The neuronal composition of area 17 of rat visual cortex. II. The nonpyramidal cells

In the preceding article the characteristics of the various types of pyramidal cells present in area 17 of rat visual cortex were described (Peters and Kara, '85). In the present article the nonpyramidal cell population of this cortex is considered. It is known from Golgi preparations that in layers II-VIa there are bipolar cells, smooth or sparsely spinous multipolar and bitufted cells with either unmyelinated local plexus or myelinated axons, and chandelier cells. Each of these cell types has been previously examined in Golgi-electron microscopic preparations. The question now being asked is whether the information about the characteristics of these different types of nonpyramidal cells derived from the Golgi-electron microscopic studies can be used to identify the cell bodies of nonpyramidal cells in tissue prepared for conventional electron microscopy. If this can be done then the neuronal composition of area 17 can be determined. It has been found that the cell bodies of bipolar cells can be readily identified because they are elongate and have nuclei with a vertical infolding and few axosomatic synapses, which are of both the symmetric and asymmetric varieties. Evidence is presented to show that there are two types of bipolar cells, small ones and large ones, the large ones being distinguished by their well-developed endoplasmic reticulum in which the cisternae are arranged parallel to the cell surface. Bipolar cells account for 6% of the neuronal profiles in layer II/III, 3% in layer IV, 5% in layer V, and 2% in layer VIa. The cell bodies of other types of nonpyramidal cells in layers II-VIa cannot be distinguished from each other in thin sections, because recognition of the different cell types depends upon the characteristics and distribution of their dendrites and axons. However, it is evident that in this group of neurons there are some with small cell bodies and others with large cell bodies, and in both size groups there are varieties of neurons which can be recognized from the characteristics of their perikaryal cytoplasm. All of these neurons have both symmetric and asymmetric axosomatic synapses. The greatest number of these nonpyramidal cells which are not bipolar in form is found within layer II/III, where they account for 7% of all neuronal profiles. These neurons comprise 4% of all neuronal profiles in layer IV, 6% in layer V, and 2% in layer VIa. Layers I and VIb contain only nonpyramidal cells, but these are different from the ones in layers II-VIa.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glutamate-positive neurons and axon terminals in cat sensory cortex: a correlative light and electron microscopic study

Immunocytochemical methods were used to perform a correlative light and electron microscopic study of neurons and axon terminals immunoreactive to the antiglutamate (Glu) serum of Hepler et al. ('88) in the visual and somatic sensory areas of cats. At the light microscopic level, numerous Glu-positive neurons were found in all layers except layer I of both cortical areas. On the basis of the dendritic staining of Glu-positive cells, two major morphological categories were found: pyramidal cells, which were the most frequent type of immunostained neuron, and multipolar neurons, which were more numerous in layer IV of area 17 than in any other layer. A large number of Glu-positive neurons, however, did not display dendritic labelling and were considered unidentified neurons. Counts of labelled neurons were performed in the striate cortex; approximately 40% were Glu-positive. Numerous lightly stained punctate structures were observed in all cortical layers: the majority of these Glu-positive puncta were in the neuropil. After resectioning the plastic sections for electron microscopy it was observed that: 1) the majority of neurons unidentifiable at light microscopic level were indeed pyramidal neurons except in layer IV of area 17, where many stained cells were probably spiny stellate neurons. Some Glu-positive neurons, however, exhibited clear ultrastructural features of nonspiny nonpyramidal cells; 2) all synaptic contacts made by Glu-positive axon terminals were of the asymmetric type, but not all asymmetric synaptic contacts were labelled. The vast majority of postsynaptic targets of Glu-positive axons were unlabelled dendritic spines and shafts. The present results provide further evidence that Glu (or a closely related compound) is probably the neurotransmitter of numerous excitatory neurons in the neocortex.     

