Development and regulation of β adrenergic receptors in kitten visual cortex: An immunocytochemical and autoradiographic study

The developmental pattern and laminar distribution of β₁ and β₂ adrenergic receptor subtypes were studied in cat visual cortex with autoradiography using [¹²⁵I]iodocyanopindolol as a ligand and also with immunocytochemistry using a monoclonal antibody directed against β adrenergic receptors. In the primary visual cortex of adult cats, the laminar distributions of both β₁ and β₂ adrenergic receptors revealed by autoradiography were very similar, with concentrations in layers I, II, III and VI. In young kittens (postnatal days 1 and 10), fewer β adrenergic receptors were present, and they were concentrated in the deep cortical layers (V–VI) and subcortical white matter. Between postnatal days 15 and 40, β adrenergic receptors increased in density more quickly in the superficial layers than they did in the deep and middle cortical layers. By postnatal day 40, the adult pattern was achieved, with two bands of intense binding in the superficial and deep cortical layers and a lower density in layer IV. Immunocytochemical techniques applied to adult cat cortex showed that β adrenergic receptor-like immunoreactivity was found in different populations of neurons and glial cells. The immunoreactive neural cells were most dense in layers II, III and VI. About 50% of these immunoreactive neural cells were glial cells, primarily astrocytes. Immunoreactive pyramidal cells were mostly located in layers III and V. In layer IV, many stellate cells were stained. Immunoreactive astrocytes in the subplate and white matter progressively increased in number during development until adulthood. The pattern of laminar distribution and the developmental process was not affected by interrupting noradrenergic innervation from locus ceruleous either before or after the critical period. However, when visual input was interrupted by lesions of the lateral geniculate nucleus in young kittens (postnatal day 10), the density of both β adrenergic receptor subtypes decreased significantly in the deep cortical layers. Lateral geniculate nucleus lesions in adult cats resulted in a pronounced decrease in β adrenergic receptor density in layer IV.     

Calcium calmodulin dependent kinase II in cat visual cortex and its development.

A monoclonal antibody against the alpha-subunit of calcium/calmodulin-dependent protein kinase II (CAM-K II) was used to visualize the kinase in developing kitten visual cortex. CAM-K II was first expressed in neurons of the deep cortical layers (V and VI) at postnatal day 1-4 and appeared in the remaining cortical layers within the first 2 weeks. The level of immunoreactivity declined in cells of layer V and upper layer VI at about 30-40 days of age. By postnatal day 90, the most densely labelled neurons were concentrated in cortical layers II, III, lower layer IV and in layer VI. This laminar pattern remained constant into adulthood. EM studies showed that the kinase was found in both pre- and postsynaptic locations. About twice as many immunopositive neurons were found in cortical layers II-IV and VI in young adult cats when geniculate input was removed by an unilateral thalamic lesion performed early in life. These results indicate that expression of CAM-K II is developmentally regulated in visual cortical neurons; the alteration of immunoreactivity after early LGN lesions suggests that the level of the kinase (or its alpha-subunit) is also regulated by cortical input.     

Laminar pattern of cholinergic and adrenergic receptors in rat visual cortex using quantitative receptor autoradiography

The laminar distribution of muscarinic acetylcholine receptors, including the M1-receptor subtype, of beta-adrenergic receptors, and noradrenaline uptake sites, was studied in the adult rat visual, frontal, somatosensory and motor cortex, using quantitative receptor autoradiography. In the visual cortex, the highest density of muscarinic acetylcholine receptors was found in layer I. From layer II/III to layer V binding decreases continuously reaching a constant binding level in layers V and VI. This laminar pattern of muscarinic receptor density differs somewhat from that observed in the non-visual cortical regions examined: layer II/III contained the highest receptor density followed by layer I and IV: lowest density was found in layer V and VI. The binding profile of the muscarinic cholinergic M1-subtype through the visual cortex shows a peak in cortical layer II and in the upper part of layer VI, whereas in the non-visual cortical regions cited the binding level was high in layer II/III, moderate in layer I and IV, and low in layer VI. Layers I to IV of the visual cortex contained the highest beta-adrenergic receptor densities, whereas only low binding levels were observed in the deeper layers. A similar laminar distribution was found also in the frontal, somatosensory and motor cortex. The density of noradrenaline uptake sites was high in all layers of the cortical regions studied, but with noradrenaline uptake sites somewhat more concentrated in the superficial layers than in deeper ones. The distinct laminar pattern of cholinergic and noradrenergic receptor sites indicates a different role for acetylcholine and noradrenaline in the functional anatomy of the cerebral cortex, and in particular, the visual cortex.     

