings in cultured hippocampal neurons, we examined synaptic changes occurring as a result of a transient activation of NMDA receptors in the network. This enhanced transient activation led to a long-lasting increase in synchrony of spontaneous activity of neurons in the network. Simultaneous long-term potentiation of excitatory synaptic strength and a pronounced long-term depression of inhibitory synaptic currents (LTDi) were produced, which were independent of changes in postsynaptic potential and Ca2+ concentrations. Surprisingly, miniature inhibitory synaptic currents were not changed by the conditioning, whereas both frequency and amplitudes of miniature EPSCs were enhanced. LTDi was mediated by activation of a presynaptic GABAB receptor, because it was blocked by saclofen and CGP55845 [(2S)-3-{[(15)-1-(3,4-dichlorophenyl)ethyl] amino-2-hydroxypropyl)(phenylmethyl)phosphinic acid]. The cAMP antagonist Rp-adenosine 3′,5′-cyclic monophosphothioate abolished all measured effects of NMDA-dependent conditioning, whereas a nitric oxide synthase inhibitor was ineffective. Finally, network-induced plasticity was not occluded by a previous spike-timing-induced plasticity, indicating that the two types of plasticity may not share the same mechanism. These results demonstrate that network plasticity involves opposite affects on inhibitory and excitatory neurotransmission.
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(2006) Cell Calcium. 40, 5-6, p. 441-449 Abstract
The extent of diffusion of a locally evoked calcium surge in dendrites of cultured hippocampal neurons was studied by flash photolysis of caged EGTA. Cells were transfected with pDsRed for visualization, preincubated with caged NP-EGTA (AM) and Fluo-4 (AM) at room temperature and imaged in a PASCAL confocal microscope. Pulses of UV laser light within an active sphere of about 1 μm2 produced a rise of Fluo-4 fluorescence transients in dendrites which peaked at 1 ms and decayed exponentially with a fast (8-10 ms) time constant. A slower decay component was uncovered following incubation with thapsigargin. Lateral diffusion of [Ca2+]i did not vary significantly among different size dendrites being symmetric and reaching about 3-3.5 μm at a diffusion rate of 0.8 μm/ms on both sides of the photolysis center. Fluo-4 was also replaced by the membrane-bound Fluo-NOMO (AM) or by the 'heavy' Calcium Green dextran (CGd) loaded through a patch pipette. Similar rates of diffusion were found in these cases, indicating that the diffusion is not of the dye complexed to calcium but of genuine free calcium ions. Interestingly, presence of a dendritic spine at the focus of photolysis significantly reduced [Ca2+]i spread while the focal transient remained unaffected. Finally, [Ca2+]i diffused about twice as far from the photolysis sphere in glass tubes of a similar diameter to that of a dendrite, indicating that intrinsic calcium uptake mechanisms in the dendrite determine the diffusion of calcium away from its original site of rise.
2005
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(2005) Journal of Neurochemistry. 95, 5, p. 1401-1410 Abstract
The Wiskott-Aldrich syndrome protein family Verprolin-homologous protein (WAVE) complex has been proposed to link Rho GTPase activity with actin polymerization but its role in neuronal plasticity has never been documented. We now examined the presence, distribution and dynamics of WAVES in cultured hippocampal neurons. WAVES was localized to dendritic spines via its N-terminal domain. Green fluorescent protein (GFP)-tagged WAVES clusters demonstrate an F-actin-dependent high rate of local motility. Constitutive Rac activation translocates WAVES (via the N-terminus), to the leading edge of lamellipodia. Also, spinogenesis is associated with actin-based motility of the WAVES protein. Brain specific WAVE1 showed similar localization and effects on spine density. Cytoplasmic fragile X mental retardation protein interacting protein (CYFIP) and non-catalytic region of tyrosine kinase adaptor protein 1 (NCK-1), proteins that are assumed to complex with WAVE, have a somewhat similar cellular distribution and motility. We propose that the WAVE complex is a downstream effector of the Rac signaling cascade, localized to sites of novel synaptic contacts by means of its N-terminal domain, to guide local actin polymerization needed for morphological plasticity of neurons.
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(2005) Journal of Neurophysiology. 94, 5, p. 3406-3416 Abstract
Dissociated neurons were cultured on lines of various lengths covered with adhesive material to obtain an experimental model system of linear signal transmission. The neuronal connectivity in the linear culture is characterized, and it is demonstrated that local spiking activity is relayed by synaptic transmission along the line of neurons to develop into a large-scale population burst. Formally, this can be treated as a one-dimensional information channel. Directional propagation of both spontaneous and stimulated bursts along the line, imaged with the calcium indicator Fluo-4, revealed the existence of two different propagation velocities. Initially, a small number of neighboring neurons fire, leading to a slow, small and presumably asynchronous wave of activity. The signal then spontaneously develops to encompass much larger and further populations, and is characterized by fast propagation of high-amplitude activity, which is presumed to be synchronous. These results are well described by an existing theoretical framework for propagation based on an integrate-and-fire model.
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(2005) Molecular and Cellular Neuroscience. 30, 1, p. 24-36 Abstract
The utilization of human embryonic stem cells (hESC) for basic and applied research is hampered by limitations in directing their differentiation. Empirical poorly defined methods are currently used to develop cultures enriched for distinct cell types. Here, we report the derivation of neural precursors (NPs) from hESC in a defined culture system that includes the bone morphogenetic protein antagonist noggin. When hESC are cultured as floating aggregates in defined medium and BMP signaling is repressed by noggin, non-neural differentiation is suppressed, and the cell aggregates develop into spheres highly enriched for proliferating NPs. The NPs can differentiate into astrocytes, oligodendrocytes, and mature electrophysiologically functional neurons. During prolonged propagation, the differentiation potential of the NPs shifts from neuronal to glial fate. The presented noggin-dependent controlled conversion of hESC into NPs is valuable for the study of human neurogenesis, the development of new drugs, and is an important step towards the potential utilization of hESC in neural transplantation therapy.
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(2005) Nature Reviews Neuroscience. 6, 4, p. 277-284 Abstract
A recent flurry of time-lapse imaging studies of live neurons have tried to address the century-old question: what morphological changes in dendritic spines can be related to long-term memory? Changes that have been proposed to relate to memory include the formation of new spines, the enlargement of spine heads and the pruning of spines. These observations also relate to a more general question of how stable dendritic spines are. The objective of this review is to critically assess the new data and to propose much needed criteria that relate spines to memory, thereby allowing progress in understanding the morphological basis of memory.
2004
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(2004) European Journal of Neuroscience. 20, 10, p. 2649-2663 Abstract
We investigated the role of dendritic spine morphology in spine-dendrite calcium communication using novel experimental and theoretical approaches. A transient rise in [Ca2+]i was produced in individual spine heads of Fluo-4-loaded cultured hippocampal neurons by flash photolysis of caged calcium. Following flash photolysis in the spine head, a delayed [Ca 2+]i transient was detected in the parent dendrites of only short, but not long, spines. Delayed elevated fluorescence in the dendrite of the short spines was also seen with a membrane-bound fluorophore and fluorescence recovery from bleaching of a calcium-bound fluorophore had a much slower kinetics, indicating that the dendritic fluorescence change reflects a genuine diffusion of free [Ca2+]i from the spine head to the parent dendrite. Calcium diffusion between spine head and the parent dendrite was regulated by calcium stores as well as by a Na-Ca exchanger. Spine length varied with the recent history of the [Ca2+]i variations in the spine, such that small numbers of calcium transients resulted in elongation of spines whereas large numbers of calcium transients caused shrinkage of the spines. Consequently, spine elongation resulted in a complete isolation of the spine from the dendrite, while shrinkage caused an enhanced coupling with the parent dendrite. These studies highlight a dynamically regulated coupling between a dendritic spine head and its parent dendrite.
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(2004) European Journal of Neuroscience. 19, 12, p. 3151-3164 Abstract
Activation of protein kinase C (PKC) produced a novel and rapid formation of lamellae over large surfaces of dendrites in cultured hippocampal neurons. This action was dendrite-specific, involved a postsynaptic locus of activation of PKC and required actin polymerization, but not activation of Erk. Over-expression of active Rho-A GTPase converted a mature, highly branched and spiny neuron into a primitive, non-branching, aspiny neuron. Overexpression of active Rac1 caused massive lamellae formation in the transfected neurons. These morphologically aberrant neurons retained synaptic connectivity with adjacent, normal neurons, as well as the ability to form lamellae in response to PKC. On the other hand, transfection with a dominant negative Rac-N17 or a toxin C3, Rho-A-inactivating plasmid blocked lamellae formation by PKC, but did not prevent PKC-induced plasticity of synaptic currents. These data indicate that PKC activates two independent molecular pathways, one of which involves Rac1 and Rho-A, to produce massive actin-based structural plasticity in dendrites and spines.
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(2004) Molecular Neurobiology. 29, 2, p. 167-178 Abstract
Reactive oxygen species (ROS) have been considered for some time only in the context of oxidative stress-induced cell damage. In this review, we discuss the growing body of evidence that implicates ROS in general, and hydrogen peroxide (H2O2) in particular, in regulatory events underlying synaptic plasticity. H2O2 is regarded in this context as a specific diffusible signaling molecule. The action of H 2O2 is assumed to be carried out via the release of calcium ions from internal stores, modulating the activity of specific calcium-dependent protein phosphatases. These phosphatases eventually affect neuronal plasticity. We discuss the role of H2O2 in these systems, stressing the importance of cellular regulation of H2O 2 levels that are altered in aging individuals, in the ability to express plasticity. These studies highlight the function of H2O 2 in processes of learning and memory and their change in elderly individuals, irrespective of neurodegeneration found in Alzheimer's patients.
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(2004) Journal of Neuroscience Methods. 133, 1-2, p. 153-159 Abstract
Using a pulsed UV laser in a confocal scanning microscope, we present a relatively cheap, accurate and efficient method for fast UV laser flash photolysis of caged molecules in two-dimensional cultured neurons. The laser light is introduced through the imaging optics, can be localized by a parallel red laser and can photolyse a sphere of less than 1μm2, and evoke local fluorescence changes in the imaged neurons. Caged glutamate and caged fluorescein are used to illustrate a disparity between spines and their parent dendrites at a sub-micron resolution.
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(2004) European Journal of Neuroscience. 19, 5, p. 1174-1190 Abstract
Down's syndrome (DS), the phenotypic manifestation of trisomy 21, involves overexpression of chromosome 21-encoded genes. The gene for amyloid precursor protein (APP), known to be involved in AD pathology, resides on chromosome 21 along with the gene for Cu/Zn superoxide dismutase (SOD1), a key enzyme in the metabolism of oxygen free radicals. We investigated the consequences of a combined increase in APP and SOD1, in a double-transgenic (tg)-APP-SOD1 mouse. These mice expressed severe impairment in learning, working and long-term memory. Expression of long-term potentiation in hippocampal slices was impaired in both tg-SOD and tg-APP-SOD mice, but not in tg-APP mice, indicating that increased APP by itself did not affect in vitro synaptic plasticity. In tg-APP-SOD mice, membrane-bound high molecular weight APP species accumulated while APP cleavage products did not increase and levels of secreted APP were unchanged. Severe morphological damage, including lipofuscin accumulation and mitochondria abnormalities, were found in aged tg-APP-SOD but not in the other mice. Thus, a combined elevation of the two chromosome 21 genes in tg-APP-SOD mice induced age-dependent alterations in morphological and behavioural functions.
2003
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(2003) Journal Of Physiology-London. 553, 3, p. 729-745 Abstract
Spontaneous activity in the central nervous system is strongly suppressed by blockers of gap junctions (GJs), suggesting that GJs contribute to network activity. However, the lack of selective GJ blockers prohibits the determination of their site of action, i.e. neuronal versus glial. Astrocytes are strongly coupled through GJs and have recently been shown to modulate synaptic transmission, yet their role in neuronal network activity was not analysed. The present study investigated the effects and site of action of the GJ blocker, carbenoxolone (CBX), on neuronal network activity. To this end, we used cultures of hippocampal or cortical neurons, plated on astrocytes. In these cultures neurons display spontaneous synchronous activity and GJs are found only in astrocytes. CBX induced in these neurons a reversible suppression of spontaneous action potential discharges, synaptic currents and synchronised calcium oscillations. Moreover, CBX inhibited oscillatory activity induced by the GABAA antagonist, bicuculline. These effects were not due to blockade of astrocytic GJs, since they were not mimicked nor occluded by endothelin-1 (ET-1), a peptide known to block astrocytic GJs. Also, these effects were still present in co-cultures of wild-type neurons plated on astrocytes originating from connexin-43 (Cx43) knockout mice, and in neuronal cultures which contain few isolated astrocytes. CBX was not likely to exert its effect through neuronal GJs either, as immunostaining for major neuronal connexins (Cx) as well as dye or electrical coupling, were not detected in the different models of cultured neurons examined. Finally while CBX (at 100 μM) did not modify presynaptic transmitter release and postsynaptic responses to glutamate, it did cause an increase in the action potential threshold and strongly decreased the firing rate in response to a sustained depolarising current. These data demonstrate that CBX does not exert its action on network activity of cultured neurons through astrocytic GJs and suggest that it has direct effects on neurons, not involving GJs.
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(2003) Journal of Neuroscience. 23, 32, p. 10359-10367 Abstract
Hydrogen peroxide (H2O2), a reactive oxygen species, is assumed to have a detrimental effect on neuronal plasticity. Indeed, H 2O2 suppresses long-term potentiation (LTP) in hippocampal slices of normal rats and wild-type (wt) mice. Transgenic mice overexpressing superoxide dismutase (SOD) 1 (tg-SOD), which maintain high ambient H2O2, have also been shown to be impaired in their ability to express hippocampal LTP. Paradoxically, H2O 2, at a concentration (50 μM) that blocks LTP in wt mice, actually enhanced LTP in slices of 2-month-old tg-SOD mice. H2O 2-dependent LTP in tg-SOD was blocked by the protein phosphatase calcineurin inhibitor FK506, but not by rapamycin, an FK-binding protein 12 (FKBP12) inhibitor or by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), a serine-kinase inhibitor. Interestingly, wt and tg-SOD mice expressed similar levels of the antioxidant enzyme catalase and similar activity of glutathione peroxidase. An opposite situation was found in 2-year-old mice. Aged wt mice were impaired in LTP in a manner that could be reversed by the addition of H2O2. Surprisingly, aged tg-SOD mice exhibited larger LTP than that found in wt mice, but this was now reduced by 50 μM H 202. Both young tg-SOD and aged control mice displayed altered protein phosphatase activity, compared with that of young controls; moreover, FK506 inhibited LTP in old tg-SOD as well as in old wt mice treated with H2O2. These data promoted a dual role for H 2O2 in the regulation of LTP, and proposed that it is mediated by the protein phosphatase calcineurin.
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(2003) Neurochemistry International. 43, 3, p. 179-189 Abstract
Reduction in GluR2 subunit expression and subsequent increases in AMPA receptor mediated Ca2+ currents were postulated to exacerbate glutamate neurotoxicity following seizures or global ischemia. To directly test the effects of shifting the GluR1/GluR2 subunit ratio on excitotoxicity, GluR2 antisense deoxyoligonucleotides (AS-ODNs) were applied to dissociated hippocampal cultures for 1-8 days. The GluR1/GluR2 protein ratio was examined immunohistochemically and by Western blotting. [Ca2+]i concentrations were determined by ratiometric imaging of Fura 2-loaded cells. The cultures were exposed to glutamate, AMPA, NMDA or kainic acid (KA) 3 days after GluR2 knockdown and cell viability was determined 1 day later by MTT reduction assay or Trypan blue exclusion. Although GluR2 AS-ODNs increased the GluR1/GluR2 protein ratio in a time dependent manner, neurons and glia appeared healthy and MTT reduction values were similar to untreated and sense controls. Basal [Ca2+]i levels were unchanged but [Ca2+]i was selectively increased by agonist stimulation of AMPA receptors. Unexpectedly, delayed neurotoxicity was attenuated at saturating doses of glutamate while little difference in cell viability was observed at lower doses or with the other excitotoxins at any concentration. Therefore, there was a dissociation between rises in AMPA receptor-mediated Ca2+ influx and neurotoxicity despite marked decreases in GluR2 but not GluR1 immunoreactivity. It is proposed that a modification of AMPA receptor stochiometry that raises agonist-stimulated Ca2+ influx during an excitotoxic insult may have eventual neuroprotective effects.
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(2003) European Journal of Neuroscience. 17, 12, p. 2573-2585 Abstract
The role of afferent innervation in the formation of dendritic spines was studied in cultured rat striatum. The striatum is a unique structure in that it contains highly spiny GABAergic projection neurons, with no known local excitation. Grown alone in culture, striatal neurons did not express spontaneous network activity and were virtually devoid of dendritic spines. Adding GFP-expressing mouse cortical neurons to the striatal culture caused the appearance of spontaneous and evoked excitatory synaptic currents in the striatal neurons and a 10-fold increase in the density of spines on their dendrites. This effect was blocked by a continuous presence of TTX in the growth medium, while removal of the drug caused a rapid appearance of spines. Exposure to glutamate, or the presence of cortex-conditioned medium did not mimic the effect of cortical neurons on formation of spines in the striatal neurons. Also, the cortical innervation did not cause a selective enhancement of survival of specific subtypes of spiny striatal neurons. These experiments demonstrate that excitatory afferents are necessary for the formation of dendritic spines in striatal neurons.
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(2003) European Journal of Neuroscience. 17, 12, p. 2529-2539 Abstract
The roles of protein kinase C and the MAP-kinase extracellular receptor kinase in structural changes associated with long-term potentiation of network activity were examined in cultured hippocampal neurons. A brief exposure to a conditioning medium that favours activation of the N-methyl-D-aspartate receptor caused a rapid and specific increase in staining of neurons for the phosphorylated form of extracellular receptor kinase as well as of cyclic AMP response element binding protein. Exposure of the cultures to the conditioning medium was followed by a protein synthesis-dependent formation of novel dendritic spines. An extracellular receptor kinase antagonist PD98059 blocked the phosphorylated form of extracellular receptor kinase response and the formation of novel spines. A selective protein kinase C agonist, phorbol 12-myristate 13-acetate, caused activation of extracellular receptor kinase and formation of novel spines. The protein kinase C antagonist GF109203x blocked the phosphorylated form of extracellular receptor kinase response and the subsequent spine formation by phorbol 12-myristate 13-acetate. Both the conditioning medium and phorbol 12-myristate 13-acetate caused a delayed increase in mean amplitude of miniature excitatory postsynaptic currents recorded in the hippocampal neurons. These results indicate that activation of extracellular receptor kinase mediates the effect of a conditioning protocol on the formation of dendritic spines. The formation of novel spines was associated with long-term increase in network activity and functional synaptic connectivity among the cultured neurons.
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Is fragile X mental retardation protein involved in activity-induced plasticity of dendritic spines?(2003) Brain Research. 972, 1-2, p. 9-15 Abstract
Dendritic morphology of 2-week-old cultured neurons, taken from postnatal day 1 fragile X mental retardation gene1 knock out (FMR1-/-) mice hippocampus, were compared with cells taken from wild type mice. Under control conditions the FMR1-/- neurons displayed significantly lower spine densities compared to wild type neurons. Pharmacological stimulation of electrical activity, induced by bicuculline, caused a reduction in dendritic spine density in both the FMR1-/- and the wild type cells. In both groups, bicuculline induced a significant shrinkage of spines that were occupied by one or more synaptophysin-immunoreactive presynaptic terminals. The concentration of FMR1 in the wild type cultures was not affected by bicuculline treatment. These experiments indicate that FMR1 is not likely to be an essential factor in activity-modulated morphological plasticity of dendritic spines in cultured hippocampal neurons.
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(2003) Hippocampus. 13, 7, p. 826-834 Abstract
The cytokine interleukin-1 (IL-1) is produced by peripheral immune cells as well as glia and neurons within the brain; it plays a major role in immune to brain communication and in modulation of neural, neuroendocrine, and behavioral systems during illness. Although previous studies demonstrated that excess levels of IL-1 impaired memory processes and neural plasticity, it has been suggested that physiological levels of IL-1 are involved in hippocampal-dependent memory and long-term potentiation (LTP). To examine this hypothesis, we studied IL-1 receptor type I knockout (IL-1rKO) mice in several paradigms of memory function and hippocampal plasticity. In the spatial version of the water maze test, IL-1rKO mice displayed significantly longer latency to reach a hidden platform, compared with wild-type controls. Furthermore, IL-1rKO exhibited diminished contextual fear conditioning. In contrast, IL-1rKO mice were similar to control animals in hippocampal-independent memory tasks; i.e., their performance in the visually guided task of the water maze and the auditory-cued fear conditioning was normal. Electrophysiologically, anesthetized IL-1rKO mice exhibited enhanced paired-pulse inhibition in response to perforant path stimulation and no LTP in the dentate gyrus. In vitro, decreased paired-pulse responses, as well as a complete absence of LTP, were observed in the CA1 region of hippocampal slices taken from IL-1rKO mice compared with WT controls. These results suggest that IL-1 contributes to the regulation of memory processes as well as short- and long-term plasticity within the hippocampus. These findings have important implications to several conditions in humans, which are associated with long-term defects in IL-1 signaling, such as mutations in the IL-1 receptor accessory protein-like gene, which are involved in a frequent form of X-linked mental retardation.
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(2003) Neuroscience. 121, 1, p. 83-98 Abstract
The generation of an Atm-/- mouse model of the human ataxia-telangiectasia (AT) opened new avenues toward a better understanding of the molecular and cellular basis of AT. We have recently reported that 5-month-old Atm-/- mice exhibit severe loss of tyrosine hydroxylase-positive, dopaminergic nigro-striatal neurons, down to 26% of age-matched controls. In the present study we analyzed development of the dopaminergic cell loss in the context of the nigro-striatal system. We found that dopaminergic neurons are formed normally in the Atm-/- mouse, and degenerate during the first few months of life; there was no difference between 1-month-old Atm-/- and control mice in the number of dopaminergic cells that were retrogradely labeled by an injection of fluorescent tracer into the striatum. On the other hand, a dramatic reduction in the number of labeled cells was found in 5-month-old Atm-/- mice. This cell loss was significant in areas A9 and A10 but not in area A9-1. These findings indicate that midbrain dopaminergic neurons in Atm-/- mice initially send normal axons to the striatum, only to degenerate later in life. In addition, an age-dependent as well as topographic, medial-to-lateral loss of GAD, metenkephaline and substance-P immunopositive cells was found in the striatum of the Atm-/- mice. This phenomenon was significant only in the 5-month-old Atm-/- mice (3 months after the beginning of detectable dopaminergic cell loss). In both the striatum and the substantia nigra, the apparent cell loss was accompanied by gliosis. In addition, alpha-synuclein immunopositive bodies were observed in the cortex, striatum and substantia nigra of these mice. The present data indicate that Atm-/- mice exhibit a progressive, age-dependent, reduction in dopaminergic cells of the substantia nigra, followed by a reduction in projection neurons of the striatum. Thus, the Atm-/- mouse may model the extrapyramidal motor deficits seen in AT patients. (C) 2003 IBRO. Published by Elsevie
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(2003) Journal of Neuroscience. 23, 1, p. 269-276 Abstract
Unlike the proposed role of reactive oxygen species in neurodegeneration, acute effects of reactive oxygen on synaptic plasticity are poorly understood. Using rat hippocampal slices, we found that exposure to a high concentration (0.5-5 mM) of H2O2 reduces EPSPs in both potentiated and nonpotentiated synapses. Exposure of the slices to 20 μM H2O2 did not affect expression of preestablished long-term potentiation (LTP) but prevented induction of new LTP and enhanced long-term depression (LTD). Surprisingly, 1 μM H2O2 caused a twofold increase in LTP compared with controls, and it further enhanced NMDA-independent LTP. A low concentration of H2O2 also suppressed LTD. Nifedipine, an L-type calcium channel blocker, did not affect control LTP but blocked effects of both 1 and 20 μM H2O2. Calcineurin inhibitors [FK506 (FR900506) and cyclosporin A but not rapamycin] acted similarly and also restored LTP in the presence of 20 μM H2O2. These results suggest that H2O2 alters NMDA-independent, voltage-gated calcium channel-mediated LTP by activating calcineurin.
