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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.

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 (

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 GABA_B 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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Keywords: Behavioral Sciences; Clinical Neurology; Psychiatry

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 GABA_B receptor or by a conventional chloride‐linked GABA_A 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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-HT_1A 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.

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.

Keywords: Neurosciences

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.