The development of somatostatin immunoreactive neurons in cat visual cortical areas

The development of somatostatin immunoreactive (SOM-ir) neurons in cat striate and extrastriate cortex was studied to determine whether temporal changes in the morphology, distribution and density of SOM-ir neurons during development would provide clues to the emergence of specific cortical areas. The visual cortical areas examined included areas 17–19 and 7, posteromedial lateral suprasylvian, posterolateral lateral suprasylvian cortex and splenial visual area. We observed that the pattern of SOM-ir neurons in the cortical plate reflects the maturation of the cortical plate. At 1 week of age, SOM-ir neurons were only found in layers V and VI of the developing cortex; by 2 weeks of age, SOM-ir neurons were found in layer IV; and by 3 weeks of age, SOM-ir neurons were located in all layers of the cortex except layer I. SOM-ir neurons in the subplate were much more numerous under lateral cortical areas than under medial areas. This difference decreased over the first 2 postnatal weeks and by the 14th day after birth (P14), the distribution and numbers of SOM-ir neurons in the subplate/white matter had reached the adult pattern. The timing of exuberant SOM expression in the subplate suggests a function in the formation of visual corticocortical connections which begin to develop during the first postnatal week in the kitten.     

Cellular and subcellular localization of protein kinase C in cat visual cortex

Polyclonal antibodies against 3 protein kinase C (PKC) subtypes (I, II and III) were applied to localize the kinase in cat visual cortex. These antibodies exclusively stained neuronal cells. Both pyramidal and non-pyramidal cells exhibiting PKC-like immunoreactivity were concentrated in layers, II, III, V and VI with relatively few cells in layer IV. Electron microscopic examination did not reveal any presynaptic localization of the kinase. PKC immunoreactivity remained normal in a zone of cortex surgically isolated from the rest of the brain by an undercut procedure. These results suggest that PKC is heterogenously distributed in adult cat visual cortex; the kinase recognized by the polyclonal antibodies is localized postsynaptically in intracortical neurons of the superficial and deep cortical layers and the expression of the kinase is not regulated by extracortical input.     

Immunoreactivity for Taurine Characterizes Subsets of Glia,GABAergic and non-GABAergic Neurons in the Neo- and Archicortex of the Rat,Cat and Rhesus Monkey: Comparison with Immunoreactivity for Homocysteic Acid

The cerebral cortex is an area rich in taurine (2-aminoethanesulphonic acid), but only limited information exists regarding its cellular distribution. We therefore examined taurine-like immunoreactivity in the cerebral cortex of the rat, cat and macaque monkey using antiserum directed against glutaraldehyde-conjugated taurine. Immunostaining was assessed at the light and electron microscopic level, and patterns obtained in light microscopic studies were compared to those produced with antiserum to gamma-aminobutyric acid (GABA) and homocysteic acid (HCA). In all three species, strong taurine-like immunoreactive perivascular endothelial cells, pericytes and oligodendrocytes were found. These cells were located throughout the neuropil, which itself showed a low level of immunoreactivity. In rats and cats, a small number of weakly taurine-enriched neurons were observed, particularly in superficial layers. In all cortical areas of the macaque, however, glial staining was matched by strong, selective staining of subpopulations of cortical neurons which were distributed in a bilaminar pattern involving layers II/III and VI. In addition, in primary visual cortex, area 17, immunopositive neurons were also present in sublayer IVCbeta, while in the hippocampus strongly taurine-positive neurons were most conspicuous in the granule cell layer of the dentate gyrus. In all regions, strongly taurine-positive neurons constituted only a subpopulation of the neurons occupying a given layer. Examination of adjacent sections for GABA immunoreactivity showed that the most strongly taurine-positive neurons in layers II/III were immunoreactive for GABA. The cells located in layers IVCbeta and VI, and the granule cells of the dentate gyrus, however, were GABA-negative. The morphological features of these latter groups suggested that the antiserum to taurine identifies subsets of spiny stellate, small pyramidal and dentate granule cells. None of these neurons showed immunoreactivity with antiserum to HCA in the primate; HCA-positive glia were found along the pial and white matter boundaries of the cortex, and showed no overlap with strongly taurine-positive glial elements. Although a transmitter role for taurine may be unlikely, particularly in view of its enrichment in subpopulations of both inhibitory and excitatory cells, the capacity of taurine to influence membrane-associated functions in excitable tissues, and its selective distribution demonstrated here, provides the potential for a contribution to communication between cortical cells.     