2002
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(2002) Nature Reviews Neuroscience. 3, 12, p. 965-974 Abstract
The neurotrophins are best known for their ability to support neuronal survival and differentiation, but a role in synapse formation and plasticity has recently emerged. For central neurons, brain-derived neurotrophic factor can increase the number of excitatory and inhibitory synapses by regulating axonal morphology or by directly promoting synapse formation. In addition, neurotrophins promote the maturation and stabilization of the cellular and molecular components that are responsible for neurotransmitter release, and this ultimately leads to an increase in the number of functional synapses. These long-term structural and molecular changes are likely to be crucial not only during development, but also during synaptic plasticity in the adult.
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(2002) Applied Physics B-Lasers And Optics. 74, SUPPL., p. S97-S101 Abstract
We demonstrate third-harmonic microscopy of thin biological objects using a Ti:sapphire laser source. A very simple modification to the collection and detection systems of the microscope allows for the detection of the third-harmonic signal near 270 nm. We show that, using a Ti:sapphire laser, we can combine third-harmonic imaging with two-photon excitation fluorescence simultaneously. Compared to other possible laser sources for third-harmonic microscopy, the Ti:sapphire laser provides better optical resolution, better power availability, and is less absorbed in water. We find that the Ti:sapphire laser is the most suitable laser source for third-harmonic microscopy for thin biological specimens.
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(2002) Changing Views Of Cajal'S Neuron. 136, p. 101-107 Abstract
Ever since dendritic spines were first described in detail by Santiago Ramón y Cajal, they were assumed to underlie the physical substrate of long term memory in the brain. Recent time-lapse imaging of dendritic spines in live tissue, using confocal microscopy, have revealed an amazingly plastic structure, which undergoes continuous changes in shape and size, not intuitively related to its assumed role in long term memory. Functionally, the spine is shown to be an independent cellular compartment, able to regulate calcium concentration independently of its parent dendrite. The shape of the spine is instrumental in regulating the link between the synapse and the parent dendrite such that longer spines have less impact on the dendrite than shorter ones. The spine can be formed, change its shape and disappear in response to afferent stimulation, in a dynamic fashion, indicating that spine morphology is an important vehicle for structuring synaptic interactions. While this role is crucial in the developing nervous system, large variations in spine densities in the adult brain indicate that tuning of synaptic impact may be a role of spines throughout the life of a neuron.
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(2002) Plasticity In The Adult Brain: From Genes To Neurotherapy. 138, p. 53-59 Abstract
Keywords: CULTURED HIPPOCAMPAL-NEURONS; LONG-TERM POTENTIATION; METHYL-D-ASPARTATE; RECEPTOR ACTIVATION; COMPLEX ENVIRONMENT; GLUTAMATE RECEPTORS; SYNAPTIC FUNCTION; CEREBRAL-CORTEX; CALCIUM; SYNAPSES
2001
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(2001) Neuron. 31, 2, p. 169-171 Abstract
Two recent studies by Sheng and associates (Pak et al., 2001; Sala et al., 2001) provide an elegant molecular analysis of the role of a spine-specific protein, SPAR, and the synaptic proteins Shank and Homer, in regulating dendritic spine morphology, and the possible functional consequences of this regulation.
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(2001) Journal of Neuroscience. 21, 16, p. 6115-6124 Abstract
Regulation of dendritic spine motility was studied in dissociated cultures of the rat and mouse hippocampus, using green fluorescent protein-labeled neurons or neurons loaded with the calcium-sensitive dye Oregon Green-1. Cells were time-lapsephotographed on a confocal laser-scanning microscope at high resolution to detect movements as well as spontaneous fluctuations of intracellular calcium concentrations in their dendritic spines. Active presynaptic terminals attached to the spines were labeled with FM4-64, which marks a subset of synaptophysin-labeled terminals. Dendritic spines were highly motile in young, 4- to 7-d-old cells. At this age, neurons had little spontaneous calcium fluctuation or FM4-64 labeling. Within 2-3 weeks in culture, dendritic spines were much less motile, they were associated with active presynaptic terminals, and they expressed high rates of spontaneous calcium fluctuations. Irrespective of age, and even on the same dendrite, there was an inverse relationship between spine motility and presence of FM4-64-labeled terminals in contact with the imaged spines. Spine motility was blocked by latrunculin, which prevents actin polymerization, and was disinhibited by blockade of action potential discharges with tetrodotoxin. It is proposed that an active presynaptic terminal restricts motility of dendritic spines.
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(2001) Neuropsychopharmacology. 24, 6, p. 608-616 Abstract
Transcranial magnetic stimulation (TMS) has been proposed as a safe and efficient treatment of human clinical depression. Although its antidepressive mechanism of action remained unknown, our previous studies indicate that TMS has a long-lasting effect on neuronal excitability in the hippocampus. We now compare the effects of chronic TMS with those of the antidepressant drugs desipramine and mianserin. The three treatments did not affect basal conduction in the perforant path to the dentate gyrus, but markedly suppressed paired-pulse and frequency-dependent inhibition, resulting from a reduction in local circuit inhibition in the dentate gyrus. Concomitantly, these treatments enhanced the expression of long-term potentiation in the perforant path synapse in the dentate gyrus. Finally, chronic TMS as well as mianserin suppressed the serotonin-dependent, potentiating action of fenfluramine on population spike in the dentate gyrus. Thus, TMS, mianserin, and desipramine are likely to affect the same neuronal populations, which may be relevant to their antidepressant action.
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(2001) European Journal of Neuroscience. 13, 7, p. 1273-1282 Abstract
We have recently demonstrated that embryonic E16 hippocampal neurons grown in cultures are unable to form fast synaptic connections unless treated with BDNF or NT-3, This experimental system offers an opportunity to define the roles of neurotrophins in processes leading to formation of functional synaptic connections. We have used ultrastructural and electrophysiological methods to explore the cellular locations underlying neurotrophin action on synaptic maturation. The rate of spontaneous miniature excitatory postsynaptic currents (mEPSCs) evoked by hyperosmotic stimulation was 7-16-fold higher in neurotrophin-treated cells than in controls. In addition, the potent neurotransmitter-releasing drug alpha -latrotoxin was virtually ineffective in the control cells while it stimulated synaptic events in neurotrophin-treated cells. Likewise, the membrane-bound dye FM1-43 was taken up by terminals in neurotrophin-treated cultures five-fold more than in controls. Both the total number and the number of docked synaptic vesicles were increased by neurotrophin treatment. Activation of synaptic responses by neurotrophins occurred even when postsynaptic glutamate receptors and action potential discharges were pharmacologically blocked. These results are consistent with a presynaptic locus of action of neurotrophins to increase synaptic vesicle density which is critical for rapid synaptic transmission. They also suggest that neurotrophins can activate synapses in the absence of pre- and postsynaptic neuronal activity.
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(2001) Neurobiology of Aging. 22, 2, p. 255-263 Abstract
Transcranial magnetic stimulation (TMS) is being proposed as a method of choice for the treatment of clinical depression, yet its action in the brain is still not well understood. In previous studies we found that TMS has a long-term effect on reactivity of the hippocampus to perforant path stimulation. Since the efficacy of antidepressants is highly age-dependent, we studied possible age-related effects of TMS on hippocampal evoked responses. Young adult (3 months), aging (10 months) and aged (24-26 months) awake rats were subjected to daily TMS for one week, followed by measurements of several parameters of reactivity to perforant path stimulation in the anesthetized rat. TMS did not affect responses of the hippocampus to single perforant path stimulation, but reduced drastically paired-pulse and frequency dependent depression in the young and aging but not the old rats. Likewise, TMS increased LTP expression in the young but not the old rats, and reduced the efficacy of serotonin modulation of reactivity of the hippocampus, in the young but not the old rats. Thus, long term effects of chronic TMS on local GABAergic inhibition are highly age dependent.
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(2001) Hormones and Behavior. 40, 2, p. 156-159 Abstract
Dissociated cultured rat hippocampal pyramidal neurons respond to estradiol with a time-dependent, two-fold increase in density of their dendritic spines. This effect is mediated by an estrogen receptor, probably of the alpha nuclear receptor type. In searching for the molecular mechanisms leading from the initial activation of the estrogen receptor to the final formation of new dendritic spines, we found that estradiol acts on GABAergic interneurons expressing the estrogen receptor by decreasing their inhibitory tone. In culture, this is assumed to cause a shift in the balance between excitation and inhibition toward enhanced excitation, over-activation of the pyramidal neurons, and subsequent formation of novel dendritic spines. The action of estradiol on spine formation is mediated by phosphorylation of cyclic AMP response element binding protein in the pyramidal neurons and is blocked when inhibition is enhanced by diazepam and when excitation is blocked by tetrodotoxin. Progesterone blocks the effect of estradiol on dendritic spines through its conversion to tetrahydroprogesterone, which enhances GABAergic inhibition. Subsequent to formation of novel dendritic spines, there is an increase in the density of glutamatergic receptors in the affected cells, an increase in the cellular calcium response to glutamate, and an increase in network synaptic activity among the cultured neurons.
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(2001) Progress in Neurobiology. 63, 1, p. 61-70 Abstract
Contrary to a century-old belief that dendritic spines are stable storage sites of long term memory, the emerging picture from a recent flurry of exciting observations using novel high resolution imaging methods of living cells in culture is that of a dynamic structure, which undergoes fast morphological changes over periods of hours and even minutes. Concurrently, the nature of stimuli which cause formation or collapse of dendritic spines has changed from a mysterious Hebbian-governed plasticity producing stimulus to the more trivial activation of the synapse by strong/weak stimulation. The molecular mechanisms underlying spine plasticity are beginning to emerge; the role of presynaptic and/or postsynaptic activity, genetic, central or local factors in the formation and retraction of spines are currently being analyzed. A common mechanism for both, formation/elongation and pruning/retraction of spines, involving changes in intracellular calcium concentration ([Ca2+](i)), is emerging. It appears that [Ca2+](i) is related to changes in spines in a bell shape form: lack of synaptic activity causes transient outgrowth of filopodia but eventual elimination of spines, a moderate rise in [Ca2+](i) causes elongation of existing spines and formation of new ones, while a massive increase in [Ca2+](i) such as that seen in seizure activity, causes fast shrinkage and eventual collapse of spines. Nuclear signals (e.g. CREB), activated by an increase in [Ca2+](i), are involved in the central regulation of spine formation, while spine shrinkage and elongation are probably triggered by local [Ca2+](i) changes. This hypothesis provides a parsimonious explanation for conflicting reports on activity-dependent changes in dendritic spine morphology. Still, the many differences between cultured neurons, with which most of current studies are conducted, and the neuron in the real brain, require a cautious extrapolation of current assumptions on the regulation of spine formation. Copyright (C) 2001 Elsevier Science Ltd.
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Functional plasticity triggers formation and pruning of dendritic spines in cultured hippocampal networks(2001) Journal of Neuroscience. 21, 1, p. 186-193 Abstract
Despite widespread interest in dendritic spines, little is known about the mechanisms responsible for spine formation, retraction, or stabilization. We have now found that a brief exposure of cultured hippocampal neurons to a conditioning medium that favors activation of the NMDA receptor produces long-term modification of their spontaneous network activity. The conditioning protocol enhances correlated activity of neurons in the culture, in a process requiring an increase in [Ca2+](i) and is associated with both formation of novel dendritic spines and pruning of others. The novel spines are likely to be touched by a presynaptic terminal, labeled with FM4-64 dye, whereas the absence of such terminals increases the likelihood of spine pruning. These results indicate that long-term functional changes are correlated with morphological modifications of dendritic spines of neurons in a network.
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(2001) Neuron. 30, 3, p. 751-758 Abstract
Dendritic spines have long been known to contain contractile elements and have recently been shown to express apparent spontaneous motility. Using high-resolution imaging of dendritic spines of green-fluorescent protein (GFP)-expressing, patch-clamped hippocampal neurons in dissociated culture, we find that bursts of action potentials, evoked by depolarizing current pulses, cause momentary contractions of dendritic spines. Blocking calcium currents with cobalt prevented these twitches. In additional experiments with neurons loaded via a micropipette with calcium-sensitive and insensitive dyes, spontaneous calcium transients were associated with a rapid contraction of the spine head. The spine twitch was prolonged by tetraethylammonium or bicuculline, which enhance calcium transients, and was blocked by the actin polymerization antagonist latrunculin-B. The spine twitch may be instrumental in modulating reactivity of the NMDA receptor to afferent stimulation, following back-propagating action potentials.
2000
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(2000) Trends in Neurosciences. 23, 2, p. 53-57 Abstract
The recent advent of novel high-resolution imaging methods has created a flurry of exciting observations that address a century-old question: what are biological signals that regulate formation and elimination of dendritic spines? Contrary to the traditional belief that the spine is a stable storage site of long-term neuronal memory, the emerging picture is of a dynamic structure that can undergo fast morphological variations. Recent conflicting reports on the regulation of spine morphology lead to the proposal of a unifying hypothesis for a common mechanism involving changes in postsynaptic intracellular Ca2+ concentration, [Ca2+](i): a moderate rise in [Ca2+](i) causes elongation of dendritic spines, while a very large increase in [Ca2+](i) causes fast shrinkage and eventual collapse of spines. This hypothesis provides a parsimonious explanation for conflicting reports on activity-dependent changes in dendritic spine morphology, and might link these changes to functional plasticity in central neurons.
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(2000) Current Opinion in Neurobiology. 10, 5, p. 582-586 Abstract
A recent series of exciting observations, using novel high-resolution time-lapse imaging of living cells, has provoked a major shift in our understanding of the dendritic spine, from a stable storage site of long-term memory to a dynamic structure that undergoes rapid morphological variations. Through these recent observations, the molecular mechanisms underlying spine plasticity are beginning to emerge. A common mechanism involving changes in intracellular Ca2+ concentration may control both the formation/elongation and the pruning/retraction of spines. Spine motility may be instrumental in the formation of synapses, may contribute to the anchoring/removing of glutamate receptors at spine heads, and may control the efficacy of existing synapses.
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(2000) Neuroendocrinology. 72, 3, p. 133-143 Abstract
Estradiol has been shown to cause an increase in dendritic spine density in cultured hippocampal neurons, an effect mediated by downregulation of brain-derived neurotrophic factor (BDNF) and glutamic acid decarboxylase (GAD), and the subsequent phosphorylation of cAMP response element binding protein (CREB) in response to enhanced activity levels. Interestingly, progesterone was shown to counteract the effects of estradiol on dendritic spine density in vivo and in vitro. The present study examined how progesterone may act to block the effects of estradiol in the molecular cascade of cellular events leading to formation of dendritic spines. Progesterone did not affect the estradiol-induced downregulation of BDNF or GAD, but it did block the effect of estradiol on CREB phosphorylation. The latter effects of progesterone on the pCREB response and spine formation were reversed by indomethacin, which prevents the conversion of progesterone to the neurosteroid tetrahydroprogesterone (THP). We therefore examined if the progesterone effects were caused by its active metabolite THP. Progesterone treatment caused a 60-fold increase in THP in the culture medium. THP itself enhanced spontaneous GABAergic activity in patch-clamped cultured neurons. Finally, THP blocked the estradiol-induced increase in spine density. These results suggest that progesterone, through conversion to THP, blocks the effects of estradiol on dendritic spines not via a direct nuclear receptor interaction but by counteracting the enhanced excitability produced by estradiol in the cultured network. Copyright (C) 2000 S. Karger AG, Basel.
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(2000) Neural Plasticity. 7, 1-2, p. 31-42 Abstract
Dissociated hippocampal neurons, grown in culture for 2 to 3 weeks, tended to fire bursts of synaptic currents at fairly regular intervals, representing network activity. A brief exposure of cultured neurons to GABA caused a total suppression of the spontaneous network activity. Following a washout of GABA, the activity was no longer clustered in bursts and instead, the cells fired at a high rate tonic manner. The effect of removing GABA could be seen as long as 1 to 2 days after GABA withdrawal and is expressed as an increase in the number of active cells in a network, as well as in their firing rates. Such striking effects of GABA removal may underlie part of the GABA withdrawal syndrome seen elsewhere.
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(2000) Cerebral Cortex. 10, 10, p. 1045-1052 Abstract
Fragile-X, the main cause of inherited human mental retardation is associated with the absence of a recently identified fragile-X mental retardation protein (FMRP). Mice in which this protein is lacking due to a knockout (KO) mutation are reported to express altered dendritic spines on their cortical neurons compared with wild type (WT) controls. We have used tissue-cultured neurons to examine differences in morphology and synaptic connectivity between WT and FMRP-deficient mice. Hippocampal neurons taken from KO mice and grown in culture for 3 weeks have shorter dendrites and fewer dendritic spines than their WT counterparts. Also, KO cells tend to express fewer functional synaptic connections, which develop more slowly and produce smaller excitatory synaptic currents than WT controls. These observations may have important implications for the understanding of mental retardation associated with the absence of FMRP.
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(2000) Hippocampus. 10, 5, p. 587-595 Abstract
The recent use of novel high-resolution imaging methods of living neurons in vitro has led to a change in the view of the dendritic spine, from a stable, long-term memory storage device to that of a dynamic structure, which can undergo fast morphological changes over periods of hours and even minutes. While the functional significance of these changes in spine dimensions is still obscure, we have obtained evidence to indicate that the length of the spine has a critical role in determining the degree of interaction between the spine head and the parent dendrite, such that longer spines are more independent of the parent dendrite than the short ones. We have now studied the role of intracellular calcium stores in determining the magnitude and time course of spine responses to a calcium surge evoked in response to glutamate, which causes an influx of calcium, and the results indicate that spine morphology has an important role in determining the involvement of the stores in calcium responses. Since spines can change their length over a rather short time, these results indicate that changes in spine length serve to fine-tune the interaction between the spine head and the parent dendrite on a continuous basis. (C) 2000 Wiley-Liss, Inc.
1999
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(1999) Journal of Neuroscience. 19, 24, p. 10977-10984 Abstract
Cu/Zn superoxide dismutase (SOD-1) is a key enzyme in oxygen metabolism in the brain. Overexpression of SOD-1 in transgenic (Tg) mice has been used to study the functional roles of this enzyme in oxidative stress, lipid peroxidation, and neurotoxicity. We found that Tg-SOD-1 mice are strikingly less sensitive to kainic acid-induced behavioral seizures than control mice. Furthermore, the hippocampus of Tg-SOD-1 mice was far less sensitive to local application of bicuculline, a GABA-A antagonist, than the hippocampus of control mice. GABAergic functions, expressed in extracellular paired-pulse depression, and in IPSCs recorded in dentate granular cells were enhanced in Tg-SOD-1 mice. Finally, long-term potentiation (LTP), not found in the dentate gyrus of Tg-SOD-1 mice, could be restored by local blockade of inhibition and could be blocked in control mice by injection of diazepam, which amplifies inhibition. These results indicate that constitutive elevation of SOD-1 activity exerts a major effect on neuronal excitability in the hippocampus, which, in turn, controls hippocampal ability to express LTP.
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(1999) Proceedings of the National Academy of Sciences of the United States of America. 96, 21, p. 12068-12072 Abstract
The ability to monitor ongoing changes in the shape of dendritic spines has important implications for the understanding of the functional correlates of the great variety of shapes and sizes of dendritic spines in central neurons. We have monitored and three-dimensionally reconstructed dendritic spines in cultured hippocampal neurons over several hours of observation in a confocal laser scanning microscope. In the absence of extrinsic stimulation, the dimensions of dendritic spines of 3-week-old cultured neurons did not change to any significant degree over 3-4 hr in the culture dish, unlike the case with younger cultures. Releasing calcium from stores with pulse application of caffeine causes a transient rise of [Ca2+](i) in dendrites and spines, monitored with the calcium dye Oregon-green. Application of caffeine to a dendrite imaged with calcein caused a fast and significant increase in the size of existing dendritic spines and could lead to formation of new ones. This effect is mediated by calcium released from the ryanodine- sensitive stores, as application of caffeine in the presence of ryanodine blocked this effect on the morphology of dendritic spines. Thus, release of calcium from stores is sufficient to produce significant changes in the shape of dendritic spines of cultured hippocampal neurons.
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(1999) NeuroReport. 10, 13, p. 2875-2877 Abstract
THE recent conflicting observations on the effects of excitatory afferent activity on dimensions of dendritic spines of central neurons led us to examine the possibility that the same spine can either increase or decrease its length in response to different stimuli. Cultured hippocampal neurons labeled with calcein, were 3D reconstructed in a confocal laser scanning microscope. Their responses to pulse application of glutamate were examined. Short pulses of glutamate caused elongation of dendritic spines, while long pulses caused fast shrinkage of the same set of spines. Thus, the same spine can undergo two opposite responses to application of glutamate, depending on the stimulation intensity/duration. These observations have important implications for understanding the roles of dendritic spines in information processing in central neurons.
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(1999) Journal of Biological Chemistry. 274, 31, p. 21673-21678 Abstract
Gaucher disease is a glycosphingolipid storage disease caused by defects in the activity of the lysosomal hydrolase, glucocerebrosidase (GlcCerase), resulting in accumulation of glucocerebroside (glucosylceramide, GlcCer) in lysosomes. The acute neuronopathic type of the disease is characterized by severe loss of neurons in the central nervous system, suggesting that a neurotoxic agent might be responsible for cellular disruption and neuronal death. We now demonstrate that upon incubation with a chemical inhibitor of GlcCerase, conduritol-B-epoxide (CBE), cultured hippocampal neurons accumulate GlcCer. Surprisingly, increased levels of tubular endoplasmic reticulum elements, an increase in [Ca2+](i) response to glutamate, and a large increase in [Ca2+](i) release from the endoplasmic reticulum in response to caffeine were detected in these cells. There was a direct relationship between these effects and GlcCer accumulation since co- incubation with CBE and an inhibitor of glycosphingolipid synthesis, fumonisin B1, completely antagonized the effects of CBE. Similar effects on endoplasmic reticulum morphology and [Ca2+](i) stores were observed upon incubation with a short-acyl chain, nonhydrolyzable analogue of GlcCer, C8- glucosylthioceramide. Finally, neurons with elevated GlcCer levels were much more sensitive to the neurotoxic effects of high concentrations of glutamate than control cells; moreover, this enhanced toxicity was blocked by pre- incubation with ryanodine, suggesting that [Ca2+](i) release from ryanodine-sensitive intracellular stores can induce neuronal cell death, at least in neurons with elevated GlcCer levels. These results may provide a molecular mechanism to explain neuronal dysfunction and cell death in neuronopathic forms of Gaucher disease.
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(1999) Journal of Neuroscience. 19, 8, p. 3198-3203 Abstract
Transcranial magnetic stimulation (TMS) has become a promising treatment of affective disorders in humans, yet the neuronal basis of its long-lasting effects in the brain is still unknown. We studied acute and lasting effects of TMS on reactivity of the rat hippocampus to stimulation of the perforant path. Application of TMS to the brain of the anesthetized rat caused a dose- dependent transient increase in population spike (PS) response of the dentate gyrus to perforant path stimulation. In addition, TMS caused a marked decrease in inhibition and an increase in paired-pulse potentiation of reactivity to stimulation of the perforant path. Also, TMS suppressed the ability of fenfluramine (FFA), a serotonin releaser, to potentiate PS response to perforant path stimulation. Chronic TMS did not affect single population spikes but caused an increase in paired-pulse potentiation, which was still evident 3 weeks after the last of seven daily TMS treatments. After chronic TMS, FFA was ineffective in enhancing reactivity to perforant path stimulation, probably because it lost the ability to release serotonin. In addition, the β adrenergic receptor agonist isoproterenol, which caused an increase in PS in the control rats, failed to do so in the TMS-treated rats. These results indicate that TMS produces a long-term reduction in efficacy of central modulatory systems.
