Synapses of double bouquet cells in monkey cerebral cortex visualized by calbindin immunoreactivity

In the monkey neocortex, immunoreactivity for the 28-kDa vitamin D-dependent calcium binding protein (Calbindin) is contained in a set of GABAergic intrinsic neurons whose small size and laminar locations render them very distinct from a second set of GABAergic intrinsic neurons that show immunoreactivity for another calcium-binding protein, parvalbumin. A conspicuous feature of many calbindin-immunoreactive cells is their possession of long, vertically oriented bundles of immunoreactive processes that descend or ascend vertically through several cortical layers. These are components of the radial fasciculi of the cortex and are here shown by correlative electron microscopic immunocytochemistry to consist of both immunoreactive dendrites and unmyelinated axons. The morphology of the bundles and the parent cells indicates that the cells are classical double bouquet cells. The calbindin-positive axons in the radial fasciculi in the present study formed symmetric synapses on unlabeled dendritic shafts (62%) and spines (38%). Despite the close-packed nature of the immunoreactive axons, relatively few terminals of the same axon converged on a single postsynaptic profile. The postsynaptic profiles were identified in certain cases as side branches of pyramidal cell apical and basal dendrites. Mainstem apical dendrites generally did not receive synapses derived from the calbindin-positive axons. These results indicate that double bouquet cells can be distinguished both by their GABAergic character and by their possession of calbindin immunoreactivity. They are probably major contributors to the vertical flow of inhibitory influences across laminae of the cerebral cortex.     

Map of the synapses formed with the dendrites of spiny stellate neurons of cat visual cortex

The synaptic input of six spiny stellate neurons in sublamina 4A of cat area 17 was assessed by electron microscopy. The neurons were physiologically characterized and filled with horseradish peroxidase in vivo. After processing the neurons were reconstructed at the light microscopic level using computer-assisted methods and analyzed quantitatively. The extensive branching of the dendritic tree about 50 μm from the soma meant that the distal branches constituted five times the length of proximal dendrite. Proximal and distal portions of a single dendrite from each neuron were examined in series of ultrathin sections (1,456 sections) in the electron microscope. The majority (79%) of the 263 synapses examined were asymmetric; the remainder (21%) were symmetric. Symmetric synapses formed 35% of synapses sampled on proximal dendrites and were usually located on the shaft. They formed only 4% of synapses sampled on distal dendrites. Spines accounted for less than half of the total asymmetric synapses (45%); the remainder were on shafts. Symmetric synapses formed with four of 92 spines. Nine spines formed no synapses. Spiny stellate neurons in cat visual cortex appear to differ considerably from pyramidal neurons in having a significant asymmetric (excitatory) synaptic input to the dendritic shaft.     

Postnatal development of neurons and synapses in the visual and motor cortex of rabbits: a quantitative light and electron microscopic study

Introductory to a morphological investigation on the effects of early visual deprivation and on the critical periods in early postnatal life we have studied quantitatively the normal postnatal growth of neurons and synapses in the visual and motor cortex of rabbits. The major results of this analytical study are: (1) rapid decrease in neuron density and a rapid increase in neuronal volume are observed. They are almost completed at postnatal Day 10, i.e., before natural eye opening. The drop in neuron density is caused to a very large extent by an increase in cortical volume and not by a considerable disappearance of neurons; (2) the formation of synaptic contact zones starts at Day 6 to 7 and is most pronounced between Day 10 and Day 21, i.e., after natural eye opening. At Day 27 synaptic density has reached adult levels in the visual cortex and is in excess of the adult level in the motor cortex. In visual area I and in the motor cortex a significant difference in synaptic increase is observed between the left and right hemisphere, resulting in a lower synaptic density in the left counterparts at Day 27 and in adult animals [56,57]. In the visual cortex a small but highly correlated increase in synaptic vesicle density is observed. In the motor cortex no correlated relation between age and vesicle density is observed. In both cortical areas synaptic vesicle density has reached about 70 percent of the adult level at Day 27; and (3) in newborn and young rabbits the motor cortex seems to be more mature than the visual cortex.     