A quantitative analysis of parvalbumin neurons in rabbit auditory neocortex

Parvalbumin (PV) is a calcium-binding protein present in GABAergic cells in the cerebral cortex and in thalamic relay neurons. In the present study, parvalbumin immunocytochemistry (PVi) and stereological methods were used to obtain estimates of cortical volume, total neuron number, laminar density, and the percentage of PV-immunoreactive neurons in auditory neocortex. PVi clearly delineated the primary auditory cortex (AI), which was characterized by two PV+ bands: dense terminal-like labeling within lamina III/IV and PV+ somata in lamina VIa. Stereological analysis of Nissl-stained sections revealed that the total number of neurons in rabbit AI was 1.48 × 10⁶ with a mean neuronal density of 55 × 10³/mm³. Based on a mean cortical thickness of 1.92 mm, there are approximately 106,000 neurons in a 1 mm² column of auditory cortex. PVi yields an extraordinary Golgi-like staining of nonpyramidal cells in all cortical layers. PV+ nonpyramidal cells constitute approximately 7.0% of the neurons in AI. There were significant differences in the morphology and density of PV+ neurons across layers. Although only 5% of cells in lamina I were PV+, three nonpyramidal cell types were present. Lamina II had the highest numerical density within AI but the lowest percentage of PV+ neurons (3.3%). Lamina II, however, contained the greatest diversity of PV+ nonpyramidal cell types, which included small multipolar cells, bipolar cells, and, less frequently, large cells of the bitufted, bipolar, and stellate varieties. Lamina IV had one of the highest numerical densities (67.6 × 10³ neurons/mm³) and contributed nearly 27% of the total neuron number in AI. The numerical density of PV+ nonpyramidal cells was also greatest within lamina IV (7.1 × 10³ /mm³) where they formed 10.4% of the neuronal population. PV+ nonpyramidal cells in lamina IV and lamina III were predominantly large basket-type cells with bitufted dendritic domains and tangentially oriented local axonal plexuses. The terminal-like label within lamina III/IV derived in part from the basket-cell axons, which formed pericellular arrays around unstained somata. Cell-sparse lamina V contained the largest PV+ nonpyramidal cells in AI. These cells, which formed 11% of the neuron population in lamina V, were notable for their tangentially oriented dendritic fields and local axonal arbors. PVi partitioned lamina VI into VIa and VIb. Large multipolar nonpyramidal cells were distributed throughout lamina VI and made up approximately 6% of the total population. Lamina VIa contained a band of lightly labeled PV+ pyramidal neurons that formed 15% of the neuronal population. Double-labeling experiments revealed that some PV+ pyramidal neurons within VIa also project to the ventral subdivision of the medial geniculate body (MGB). PVi demarcated the three major subdivisions of the MGB: the ventral (vMGB), dorsal (dMGB), and internal (iMGB) nuclei. The vMGB was strongly PV immunoreactive due to dense labeling of the neuropil and moderately labeled somata. The dMGB was characterized by scattered large PV+ cells and coarse PV+ axons. Relative to the vMGB, the neuropil of the dMGB contained only light terminal-like labeling. The internal MGB contained few, if any, PV+ somata and had the least terminal-like labeling of all MGB subdivisions. Because calcium-binding proteins delineate functionally distinct, parallel pathways to sensory neocortex, they will be useful chemoarchitectonic tools for guiding future connectional studies of the MGB with the auditory neocortex and brainstem. © 1994 Wiley-Liss, Inc.     

VIP- and CCK-like-immunoreactive neurons in the hedgehog (Erinaceus europaeus) and sheep (Ovis aries) brain

The distribution pattern and the morphology of vasoactive intestinal polypeptide (VIP)- and cholecystokinin (CCK)-like-immunoreactive neurons were studied in the brain of the hedgehog and the sheep by means of the peroxidase-antiperoxidase immunocytochemical method. A total of 34 hedgehogs and 26 sheep of both sexes were used. Fourteen hedgehogs and 13 sheep received an intracerebroventricular injection of colchicine that enhanced the immunostaining and revealed "new" immunoreactive cell bodies. VIP-immunoreactive bipolar and multipolar neurons were observed in both species in the cerebral cortex, hippocampal formation, amygdaloid complex, hypothalamus, and central gray substance of the midbrain. CCK-immunoreactive bipolar, bitufted, and multipolar neurons displayed a broader distribution in both mammals than VIP neurons and were found in the cerebral cortex, the hippocampal formation, the amygdaloid complex, the hypothalamus, the mesencephalon, and the pons. In the cortex, in both the hedgehog and the sheep, VIP neurons were located in all layers but were concentrated in layers II and III, with the majority being typical bipolar. CCK neurons were more numerous in the superficial layers (I-III) but were found in the deep layers as well. They were bipolar, bitufted, or multipolar in morphology. From these neurons a small percentage, which were located almost exclusively in layers II and III of the visual cortex, exhibited also VIP immunoreactivity. Perikarya of such double-labeled cells were ovoid or round in shape with one or two main processes emanating from each pole of the cell body and oriented perpendicularly to the pia. The coexistence of the two peptides within individual neurons of the cortex has not been reported in other species and its physiological significance is discussed in relation to the GABAergic neurons of the cortex.     