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(1999) Hippocampus. 9, 5, p. 534-541 Abstract
Vasoactive intestinal peptide (VIP) expression is restricted to interneurons in the hippocampus of normal adult rats. However, 3-6 hours after a 60-minute walk in an activity wheel, VIP was transiently expressed in most pyramidal and granular neurons of the hippocampus. Locomotion was also associated with a dramatic increase in VIP immunoreactivity in the motor cortex, primarily in bipolar cells. Reverse transcriptase-polymerase chain reaction analysis indicated that VIP mRNA increases transiently by more than twofold, before the increases in peptide immunoreactivity in both the hippocampus and motor cortex. By comparison, another marker of inhibitory interneurons, glutamate decarboxylase, did not change its expression pattern after locomotion. The calcium binding protein, calbindin-D28K, normally expressed in interneurons, was now found also in glial cells of the hippocampus and motor cortex. Another marker of enhanced electrical activity, the immediate early gene, c-Fos, was expressed in pyramidal and granular neurons at 3 hours but not at 6 hours after locomotion. These results suggest that mapping of peptide expression in the brain of a docile, inactive rat may not reflect the real distribution and functions of a peptide in an active animal.
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(1999) Journal of Neuroscience. 19, 2, p. 520-528 Abstract
Calcium waves represent a widespread form of intercellular communication. Although they have been thought for a long time to require gap junctions, we recently demonstrated that mouse cortical astrocytes use an extracellular messenger for calcium wave propagation. The present experiments identify ATP as a major extracellular messenger in this system. Medium collected from astrocyte cultures during (but not before) calcium wave stimulation contains ATP. The excitatory effects of medium samples and of ATP are blocked by purinergic receptor antagonists and by pretreatment with apyrase; these same purinergic receptor antagonists block propagation of electrically evoked calcium waves. ATP, applied at the concentration measured in medium samples, evokes responses that are qualitatively and quantitatively similar to those evoked by those medium samples. These data implicate ATP as an important transmitter between CNS astrocytes.
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Geometry of dendritic spines affects calcium dynamics in hippocampal neurons: Theory and experiments(1999) Journal of Neurophysiology. 82, 1, p. 450-462 Abstract
The role of dendritic spine morphology in the regulation of the spatiotemporal distribution of free intracellular calcium concentration ([Ca2+])(i)) was examined in a unique axial-symmetrical model that focuses on spine-dendrite interactions, and the simulations of the model were compared with the behavior of real dendritic spines in cultured hippocampal neurons. A set of nonlinear differential equations describes the behavior of a spherical dendritic spine head, linked to a dendrite via a cyclindrical spine neck. Mechanisms for handling of calcium (including internal stores, buffers, and efflux pathways) are placed in both the dendrites and spines. In response to a calcium surge, the magnitude and time course of the response in both the spine and the parent dendrite vary as a function of the length of the spine neck such that a short neck increases the magnitude of the response in the dendrite and speeds up the recovery in the spine head. The generality of the model, originally constructed for a case of release of calcium from stores, was tested in simulations of fast calcium influx through membrane channels and verified the impact of spine neck on calcium dynamics. Spatiotemporal distributions of [Ca2+](i) measured in individual dendritic spines of cultured hippocampal neurons injected with Calcium Green-1, were monitored with a confocal laser scanning microscope. Line scans of spines and dendrites at a
1998
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(1998) Proceedings of the National Academy of Sciences of the United States of America. 95, 21, p. 12653-12656 Abstract
Ataxia-telangiectasia (AT) is a human disease caused by mutations in the ATM gene. The neural phenotype of AT includes progressive cerebellar neurodegeneration, which results in ataxia and eventual motor dysfunction. Surprisingly, mice in which the Atm gene has been inactivated lack distinct behavioral ataxia or pronounced cerebellar degeneration, the hallmarks of the human disease. To determine whether lack of the Atm protein can nonetheless lead to structural abnormalities in the brain, we compared brains from male Atm-deficient mice with male, age-matched controls. Atm-deficient mice exhibited severe degeneration of tyrosine hydroxylase-positive, dopaminergic nigro-striatal neurons, and their terminals in the striatum. This cell loss was accompanied by a large reduction in immunoreactivity for the dopamine transporter in the striatum. A reduction in dopaminergic neurons also was evident in the ventral tegmental area. This effect was selective in that the noradrenergic nucleus locus coeruleus was normal in these mice. Behaviorally, Atm-deficient mice expressed locomotor abnormalities manifested as stride-length asymmetry, which could be corrected by peripheral application of the dopaminergic precursor L-dopa. In addition, these mice were hypersensitive to the dopamine releasing drug D-amphetamine. These results indicate that ATM deficiency can severely affect dopaminergic neurons in the central nervous system and suggest possible strategies for treating this aspect of the disease.
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(1998) Proceedings of the National Academy of Sciences of the United States of America. 95, 19, p. 11412-11417 Abstract
Dendritic spines are of major importance in information processing and memory formation in central neurons. Estradiol has been shown to induce an increase of dendritic spine density on hippocampal neurons in vivo and in vitro. The neurotrophin brain-derived neurotrophic factor (BDNF) recently has been implicated in neuronal maturation, plasticity, and regulation of GABAergic interneurons. We now demonstrate that estradiol down-regulates BDNF in cultured hippocampal neurons to 40% of control values within 24 hr of exposure. This, in turn, decreases inhibition and increases excitatory tone in pyramidal neurons, leading to a 2-fold increase in dendritic spine density. Exogenous BDNF blocks the effects of estradiol on spine formation, and BDNF depletion with a selective antisense oligonucleotide mimics the effects of estradiol. Addition of BDNF antibodies also increases spine density, and diazepam, which facilitates GABAergic neurotransmission, blocks estradiol-induced spine formation. These observations demonstrate a functional link between estradiol, BDNF as a potent regulator of GABAergic interneurons, and activity-dependent formation of dendritic spines in hippocampal neurons.
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(1998) Journal of Neuroscience. 18, 18, p. 7256-7271 Abstract
Cell cultures were used to analyze the role of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the development of synaptic transmission. Neurons obtained from embryonic day 18 (E18) rat hippocampus and cultured for 2 weeks exhibited extensive spontaneous synaptic activity. By comparison, neurons obtained from E16 hippocampus expressed very low levels of spontaneous or evoked synaptic activity. Neurotrophin treatment produced a sevenfold increase in the number of functional synaptic connections in the E16 cultures. BDNF induced formation of both excitatory and inhibitory synapses, whereas NT-3 induced formation of only excitatory synapses. These effects were independent of serum or the age of the gila bed used for the culture. They were not accompanied by significant changes in synaptic-vesicle-associated proteins or glutamate receptors. Treatment of the cultures with the neurotrophins for 3 d was sufficient to establish the maximal level of functional synapses. During this period, neurotrophins did not affect the viability or the morphology of the excitatory neurons, although they did produce an increase in the number and length of dendrites of the GABAergic neurons. Remarkably, only BDNF caused an increase in the number of axonal branches and in the total length of the axons of the GABAergic neurons. These results support a unique and differential role for neurotrophins in the formation of excitatory and inhibitory synapses in the developing hippocampus.
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(1998) Neurobiology of Aging. 19, 4, p. 317-324 Abstract
Local modulation of hippocampal-evoked responses to perforant path stimulation was studied by leaking drugs from the recording pipette placed in the dentate gyrus of anesthetized young (3 months old), aging (17 months old) and old (28 months old) rats. In old rats, the excitatory postsynaptic potential (EPSP) slope was much reduced compared to young: and aging rats. The population spike (PS) size was similar in all age groups. Bicuculline caused a marked increase in PS size relative to population EPSP, and reversed the response to the second pulse in a paired-pulse paradigm from inhibition to facilitation. The effect of bicuculline was only slightly reduced in old rats. The 5-HT1a agonist 8-OH-DPAT potentiated PSs in the dentate gyrus, while not affecting paired-pulse inhibition. The effect of 8-OH-DPAT was slightly reduced in old rats. Carbachol, a cholinergic agonist, reversed paired-pulse inhibition into facilitation in the young brain, but not in aging and old rats. These results demonstrate that age affects differentially the action of biogenic amines on hippocampal reactivity to efferent stimulation. (C) 1998 Elsevier Science Inc.
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(1998) European Journal of Neuroscience. 10, 6, p. 2192-2198 Abstract
Physiological and morphological properties of cultured hippocampal neurons were measured to investigate whether NMDA receptors play a role in survival and differentiation. Neurons dissociated from mouse embryos with different NMDAR1 genotypes were grown in culture. Electrophysiological analysis verified the absence of NMDA receptor-mediated currents in neurons taken from homozygous mutant (NR1-/-) embryos. The number of surviving hippocampal neurons was 2.5-fold higher in cultures from the NR1-/- embryos compared with wild type (NR1+/+) and heterozygous (NR1+/-) controls. Despite the lack of NMDA receptor function, NR1-/- neurons formed synapsin I-positive presynaptic boutons associated with MAP2ab-positive dendrites in culture. Confocal microscopic analysis of Dil labelled neurons confirmed the presence of dendritic spines on NR1-/- neurons with 80% of the density found in NR1+/+ neurons. These results suggest that the NMDA receptor has little effect on general features of neuronal differentiation. In contrast, there is clear effect on neuronal survival. This finding establishes neuron number in standard culture conditions as a measure of NMDA receptor activity.
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(1998) Journal of Neuroscience. 18, 11, p. 4177-4188 Abstract
The effects of changing NMDA receptor subunit composition on synaptic plasticity in the hippocampus were analyzed by creating transgenic mice overexpressing NR2D, a predominantly embryonic NMDA receptor subunit. NMDA- evoked currents in the transgenic mice had smaller amplitudes and slower kinetics. The transgenics also displayed age-dependent deficits in synaptic plasticity in area CA1 of the hippocampus. Long-term depression was selectively impaired in juvenile mice when NR2D overexpression was moderate. In mature mice, overexpression of NR2D was associated with a reduction of both NR2B and Ca2+-independent activity of Ca2+- and calmodulin-dependent protein kinase II. These biochemical changes were correlated with a marked impairment of NMDA-dependent long-term potentiation, but spatial behavior was normal in these mice. These results show that the developmental regulation of NMDA receptor subunit composition alters the frequency at which modification of synaptic responses occur after afferent stimulation.
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(1998) Journal of Neuroscience. 18, 7, p. 2550-2559 Abstract
We have previously shown that estradiol causes a twofold rise in dendritic spine density in cultured rat hippocampal neurons, as it does in vivo. More recently, estrogen receptors have been localized to aspiny inhibitory hippocampal interneurons, indicating that their effect on spiny pyramidal neurons may be indirect. We therefore examined the possibility that estradiol affects spine density by regulating inhibition in cultured hippocampal interneuron. Immunocytochemically, estrogen receptors were found to be co-localized with glutamate decarboxylase (GAD)-positive neurons (~21% of total neurons in the culture). Exposure of cultures to estradiol for 1 d caused a marked decrease (up to 80%) in the GAD content of the interneurons, measured both by immunohistochemistry and Western blotting. Also, the number of GAD-positive neurons in the cultures decreased to 12% of the total cell population. Moreover, GABAergic miniature IPSCs were reduced in both size and frequency by estradiol, whereas miniature EPSCs increased in frequency. We then mimicked the proposed effects of estradiol by blocking GABA synthesis with mercaptopropionic acid (MA). Cultures treated with MA expressed a dose- dependent decrease in GABA immunostaining that mimicked that seen with estradiol. MA-treated cultures displayed a significant 50% increase in dendritic spine density over controls, similar to that produced by estradiol. These results indicate that estradiol decreases GABAergic inhibition in the hippocampus, which appears to effectively increase the excitatory drive on pyramidal cells, and thus may provide a mechanism for formation of new dendritic spines.
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(1998) European Journal of Neuroscience. 10, 2, p. 538-544 Abstract
Copper/zinc superoxide dismutase (CuZn-SOD) is a key enzyme in the metabolism of oxygen free radicals. The gene encoding CuZn-SOD resides on human chromosome 21 and is overexpressed in Down syndrome (DS) patients. Overexpression of CuZn-SOD in transgenic (Tg) mice and cultured cells creates chronic oxidative stress leading to enhanced susceptibility to degeneration and apoptotic cell death. We have now found that three lines of Tg-CuZn-SOD mice, one of which also overexpresses S100β, a glial calcium binding protein, are deficient in spatial memory. Furthermore, hippocampal slices taken from these mice have an apparently normal synaptic physiology, but are impaired in the ability to express long-term potentiation (LTP). This effect on hippocampal LTP was abrogated by treatment of slices with the H2O2 scavenger catalase or the antioxidant N-t-butyl-phenylnitrone (BPN). It is proposed that elevated CuZnSOD causes an increase in tetanic stimulation-evoked formation of H2O2 which leads to diminished LTP and cognitive deficits in these mice.
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(1998) Proceedings of the National Academy of Sciences of the United States of America. 95, 4, p. 1888-1893 Abstract
Neu differentiation factor (NDF/neuregulin) is widely expressed in the central and peripheral nervous systems, where it functions as a mediator of the interactions between nerve cells and Schwann, glia, oligodendrocyte, and muscle cells, to control cellular proliferation, differentiation, and migration. NDF binds to two receptor tyrosine kinases, ErbB-3 and ErbB-4. Here we demonstrate that NDF and its ErbB-4 receptor are highly reactive to changes in ambient neuronal activity in the rodent brain in a region- selective manner. Generation of epileptic seizures by using kainic acid, a potent glutamate analog, elevated levels of NDF transcripts in limbic cortical areas, hippocampus, and amygdala. Concomitantly, ErbB-4 mRNA was increased with a similar spatial distribution, but transcription of the other NDF receptor, ErbB-3, did not change. A more moderate stimulation, forced locomotion, was accompanied by an increase in NDF transcripts and protein in the hippocampus and in the motor cortex. Similar changes were found with ErbB-4, but not ErbB-3. Last, a pathway-specific tetanic stimulation of the perforant path, which produced long-term potentiation, was followed by induction of NDF expression in the ipsilateral dentate gyrus and CA3 area of the hippocampus. Taken together, these results indicate that NDF is regulated by physiological activity and may play a role in neural plasticity.
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Age dependence of muscarinic plasticity in the rat hippocampus(1998) Progress In Alzheimer'S And Parkinson'S Diseases. 49, p. 661-666 Abstract
Keywords: Behavioral Sciences; Clinical Neurology; Psychiatry
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(1998) Neural Plasticity. 6, 3, p. 1-7 Abstract
Activation of cyclic AMP dependent kinase is believed to mediate slow onset, long-term potentiation (LTP) in central neurons. Cyclic-AMP activates a cascade of molecular events leading to phosphorylation of the nuclear cAMP responsive element binding protein (pCREB). Whereas a variety of stimuli lead to activation of CREB, the molecular processes downstream of CREB, which may be relevant to neuronal plasticity, are yet largely unknown. We have recently found that following exposure to estradiol, pCREB mediates the large increase in dendritic spine density in cultured rat hippocampal neurons. We now extend these observations to include other stimuli, such as bicuculline, that cause the formation of new dendritic spines. Such stimuli share with estradiol the same mechanism of action in that both require activity-dependent CREB phosphorylation. Our observations suggest that CREB phosphorylation is a necessary, but perhaps not sufficient, step in the process leading to the generation of new dendritic spines and perhaps to functional plasticity as well.
1997
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(1997) Journal of Neuroscience. 17, 5, p. 1670-1682 Abstract
Cultured rat hippocampal neurons grown on glass coverslips for 1-3 weeks were loaded with the calcium-sensitive fluorescent dye Fluo-3 and viewed with a confocal laser scanning microscope. Large pyramidal-shaped neurons were found to contain dye-accumulating organelles in their somata, primarily around nuclei and near the base of their primary dendrites. These organelles varied in size and increased in density over weeks in culture, and were not colocalized with the endoplasmic reticulum or with mitochondria. The Fluo-3 fluorescence in these calcium-containing organelles (COOs) was transiently quenched by exposure to Mn2+, indicating that the dye is a genuine [Ca2+] reporter and is not just a site of accumulating Fluo-3 dye. Recovery of fluorescence in the COOs after washout of Mn2+ involved activation of a thapsigargin-sensitive process. COOs responded to stimuli that evoke a rise of cytosolic [Ca2+] ([Ca](i)) in a unique manner; perfusion of caffeine caused a prolonged rise of [Ca] in the COOs ([Ca](C)), whereas it caused only a transient rise of [Ca](i). Pulse application of caffeine also caused a faster effect on [Ca](C) than on [Ca](i). Glutamate caused a transient rise of both [Ca](i) and [Ca](C), followed by a prolonged fall of only [Ca](C) to below rest level. This fall was blocked by preincubation with thapsigargin. Ryanodine blocked the cytosolic effects of caffeine but not its effect on [C](C). A clear distinction between COOs and the known calcium stores was seen in digitonin-permeabilized cells; in these, remaining Fluo-3 reported changes in store calcium, i.e., caffeine caused a reduction in Fluo-3 fluorescence in permeabilized cells, whereas it still caused an increase in [Ca](C). A possible role of CCOs in regulation of release of calcium from ryanodine-sensitive stores was indicated by the observation that COO- containing cells exhibited a larger and faster response to caffeine than cells that did not have them. We propose that COOs constitute a unique functional compartment involved in release of calcium from calcium-sensitive stores.
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(1997) Life Sciences. 60, 13-14, p. 1085-1091 Abstract
The cholinergic septohippocampal system has been associated with learning and memory, as evidenced by the severe loss of these functions in lesioned animals as well as in senile demented patients. In an attempt to comprehend the physiological basis of the cholinergic innervation for hippocampal functions, numerous studies employed the in-vitro hippocampal slice preparation and analyzed the consequences of exposing the cells to cholinergic ligands. Many effects of activating a cholinergic receptor in the hippocampus were thus described, including blockade of several types of potassium conductances, yet few of these effects are intuitively related to the involvement of the cholinergic system in hippocampal plasticity. An alternative approach involves focusing on the possible effect of low concentration of cholinergic ligands on reactivity of the hippocampus to afferent stimulation. We found two new actions of acetylcholine (ACh); The first one is a fast onset, short lived increase in cellular responses to activation of the N-methyl-D-aspartate (NMDA) receptor, and the second one is a slow onset, long lasting increase in reactivity to afferent stimulation, resembling that produced by a tetanic stimulation, which we called muscarinic long term potentiation (LTPm). The latter effect is mediated by a postsynaptic M2 receptor, and it shares several properties with the more familiar tetanic LTP. In addition, LTPm involves a rise of intracellular calcium concentration and an activation of both a tyrosine kinase and a serine/threonine kinase. Intuitively, LTPm is better related to hippocampal plasticity than the other reported effects of ACh in the hippocampus. Indeed, aged rats, which are cognitively impaired, lack LTPm while they do express other muscarinic actions. It is proposed that LTPm is an important link between the cholinergic action and function in the hippocampus.
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(1997) Proceedings of the National Academy of Sciences of the United States of America. 94, 4, p. 1482-1487 Abstract
While evidence has accumulated in favor of cAMP-associated genomic involvement in long-term synaptic plasticity, the mechanisms downstream of the activated nucleus that underlie these changes in neuronal function remain mostly unknown. Dendritic spines, the locus of excitatory interaction among central neurons, are prime candidates for long-term synaptic modifications. We now present evidence that links phosphorylation of the cAMP response element binding protein (CREB) to formation of new spines; exposure to estradiol doubles the density of dendritic spines in cultured hippocampal neurons, and concomitantly causes a large increase in phosphorylated CREB and in CREB binding protein. Blockade of cAMP-regulated protein kinase A eliminates estradiol-evoked spine formation, as well as the CREB and CREB binding protein responses. A specific antisense oligonucleotide eliminates the phosphorylated CREB response to estradiol as well as the formation of new dendritic spines. These results indicate that CREB phosphorylation is a necessary step in the process leading to generation of new dendritic spines.
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(1997) Journal of Neuroscience. 17, 22, p. 8695-8701 Abstract
Exposure of rat hippocampal slices to low concentrations of the muscarinic agonist carbachol (CCh) has been shown to produce a slow onset long-term potentiation (LTP) of reactivity to afferent stimulation in CA1 neurons. Although this potentiation shares a number of properties with tetanic LTP, muscarinic LTP (LTP(m)) is independent of activation of the NMDA receptor. We now demonstrate that low levels of hydrogen peroxide (H2O2) cause hippocampal slices to lose the ability to express LTP(m). This powerful effect of H2O2 is selective in that it does not affect the reactivity of hippocampal neurons to higher concentrations of CCh. In fact, H2O2 also blocks induction of a slow onset, non-NMDA-dependent tetanic LTP (NN-LTP). The functional relevance of this action of H2O2 is exemplified by the fact that the hippocampus of aged rats, which produces higher levels of endogenous H2O2 than that of young rats, lacks LTP(m) and expresses a markedly reduced NN-LTP. In aged rats, the lack of LTP(m) contrasts with an apparently normal muscarinic suppression of the EPSP slope induced by higher concentrations of CCh. When hippocampal slices from aged animals are treated with catalase, an enzyme that breaks down H2O2, LTP(m) is restored, and NN-LTP is enhanced. Thus, our study proposes a unique and novel age-dependent peroxide regulation of LTP(m) in the brain and provides a link between the cholinergic system, aging, and memory functions.
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(1997) Journal of Neurophysiology. 77, 3, p. 1614-1623 Abstract
Transverse hippocampal slices were cut from 8- to 9-day-old rats and maintained in an interface chamber for periods of 1-4 wk. in tissue culture conditions. Neurons in the slice preserved their spatial organization and connectivity. Dendritic spine density in CA1 neurons was very low at 1 wk in culture, and long, filopodia-like structures were abundant. Spine density increased in these neurons nearly threefold during the course of 3 wk in vitro, to approach values of those of the normal, in vivo hippocampus. The magnitude of long-term potentiation (LTP) of reactivity of CA1 to stimulation of CA3 neurons also increased during weeks in culture in parallel with the change in spine density. Chronic exposure of slices to drugs that interact with synaptic activity caused changes in their dendritic spine density. Blockade of the N-methyl-D-aspartate (NMDA) receptors with the receptor antagonist 2-aminophosphonovalerate (D-APV) or blockade of action potential discharges with tetrodotoxin (TTX) prevented dendritic spine development in immature cultures. Enhancing synaptic activity by blockade of GABAergic inhibition with picrotoxin did not affect spine density to a significant degree. D-APV-treated slices expressed larger LTP than controls. TTX-treated slices expressed smaller LTP than controls. Picrotoxin treated slices did not express LTP. It is proposed that LTP and dendritic spine density are correlated strongly during development, whereas they are not correlated in the more mature slice/culture of the hippocampus where spine density can be modulated by chronic exposure to blockers of synaptic activity, which will not affect LTP in a similar manner.