Immunohistochemical localization of a membrane-associated, 4.1-like protein in the rat visual cortex during postnatal development

Expression and localization of a membrane-associated protein, an analog of erythrocyte protein 4.1, in the visual cortex were immunohistochemically studied in the rat, ranging in age from newborn to adult. In the adult, dendrites and somas of layer V pyramidal cells were stained by the antiprotein 4.1 antibody. In most of these immunoreactive neurons, the plasma membrane seemed to be preferentially stained. Neurons located in layers II and III of the cortex were only faintly stained, and those in layers IV and VI were not stained. At birth, the immunoreactivity was already present in pyramidal cells located in the upper part of the cortical subplate. Immature neurons located in the cortical plate were not stained by the antibody, suggesting that the 4.1-like protein is expressed only in the neurons that have differentiated or are differentiating. At postnatal days 2-8, immunoreactive neurons were dramatically increased in layers V and VI and intense labeling was seen at the apical dendrites of layer V pyramidal cells. Most of the stained processes of these and other neurons showed a sign of rapid dendritic growth, i.e., growth cones and filopidia. At days 10-17, the basal dendrites of pyramidal cells in layers II and III became detectable, although still slender. At days 20-37, these dendrites in layers II, III, and V became intensely immunoreactive, and dendritic spines were visualized by the antibody. Throughout all the ages, axons of neurons and neuroglia were not stained by the antibody. Also, most of the neurons in layer IV of the cortex were not immunoreactive. These results suggest that the 4.1-like protein is abundantly expressed in growing parts of the dendrites and spines. A hypothesis that this protein may play a role in synaptic plasticity in the developing visual cortex is discussed.     

Glutamic acid decarboxylase and somatostatin immunoreactivities in rat visual cortex

Antibodies to glutamic acid decarboxylase (GAD) and somatostatin (SS) were used to determine the laminar distribution and morphology of GAD- and SS-immunoreactive neurons and terminals in rat visual cortex. The present study demonstrates that GAD-immunoreactive neurons constitute several morphologically distinct subclasses of neurons in rat visual cortex. These subclasses of neurons can be distinguished by differences in soma size, soma shape, dendritic branching patterns, axonal arborizations, and location in the neuropil. GAD-immunoreactive neurons are found throughout all layers of visual cortex. They have nonpyramidal morphology and constitute roughly 15% of the total neuronal population. The laminar pattern of GAD-immunoreactive puncta is uneven, with a prominent band of terminals in layer IV. Numerous large GAD-positive puncta surround the somata and proximal dendrites of pyramidal cells in layers II, III, and V. SS-immunoreactive neurons constitute a less numerous and more restricted population of nonpyramidal neurons. Their somata are located mainly in layers II, III, V, and VI. Very few, if any, SS-immunoreactive neurons are found in layers I and IV. SS-immunoreactive terminals are arranged along vertical and diagonal collateral branches that have a beaded appearance. Finally, many neurons in the supra- and infragranular layers and in the white matter are immunoreactive to both glutamic acid decarboxylase and somatostatin. This coexistence of immunoreactivity to both GAD and SS may characterize a broad subclass of cortical nonpyramidal neurons.     