A reassessment of the forms of nonpyramidal neurons in area 17 of cat visual cortex

The nonpyramidal neurons in area 17 of cat visual cortex have been examined in Golgi preparations. From their dendritic patterns, neurons are classified as being multipolar, bitufted, or bipolar, and on the basis of the abundance of dendritic spines as spinous, sparsely spinous, or smooth. When neurons are so classified seven different types of nonpyramidal neurons are encountered in layers II through V. Three of the types of multipolar neurons in layers II through V have spherical dendritic trees. The small multipolar cells have smooth dendrites and are the smallest neurons in the cortex. They have short dendrites and dense local axonal plexuses and occur throughout layers II to V The sparsely spinous stellate cells have longer dendrites, are confined to layer II/III, and have local axonal arborizations, whereas the spinous stellate cells are limited to layer IV. A fourth type of multipolar neuron in layers II through V is the basket cell. Such neurons have elongate dendritic trees and either smooth or sparsely spinous dendrites. Depending upon the orientation of the neurons in the sections, their axons appear to form arcades or long, horizontally extended branches, or a mixture of these two axonal patterns. The terminal portions of the axons of these basket cells pass around the cell bodies of adjacent neurons. The two types of bitufted neurons in layers II through V have vertically oriented dendritic trees. One type, the chandelier cell, has smooth dendrites and a characteristic axon forming vertical strings of terminals. The other sparsely spinous bitufted neurons have axons producing vertically oriented plexuses. The remaining type of neuron encountered in layers II through V is a bipolar cell. The bipolar cell has a single major dendritic trunk arising from each pole of the cell body, and each of these gives rise to a very narrow, long, and vertically oriented dendritic tree. The axon usually takes origin from one of the primary dendrites. In layer I are horizontally oriented, bitufted cells with smooth dendrites. The axons of these horizontal cells of layer I arise from one of the primary dendritic trunks and appear to form a plexus confined to layer I. Horizontally oriented neurons are also present in deep layer VI, but the horizontal cells of layer VI are bipolar. The other two neuronal types in layer VI are multipolar cells with sparsely spinous dendrites. The larger of these two types resembles the basket cells in layers II through V, the only important difference between them being that in addition to the long horizontal branches, the axons of the basket cells of layer VI have a long ascending branch which reaches at least as far as layer IV. The other sparsely spinous cells of layer VI are medium sized. Their axons take a descending and oblique course before elaborating a locally distributed plexus. The various types of neurons defined in this study are compared with neurons described by previous authors who have examined the populations of nonpyramidal cells in area 17 of cat visual cortex and in other visual and nonvisual cortical areas of cats, monkeys, and rodents. In some cases it has been possible to postulate the functional roles that particular types of neurons might play within cat visual cortex.     

High-Resolution Light and Electron Microscopic Immunocytochemistry of Colocalized GABA and Calbindin D-28k in Somata and Double Bouquet Cell Axons of Monkey Somatosensory Cortex

A simple method for high-resolution immunocytochemical colocalization of different antigens in semithin sections 1 - 3 μm thick was used to study the colocalization of the calcium binding protein calbindin D-28k (calbindin) with γ-aminobutyric acid (GABA) in double bouquet cells of monkey (Macaca fuscata) somatosensory cortex. Double bouquet cells were first visualized in vibratome sections by pre-embedding immunocytochemical staining for calbindin. Sections containing calbindin-immunoreactive somata and double bouquet cell axons were then osmicated, embedded in Araldite, resectioned at 1–3μm and stained for GABA by postembedding immunocytochemistry after elution of the bound anti-calbindin antibodies. Other semithin sections adjacent to those eluted and still containing calbindin immunoreactive somata and processes were resectioned at 60–70 nm for electron microscopy and stained immunocytochemically for GABA by the postembedding immunogold procedure. Calbindin-positive cells are most numerous in layer II and upper layer III, where they outnumber those in all other layers combined. In layers II and upper III, -30% of the stained cells are pyramidal and do not colocalize GABA. Only approximately two-thirds of the calbindin-stained nonpyramidal cells in these layers colocalize GABA, but among these virtually all the calbindin-positive double bouquet cells and their axons are GABA-immunoreactive. In deeper layers all calbindin-positive cells are nonpyramidal and all colocalize GABA. At the electron microscopic level, however, significant numbers of calbindin-positive axon terminals making symmetrical synapses are not GABA-immunoreactive. These results suggest the calbindin cells of monkey somatosensory cortex are a heterogeneous population that includes GABAergic and non-GABAergic cell types.     