Distribution of synapses on fast and slow pyramidal tract neurons in the cat. An electron microscopic study

Distributions of synapses on various portions of fast and slow pyramidal tract neurons (PTNs) in cat motor cortex were studied with electron microscopy. PTNs were identified by their antidromic invasion following stimulation of the medullary pyramid and were classified into fast and slow PTNs according to conduction velocities of their axons. Two fast and two slow PTNs were intracellularly labeled and, by systematic sampling, electron micrographs from various portions of these neurons were examined to compare the distributions of different types of synapses. It was found that most synapses formed on apical and basal dendrites of fast PTNs were with the dendritic shafts. In slow PTNs, while synapses on apical dendrites were mostly axospinous, about 70% of the sampled synapses on basal dendrites of slow PTNs were established with the dendritic shafts. Virtually all synapses on apical dendrites of slow PTNs belonged to asymmetrical type and most of the synapses sampled from basal dendrites of fast PTNs were also asymmetrical. On the other hand, about 29% of the synapses found on apical dendrites of fast PTNs were symmetrical and a trend was observed for this type of synapses to increase their number with increasing proximity to the cell body. Over 28% of the synapses on basal dendrites of slow PTNs were also symmetrical and seemed to be mainly distributed in layer VI. All synapses formed on the soma were symmetrical both for the fast and slow PTNs.     

Synaptic patterns in the visual cortex of turtle: An electron microscopic study

The part of turtle general cortex that receives afferent fibers from the dorsal lateral geniculate nucleus and that shows evoked potentials to light stimuli has been studied with the electron microscope. This cortex consists of an outer molecular layer, a perikaryal layer, and a subcellular layer lying on a row of ependymal cell bodies. Neurons in the perikaryal lamina are characterized by long spine-bearing apical dendrites ascending through the outer molecular layer and short finer basal dendrites in the subcellular zone. Scattered neurons without apical dendrites occur in both the molecular and subcellular zones. Two types of dendritic spines can be distinguished. Some are large, have a complex irregular shape, contain a variety of membranous sacs and mitochondria, and occasionally, a single bundle of microtubules embedded in an electron-dense background opacity. These large spines are the most common postsynaptic element in the outer third of the molecular layer, where they are located on the distal tips of the apical dendrites. Other spines are small, with a simple spherical distal enlargement that contains only electron-dense fuzz. They are the most common post-synaptic element in the lower two-thirds of the molecular layer where they arise from the proximal portion of apical dendrites. Most synaptic contacts are found on the dendritic spines and are of the “round-asymmetrical” type. Not infrequently “flat-symmetrical” synapses are seen coupled to “round-asymmetrical” contacts on individual large spines. The few contacts present on spine-bearing dendritic shafts are of both types. Axo-somatic contacts are mainly of the “flat-symmetrical” variety. Thus the synaptic patterns on the principal cells of turtle visual cortex are remarkably similar to those found on pyramidal cells of mammalian neocortex. In addition, however, axon terminals, dendrites and glial (ependymal) processes were often seen to give rise to membranous pouches containing large vacuoles and invaginating into dendritic shafts or spines. Rarely, axon terminals were seen to form contacts, identical in appearance to synaptic contacts, on cell bodies in the ependymal lining. More frequently, unusual types of membrane differentiations were present at the site of apposition of the membranes of axon terminals and ependymal processes. They are interpreted as functional neuroependymal contacts.     

