Physiological, morphological, and cytochemical characteristics of a layer 1 neuron in cat striate cortex.

We have recorded from a small neuron in layer 1 of the striate visual cortex in a 34-day-old kitten. It had a simple, orientation-selective receptive field that was nondirectional and showed length summation. The neuron was injected intracellularly with horseradish peroxidase. Computer-aided reconstruction revealed that it had a dense axonal plexus confined to layer 1, elongated in the anteroposterior dimension. By means of an antibody directed against a GABA-like antigen, and postembedding immunocytohemistry, the neuron was found to be strongly immunoreactive. The main input to soma and dendrites of the neuron was from synapses that were not GABA-L-immunoreactive, and probably originated from pyramidal cells. The axon of the cell formed synapses on dendritic shafts and spines, whose most likely sources were the apical tufts of pyramidal cell dendrites. These data suggest that such neurons may be involved in local circuits that contribute to the formation of pyramidal cell receptive fields.

Synaptic organization of serotonin-immunoreactive fibers in primary visual cortex of the macaque monkey.

The macaque neocortex is very densely innervated by serotonin-containing fibers. The highest density of these fibers is in primary sensory regions such as the primary visual cortex. By using an antibody against serotonin, we analyzed the distribution and morphology of serotonin-immunoreactive fibers and synapses in the primary visual cortex of the adult cynomolgus monkey. In addition, we quantified the laminar distribution of labeled varicosities and the distances between varicosities in single fibers. While serotonin-immunoreactive fibers are found in all cortical layers, at least three bands of heightened density of innervation were readily recognized that were coincident with 1) layer IIIB to IVC alpha, 2) layer VA, and 3) layer VIB. Layer IVC alpha of area 17 contained more varicosities per unit area than any other sublayer. There was a high degree of variability in the intervaricosity distances along single fibers; more than half were longer than 10 microns. At the electron microscopic level, synaptic contacts were also observed throughout the entire thickness of area 17, with the highest frequency in layer IV. The labeled varicosities were packed with electron-lucent synaptic vesicles and formed synaptic complexes with small, but conspicuous, post-synaptic densities. Dendritic shafts were the most common postsynaptic target of the labeled synapses. Among these characteristically slender post-synaptic shafts, profiles with structural features of both spiny and smooth dendrites were observed. The small diameter of most of the postsynaptic dendrites indicated that distal dendrites were preferentially contacted by serotonin-immunoreactive varicosities. Although direct identification of the postsynaptic neurons will be required for complete characterization of this circuitry, the distribution of serotonin-immunoreactive varicosities suggests that serotoninergic interactions in the primary visual cortex of the cynomolgus monkey are directed predominantly at the distal dendrites of granular and infragranular neurons rather than at targets in the supragranular layers.

Enigmatic bipolar cell of rat visual cortex.

Our earlier Golgi-electron microscopic study of bipolar cells in the rat visual cortex showed the axons of these neurons as forming asymmetric synapses (Peters and Kimerer; J. Neurocytol, 10:921-946, '81) in which the most common postsynaptic elements were dendritic spines. This result was unexpected, since Parnavelas (Parnavelas, Sullivan, Lieberman, and Webster: Cell Tissue Res. 183:499-517, '77) had earlier shown a bipolar cell from the same cortex to have an axon forming symmetric synapses with dendritic shafts. Here then was an enigma, strengthened by examination of neuronal components labelled by antibodies to two compounds in particular--namely, vasoactive intestinal polypeptide (VIP) and choline acetyltransferase (ChAT). Antibodies to these compounds preferentially label bipolar cells in the rat cerebral cortex, and the labelled axon terminals form symmetric synapses. Against this background the present study was performed, and it has been shown that the resolution to the enigma is that there are two different populations of bipolar cells in the rat visual cortex. Thus some Golgi-impregnated bipolar cells examined by electron microscopy after gold toning have been found to possess axons forming asymmetric synapses, and others have been found to have axons forming symmetric synapses. The axons of the bipolar cells forming asymmetric synapses most commonly synapse with dendritic spines (67%), although other terminals synapse with dendritic shafts (33%). In contrast, the bipolar cells with axons forming symmetric synapses preferentially synapse with dendritic shafts (100%). The population of bipolar cells that form symmetric synapses includes the ones that label with antibodies to vasoactive intestinal polypeptide (VIP), for the axons of VIP-labelled bipolar cells have been traced to labelled terminals forming symmetric synapses. However, examination of the population of VIP-labelled axon terminals shows that in addition to dendritic shafts, some of the labelled terminals synapse with the cell bodies of pyramidal and nonpyramidal cells. This includes bipolar cells, some of which receive large numbers of VIP-labelled axon terminals. It is also shown that some VIP-positive bipolar cells have myelinated axons. Analysis of tissue labelled with VIP antibody reveals that about 50% of the total population of bipolar cells in the rat visual cortex is VIP positive. These results are discussed in the light of information about labelling of bipolar cells with antibodies to gamma-aminobutyric acid (GABA) and to other peptides, and it is suggested that most VIP-positive bipolar cells also contain GABA.

