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Critical dynamics are thought to play an important role in neuronal information-processing: near critical networks exhibit neuronal avalanches, cascades of spatiotemporal activity that are scale-free, and are considered to enhance information capacity and transfer. However, the exact relationship between criticality, awareness, and information integration remains unclear. To characterize this relationship, we applied multi-scale avalanche analysis to voltage-sensitive dye imaging data collected from animals of various species under different anesthetics. We found that anesthesia systematically varied the scaling behavior of neural dynamics, a change that was mirrored in reduced neural complexity. These findings were corroborated by applying the same analyses to a biophysically realistic cortical network model, in which multi-scale criticality measures were associated with network properties and the capacity for information integration. Our results imply that multi-scale criticality measures are potential biomarkers for assessing the level of consciousness.

Keywords: Ophthalmology

The development of functional imaging techniques applicable to neuroscience and covering a wide range of spatial and temporal scales has greatly facilitated the exploration of the relationships between cognition, behaviour and electrical brain activity. For mammals, the neocortex plays a particularly profound role in generating sensory perception, controlling voluntary movement, higher cognitive functions and planning goal-directed behaviours. Since these remarkable functions of the neocortex cannot be explored in simple model preparations or in anesthetised animals, the neural basis of behaviour must be explored in awake behaving subjects. Because neocortical function is highly distributed across many rapidly interacting regions, it is essential to measure spatiotemporal dynamics of cortical activity in real-time. Extensive work in anesthetised mammals has shown that in vivo Voltage-Sensitive Dye Imaging (VSDI) reveals the neocortical population membrane potential dynamics at millisecond temporal resolution and subcolumnar spatial resolution. Here, we describe recent advances indicating that VSDI is also already well-developed for exploring cortical function in behaving monkeys and mice. The first animal model, the non-human primate, is well-suited for fundamental exploration of higher-level cognitive function and behavior. The second animal model, the mouse, benefits from a rich arsenal of molecular and genetic technologies. In the monkey, imaging from the same patch of cortex, repeatedly, is feasible for a long period of time, up to a year. In the rodent, VSDI is applicable to freely moving and awake head-restrained mice. Interactions between different cortical areas and different cortical columns can therefore now be dynamically mapped through VSDI and related to the corresponding behaviour. Thus by applying VSDI to mice and monkeys one can begin to explore how behaviour emerges from neuronal activity in neuronal networks residing in different cortical areas.

Purpose/Aim of the study: To study changes in retinal blood flow velocity in patients with early and neovascular age-related macular degeneration (AMD). We used the Retinal Function Imager (RFI, Optical Imaging Ltd., Rehovot, Israel), a noninvasive diagnostic approach for measuring blood flow velocity. Materials and Methods: Sixty eyes of 43 AMD patients and 53 eyes of 35 healthy individuals over the age of 50 were recruited for this study. All patients were scanned by the RFI with analysis of blood flow velocity of secondary and tertiary branches of arteries and veins. Differences among groups were assessed by mixed linear models. Results: The average velocity in AMD patients was significantly lower compared to controls in arteries (3.6 ± 1.4 versus 4.3 ± 1.0 mm/sec, p = 0.009) but not in veins (2.6 ± 0.9 versus 3.1 ± 0.6 mm/sec, p = 0.08). When comparing the velocity between low-and high-grade AMD eyes, venous velocity was slower in the high grade AMD eyes only in the "narrow" group of vessels. Conclusions: Decreased blood flow velocity in retinal arteries in patients with AMD was found. Despite the fact that AMD is essentially a choroidal disease, retinal vessels show a functional abnormality, which may suggest that the vascular abnormality in this disease is more generalized.

Fundamental understanding of higher cognitive functions can greatly benefit from imaging of cortical activity with high spatiotemporal resolution in the behaving non-human primate. To achieve rapid imaging of high-resolution dynamics of cortical representations of spontaneous and evoked activity, we designed a novel data acquisition protocol for sensory stimulation by rapidly interleaving multiple stimuli in continuous sessions of optical imaging with voltage-sensitive dyes. We also tested a new algorithm for the "temporally structured component analysis" (TSCA) of a multidimensional time series that was developed for our new data acquisition protocol, but was tested only on simulated data (Blumenfeld, 2010). In addition to the raw data, the algorithm incorporates prior knowledge about the temporal structure of the data as well as input from other information. Here we showed that TSCA can successfully separate functional signal components from other signals referred to as noise. Imaging of responses to multiple visual stimuli, utilizing voltage-sensitive dyes, was performed on the visual cortex of awake monkeys. Multiple cortical representations, including orientation and ocular dominance maps as well as the hitherto elusive retinotopic representation of orientation stimuli, were extracted in only 10. s of imaging, approximately two orders of magnitude faster than accomplished by conventional methods. Since the approach is rather general, other imaging techniques may also benefit from the same stimulation protocol. This methodology can thus facilitate rapid optical imaging explorations in monkeys, rodents and other species with a versatility and speed that were not feasible before.

