Living Brain Slices Research Articles

Understanding of cerebral energy metabolism by dynamic living brain slice imaging system with [ 18F]FDG

Recently, lactate has been receiving great attention as an energy substrate in the brain. In this study, the role of lactate was evaluated by “bioradiography” system with 2-deoxy-2-[ 18F]fluoro- d-glucose ([ 18F]FDG), which is a positron emitting radiotracer for glucose uptake quantification. “Bioradiography” is the dynamic living tissue slice imaging system for positron-emitter labeled compounds. We investigated the brain energy metabolism under resting state and neural activated conditions induced by KCl addition. The monocarboxylate transporter inhibitor, α-cyano-4-hydroxycinnamate (4-CIN), had no effect on [ 18F]FDG uptake rate in rat brain slices before KCl addition. On the other hand, addition of 4-CIN induced larger [ 18F]FDG uptake rates under the activated condition in comparison with the control condition. Because neurons cannot utilize lactate under the 4-CIN loaded conditions, this indicates that activated neurons consume lactate as an energy substrate. The lactate concentration in the incubation medium was increased with KCl treatment in both groups and the extent was slightly greater in 4-CIN group. These results suggested that: (1) the brain mainly uses glucose, not lactate, as an energy substrate in resting state; (2) when neuron is stimulated, excess amounts of lactate might be produced in astrocytes and the lactate is mobilized as an energy substrate.

Demonstration of hyperaccumulation of [18F]2‐fluoro‐2‐deoxy‐D‐glucose under oxygen deprivation in living brain slices using bioradiography

To clarify the mechanism of hyperaccumulation of glucose in acute brain ischemia by PET, changes of glucose metabolism and mitochondrial electron transfer function were examined in living brain slices in vitro during control, hypoxic, and anoxic conditions by positron autoradiography using [(18)F]2-fluoro-2-deoxy-D-glucose ([(18)F]FDG) and [(15)O]oxygen. [(15)O]Oxygen fixation reflecting mitochondrial electron transfer function was reduced and [(18)F]FDG uptake reflecting glucose metabolism was increased in proportion to the strength of oxygen deprivation during anoxia and hypoxia. Mitochondrial electron transfer function decreased with no regional differences, whereas the glucose metabolism was the most enhanced in the hippocampus and thalamus. The enhanced glucose metabolism was associated with an increased glutamate efflux after hypoxia and anoxia. Glucose metabolism was also increased by the addition of glutamate and was attenuated by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 in the hippocampus and thalamus. The hyperaccumulation of glucose in acute brain ischemia was demonstrated in living brain slices using bioradiography with reduced mitochondrial electron transfer. The activation of NMDA receptors by glutamate during acute brain ischemia might be responsible for hyperutilization of glucose in the hippocampus and thalamus.

Dendritic morphology, local circuitry, and intrinsic electrophysiology of neurons in the rat medial and lateral habenular nuclei of the epithalamus

The habenular complex of the epithalamus in the mammalian brain receives input from the limbic forebrain and pallidum and, in turn, projects to numerous midbrain structures. Traditionally, the habenular complex is divided into the medial nucleus and two divisions of the lateral nucleus. Based on their distinct input and output pathways, the habenula is considered to constitute three, partially overlapping channels that regulate information flow from the limbic forebrain and pallidum to the midbrain. As a step to improve our understanding of how information delivered from the limbic forebrain and pallidum is processed in the habenula, we examined the electrical property and morphology of medial and lateral habenular cells. For this study, we generated live brain slices from rat habenula and performed whole cell recording. During recording, we filled habenular cells with biocytin. Medial habenular cells generate tonic trains of action potentials, whereas lateral habenular cells are capable of producing action potentials in burst mode. Lateral habenular cells produce dendrites that are much longer than those of medial habenular cells. Two distinct intrinsic circuits exist in the medial habenular nucleus, whereas in the lateral habenular nucleus, intrinsic axons travel largely from medial to lateral direction. The connection between the two habenular nuclei is asymmetrical in that only the medial habenula sends projection to the lateral habenula. The differences in the electrical and morphological properties of medial and lateral habenular cells indicate that the two nuclei process and integrate information in distinct fashions that is delivered from the limbic forebrain and pallidum.