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(1997) Journal of Neuroscience. 17, 14, p. 5591-5598 Abstract
Hippocampal dentate gyrus reactivity to perforant path (PP) stimulation in the anesthetized rat was enhanced after systemic administration of the serotonin-releasing drug fenfluramine (FFA). This effect of FFA was mimicked by local application of the drug via the recording pipette, indicating that the effect of FFA is mediated by release of serotonin from intrahippocampal serotonergic terminals. The 5-HT1a antagonist NAN-190 and the 5-HT1b agonist CGS-12066-B, applied both systemically and locally, blocked the effect of FFA. This blocking action was not shared by the 5-HT2-4 receptor agonists or antagonists tested. The 5-HT1a receptor agonist 8-OH-DPAT, applied systemically, caused a marked reduction in population spike responses to PP stimulation, whereas an opposite effect was produced by local application of this drug. The effect of peripheral application of 8-OH-DPAT was blocked by depletion of serotonin. The local effect of FFA was blocked by a reducing neurotransmitter release with a pipette containing 10 mM Mg2+. Finally, local application of the GABA antagonist picrotoxin also enhanced population spike response to PP stimulation, and the effects of picrotoxin and FFA occluded. These results indicate that serotonin released from terminals in the hippocampus activates a 5-HT1a receptor on interneurons that suppresses their activity and thus enhances dentate granular cell population spike response to PP stimulation.
1996
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Lasting effects of glutamate on nuclear calcium concentration in cultured rat hippocampal neurons: Regulation by calcium stores(1996) Journal Of Physiology-London. 496, 1, p. 39-48 Abstract
1. Changes in free intracellular calcium concentrations ([Ca2+](i)) were measured in the nucleus and perinuclear regions of cultured rat hippocampal neurons using either fura-2 or fluo-3 calcium indicators. 2. Brief application of glutamate caused a transient rise of [Ca2+](i) in all cell compartments, which recovered to pre-drug levels in all but the nuclear region. The new, higher level of nuclear calcium ([Ca2+](n)) was sustained for as long as the cell was monitored. 3. The new level of [Ca2+](n) was dependent on the magnitude of the calcium transient, and was higher in older cells in culture, but it did not affect responses to subsequent applications of glutamate. 4. The sustained elevation of [Ca2+](n) was prevented by drugs which affect calcium stores (caffeine, ryanodine and Ruthenium Red), indicating that an extranuclear calcium store interacts with [Ca2+](n).
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(1996) Mechanisms of Development. 59, 1, p. 89-102 Abstract
To understand the mechanism of the sequential restriction of multipotency of stem cells during development, we have established culture conditions that allow the differentiation of neuroepithelial precursor cells from embryonic stem (ES) cells. A highly enriched population of neuroepithelial precursor cells derived from ES cells proliferates in the presence of basic fibroblast growth factor (bFGF). These cells differentiate into both neurons and glia following withdrawal of bFGF. By further differentiating the cells in serum-containing medium, the neurons express a wide variety of neuron-specific genes and generate both excitatory and inhibitory synaptic connections. The expression pattern of position-specific neural markers suggests the presence of a variety of central nervous system (CNS) neuronal cell types. These findings indicate that neuronal precursor cells can be isolated from ES cells and that these cells can efficiently differentiate into functional post-mitotic neurons of diverse CNS structures.
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(1996) Proceedings of the National Academy of Sciences of the United States of America. 93, 16, p. 8530-8535 Abstract
Cu/Zn superoxide dismutase (Cu/Zn SOD) is a key enzyme in the metabolism of oxygen free radicals. The gene resides on chromosome 21 and is overexpressed in patients with Down syndrome. Cultured neurons of transgenic Cu/Zn SOD (Tg-Cu/Zn SOD) mice with elevated activity of Cu/Zn SOD were used to determine whether constitutive overexpression of Cu/Zn SOD creates an indigenous oxidative stress that predisposes the Tg-Cu/Zn SOD neurons to added insults. Neurons from three independently derived Tg-Cu/Zn SOD strains showed higher susceptibility than nontransgenic neurons to kainic acid (KA)- mediated excitotoxicity, reflected by an earlier onset and enhanced apoptotic cell death. This higher susceptibility of transgenic neurons to KA-mediated apoptosis was associated with a chronic prooxidant state that was manifested by reduced levels of cellular glutathione and altered [Ca2+](i) homeostasis. The data are compatible with the thesis that overexpression of Cu/Zn SOD creates chronic oxidative stress in the transgenic neurons, which exacerbates their susceptibility to additional insults such as KA-mediated excitotoxicity.
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(1996) Brain Research. 719, 1-2, p. 234-238 Abstract
In previous studies we have demonstrated that raphe grafts, implanted into serotonin-depleted rat hippocampus can restore behavioral and physiological functions impaired by serotonin depletion. Since aging is associated with a reduction in serotonergic functions, we explored the possibility that grafting embryonic raphe tissue will ameliorate age-associated reduction of serotonergic functions in the hippocampus. Aged rats were implanted with E14 embryonic neural tissue, containing the raphe, or part of the parietal cerebral cortex. Three months later, the rats were anesthetized, and the responses of the dentate gyrus to perforant-path stimulation were measured. Serotonin-containing neurons were found in the raphe-grafted hippocampi. No differences were found between the two groups in the volume of the graft in the host brain. Raphe-grafted rats were not different from the cortex-grafted rats in reactivity to perforant path stimulation or in the response to a second of a pair of stimuli to the perforant path. They did, however, express a pronounced commissural inhibition, unlike the cortex-grafted rats. These results are similar to those found previously with a pharmacological enhancement of serotonergic neurotransmission. It is suggested that a graft of serotonergic neurons can ameliorate age-associated reduction in serotonergic functions in the hippocampus.
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(1996) Neuroscience. 71, 4, p. 1005-1011 Abstract
Rat hippocampal neurons, grown in dissociated culture for about 18 days, were exposed for 6 h to three days to stimuli which cause either an increase (GABA(A) antagonists, bicuculline or picrotoxin), or decrease (tetrodotoxin) in spontaneous neuronal activity. Individual neurons were stained with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate and visualized with a confocal laser scanning microscope. GABA antagonists caused a marked, up to 60%, increase in spine density on secondary dendrites or cultured hippocampal neurons. This was associated with a small decrease in spine length. The rise in spine density was partially prevented by treatment with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetra-acetate, or by blockade of protein synthesis with cycloheximide. Tetrodotoxin caused a marked elongation of dendritic spines (but did not cause a decrease in spine density comparable to the increase caused by picrotoxin). This effect was seen primarily but not exclusively in spines with no distinct head. Both treatments were most effective within 24 h of exposure. There were no other systematic effects of the drugs on the morphology of the dendritic spines. These results indicate that dendritic spines in cultured neurons depend on ongoing synaptic activity to maintain their shape, and that neurons respond to an increase in synaptic demand by an increase in spine density. Thus, dendritic spines are likely to have a role in short-term synaptic interaction rather than to constitute a long-term memory storage device.
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Serotonin, aging and cognitive functions of the hippocampus(1996) Reviews in the Neurosciences. 7, 2, p. 103-113 Abstract
The cholinergic hypothesis of senile dementia /18/ does not provide a sufficient explanation for age-dependent spatial learning deficits; these are observed before an appreciable reduction of cholinergic markers can be detected. Behavioral deficits similar to those observed in old rats cannot be induced in young rats by comparable cholinergic lesions but do occur following combined cholinergic/serotonergic lesions. Serotonergic raphe grafts in the hippocampus (but not in the entorhinal cortex or hypothalamus) prevent such combined lesion-induced spatial learning deficits. The behavioral deficits are associated with a reduction of hippocampal commissure feed-forward inhibition. Similar reduced inhibition is found in old rats, deficient in their performance of a spatial learning water-maze task. Finally, treating old rats with the serotonergic precursor 5-hydroxytryptophan (5-HTP) reduces the age-dependent spatial learning deficits and restores hippocampal commissure feed-forward inhibition. Serotonin may act in parallel to the cholinergic innervation of the hippocampus by affecting inhibitory interneurons but in addition it may act by modulating acetylcholine release. Acetylcholine release is modulated by serotonin and the enhancing effects of serotonin releasing drugs on dentate granule cell excitability are mediated by acetylcholine. We thus propose that a reduction of serotonergic modulation of hippocampal interneuron activity and impaired modulation of cholinergic effects in the hippocampus contribute to age-dependent cognitive deficits.
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(1996) Journal Of Physiology-London. 492, 2, p. 479-493 Abstract
1. Two concentration-dependent effects of the muscarinic agonist carbachol (CCh) were characterized in submerged slices of rat hippocampus using extracellular recordings of excitatory postsynaptic potentials (EPSPs): muscarinic long-term potentiation (LTP(m)) and depression. 2. LTP(m) of the EPSP slope was seen following long exposure (20 min) of the slice to low concentrations of CCh (0.2-0.5 μM). This LTP(m) was not accompanied by a change in the size of the afferent fibre volley or by a change in paired-pulse potentiation, consistent with a postsynaptic locus of CCh action. 3. Intracellular recordings from voltage-clamped neurons of inward current evoked by iontophoretically applied α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) revealed that, while cellular responses to NMDA rose transiently upon superfusion with 0.5 μM CCh, responses to AMPA increased gradually and remained potentiated after washout of CCh. 4. LTP(m) is mediated by an M2 muscarinic receptor. Two M2 muscarinic receptor antagonists, methoctramine and AFDX-116, blocked LTP(m). The M2 agonist oxotremorine induced LTP(m) at low agonist concentrations. None of the M1 and M3 receptor agonists and antagonists tested affected LTP(m). 5. Muscarinic fast onset depression of the EPSP was seen in response to higher concentrations of CCh (2-5 μM). This depression was accompanied by an increase in paired-pulse potentiation, indicating a possible presynaptic locus of action. The M3 muscarinic receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) blocked the muscarinic depression of the EPSP slope. M1, M2 and M4 muscarinic antagonists did not block this response. 6. Blockade of the muscarinic depression by 4-DAMP did not uncover a suppressed LTP(m). However, addition of picrotoxin facilitated the expression of LTP(m) induced by high concentrations of CCh, indicating an involvement of interneurons in regulation of LTP(m). 7. Cholinergic denervation produced by fimbria-fornix transection resulted in supersensitivity of both M2- and M3-mediated effects, indicating that the receptors mediating these effects are not located on presynaptic cholinergic fibres. In the presence of 4-DAMP and picrotoxin the dose-response curve for CCh-induced effects in slices from lesioned animals was shifted to the left relative to that of normal animals, indicating a supersensitivity of both receptor types.
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(1996) Journal of Neuroscience. 16, 13, p. 4059-4068 Abstract
The effects of gonadal steroid hormones on dendritic spines were studied in hippocampal neurons that were dissociated and grown in culture for 2-3 weeks. Exposure to estradiol caused up to a twofold increase in dendritic spine density in these neurons. The effect of estradiol was stereospecific and blocked by the steroid antagonist tamoxifen. The estradiol-induced rise in spine density was blocked by the NMDA antagonist APV, but not by the AMPA/KA antagonist DNQX. The estradiol-induced rise in spine density was blocked by the serine/threonine kinase inhibitor H7, but not by the tyrosine kinase inhibitor genestein, and was partially mimicked by PMA, an activator of protein kinase C. Estradiol also caused an increase in the fluorescence intensity of synaptophysin-immunoreactive terminals, corresponding to presynaptic boutons. Finally, estradiol caused a rise in [Ca](i) reactivity of the cultured neurons to topical application of glutamate. These studies are the first to examine receptor and second messenger regulation of dendritic spines, and they illustrate the viability of cultured neurons as a powerful test system to address issues related to the regulation of dendritic spine maturation.
1995
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(1995) Trends in Neurosciences. 18, 11, p. 468-471 Abstract
Ever since their first description in neurons, dendritic spines could be visualized only in fixed tissue, using high-power light and electron microscopy. Recent studies have been able to measure the free intracellular Ca2+ concentration ([Ca2+]i) in dendritic spines of live neurons, and the results suggest that the spine is an independent cellular Ca2+ compartment. Other recent observations have indicated that the density of spines on dendrites changes in a dynamic fashion depending on ongoing neuronal activity. Together, these findings have led to the proposal that the dendritic spine is not only a storage device for long-term memory but perhaps a means for isolating the cell from the harmful consequences of synaptically evoked surges in [Ca2+]i. In other words, the dendritic spine is a neuroprotectant. This hypothesis has specific testable implications, including relating cell activity to spine density.
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''Sodium overload'' of neurons after glutamate and NMDA toxic treatment in cultured hippocampal neurons(1995) Biologicheskie Membrany. 12, 6, p. 585-590 Abstract
Changes in cytosolic Na+ ([Na+](i)) caused by a toxic glutamate (GLU) or NMDA treatment of cultured hippocampal neurons were monitored using SBFI fluorescent probe and imaging microscopy. Both GLU and NMDA (50 or 100 mu M in Mg2+-free solution, 15 min) induced a considerable Increase in [Na+](i) (from 6-8 to 30-45 mM) which persisted after the termination of the treatment. APV (100 mu M), a competitive NMDA antagonist, failed to decrease the elevated [Na+](i) when applied in the post-NMDA period. The results obtained suggest that the main reason for the impairment of Na+/Ca2+ exchange in the post-glutamate period (see FEBS Letters, 1993, 324, 271-273) is a reduction of the transmembrane NE gradient caused apparently by inhibiting Na+-K+ pump.
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(1995) Journal Of Physiology-London. 486, 2, p. 283-295 Abstract
1. Cultured rat hippocampal neurons were loaded with the Ca2+ indicator fura‐2 through micropipettes and visualized with an inverted microscope equipped with a high power objective and a cooled CCD camera. The responses of dendritic spines and their parent dendrites to stimuli which evoke a rise of [Ca2+]i were monitored. 2. NMDA caused a rapid and transient rise in [Ca2+]i, which was more evident in the spine than in the parent dendrite. The recovery in both compartments had the same time course, and was dependent on normal [Na+]o. 3. Application of alpha‐latrotoxin, which causes release of neurotransmitters from terminals, produced a rise of [Ca2+]i in the dendritic spines, more than in their parent dendrites. Prolonged exposure to the drug eliminated the spine/dendrite disparity. 4. The presence of voltage‐gated calcium channels in dendritic spines is indicated by the enhanced calcium rise in spines rather than dendrites of cells depolarized by either intracellular current injection or by raising [K+]o. This rise was attenuated by nifedipine or verapamil, both L‐type channel blockers. 5. It is suggested that the dendritic spine constitutes an independent calcium compartment that is closely linked to the parent dendrite.
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(1995) Neuroscience Letters. 193, 2, p. 73-76 Abstract
Individual cultured rat hippocampal neurons grown on glass coverslips were loaded with lucifer yellow (LY) and segments of their dendrites, containing dendritic spines, were exposed to the glutamate agonist N-methyl-D-aspartate (NMDA). Repeated pulse application of NMDA caused a reduction of spine length by 20%, within 1-2 h of exposure to the drug. No such changes were seen in spines exposed to NMDA in presence of the antagonist 2-aminophosphonovalerate or in spines on sister dendrites which were not exposed to NMDA. It is suggested that dendritic spines shrink following exposure to excessive synaptic stimulation.
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(1995) Journal of Neuroscience Methods. 59, 1, p. 25-29 Abstract
The growing interest in dendritic spines in recent years originates from both the realization that the spine is likely to be the site where long-term plastic changes in synaptic properties take place, and that imaging methods are available which allow, for the first time, the study of these changes in the living dendritic spine. This report briefly summarizes methodological and biological issues associated with the study of dendritic spines in living tissue. The combined use of electrical and imaging methods for the study of dendritic spines certainly will contribute to a better understanding of synaptic integration and plasticity.
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(1995) Journal of Neuroscience. 15, 1 I, p. 1-11 Abstract
We monitored developmental alterations in the morphology of dendritic spines in primary cultures of hippocampal neurons using confocal laser scanning microscopy (CLSM) and the fluorescent marker Dil. Dissociated rat hippocampal neurons were plated on polylysine-coated glass cover slips end grown in culture for 1-4 weeks. Fixed cultures were stained with Dil and visualized with the CLSM. Spine density, spine length, and diameters of spine heads and necks were measured. Some cultures were immunostained for synaptophysin and others prepared for EM analysis. In the 1-3 week cultures, 92-95% of the neurons contained spiny dendrites. Two subpopulations of spine morphologies were distinguished. At 1 week in culture, 'headless' spines constituted 50% of the spine population and were equal in length to the spines with heads. At 2, 3, and 4 weeks in culture headless spines constituted a progressively smaller fraction of the population and were, on average, shorter than spines with heads. Spines with heads had narrower necks than headless spines. At 3 weeks in culture, spines were associated with synaptophysin-immunoreactive labeling, resembling synaptic terminals. At 4 weeks in culture, only 70% of the Dil-filled cells had spiny dendrites, and the density of spines decreased. Ultrastructurally, the majority of dendritic spine-like structures at I week resembled long filopodia without synaptic contacts. The majority of axospinous synapses were on short 'stubby' spines. At 3 weeks in culture, the spines were characteristic of those seen in vivo. They contained no microtubules or polyribosomes, were filled with a characteristic, filamentous material, and formed asymmetric synapses. These studies provide the basis for further analysis of the rules governing the formation, development, and plasticity of dendritic spines under controlled, in vitro conditions.
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(1995) Journal of Neurophysiology. 74, 1, p. 484-488 Abstract
1. Cultured hippocampal neurons were recorded with a patch pipette containing 100 μM of the calcium indicator Fluo-3, and one of their dendrites, carrying dendritic spines, was visualized with a x100, 1.3- numerical aperture oil objective. Calcium spikes evoked by depolarizing the somata and changes in free dendrite and spine calcium concentrations ([Ca](d) and [Ca](s), respectively) were monitored with a cooled charge-coupled device (CCD) camera, acquiring images at a rate of 17-20 ms per frame. In the majority of spine-dendrite pairs, [Ca](s) rose faster and to a higher level than the adjacent [Ca](d). Likewise, topical application of glutamate evoked a faster and larger change in [Ca](s) than in [Ca](d). The rise of intracellular calcium concentration in response to a depolarizing current pulse, but not in response to glutamate, was reduced in the presence of the calcium antagonist verapamil in both dendrites and spines. It is suggested that dendritic spines possess voltage-gated calcium channels.
1994
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(1994) European Journal of Pharmacology. 264, 3, p. 279-284 Abstract
Reactivity of the hippocampal system to stimulation of its main afferent, the perforant path, was studied in the intact, anesthetized rat. Parental administration of fenfluramine caused a marked elevation of population spike response to perforant path stimulation. An injection of atropine before, but not after fenfluramine, blocked the potentiating effect of fenfluramine. The atropine blockade was dose-dependent and not mimicked by the peripheral muscarinic receptor antagonist methyl atropine. This effect of fenfluramine was also prevented by an injection of the 5-HT receptor antagonist spiperone. The effect of fenfluramine was mimicked by the anticholinesterase physostigmine, which was not affected by spiperone pretreatment. It is proposed that release of 5-HT (5-hydroxytryptamine) by fenfluramine potentiates reactivity to afferent stimulation by interacting with cholinergic terminals in the hippocampus.
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(1994) European Journal of Neuroscience. 6, 11, p. 1720-1728 Abstract
Combined with a partial cholinergic deficiency, serotonergic lesions induce severe spatial learning deficits. Serotonergic lesions, however, have additional effects, such as reduced body weight and disruption of thermoregulation, which may be the cause of the observed learning deficits. Restoration of the serotonergic innervation of the hippocampus by raphe grafts reduces these learning deficits. The effects of the grafts may result from a direct support of spatial learning but may also be an indirect result of preventing some of the other effects of serotonergic lesions. In the present study we used raphe grafts to examine the selectivity and specificity of the effects of serotonergic lesions in the rat, and used the behavioural effects as an indication of successful transplantation in order to examine the fine details of such grafts. Raphe grafts in the hippocampus did not prevent the effects of the lesions on body weight, thermoregulation and exploratory behaviour but did minimize the effects of the lesions on spatial learning. In contrast, raphe grafts in the hypothalamus reduced the effects of the lesions on thermoregulation but failed to support learning. The grafted fibres showed termination specificity with the interneurons, which is typical of the serotonergic innervation of the normal hippocampus. The results indicate that the serotonergic innervation of the hippocampus functions locally to support spatial learning. This role of serotonin is independent of its involvement in modulation of body weight, thermoregulation or exploratory behaviour. The results confirm that the modes of serotonergic action in the hippocampus include the selective innervation of specific interneuron subpopulations.
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Selective elimination of hypothalamic neurons by grafted hypertension-inducing neural tissue(1994) Journal of Neuroscience. 14, 8, p. 4891-4902 Abstract
Embryonic hypothalamic tissue originating from spontaneously hypertensive rats (SHR) was implanted in young normotensive Wistar Kyoto rats in an attempt to localize hypothalamic regions directly responsible for the induction of hypertension. A 25% increase in host systolic blood pressure as compared with the controls was recorded 3 months after implantation in the animals receiving rostral hypothalamic tissue (R-SHR), whereas blood pressure was not affected in the animals grafted with caudal hypothalamic tissue (C-SHR). The hypertension in the R-SHR group was accompanied by hypertrophy of the heart and kidneys. The number of vasopressin-immunopositive (VPi) parvocellular cells in the hypothalamic paraventricular nucleus (PVN) of the R-SHR group was massively reduced (by 72%), while that of the tyrosine hydroxylase-immunopositive cells displayed no change. In the suprachiasmatic nucleus of these animals the VPi cell number was unaltered. In the C-SHR, the amount of parvocellular VPi cells was also unaltered. Likewise, oxytocin-containing cells were the same in all groups. DNA nick-end labeling of the tissue revealed that PVN cells are undergoing programmed cell death. These results implicate a selective degeneration by hypothalamic PVN cells in the pathogenesis of hypertension.
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(1994) Journal Of Physiology-London. 478, 2, p. 251-263 Abstract
1. Variations in intracellular free Ca2+ concentration ([Ca2+]i) induced by alteration of the extracellular concentrations of Ca2+ ([Ca2+]o) and K+ ([K+]o) were imaged in single fluo‐3‐loaded C6 glioma cells. In addition, the effect of membrane potential on [Ca2+]i was investigated in fura‐2‐loaded, voltage‐clamped cells. 2. Step alterations of [Ca2+]o from 0 to 10 nM were followed by proportional variations in [Ca2+]i, with a maximum 7‐fold increase and an apparent half‐maximum at [Ca2+]o of 1.5 mM. 3. The time to half‐maximum change (t1/2) of [Ca2+]o‐associated [Ca2+]i variations ranged between 10 and 50 s, and was inversely related to the amplitude of [Ca2+]o steps. 4. Transient, serotonin‐induced [Ca2+]i elevations, used as a measure of Ca2+ availability in inositol 1,4,5‐trisphosphate‐sensitive stores, were diminished within 10 min in 0 mM [Ca2+]o, but were unaffected by [Ca2+]o changes in the 1‐5 mM range. 5. Restoration of normal [Ca2+]i following its elevation by serotonin was delayed by removal of external Na+ or Cl‐ and was enhanced by warming the medium to 37 degrees C. These conditions did not affect [Ca2+]o‐associated [Ca2+]i variations. 6. [Ca2+]o‐associated [Ca2+]i variations were depressed by La3+ and Ba2+, while blockers of voltage‐activated Ca2+ channels were ineffective. 7. Elevated [K+]o depressed the basal level of [Ca2+]i, and in high concentrations (70‐140 mM) also diminished the response to serotonin. 8. Depolarizing the membrane potential of voltage‐clamped cells reversibly reduced [Ca2+]i. These membrane‐potential associated [Ca2+]i variations were blocked by La3+, Ba2+ and TEA, all of which also depolarized membrane resting potential. 9. Apamin (at 1‐10 microM), a blocker of [Ca2+]i‐activated K+ channel, totally and reversibly prevented [Ca2+]o‐associated [Ca2+]i variations. 10. These studies indicate that C6 cells are responsive to variations in [Ca2+]o, and that a K+ channel is a possible path through which Ca2+ penetrates into the cell.