The brainstem projection to the lateral geniculate nucleus in the cat: identification of cholinergic and monoaminergic elements.

The pontomesencephalic projection to the dorsal lateral geniculate nucleus (dLGN) of the cat was analyzed by combining retrograde transport of rhodamine-labeled latex spheres and immunohistochemistry. After injections of latex beads into the dLGN, sections of the brainstem were treated immunohistochemically for choline acetyltransferase (ChAT), serotonin (Ser), tyrosine hydroxylase (TH), and dopamine-beta-hydroxylase (DBH). Essentially, six regions in the brainstem contained retrogradely labeled cells: the superior colliculus, the parabigeminal nucleus, the dorsal raphe nuclei, the parabrachial area of the central tegmental field, the marginal nucleus of the brachium conjunctivum, and the nucleus coeruleus. Furthermore, isolated retrogradely labeled cells were present in the central nucleus of the raphe, in the cuneiform nucleus, and in the periaqueductal gray. Most serotoninergic double-labeled cells were found in the medial and lateral divisions of the dorsal raphe nuclei, but a few were also present in the central nucleus of the raphe. In the sections immunostained for ChAT, double-labeled cells were located in the central tegmental field, in the marginal nucleus of the brachium conjunctivum, and in the nucleus coeruleus. In the sections treated for TH and DBH, double-labeled cells showed a similar distribution, and like the ChAT(+) cells, they were located mainly in the central tegmental field, in the marginal nucleus of the brachium conjunctivum, and in the nucleus coeruleus. In these regions the cholinergic and noradrenergic cells that projected to the lateral geniculate nucleus were intermingled, the former predominating rostrally and the latter caudally. The majority of retrogradely labeled cells were located in the region of the central tegmental field in the vicinity of the brachium conjunctivum, and most of these cells were also ChAT-immunoreactive. We, therefore, conclude that the cholinergic projection is the most important of the central core projections ascending to the dLGN.

Innervation of cat visual areas 17 and 18 by physiologically identified X- and Y- type thalamic afferents. II. Identification of postsynaptic targets by GABA immunocytochemistry and Golgi impregnation.

The precise location of physiologically identified specific afferent input on the different types of cell in the visual cortex and the identification of the neurotransmitters of these cells are essential to a better understanding of the first stage of cortical processing. A combination of anatomical, neurochemical, and physiological methods was used to identify the cortical neurones that receive synaptic input from X- and Y-type afferents, which are thought to originate from cells of the lateral geniculate nucleus. One method relied on chance contacts made between single physiologically characterised axons, which had been injected with horseradish peroxidase (HRP), and the processes of cells impregnated by the Golgi method. These experiments revealed that both X and Y axons formed synapses on the dendrites of spiny stellate cells in layer 4. Y axons in both areas 17 and 18 established multiple synaptic contacts on basal dendrites of layer 3 pyramidal cells. One X axon contacted the apical dendrite of a layer 5 pyramidal cell and one Y axon contacted the dendrite of a large cell with smooth dendrites in layer 3. The maximum number of synapses made between one axon and a single postsynaptic cell was eight, although in most cases it was only one. It was concluded that one axon only provides a small fraction of the geniculate afferent input to an individual cell. A second method revealed that the somata in layer 4 in synaptic contact with the HRP-filled axon terminals were GABA-immunoreactive, and therefore might be involved in inhibitory processes. From light microscopic data it was found that somata receiving contacts from X axons in area 17 were significantly smaller (average diameter 15 microns) than those contacted by the Y axons in areas 17 and 18 (average diameter 24 microns). Somatic contacts were extremely rare in layer 6. These data show that the X and Y afferents may activate separate subsets of inhibitory neurones.

