Comparative electron microscopic study of the visual cortex neurons of the cat in postnatal ontogeny

The maturation of layers II-VI of neurons and perineuronal neuropil of the cat visual cortex (field 17) was studied from postnatal day 1 to day 21. The differentiation of large, small (associate) pyramid and stellate neurons was described. During the first postnatal week, the somata of layers II-VI of neurons undergo significant changes, the perikaryal cytoplasm increases in volume. Cell bodies of large pyramidal neurons mature by day 15. During the second postnatal week and almost till day 15, the rough endoplasmic reticulum of small pyramidal and stellate neurons undergoes proliferation; dendritic processes are branching. In stellate neurons the amount of cytoplasmic organelles increases dramatically only after the second postnatal week, and this is presumably induced by the opening of eyes on day 12. The second postnatal week is the period of greatest growth of dendritic, axonal and glial processes in perineural neuropil of layers V-VI. In the perineuronal neuropil of large pyramidal neurons (layers V-VI) there appear symmetric synapses with pyramidal cells, dendritic processes and dendritic spines. This occurs just at the time when kittens first open the eyes. From this time and during postnatal days 15-21, asymmetric synapses appear in the perineuronal neuropil of large pyramidal neurons. In the perineuronal neuropil of small pyramidal and stellate neurons. (layers II-IV), synapses reveal the mature appearance by day 15. After the opening of the eyes and up to postnatal day 21, dendritic growth and spine production occur in the perineuronal neuropil of small pyramidal and stellate neurons.     

Role of intracortical inhibition in orientation tuning dynamics in the cat striate cortex neurons

A suggestion about the leading role of GABA-induced intracortical inhibition in the dynamics of orientation tuning (OT) of the cat striate cortical neurons was tested in acute experiments before and during the local blockade of their inhibition by iontophoretic application of bicucculine. In the course of the investigation of these dynamics, with the use of a temporal scanning method, two types of neurons differing in the inhibition blockade-induced OT changes were found. In the neurons of the first type (57%), bicuculline induced the OT dynamics or enhanced it, if it pre-existed before the bicuculline application. In the neurons of the second type (43%), bicuculline strongly reduced or eliminated the dynamic shift of a preferred orientation. These results mean that under normal conditions the inhibition stabilizes and sharpens OT in some cells, while in other cells, in contrast, it causes the OT dynamics. The following mechanisms may underlie the observed effects: an elimination of the inhibition originating from lateral non-isoorientational excitatory inputs of a receptive field; an inhibition of these inputs via the adjacent interneurons activated by a powerful discharge of the examined neuron; a long-term afterhyperpolarization of the neuron, and the dynamics of the excitatory and inhibitory zones of the receptive field.Neirofiziologiya/Neurophysiology, Vol. 27, No. 2, pp. 100–109, March–April, 1995.     

Antisera to gamma-aminobutyric acid. I. Production and characterization using a new model system

Antisera to the amino acid gamma-aminobutyric acid (GABA) have been developed with the aim of immunohistochemical visualization of neurons that use it as a neurotransmitter. GABA bound to bovine serum albumin was the immunogen. The reactivities of the sera to GABA and a variety of structurally related compounds were tested by coupling these compounds to nitrocellulose paper activated with polylysine and glutaraldehyde and incubating the paper with the unlabeled antibody enzyme method, thus simulating immunohistochemistry of tissue sections. The antisera did not react with L-glutamate, L-aspartate, D-aspartate, glycine, taurine, L-glutamine, L-lysine, L-threonine, L-alanine, alpha-aminobutyrate, beta-aminobutyrate, putrescine, or delta-aminolevulinate. There was cross-reaction with gamma-amino-beta-hydroxybutyrate, 1-10%, and the homologues of GABA: beta-alanine, 1-10%, delta-aminovalerate, approximately 10%, and epsilon-amino-caproate, approximately 10%. The antisera reacted slightly with the dipeptide gamma-aminobutyrylleucine, but not carnosine or homocarnosine. Immunostaining of GABA was completely abolished by adsorption of the sera to GABA coupled to polyacrylamide beads by glutaraldehyde. The immunohistochemical model is simple, amino acids and peptides are bound in the same way as in aldehyde-fixed tissue and, in contrast to radioimmunoassay, it uses an immunohistochemical detection system. This method has enabled us to define the high specificity of anti-GABA sera and to use them in some novel ways. The model should prove useful in assessing the specificity of other antisera.     