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(1994) Brain Research. 642, 1-2, p. 111-116 Abstract
Depletion of the forebrain serotonergic system was found in previous studies to induced an increased excitability of the dentate gyrus (DG) granule cells and, when combined with a cholinergic deficiency, to impair spatial learning. We now compared the effects of general forebrain serotonergic lesions induced by intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT), to those of a more restricted injection of 5,7-DHT into fornix-fimbria and cingulum, to eliminate hippocampal serotonergic innervation. Control and lesioned rats were injected with atropine and tested in the spatial learning water-maze task. Following the behavioral tests, rats were anesthetized and the responsiveness of the DG to perforant path (PP) stimulation was measured. To assess the lesions functionally, responses to application of the serotonin releasing drug fenfluramine (FFA) were measured. Finally, the reduction, in the hippocampus of serotonergic innervation was evaluated by [3H]imipramine binding. The effects of the lesions on the responsiveness to FFA confirmed that the ICV lesions were functionally more general than the FF lesions. [3H]Imipramine binding indicated that both lesions reduced the sertonergic innervation of the hippocampus significantly. Behaviorally, both lesioned groups were impaired in the water-maze. Electrophysiologically, in both DG excitability was higher than in controls and in both hyperexcitability was associated with an increase in feed-forward inhibition. The results suggest that the serotonergic innervation of the hippocampus proper is involved in cognitive functions associated with the hippocampus.
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(1994) Neurobiology of Aging. 15, 5, p. 635-641 Abstract
Spatial memory ability, tested in a water maze, was severely impaired in control, 24-month-old hooded rats. A daily injection of the serotonin precursor, 5-hydroxytryptophane (5-HTP), prior to training sessions, had no effect on the behavior of the young rats but improved considerably the performance of the old rats in the watermaze. In the same groups of young and aged rats, the response properties of the hippocampal dentate gyrus to perforant path stimulation was assessed before and after parenteral administration of 5-HTP. The dentate gyrus of aged rats produces a smaller EPSPs in response to perforant path stimulation but a larger population spike for a given EPSP than that produced in young rat brains. These differences are not affected by 5-HTP. In young brains, priming commissural stimulation suppresses subsequent reactivity to perforant path stimulation. This priming effect is nearly absent in aged rat hippocampus but reappears when the rat is injected with 5-HTP. It is suggested that the serotonergic innervation of the rat hippocampus plays a major role in regulation of the excitability of the hippocampus and in behavioral functions associated with this structure.
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(1994) Brain Research Bulletin. 35, 5-6, p. 397-402 Abstract
The epigenetic stimuli that regulate the development of noradrenergic LC neurons were studied in an vitro system of LC primary cultures. Noradrenergic cells were identified using immunocytochemical staining for tyrosine hydroxylase (TH). Maturation of noradrenergic neurons was assessed by measuring the high affinity uptake of norepinephrine (NE). Coculturing target cells with LC neurons exerts both stimulatory and inhibitory effects on NE uptake, depending on the density of plated cells. The target stimulatory effect may be mediated by glial soluble factors, whereas the inhibitory effect may be mediated by glial membranal molecules. In addition to target derived trophic factors, the effect of elevated cAMP levels was examined. cAMP analogs and forskolin dramatically increase the number of TH+ cells, possibly by supporting their survival. This phenomenon is not dependent on calcium or calcium requiring processes and is not mediated by glial cells. The trophic activity of cAMP appears to be exerted by protein phosphorylation via cAMP dependent protein kinase. Norepinephrine is suggested to be one signal that triggers cAMP elevation through the β-adrenergic receptor and thereby affects LC development. Morphine, which is known to inhibit adenylate cyclase, reduces NE uptake and number of TH+ neurons. Morphine also inhibits the NT-3 induced increase in noradrenergic survival. We hypothesize that morphine exerts these effects by modulating the cAMP cascade.
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Changes in cytosolic sodium caused by a toxic glutamate treatment of cultured hippocampal neurons(1994) Biochemistry and Molecular Biology International. 32, 3, p. 475-482 Abstract
Changes in cytosolic Na+ ([Na+](i)) caused by a toxic glutamate (GLU) or NMDA treatment of cultured hippocampal neurons were monitored by using SBFI fluorescent probe and imaging microscopy. Both GLU and NMDA (50 or 100 μM in Mg2+-free solution, 15 min) induced a marked increase in [Na+](i) (from 6-8 to 30-45 mM) which persisted after the termination of a treatment. The competitive NMDA antagonist, APV (100 μM) when applied in the post-NMDA period failed to decrease the elevated [Na+](i). The results obtained strongly suggest that the main reason for an impairment of Na+/Ca2+ exchange in the post-glutamate period is a reduction of the transmembrane Na+ gradient caused apparently by inhibition of Na+-K+ pump.
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(1994) Journal of Neurophysiology. 72, 4, p. 2034-2040 Abstract
1. We studied long-term cholinergic effects on synaptic transmission in submerged hippocampal slices using intra- and extracellular recording techniques. 2. Bath application of submicromolar concentrations of carbachol (CCh) produced a gradually developing, long-lasting increase in the CA1 excitatory postsynaptic potential and population spike. This potentiation was blocked by atropine and, hence, named muscarinic long-term potentiation (LTP(m)). Application of DL-2-amino-5-phosphonovaleric acid had no effect on LTP(m), indicating that this phenomenon is N-methyl-D-aspartate receptor independent. 3. These effects of CCh were not likely to be due to the blockade of one of several K+ conductances by the drug; the time and concentration dependence of LTP(m) were different from those associated with cholinergic blockade of K+ conductances. 4. Removal of extracellular calcium (Ca(o)/2+) from the bath blocked synaptic transmission. CCh added in calcium-free medium induced LTP(m), which was revealed upon removal of the drug by washing with normal calcium-containing medium. Neither cutting CA1- CA3 connections nor cessation of synaptic stimulation interfered with LTP(m) induction. 5. Application of thapsigargin or H-7 together with CCh blocked LTP(m), suggesting the involvement of intracellular calcium (Ca(i)/2+) stores and protein kinases, respectively, in the LTP(m) mechanism. 6. Subthreshold cholinergic stimulation coupled with subthreshold tetanic stimulation caused LTP. CCh had no effect when administered after the LTP mechanism had been saturated by repeated suprathreshold tetani. Tetanic stimulation failed to cause LTP when applied after LTP(m) had been induced by CCh. These experiments indicate that tetanus-induced potentiation and LTP(m) share a common mechanism and provide a direct link between ACh and mechanisms of synaptic plasticity.
1993
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(1993) Brain Research. 625, 1, p. 63-74 Abstract
Grafts of fetal dentate gyrus (DG) and CA1 hippocampal subfield tissue were extruded into the dentate gyri of adult male Sprague-Dawley rats, 7-10 days after lesioning the granule cells with colchicine (0.06 μl of 7 mg/ml solution at each of 5 sites/hippocampus). Graft area-host and host-graft area connectivities were investigated 4-6 months post-transplantation by recording extracellular evoked responses in hippocampal slice preparations. Following stimulation of the host mid-molecular layer, evoked field potential responses, showing considerable variation, were recorded in both types of graft. Evoked responses in the lesioned DG without grafts were recorded in very few slices. Stimulation of the area of DG tissue grafts occasionally evoked responses in the host CA3/CA4 and there was no evidence for CA1 graft area-CA3/CA4 connectivity; stimulation of DG and CA1 graft areas occasionally evoked responses in the host CA1. Responses in the area of both DG and CA1 grafts supported short-term potentiation following stimulation of the host mid-molecular layer but only DG graft areas supported long-term potentiation of the population spike amplitude. In the area of both types of transplant a tonic bicuculline-sensitive inhibition was present and paired-pulse stimulation paradigms provided some evidence for inhibition. It is possible that responses recorded within the area of grafted tissue to stimulation of the host are attributable to host-graft connectivity and similarly, responses recorded in the host to stimulation of the area of the graft may be attributable to graft-host connectivity. Only DG graft areas received host inputs which were capable of sustaining a long-term potentiation and establishing efferent contacts with the host CA3/CA4 subfield, suggesting that these would be more likely than CA1 grafts to reinstate normal functional circuitry.
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(1993) Annals of the New York Academy of Sciences. 695, 1, p. 254-257 Abstract
The cholinergic hypothesis of senile dementia proposes that an age‐dependent reduction of central cholinergic functions accounts for the severe cognitive deficits seen in aged rats. A careful examination of the experimental evidence cited in support of this hypothesis reveals that it cannot account for some behavioral observations. We have modified this hypothesis and wish to propose that serotonin and acetylcholine interact to allow normal cognitive functions in the brain. Thus, a partial reduction in both cholinergic and serotonergic functions will cause severe memory impairment in young as well as in aged rats. We found that restoration of the serotonergic innervation in the hippocampus of serotonin depleted rats, using tissue transplants, can restore impaired behavior. We have localized a memory‐related interaction between serotonin and acetylcholine in the hippocampus and are in the process of identifying a physiological function which may underly this interaction.
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(1993) Annals of the New York Academy of Sciences. 695, 1, p. 300-303 Abstract
Rigid analogs of acetylcholine (ACh) were designed for selective actions at muscarinic receptor (mAChR) subtypes and distinct second messenger systems. AF102B, AF150, and AF151 are such rigid analogs of ACh. AF102B, AF150 and AF151 are centrally active M1 agonists. AF102B has a unique agonistic profile showing, inter alia: only part of the M1 electrophysiology of ACh and unusual binding parameters to mAChRs. AF150 and AF151 are more efficacious agonists than AP102B for M1 AChRS in rat cortex and in CHO cells stably transfected with the ml AChR subtype. Notably, the selectivity of the new ml agonists is reflected also by activation of select second messenger systems via distinct G‐proteins. These compounds reflect a new pharmacological concept, tentatively defined as ligand‐selective signaling. Thus, agonist/m1AChR complexes may activate different combinations of signaling pathways, depending on the ligand used. Rigid agonists may activate a limited repertoire of signaling systems. In various animal models for Alzheimer's disease (AD) the agonists AF102B, AF150 and AF151, exhibited positive effects on mnemomic processes and a wide safety margin. Such agonists, and especially AF102B, can be considered as a rational treatment strategy for AD.
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(1993) Brain Research. 614, 1-2, p. 257-269 Abstract
The environmental signals which regulate the development of central noradrenergic neurons are largely unknown. The aim of the present study was to search for factors affecting the development of these cells. Dissociated cultures of embryonic dorsal brainstem tissue, containing the nucleus locus coeruleus (LC), were established; norepinephrine (NE) and GABA uptake were assessed, and noradrenergic versus total neurons were identified and counted following immunocytochemical staining with tyrosine hydroxylase (TH) and neuron specific enolase (NSE) antibodies, respectively. Application of dibutyryl cAMP (dbcAMP), other cAMP analogs, or forskolin, to LC cultures resulted in a significant increase in NE uptake which was associated with up to a 4-fold increase in the number of TH immunoreactive cells (TH+). dbcAMP treatment caused an increase in the number of TH+ cells in LC cultures by enhancing their survival and/or by upregulating their phenotypic differentiation. A possible effect of dbcAMP on cell proliferation and transformation of non-noradrenergic cells to noradrenergic TH+ cells were examined and suggested not to underlie this effect of cAMP. Glial cells may mediate the effect of cAMP on noradrenergic neurons. Calcium was not involved in the trophic activity of dbcAMP, which was probably mediated by protein phosphorylation via cAMP dependent protein kinase. Insulin (25 μg/ml) was found to increase the number of TH+ cells by 73%. The β-adrenergic agonist isoproterenol also increased the number of TH+ cells by 53%. We propose a neurotrophic role for NE during development of central noradrenergic neurons.
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(1993) Experimental Neurology. 121, 2, p. 256-260 Abstract
We compared the effects of embryonic raphe grafted into either the hippocampus or the entorhinal cortex on the ability of rats to perform a spatial memory watermaze task. Serotonin depletion or partial cholinergic lesion of the hippocampus (by injection of colchicine into the septum) did not affect the ability of rats to perform the task, but the combined treatment did. Double-lesioned rats, with raphe grafts in the hippocampus, but not in the entorhinal cortex, performed similar to control or single-lesioned rats. The results suggest that the functioning of the serotonergic innervation of the hippocampus, and not of its afferents, is crucial for the ability of rats to perform spatial memory tasks, especially when the septohippocampal cholinergic connection is disrupted.
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(1993) Hippocampus. 3, 2, p. 229-238 Abstract
GABA evoked a reversible rise of free intracellular calcium concentration ([Ca]i) in cultured rat hippocampal neurons, detected with Fluo‐3 fluorescence in a confocal laser scanning microscope. The GABA‐evoked change of [Ca]i was mimicked by muscimol and not by baclofen, but was only minimally affected by picrotoxin or bicuculline, indicating that this effect of GABA is not likely to be mediated by activation of GABAB receptor or by a conventional chloride‐linked GABAA receptor. GABA‐evoked rise of [Ca]i expressed a marked desensitization; only 10–20 minutes after a previous exposure to GABA was the response to a subsequent application fully expressed. This desensitization was not seen in electrophysiological responses to GABA or in [Ca]i changes evoked by NMDA in the same neurons. The GABA response appeared to be developmentally regulated and was seen in 1–7‐day‐old more than in 21–28‐day‐old cells. It is suggested that GABA evokes a unique change of [Ca]i in young hippocampal neurons.
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Calcium and neuronal plasticity(1993) Israel Journal of Medical Sciences. 29, 9, p. 543-548 Abstract
The proposed involvement of free intracellular calcium concentration ([Ca]i) in neuronal plasticity is examined. While it is generally believed that a rise of [Ca]i is necessary for the triggering of long-term modification of synaptic connections, there are many unresolved issues related to this dogma; it is not entirely clear what is the source of the elevated calcium, how much of a calcium rise is sufficient to produce the synaptic potentiation, where and for how long, and what are the relevant chemical consequences of the transient rise of [Ca]i. It is generally believed that the dendritic spine is the locus of synaptic modification, yet little evidence exists to support this view High resolution calcium imaging studies may contribute to the clarification of some key issues in the field of neuronal plasticity.
1992
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(1992) European Journal of Pharmacology. 220, 1, p. 103-106 Abstract
The putative muscarinic M1 receptor agonist, AF102B, was applied to rat hippocampal slices and the responses of intracellularly recorded pyramidal cells were examined. AF102B mimicked some effects of acetylcholine on these cells as follows: at low concentration, AF102B attenuated a slow after-hyperpolarization in response to a long depolarizing current pulse. This effect was blocked by the M1 antagonist, pirenzepine. At higher concentrations, AF102B also depolarized the cells and caused an increase in their input resistance. AF102B did not affect local excitatory postsynaptic potentials or reactivity to topically applied excitatory amino acid substances. These experiments indicate that AF102B acts as an agonist at some muscarinic M1 receptor subtypes in mammalian brain.
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(1992) Hippocampus. 2, 3, p. 279-286 Abstract
The therapeutic action of lithium in affective disorders is still unclear. One effect lithium is to deplete membrane inositol and consequently to exhaust the phosphoinositide (PI) pathway. Under chronic lithium treatment, rats showed persistent performance deficits in an active avoidance task and in a visually cued maze. The same treatment, however, resulted in only a transient deficit in the performance of rats in a spatial memory task. Lithium treatment caused a similarly transient deficit in the ability of acetylcholine to potentiate responses to N‐methyl‐D‐aspartate (NMDA) in neurons of the hippocampal slice. The authors propose that the development of compensatory mechanisms may account for the lack of severe memory impairments during lithium treatment. It is suggested that the effects of lithium on the PI pathway are not sufficient to explain the behavioral consequences of chronic lithium treatment.
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(1992) Brain Research. 587, 1, p. 83-87 Abstract
The effects of acetylcholine (ACh) on changes in [Ca]i produced by the glutamate agonist N-methyl-d-aspartate (NMDA) were measured in cultured rat hippocampal neurons loaded with the fluorescent calcium indicator Fluo-3 in a confocal laser scanning microscope. NMDA produced a dose-dependent reversible rise in [Ca]i. ACh had a smaller and less consistent effect on [Ca]i but could cause a marked enhancement of the reactivity of neurons to NMDA. This effect was reversed by the presence of the muscarinic antagonist atropine. AMPA, another glutamate agonist which causes a rise in [Ca]i by activating voltage gated calcium influx was less affected by ACh. Caffeine, which releases calcium from intracellular stores also enhanced reactivity of these neurons to NMDA. It is suggested that ACh can enhance reactivity to NMDA by releasing calcium from internal stores.
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(1992) Neuroscience Letters. 140, 2, p. 243-246 Abstract
Changes in intracellular calcium concentration ([Ca2+]i) in response to topical application of the glutamate agonists N-methyl-d-aspartate (NMDA), or amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were measured in cultured rat hippocampal neurons loaded with Fluo-3 and visualized in a confocal laser scanning microscope. These neurons were subsequently stained for the presence of the enzyme marker for γ-amino butyric acid (GABA), glutamate decarboxylase (GAD). GAD-positive, putative interneurons were less responsive to NMDA and AMPA than GAD-negative neurons. The time course of the rise and decay of [Ca2+]i was similar in the two groups of neurons. Also, there was no clear difference in the shape and size of these two neuron groups indicating that the difference between them is not due to diffusion distances. These data indicate that interneurons are probably more able to handle a calcium load than other neurons, a difference that may underly their resistance to treatments which cause degeneration of other neurons in the hippocampus.
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(1992) Journal Of Physiology-London. 448, 1, p. 655-676 Abstract
1. The confocal laser scanning microscope (CLSM) was used in conjunction with the calcium indicator dye Fluo‐3 to record changes in free intracellular calcium concentration ([Ca2+]i) in cultured hippocampal neurons in response to superfusion of N‐methyl‐D‐aspartate (NMDA). 2. NMDA caused a rapid rise in [Ca2+]i in all parts of the neuron. The rise in [Ca2+]i was dependent on activation of an NMDA receptor, was enhanced by the removal of Mg2+ and addition of glycine to the superfusion medium, and was dependent on normal [Ca2+]o. 3. The rise of [Ca2+]i was seen first near the membrane. A wave of elevated [Ca2+]i moved centripetally at a rate of 117 microns/s. 4. Dantrolene pre‐incubation caused a significant reduction in the efficacy of the NMDA‐induced rise in [Ca2+]i, indicating that at least part of the rise is caused by intracellular release of calcium. 5. The replacement of calcium by barium caused a reduction in the response to NMDA, but a significant response was still present in these cells, supporting the assumption that NMDA causes release of calcium from intracellular stores. 6. The removal of sodium from the superfusion medium prolonged the [Ca2+]i rise in response to NMDA indicating that the Na‐Ca antiporter is instrumental in reducing [Ca2+]i. 7. These studies demonstrate the multiplicity of regulating mechanisms of [Ca2+]i following activation of NMDA receptors.
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(1992) Brain Research. 574, 1-2, p. 147-156 Abstract
Cells in layers II-III or VI were activated by microdrop application of acetylcholine (ACh), while monitoring the intracellular response of layer V pyramidal cells. This enabled the tracing of functional connections between the cells of layers II-III or VI with those of layer V. ACh activation of layer II-III or VI cells resulted in a small depolarization of these cells, accompanied by a burst of excitatory postsynaptic potentials (EPSPs) from layer V pyramidal cells. These effects of ACh were blocked by tetrodotoxin (TTX), suggesting the involvement of actions potentials in their production. The input resistance of layer V pyramidal cells during and after the EPSP burst was not significantly different from control values, further suggesting an indirect effect of ACh on layer V pyramidal cells. Isolation of the supragranular layer, by horizontal cutting, did not prevent the EPSP burst evoked by ACh application to the lower layer VI, suggesting a direct input from layer VI to layer V pyramidal cells. ACh applied near pyramidal cells in layers II-III, V or VI caused transient hyperpolarization associated with a decrease in input resistance followed by a large depolarization, an increase in input resistance, and action potential discharges. The ACh- mediated hyperpolarization and the train of action potentials of layer II-III pyramidal cells were blocked by TTX. Thus the ACh-activated cells in layers II-III and VI make an excitatory synaptic contact with layer V pyramidal cells, producing the EPSP burst observed in layer V.
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(1992) Journal Of Physiology-London. 447, 1, p. 513-533 Abstract
1. The cellular mechanism by which acetylcholine (ACh) potentiates neuronal responses to N‐methyl‐D‐aspartate (NMDA) was investigated in CA1 neurones of hippocampal slices using current‐ and voltage‐clamp techniques. 2. Loading cells with 5'‐guanylylimidodiphosphate (GppNHp) caused a gradual increase in response to NMDA. Pulses of ACh accelerated this increase. Guanosine 5'‐O‐(2‐thiodiphosphate) (GDP beta S) blocked the potentiating effect of ACh on responses to NMDA. 3. Acute LiCl caused a gradual decrease in the potentiating effect of ACh, while the potentiation was completely prevented by 3 day chronic 6 mequiv/kg (I.P.) LiCl treatment and restored by acute treatment with 10 mM‐inositol. 4. Loading cells with a general protein kinase inhibitor, H‐7, enhanced the potentiating effect of ACh on responses to NMDA and blocked the effect of ACh on the after‐hyperpolarization (AHP). 5. Ultraviolet irradiation of cells loaded with a photolabile inositol 1,4,5‐trisphosphate (InsP3) caused a transient increase in responses to NMDA, while penetrating cells with active InsP3‐containing pipettes caused a gradual BAPTA‐sensitive increase in responses to NMDA. 6. Reducing the rate of InsP3 metabolism, with 2,3‐diphosphoglyceric acid (DPG), caused an increase and prolongation of the potentiating effect of ACh, while blocking the InsP3 receptor with heparin prevented the cholinergic potentiation. 7. NMDA, by itself, potentiated subsequent responses to NMDA, an effect that was blocked when [Ca2+]i was chelated with BAPTA. NMDA and ACh were also found to compete in potentiating responses to NMDA. Finally, the cholinergic potentiation was blocked when cells were loaded with BAPTA. 8. We propose that activation of the InsP3 branch of the phosphoinositide pathway potentiated responses to NMDA and that InsP3 exerted this effect by elevating [Ca2+]i.
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(1992) GLIA. 6, 2, p. 118-126 Abstract
Whole‐cell membrane currents and imaging of intracellular calcium concentrations ([Ca2+]i) were used to investigate the role of calcium in a response to serotonin of C6 glioma cells. Activation of a high‐affinity serotoinin receptor induced a transient rise in calcium concentration in these cells and activated a predominantly potassium conductance, with a small chloride component. Perfusion of the cytoplasm with an internal solution containing high calcium concentration induced similar but prolonged increase of membrane conductance. The responsiveness of C6 cells to serotonin was negatively correlated with the concentration of the unbound calcium chelator BAPTA when BAPTA‐buffered calcium‐containing intracellular solutions were used. Responses to serotonin persisted in the absence of external calcium, decreased gradually, and then recovered partially after replenishment of extracellular calcium. These findings substantiate the direct role of intracellular calcium in mediating the serotonin response, and indicate that serotonin‐induced release of calcium from intracellular stores is sufficient for the activation of conductance in the C6 glioma cell line. © 1992 Wiley‐Liss, Inc.
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(1992) Developmental Neuroscience. 14, 2, p. 166-172 Abstract
We compared the effects of embryonic raphe grafted into either the hippocampus or the entorhinal cortex, on the responsiveness of dentate granule cells to stimulation of the perforant path. Raphe grafts in the hippocampus reversed the hyperexcitability of granule cells, resulting from depletion of the serotonergic innervation. Such grafts also restored the responsiveness of the granule cells to application of a serotonin releasing drug, fenfluramine (FFA). In contrast, hyperexcitability was not reversed when the graft was placed in the entorhinal cortex. Furthermore, although some increase in population spike size was observed in these rats after application of FFA, this increase had a response profile which was different from that of control and of lesioned rats that were grafted in the hippocampus. These results suggest that the serotonergic innervation, within the hippocampus and not in the entorhinal cortex, modulates granule cells excitability.