Age-related changes of catecholamines and their metabolites content in the visual system of the rat.

The changes in the content of the catecholamines in each structure of the geniculate and extrageniculate visual system of the rat during the aging period (6-30 months) have been studied. Dopamine was found at lower levels than noradrenaline in all the structures. The dopamine and noradrenaline showed different developmental profiles. Dopamine and its metabolite levels decreased in the lateral geniculate and visual cortex and increased in superior colliculus and posterior thalamus. Noradrenaline and its metabolites increased in all structures during the aging period. However, 3-methoxy-4-hydroxyphenylglycol/noradrenaline and normetanephrine/noradrenaline ratios decreased in all structures except in superior colliculus. These results suggest age-related changes in the catecholamines in the visual system of the rat.

New dopaminergic terminal fields in the motor, visual (area 18b) and retrosplenial cortex in the young and adult rat. Immunocytochemical and catecholamine histochemical analyses.

New dopaminergic terminal fields have been visualized in the rat cerebral neocortex, using two morphological methods based on distinct properties of the dopaminergic system: presence of the first synthetic enzyme, tyrosine hydroxylase, and high-affinity uptake of amines. Tyrosine hydroxylase was used as an immunocytochemical marker after destruction of the cortical noradrenergic system, induced either neonatally by 6-hydroxydopamine or later on by DSP4, and controlled by the absence of dopamine beta-hydroxylase immunoreactivity. The uptake and storage of exogenous amines in tissue sections, in the presence of selective high-affinity transport inhibitors, enabled the specific visualization of the dopaminergic system with fluorescence histochemistry. A dopaminergic innervation of low density was observed along a dorsal sagittal strip which extended from the genu of corpus callosum until about 2 mm behind the splenium and encompassed several distinct cytoarchitectonic areas in the sensorimotor and visual cortex (medial and lateral agranular field, area 18b), as well as in discrete zones of the retrosplenial granular 29c,b, and agranular 29d areas. The distribution of these dopaminergic fields suggested a columnar organization. Several characteristics of the dopaminergic innervation were similar to that of the superficial anterior cingulate cortex (area 24): the laminar distribution to the superficial I-III layers, the secondarily developed varicose aspect in catecholamine fluorescence histochemistry and the delayed postnatal ingrowth in contrast with the early prenatal dopaminergic input to the prefrontal cortex. These similarities suggested that the subpopulation of dopaminergic neurons which provides projections to the anterior cingulate cortex could also contribute to the motor and visual cortex and thus play a role in sensorimotor integration. The predictive value of these results in the ascent of the phylogenetic scale are further considered.

Antisera to gamma-aminobutyric acid. III. Demonstration of GABA in Golgi-impregnated neurons and in conventional electron microscopic sections of cat striate cortex.

Two methods are described for the immunocytochemical demonstration of immunoreactive gamma-aminobutyric acid (GABA) in the visual cortex of the cat, an area that contains several types of GABAergic neurons and requires combined methods for their characterization. The first method is illustrated by a representative example of a Golgi-impregnated and gold-toned interneuron of the "bitufted" type situated in layer VI and having an ascending axon. After recording the three-dimensional features of the cell, semithin (0.5 micron) sections of the perikaryon were cut and GABA was demonstrated in the cell body by the unlabeled antibody enzyme method. While immunocytochemistry was used to determine the probable transmitter of the neuron, Golgi-impregnation of the same cell was used to identify its neuronal type. Since aldehyde-osmium fixation was used, further electron microscopic (EM) analysis of the neuron's synaptic connections was possible. The second procedure demonstrated GABA in EM sections of aldehyde-osmium-fixed cortex using protein A-gold as an immunocytochemical marker. Immunoreactivity was found in certain neurons, dendrites, axons, and boutons forming type II synaptic contacts that from previous studies have been thought to be GABAergic. Thus ultrastructural analysis using optimal conditions can now be supplemented with the identification of the transmitter in the same section.