Propagation of γPKC translocation along the dendrites of Purkinje cell in γPKC‐GFP transgenic mice

To elucidate spatial and temporal profiles of the protein kinase C (PKC) activation in relation to neuronal functions including synaptic plasticity, we tried to detect PKC translocation in living brain slices. We first developed brain region-specific and inducible gammaPKC-GFP transgenic mice using a tetracycline (tet)-regulated system. In the transgenic mice, the expression of gammaPKC-GFP was region-specifically regulated by the promoter and abolished by the administration of doxycycline. Cerebellar slices from the mice were utilized for intracellular recording and fluorescence imaging of gammaPKC-GFP in Purkinje cells. GFP fluorescence was uniformly distributed from soma to dendritic arbor. When mGluR agonists were applied, the intensity was transiently increased at the edge of the dendrite and concomitantly decreased in the cytoplasm, indicating that gammaPKC translocated to the plasma membrane. This transient change in the pattern of GFP fluorescence simultaneously occurred throughout the Purkinje cell dendrites by agonist stimulation. Translocation of gammaPKC-GFP was also induced by electrical stimulation of parallel fibres. However, the event was not restricted at the distal dendrites, propagated forwardly along the dendritic tree and reached to the proximal trunk close to the soma. Time course of the propagation was slower than the electrical signal and Ca(2+) waves and faster than conveying molecules through microtubules. The present results indicate that PKC signals activated locally by parallel fibre input could propagate to the soma through dendrites in living Purkinje neurones. The findings may provide us with a new insight for understanding molecular mechanisms of the synaptic plasticity including cerebellar long-term depression.

Light-induced reduction of cytoplasmic free calcium in neurons proposed to be encephalic photoreceptors in chick brain

A population of cerebrospinal fluid-contacting neurons (CSFcn) in the lateral septal organ (LSO) may serve as encephalic photoreceptors (EPRs) functioning to signal the onset of seasonal reproductive development in birds. Previous studies on CSFcn in the LSO have focused on identification of retinal protein components in fixed brain tissue. In order to understand better the mechanisms underlying the light-induced photosexual response in birds, a physiological characterization is required. In this study, changes of intracellular free calcium concentration ([Ca 2+] i) were monitored during light stimulation of CSFcn in the LSO in live brain slices from embryonic chicks. Using the fluorescent calcium indicator fluo-4, a reduced [Ca 2+] i within CSFcn was recorded in response to photostimulation, which is consistent with what has been demonstrated in rods and cones following illumination. Results support the hypothesis that CSFcn in the LSO function as EPRs in the avian brain.

An ex vivo method for evaluating the biocompatibility of neural electrodes in rat brain slice cultures

Failure of neural recording electrodes implanted in the brain is often attributed to the formation of glial scars around the implant. A leading cause of scar formation is the electrode material. Described below is an approach to evaluate the biocompatibility of novel electrode materials in a representative three-dimensional model. The model, brain slice culture, accounts for the response of the neural tissue in the absence of the systemic response. While limitations of any in vitro model exist, brain slice culture provides an indication of the response of neurons and glia in an environment more indicative of the in vivo environment than two-dimensional cell culture of glia or neurons alone. Polybenzylcyclobutene (BCB) electrodes were developed as test materials for flexible electrodes due to ease of processing, low water uptake, and inherent flexibility when formed in thin sheets. Biocompatibilty of the BCB neural electrodes was evaluated using living brain slices derived from the hippocampal regions of 100 g CD rats. Importantly, fewer animals can be used in brain slice culture to evaluate the neural tissue response than when using live animals, since several slices can be obtained per animal. Cellular response to the electrodes was evaluated at 0, 7, and 14 days. At all time points living cells, both neurons and glia, were observed in the vicinity of the electrode. In addition, cells were observed migrating out from the brain slices onto the shank of the BCB electrode. Brain slice culture is shown to be a viable alternative to in vivo evaluation, in that the response of both neurons and glia can be evaluated in a native three-dimensional state, while sacrificing fewer animals. Future in vivo evaluation with BCB will provide definitive answers on the degree of glial scarring in response to this new and biocompatible electrode material.