Excitatory inputs to spiny cells in layers 4 and 6 of cat striate cortex

The principal target of lateral geniculate nucleus in the cat visual cortex is the stellate neurons of layer 4. In previously reported work with intracellular recording and extracellular stimulation in slices of visual cortex, three general classes of fast excitatory synaptic potentials (EPSPs) in layer 4a spiny stellate neurons were identified. One of these classes, characterized by large and relatively invariant amplitudes (mean 1.7 mV, average coefficient of variation (CV) 0.083) were attributed to the action of geniculate axons because, unlike the other two classes, they could not be matched by intracortical inputs, using paired recording. We have examined in detail the properties of this synaptic input in twelve examples, selecting for study those EPSPs where there was secure extracellular stimulation of the single fibre input to a pair of stimuli 50 ms apart. In our analysis, we conclude that the depression that these inputs show to the second stimulus is entirely postsynaptic, since the evidence strongly suggests that the probability of transmitter release at the synaptic site(s) remains 1.0 for both stimuli. We argue that the most plausible explanation for this postsynaptic depression is a reduction in the average probability of opening the synaptic channels. Using a simple biochemical analysis (c.f. Sigworth plot), it is then possible to calculate the number of synaptic channels and their probability of opening, for each of the 12 connections. The EPSPs had a mean amplitude of 1.91 mV (+/- 1.3 mV SD) and a mean CV of 0.067 (+/- 0.022). The calculated number of channels ranged from 20 to 158 (59.4 +/- 48.7) and their probability of opening to the first EPSP had an average of 0.83 (+/- 0.09), with an average depression of the probability to 0.60 for the second EPSP. Geniculate afferents also terminate in layer 6. Intracellular recordings were also made in the upper part of this layer and a total of 51 EPSPs were recorded from pyramidal cells of three principal types. Amongst this dataset we sought EPSPs with similar properties to those characterized in layer 4a. Three examples were found, which is a much lower percentage (6%) than the incidence of putative geniculate EPSPs found in layer 4a (42%).     

The neurons in layer 1 of cat visual cortex.

We have examined the morphology of neurons in layer 1 by injecting them intracellularly with lucifer yellow in lightly fixed brain slices (250 microns thick) taken from the medial bank of area 17 in adult cats. Of 22 neurons with well-filled dendrites, 16 had smooth dendrites, two had sparsely spiny dendrites (less than 200 spines) and, unexpectedly, four had spiny dendrites typical of pyramidal cells. The axon was generally not well filled. Computer reconstructions showed that parts of the dendritic tree had been lost in the sectioning. Nevertheless, measurements of the length of intact dendrites suggested an average diameter of the dendritic tree of 220 microns. The density of the neurons was such that the dendritic trees of about six neurons cover each point in layer 1. Thus, despite the very low density of neurons that characterizes layer 1, there are more than sufficient neurons to sample from the entire representation of the visual field in area 17.     