Purpose. To study the short-term effects of intravitreal bevacizumab (Avastin) on retinal blood flow velocity and compare them to clinical outcomes assessed by optical coherence tomography (OCT) and tests of visual acuity. Methods. The Retinal Function Imager (RFI) was used noninvasively and quantitatively to measure retinal blood flow velocity. Eight patients receiving intravitreal injection of Avastin for choroidal neovascularization (CNV) were included in this study. All were imaged by the RFI preinjection and 1 and 7 days postinjection. Visual acuity (VA) and OCT were recorded preinjection and 1 month postinjection. Comparisons were performed using paired Student t test and correlation using Spearman rank test. Results. A good correlation was found between the 1-month change in VA and OCT measurements and the short-term change induced in blood flow velocity. Arterial and venous velocity changes 1 day after the injection correlated with the VA change (p

Pyramidal cells in layers 2 and 3 of the neocortex of many species collectively form a clustered system of lateral axonal projections (the superficial patch system - Lund JS, Angelucci A, Bressloff PC. 2003. Anatomical substrates for functional columns in macaque monkey primary visual cortex. Cereb Cortex. 13:15-24. or daisy architecture - Douglas RJ, Martin KAC. 2004. Neuronal circuits of the neocortex. Annu Rev Neurosci. 27:419-451.), but the function performed by this general feature of the cortical architecture remains obscure. By comparing the spatial configuration of labeled patches with the configuration of responses to drifting grating stimuli, we found the spatial organizations both of the patch system and of the cortical response to be highly conserved between cat and monkey primary visual cortex. More importantly, the configuration of the superficial patch system is directly reflected in the arrangement of function across monkey primary visual cortex. Our results indicate a close relationship between the structure of the superficial patch system and cortical responses encoding a single value across the surface of visual cortex (self-consistent states). This relationship is consistent with the spontaneous emergence of orientation response-like activity patterns during ongoing cortical activity (Kenet T, Bibitchkov D, Tsodyks M, Grinvald A, Arieli A. 2003. Spontaneously emerging cortical representations of visual attributes. Nature. 425:954-956.). We conclude that the superficial patch system is the physical encoding of self-consistent cortical states, and that a set of concurrently labeled patches participate in a network of mutually consistent representations of cortical input. The Authors 2011. Published by Oxford University Press.2011This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

A neural field model is presented that captures the essential non-linear characteristics of activity dynamics across several millimeters of visual cortex in response to local flashed and moving stimuli. We account for physiological data obtained by voltage-sensitive dye (VSD) imaging which reports mesoscopic population activity at high spatio-temporal resolution. Stimulation included a single flashed square, a single flashed bar, the line-motion paradigm - for which psychophysical studies showed that flashing a square briefly before a bar produces sensation of illusory motion within the bar - and moving squares controls. We consider a two-layer neural field (NF) model describing an excitatory and an inhibitory layer of neurons as a coupled system of non-linear integro-differential equations. Under the assumption that the aggregated activity of both layers is reflected by VSD imaging, our phenomenological model quantitatively accounts for the observed spatiotemporal activity patterns. Moreover, the model generalizes to novel similar stimuli as it matches activity evoked by moving squares of different speeds. Our results indicate that feedback from higher brain areas is not required to produce motion patterns in the case of the illusory line-motion paradigm. Physiological interpretation of the model suggests that a considerable fraction of the VSD signal may be due to inhibitory activity, supporting the notion that balanced intra-layer cortical interactions between inhibitory and excitatory populations play a major role in shaping dynamic stimulus representations in the early visual cortex.