Golgi, histochemical, and immunocytochemical analyses of the neurons of auditory-related cortices of the rhesus monkey

Morphological characteristics of the neurons of the auditory cortical areas of the rhesus monkey were investigated using Golgi and horseradish peroxidase methods. Neurons of the auditory cortices can be segregated into two categories, spinous and nonspinous, which can be further subclassified according to their dendritic arrays. The spinous neurons include pyramidal, "star pyramid," multipolar, and bipolar cells. As in other cortices, pyramidal cells are found in layers II-VI and appear to be the most numerous of all cortical neurons. The "star pyramids" have radially oriented dendrites with a less prominent apical shaft and are found mainly in the middle cortical layers. The spinous multipolar neurons are also found in the middle cortical layers and have their dendrites radially arrayed but have no apical dendrite. The spinous bipolar cells, found in the infragranular layers, occur most frequently in the lateral auditory association cortex. The nonspinous neurons include neurogliaform, multipolar, bitufted, and bipolar cells and are found in all cortical layers. The neurogliaform cells are the smallest of all neurons and have radially arrayed, recurving dendrites. The nonspinous multipolar cells also have radially arrayed dendrites but vary in size from being confined to one cortical layer to extending across four laminae. The bitufted neurons are subclassified into three groups: neurons whose primary dendrites arise radially from their somata, those whose dendrites arise from two poles of their somata, and those that have a single primary dendrite arising from one pole and multiple dendrites from another pole of their somata. The nonspinous bipolar cells also have several variants but usually have dendrites arising from two poles of the somata. The chemical characteristics of the auditory neurons were investigated using histochemical and immunocytochemical methods. Peptidergic neurons, i.e., cholecystokinin-, vasoactive intestinal polypeptide-, somatostatin-, and substance P-reactive neurons are found in the various subregions of the auditory cortices and are distributed differentially in the cortical laminae. These neurons are of the nonpyramidal type. Gamma aminobutyric acid-reactive neurons are also nonpyramidal cells and they are found in all cortical layers. Their numbers varied among the cortical laminae in the different auditory regions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neurons of the lateral entorhinal cortex of the rhesus monkey: a Golgi, histochemical, and immunocytochemical characterization.

This study identifies the neuronal types of the rhesus monkey lateral entorhinal cortex (LEC) and discusses the importance of these data in the context of the connectional patterns of the LEC and the possible role of these cells in neurodegenerative diseases. These neuronal types were characterized with the aid of Golgi impregnation techniques. These characterizations were based upon their spine densities, dendritic arrays, and, where possible, axonal arborizations. The cells could be segregated into only spinous and sparsely spinous types. The most numerous spinous types were pyramidal neurons. Other spinous types included multipolar, vertical bipolar and bitufted, and vertical tripolar neurons. The sparsely spinous neuronal types consisted of multipolar, horizontal bipolar and bitufted, and neurogliaform cells. These cells were further classified with the aid of histochemical stains and immunocytochemical markers. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry stained multipolar, bipolar, and bitufted neurons. Stain for cytochrome oxidase (CO) was found in pyramidal and nonpyramidal cell types. Immunocytochemical techniques revealed several nonpyramidal neurons that contain somatostatin (Som) or substance P (SP). This study complements previous analyses of the neuronal components described in the LEC and adds further information about the distribution of selected neurochemicals within this cortex.     

Somatostatin (SRIF)-like immunoreactivity in subcortical and cortical visual centers of the rat

The distribution of neuronal elements containing immunoreactive somatostatin (I-SRIF) in the rat central visual pathway was examined by light-microscopic immunocytochemistry. These studies were concerned with the location and morphology of neurons and innervated cells and the distribution of fiber and terminal plexuses in the primary visual cortex (area 17), visual association areas 18 and 18a, the superior colliculus, the lateral geniculate nucleus, and the pretectum. In the superior colliculus, I-SRIF-containing fibers and perikarya were distributed predominantly in the superficial, or visual, layers; these elements were moderately dense and occupied the entire mediolateral extent of these layers. In the intermediate and deep layers, immunoreactive neurons were widely scattered, and fibers were located mainly in the medial third. Immunoreactive cell populations in the superior colliculus included small bipolar neurons with fusiform perikarya and multipolar neurons with round to ovoid perikarya. In the pretectum, the peptide was demonstrable in large and small multipolar neurons of the nucleus of the optic tract and in the posterior and olivary pretectal nuclei. I-SRIF-containing neurons were also present in the nucleus of the posterior commissure, the nucleus of Edinger-Westphal, and the ventral division of the lateral geniculate nucleus. In the visual cortex, the peptide was present in all layers and in a variety of morphologically defined cell populations, including some which are presumed excitatory (pyramidal and bipolar cells) and others which are presumed inhibitory (bitufted and stellate cells). Our data suggest that somatostatin is involved in visual and visuomotor reflex pathways and in the horizontal optokinetic nystagmus reflex pathway. These results provide a foundation for further studies to evaluate the role of this peptide in visual processes.     