Postnatal development of the cholecystokinin innervation of monkey prefrontal cortex

Although the structure and function of primate prefrontal cortex undergo substantial modifications during postnatal development, relatively little is known about the maturation of neurotransmitter systems in these cortical regions. In the primate brain, cholecystokinin is present in the greatest concentrations in prefrontal regions. Thus, in this study, we used immunohistochemical techniques to investigate the postnatal development of the cholecystokinin innervation of monkey prefrontal cortex. In animals aged 4 days through adult, cholecystokinin immunoreactivity was present in nonpyramidal neurons that appeared to represent at least two distinct cell types. The most common type was a vertically oval bitufted neuron, located in layers II-superficial III, which typically had a radially descending axon that gave rise to short collaterals in layer IV. Another frequently observed cell type was a larger multipolar neuron located in the superficial half of layer III. The axon of these neurons branched locally in the vicinity of the cell body. The greatest density of cholecystokinin-containing neurons and processes was present in monkeys less than 1 month of age. The density of immunoreactive structures in every prefrontal region then progressively declined with increasing age, with the most marked changes occurring during the first postnatal year. As a result, the density of labeled neurons in adult monkeys was less than one-third of that in neonatal monkeys. However, labeled structures were significantly more dense in some ventromedial and orbital regions than in dorsal regions of the prefrontal cortex in neonatal, but not in older animals. In all animals, cholecystokinin-containing neurons were present in highest density in layers II-superficial III, and labeled terminal fields were observed in layers II, IV, and VI. In animals less than 1 month of age, fascicles of radial fibers traversed through layers III and V, whereas in animals 1 to 3 months of age, individual radial fibers rather than fiber bundles were present in layers III and V. In addition, immunoreactive pericellular arrays, which appeared to surround unlabeled nonpyramidal cells, were present in layers V and VI and the subcortical white matter in the youngest monkeys. Although many aspects of the cholecystokinin innervation of monkey prefrontal cortex remain constant during postnatal life, the distinct developmental changes in the cholecystokinin innervation of these regions suggest that it may play an important role in the maturation of the cortical circuitry that mediates the acquisition of certain cognitive abilities. © 1993 Wiley-Liss, Inc.     

Morphology, distribution, and synaptic relations of somatostatin- and neuropeptide Y-immunoreactive neurons in rat and monkey neocortex

Neurons in the monkey and rat cerebral cortex immunoreactive for somatostatin tetradecapeptide (SRIF) and for neuropeptide Y (NPY) were examined in the light and electron microscope. Neurons immunoreactive for either peptide are found in all areas of monkey cortex examined as well as throughout the rat cerebral cortex and in the subcortical white matter of both species. In monkey and rat cortex, SRIF-positive neurons are morphologically very similar to NPY-positive neurons. Of the total population of SRIF-positive and NPY-positive neurons in sensory-motor and parietal cortex of monkeys, a minimum of 24% was immunoreactive for both peptides. Most cell bodies are small (8 to 10 micron in diameter) and are present through the depth of the cortex but are densest in layers II-III, in layer VI, and in the subjacent white matter. From the cell bodies several processes commonly emerge, branch two or three times, become beaded, and extend for long distances through the cortex. The fields formed by these processes vary from cell to cell; therefore, the usual morphological terms bipolar, multipolar, and so on do not adequately characterize the full population of neurons. Virtually every cell, however, has at least one long vertically oriented process, and most processes of white matter cells ascent into the cortex. No processes could be positively identified with the light microscope as axons. The processes of the peptide-positive neurons form dense plexuses in the cortex. In each area of monkey cortex, SRIF-positive and NPY-positive processes form a superficial plexus in layers I and II and a deep plexus in layer VI. These plexuses vary in density from area to area. All appear to arise from cortical or white matter cells rather than from extrinsic afferents. In some areas such as SI and areas 5 and 7, the superficial plexus extends deeply into layers III and IV; and in area 17, two very prominent middle plexuses occur in layers IIIB through IVB and in the upper one-third of layer V; these are separated by layer IVC, a major zone of thalamic terminations, which contains very few SRIF- or NPY-positive processes. The density of the plexuses is greater for NPY-positive processes than for SRIF-positive processes in all areas. In the rat, the plexuses do not display a strict laminar organization but generally are densest in the supragranular layers (I to III) and decline steadily in the deeper layers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Short-axon neurons with two axon-like processes in the visual cortex of the cat. A Golgi study

In the visual cortex (areas 17, 18 and 19) of adult cats and a 40-day-old kitten, short-axon cells with two axon-like processes are described. These neurons belong to different cell types (spineless stellates of layers II/III and IV, fusiform cells of layers III, IV and V, and a large multipolar spineless cell of layer III). The axon-like processes present the same morphological characteristics as single axons of the corresponding neuronal type.     