Selective sensitivity of the cat striate neurons to cruciform and angular figures of various orientations

Selectivity and invariability of tuning were studied in 51 neurons of the primary visual cortex (area 17); cruciform and angular figures (CF and AF, respectively) of different configurations and orientations were presented in their receptive fields. Twenty-three neurons, or 45% of the studied cells, demonstrated selective sensitivity to these figures. Their responses considerably (2.38±0.36 times, on average) increased, as compared with those evoked by presentation of a single bar of preferred orientation. In the examined group, 2 cells demonstrated sensitivity both to the CF and AF. A wide range of detector properties related to the CF and AF analysis was found in the analyzed neuronal population. Detectors of configuration of these figures are described. Selective sensitivity to the angle between branches of these figures was observed in 17 neurons, and responses of 2 neurons among them showed invariability to orientation of these figures. Four cells were selective for orientation and were insensitive to configuration, and 4 other cells showed no specific sensitivity to either of these properties, but were sensitive to the appearance of a CF itself in their receptive field (these cells were regarded as invariant detectors of crossing nodes). Data inconsistent with the hierarchic principle of detection of the above properties are presented. Possible mechanisms and functional significance of selective sensitivity of striate neurons to the CF and AF are discussed.Neirofiziologiya/Neurophysiology, Vol. 27, No. 5/6, pp. 403–412, September–December, 1995.     

GABA-immunoreactive neurons and terminals in the cat periaqueductal gray matter: A light and electron microscopic study

Immunocytochemical and electron microscopic methods were used to study the GABAergic innervation in adult cat periaqueductal gray matter (PAG). A mouse monoclonal antibody against γ -aminobutyric acid (GABA) was used to visualize the inhibitory neuronal system of PAG. At light microscopy, GABA-immunopositive (GABA_IP) neurons formed two longitudinally oriented columns in the dorsolateral and ventrolateral PAG that accounted for 36% of the neuronal population of both PAG columns; their perikaryal cross-sectional area was smaller than that of unlabeled (UNL) neurons found in the same PAG subdivisions. At electron microscopic level, patches of GABA immunoreactivity were readily detected in neuronal cell bodies, proximal and distal dendrites, axons and axon terminals. Approximately 35–36% of all terminals were GABA_IP; they established symmetric synapses with dendrites (84.72% of the sample in the dorsolateral PAG and 86.09% of the sample in the ventrolateral PAG) or with cell bodies (7–10% of the sample). Moreover, 49.15% of GABA_IP axon terminals in the dorsolateral and 52.16% in the ventrolateral PAG established symmetric synapses with GABA_IP dendrites. Immunopositive axon terminals and unlabeled terminals were also involved in the formation of a complex synaptic arrangment, i.e. clusters of synaptic terminals in close contact between them that were often observed in the PAG neuropil. Moreover, a fair number of axo-axonic synapses between GABA_IP and/or UNL axon terminals were present in both PAG subdivisions. Several dendro-dendritic synapses between labeled and unlabeled dendrites were also observed in both PAG subdivisions. These results suggest that in the cat PAG there exist at least two classes of GABArgic neurons. The first class could exert a tonic control on PAG projecting neurons, the second could act on those GABAergic neurons that in turn keep PAG projecting neurons under tonic inhibition. The functional implications of this type of GABAergic synapse organization are discussed in relation to the dishinibitory processes that take place in the PAG.     

Re-evaluation of epoxy resin sections for light and electron microscopic immunostaining.

Epoxy resins provide optimal tissue morphology at both the light and the electron microscopic level and therefore enable correlative studies on semithin and thin sections from the same tissue block. Here we report on an approach to retain these advantages for immunolabeling studies by adapting and combining well-known techniques, i.e., surface etching with sodium ethoxide and heat-mediated antigen retrieval. We propose a simple procedure for immunostaining semithin and thin epoxy resin sections. To check its applicability, well characterized, commercially available antibodies (against E-cadherin, alpha-catenin, and beta-catenin) were used on sections of human small intestine. By light microscopy, the immunostaining efficiency was compared on cryo-, paraffin, and epoxy semithin sections processed in parallel. The most detailed results were obtained on semithin sections, where the labeling precisely delineated the lateral plasma membrane of the enterocytes. At the electron microscopic level the procedure did not damage the structures and allowed an efficient, reproducible immunogold labeling extending homogeneously over exceptionally wide tissue areas. The three antibodies specifically labeled the zonula adherens of the junctional complex between epithelial cells and, in agreement with light microscopic observations, the lateral plasma membrane.     