Different populations of GABAergic neurons in the visual cortex and hippocampus of cat contain somatostatin- or cholecystokinin-immunoreactive material

The coexistence of gamma-aminobutyric acid (GABA), glutamate decarboxylase (GAD), and cholecystokinin (CCK)- or somatostatin-immunoreactive material in the same neurons was studied in the hippocampus and visual cortex of the cat. One-micrometer-thick serial sections of the same neuron were reacted to reveal different antigens by the unlabeled antibody enzyme method. All CCK- and somatostatin-immunoreactive neurons in the cortex and all CCK-immunoreactive and the majority of somatostatin-immunoreactive neurons in the hippocampus that could be examined in serial sections were also immunoreactive for GABA. In neurons that were immunoreactive for GAD it was often possible to demonstrate immunoreactivity for one of the peptides as well as for GABA. GABA-immunoreactive neurons, as revealed by an antiserum to GABA, were present in all layers of the cortex and hippocampus, and their shape, size, and distribution were similar to GAD-immunoreactive neurons. All GAD-immunoreactive neurons were also positive for GABA, but the latter staining revealed additional neurons. CCK/GABA- and somatostatin/GABA-immunoreactive neurons were present mainly in layers II and upper III and in layers V and VI in the visual cortex. CCK/GABA-immunoreactive neurons were most frequently present in the strata oriens, pyramidale, and moleculare of the hippocampus and in the polymorph cell layer of the dentate gyrus. Somatostatin/GABA-immunoreactive neurons were localized mainly in the stratum oriens and in the hilus of the fascia dentata. The two peptides could not be found in the same neuron. The majority of neurons that were GABA immunoreactive did not stain for either peptide. The presence of CCK- and somatostatin-immunoreactive material in GABAergic cortical neurons raises the possibility that neuroactive peptides affect GABAergic neurotransmission.     

Calretinin in the entorhinal cortex of the rat: distribution, morphology, ultrastructure of neurons, and co-localization with gamma-aminobutyric acid and parvalbumin

Calretinin is a marker that differentially labels neurons in the central nervous system. We used this marker to distinguish subtypes of neurons within the general population of neurons in the entorhinal cortex of the rat. The distribution, morphology, and ultrastructure of calretinin-immunopositive neurons in this cortical area were documented. We further analyzed the co-localization of the marker with gamma-aminobutyric acid (GABA) and studied whether calretinin-positive neurons project to the hippocampal formation. Methods used included single-label immunocytochemistry at the light and electron microscopic level, retrograde tracing combined with immunocytochemistry, and double-label confocal laser scanning microscopy (CLSM). The entorhinal cortex contained calretinin-positive cells in a scattered fashion, in all layers except layer IV (lamina dissecans). Bipolar and multipolar dendritic configurations were present, displaying smooth dendrites. Bipolar cells had a uniform morphology whereas the multipolar calretinin cell population consisted of large neurons, cells with long ascending dendrites, horizontally oriented neurons, and small spherical cells. Retrograde tracing combined with immunocytochemistry showed that calretinin is not present in cells projecting to the hippocampus. Few synapic contacts between calretinin-positive axon terminals and immunopositive cell bodies and dendrites were seen. Most axon terminals of calretinin fibers formed asymmetrical synapses, and immunopositive axons were always unmyelinated. Results obtained in the CLSM indicate that calretinin co-exists in only 18-20% of the GABAergic cell population (mostly small spherical and bipolar cells). Thus, the entorhinal cortex contains two classes of calretinin interneurons: GABA positive and GABA negative. The first class is presumably a classical, GABAergic inhibitory interneuron. The finding of calretinin-immunoreactive axon terminals with asymmetrical synapses suggests that the second class of calretinin neuron is a novel type of a (presumably excitatory) interneuron.     

Synaptic organization of GABAergic neurons in the mouse SmI cortex

Immunocytochemical methods were used to examine GABAergic neurons in the barrel region of the mouse primary somatosensory cortex. GABAergic neurons occur in all layers of the barrel cortex but are more concentrated in the upper portion of layers II/III and in layers IV and VI. Nine cells in layer IV were examined with the electron microscope, and portions of their dendrites were reconstructed from serial thin sections. These cells are of the nonspiny, multipolar or bitufted varieties, and some of them have beaded dendrites. The labeled cell bodies and their reconstructed dendrites were postsynaptic at asymmetrical synapses with thalamocortical axon terminals labeled by lesion-induced degeneration and with unlabeled axon terminals. Each cell also received symmetrical synapses from GABAergic axon terminals and from unlabeled axon terminals. Our results indicate that GABAergic cell bodies and processes receive synapses from thalamocortical axon terminals but that different cells display marked differences in the proportion of thalamocortical and other synapses they receive. These results indicate that GABAergic cells form a heterogeneous population with respect to their morphologies and patterns of synaptic inputs. The synaptic sequences revealed here for GABAergic neurons represent an anatomical substrate for various inhibitory processes known to occur within the cerebral cortex.     