The Retinal Function Imager (RFI; Optical Imaging, Rehovot, Israel) is a unique, noninvasive multiparameter functional imaging instrument that directly measures hemodynamic parameters such as retinal blood-flow velocity, oximetric state, and metabolic responses to photic activation. In addition, it allows capillary perfusion mapping without any contrast agent. These parameters of retinal function are degraded by retinal abnormalities. This review delineates the development of these parameters and demonstrates their clinical applicability for noninvasive detection of retinal function in several modalities. The results suggest multiple clinical applications for early diagnosis of retinal diseases and possible critical guidance of their treatment.

Little is known about the "inverse" of the receptive field - the region of cortical space whose spatiotemporal pattern of electrical activity is influenced by a given sensory stimulus. We refer to this activated area as the cortical response field, the properties of which remain unexplored. Here, the dynamics of cortical response fields evoked in visual cortex by small, local drifting-oriented gratings were explored using voltage-sensitive dyes. We found that the cortical response field was often characterized by a plateau of activity, beyond the rim of which activity diminished quickly. Plateau rim location was largely independent of stimulus orientation. However, approximately 20 ms following plateau onset, 1-3 peaks emerged on it and were amplified for 25 ms. Spiking was limited to the peak zones, whose location strongly depended on stimulus orientation. Thus, alongside selective amplification of a spatially restricted suprathreshold response, wider activation to just below threshold encompasses all orientation domains within a well-defined retinotopic vicinity of the current stimulus, priming the cortex for processing of subsequent stimuli.

During the last few decades, neuroscientists have benefited from the emergence of many powerful functional imaging techniques that cover broad spatial and temporal scales. We can now image single molecules controlling cell differentiation, growth and death; single cells and their neurites processing electrical inputs and sending outputs; neuronal circuits performing neural computations in vitro; and the intact brain. At present, imaging based on voltage-sensitive dyes (VSDI) offers the highest spatial and temporal resolution for imaging neocortical functions in the living brain, and has paved the way for a new era in the functional imaging of cortical dynamics. It has facilitated the exploration of fundamental mechanisms that underlie neocortical development, function and plasticity at the fundmental level of the cortical column.

The rodent primary somatosensory cortex is spontaneously active in the form of locally synchronous membrane depolarizations (UP states) separated by quiescent hyperpolarized periods (DOWN states) both under anesthesia and during quiet wakefulness. In vivo whole-cell recordings and tetrode unit recordings were combined with voltage-sensitive dye imaging to analyze the relationship of the activity of individual pyramidal neurons in layer 2/3 to the ensemble spatiotemporal dynamics of the spontaneous depolarizations. These were either brief and localized to an area of a barrel column or occurred as propagating waves dependent on local glutamatergic synaptic transmission in layer 2/3. Spontaneous activity inhibited the sensory responses evoked by whisker deflection, accounting almost entirely for the large trial-to-trial variability of sensory-evoked postsynaptic potentials and action potentials. Subthreshold sensory synaptic responses evoked while a cortical area was spontaneously depolarized were smaller, briefer and spatially more confined. Surprisingly, whisker deflections evoked fewer action potentials during the spontaneous depolarizations despite neurons being closer to threshold. The ongoing spontaneous activity thus regulates the amplitude and the time-dependent spread of the sensory response in layer 2/3 barrel cortex.

A novel method of chronic optical imaging based on new voltage-sensitive dyes (VSDs) was developed to facilitate the explorations of the spatial and temporal patterns underlying higher cognitive functions in the neocortex of behaving monkeys. Using this system, we were able to explore cortical dynamics, with high spatial and temporal resolution, over period of ≤1 yr from the same patch of cortex. The visual cortices of trained macaques were stained one to three times a week, and immediately after each staining session, the monkey started to perform the behavioral task, while the primary and secondary visual areas (V1 and V2) were imaged with a fast optical imaging system. Long-term repeated VSD imaging (VSDI) from the same cortical area did not disrupt the normal cortical architecture as confirmed repeatedly by optical imaging based on intrinsic signals. The spatial patterns of functional maps obtained by VSDI were essentially identical to those obtained from the same patch of cortex by imaging based on intrinsic signals. On comparing the relative amplitudes of the evoked signals and differential map obtained using these two different imaging methodologies, we found that VSDI emphasizes subthreshold activity more than imaging based on intrinsic signals, that emphasized more spiking activity. The latency of the VSD-evoked response in V1 ranged from 46 to 68 ms in the different monkeys. The amplitude of the V2 response was only 20-60% of that in V1. As expected from the anatomy, the retinotopic responses to local visual stimuli spread laterally across the cortical surface at a spreading velocity of 0.15-0.19 m/s over a larger area than that expected by the classical magnification factor, reaching its maximal anisotropic spatial extent within ∼40 ms. We correlated the observed dynamics of cortical activation patterns with the monkey's saccadic eye movements and found that due to the slow offset of the cortical response relative to its onset, there was a short period of simultaneous activation of two distinct patches of cortex following a saccade to the visual stimulus. We also found that a saccade to a small stimulus was followed by direct transient activation of a cortical region in areas of V1 and V2, located retinotopically within the saccadic trajectory.