Prenatal development of GABA-immunoreactive neurons in the human striate cortex.

The prenatal development of neurons immunoreactive to gamma-aminobutyric acid (GABA) in the striate cortex (area 17) of human foetuses aged from 14 weeks to term was studied immunocytochemically. In the 14 week foetus GABA-immunoreactive cells occurred in all layers of area 17 with the highest density in the marginal zone (MZ), subplate (SP), deep intermediate zone (IZ) and ventricular zone (VZ). The cortical plate (CP), which gives rise to most of the definitive adult cortical layers, had relatively low concentrations of GABAergic cells. By 17 weeks the density in the proliferative VZ had declined. At 20 weeks some of the adult layers were recognisable; the density of GABA-positive neurons was now highest in the definitive cortex, especially the deep layers (layers VI and V), was lower in the superficial cortical plate, and was lowest in IZ, where the white matter would form. The peak of GABA-immunoreactive neuronal density continued to move superficially during development, and was in layer IVc by 30 weeks. The laminar distribution stabilised from 30 weeks with three dense bands: in layer IVc and superficial V, layer IVa, and layers II and superficial III. The tangential distribution of GABAergic neurons was determined in two older brains (32 and 39 weeks) and no unequivocal spatial periodicity was observed in this plane. The mean cross-sectional area of GABAergic neurons in area 17 increased with foetal age, and also increased from superficial to deep layers at each age. Most GABA-immunoreactive neurons in younger brains contained immunonegative or weakly positive nuclei and had few visible processes, while in the older brains most neurons contained positive nuclei and had more visible processes. The proportion of GABA-immunoreactive bipolar cells declined during development while that of multipolar cells increased. GABAergic neurons thus differentiate early in human foetal striate cortex. They are initially most numerous in the proliferative layers deep to the developing definitive cortex; from 20 weeks of gestation, their peak moves superficially into the maturing deep layers (VI and V) and a stable laminar distribution is attained by 30 weeks, with peaks in layers II/IIIm, IVa and IVc/V. There is no obvious horizontal periodic distribution before term.     

Postnatal development of area 17 callosal connections in Tupaia.

The goal of the present study was to investigate the pattern of maturation of callosal projecting neurons in a well-studied mammalian visual system with unique structural and functional properties. Studies of the distribution pattern of interhemispheric connections in the adult tree shrew primary visual cortex reveal not only a high concentration of labeled neurons along the area 17/18 border, as in standard experimental animals such as the cat and monkey, but also numerous callosal projecting neurons in the adjacent dorsal part of area 17, which largely corresponds to the binocular visual field (Kretz and Rager, Exp. Brain Res. 82:271, '90). Callosal projections were anatomically traced in 11 tree shrews (Tupaia belangeri) at various ages between postnatal day 7 (7, 9, 10, 13, 15, 17, 19, and 26 days old) and adulthood (107 days old). In each animal, four injections of wheat germ agglutinin conjugated to horseradish peroxidase were made in a standard configuration into the striate cortex of one hemisphere. In young tree shrews only 7 and 9 days old, heavily labeled terminal axon structures could be seen in the white matter and in layer VI of the opposite hemisphere. Only a few labeled neurons, however, were detected in layer III. The small number of labeled neurons indicated that early in postnatal development, only a few callosal axons had invaded the upper cortical layers. By 10 days of age, the number of supragranular neurons was increasing and the maximal value was counted in a 13-day-old tree shrew. A sharp decline in the number of labeled supragranular neurons was noticed--about 94% in our case--between days 13 and 15. In animals more than 15 days old, the distribution pattern and the density of the neurons looked like the pattern seen in the adult Tupaia brain. The labeled cells were mostly concentrated in layers II and III. The majority of neurons resembled typical pyramidal cells. However, some of the neurons in sublayer IIIc had elongated cell bodies oriented parallel to the laminar boundaries. In contrast to the supragranular cells found in all stages investigated, small populations of labeled cells in layer VI were observed in 9- to 17-day-old tree shrews only. In young postnatal animals 7 to 13 days old, a peculiar cell type was labeled on the ipsilateral side. In coronal sections these cell bodies formed a continuous band that extended from the ventricular wall to the subcortical white matter. These cells might belong to a population of cells still in migration.     