1991
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(1991) Brain Research. 566, 1-2, p. 21-25 Abstract
It has been previously demonstrated that an embryonic raphe grafted into a serotonin-depleted hippocampus restores normal serotonin innervation of the hippocampus10,23,24 and behaviors associated with serotonin14,18,20. To test the possibility that the behavioral effects of these grafts result from non-specific actions of the grafted tissue or the grafting procedure itself8,11, we compared raphe grafts with septal grafts, in serotonin-depleted rats. We also compared the effects of a serotonin synthesis inhibitor, p-chlorophenylalanine, on the behavior of normal, serotonin-depleted and raphe-grafted rats. The results indicate that the bulk of behavioral effects of raphe grafts are due to the serotonergic nature of the graft.
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(1991) Brain Research. 540, 1-2, p. 322-324 Abstract
We examined the effect of elevating intracellular calcium ([Ca2+]i) on responses to iontophoretically applied N-methyl-d-aspartate (NMDA), and quisqualate in CA1 neurons of the hippocampal slice. Topical application of calcimycin (A23187), a calcium ionophore, potentiated responses to NMDA but not to quisqualate. This potentiation was prevented by loading cells with the calcium chelator, BAPTA, suggesting that the action of calcimycin on NMDA receptors was mediated by an elevation of [Ca2+]i in the recorded cell. The potentiation was also recorded in voltage-clamped and in cesium-loaded cells, suggesting that it was not mediated by non-specific changes in voltage or input resistance of the cell that may have resulted from the rise in [Ca2+]i. We propose that intracellular calcium plays a crucial role in regulating the activity of the NMDA subtype of l-glutamate receptor.
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(1991) Brain Research. 540, 1-2, p. 291-294 Abstract
the effect of the α2 antagonist, idazoxan (IDA), on the excitability of neurons in the dentate gyrus of the hippocampus was studied. Population field potentials (PS) evoked by stimulation of the perforant pathway were measured before and after drug treatment. IDA enhanced the amplitude of the PS, while decreasing the slope of the EPSP. Neurotaxic destruction of noradrenergic nerve terminals completely abolished the IDA effect, arguing that its mechanism of action is through enhanced release of noradrenaline (NA). It is proposed that NA enhances the EPSP-to-spike coupling component of the PS.
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Hypertension induced by hypothalamic transplantation from genetically hypertensive to normotensive rats(1991) Journal of Neuroscience. 11, 2, p. 401-411 Abstract
The role of the hypothalamus (HTH) in the pathogenesis of genetic hypertension was studied in spontaneously hypertensive rats (SHR). It is currently believed that, in this strain, the genetic defect manifests itself mainly in the HTH. We examined this hypothesis by grafting HTH neurons from embryos of SHR or control Wistar Kyoto (WKY) rats into the HTH of adult normotensive WKY rats. Changes in host systolic blood pressure (SBP) were monitored, and alterations in vasoactive intestinal polypeptide (VIP) gene expression of the host brain were studied. In rats grafted with HTH tissue from SHR embryos (G-SHR), the blood pressure rose by 31% as compared with that in the grafted control group. The blood pressure climbed gradually over a period of 6 weeks to its highest level, which was maintained for at least 3 months following grafting. Along with the elevated blood pressure, the heart weight increased by 80% compared to controls. Behavioral changes were also evident in the G-SHR rats, and these were similar to those of the native SHR strain. In situ hybridization histochemistry showed a 40% elevation in VIP transcripts in the suprachiasmatic nucleus of the host G-SHR brain compared to controls. These studies demonstrate that transplantation of embryonic SHR HTH tissue into brains of adult normotensive rats results in the development of hypertensive characteristics in the host. It thus appears that the HTH is a prime candidate for the source of changes leading to spontaneous hypertension in mammals.
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(1991) International Journal of Developmental Neuroscience. 9, 3, p. 251-258 Abstract
Specific and localized lesions of the 5-HT fibers in the hippocampus induce homotypic collateral sprouting and enhance serotonergic fiber outgrowth from adult neurons and transplanted fetal tissue. In this study, hippocampal extracts were prepared and applied to primary cultures of fetal serotonergic neurons. The effects of plating density and serum additives were examined. The growth of the serotonergic neurons in the rostral brainstem dissociated cultures were estimated by measuring the specific uptake of [3H]5-HT. The results indicate the presence of a trypsin-sensitive factor which is active when prepared fresh at dilutions up to 1 10,000. The factor is higher in hippocampus than cerebellum. Young male tissue contained more activity than either female or aged hippocampus. Although both positive and negative effects are described, higher dilutions of factor ( 1 1,000) were generally stimulatory in high density cultures while lower dilutions ( 1 10) were inhibitory in low density cultures. Specific removal of 5-HT hippocampal afferents with fornix-fimbria microinjections of 5,7-dihydroxytryptamine resulted in an initial loss of activity (2 days and 2 weeks) followed by an enhanced activity (2 months) compared to normal hippocampal extract. Several possibilities are discussed as to the identity of the serotonergic growth factor from hippocampal supernatant.
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(1991) Neurobiology of the Locus Coeruleus. Barnes C. D. & Pompeiano O.(eds.). C ed. p. 323-330 (trueProgress in Brain Research). Abstract
Acting at postsynaptic alpha-1 and beta-1-receptors, norepinephrine (NE) exerts a complex action in rat hippocampus. It is currently believed that beta-1-receptor activation enhances excitability of recorded neurons, whereas alpha-1 activation suppresses reactivity to afferent stimulation. These reported effects of alpha-agonists are not consistent with alpha-1 effects found elsewhere in the brain. We have conducted experiments in the anesthetized rat and found that an amphetamine-induced increase in the dentate gyrus population spike can be blocked by a beta-antagonist but also by an alpha-1-antagonist. We have conducted experiments in the brain slide preparation and found that an alpha-agonist, phenylephrine (PHE), selectively enhances responses to N-methyl-D-aspartate (NMDA) but not to quisqualate. We propose that the product of activation of both alpha- and beta-receptor types will enhance reactivity of hippocampal cells to afferent stimulation.
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The effects of serotonin depletion and raphe grafts on hippocampal electrophysiology and behavior(1991) Journal of Neuroscience. 11, 6, p. 1585-1596 Abstract
The involvement of the serotonergic system in spatial learning and a possible correlation between serotonergic modulation of hippocampal electrical activity and spatial learning were studied in rats. Control, partial septal-lesioned (SL), 5,7-dihydroxytryptamine (5,7-DHT)-injected (DHT), double-lesioned (5,7-DHT and SL; DL), and DL rats that were transplanted with embryonic raphe grafts into the hippocampus (RG) were tested in a spatial task in a water maze and in an active avoidance shuttle-box task. The responses of the dentate gyrus (DG) to perforant-path (PP) stimulation were examined in the same rats, under the following conditions: (1) priming stimulation of the PP (testing feedback inhibition), (2) priming stimulation of the commissural pathway (testing feedforward inhibition), (3) during repeated stimulation of the PP at 7 Hz (frequency potentiation), and (4) following tetanic stimulation [long-term potentiation (LTP)]. DL, but not DHT or SL, treatment severely impaired the performance of rats in both reference- and working-memory tasks in the water maze. This effect was not seen in the shuttle box. The ability of the DG to exhibit LTP, which was reduced in the DHT and SL rats, was apparently similar to controls in the DL group. DL, but not DHT or SL alone, resulted in a reduction of inhibition in the DG. Both the behavioral deficits and the reduction in hippocampal inhibition were ameliorated by intrahippocampal raphe grafts. These results indicate that the serotonergic innervation of the hippocampus plays a role in spatial learning when the septohippocampal neurotransmission systems are disrupted. Furthermore, these results suggest that restoration of modulation of hippocampal inhibition, by raphe grafts, underlies the behavioral recovery observed in these rats.
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(1991) Progress in Brain Research. C ed. p. 571-585 Abstract
The gating and tuning actions of noradrenaline (NA) at post-synaptic sites have been highly suggestive of an important role for the locus coeruleus (LC) in attention, learning and memory. By recording the activity of single units in the LC in behaving rats in a strictly controlled conditioning paradigm, direct evidence was provided that this nucleus is engaged during specific aspects of learning. The neuronal response to a discrete sensory stimulus was monitored as a function of the changing significance of the stimulus i.e., when it was novel, during habituation, associative learning, reversal and extinction. Both appetitive and aversive paradigms were used. We consistently observed differential conditioned responding with food reinforcement, while when footshock reinforcement was used, there was an increase in response to both CS+ and CS–. In both paradigms, the LC response disappeared when the conditioning was expressed at a behavioral level, to reappear vigorously as soon as the stimulus reinforcement contingencies were changed, i.e., during reversal or extinction. These results suggest that the LC does not mediate specific sensory or associative information necessary for ongoing performance but shows remarkable plasticity of sensory responding as a function of changing cognitive significance of the stimulus.
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Control of the maturation and the survival of central noradrenergic neurons in culture(1991) Journal de Physiologie. 85, 2, p. 84-89 Abstract
Central noradrenergic neurons from the locus coeruleus express unique plastic properties. The aim of this study was to identify factors that specifically regulate the development and the survival of the noradrenergic cells. Primary dissociated cultures of embryonic locus coeruleus (EC) neurons were established. Norepinephrine (NE) uptake was used as as index of maturation of the noradrenergic neurons. The noradrenergic cells were identified and quantified following immunocytochemical staining for tyrosine hydroxylase antibody. We have examined the effect of hippocampal target tissue and of cyclic-AMP (cAMP) on the development of these cells. Coculturing EC cells with a low density of hippocampal target cells, resulted in a significant increase in NE uptake. However, when the amount of hippocampal target cells was doubled an enormous decrease in NE uptake occurred the target stimulatory effect was mediated by both neurons and glia, whereas the inhibitory effect was mediated by direct contact between target glia and LC neurons and detected only in the presence of serum. In addition to target effect, we also tested the effect of elevated intracellular cAMP level on NE uptake versus GABA uptake. GABA uptake served as a developmental index of the non noradrenergic cells. Increasing the intracellular cAMP level, by application of the membrane permeable analog dibutyryl cyclic AMP (DbcAMP), resulted in a selective stimulation of NE uptake, due to enhanced survival of noradrenergic neurons. GABA uptake and the number of non-noradrenergic cells were not changed in the presence of DbcAMP. DbcAMP could maintain the survival of EC neurons in the absence of glial cells. Galcium was no required for the expression of DbcAMP effect. Protein phosphorylation by cAMP dependent protein kinase probably mediated the DbcAMP survival effect. Among several growth factors that were examined, insulin selectively increased the number of noradrenergic cells. We assume that the is a physiological primary signal, specific for the EC cells, that acts via the cAMP pathway. This physiological factor is responsable for maintenance of survival of noradrenergic neurons.
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(1991) Nature. 354, 6348, p. 76-80 Abstract
THE dendritic spine is a basic structural unit of neuronal organization. It is assumed to be a primary locus of synaptic plasticity, and to undergo long-term morphological and functional changes1-6, at least some of which are regulated by intracellular calcium concentrations7-11. It is known that physiological stimuli can cause marked increases in intracellular calcium levels in hippocampal dendritic shafts12,13, but it is completely unknown to what extent such changes in the dendrites would also be seen by calcium-sensing structures within spines. Will calcium levels in all spines change in parallel with the dendrite or will there be a heterogeneous response? This study, through direct visualization and measurement of intracellular calcium concentrations in individual living spines, demonstrates that experimentally evoked changes in calcium concentrations in the dendritic shaft ([Ca2+]d) are frequently not parallelled in the spine ([Ca2+]s). This isolation is not caused by a physical diffusion barrier. This report provides, to our knowledge, the first direct demonstration of autonomous spine function.
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(1991) Neurobiology of the Locus Coeruleus. C ed. p. 617-623 Abstract
Primary dissociated cultures of embryonic locus coeruleus (LC) neurons were established. Norepinephrine (NE) uptake was used as an index of maturation of the noradrenergic (NA) neurons from the LC. When LC cells were cocultured with a low density of hippocampal target cells, NE uptake was stimulated. However, increasing the concentration of hippocampal cells resulted in a significant decrease in NE uptake. The target stimulatory effect was mediated by both neurons and glia, whereas the inhibitory effect was mediated by direct contact between target glia and LC neurons and detected only in the presence of serum. In addition to the effect of target, we also tested the effect of elevated intracellular cAMP levels on NE uptake versus GABA uptake. GABA uptake was an index of development of non-NA cells. Increasing intracellular cAMP resulted in selective stimulation of NE uptake. These studies illustrate the potential of dissociated LC cultures in studying the regulation of NA axonal outgrowth.
1990
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(1990) Experimental Brain Research. 82, 1, p. 199-207 Abstract
Serotonin modulating effects on hippocampal electrical activity were studied using serotonin releasing drugs (e.g. d-fenfluramine, FFA, and p-chloroamphetamine, PCA). FFA and PCA enhanced the reactivity of the dentate gyrus to stimulation of the perforant path (PP) in the anesthetized rat. The population spike (PS) but not the population EPSP (EPSP) was enhanced by FFA indicating that the drug effect is not exerted at the PP synapse, but at some postsynaptic site between the synapse and the spike generation mechanism. A depth profile of the response to PP stimulation indicated that the largest effect of FFA was present just below the granular cell layer. There were no systematic effects of FFA on the EPSP at any depth tested. The effect of FFA was much reduced in rats depleted of serotonin by p-chlorophenylalanine (PCPA) and restored when serotonin stores were repleted by the serotonin precursor 5-hydroxytryptophane (5-HTP). d-FFA was at least twice as effective as 1-FFA in enhancing responses in the dentate gyrus (DG). In noradrenaline (NA) depleted rats the increase in PS size was as in control rats. The effects of FFA were blocked by the 5-HT1a antagonist spiperone but not by the 5-HT2 antagonist mianserin. These results suggest that the effect of FFA is primarily due to release of serotonin from its terminals. At the gross electrographic level, FFA suppressed spontaneous sharp wave activity and reduced the magnitude of hippocampal EEG. Spontaneous extracellular single unit activity, recorded in the DG, was also inhibited by FFA concomitantly with the increase in the PS size. FFA did not affect paired-pulse depression. Transection of the commissural connection to the hippocampus (stimulation of which elicits feed forward inibition) markedly attenuated the effects of FFA. It is suggested that serotonin exerts a dual effect on DG granule cells; it suppresses spontaneous activity while enhancing excitability to afferent stimulation. Possibly, the effects of serotonin are exerted by modulation of commissural feed-forward inhibition.
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(1990) Journal Of Physiology-London. 427, 1, p. 381-393 Abstract
1. The effects of acetylcholine (ACh) on excitatory postsynaptic potentials (EPSPs) evoked by stimulating Schaffer‐commissural afferents and on ionophoretically applied L‐glutamate ligands, were investigated in CA1 neurones of hippocampal slices using current‐ and voltage‐clamp techniques. 2. ACh produced a transient suppression followed by a long‐lasting facilitation of EPSPs. The facilitation was also seen in Cs(+)‐filled cells under voltage‐clamp conditions. Both suppressing and facilitating effects were blocked by atropine. 3. All components of the EPSP were reduced in the initial phase of ACh action, while only the slow component was enhanced during the later phase. The facilitation was blocked by an N‐methyl‐D‐aspartate (NMDA) receptor antagonist, d‐2‐amino‐5‐phosphonovalerate (2‐APV) and by hyperpolarization. 4. ACh also facilitated responses to ionophoretically applied NMDA in voltage‐clamped, Cs(+)‐filled cells in Ba2(+)‐treated slices. ACh facilitated responses to L‐glutamate which was blocked by 2‐APV. ACh failed to affect responses to kainate or quisqualate. 5. We conclude that ACh, acting on muscarinic receptors, exerts a primary effect in the hippocampus to specifically amplify NMDA receptor‐mediated synaptic responses and thereby facilitate EPSPs.
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(1990) Brain Research. 521, 1-2, p. 1-6 Abstract
The ability of midbrain raphe (MR) cells grafted into serotonin-depleted brain to restore hippocampal responses to synaptically released serotonin was studied using a serotonin-releasing drug, d-fenfluramine (FFA). In normal rats, FFA enhances reactivity of the dentate gyrus (DG) to perforant-path stimulation, while suppressing spontaneous activity. These effects of the drug were dependent on the presence of normal serotonin neutransmission in the hippocampus. Depletion of brain serotonin markedly attenuated DG reactivity to FFA. MR grafts (4-5 months after transplantation) restored the reactivity of the hippocampus to FFA. Immature MR grafts (3 weeks after transplantation) or septal control grafts could not reproduce the effects of mature grafts. These experiments demonstrate the possible functional incorporation of neural transplants in a host brain and the possible involvement of these grafts in regulation of normal host electrical activity. The combined utilization of a serotonergic graft and a releasing drug (i.e. FFA), in the serotonin-deprived brain, allows the study of serotonin functions in restricted parts of the brain.
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(1990) European Journal of Pharmacology. 181, 3, p. 299-301 Abstract
Intracellular activity was recorded from CA1 neurons in the slice preparation, by using K-acetate-, Bapta- or EGTA-filled micropipettes. The calcium chelator, Bapta, clamps [Ca]i more rapidly than EGTA and is less associated with pH changes. Neurons recorded with Bapta-filled micropipettes were much less sensitive to 5-HT and baclafen than those recorded with the standard K-acetate- or EGTA-filled micropipetes. These results suggest that [Ca]i is important for the generation of the 5-HT-dependent potassium current.
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(1990) Neuroscience Letters. 113, 1, p. 62-65 Abstract
The effect of acetylcholine (ACh) on intracellular responses to ionophoretic application of N-methyl-d-aspartate (NMDA) was examined in rat hippocampal slice. Recordings were obtained from CA1 neurons under current- and voltage-clamp conditions. Drugs were applied topically by ionophoretic and microdrop techniques. ACh produced an atropine-sensitive potentiation of responses to NMDA. The effect of ACh on NMDA receptor-mediated responses was independent of changes in voltage or potassium conductances caused by ACh. ACh also potentiated responses to l-glutamate but not to kainate or quisqualate. This effect was blocked by dl-2-amino-5-phosphonovalerate (2-APV), an NMDA receptor antagonist. We conclude that ACh, acting on muscarinic receptors, potentiates selectively, the NMDA subclass of l-glutamate receptor.
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(1990) Brain Research. 513, 1, p. 171-174 Abstract
We examined the electrophysiological properties of cholinergic and non-cholinergic neurons in the medial septum-diagonal band complex (MSDB) of the rat in the in vitro slice preparation. Cells were identified electrophysiologically, filled with Lucifer yellow, fixed and processed immunohistochemistry with fluorescent labeled anti-choline acetyltransferase (ChAT) antibody. Cholinergic and non-cholinergic neurons differed in action potential parameters, spike afterpotentials and in current-voltage relationships. In addition, cholinergic neurons expressed a potent transient outward rectification in response to a depolarizing current pulse.
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(1990) Developmental Brain Research. 52, 1-2, p. 247-252 Abstract
Intracellular activity was recorded from neurons in immature rat hippocampal slices. The presence of intrinsic inhibitory synaptic potentials as well as responses to serotonin were assessed in slices of 1, 2 or 3 postnatal weeks of age. Young (1 week) cells had only a marginal hyperpolarizing response to serotonin and no detectable intrinsic inhibitory synaptic potentials. At 2 weeks of age neurons already expressed a fast IPSP (inhibitory postsynaptic potential). The responses to serotonin were different from those of adult cells in that they involved primarily a large decrease in input resistance with only small potential changes. In cells of this age serotonin caused a marked increase in spontaneous IPSP discharges and a blockade of a slow afterhyperpolarization. In 3-week-old rats the fast and slow components of the IPSP were present as in adult and the responses to serotonin included a large hyperpolarization associated with an increase in K conductance, a blockade of slow afterhyperpolarization and a blockade of a slow IPSP, as seen in adult cells. These results indicate that the complex pattern of reactivity to serotonin is differentially regulated in the developing brain.
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(1990) Brain Research. 511, 1, p. 163-164 Abstract
Topical application of 4-aminopyridine (4-AP) onto hippocampal slices produced spontaneous repetitive large hyperpolarizing potentials in CA1 neurons. This effect of 4-AP was blocked by a new GABAB receptor antagonist, 2-hydroxy-saclofen. 2-Hydroxy-saclofen also blocked slow IPSPs evoked by stimulation of stratum radiatum. It is suggested that 4-AP-evoked slow hyperpolarizing potentials are in fact slow IPSPs evoked by activation of a selective subset of interneurons which do not produce fast IPSPs.
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(1990) Developmental Brain Research. 52, 1-2, p. 191-199 Abstract
Primary cultures from dissociated locus coeruleus (LC) neurons of 14-day-old (E14) fetal rats were grown in vitro in serum complemented medium. Noradrenergic cells were identified using immunocytochemical staining for tyrosine hydroxylase (TH) antibody. Maturation of noradrenergic neurons was assessed by measuring the high-affinity uptake of [3H]norepinephrine (NE). The presence of hippocampal cells stimulated the specific uptake of [3H]NE by LC cells only when plated at low density. Increasing the concentration of hippocampal cells resulted in a 50% decrease in NE uptake by LC cells. A similar inhibitory effect was observed with striatal cells. The inhibition exerted by striatal cells appears to be developmentally regulated, with E18 exerting a stronger inhibitory effect than E15 striatum. The decrease in [3H]NE uptake in hippocampal-LC cocultures was due to a decrease in uptake by individual noradrenergic neurons. For a given plating density, the decrease in uptake of [3H]NE per noradrenergic cell in LC culture was only half the decrease in the cocultures, suggesting a target-associated effect rather than density-derived toxic effect. In culture conditions which favored neuronal but not glial survival, the stimulatory target effect was evident, and the inhibitory effect was absent. Medium conditioned by target glial cells had a marked stimulatory effect on [3H]NE uptake. Glial feader-layer had a strong inhibitory effect on [3H]NE uptake in serum-containing medium. We suggest that both neurons and glia mediate the target-stimulatory effect, whereas the inhibitory effect is mediated by direct contact between target glia and LC neurons.
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(1990) Neuroscience. 36, 3, p. 631-641 Abstract
Activity of hippocampal neurons was recorded in an in vitro slice preparation. Topical application of serotonin produced hyperpolarization, blockade of a slow afterhyperpolarization which follows a burst discharge and blockade of a slow inhibitory postsynaptic potential. The slow inhibitory postsynaptic potential evoked by stimulation of the apical dendritic region of the hippocampus is more sensitive to serotonin than the membrane potential or conductance. The effects of serotonin on the inhibitory postsynaptic potentials are blocked by the 5-HT1A antagonist spiperone, and not by mianserin, a 5-HT2 antagonist. The attenuation of the inhibitory postsynaptic potentials is not accompanied by a change in postsynaptic reactivity to GABA or baclofen. Serotonin blocks repetitive large inhibitory postsynaptic potentials evoked in hippocampal neurons by topical application of 4-aminopyridine. Putative interneurons are more sensitive to topical application of serotonin than pyramidal neurons. Fenfluramine, a serotonin releaser mimics the effects of topical application of serotonin indicating that synaptically released serotonin can produce the changes in membrane potential and reactivity to afferent stimulation. It is suggested that serotonin attenuates slow inhibitory postsynaptic potentials by inhibiting feed forward inhibitory interneurons which impinge upon the recorded pyramidal neurons.
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(1990) Neurobiology of Aging. 11, 4, p. 481-484 Abstract
A monoclonal antibody to the nerve growth factor receptor (NGFR) (IgG 192) was used to visualize differences in immunohistochemical labeling of young (10 months) and old (35 months) rats. Three parameters were analyzed; cell counts, immunoreactive cross-sectional surface area (SA) and optical density (OD) of labeled cells. Large reductions in all three parameters were recorded in the medial septum (MS). Both OD and immunoreactive SA were reduced in the VDB, while only OD was reduced in the HDB. This observation confirms that NGFR labeling is reduced in the aged rat septum and adds that the loss of labeling is differential, with greater deficits in the MS-VDB complex than in the HDB.