High-resolution images of the somatotopic hand representation in macaque monkey primary somatosensory cortex (area S-I) were obtained by optical imaging based on intrinsic signals. To visualize somatotopic maps, we imaged optical responses to mild tactile stimulation of each individual fingertip. The activation evoked by stimulation of a single finger was strongest in a narrow transverse band (∼1 × 4 mm) across the postcentral gyrus. As expected, a sequential organization of these bands was found. However, a significant overlap, especially for the activated areas of fingers 3-5, was found. Surprisingly, in addition to the finger-specific domains, we found that for each of the fingers, weak stimulation activated also a second "common patch" of cortex, located just medially to the representation of the finger. These results were confirmed by targeted multiunit and single-unit recordings guided by the optical maps. The maps remained very stable over many hours of recording. By optimizing the imaging procedures, we were able to obtain the functional maps extremely rapidly (e.g., the map of five fingers in the macaque monkey could be obtained in as little as 5 min). Furthermore, we describe the intraoperative optical imaging of the hand representation in the human brain during neurosurgery and then discuss the implications of the present results for the spatial resolution accomplishable by other neuroimaging techniques, relying on responses of the microcirculation to sensory-evoked electrical activity. This study demonstrates the feasibility of using high-resolution optical imaging to explore reliably short- and long-term plasticity of cortical representations, as well as for applications in the clinical setting.

Explorations of learning and memory, other long-term plastic changes, and additional cognitive functions in the behaving primate brain would greatly benefit from the ability to image the functional architecture within the same patch of cortex, at the columnar level, for a long period of time. We developed methods for long-term optical imaging based on intrinsic signals and repeatedly visualized the same functional domains in behaving macaque cortex for a period extending over 1 year. Using optical imaging and imaging spectroscopy, we first explored the relationship between electrical activity and hemodynamic events in the awake behaving primate and compared it with anesthetized preparations. We found that, whereas the amplitude of the intrinsic signal was much larger in the awake animal, its temporal pattern was similar to that observed in the anesthetized animals. In both groups, deoxyhemoglobin concentration reached a peak 2-3 sec after stimulus onset. Furthermore, the early activity-dependent increase in deoxyhemoglobin concentration (the 'initial dip') was far more tightly colocalized with electrical activity than the delayed increase in oxyhemoglobin concentration, known to be associated with an increase in blood flow. The implications of these results for improvement of the spatial resolution of blood oxygenation level-dependent functional magnetic resonance imaging are discussed. After the characterization of the intrinsic signal in the behaving primate, we used this new imaging method to explore the stability of cortical maps in the macaque primary visual cortex. Functional maps of orientation and ocular dominance columns were found to be stable for a period longer than 1 year.

The relation between the activity of a single neocortical neuron and the dynamics of the network in which it is embedded was explored by single-unit recordings and real-time optical imaging. The firing rate of a spontaneously active single neuron strongly depends on the instantaneous spatial pattern of ongoing population activity in a large cortical area. Very similar spatial patterns of population activity were observed both when the neuron fired spontaneously and when it was driven by its optimal stimulus. The evoked patterns could be used to reconstruct the spontaneous activity of single neurons.

Modern functional neuroimaging methods, such as positron-emission tomography (PET), optical imaging of intrinsic signals, and functional MRI (MRI) utilize activity-dependent hemodynamic changes to obtain indirect maps of the evoked electrical activity in the brain. Whereas PET and flow- sensitive MRI map cerebral blood flow (CBF) changes, optical imaging and blood oxygenation level-dependent MRI man areas with changes in the concentration of deoxygenated hemoglobin (HbR). However, the relationship between CBF and HbR during functional activation has never been tested experimentally. Therefore, we investigated this relationship by using imaging spectroscopy and laser-Doppler flowmetry techniques, simultaneously, in the visual cortex of anesthetized cats during sensory stimulation. We found that the earliest microcirculatory change was indeed an increase in HbR, whereas the CBF increase lagged by more than a second after the increase in HbR. The increased HbR was accompanied by a simultaneous increase in total hemoglobin concentration (Hbt), presumably reflecting an early blood volume increase. We found that the CBF changes lagged after Hbt changes by 1 to 2 sec throughout the response. These results support the notion of active neurovascular regulation of blood volume in the capillary bed and the existence of a delayed, passive process of capillary filling.