Selective cytochemical demonstration of glycoconjugate-containing terminal N-acetylgalactosamine on some brain neurons

Paraffin embedded sections of rat, mouse, dog, and human brain were stained with a battery of lectin-horseradish perioxidase conjugates to localize and characterize glycoconjugates. In the rat and mouse cerebral cortex, a subpopulation of nonpyramidal neurons stained selectively with three lectins with specific affinity for terminal N-acetylgalactosamine (GalNAc). These and only these lectins stained the surface of the cell body, dendritic shafts, and proximal parts of the dendritic arborization. Most reactive, nonpyramidal neurons revealed a multipolar dendritic pattern, but some possibly belonged to the bitufted and bipolar types of neuron. The GalNAc-containing neurons appeared widely distributed in layers II-VI with relatively greater abundance in layers IV and V. In the cortex of rats and mice the stained neurons occurred in moderate numbers in the frontal, frontoparietal, striate, retrosplenial, and entorhinal regions, but were less numerous in the hippocampal gyrus, dentate gyrus, and olfactory area. Other neurons in the basal ganglia and brain stem stained weakly for GalNAc. Examination of the frontal cortex of human and canine brains showed a similar distribution of nonpyramidal neurons with affinity for GalNAc-binding lectins. At high magnification, the surface staining of neurons in the cerebral cortex, deep cerebellar nucleus, and other sites appeared periodic rather than continuous. The periodic character of the neuronal surface staining suggested a location for the reactive glycoconjugate in or between the synapses. The GalNAc-containing glycoconjugate occurred in a selected cell type, failed to bind the other lectin conjugates, and differed from biochemically detected glycoconjugates. It is, therefore, considered a newly recognized entity of possible physiologic significance for a population of cortical neurons.     

Selective staining of a subset of GABAergic neurons in cat visual cortex by monoclonal antibody VC1.1

VC1.1 is a monoclonal antibody generated against cat area 17, which selectively outlines subsets of cortical neurons (Arimatsu et al., 1987). This study was conducted to determine the ultrastructural distribution of the VC1.1 antigen and to identify the particular subclasses of cortical neurons that were labeled. In the light microscope, VC1.1 delineated the surfaces of neurons located mainly in layer IV but also in other layers. The staining surrounded neuronal cell bodies and dendrites in a periodic or meshwork pattern but did not label axons. VC1.1-labeled neurons were morphologically heterogeneous and included multipolar, bipolar, and bitufted classes. In the electron microscope, VC1.1 immunoreactivity surrounded presynaptic membranes of terminal boutons and intersynaptic sections of postsynaptic membranes, but was not present within terminal boutons or synaptic clefts. Both asymmetric and symmetric synapses were immunoreactive. Labeling was also observed intracellularly on VC1.1-outlined neurons, associated with perisynaptic portions of plasma membranes. Tract-tracing methods were used in conjunction with immunocytochemistry to determine whether VC1.1 identified projection neurons, local circuit neurons, or a combination of both types. Layer V and VI corticogeniculate and corticotectal projection neurons were retrogradely labeled with rhodamine fluorescent latex microspheres. In a large sample of retrogradely labeled neurons, none were VC1.1-positive, suggesting that VC1.1 stained a population of local circuit neurons. Additional immunocytochemical double-labeling studies with an antiserum to GABA and VC1.1, revealed that VC1.1-positive neurons were immunoreactive to GABA. These were a major subset of the GABAergic neurons in area 17 and tended to have medium to large cell bodies. It is concluded that VC1.1 identifies a new, immunologically distinct subset of GABAergic neurons in area 17. The restricted distribution of this antigen on perisynaptic portions of GABA-containing cells and surrounding terminal boutons onto these cells suggests that this antigen may play an important role in inhibitory cortical circuits.     

Properties of entorhinal cortex deep layer neurons projecting to the rat dentate gyrus

Medial entorhinal cortex (EC) deep layer neurons projecting to the dentate gyrus (DG) were studied. Neurons, retrogradely-labelled with rhodamine-dextran-amine were characterized electrophysiologically with the patch clamp technique and finally labelled with biocytin. Pyramidal and nonpyramidal neurons form projections from the deep layers of the EC to the molecular layer of the DG. In addition, both classes of projection neurons send ascending axon collaterals to the superficial layers of the EC. Both classes of neurons were characterized physiologically by regular action potential firing upon depolarizing current injection. While a substantial number of pyramidal projection cells showed intrinsic membrane potential oscillations, none of the studied nonpyramidal cells exhibited oscillations. Despite the morphological similarity of bipolar and multipolar cells to those of GABAergic interneurons in the EC, their electrophysiological characteristics were similar to those of principal neurons and immunocytochemistry for GABA was negative. We conclude, that neurons of the deep layers of the medial EC projecting to the DG may function as both local circuit and projecting neurons thereby contributing to synchronization between deep layers of the EC, superficial layers of the EC and the DG.     