Glutamate receptor subunits at mossy fiber-unipolar brush cell synapses: Light and electron microscopic immunocytochemical study in cerebellar cortex of rat and cat

The present study provides a survey of the immunolocalization of ionotropic glutamate receptor subunits throughout the rat and cat cerebellar cortex, with emphasis on the unipolar brush cell (UBC), a hitherto neglected cerebellar cell that is densely concentrated in the granular layer of the vestibulocerebellum and that forms giant synapses with mossy fibers. An array of nine previously characterized antibodies has been used, each of which stained a characteristic profile of cerebellar cells. The UBCs of both rat and cat were strongly immunostained by an antibody against the α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptor subunits, GluR2 and GluR3; were moderately immunostained by a monoclonal antibody to kainate receptor subunits, GluR5/6/7; were weakly immunostained by antibodies to NR1 subunits; and were not stained by antibodies to GluR1, GluR4, GluR6/7, KA-2, and NR2A/B. Postsynaptic densities of the giant mossy fiber-UBC synapses were GluR2/3, GluR5/6/7, and NR1 immunoreactive. The other cerebellar neurons were all immunolabeled to some extent with the GluR2/3 and NR1 antibodies. In addition, Purkinje cells were immunopositive for GluRl and GluR5/6/7; granule cells were immunopositive for GluR5/6/7, GluR6/7, KA-2, and NR2A/B. The Golgi-Bergmann glia was densely stained by GluRl and GluR4 antibodies, whereas astrocytes of the granular layer were stained by the GluR4 antiserum. Data provided herein may guide further electrophysiological and pharmacological studies of cerebellar cells in general and the UBCs in particular. © 1995 Wiley-Liss, Inc.     

Map of the synapses onto layer 4 basket cells of the primary visual cortex of the cat

The pattern of excitatory and inhibitory inputs to the inhibitory neurons is largely unknown. We have set out to quantify the major excitatory and inhibitory inputs to layer 4 basket cells from the primary visual cortex of the cat. The synapses formed with the soma, and proximal and distal dendrites, were examined at the light and electron microscopic levels in four basket cells, recorded in vivo and filled with horseradish peroxidase. The major afferents of layer 4 have been well characterised, both at the light and electron microscopic levels. The sizes of the synaptic boutons of the major excitatory inputs to layer 4 from the thalamic relay cells, spiny stellate cells, and layer 6 pyramidal neurons are statistically different. Their distributions were compared to those of the boutons forming asymmetric contacts onto the basket cells, which were assumed to be provided by the same set of excitatory afferents. The best-fit results showed that about equal numbers of synapses were provided by the layer 6 pyramids (43%) and the spiny stellates (44%), whereas the thalamic afferents contributed only 13%. A similar analysis on the symmetric synaptic input to the basket cells indicated that as much as 79% of the symmetric synapses could have originated from other layer 4 basket cells. Thalamic and spiny stellate synapses were preferentially located on the soma and proximal dendrites, regions that also had 76% of all the symmetric contacts. J. Comp. Neurol. 380: 230–242, 1997. © 1997 Wiley-Liss, Inc.     

The distribution of recurrent Purkinje collateral synapses in the mouse cerebellar cortex: an electron microscopic study

Myelinated fibers found in the lower molecular layer of the mouse cerebellum form synaptic enlargements of a uniform morphological appearance. Similar synapses have been observed to come from myelinated fibers in the upper granularlower Purkinje cell layer. Both have been interpreted as Purkinje collateral boutons. The study of the distribution of Purkinje collateral synapses revealed that one third formed the supraganglionic plexus in the lower molecular layer and two thirds formed an infraganglionic plexus in the lower Purkinje upper granular zone. In the molecular layer, basket cell somata and dendrites received the gratest number of Purkinje collaterals, while Purkinje dendrites and branchlets received relatively few. In the infraganglionic plexus, the Purkinje collaterals terminated on “very low basket” cells and upon a dense dendritic “thicket” belived to be contributed largely by basket cells.     