The goal of this study was to explore the functional organization of direction of motion in cat area 18. Optical imaging was used to record the activity of populations of neurons. We found a patchy distribution of cortical regions exhibiting preference for one direction over the opposite direction of motion. The degree of clustering according to preference of direction was two to four times smaller than that observed for orientation. In general, direction preference changed smoothly along the cortical surface; however, discontinuities in the direction maps were observed. These discontinuities formed lines that separated pairs of patches with preference for opposite directions. The functional maps for direction and for orientation preference were closely related; typically, an iso-orientation patch was divided into regions that exhibited preference for opposite directions, orthogonal to the orientation. In addition, the lines of discontinuity within the direction map often connected points of singularity in the orientation map. Although the organization of both domains was related, the direction and the orientation selective responses were separable; whereas the selective response according to direction of motion was nearly independent of the length of bars used for visual stimulation, the selective response to orientation decreased significantly with decreasing length of the bars. Extensive single and multiunit electrical recordings, targeted to selected domains of the functional maps, confirmed the features revealed by optical imaging. We conclude that significant processing of direction of motion is performed early in the cat visual pathway.

Processing of retinal images is carried out in the myriad dendritic arborizations of cortical neurons. Such processing involves complex dendritic integration of numerous inputs, and the subsequent output is transmitted to multiple targets by extensive axonal arbors. Thus far, details of this intricate processing remained unexaminable. This report describes the usefulness of real-time optical imaging in the study of population activity and the exploration of cortical dendritic processing. In contrast to single- unit recordings, optical signals primarily measure the changes in transmembrane potential of a population of neuronal elements, including the often elusive subthreshold synaptic potentials that impinge on the extensive arborization of cortical cells. By using small visual stimuli with sharp borders and real-time imaging of cortical responses, we found that shortly after its onset, cortical activity spreads from its retinotopic site of initiation, covering an area at least 10 times larger, in upper cortical layers. The activity spreads at velocities from 100 to 250 μm/msec. Near the V1/V2 border the direct activation is anisotropic and we detected also anisotropic spread; the 'space constant' for the spread was ~2.7 mm parallel to the border and ~1.5 mm along the perpendicular axis. In addition, we found cortical interactions between cortical activities evoked by a small 'center stimulus' and by large 'surround stimuli' positioned outside the classical receptive field. All of the surround stimuli used suppressed the cortical response to the center stimulus. Under some stimulus conditions iso- orientation suppression was more pronounced than orthogonal-orientation suppression. The orientation dependence of the suppression and its dependency on the size of some specific stimuli indicate that at least part of the center surround inhibitory interaction was of cortical origin. The findings reported here raise the possibility that distributed processing over a very large cortical area plays a major role in the processing of visual information by the primary visual cortex of the primate.

In primate primary visual cortex, neurons sharing similar response properties are clustered together forming functional domains that appear as a mosaic of patches or bands, often traversing the entire cortical depth from the pia to the white matter. Similarly, each cortical site connects laterally through an extensive network of intrinsic projections that are organized in multiple clusters (patches) and reach distances of up to a few millimeters. The relationship between the functional domains and these laterally connected patches has remained a controversial issue despite intensive research efforts. To investigate this relationship, we obtained high-resolution functional maps of the cortical architecture by in vivo optical imaging. Subsequently, extracellular injections of the sensitive anterograde tracer biocytin were targeted into selected functional domains. Within the ocular dominance system, we found that long-range intrinsic connections tended to link the monocular regions of same-eye ocular dominance columns. Furthermore, we discovered that binocular domains formed a separate set of connections in area V1; binocular regions were selectively connected among themselves but were not connected to strictly monocular regions, suggesting that they constitute a distinct columnar system. In the other subsystem subserving orientation preference, patches of intrinsic connections tended to link domains sharing similar orientation preferences. Analyses of the precision of these connections indicated that in both functional subsystems,