Hypocretin/orexin innervation and excitation of identified septohippocampal cholinergic neurons.

Hypothalamic fibers containing the wake-promoting peptides, hypocretins (Hcrts) or orexins, provide a dense innervation to the medial septum-diagonal band of Broca (MSDB), a sleep-associated brain region that has been suggested to show intense axonal degeneration in canine narcoleptics. The MSDB, via its cholinergic and GABAergic projections to the hippocampus, controls the hippocampal theta rhythm and associated learning and memory functions. Neurons of the MSDB express very high levels of the Hcrt receptor 2, which is mutated in canine narcoleptics. In the present study, we investigated the electrophysiological effects of Hcrt peptides on septohippocampal cholinergic neurons that were identified in living brain slices of the MSDB using a selective fluorescent marker. Hcrt activation of septohippocampal cholinergic neurons was reversible, reproducible, and concentration dependent and mediated via a direct postsynaptic mechanism. Both Hcrt1 and Hcrt2 activated septohippocampal cholinergic neurons with similar EC(50) values. The Hcrt effect was dependent on external Na(+), reduced by external Ba(2+), and also reduced in recordings with CsCl-containing electrodes, suggesting a dual underlying ionic mechanism that involved inhibition of a K(+) current, presumably an inward rectifier, and a Na(+)-dependent component. The Na(+) component was dependent on internal Ca(2+), blocked by replacing external Na(+) with Li(+), and also blocked by bath-applied Ni(2+) and KB-R7943, suggesting involvement of the Na(+)-Ca(2+) exchanger. Using double-immunolabeling studies at light and ultrastructural levels, we also provide definitive evidence for a hypocretin innervation of cholinergic neurons. Thus Hcrt effects within the septum should increase hippocampal acetylcholine release and thereby promote hippocampal arousal.

Shining light on neurons--elucidation of neuronal functions by photostimulation.

Many neuronal functions can be elucidated by techniques that allow for a precise stimulation of defined regions of a neuron and its afferents. Photolytic release of neurotransmitters from 'caged' derivates in the vicinity of visualized neurons in living brain slices meets this request. This technique allows the study of the subcellular distribution and properties of functional native neurotransmitter receptors. These are prerequisites for a detailed analysis of the expression and spatial specificity of synaptic plasticity. Photostimulation can further be used to fast map the synaptic connectivity between nearby and, more importantly, distant cells in a neuronal network. Here we give a personal review of some of the technical aspects of photostimulation and recent findings, which illustrate the advantages of this technique.

Neurophysiological effects of corticotropin-releasing factor in living slices of the olfactory area of the rat cortex.

Application of corticotropin-releasing factor (CRF) at concentrations of 10(-9) and 10(-8) M to living brain slices induced activation of the pre- and postsynaptic excitatory components of focal potentials recorded in the slices. The amplitudes and durations of the AMPA and NMDA components of EPSP increased during exposure to CRF, while the amplitude of the GABA(B)-mediated IPSP was suppressed. At the higher CRF concentration (10(-8) M), cells in slices showed epileptiform discharges. The effects of CRF were reversible and disappeared on washing. Long-term treatment with CRF (90 min) induced changes in cells in slices similar to those associated with long-term post-tetanic potentiation. These data provide evidence that CRF has marked activatory properties and influences the glutamatergic and GABAergic systems.