A golgi and electron microscopic study of a dysplastic gangliocytoma of the cerebellum

Summary The fine structure of a dysplastic gangliocytoma of the cerebellum is studied by means of the Golgi method and electron microscopic examination.Thick proximally unbranched dendrites with terminal arborizations and varicose influorescences in the form of a basket are stained with the Golgi method. Axons are always descendant to the inner myelinated layer of the redistributed cerebellar cortex, while ascendant collaterals are observed at the level of the outer myelinated layer.Clear and dense-core vesicles and synapses are common in the cellular profiles under electron microscopic examination. From these data and because of the lack of putative connections through the white matter, an organized, self-regulated, catecholamine-mediated complex may be postulated.     

The pharmacology of synapses formed by identified corticocollicular neurons in primary cultures of rat visual cortex

Primary cultures of neurons from the visual cortex of 7-10-d-old Long Evans rats were used to study the pharmacology of synaptic transmission. Dissociated cells were grown either in mass cultures, which contained 8000-10,000 neurons, or in miniature island cultures of 50-100 cells. Prior to dissociation, cells in layer V of visual cortex that project to the superior colliculus were labeled in vivo by retrograde transport of fluorescent latex microspheres-a permanent fluorescent marker. After 2 d to 8 weeks in culture, labeled neurons were identified by epifluorescent illumination, and electrophysiological recordings were obtained from a labeled cell and, simultaneously, from a nearby unlabeled neuron in the same field of view. The 2 neurons were stimulated sequentially by current injection and the pharmacology of evoked postsynaptic potentials (PSPs) was investigated. In mass cultures, relatively few pairs of neurons from which we recorded were synaptically connected, although nearly every cell exhibited abundant spontaneous EPSPs and IPSPs. Neurons grown on island cultures generally did not exhibit spontaneous synaptic activity; however, stimulation of one of the cells in a pair frequently elicited a short-latency PSP in the follower neuron. Retrogradely labeled corticocollicular neurons produced only excitatory PSPs in follower cells, while unlabeled neurons were either excitatory or inhibitory. Three antagonists of excitatory amino acid receptors, kynurenic acid, piperidine dicarboxylic acid, and gamma-D-glutamylglycine, completely blocked EPSPs produced by labeled corticocollicular neurons, as well as EPSPs produced by nearly all of the unlabeled excitatory cells. We have previously shown that these compounds block both N-methyl-D-aspartate (NMDA)-type and non-NMDA receptors on cultured cortical neurons (Huettner and Baughman, 1986). The specific NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV) did not alter short-latency EPSPs recorded in 1 mM Mg2+, but did reduce longer-latency EPSPs polysynaptic activity. Since responses mediated by the NMDA receptor are known to be antagonized by Mg2+ (Mayer and Westbrook, 1985), we perfused cultures with Mg2+-free medium and found that the falling phase of some monosynaptic EPSPs was prolonged. Addition of APV to Mg2+-free medium reduced the duration of the falling phase of EPSPs such that they returned to the time course obtained in 1 mM Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heterogeneity of GABAergic cells in cat visual cortex

Antibodies against neuropeptides and against a vitamin D-dependent calcium-binding protein (CaBP) label small cells with nonpyramidal-like morphology in the cat visual cortex (areas 17, 18, and 19). Since GABAergic cells are interneurons, a double-staining procedure was used to test for the coexistence of cholecystokinin (CCK), somatostatin (SRIF), neuropeptide Y (NPY), corticotropin-releasing factor (CRF), vasoactive intestinal polypeptide (VIP), and CaBP with glutamic acid decarboxylase (GAD). Our results show that CRF and VIP do not coexist with GAD, while the 3 other peptides and CaBP do. Hence GAD-positive cells can be subdivided into 4 broad groups: (1) cells that are only GAD-positive, (2) cells that are GAD- and CaBP-positive, (3) GAD-positive neurons also containing CCK, and (4) GAD-positive cells that also contain SRIF. A small subset of class 2 also contains SRIF and most cells of class 4 also contain NPY. The 4 classes of GAD-positive cells differ in laminar position: class 1 predominates in layers IV and V, classes 2 and 3 in the upper laminae (II and III), and class 4 in the deepest layer (VI).     