Long standing questions related to brain mechanisms underlying perception can finally be resolved by direct visualization of the architecture and function of mammalian cortex. This advance has been accomplished with the aid of two optical imaging techniques with which one can literally see how the brain functions. The upbringing of this technology required a multi-disciplinary approach integrating brain research with organic chemistry, spectroscopy, biophysics, computer sciences, optics and image processing. Beyond the technological ramifications, recent research shed new light on cortical mechanisms underlying sensory perception. Clinical applications of this technology for precise mapping of the cortical surface of patients during neurosurgery have begun. Below is a brief summary of our own research and a description of the technical specifications of the two optical imaging techniques. Like every technique, optical imaging also suffers from severe limitations. Here we mostly emphasize some of its advantages relative to all alternative imaging techniques currently in use. The limitations are critically discussed in our recent reviews. For a series of other reviews, see Cohen (1989).

The design of a macroscope constructed with photography lenses is described and several applications are demonstrated. The macroscope incorporates epi-illumination, a 0.4 numerical aperture, and a 40 mm working distance for imaging wide fields in the range of 1.5-20 mm in diameter. At magnifications of 1 × to 2.5 ×, fluorescence images acquired with the macroscope were 100-700 times brighter than those obtained with commercial microscope objectives at similar magnifications. In several biological applications, the improved light collection efficiency (20-fold, typical) not only minimized bleaching effects, but, in concert with improved illumination throughput (15-fold, typical), significantly enhanced object visibility as well. Reduced phototoxicity and increased signal-to-noise ratios were observed in the in vivo real-time optical imaging of cortical activity using voltage-sensitive dyes. Furthermore, the macroscope has a depth of field which is 5-10 times thinner than that of a conventional low-power microscope. This shallow depth of field has facilitated the imaging of cortical architecture based on activity-dependent intrinsic cortical signals in the living primate brain. In these reflection measurements large artifacts from the surface blood vessels, which were observed with conventional lenses, were eliminated with the macroscope.

We have shown previously the existence of small, activity-dependent changes in intrinsic optical properties of cortex that are useful for optical imaging of cortical functional architecture. In this study we introduce a higher resolution optical imaging system that offers spatial and temporal resolution exceeding that achieved by most alternative imaging techniques for imaging cortical functional architecture or for monitoring local changes in cerebral blood volume or oxygen saturation. In addition, we investigated the mechanisms responsible for the activity-dependent intrinsic signals evoked by sensory stimuli, and studied their origins and wavelength dependence. These studies enabled high-resolution visualization of cortical functional architecture at wavelengths ranging from 480 to 940 nm. With the use of near-infrared illumination it was possible to image cortical functional architecture through the intact dura or even through a thinned skull. In addition, the same imaging technique proved useful for imaging and discriminating sensory-evoked, activity-dependent changes in local blood volume and oxygen saturation (oxygen delivery). Illumination at 570 nm allowed imaging of activity-dependent blood volume increases, whereas at 600-630 nm, the predominant signal probably originated from activity-dependent oxygen delivery from capillaries. The onset of oxygen delivery started prior to the blood volume increase. Thus, optical imaging based on intrinsic signals is a minimally invasive procedure for monitoring short- and long-term changes in cerebral activity.

The interactions between myelinated axons and surrounding glia cells, in rat optic nerve, were investigated by optical recording with voltage-sensitive dyes. Electrical stimulation of the nerve evoked an optical signal revealing two clearly distinct components: a fast propagating compartment, corresponding to the compound action potential, and a prominent slow component. Several lines of evidence suggest that part of the slow component originated from depolarization of the oligodendrocytes by potassium accumulation in the paranodal or internodal region. In addition, the experiments suggest that in this preparation axons also have voltage-dependent Ca²⁺ channels, and a Ca²⁺-dependent K⁺ conductance involved in the depolarization of oligodendrocytes. Thus, axons and oligodendrocytes communicate in an intimate, ionically-mediated fashion, and oligodendrocytes may play an important functional role beyond that of providing the myelin sheath.

We have investigated the use of optical methods for monitoring neuron activity in mammalian cortex. The cortex was stained with a voltage-sensitive dye and fluorescence was simultaneously measured from 124 areas using a photodiode array. Optical signals were detected in rat somatosensory cortex in response to small whisker movements and in visual cortex in response to light flashes to the eye. Relatively large signals were obtained during focal interictal epileptiform discharges induced by bicuculline. The measuring system had a time resolution of milliseconds and a spatial resolution of a few hundred micrometers. Simultaneous, multi-site optical recordings of activity may provide a new and potentially powerful method for studying function and dysfunction in mammalian cortex.