Use of YFP to study amyloid-β associated neurite alterations in live brain slices

Neuritic plaques are one of the defining neuropathological features of Alzheimer’s disease (AD). These structures are composed of a buildup of fibrils of the amyloid-β (Aβ) peptide (amyloid) surrounded by activated glial cells and degenerating nerve processes (dystrophic neurites). To study neuritic plaques and possible abnormalities associated with dendrites, axons, and synaptic structures, we have developed an acute slice preparation model using PDAPP, yellow fluorescent protein (YFP) double transgenic mice (a mouse model with AD-like pathology that stably expresses YFP in a subset of neurons in the brain). With laser scanning confocal microscopy, we have imaged living brain slices from PDAPP, YFP double transgenic mice as old as 20 months and have been able to visualize axons, dendrites, dendritic spines, and dystrophic neurites for many hours. Our initial studies suggest that dystrophic axons and dendrites within neuritic plaques are fairly stable structures in the absence of exogenous perturbations. This acute slice preparation model should prove to be a useful tool to explore the pathophysiology of Aβ-related axonal, dendritic, and synaptic dysfunction.

Multiple Cell Populations in the Early Postnatal Subventricular Zone Take Distinct Migratory Pathways: A Dynamic Study of Glial and Neuronal Progenitor Migration

Neural progenitors in the subventricular zone (SVZ) of the postnatal rat forebrain give rise to either olfactory interneurons or glia. To investigate the overall patterns of progenitor movement, we labeled neonatal rat SVZ cells by stereotactic injection of a GFP-encoding retrovirus into the SVZ at various coronal levels. We then studied the movements of labeled cells by time-lapse videomicroscopy in living brain slices cut in different orientations. We observed two migration patterns: (1) progenitors migrated radially into the overlying white matter and cortex, but only at the level of viral injection; these were previously shown to give rise to astrocytes and oligodendrocytes, (2) progenitors migrated in a bidirectional, rostrocaudal pattern along the entire extent of the SVZ; many of these cells eventually migrated into the olfactory bulb and developed into interneurons, but they did not turn to migrate radially out of the SVZ until they reached the olfactory bulb. Video imaging showed apparent boundaries to migration between the SVZ and adjacent structures. These observations indicate that there are at least two distinct migratory pathways within the SVZ used differentially by immature neurons and glia.

Dynamic confocal imaging in acute brain slices and organotypic slice cultures using a spectral confocal microscope with single photon excitation.

Confocal imaging in living brain slices allows the resolution of submicrometre structures of nerve cells, glia and brain vessels. Imaging living brain slices is in many respects different from conventional fixed histological preparations for which confocal microscopes were designed originally. Several problems (i.e. mechanical and thermal drift, and autofluorescence) resulting from the optical and structural properties of brain slices are discussed. Fluorescent indicators may be used to monitor numerous intracellular parameters such as pH and Ca(2+) concentration, but not all of them are equally suitable for this type of work. Genetically engineered fluorescent proteins can be used to visualise the fine dendritic structure of neurones or track particular intracellular structures and proteins. They have also been used to generate indicators for Ca(2+), cAMP and other molecules. While conventional chemical indicators can be either loaded into neurones via patch pipettes or as membrane-permeable esters, protein indicators can be expressed in various types of cells using adenoviral vectors. Adenoviral transgenesis can be performed in vitro in both acute slices and organotypic slice cultures. Organotypic slice cultures give excellent optical access to neurones loaded with either conventional fluorescent indicators or transfected with adenovirus to express fluorescent proteins. They are most suitable for experiments where both conventional and genetically engineered indicators are combined. Single photon imaging in brain slices is limited to the superficial layers (approximately<or=50 microm), while multiphoton excitation has a much greater depth of penetration. However, the overall optical resolution achievable in single photon mode is at least as good as when using multiphoton excitation.

Enhanced cytomegalovirus infection of developing brain independent of the adaptive immune system.