Antisera to gamma-aminobutyric acid. II. Immunocytochemical application to the central nervous system.

An antiserum to gamma-aminobutyric acid (GABA) was tested for the localization of GABAergic neurons in the central nervous system using the unlabeled antibody enzyme method under pre- and postembedding conditions. GABA immunostaining was compared with glutamate decarboxylase (GAD) immunoreactivity in the cerebellar cortex and in normal and colchicine-injected neocortex and hippocampus of cat. The types, distribution, and proportion of neurons and nerve terminals stained with either sera showed good agreement in all areas. Colchicine treatment had little effect on the density of GABA-immunoreactive cells but increased the number of GAD-positive cells to the level of GABA-positive neurons in normal tissue. GABA immunoreactivity was abolished by solid phase adsorption to GABA and it was attenuated by adsorption to beta-alanine or gamma-amino-beta-hydroxybutyric acid, but without selective loss of immunostaining. Reactivity was not affected by adsorption to glutamate, aspartate, taurine, glycine, cholecystokinin, or bovine serum albumin. The concentration (0.05-2.5%) of glutaraldehyde in the fixative was not critical. The antiserum allows the demonstration of immunoreactive GABA in neurons containing other neuroactive substances; cholecystokinin and GABA immunoreactivities have been shown in the same neurons of the hippocampus. In conclusion, antisera to GABA are good markers for the localization of GABAergic neuronal circuits.

Distribution of neuropeptide Y immunoreactivity in human visual cortex and underlying white matter.

Immunocytochemical techniques have been used to study neuropeptide Y (NPY) distribution in the human visual cortex (Brodman's areas 17, 18 and 19) NPY cell bodies belong mostly to inhibitory (multipolar and bitufted) but also to excitatory (bipolar and some pyramidal) neuronal types. Their distribution is similar in the three cortical areas studied: 20 to 40% of the NPY perikarya are located in the cortical gray matter, mostly in the deep layers, while the remaining 60 to 80% are located in the underlying white matter. Immunoreactive NPY processes form a rich network of intersecting fibers throughout the entire visual cortex. A superficial plexus (layers I and II) and a deep plexus (deep layer V and layer VI) of NPY fibers are present in areas 17, 18 and 19. In area 17, an additional well developed plexus is present in layers IVb and IVc. These plexuses receive branches from long parallel fibers arising from deep cortical layers or underlying white matter and terminating in superficial layers. Local or extrinsic NPY terminals wind around vessels in the cortex as well as in the white matter, and either penetrate them or form clusters of club endings on their walls. Our results suggest a role for NPY in human visual circuitry and in cortical blood flow regulation.

Picture processing techniques applied to autoradiographic studies of visual cortex.

Picture processing techniques are applied to 2-deoxyglucose autoradiographs of sections from striate cortex and to patterns resulting from staining these sections for cytochrome oxidase. This procedure allows computer identification of deoxyglucose active and inactive regions in the autoradiographs and cytochrome active and inactive regions in the stain patterns. Subsequently, the topographical relationship between these patterns can be quantitatively analyzed by means of overlap and density distribution measures and can be displayed using color enhanced graphics. The processing techniques have been applied in studies of the functional organization of visual cortex in primates. Computer graphic techniques have allowed implementation of split-field presentations of stimuli in deoxyglucose experiments. An application of this split-field technique for presenting multiple-stimuli to distinct parts of the visual field is described and an autoradiograph from a split-field experiment is shown.

The distribution and ontogenesis of [3H]nicotine binding sites in cat visual cortex.