A study of tachykinin-immunoreactivity in the cat visual cortex

The localization of tachykinin-immunoreactivity in the cat visual cortex (area 17) was investigated using immunohistochemical methods. Strong laminar specificity was observed, with immunoreactivity highest in layer V, followed by layers I, VI, II and III, and the lowest density in layer IV. Most of the immunoreactive product was localized in neuronal processes. A few immunopositive cell bodies were also present. The immunopositive neurons were non-pyramidal, multipolar, or bipolar in shape, and mostly found in layer V. There were particularly dense immunopositive fibers and varicosities around somata in layer V. These may represent tachykinin-containing presynaptic terminals (boutons). The results provide anatomical evidence that tachykinin may primarily affect layer V neurons in the cat visual cortex.     

A golgi study of the class V cell in the visual thalamus of the cat

Golgi methods were used to study class V cells within the cat visual thalamus. Counterstaining was combined with Golgi staining to assess the distribution of dendrites relative to cytoarchitectural boundaries. Class V cells were encountered within all laminae of the lateral geniculate nucleus, the medial interlaminar nucleus, and the lateral posterior complex. The cells possess medium-sized perikarya and smooth and varicose or moniliform dendrites. Dendritic appendages are sparse and occur as single or serial swellings on thin processes. Many class V cells exhibit large, sparse dendritic arbors which span laminar or nuclear borders; dendrites were seen to lie within and to cross the interlaminar zones of the lateral geniculate nucleus, and extend beyond this nucleus into the perigeniculate nucleus and medial interlaminar nucleus. Class V cells of the lateral posterior complex send dendrites into the external medullary lamina. Indirect evidence favors the interpretation that the class V cells are thalamo-cortical relay cells.     

A specialized type of neuron in the visual cortex of cat: A Golgi and electron microscope study of chandelier cells

The axonal arborization of chandelier cells is characterized by its conspicuous, vertically oriented, bouton aggregates. The efferent synaptic relationships established by these terminal formations were investigated by electron microscopy of Golgi preparations after gold toning and deimpregnation. In all cases examined form layers II and III of cat areas 17 and 18, the terminal formations, here denominated specific terminal portions (stp), make symmetric synapses upon axon initial segments of pyramidal neurons. Some identified stp's were reconstructed from ultrathin serial sections with the aid of a microcomputer-based system, and the number of synaptic contacts established on axon initial segments was evaluated. No evidence was found that parts of the axonal tree other than stp's also engage in synaptic contacts. Specific terminal portions are rather variable in complexity. However, the synaptic contacts they engage in are constant and the complexity of stp's from the same axonal arborization varies. It is, therefore, clear that all stp's are terminal axonal formations of a unique, specialized type of neuron. Computer techniques and conventional Golgi observations were used to study further details of chandelier cell morphology. Axonal plexuses are preferentially, although not exclusively, local and distribute within spheric, ovoid, or disk-shaped spaces In most chandelier cells, the main axonal trunk descends to the white matter, where we have been unable to follow it further.     

Distribution of corticocortical and thalamocortical synapses on identified motor cortical neurons in the cat: Golgi, electron microscopic and degeneration study

Details of the terminal connection of corticocortical and thalamocortical fibers on pyramidal and stellate neurons in the cat motor cortex were studied using the electron microscope in combination with the Golgi and axonal degeneration techniques. Corticocortical terminals were examined in 23 identified neurons of which 11 were pyramidal and 12 were stellate. Stellate neurons located in layer III received many degenerating terminals (average 8.4 +/- 2.2 per unit length of dendrite (ULD)) and the majority of these (95%) were found on the proximal dendrites or on the cell bodies. The pyramidal neurons received fewer degenerating terminals (average 2.1 +/- 0.27/ULD) and these were located on more distal dendritic shafts or on dendritic spines. The majority of these synapses were of the asymmetric type. Thalamocortical terminals were examined in 9 pyramidal and 9 stellate neurons. Pyramidal neurons received many terminals (average 6.0 +/- 1.23/ULD) and these were found on the basal as well as the apical dendrites and on dendrite spines. Stellate neurons received fewer terminals (average 4.2 +/- 0.64/ULD) and were located primarily on proximal dendritic shafts. The majority of these synapses were of the asymmetric type. The functional role of these synapses is discussed in relation to the physiological results reported in the preceding paper.     