Autoradiographic analysis of adrenergic receptors in the mammalian brain

Noradrenaline (NA) exerts its physiological and pharmacological effects in the central nervous system by interacting with specific receptor sites which are divided into four subtypes, namely alpha-1, alpha-2, beta-1 and beta-2 adrenoceptors. Alpha-1 and beta-1 receptors are thought to be neuronal and post-synaptic, whereas alpha-2-R are neuronal pre- and postsynaptic and beta-2-R have a non neuronal (glial, vascular) localization. The autoradiographic localization of adrenergic receptors is requisite to a better understanding of adrenergic modulation in the nervous system. It complements analyses of adrenergic fibers and terminals and allows comparisons between afferent transmission and various receptor systems. In addition, receptor autoradiography is a preliminary step towards non invasive, in vivo receptor imaging using positron emission tomography (PET). Classical autoradiographic methods using tritium-labeled ligands are relatively tedious, as they require exposure times of several months. In order to circumvent these difficulties, an autoradiographic procedure was developed for visualization of I-125-labeled ligands. The method is validated by its application to the analysis of neuronal postsynaptic (alpha-1 and beta-1) adrenoceptors, in ferret visual cortex, in the forebrain of normal and reeler mutant mice and in the embryonic mouse brain. Distributions of alpha-1 and beta-1 adrenoceptors are studied using revelation of HEAT and ICYP binding sites, respectively. The cerebral cortex of ferret was chosen because it is widely used in vision research. The density of both alpha-1 and beta-1 adrenoceptors shows laminar heterogeneities. Beta-receptors are most heavily concentrated in cortical layers I, II and III, but very low in layer IV and moderately represented in layers V and VI. In contrast, alpha-1 receptors are more diffusely distributed, although preferentially concentrated in layer IV and, to a lesser extent, in upper cortical laminae. The two adrenoceptors are thus segregated in the radial dimension of the cortex, following distributions which are nearly complementary. These observations suggest that alpha- and beta-adrenoceptors might be associated with different stages and/or modes of information processing in the primary visual area. Adrenoceptor distribution was mapped in normal and reeler mice, in order to correlate receptor patterns with architectonic anomalies known to exist in reeler mutant mice. In normal mice, beta-1-receptors predominate in striatum, cortical layers I to III, hippocampal regio superior and some thalamic nuclei; they are moderately concentrated in cortical layers V and VI and poorly represented in lamina IV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Widespread projections from subgriseal neurons (layer VII) to layer I in adult rat cortex

Theories of information processing and plasticity in mammalian cortex often rely on knowledge of intracortical networks studied in rodent cortex. Accordingly, the contribution of all cells involved in this circuitry is potentially significant, including connections from a subset of neurons that persist from the developmental subplate, called subgriseal neurons in the present study. Ascending corticocortical connections from subgriseal neurons were identified by using in vivo transport of fluorescent retrograde tracers from discrete applications confined to cortical layer I (approximately 1 mm2) or from injections placed into superficial cortical layers. Applications restricted to cortical layer I can be identified by a subsequent retrograde labeling pattern that includes neurons in layers II/III and V but not those in layer IV. In contrast, when retrograde tracer is deposited in layers II/III, layer IV cells are also labeled. By using this identification technique in juvenile and adult rats, widespread interareal projections to superficial layers, including unequivocal connections to cortical layer I, were found to originate from a tangential band of neurons directly below the conventionally identified gray matter (i.e., subgriseal) and from a smaller number of cells in the white matter (WM) proper. Subgriseal and WM neurons were labeled below application and injection sites in somatosensory, auditory, visual, motor, frontal, and adjacent areas at distances of more than 4 mm. However, the subgriseal-to-superficial pathway was not sensitive to nonfluorescent retrograde tracers including horseradish peroxidase. Because neurons in the deeper cortical layers can be strongly influenced through input to their apical dendritic extensions in cortical layer I, the widespread connections described in the present study indicate that the ascending subgriseal projections should be considered in models of mature cortical function.     