In vitro autoradiographic techniques using [3H]nicotine were used to characterise nicotine binding sites in developing kitten visual cortex. These binding sites in adult animals have a Bmax of 3.91 fmol/mg protein and a Kd of 4.40 nM. Displacement experiments indicate that [3H]nicotine binds to a nicotinic receptor site that is similar to central nicotinic sites described by investigators in other mammals. The number of binding sites increases during postnatal development, peaking near 60 days of age and levelling-off thereafter. There is no evidence for large changes in affinity during postnatal development for this binding site. [3H]Nicotine binding sites are densely concentrated in layer IV in the visual cortex of adult animals, with sharply reduced binding outside of cortical areas 17 and 18. This laminar pattern does not change during postnatal development, but an increase in the number of binding sites in layer IV as well as in layers I and VI occurs during early postnatal life. These binding sites disappear when extrinsic cortical inputs are severed. However, they survive when neurons in the visual cortex are selectively destroyed with a cell-specific neurotoxin. Unilateral destruction of the lateral geniculate nucleus eliminates [3H]nicotine binding sites in the visual cortex ipsilateral to the lesion, suggesting that they are located presynaptically on the terminals of lateral geniculate nucleus afferent fibres. The laminar pattern of binding of [3H]nicotine during early development of the visual cortex is complimentary to that for muscarinic acetylcholine receptors. These latter receptors redistribute during postnatal development becoming less prominent in layer IV at the same time as the [3H]nicotine binding sites are increasing in number in this layer. For a short period of time at the height of the critical period for cortical plasticity, both populations of binding sites are located in layer IV.

A selective decrease of cholinergic muscarinic receptors in the visual cortex of adult rats following developmental lead exposure.

The effects of low-level developmental lead (Pb) exposure (postnatal days 0-21) on the binding of [3H]quinuclidinylbenzilate (QNB) and on acetylcholinesterase (AChE) activity in the retina, superior colliculus, lateral geniculate nucleus and visual cortex (VC) were studied in the adult rat. Maximal blood and tissue Pb concentrations (50-60 micrograms %) were reached on day 21 and decreased to control levels (4-5 micrograms %), except in the retina (12 micrograms %) and VC (18 micrograms %; 0.87 microM), by day 90. A large decrease in [3H]QNB binding (-38%) and AChE activity (-29%) was found only in the VC of Pb-exposed rats. Scatchard plots of saturation binding data revealed a decrease in the density (Bmax), but not in the affinity (Kd), of the muscarinic receptors. Pb (10(-4)-10(-9)M) had no effect on [3H]QNB binding or AChE activity in VC membrane preparations from control rats. The mechanism accounting for this selective decrease of cholinergic muscarinic receptors in the VC is presently unknown. These results, in combination with those from our psychophysical and pharmacological studies demonstrating a scopolamine supersensitivity in Pb-exposed rats, suggest that the long-term effects of developmental Pb exposure are due to a direct action of Pb on visual cortex cholinergic neurons.

Benzodiazepine receptors in the visual structures of monocularly deprived rats. Effect of light and dark adaptation.

In 25-day-old rats with one eyelid sutured at the age of 10 days, the binding of [3H]flunitrazepam in the visual structures (retina, lateral geniculate nucleus, superior colliculus, visual cortex) and frontal cortex was determined. Monocular visual deprivation (MD) resulted in a significant decrease of the [3H]flunitrazepam binding in the retina of the open eye to about 76% of the control value. No changes in [3H]flunitrazepam binding were detectable under these conditions in the central visual structures examined and the non-visual cortical region. Scatchard analysis indicated that the changes found in the retina of the open eye of MD rats are due to a decreased binding affinity only, the maximum receptor number being unaffected. Eight hours after re-opening the sutured eyelid of 25-day-old MD rats, benzodiazepine binding in the open eye was increased to the control level, whereas the binding in the retina of the re-opened eye remained unchanged in comparison to control animals. Dark adaptation of 25-day-old control rats resulted in an increased [3H]flunitrazepam binding in the retina by 28% compared to that detectable in the retina of light-adapted animals. In contrast, dark-adaptation of MD rats did not affect [3H]flunitrazepam binding in the retina of both eyes in comparison to that found in the corresponding retina of light-adapted MD animals. The data obtained suggest a physiological coupling between both retinas, possibly mediated through centres inside of the central nervous system.

GAP-43 in adult visual cortex.