1989
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(1989) Brain Research. 502, 1, p. 67-74 Abstract
The responses of CA1 neurons to topical application of serotonin (5-HT) and selective 5-HT1a and 5-HT1b agonists were examined with intracellular recording in the hippocampal slice. 5-HT produced a uniform hyperpolarizing response associated with an increase in K conductance as previously reported. In addition a marked reduction was reorded in slow after hyperpolarization (AHP) which follows a burst discharge. 8-OH-DPAT, ipsapirone and LY165, 163 partially mimicked the hyperpolarizing response to 5-HT when first applied to the slice. However, these 5-HT1a ligands antagonized responses to subsequent applications of 5-HT. Topical application of the 5-HT1b ligand TFMPP on the slice did not produce the direct or antagonistic action seen with the 5-HT1a ligands. It is suggested that the physiological response to 5-HT in the rat hippocampus is mediated by a 5-HT1a receptor. The currently available 5-HT1a ligands show a low agonist potential and a high antagonist action towards the responses of hippocampal neurons to 5-HT. Definite classification of the hyperpolarizing response to 5-HT awaits development of more specific ligands having a pure agonistic activity.
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(1989) Brain Research. 500, 1-2, p. 139-148 Abstract
Behavioral effects of septal lesion and fornix-fimbria transection were compared in absence and presence of a septal transplant in the hippocampus. The transplant grew in the hippocampus and projected acetylcholinesterase (AChE)-containing fibers throughout the extent of the denervated hippocampus. There were no differences in graft size or AChE reinnervation pattern after septal lesion or fornix transection. An increase in the density of M1 binding sites seen in hippocampal CA3 region after a cholinergic lesion, was restored back to normal after reinnervation of the hippocampus by the graft. Fornix-transected rats were more impaired in water maze acquisition than septal-lesioned rats which were impaired compared to controls. Septal-grafted rats were not different from lesioned rats in the behavioral tasks. However, an injection of physostigmine improved their performance relative to lesioned non-grafted rats. These experiments indicate that grafts can ameliorate behavioral deficits when the efficacy of acetylcholine of graft origin is enhanced.
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(1989) Neuroscience Letters. 101, 2, p. 169-174 Abstract
Synaptic connections between rat hippocampal neurons were studied in dissociated cell culture. Activation of a cultured neuron by pulse application of glutamate could produce postsynaptic currents (PSCs) in other neurons in the culture dish. Activation of protein kinase C (PKC) by a phorbol ester caused an enhancement of the magnitude of the PSCs without affecting much the delay and decay time constant of the recorded PSCs. The increased reactivity to synaptic activation was not accompanied by a postsynaptic change in sensitivity to topical application of an excitatory amino acid, glutamate. A PKC inhibitor polymyxin B reduced the effects of the phorbol ester. It is suggested that PKC activation plays an important role in the regulation of release of neurotransmitters from cultured central neurons.
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(1989) Synapse. 4, 4, p. 305-312 Abstract
Activity of hippocampal neurons was recorded in a dissociated culture under patch‐clamp conditions. Excitatory and inhibitory postsynaptic currents (PSCs) were evoked in response to short pulse application of glutamate to other neurons in the culture dish. These PSCs were suppressed by topical application of acetylcholine (ACh) near the recorded neuron. The dose‐dependent effect of the muscarinic antagonist pirenzepine indicates that the effect of ACh is mediated by an M2 receptor. ACh did not affect inward current responses to direct application of glutamate onto postsynaptic neurons. This indicates that ACh may interfere with the release process and not with the postsynaptic response to the neurotransmitter. In some cells, ACh reduced inward Ca currents recorded in the presence of Na and K channel blockers. This effect was atropine sensitive and may underly the reduced PSCs. It is suggested that ACh modulates release of neurotransmitters by reducing presynaptic Ica and thereby reducing evoked PSCs.
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(1989) Brain Research. 478, 1, p. 184-186 Abstract
The ability of embryonic raphe cells grafted into the hippocampus to restore spatial learning ability was tested in rats with combined serotonergic/cholinergic deficits. Embryonic raphe cells (E14) were transplanted into the hippocampus of serotonin-depleted rats. Two to 3 months after transplantation, control, lesioned and grafted rats were tested in a spatial memory task (a water maze) with and without the addition of atropine. All 3 groups could negotiate the water maze equally well, in non-drug conditions. The injection of atropine cause a severe distruption of performance only in the serotonin depleted rats. The presence of an active serotonergic graft was examined in the intact hippocampus using the serotonin releasing drug fenfluramine (FFA). A pronounced depression of hippocampal EEG was observed in control and grafted but not in lesioned rats 15 min after the injection of FFA. These results suggest the involvement of serotonin in cognitive functions in the rat. Furthermore, it is suggested that an interaction between serotonergic and cholinergic neurotransmission occurs in the hippocampus.
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(1989) Neurobiology of Aging. 10, 4, p. 305-310 Abstract
Cholinergic M1 and M2 muscarinic receptors in aged and young rat brains were studied by quantitative autoradiography of tritiated QNB in the presence of pirenzepine or carbachol. A selective pattern of decreased binding density was observed in the aged rat. A large number of regions showed no effect of aging; these include subdivisions of the hippocampal formation and most thalamic and hypothalamic nuclei. M1 and M2 receptors showed small but significant decreases in cortical regions and in the striatum. The largest effects were seen in M2 receptors of the ventral forebrain cholinergic nuclei where binding was reduced by up to 40%. No similar reductions were seen in the M1 receptor population in these regions. The results suggest that both muscarinic receptor subtypes show an anatomically selective pattern of decrease with age, with the M2 receptor subtype in the basal forebrain nuclei being specially vulnerable to the effects of aging.
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(1989) Brain Research. 477, 1-2, p. 404-407 Abstract
A possible interaction between serotonergic and cholinergic neurotransmission was examined in relation to the performance of a spatial memory task. Blockade of cholinergic transmission with a high dose of atropine was sufficient to impair performance of a water maze task. A partial reduction of cholinergic transmission using a low dose of atropine had no effect on this performance. Reducing serotonin synthesis, using a specific inhibitor of tryptophane hydroxylase p-chlorophenylalanine (PCPA) also had no effect on performance of such a task. However, a combined treatment with a low dose of atropine and PCPA severely impaired the performance of rats in the water maze. The rats were impaired in both acquisition of the initial spatial task and in reacquisition of a new spatial position (= working memory). These findings suggest an interaction between cholinergic and serotonergic transmission in acquisition and retention of spatial information. Furthermore they propose that deficits in cognitive abilities, observed in aging or Alzheimer's disease, may result from the combined reduction in cholinergic and serotonergic transmission.
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NEURAL GRAFTS AND NEUROTRANSMITTER INTERACTIONS IN COGNITIVE DEFICITS(1989) Neuronal Grafting And Alzheimers Disease. p. 141-149 Abstract
Keywords: Neurosciences
1988
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(1988) Neuroscience. 27, 3, p. 905-909 Abstract
Activity of CA1 neurons was recorded in rat hippocampal slices with a lidocaine-derived QX-572-filled micropipette. The QX compound abolished Na action potentials as reported earlier. In addition it reduced markedly the burst slow afterhyperpolarization seen in these neurons in response to a depolarizing current pulse. It also modified responses to neurotransmitter substances associated with changes in K currents, acetylcholine and serotonin. QX-filled micropipettes can therefore provide some insight into mechanisms of action of certain neurotransmitters in the brain but they cannot be used as selective blockers of Na currents.
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(1988) Journal of Neurochemistry. 51, 5, p. 1381-1385 Abstract
Abstract: Cholinergic muscarinic receptors in aged and young rat brains were studied by quantitative autoradiography of tritiated quinuclidinyl benzilate. A selective pattern of decreased binding density was observed in the aged rat. A large number of regions showed no effect of aging; these include subdivisions of the hippocampal formation and most tha‐lamic and hypothalamic nuclei. Small but significant decreases were found in cortical regions and in the striatum. The largest effects were seen in ventral forebrain cholinergic nuclei, where 40‐60% depletions were found in the diagonal band, nucleus basalis magnocellularis, ventral pallidum, and substantia innominata.
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(1988) Neuroscience Letters. 94, 1-2, p. 173-176 Abstract
Electrical stimulation of the perforant path produces a characteristic population EPSP and population spike in the dentate gyrus of the anesthetized rat. Parenteral administration of a serotonin releasing drug d-fenfluramine (FFA) caused a marked (30-100%) and highly significant increase in dentate gyrus population spike response to perforant path stimulation without affecting the slope of the population excitatory postsynaptic potential (EPSP). This indicates that FFA modifies granular cell excitability to afferent stimulation. The facilitatory effect of FFA was not present in rats depleted of serotonin following treatment with the synthesis inhibitor p-chlorophenylalanine (PCPA) but was restored after restoration of serotonin synthesis with the precursor 5-hydroxytryptophan indicating that presence of serotonin in terminals is required for the action of FFA.
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(1988) Behavioural Brain Research. 30, 2, p. 215-219 Abstract
The acquisition and retention of a water maze task was examined in 12 intact, young Wistar rats. Acetylcholinesterase activity in 43 discrete brain regions was then measured in the same rats by quantitative histochemistry. Individual learning and retention indices were found to be significantly correlated with acetylcholinesterase (AChE) levels in specific regions, e.g. cholinergic nuclei; the ventral pallidum and nucleus basalis; and in the dentate gyrus of the hippocampus. High levels of AChE in these regions predicted poor performance in the water maze. Thus cholinergic activity in selected regions of the rat brain might be involved in the performance of spatial memory tasks.
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(1988) Brain Research. 452, 1-2, p. 79-86 Abstract
Tetanic stimulation of the basal dendritic field (stratum oriens) of CA1 area of rat hippocampal slice can produce a slow depolarization associated with an increase in input resistance, decrease in accomodating properties of the cell to a depolarizing drive and an increase in synaptic activity. These effects are enhanced by an acetylcholinesterase inhibitor, eserine, and blocked by the muscarinic antagonists, pirenzepine and atropine. The accommodating property of the cell is more sensitive to the stimulation than the membrane potential or input resistance. The effects of oriens stimulation can be obtained also in fornix-fimbria transected hippocampal slice indicating that it may activate a local cholinergic or non-cholinergic pathway. The stimulation causes a heterosynaptic enhancement of reactivity of neurons to afferent stimulation indicating that acetylcholine may cause an enhanced excitability of hippocampal neurons.
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Behavioral and physiological effects of trimethyltin in the rat hippocampus(1988) NeuroToxicology. 9, 3, p. 481-489 Abstract
An injection of trimethyltin (TMT) into adult male Wistar rats produced a transient hyperirritability and aggressive behavior. When these effects of the drug subsided, the rats were still markedly impaired in performance of a spatial memory task examined in a watermaze. Activity of neurons in the hippocampus of these TMT-treated rats was studied in a slice preparation. A large (8 mg/kg) or double (7.5 and 3.5 mg/kg, two weeks apart) i.p. injection of TMT caused a loss of slow inhibitory postsynaptic potentials recorded in CA1 neurons in response to stimulation of stratum radiatum. A loss of accomodating properties of the neurons in response to a prolonged depolarizing current pulse was also seen in some of these cells. Other resting properties of the neurons were within the normal range. Topical application of acetylcholine (ACh) produces in normal hippocampal neurons a depolarizing response and a blockade of after-hyperpolarization. Only the latter response to ACh was seen in cells from TMT treated rats. No effect of the drug on reactivity to serotonin was detected. These effects of TMT were not seen when the drug was applied acutely while recording from cells in the slice indicating that it involves a degeneration process. These results suggest that the cognitive deficits in TMT treated rats might be associated with deficits in cholinergic functions.
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(1988) Progress in Brain Research. 78, C, p. 95-101 Abstract
The transplanted tissue can promote local processes of regeneration, prevent retrograde degeneration, or simply serve as a slow-release biological capsule, which discharges the appropriate neurotransmitter in a random fashion into the intercellular space. This chapter describes the physiology of graft-host interactions in the rat hippocampus. The chapter also addresses two main questions that guide the research on the physiology of graft-host interactions, (1) can the graft substitute for the damaged host tissue, be incorporated into host circuits and function as expected of a normal host tissue; and (2) can the graft serve as a simple model system for asking questions relevant to normal brain operation. Independent of the possible restoration of functions, the graft can be used as a simple model for complex networks in the brain. The chapter indicates that given the existing limitation of resolution of electrophysiological techniques in the in vitro slice preparation, the heterogeneity of grafted tissue and synapse formation with selective populations of host neurons, the graft should be useful for examining basic questions relevant to central neurotransmission.
1987
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(1987) Developmental Brain Research. 35, 1, p. 158-160 Abstract
The ontogeny of muscarinic cholinergic receptors was studied in primary cultures of dissociated rat hippocampal cells. The specific [3H]quinuclidinyl benzilate ([3H]QNB) to intact cells was detected after two days in culture. Scatchard analysis revealed only a unique binding site at all of the days tested, with a very high affinity (0.205-0.253 nM) for the substrate. Specific binding kept increasing up to a level of 2035 fmol/mg protein after 15 days in culture. Protein content by itself also increased over time, reaching the highest level at 15 days (286 μg protein/35 mm plate). The cultured hippocampus can thus serve as a model system for the study of the development of muscarinic receptors in vitro.
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(1987) Brain Research. 414, 2, p. 285-293 Abstract
The effects of topical application of microdrops containing 4-aminopyridine (4-AP) on properties of CA1 neurons were examined in the hippocampal slice preparation. 4-AP triggered repetitive large (4-10 mV) hyperpolarizing potentials (HPs) having a short rise time and slow (3-4 s) decay. There was a marked decrease in input resistance during the HPs. The HPs are likely to be caused by an increase in potassium conductance; their reversal potential was 15-20 mV negative to rest, the reversal potential shifted in the depolarizing direction when the slice was bathed in high potassium medium, and it was the same with KCl or potassium acetate recording electrodes. The HPs were not generated by release of neurotransmitter substances from terminals of extrinsic afferents since they were present in slices taken from deafferented hippocampus but they were blocked by tetrodotoxin (TTX) or Cd and Mn, indicating that they are synaptic potentials of local origin. HPs were still present when Ca-dependent K currents were blocked by acetylcholine and noradrenaline. Three of 56 cells recorded in the hippocampus could be classified as interneurons. They emitted high frequency trains of action potentials in response to 4-AP, at a rate corresponding to the HPs recorded in all other neurons. It is suggested that 4-AP excites a specific type of interneuron which in turn generates large K-mediated inhibitory postsynaptic potentials in the pyramidal neurons of CA1 region of the hippocampus.
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(1987) Progress In Brain Research
. 71, C, p. 349-357 AbstractThis chapter discusses the efficacy of using neural grafting to restore physiological and behavioral functions following damage to the cholinergic septohippocampal system. It presents a study in which embryonic septal regions were injected into the hippocampi of fimbria\u2013fornix (FF) transected rats. The grafts developed an extensive innervation of the host hippocampus and, when stimulated, produced a slow depolarizing response that was blocked by atropine and facilitated by physostigmine. There is evidence that the graft restored hippocampal theta rhythm, which had been eliminated by FF transactions or septal lesions. However, unlike unlesioned controls, theta in grafted animals is not correlated with movement. Performance in a water maze, which was markedly impaired by fornix transection, showed no improvement 6 months after transplantation, but was restored in septal lesioned, septal grafted rats. The results indicate that grafting may provide a useful tool for studying the action of neurotransmitter systems and their involvement in higher brain functions.
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Chloride conductances in central neurons(1987) Israel Journal of Medical Sciences. 23, 1-2, p. 95-100 Abstract
1986
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(1986) Neuroscience Letters. 71, 3, p. 306-310 Abstract
Activity of serotonergic dorsal raphe neurons was recorded intracellularly in a brainstem slice preparation from rats before and after local microperfusion of the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). The initial effect of the drug consisted of a transient hyperpolarization. Over 1-2 h after drug application there was a gradual decrease in efficacy of a hyperpolarizing current pulse to evoke a transient rectification. Also, action potentials in some cells failed to evoke a spike after-hyperpolarization. These effects are related to the neurodegenerative action of 5,7-DHT.
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PROPERTIES OF RAT MEDIAL SEPTAL NEURONS RECORDED INVITRO(1986) Journal Of Physiology-London. 379, p. 309-330 Abstract
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Real-time optical mapping of neuronal activity in vertebrate CNS in vitro and in vivo.(1986) Society of General Physiologists Series. 40, p. 165-197 Abstract
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(1986) Brain Research. 364, 1, p. 162-166 Abstract
Embryonic midbrain raphe was grafted into serotonin-deficient adult rat hippocampus. Serotonin-containing neurons in the graft survive for at least 6 months after grafting. Grafted neurons develop physiological properties, not present on the day of grafting, identical to those of normal adult serotonin-containing neurons. These include (a) high input resistance and slow membrane time constant, (b) lack of inward rectification in response to hyperpolarizing current pulses and (c) a potent, 4-aminopyridine-sensitive transient outward rectification. The grafted neurons innervate the host tissue with axons that have a slow conduction velocity and refractoriness. It is suggested that grafted CNS neurons may possess normal physiological properties.
1985
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(1985) Brain Research. 359, 1-2, p. 347-350 Abstract
Activity of neurons in the dorsal raphe nucleus of the rat was recorded intracellularly in a brainstem slice. The neurons had a high input resistance, a linear I-V curve in the hyperpolarizing direction and a long membrane time constant. Broad action potentials were followed by a large afterhyperpolarization (AHP). This AHP removes partial inactivation of a potent transient outward rectifier that is activated by a subsequent depolarization of the neurons; it clamps the cell membrane at a potential slightly below firing level and blocks generation of action potentials for up to 100 ms. The transient rectification was sensitive to 4-aminopyridine (4-AP) but not to tetraethylammonium (TEA). It appears to share similar properties with those of IA seen elsewhere and to function in the regulation of interspike intervals of dorsal raphe (DR) neurons in vitro.
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(1985) Physiological Psychology. 13, 3, p. 172-178 Abstract
Noradrenaline(NA)-containing fibers originate in the nucleus locus coeruleus (LC) and innervate wide cortical and subcortical structures. Extensive evidence indicates that NA of LC origin can suppress apparent spontaneous activity of many neurons in the brain, yet increase their reactivity to certain stimuli. The type of stimuli as well as the nature of the enhancement are region specific. The observed effects of activation of LC are a product of an effect on remote afferents, on local interneurons, and on the projection neuron that is being monitored. The cellular mechanisms underlying these actions have been studied with intracellular recording techniques in the slice preparation. NA hyperpolarizes hippocampal neurons probably by activating an electrogenic Na-K pump. It enhances reactivity to afferent stimulation probably by acting presynaptically and also by suppressing a postsynaptic hyperpolarizing Ca-dependent K current. This results in enhancement of the signal-to-noise ratio of the monitored system and in a more efficient response to significant environmental simuli.
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(1985) Brain Research. 336, 2, p. 302-307 Abstract
Cell suspensions from the fetal septal region were injected stereotaxically into the hippocampus of fornix-fimbria-transected adult rats. The host rats were sacrificed up to 3 months after the operation and the hippocampus sliced into 350 μm transverse slices. Intracellular recording was made from CA1 neurons adjacent to the graft. Electrical stimulation of the graft produced a voltage-dependent depolarization in some recorded neurons. This was associated with an increase in spontaneous and anodal break action potential discharges. In addition, a slow after-hyperpolarization (AHP) which typically follows a burst discharge was blocked during the depolarization indicating that the stimulation may block a Ca2+-dependent K+ current. The effects of the stimulation were antagonized by atropine. A response to the stimulation was seen 2 weeks but not 1 week after grafting. Over time, cells that were located away from the graft became activated by the stimulation. This was correlated with the extent of proliferation of acetylcholinesterase-containing fibers around the graft. These results suggest that grafted septal neurons make viable cholinergic connections with a host hippocampus.
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(1985) Neuroscience. 15, 1, p. 47-53,55-60 Abstract
Extracellular single unit recordings were made in the median raphe nucleus from rats anaesthetized with urethane. Spontaneous firing as well as orthodromic and antidromic responses to stimulation of the fornix and the medial septum were studied. One hundred and twelve units (out of a total of 355) with a regular spontaneous firing rate of 0.2-3 spikes/s were classified as serotonin-containing neurons. Fifty nine of them were antidromically invaded from either the fornix or the medial septum (conduction velocity, 0.8 m/s) and 7 additional neurones from both the fornix and the medial septum. Antidromic action potentials were followed by a period of decreased probability of firing, that was already present below threshold for antidromic invasion, were proportional to the stimulation intensity and had a latency similar to orthodromic inhibition. No preferential topographical distribution within the median raphe nucleus was observed for the serotonin neurones, even those invaded antidromically. Twenty six neurones with a clear-cut anatomical location around the borders of the median raphe nucleus showed a spontaneous rhythmic activity (4-20 spikes/s) characterized by the presence of extremely prolonged silent periods (up to 5 min). Only one of these neurones was invaded antidromically from the medial septum and none from the fornix. Of the remaining non-serotonin neurones, 28 showed a very low firing rate consisting of single action potentials every 10-60 s while 189 had a spontaneous activity of 6-30 spikes/s. Regardless of their firing rate they were all antidromically invaded from the fornix and/or the medial septum and had a conduction velocity of 5 m/s. These experiments demonstrate the electrophysiological heterogeneity of the neuronal population of the median raphe nucleus, the presence of strong projections of both putative serotonin and non-serotonin neurones to the medial septum and, via the fornix, to the hippocampus, and the existence of axonal branching in both types of neurones.
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1984
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(1984) Brain Slices. Dingledine R.(eds.). Boston, MA: . p. 227-261 Abstract
This chapter describes a novel approach to investigate the spatiotemporal distribution of electrical activity in nervous systems. Using voltage-sensitive dyes and an electro-optical measuring system, it has recently become possible to monitor electrical activity simultaneously from multiple sites on the processes of single nerve cells, either in culture or in an intact central nervous system (CNS) in vitro, to detect the activity of many individual neurons controlling a behavioral response in invertebrate ganglia, or to follow the activity of populations of neurons at many neighboring loci in mammalian brain slices or in the intact brain. Employing optical recordings and a display processor, the images of nerve cells light up on a TV monitor when they are electrically active. Thus, the spread of electrical activity can literally be visualized in slow motion. This chapter describes recent progress in the implementation of this new technique.
1983
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(1983) Brain Research. 274, 2, p. 381-387 Abstract
Discharge activity was recorded extracellularly from individual neurons of the nucleus locus coeruleus in anesthetized squirrel monkeys. These cells exhibited long-duration (2-3 ms) action potentials and discharged spontaneously in a slow (0.2-2 Hz) irregular fashion. Stimulation of the lateral hypothalamus evoked antidromic responses at latencies of 10-20 ms, indicating conduction velocities of over 1 m/s in some cases. The mean refractory period for these axons was 2.6 ms. When the rate of hypothalamic stimulation was increased from 1 to 10 Hz there was a 15-20% increase in antidromic latencies. These properties are similar to those previously observed for rat LC neurons, except that conduction velocities are higher in monkey.
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Excitable membrane properties of cultured central nervous system neurons and clonal pituitary cells.(1983) Cold Spring Harbor Symposia on Quantitative Biology. 48 Pt 1, p. 259-268 Abstract
1982
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(1982) Experimental Neurology. 77, 1, p. 86-93 Abstract
Intracellular activity was recorded from CA1 neurons in rat hippocampal slices. Iontophoretic application of glutamate produced a fast and reversible depolarization associated with an increase in action potential discharge rate and only minimal conductance changes. Concurrent application of norepinephrine (NE) or isoproterenol with moderate iontophoretic currents or in a microdrop potentiated the depolarizing responses to glutamate. This effect was not reproduced by serotonin. It is suggested that NE acts postsynaptically to increase the efficacy of the depolarizing action of excitatory neurotransmitters.