Cytomegalovirus (CMV) has been suggested as the most prevalent infectious agent causing neurological dysfunction in the developing brain; in contrast, CMV infections are rare in the adult brain. One explanation generally given for the developmental susceptibility to the virus is that the developing immune system is too immature to protect the central nervous system from viral infection, but as the immune system develops it can protect the brain. We suggest an alternate view: that developing brain cells are inherently more susceptible to CMV infection, independent of the immune system. We used a recombinant mouse CMV that leads to green fluorescent protein expression in infected cells. Control experiments demonstrated a high correlation between the number of cells detected with the viral GFP reporter gene and with immunocytochemical detection of the virus. After intracerebral inoculation, the number of CMV-infected cells in neonatal brains was many times greater than in mature control or mature immunodepressed SCID mice, and the mortality rate of neonates was substantially greater than SCID or control adults. Parallel experiments with live brain slices inoculated in vitro, done in the absence of the systemic immune system, generated similar data, with immature hippocampus, hypothalamus, cortex, striatum, and cerebellum showing substantially greater numbers of infected cells (100-fold) than found in adult slices in these same regions. Interestingly, in the cerebellar cortex, CMV-infected cells were more prevalent in the postmitotic Purkinje cell layer than in the mitotic granule cell layer, suggesting a selective infection of some cell types not dependent on cell division. Together, these data support the view that CMV has an intrinsic preference for infection of developing brain cells, independent, but not mutually exclusive, of the developmental status of the systemic immune system in controlling CMV infection.

Functionally Independent Columns of Rat Somatosensory Barrel Cortex Revealed with Voltage-Sensitive Dye Imaging

Whisker movement is somatotopically represented in rodent neocortex by electrical activity in clearly defined barrels, which can be visualized in living brain slices. The functional architecture of this part of the cortex can thus be mapped in vitro with respect to its physiological input and compared with its anatomical architecture. The spatial extent of excitation was measured at high temporal resolution by imaging optical signals from voltage-sensitive dye evoked by stimulation of individual barrels in layer 4. The optical signals correlated closely with subthreshold EPSPs recorded simultaneously from excitatory neurons in layer 4 and layer 2/3, respectively. Excitation was initially (<2 msec) limited to the stimulated barrel and subsequently (>3 msec) spread in a columnar manner into layer 2/3 and then subsided in both layers after approximately 50 msec. The lateral extent of the response was limited to the cortical column defined structurally by the barrel in layer 4. Two experimental interventions increased the spread of excitation. First, blocking GABA(A) receptor-mediated synaptic inhibition caused excitation to spread laterally throughout wide regions of layer 2/3 and layer 5 but not into neighboring barrels, suggesting that the local excitatory connections within layer 4 are restricted to single barrels and that inhibitory neurons control spread in supragranular and infragranular layers. Second, NMDA receptor-dependent increase of the spread of excitation was induced by pairing repetitive stimulation of a barrel column with coincident stimulation of layer 2/3 in a neighboring column. Such plasticity in the spatial extent of excitation in a barrel column could underlie changes in cortical map structure induced by alterations of sensory experience.

Imaging synaptic zinc release in living nervous tissue

Zinc enriched neurons have a pool of synaptic vesicles which contain free or loosely-bound zinc ions. The movement of the vesicular zinc ions into the synaptic clefts has been previously studied by microdialysis, fluorescence postmortem staining for zinc and radioactive zinc isotope. In this study the zinc fluorescence probe N-6-metoxy- p-toluensulfonamide quinoline (TSQ) has been applied as a tracer of synaptic release of zinc ions. This fluorochrome permeates cell membranes and when exposed to living brain slices gives rise to a staining pattern similar to that seen with autometallography. In the living brain slices, fluorescence emission persists after exposure to calcium saturated ethylen diamino-tetra-acetic acid (Ca-EDTA) because this chelator does not penetrate cell membranes, while sodium dethyldithiocarbamate (DEDTC), that does penetrate membranes, partially suppressed the fluorescence emission. Stimulation of slices bathed in the non-permeant chelator Ca-EDTA with 50 mM potassium chloride leads to a rapid and complete disappearance of fluorescence. In the absence of Ca-EDTA, however, potassium stimulation induces a sudden transitory increase in fluorescence. This increase is caused by a translocation of the fluorochrome (TSQ) zinc molecules from the weakly acid interior of the synaptic vesicles to the neutral extracellular space, whereby the fluorescence emission of the molecules is enhanced sufficiently to be recorded by a high sensitivity TV camera.