Immunocytochemical localization of somatostatin and cholecystokinin in the cat visual cortex

Immunocytochemistry was used to examine the morphology and distribution of cholecystokinin-like and somatostatin-like neurons in areas 17, 18 and 19 of cat visual cortex as a function of lamination. Immunoreactive cells of both peptides were observed in all layers of cat visual cortex. While somatostatin-like cells occurred mainly in layers II + III and VI, cholecystokinin-like cells were observed chiefly in the superficial layers (I + II + III). Somatostatin-like cells displayed morphological features of multipolar and bipolar varieties, and cholecystokinin-like cells displayed morphological features of multipolar and bitufted varieties. Similar results were obtained for all 3 areas.     

Form and distribution of neurons in rat cingulate cortex: Areas 32, 24, and 29

The cytoarchitecture of rat cingulate cortex is described. This includes the topographical distribution and layering patterns of Brodmann's areas 25, 32, 24, and 29a, b, c, and d. Area 24 is subdivided into a ventral area 24a and a dorsal area 24b, but an area 23 could not be identified between areas 24 and 29 An analysis of Golgi impregnations in areas 32, 24, and 29 demonstrates that most neuronal types recognized in neocortical areas are also present in cingulate cortex. Besides typical and inverted pyramidal cells, there is a wide variety of nonpyramidal cells, including multipolar, bitufted, and bipolar cells. Small multipolar cells with small somata, a dendritic tree limited to one or two layers, sparse to moderately spinous dendrites and one of two varieties of short axonal trajectories are present in layers I and II of areas 32, 24, and 29d. Medium multipolar cells occur mainly in layers III and V; they have extensive dendritic trees which traverse three or more layers, moderately spinous dendrites, and an axonal plexus which either ascends or descends in the cortex. Large multipolar cells are also frequent in layers III and V; their extensive dendritic trees are essentially spine free and they have axons which form dense terminations, particularly in the layer above the one in which the cell body is located Neurons with elongated somata and a primarily vertical orientation of the dendritic tree are either bitufted or bipolar. Bitufted cells are most frequent in layers II and III of areas 32, 24, and 29d. These cells have dendritic trees which form “hourglass shaped” fields, dendrites which are moderately spinous, and axons which form either extensive horizontal and vertical projections or are “chandelier” in form. Bipolar cells, in contrast, are found in layers II–V; their sparsely spinous dendrites form narrow dendritic trees which are oriented vertically and extend across four or more layers, and their axons have the same vertical orientation as the dendritic tree It is concluded that the form of the axonal arbors of nonpyramidal cells frequently mimics the extent and shape of their dendritic trees. Thus, small multipolar cells with limited, spherical dendritic trees may have axons which arch sharply and emit short, terminal branches. In contrast, medium and large multipolar cells have more extensive dendritic and axonal arbors which traverse two, three, or more layers. Of the fusiform cells, bitufted ones with their “hourglass” dendritic trees have extensive vertical and horizontally oriented axonal branches, while bipolar cells have narrow, vertically oriented dendritic and axonal arbors The granular layers II–IV of area 29c contain the following types of neurons: small and fusiform pyramids, medium-sized pyramids, large stellate cells, and medium multipolar cells. Fusiform pyramids are the only neurons unique to cingulate cortex. They are similar to the variety of pyramidal cells, but have an oval soma and only one basal dendrite which extends from the base of the cell body to arborize in layer IV. Large stellate cells differ from large multipolar cells in that they have densely spinous dendrites and axons which enter the white matter.     

Distribution of GABAergic neurons and axon terminals in the macaque striate cortex

Antisera to glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid (GABA) have been used to characterize the morphology and distribution of presumed GABAergic neurons and axon terminals within the macaque striate cortex. Despite some differences in the relative sensitivity of these antisera for detecting cell bodies and terminals, the overall patterns of labeling appear quite similar. GABAergic axon terminals are particularly prominent in zones known to receive the bulk of the projections from the lateral geniculate nucleus; laminae 4C, 4A, and the cytochrome-rich patches of lamina 3. In lamina 4A, GABAergic terminals are distributed in a honeycomb pattern which appears to match closely the spatial pattern of geniculate terminations in this region. Quantitative analysis of axon terminals that contain flat vesicles and form symmetric synaptic contacts (FS terminals) in lamina 4C beta and in lamina 5 suggest that the prominence of GAD and GABA axon terminal labeling in the geniculate recipient zones is due, at least in part, to the presence of larger GABAergic axon terminals in these regions. GABAergic cell bodies and their initial dendritic segments display morphological features characteristic of nonpyramidal neurons and are found in all layers of striate cortex. The density of GAD and GABA immunoreactive neurons is greatest in laminae 2-3A, 4A, and 4C beta. The distribution of GABAergic neurons within lamina 3 does not appear to be correlated with the patchy distribution of cytochrome oxidase in this region; i.e., there is no significant difference in the density of GAD and GABA immunoreactive neurons in cytochrome-rich and cytochrome-poor regions of lamina 3. Counts of labeled and unlabeled neurons indicate that GABA immunoreactive neurons make up at least 15% of the neurons in striate cortex. Layer 1 is distinct from the other cortical layers by virtue of its high percentage (77-81%) of GABAergic neurons. Among the other layers, the proportion of GABAergic neurons varies from roughly 20% in laminae 2-3A to 12% in laminae 5 and 6. Finally, there are conspicuous laminar differences in the size and dendritic arrangement of GAD and GABA immunoreactive neurons. Lamina 4C alpha and lamina 6 are distinguished from the other layers by the presence of populations of large GABAergic neurons, some of which have horizontally spreading dendritic processes. GABAergic neurons within the superficial layers are significantly smaller and the majority appear to have vertically oriented dendritic processes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Muscarinic receptor M(2) in cat visual cortex: laminar distribution, relationship to gamma-aminobutyric acidergic neurons, and effect of cingulate lesions.