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(1982) Journal Of Physiology-London. 333, DEC, p. 269-291 Abstract
1. Voltage-sensitive membrane-bound dyes and a matrix of 100 photodetectors were used to detect the spread of evoked electrical activity at the CA1 region of rat hippocampus slices. A display processor was designed in order to visualize the spread of electrical activity in slow motion.2. The stimulation of the Schaffer collateral-commissural path in the stratum radiatum evoked short latency (2-4 msec) fast optical signals, followed by longer latency (4-15 msec) slow signals which decayed within 20-50 msec. Multiple fast signals were frequently detected at the stratum pyramidale; they propagated toward the stratum oriens with an approximate conduction velocity of 0.1 m/sec.3. The fast signals were unaltered in a low Ca2+ high Mg2+ medium but were blocked by tetrodotoxin. These signals probably represent action potentials in the Schaffer collateral axons. Their conduction velocity was about 0.2 m/sec and their refractory period about 3-4 msec.4. The slow signals were absent in a low Ca2+ medium and probably represent excitatory post-synaptic potentials (e.p.s.p.s) generated in the apical dendrites of the pyramidal cells. They were generated in the stratum radiatum, where the presynaptic signals were seen, and spread into somata and basal dendrites (the stratum pyramidale and oriens, respectively).5. The timing of the signals with fast rise-time, which were detected at the statum pyramidale, approximately coincided with the timing of the extracellularly recorded field potentials. These multiple discharges probably represent action potentials of the pyramidal cells. They spread back into the apical dendrites but with significant attenuation of the amplitudes of the high frequency components of the pyramidal action potentials.6. Hyperpolarizing potentials could be detected when strong stimuli were applied to the stratum radiatum or alveus. The net hyperpolarizations were detected only in the stratum pyramidale and the border region between the stratum pyramidale and radiatum. Frequently the inhibition was masked by the large e.p.s.p.s. However, its existence could be demonstrated by treatment of the slice with picrotoxin or a low Cl- medium. Under these conditions a long-lasting depolarization of the apical dedrites was evoked by the stimulation. This was associated with an increase of the multiple discharges in the stratum pyramidale and oriens.7. These studies illustrate the usefulness of voltage-sensitive dyes in the analysis of passive and active electrical properties, pharmacological properties and synaptic connexions in mammalian brain slices, at the level both of small neuronal elements (dendrites, axons) and of synchronously active neuronal populations.
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1981
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(1981) Brain Research. 213, 2, p. 351-364 Abstract
Properties of the norepinephrine- (NE) stimulated, cAMP-generating system were studied in rat hippocampal slices. NE but not other putative neurotransmitters, caused a 3-4-fold rise in cAMP levels in the slices. All 3 main subdivisions of the hippocampus (HPC), the dentate gyrus, areas CA3 and CA1, possessed the capacity to produce cAMP. The latency to the NE stimulation of cAMP formation was about 20 sec but maximal stimulation was reached only after 5-10 min of incubation. Intrahippocampal injection of kainic acid (KA) caused a nearly complete destruction of hippocampal neurons and a marked increase in number of glial cells. NE caused a 12-15-fold rise in cAMP levels in KA-treated HPC. Compared to normal HPC where potency order of noradrenergic agonists indicated activation of a beta-1 receptor type, the pattern for the KA-treated HPC indicated the dominance of beta-2 receptors. The beta-1 antagonist, practolol, and the beta-2 antagonist, H35/25, were about equipotent in blocking the NE-stimulated cAMP formation in normal HPC. In KA-treated HPC, on the other hand, H35/25 was much more potent than practolol in inhibiting NE-stimulated cAMP formation. It is suggested that in the HPC beta-1 adrenergic receptors are primarily neuronal and beta-2 receptors, glial, and that activation of both receptor species results in activation of a cAMP-generating system.
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(1981) Brain Research. 213, 2, p. 443-448 Abstract
Intracellular activity was recorded from hippocampal CA3 pyramidal cells maintained in vitro. Histamine (HA) produced a slow depolarization associated with minimal conductance changes. In addition, there was an increase in action potential discharge rates and the emergence of bursting firing patterns. EPSP size increased by about 50% and spontaneous dendritic spikes were observed. These effects were markedly reduced by retrodotoxin. Extracellular recording of population spikes revealed a marked difference between CA1 and CA3 regions; in the former HA produced an increase in population spike size whereas in the latter this increase was larger and was associated with the appearance of secondary and tertiary population spikes. It is suggested that HA produces its effects by enhancing release of neurotransmitters from excitatory synapses on the recorded neurons.
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(1981) Brain Research. 206, 1, p. 107-128 Abstract
The ionic basis of norepinephrine (NE) action was studied with intracellular recording techniques in the rat hippocampal slice. Topical application of NE caused, in CA1 neurons, a 3-4 mV hyperpolarization associated with a 10-20% decrease in input resistance. This effect was accompanied by a decrease in spontaneous action potential discharges and, in some cells, by a reduction in EPSPs produced by stimulation of the excitatory Schaffer collateral-commissural pathway. An analysis of the voltage and concentration dependency revealed that NE may activate two different mechanisms. Experiments performed to test this hypothesis have demonstrated that a short duration hyperpolarizing action of NE was still present in a low C1- medium. The hyperpolarizing responses to NE were absent in ouabain-treated slices and in low temperature. Cyclic AMP produced a 3-4 mV hyperpolarization associated with minimal changes in input resistance. This effect of cAMP was blocked by ouabain. IBMX potentiated responses to low concentrations of NE. It is proposed that NE activates two mechanisms; one involves activation of C1- conductance and the other activation of a Na+K+ pump. This latter effect might be mediated by cAMP.
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Regional differences in neuronal responses to 5-HT: Intracellular studies in hippocampal slices(1981) Journal de Physiologie. 77, 2-3, p. 373-375 Abstract
Intracellular recordings were obtained from cells in the regions CA1, CA3 and the dentate gyrus (DG) of rat hippocampal slices. Topical application of 5-HT induced a 2-10 mV hyperpolarization in all cells tested. The hyperpolarization was accompanied by a marked reduction in input resistance in CA1 and DG cells, but by a much smaller resistance change in CA3 cells. These effects of 5-HT were not abolished by tetrodotoxin. It is suggested that 5-HT causes an increase in K+ conductance in regions CA1 and DG and that this results in hyperpolarization. A different mechanism might be activated by 5-HT in cells of the CA3 region.
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1980
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(1980) Brain Research. 202, 2, p. 387-399 Abstract
The oxidative metabolic activity of restricted regions of hippocampal slices was assessed by a continuous measurement of the fluorescence of intramitochondrial nicotinamide-adenine dinucleotide (NADH). A large increase in NADH fluorescence was triggered by sunstituting the oxygen supply to the slice by nitrogen gas. A large and transient increase in NADH fluorescence was also produced by superfusion of the slice with a high (50 mM) potassium-containing medium. Addition of norepinephrine (NE) to the superfusion medium caused a propranolol-inhibited increase in NADH fluorescence. Furthermore, ouabain, which inhibits the Na-K pump, blocked the effects of NE. An analog of cyclic adenosine monophosphate (cAMP), 8-bromo cAMP, mimicked the effect of NE. Finally, effects of NE could still be produced in a kainic acid-treated hippocampus, where most neurons were previously destroyed by the drug. It is suggested that NE activates a Na-K-ATPase pump, that this effect might be mediated by cAMP, and that these interrelations may underly the physiological action of NE in the brain.
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(1980) Brain Research. 195, 2, p. 389-401 Abstract
The effects of serotonin (5-HT) on extracellular potassium concentration ([K+]0) were measured with ion-selective microelectrodes in rat hippocampal slices. Electrical stimulation of an excitatory afferent system, the Schaffer collateral commissural pathway, caused a 2-4 mM rise in [K+]0 in the stratum pyramidale of area CA1. 5-HT caused a 0.6-1.1 mM rise in [K+]0. This rise was associated with hyperpolarization of neurons and cessation of their spontaneous spike discharge. Methysergide, a 5-HT antagonist, reduced the 5-HT effect. The change in [K+]0 was highest in stratum moleculare and lowest in stratum pyramidale, the opposite gradient to that found with excitatory electrical stimulation. The 5-HT-induced [K+]0 changes were maximal in CA1 stratum moleculare, intermediate in the dentate stratum granulare and almost non-existent in the CA3 stratum pyramidale. GABA, but not norepinephrine, produced a small (up to 0.5 mM) rise in [K+]0 in stratum pyramidale. Extracellular calcium concentration measured with a Ca2+-sensitive microelectrode was reduced by electrical stimulation but unchanged by 5-HT or norepinephrine. It is suggested that 5-HT hyperpolarizes hippocampal cells by activation of sodium- and calcium-independent potassium channels, which cause a rise in [K+]0.
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(1980) Brain Research. 195, 1, p. 215-222 Abstract
Impulses in rat locus coeruleus neurons exhibit pronounced conduction latency decreases, followed by even larger latency increases (of over 20 msec in some cases) during a single train of antidromic activation. The magnitude of latency fluctuation varies as a function of basal antidromic latency, frequency of stimulation, and number of stimuli in a train. These and additional data indicate that this variability in latency is a consequence of altered impulse conduction velocity along the axons, perhaps reflecting reduced ion concentration gradients resulting from impulse propagation. These latency changes may allow thin unmyelinated axons to influence target cells most effectively with short bursts of activity, and suggest that myelination and large axon diameter provide for high fidelity as well as for high velocity of impulse flow in nervous tissue.
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(1980) Journal Of Physiology-London. 303, 1, p. 423-439 Abstract
1. Intracellular activity was recorded from neurones in the CA1 pyramidal layer of slices of rat hippocampus maintained in vitro. 2. Application of 5‐HT in a droplet or via ionophoresis produced a 3‐5 mV hyperpolarization associated with a 30% decrease in input resistance. 3. The response to 5‐HT was minimal with a drop concentration of 1 microM and maximal with 100 microM. The responses appeared to be blocked by methysergide applied in the superfusion medium. 4. The responses to 5‐HT were minimal when the drug was applied in the apical dendritic region and maximal when it was applied near the soma. 5. 5‐HT produced no substantial changes in e.p.s.p.s evoked by stimulation of the Schaffer collateral‐commissural system or in i.p.s.p.s which were occasionally encountered following stimuli to the stratum radiatum. 6. The responses to 5‐HT are true post‐synaptic responses and are not indirect effects since they are present in a Ca2+‐deficient Mg2+‐enriched medium which blocks synaptic transmission. 7. The responses to 5‐HT were not dependent on extracellular Cl‐ concentration. 8. These experiments indicate that 5‐HT produces its effects in the rat hippocampus by activating K+ channels.
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(1980) Neuroscience Letters. 19, 1, p. 67-71 Abstract
Electrophysiological activity was recorded intracellularly from pyramidal neurons in rat hippocampal slices. Topical application of histamine produced a slow depolarization that was not associated with conductance changes. The depolarization was accompanied by an increase in the rate of action potential discharges. These effects were markedly reduced in slices maintained in a low Ca2+, high Mg2+ medium, indicating that histamine may act presynaptically on hippocampal pyramidal neurons.
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Optical methods to elucidate electrophysiological parameters(1980) Neurotransmitters and their receptors. Silman I., Littauer U. Z., Teichberg V. I., Dudai Y. & Vogel Z.(eds.). p. 531-546 Abstract
Optical methods to monitor electrical activity were developed for mammalian neurons. Most of the experiments were carried out on monolyaer cultures of neuroblastoma cells. Preliminary experiments were done with rat hippocampal slices. For transmission experiments we have found a membrane potential probe which has satisfactory sensitivy: WW 401, a merocyanine rhodanine derivative. A 10x10 array of photodiodes was positined in the real magnified image formed by a microscope objective. It was used to detect voltage changes simultaneously rom many points along the arborization of cultured neurons. Action potentials were detected from the cell body with a signal to noise ration of 20:1 without signal-averaging. With averaging, signals could be detected from every diode on which the image of the cell or its processes fell. A He-Ne laser was stabilized for fluorescence experiments. It was focused onto a small spot and changes in fluorescence from cells stained with an oxonol dye (WW 802) were monitored with a phototube. Action potentials were detected without signal-averaging even from 5μc processes or growth cones of these cells. Fluorescence proved to be by far more sensitive; up to 7% change in fluorescence for 100mV membrane potential change, two orders of magnitude larger relative to the absorption change.Different sites on the same cell could be examined by changing the position of the cell relative to the laser microbeam. A conduction velocity of 0.2 m/sec in the 7μm process was determined by the simultaneous measurement of the action potential in the cell body, with an electrode, and a remote growth cone with the laser microbeam. The same type of transmission measurements was to detect simultaneous activity from hundred of neuronsin rat hippocampal slices
1979
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(1979) Behavioral and Neural Biology. 26, 4, p. 497-501 Abstract
The effects of fornix transection on "learned helplessness," the phenomenon where prior experience with uncontrollable aversive stimulation interferes with later adaptive responding, were studied. Rats were subjected to fornix transection or control procedures, exposed to unavoidable preshock or not, and then tested for escape/avoidance performance in a shuttlebox. As in earlier studies, control rats receiving unavoidable preshock exhibited longer escape latencies in the helplessness test than non-preshocked animals. However, fornix-transected rats did not show this effect of preshock. These findings suggest that the hippocampal system is involved in learned helplessness.
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(1979) Brain Research. 170, 3, p. 409-416,IN1-IN4,417-426 Abstract
Transplants of the embryonic locus coeruleus (LC) region were implanted into the circuity of the hippocampal formation in adult rats in which the normal adrenergic afferents to the hippocampus had been removed. The growth of new adrenergic axons from the implant in the denervated hippocampus was followed for 1-14 months after surgery by means of fluorescence histochemistry, and the function of the implant-hippocampal connections was tested electrophysiologically after 2-3 months survival. In the successful cases the entire hippocampal formation was reinnervated from the LC implant within 3-6 months after operation, and the newly formed innervation still persisted unchanged by 14 months. The reinnervation was equally effective irrespective of the route by which the axons entered the hippocampus, i.e. along the lesioned fornix-fimbria or along a retrosplenial route. The pattern formed by the ingrowing LC axons mimicked to a large extent that of the normal LC afferents. Little growth was seen into denervated terminal fields of the commissural, septal or entorhinal afferents, pointing to a preference of the ingrowing LC fibers for the areas normally innervated by adrenergic afferents. In the electrophysiological experiments, stimulation of the LC implants caused (in 20 out of 29 cells monitored) an inhibition of the spontaneous activity of neurons in the host hippocampus. This inhibition had a relatively long latency and a long duration, similar to that observed after stimulation of the innate LC in the intact rat. As in the normal rat, the inhibitory responses were blocked by systemic or local application of the beta-adrenergic receptor blockers propranolol or sotalol. It is concluded that the adult rat brain is capable of carrying out all steps involved in correct functional reinnervation of a denervated region. Moreover, the implant-hippocampal preparation should be a highly suitable model system for functional studies of a central noradrenergic connection.
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(1979) Psychopharmacology. 62, 2, p. 169-173 Abstract
Pharmacologic studies of analgesia produced by stimulation of the nucleus locus coeruleus (LC) were conducted using the rat hot-plate test. A correlation between self-stimulation and analgesia produced by stimulation of LC was found. Analgesia produced by LC stimulation was attenuated by naloxone, a morphine antagonist, cyproheptidine, a serotonin antagonist, and WB-4101, an alpha-adrenergic antagonist. The analgesia was absent in 6-OHDA-treated rats. Catecholamine synthesis inhibition by a combination of reserpine and AMT or more specific inhibition of noradrenaline synthesis by DDC elevated latency to paw lick and yet did not affect stimulation-produced analgesia. It is suggested that morphinergic, serotonergic, and alpha-adrenergic mechanisms mediate LC stimulation produced analgesia.
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SEROTONERGIC INNERVATION OF THE LOCUS COERULEUS FROM THE DORSAL RAPHE AND ITS ACTION ON RESPONSES TO NOXIOUS STIMULI(1979) Journal Of Physiology-London. 286, JAN, p. 401-& Abstract
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1978
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(1978) Electroencephalography and Clinical Neurophysiology. 45, 3, p. 409-411 Abstract
The spontaneous electrical activity of the hippocampus as well as its responses to commissural stimulation were recorded in waking rats. Changes in magnitude of the early and late components of the response to commissural stimulation were associated with the behavioural state of the rat. The magnitude of the initial potential was higher in non-theta 'states' than in theta 'states', whereas the opposite was true for the late potential.
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(1978) Brain Research. 152, 3, p. 529-542 Abstract
The relationship between intracranial self-stimulation (ICSS) and stimulation-produced analgesia (SPA) was investigated in the rat employing an operant bar-press response and a modification of the hot-plate test. ICSS and SPA were elicited through bipolar electrodes chronically implanted in two catecholamine nuclei; the nucleus locus coeruleus (LC) and the substantia nigra (SN). These sites have previously been shown to yield both phenomena. SPA was shown to be of a magnitude similar to that of morphine. In addition, SPA deriving from both LC and SN was significantly reversed by the specific opiate antagonist, naloxone. The intensity of the stimulating current sufficient to induce SPA was found to be higher than that required for ICSS. The pharmacological susceptibilities of the two phenomena were tested by administering a number of drugs: haloperidol, propanolol, pimozide and AMPT attenuated ICSS in both LC and SN, while leaving SPA unaffected. In contrast, methysergide and PCPA blocked SPA and simultaneously facilitated ICSS. The present results indicate a dissociation of ICSS and SPA from LC and SN at both physiological and neurochemical levels; ICSS rates appeared to be a function of catecholaminergic tone, while SPA depended upon the integrity of serotonin transmission.
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(1978) Neuropharmacology. 17, 8, p. 619-623 Abstract
Responses of hippocampal neurones to the iontophoretic application of acetylcholine (ACh) and nicotine were measured in urethane-anaesthetized rats. The majority of bursting-type cells was excited by ACh and about half of them were depressed by nicotine. The excitation was reversed by atropine and in some cases by gallamine. The majority of non-bursting θ cells was depressed by nicotine but unaffected by ACh. The depression was antagonized by d-tubocurarine. It is suggested that the hippocampus contains both muscarinic and nicotinic receptors that have different anatomical distributions and physiological significance.
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Effects of priming stimulation of catecholamine containing nuclei in rat brain on runway performance(1978) Brain Research Bulletin. 3, 3, p. 203-206 Abstract
Rats were implanted with bipolar fine wire stimulating electrodes in the region of the pontine nucleus locus coerulus (LC) and the pars compacta of the substantia nigra (SN). The reinforcing properties of stimulation of each electrode were determined. Subsequently, rats were run in a two alley runway and the effects of priming stimulation on performance in acquisition, discrimination and reversals were measured. It was found that LC priming caused an increased latency to exit from start box. Also, LC priming reduced running time in the rewarded alley in a reversal task whereas SN priming affected both performance in the rewarded and non-rewarded alley.
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1977
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(1977) Experimental Neurology. 57, 3, p. 750-765 Abstract
The afferents to the parahippocampal area of the rat were studied with retrograde transport of horseradish peroxidase injected into the medial entorhinal cortex, lateral entorhinal cortex, parasubiculum, presubiculum, or a large injection which stained all these structures as well as the ventral hippocampus. Control rats were injected with horseradish peroxidase into the overlying visual cortex. Labeled neurons in brains with injections into the medial entorhinal cortex and the adjacent parasubicular region were found in the ipsilateral and contralateral presubicular region, the medial septal nucleus, the thalamic nucleus reuniens, the dorsal part of the lateral nucleus of thalamus, the anterior periventricular nucleus of the thalamus, and the dorsal raphe nucleus. Brains with injections into the lateral entorhinal cortex yielded labeled neurons in the medial septal nucleus, nucleus reuniens, dorsal raphe nucleus, and nucleus locus ceruleus. Injections into the presubiculum resulted, in addition, in labeling of neurons in the lateral nucleus of the thalamus. Control injections aimed at the sensory cortex overlying the parahippocampal area yielded labeled neurons in the medial septal nucleus, the dorsal lateral geniculate nucleus, and the nucleus locus ceruleus.
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(1977) Experimental Brain Research. 29, 3-4, p. 553-565 Abstract
The responses of the hippocampus of the awake rat to stimulation of one of its afferent pathways were measured during classical conditioning. It was found that when the contralateral hippocampus is stimulated concurrently with the presentation of a conditioned stimulus preceding either food or an aversive shock, a late (30-40 msec) negative component in the averaged evoked response can be recorded. This late component was absent when the interhemispheric stimulation was applied prior to presentation of a conditioned stimulus or when the rat was satiated or pretreated with drugs which interfere with noradrenergic or serotonergic neurotransmission. It is suggested that classical conditioning changes neurotransmission in certain pathways in the brain and that the monoamines are involved in mediation of this change.
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(1977) Experimental Brain Research. 28, 5, p. 529-541 Abstract
Evoked hippocampal responses to stimulation of the contralateral hippocampus were recorded in the awake rat. The effects of priming stimulation applied to the nucleus locus coeruleus and the raphe nuclei on averaged evoked hippocampal responses to the interhemispheric stimulation were measured. It was found that priming stimulation of these monoamine-containing nuclei caused the formation of a late component in the interhemispheric potential without affecting the magnitude of the initial response component. Drugs which selectively interfere with noradrenergic or serotonergic transmission, antagonized the locus coeruleus or raphe primed late response component, respectively. Parenteral administration of d-amphetamine or l-amphetamine caused the appearance of a late response component which was similar to that seen after brainstem priming stimulation. It is suggested that the monoamines modify responses to afferent stimulation of particular pathways in the brain.
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(1977) Experimental Neurology. 55, 1, p. 67-73 Abstract
Averaged evoked responses to commissural stimulation was recorded in the hippocampus of the awake rat. When tested with a twin pulse stimulation, the evoked response to the second test pulse was larger than the response to the first, conditioning pulse. When the twin pulse stimulation sequence was applied during presentation of a conditioned stimulus in a behavioral conditioning experiment, the increase in the response to the test pulse was larger than that recorded in control periods. It is suggested that excitability of the hippocampus is increased during learning.
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1976
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(1976) British Journal of Pharmacology. 58, 3, p. 341-345 Abstract
Hippocampal cellular responses to acidic amino acids and some of their antagonists were measured in the rat anaesthetized with urethane. The effects of these antagonists on the field responses of the rat hippocampus to afferent stimulation were measured in acute as well as chronically prepared rats. Hippocampal pyramidal cells were excited by microiontophoretic application of glutamate and aspartate. These responses were antagonized by glutamic acid diethyl ester (GDEE), glutamic acid dimethyl ester (GDME) and by proline. Partial specificity could be seen as excitatory responses to acetylcholine were less susceptible to the antagonists. Field responses of the hippocampus to commissural stimulation were reduced significantly in both an acutely prepared or the conscious rat following parenteral administration of GDEE and GDME but not proline. Responses to perforant path stimulation were not affected by these drugs. It is suggested that an acidic amino acid may serve as a neurotransmitter in the commissural path to area CA1 of the dorsal hippocampus. 1976 British Pharmacological Society
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BRAIN-STEM AFFERENTS TO RAT MEDIAL SEPTUM(1976) Journal Of Physiology-London. 261, 3, p. 617-631 Abstract
1972
1971
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RAPID MODIFICATION OF AMPHIBIAN BEHAVIOR BY PUNISHMENT(1971) Psychonomic Science. 24, 5, p. 249-250 Abstract