Characterization of transient cortical projections from auditory, somatosensory, and motor cortices to visual areas 17, 18, and 19 in the kitten

We have examined the anatomical features of ipsilateral transient cortical projections to areas 17, 18, and 19 in the kitten with the use of axonal tracers Fast Blue and WGA-HRP. Injections of tracers in any of the three primary visual areas led to retrograde labeling in frontal, parietal, and temporal cortices. Retrogradely labeled cells were not randomly distributed, but instead occurred preferentially at certain loci. The pattern of retrograde labeling was not influenced by the area injected. The main locus of transiently projecting neurons was an isolated region in the ectosylvian gyrus, probably corresponding to auditory area A1. Other groups of transiently projecting neurons had more variable locations in the frontoparietal cortex. The laminar distribution of neurons sending a transient projection to the visual cortex is characteristic and different from that of parent neurons of other cortical pathways at the same age. In the frontoparietal cortex, transiently projecting neurons were located mainly in layer 1 and the upper part of layers 2 and 3. In the ectosylvian gyrus, nearly all the neurons are located in layers 2 and 3. In addition, a few transiently projecting neurons are found in layer 6 and in the white matter. Transiently projecting neurons have a pyramidal morphology except for the occasional spindle-shaped cell of layer 1 and multipolar cells observed in the white matter. Anterograde studies were used to investigate the location of transient fibers in the visual cortex. Injections of WGA-HRP at the site of origin of transient projections gave rise to few retrogradely labeled cells in areas 17, 18, and 19, demonstrating that transient projections to these areas are not reciprocal. Although labeled axons were found over a wide area of the posterior cortex, they were more numerous over certain regions, including areas 17, 18, and 19, and absent from other more lateral cortical regions. Transient projecting fibers were present in all cortical layers at birth. Plotting the location of transient fibers in numerous sections and at all ages showed that these fibers are not more plentiful in the white matter than they are in the gray matter. We found no evidence that the white/gray matter border constituted a physical barrier to the growth of transient axons. Comparison of the organization of this transient pathway to that of other transient connections is discussed with respect to the development of the cortex.     

GABAA/benzodiazepine receptor-like immunoreactivity in rat and monkey cerebellum

The cellular and subcellular localization of GABAA/benzodiazepine receptor-like immunoreactivity in the rat and monkey cerebellum has been studied with a monoclonal antibody (E9) directed against the alpha-subunit of purified GABAA/benzodiazepine receptors. At both the light and electron microscopic level E9 immunoreactivity is located in all 3 layers of the cerebellar cortex and within the deep cerebellar nuclei. The reaction product accumulates within the cytoplasm of neurons and their dendrites but axons are not immunoreactive. Glial cells in the white matter and the cortical layers are also unlabeled, although in some instances Bergmann glia do contain reaction product. The overall distribution and cellular and subcellular localization of E9 immunoreactivity is identical for both monkey and rat cerebellum. On the basis of cell size, morphology, and location it is evident that E9 immunoreactivity occurs in examples of all 5 neuronal types in the cerebellar cortex: Purkinje cells, Golgi type II cells, granule cells, and stellate and basket cells. However, the distribution of the reaction product within the cells is more selective. For example, electron microscopy demonstrates that axonal processes and terminals are not E9 immunoreactive with the single exception of the mossy fiber terminals in the granular layer. Also, examples of unlabeled axon terminals resembling those derived from Golgi type II cells, basket cells, and stellate cells form synapses with immunoreactive dendrites and cell bodies in the cortical layers. Finally, in the deep cerebellar nuclei unreactive axon terminals make symmetric synapses with immunostained neurons and dendrites. These results show that E9 monoclonal antibodies label neurons and portions of their processes which are postsynaptic in GABA-mediated inhibitory circuits, and demonstrates that this antiserum can be used as a morphological marker for cells which make GABAA/benzodiazepine receptors.     