A Role of Netrin-1 in the Formation of the Subcortical Structure Striatum: Repulsive Action on the Migration of Late-Born Striatal Neurons

The mammalian striatum arises in the basal telencephalon and contains morphologically homogenous neurons that can be divided into two distinct compartments, patches and the matrix. During development, patch neurons are generated first to form a striatal primordium. After a large influx of later-born matrix neurons into this region, the unique mosaic arrangement of these two neuronal phenotypes is established. The massive migration of matrix neurons continues during the embryonic period, and they eventually comprise 80-85% of the mature striatum. To elucidate the cellular mechanism or mechanisms underlying this critical event in striatal histogenesis, we examined the migration characteristics of striatal subventricular zone (SVZ) cells at embryonic day 18 when neurogenesis peaks for matrix neurons. Using gel cultures, we show that netrin-1, one of the diffusible guidance cues expressed in the striatal ventricular zone (VZ), exerts a repulsive action on migrating SVZ cells. This effect is blocked in the presence of antibodies against Deleted in colorectal cancer (DCC), a putative receptor for netrin-1. The expression patterns of netrin-1 and DCC strongly suggest the involvement of this effect in the outward migration of SVZ cells into the striatal postmitotic region. Our cell tracing study using living brain slices demonstrates that striatal SVZ cells migrate toward and disperse throughout the striatum, in which they differentiate into phenotypes of striatal projection neurons. We suggest that netrin-1 expressed in the striatal VZ serves to guide the large influx of striatal matrix neurons into the striatal primordium and is thereby involved in the initial formation of fundamental striatal structures.

Nuclear shrinkage in live mouse hippocampal slices.

Brain slices are used extensively for biochemical, electrophysiological and molecular investigations. However, only the time frame for electrophysiological and biochemical investigations has as yet been defined. The goal of the present study was to investigate the time course of nuclear structure in live brain slices. Hippocampal slices (300 microm) were prepared from male CD1 mice (25-30 g), stained with Hoechst 33342 (10 microM), calcein-AM (2 microM) and ethidium homodimer (4 microM), and imaged with single- and dual-photon microscopy. The volume of CA1 pyramidal cell nuclei decreased from 759+/-229 microm3 in 40-50 microm depth 25 min after preparation to 453+/-169 microm3 (P<0.001) after 60 min, 315+/-112 microm3 (P<0.001) after 120 min and 128+/-71 microm3 (P<0.001) after 8 h. Similar results were obtained on a prolonged time scale in 70-80 microm depth and with an accelerated time scale in 20-30 microm depth. Live-dead staining showed that cell damage is progressing from the surface to deeper layers of the slices in a time-dependent fashion. We conclude that nuclei of CA1 hippocampal pyramidal cells show a time- and depth-dependent shrinkage converging 8 h after slice preparation to a volume of 90-130 microm; in any depth between 20 and 80 microm. The nucleus in the superficial 80 microm of each side appears dysfunctional even at times suitable for electrophysiological and biochemical experimentation in hippocampal slices. Molecular analysis of cell regulation in brain slices may, therefore, be time-dependently distorted by progressing cell death in at least half of the tissue under investigation.

Dynamics of microglial activation: A confocal time-lapse analysis in hippocampal slices