Ultrastructural characteristics of layer III pyramidal neurons in the cat primary auditory cortex (Al)

Electron microscope studies were made of retrogradely horseradish peroxidase-labeled pyramidal neurons forming transcallosal projections in layer III of the cat primary auditory cortex (Al). These showed a significant proportion of the somatic membrane to be covered with processes of astroglia, while synapses occupy 20% of the synaptic surface on average. Between 4 and 10 axosomatic synapses were identified on the profiles of callosal cell somata. All these were formed by axonal terminals containing small, flattened synaptic vesicles and had symmetrical contacts. Average length of these synaptic contacts equaled 1.6 µm. Numerous anterogradely horseradish peroxidase-labeled axonal terminals of callosal fibers were found in cortical area Al in amongst retrogradely HP-labeled neurons. The ultrastructural pattern of these is described.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 520–526, July–August, 1990.     

The availability of surface GABA B receptors is independent of gamma-aminobutyric acid but controlled by glutamate in central neurons

The efficacy of synaptic transmission depends on the availability of ionotropic and metabotropic neurotransmitter receptors at the plasma membrane, but the contribution of the endocytic and recycling pathways in the regulation of gamma-aminobutyric acid type B (GABA(B)) receptors remains controversial. To understand the mechanisms that regulate the abundance of GABA(B) receptors, we have studied their turnover combining surface biotin labeling and a microscopic immunoendocytosis assay in hippocampal and cortical neurons. We report that internalization of GABA(B) receptors is agonist-independent. We also demonstrate that receptors endocytose in the cell body and dendrites but not in axons. Additionally, we show that GABA(B) receptors endocytose as heterodimers via clathrin- and dynamin-1-dependent mechanisms and that they recycle to the plasma membrane after endocytosis. More importantly, we show that glutamate decreases the levels of cell surface receptors in a manner dependent on an intact proteasome pathway. These observations indicate that glutamate and not GABA controls the abundance of surface GABA(B) receptors in central neurons, consistent with their enrichment at glutamatergic synapses.     

Morphological bases of suppressive and facilitative spatial summation in the striate cortex of the cat

In V1 of cats and monkeys, activity of neurons evoked by stimuli within the receptive field can be modulated by stimuli in the extra-receptive field (ERF). This modulating effect can be suppressive (S-ERF) or facilitatory (F-ERF) and plays different roles in visual information processing. Little is known about the cellular bases underlying the different types of ERF modulating effects. Here, we focus on the morphological differences between the S-ERF and F-ERF neurons. Single unit activities were recorded from V1 of the cat. The ERF properties of each neuron were assessed by area-response functions using sinusoidal grating stimuli. On completion of the functional tests, the cells were injected intracellularly with biocytin. The labeled cells were reconstructed and morphologically characterized in terms of the ERF modulation effects. We show that the vast majority of S-ERF neurons and F-ERF neurons are pyramidal cells and that the two types of cells clearly differ in the size of the soma, in complexity of dendrite branching, in spine size and density, and in the range of innervations of the axon collaterals. We propose that different pyramidal cell phenotypes reflect a high degree of specificity of neuronal connections associated with different types of spatial modulation.     

Increase in the sensitivity of rabbit sensomotor cortex neurons to gamma-aminobutyric acid under the influence of diazepam (microiontophoretic study)]

A study was made of the action of diazepam on the effects of the gamma-aminobutyric acid (GABA) applied electrophoretically to the neurons of the sensory-motor rabbit cortex. It was shown that diazepam intensified the depressive action of GABA on the spontaneous neuronal activity and the prolonging action of GABA on the duration of the inhibitory phase in the neuron responses to the afferent and direct stimulation of the cortex. Diazepam failed to alter the neuron response to glycine, glutamate and acetylcholine applied microelectrophoretically. It is supposed that diazepam increased the sensitivity of the receptors of the post-synaptic membrane of the neuron to GABA.