Acetylcholine can have diverse effects on visual cortical neurons as a result of variations in postsynaptic receptor subtypes as well as the types of neurons and subcellular sites targeted. This study examines the cellular basis for cholinergic activation in visual cortex via M(2) type muscarinic receptors in gamma-aminobutyric acid (GABA)-ergic and non-GABAergic cells, using immunocytochemical techniques. At light microscopic resolution, M(2) immunoreactivity (-ir) was seen in all layers except area and sublayer specific bands in layer 4. Subcellularly, M(2)-ir occurred in both dendrites and terminals that form symmetric and asymmetric junctions. Layers 5 and 6 were characterized by axosomatic contacts that displayed labeling in the presynaptic component, and layer 6 displayed perikaryal postsynaptic staining, suggesting that corticofugal output neurons may be modulated particularly strongly via M(2). Infragranular layers differed from the supragranular layers in that more labeled profiles were axonal than dendritic, indicating a dominant presynaptic effect by acetylcholine via M(2) there. Unilateral cingulate cortex cuts caused reduction of cholinergic and noradrenergic fibers in the lesioned hemisphere at light microscopic resolution; at electron microscopic resolution, the synapse density and axonal M(2) labeling were reduced, suggesting that M(2) was localized presynaptically on extrathalamic modulatory inputs. Dual labeling with GABA in visual cortex layer 5 showed that half of M(2)-labeled dendrites originated from GABAergic neurons. Given that only one-fifth of all cortical dendritic profiles are GABAergic, this prevalence of dual labeling indicates an enrichment of M(2) within GABAergic dendrites and, thus, implicates abundant postsynaptic action on GABAergic neurons via M(2). In contrast, only one-tenth of M(2)-labeled terminals originated from GABAergic neurons, suggesting that the presynaptic action of acetylcholine via M(2) receptors would be more selective for non-GABAergic terminals.     

GABA neurons in the superficial layers of the rat dorsal cochlear nucleus: light and electron microscopic immunocytochemistry

This article is an application of light and electron microscopic immunocytochemistry to the study of the neuronal circuit of the superficial layers in the rat dorsal cochlear nucleus (DCN). An antiserum against the intrinsic marker glutamate decarboxylase (GAD) is used to identify and map axon terminals and neurons that use gamma aminobutyric acid (GABA) as a neurotransmitter. It is demonstrated that layers 1 and 2 of the DCN contain a very high density of GABAergic boutons, matched only by the granule cell domains of the ventral cochlear nucleus, especially the superficial granule cell domain. These two layers also contain much higher concentrations of GABAergic cell bodies than all other magnocellular regions of the cochlear nuclear complex. Cartwheel and stellate neurons, and probably also Golgi cells, previously characterized in Golgi and electron microscopic investigations, appear immunostained and, therefore, are presumably inhibitory. The synaptic relations between parallel fibers, the axons of granule cells, and cartwheel and stellate neurons are confirmed. The present study also supports the conclusion that stellate cells are coupled to one another by gap junctions. Also scattered in layer 1 are large, GABAergic neurons that occur with irregular frequency and presumably represent displaced Purkinje cells, previously identified with a Purkinje-cell-specific marker. Granule neurons and pyramidal neurons remain unstained, even after topical injection of colchicine, which enhances immunostaining of the other glutamate-decarboxylase-positive cells, and therefore must use transmitters different from GABA. The possible analogies between the spiny cartwheel and the aspiny stellate cells of the DCN and the cerebellar Purkinje and stellate/basket cells are discussed in the light of data from Golgi, electron microscopy, and transmitter imunocytochemistry.