Neurovirulent simian immunodeficiency virus induces calbindin-D-28K in astrocytes

Astrocyte activation has been postulated to be a major contributor to functional changes in the brain of AIDS patients. We assessed astrocyte activation in the simian immunodeficiency virus (SIV) model. Four groups of macaque brains were examined: uninoculated controls, animals inoculated with virus that did not cause disease, animals inoculated with virus that caused AIDS but did not cause encephalitis, and animals with SIV encephalitis. We examined expression of calbindin-D-28K, a calcium binding protein that is upregulated in astrocytes during excitotoxic events, as well as glial fibrillary acidic protein (GFAP). The presence of calbindin in astrocytes was confirmed by double-labeling using confocal microscopy. Increases in calbindin staining were most apparent in the white matter, but increases in GFAP staining were most apparent in middle layers of the cerebral cortex. Six of the seven animals with SIV encephalitis had calbindin immunoreactive astrocytes in the subcortical white matter, corpus callosum, internal capsule, cerebral peduncle, pontine white matter, and cerebellar white matter. Very rarely, a few, very lightly calbinding-immunoreactive astrocytes were present in the uninoculated control brains. The increase in calbindin expression by astrocytes in SIV encephalitis suggests that these cells are subject to calcium toxicity. In uninoculated control macaques, and in macaques inoculated with virus that did not cause disease, GFAP-immunoreactive astrocytes were present throughout the subcortical white matter and in layer I, but very few were found in layers III–V of the cerebral cortex. Two animals that died of AIDS without encephalitis had somewhat higher numbers of GFAP immunoreactive astrocytes in middle cortical layers. In seven animals that received passaged neurovirulent virus and developed both AIDS and encephalitis, the number of GFAP-immunoreactive astrocytes in middle cortical layers was high, indicating widespread astrocyte activation.     

A study of tachykinin-immunoreactive neurons in monkey cerebral cortex

Immunocytochemical methods were used to localize tachykinin-like immunoreactivity within neurons of the monkey cerebral cortex. Three primary antibodies were used: polyclonal antisera raised against fragments of substance P and substance K that excluded the carboxyl termini of these peptides, and a monoclonal antibody that recognized the carboxyl terminus of the tachykinin family. Each antibody stained 2 populations of cortical nonpyramidal neurons: (1) A small number of large, intensely stained cells that give rise to long, coarsely beaded processes; (2) a relatively large number of small, lightly stained cells that are embedded in dense plexuses of stained punctate profiles. The large, dark cells are present in a superficial band that includes layers II and III, and in a deep band that includes layer VI and the subjacent white matter. The smaller, pale cells are present in the middle layers of cortex (layers IV and/or V). Colocalization studies indicate that virtually all the small tachykinin-immunoreactive neurons also display GABA immunoreactivity. The larger cells are not GABA-positive, but display both somatostatin-like and neuropeptide Y-like immunoreactivity. The immunocytochemically stained beaded processes and punctate profiles from plexuses that vary in density and laminar distribution among different areas of monkey cortex. The coarsely beaded processes form a basic quadrilaminar pattern, with relatively dense plexuses in layers I and VI and in 2 middle layers, usually III and V. However, this pattern varies considerably from area to area. Electron microscopically, the large cells contain a rich collection of cytoplasmic organelles, particularly Golgi complex, while the small cells contain relatively few organelles. Both types of cells, including large neurons in the white matter, receive symmetric and asymmetric synaptic contacts on their somata and proximal dendrites. The numbers of these axosomatic contacts are low. Virtually all synaptic contacts formed by immunoreactive terminals possess symmetric membrane thickenings. In 2 areas examined in detail (areas 2 and 4), pyramidal cell somata and dendrites are the major targets of the immunoreactive synaptic terminals.     

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.