The dynamics of microglial cell activation was studied in freshly prepared rat brain tissue slices. Microglia became activated in the tissue slices, as evidenced by their conversion from a ramified to amoeboid form within several hours in vitro. To define better the cytoarchitectural dynamics underlying microglial activation, we performed direct three-dimensional time-lapse confocal imaging of microglial cells in live brain slices. Microglia in tissue slices were stained with a fluorescent lectin conjugate, FITC-IB(4), and stacks of confocal optical sections through the tissue were collected repeatedly at intervals of 2-5 min for several hours at a time. Morphometric analysis of cells from time-lapse sequences revealed that ramified microglia progress to amoeboid macrophages through a stereotypical sequence of steps. First, in the withdrawal stage, the existing ramified branches of activating microglia do not actively extend or engulf other cells, but instead retract back (mean rate, 0.5-1.5 microm/min) and are completely resorbed into the cell body. Second, in the motility stage, a new set of dynamic protrusions, which can exhibit cycles of rapid extension and retraction (both up to 4 microm/min), abruptly emerges. Sometimes new processes begin to emerge even before the old branches are completely withdrawn. Third, in the locomotory stage, microglia begin translocating within the tissue (up to 118 microm/h) only after the new protrusions emerge. We conclude that the rapid conversion of resting ramified microglia to active amoeboid macrophages is accomplished not by converting quiescent branches to dynamic ones, but rather by replacing existing branches with an entirely new set of highly motile protrusions. This suggests that the ramified branches of resting microglia are normally incapable of rapid morphological dynamics necessary for activated microglial function. More generally, our time-lapse observations identify changes in the dynamic behavior of activating microglia and thereby help define distinct temporal and functional stages of activation for further investigation.

GFAP promoter-controlled EGFP-expressing transgenic mice: A tool to visualize astrocytes and astrogliosis in living brain tissue

We have generated transgenic mice in which astrocytes are labeled by the enhanced green fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promoter. In all regions of the CNS, such as cortex, cerebellum, striatum, corpus callosum, hippocampus, retina, and spinal cord, EGFP-positive cells with morphological properties of astrocytes could be readily visualized by direct fluorescence microscopy in living brain slices or whole mounts. Also in the PNS, nonmyelinating Schwann cells from the sciatic nerve could be identified by their bright green fluorescence. Highest EGFP expression was found in the cerebellum. Already in acutely prepared whole brain, the cerebellum appeared green-yellowish under normal daylight. Colabeling with GFAP antibodies revealed an overlap with EGFP in the majority of cells. Some brain areas, however, such as retina or hypothalamus, showed only low levels of EGFP expression, although the astrocytes were rich in GFAP. In contrast, some areas that were poor in immunoreactive GFAP were conspicuous for their EGFP expression. Applying the patch clamp technique in brain slices, EGFP-positive cells exhibited two types of membrane properties, a passive membrane conductance as described for astrocytes and voltage-gated channels as described for glial precursor cells. Electron microscopical investigation of ultrastructural properties revealed EGFP-positive cells enwrapping synapses by their fine membrane processes. EGFP-positive cells were negative for oligodendrocyte (MAG) and neuronal markers (NeuN). As response to injury, i.e., by cortical stab wounds, enhanced levels of EGFP expression delineated the lesion site and could thus be used as a live marker for pathology.

The Excitatory Neuronal Network of Rat Layer 4 Barrel Cortex

Sensory whiskers are mapped to rodent layer 4 somatosensory cortex as discrete units termed barrels, which can be visualized at high resolution in living brain slices. Both anatomical and physiological properties of the layer 4 neuronal network can thus be investigated in the context of the functional boundaries of this sensory map. Large-scale confinement of neuronal arbors to single barrels was suggested by restricted lateral diffusion of DiI across septa between barrels. Morphological analysis of dendritic and axonal arborizations of individual excitatory neurons showed that neuronal processes remain within the barrel of origin through polarization toward the center of the barrel. Functionally, the large-scale properties of the neuronal network were investigated through mapping the spatial extent of field EPSPs, which were found to attenuate at barrel borders. This ensemble property of a layer 4 barrel was further investigated by analyzing the connectivity of pairs of excitatory neurons with respect to the locations of the somata. Approximately one-third of the excitatory neurons within the same barrel were synaptically coupled. At the septum between adjacent barrels the connectivity dropped rapidly, and very few connections were found between neurons located in adjacent barrels. Each layer 4 barrel is thus composed of an excitatory neuronal network, which to a first order approximation, acts independently of its neighbors.