The availability of suitable voltage-sensitive dyes and arrays of photodetectors has facilitated the optical monitoring of electrical activity simultaneously from hundreds of sites on the processes of single nerve cells, both in culture and in invertebrate ganglia. This method also provides a unique ability to detect activity in many individual neurons in an entire invertebrate ganglion controlling particular behavioral responses. The in-vitro activity of individual populations of neuronal elements (cell bodies, axons, dentrites or nerve terminals) at many neighboring loci in mammalian brain slices or isolated brain structures has been investigated. Recently dynamic patterns of electrical activity evoked in the intact vertebrate or mammalian brain by natural stimuli have also been monitored. By employing computerized optical recording and a display processor, video-displayed images of neuronal elements can be superimposed on the corresponding patterns of the optically detected electrical activity, thus allowing the spatio-temporal patterns of intracellular activity to be visualized in slow motion.

An optical method involving the use of a laser and a novel fluorescent dye as a photostimulation probe has been developed to identify presynaptic neurons in a large ensemble of cells. Illumination of an extracellularly stained neuron by the laser microbeam evokes action potentials. With this technique an interneuron connecting identified leech neurons was quickly located. The method speeds up the elucidation of neuronal networks, especially when small cells are involved.

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.

Publisher Summary Optical signals related to membrane potential have been obtained with several hundred different dyes and thus, one might not expect a single mechanism to be responsible for all of the signals. The dye signals can be divided into two types with basically different mechanisms. It is convenient that dyes that work through the two types of mechanism have in turn been used in separate kinds of applications. In one situation, the optical signals appear to arise from dye that is membrane bound. These optical signals follow changes in membrane potential with time constants of less than 0.01 ms and are, thus, suited for optical monitoring of action potentials and other rapid potential changes that occur in excitable cells. This kind of signal has been termed as fast signal. Optical signals from a different group of dyes originate from the re-equilibration of charged, permeant dye molecules across the cell membrane when the membrane potential changes.

Recent improvements in the performance of decay fluorimeters permit the acquisition of high quality decay data; the random noise level can be reduced to a desired level, and it is primarily the inevitable systematic deviations that set up a limit for the accuracy of the analysis. A systematic general approach characterizing the quality of such data, making use of fluorescence standards is suggested: Fluorophores which obey a monoexponential decay law are proposed as fluorescence decay-standards to check the quality of data obtained in real experiments. Multicomponent decay curves serve to indicate whether reliable analysis of complex decay functions can be obtained. Criteria for the quality of the fit for such experimental data are discussed, and two different methods of analysis are tested and compared. The method of weighted nonlinear least squares yielded excellent results, and its advantages and limitations are discussed. Provisions for the evaluation of a time shift and a scattered light component are presented, and a modified general procedure for error analysis of real decay data is suggested.

The fluorescence decay of chicken pepsinogen is not monoexponential throughout the emission spectrum. For light emitted at the long wavelength region of the fluorescence spectrum, the decay can be described by two exponential terms, one of them exhibiting a negative amplitude. This behavior shows that in this region of the spectrum the fluorescence builds up before it decays, indicating that the electronically excited species involved has been created during the fluorescence lifetime. For a variety of reasons it seems very unlikely that this emitting species was formed by energy transfer from other entities in the protein. The buildup of the fluorescence at the red edge of the spectrum prior to emission thus reflects a genuine relaxation process in the protein molecule in the nanosecond time scale, the excited chromophore shifting its emission spectrum to the red in the course of the relaxation process. The reaction involved in the relaxation may be a nonspecific orientation of various groups around the excited chromophore or a formation of a more specific excited state complex, an exciplex. This nanosecond relaxation process is conformation dependent and disappears upon denaturation. Similarly, chicken pepsin at neutral pH fails to show it. This seems to be the first case in which a relaxation process in the nanosecond time range has been demonstrated with a native chromophore in a protein and complements the observations of Brand and Gohlke and of Lakowicz and Weber of other nanosecond relaxation processes in proteins. Fast relaxation reactions demonstrated in the present study for tryptophans are not necessarily limited to indole side chains, except that these residues permit the study of the relaxation processes by perturbation of interactions with the environment through electronic excitation.