GAP-43 was purified from cat brain by a rapid isolation procedure and was used to raise highly specific polyclonal antibodies in rabbits. Immunoblots of proteins from adult cat, monkey and human visual cortex as well as bovine cortex also showed specific staining of a single protein that was present in both soluble and membrane fractions. Immunocytochemistry of both cat and human adult visual cortex showed that GAP-43 has a laminar distribution.

Modification of neurotransmitter receptor sensitivity in cat visual cortex during the critical period.

We have examined the characteristics of various receptors in cat visual cortex during postnatal development. These included beta-adrenergic, GABA, benzodiazepine and acetylcholine receptors. For each population of receptor the number (Bmax) and affinity (Kd) were examined as a function of postnatal age (3 days-adult). For all receptors examined, the Bmax increased during development from low early values to a peak within the critical period. The Kd also changed during development for most receptors. The simultaneous alterations in Bmax and Kd necessitate defining a term which takes both of these receptor properties into consideration. This term, called receptor sensitivity (RS), provides a more comprehensive measure of receptor function than either Bmax or Kd alone. Using this measure, we find that receptor sensitivity is low near birth for the 4 receptor populations studied, rises to a peak within the first two months of life, and then declines to near-neonatal levels for 3 of the 4 receptor populations.

Extensive co-existence of neuropeptides in the rat visual cortex.

The peroxidase-antiperoxidase immunohistochemical technique has been used to examine the co-existence of peptides within individual neurons of the rat visual cortex. Pairs of consecutive paraffin sections were stained alternately for 2 of the 4 peptides: somatostatin (SRIF), vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK) and neuropeptide Y (NPY). Analysis revealed the co-existence of SRIF with VIP, CCK and NPY and between VIP and CCK. These results show that the co-localization of neuropeptides in cortical neurons is more widespread than previously demonstrated.

Effect of desmethylimipramine on norepinephrine content and plasticity of kitten visual cortex.

Because norepinephrine (NE) reuptake inhibitors have been reported to block 6-hydroxydopamine (6-OHDA) induced NE depletion, we wondered whether a reuptake inhibitor would block 6-OHDA's effects on visual cortical plasticity. We found, however, that desmethylimipramine (DMI) did not reduce 6-OHDA-induced NE depletion at 6-OHDA doses sufficient to prevent the effects of monocular deprivation. We also found that DMI itself induced transient NE depletion. We used this last result to further examine the NE hypothesis of depletion. In contrast to 6-OHDA-induced NE depletion, DMI-induced NE depletion was not accompanied by changes in visual cortical plasticity.

Noradrenaline neuron innervation of the neocortex in the rat.

Noradrenaline innervation of the rat neocortex is studied by glyoxylic acid histochemistry and radioisotopic biochemical analysis. The data indicate that all neocortical areas receive a noradrenergic innervation which is identical in organization but varies in density from area to area. Radioisotopic analysis of catecholamines in the cortical areas studied reveals only the presence of significant levels of noradrenaline. Unilateral locus coerulus ablation greatly diminishes ipsilateral noradrenaline content and fiber innervation in all neocortical areas studied. Detailed histochemical analysis reveals a diffuse plexus-like arrangement of noradrenaline fibers, with each cortical layer having a distinctive pattern of innervation. Single noradrenergic fibers enter layer VI of cortex and branch at all levels to undergo extensive collateralization. Terminal horizontal branching in the molecular layer results in the most dense fiber plexus of all cortical layers. This pattern of noradrenaline innervation is similar to that of other non-specific afferent systems innervating neocortex.

Visual and auditory pathways contain cholecystokinin: evidence from immunofluorescence and retrograde tracing.

The distribution of cholecystokinin (CCK) in the visual and auditory systems of the rat was studied with combined immunofluorescence and fluorescence retrograde tracing techniques. Double-labeled projection neurons in the pathway from the dorsal lateral geniculate nucleus to area 17 of visual cortex, and from the superior olive to the inferior colliculus demonstrated the presence of CCK-containing pathways in the ascending visual and auditory systems. Thus, CCK can be viewed as a neurotransmitter/neuromodulator candidate in ascending sensory systems.