Developmental changes in the relationship between type 2 synapses and spiny neurons in the monkey visual cortex

This study continues an exploration of synaptic development in the primary visual cortex of the monkey (Macaca nemestrina). In a prior study (Mates and Lund, '83a), we observed that type 2 synapses on the cell bodies of spiny stellate neurons of lamina 4C appeared not only to increase in number during early postnatal development but also subsequently decreased during maturation. Using quantitative, stereological electron microscopic methods, we examined the maturation of this synapse population from embryonic day 159 to adult, on spiny stellate neurons of 4C alpha and beta and, for comparison, on pyramidal neurons in upper and lower lamina 6. Tissue was also taken for comparison from two animals reared to 8 weeks of age with binocular eyelid closure from birth. We confirmed that a marked increase and subsequent decrease occurred in this somal type 2 synapse population on both neuron populations. However, due to the infrequency of the smooth dendritic neurons (approximately 5% of the neuron population) giving rise to the type 2 contacts, and due to expansion of the neuropil during maturation increasing intercell distances against constant volume of the type 2 axon arbors, it is concluded that the decrease in type 2 somal synapses may represent a redistribution to dendrites rather than loss from the neuropil. Cells of lamina 4C beta (receiving input from the parvocellular lateral geniculate nucleus-LGN) show a slower initial accumulation of type 2 contacts compared to neurons of lamina 4C alpha (receiving input from magnocellular LGN), or to pyramidal neurons of lamina 6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of somatostatin receptors in the cat and monkey visual cortex demonstrated by in vitro receptor autoradiography.

Somatostatin (SRIF, S14) receptors in the cat and monkey visual cortex were visualized by means of in vitro autoradiography with an iodinated agonist of SRIF, [125I-Tyr0,DTrp8]S14. The kinetics, performed on tissue sections, revealed an apparently single, saturable site (KD = 3.92 +/- 0.31 10(-10) M for the cat, and 3.82 +/- 0.28 10(-10) M for the monkey visual cortex) with pharmacological specificity for S14 and [DTrp]-substituted S14. Autoradiography, performed on frontal sections of the cat and monkey visual cortex, revealed a heterogeneous regional and laminar distribution of SRIF receptors. In cat areas 17, 18, and 19, SRIF receptors occur mainly in the supragranular layers, although small interareal and intra-areal differences are observed. The infragranular layers (V-VI) in area 19 contain a significantly higher proportion of SRIF receptors compared to both areas 17 and 18. In the antero- (AMLS) and posteromedial lateral suprasylvian area (PMLS), layers V and VI contain the highest proportion of SRIF receptors. This latter pattern is also observed in the area prostriata medially adjoining area 17 in the splenial sulcus. In the monkey visual cortex, areas 17 and 18 exhibit similar distribution patterns, SRIF receptors being primarily concentrated in layers V and VI. Neither in the cat nor the monkey visual cortex could we observe significant differences in SRIF receptor distribution between different retinotopic subdivisions within one area.     

Principal components analysis of cells in cat visual cortex

Cortical cells differ along many dimensions, and statistical techniques are needed to study the relationships among them. We have used principal components analysis to study variation within a population of cells in the visual cortex of the cat. This procedure transforms the data which may then be projected onto a small number of independent axes. The axes are hierarchically ordered in terms of the sample variance they account for so that subsequent components account for decreasing proportions of the total sample variance.Four principal components (PCs) were identified by the analysis. The contributing variables on these components were respectively: (1) cutoff velocity, peak response, receptive field area and spontaneous activity; (2) tuning width, orientation selectivity, occular dominance ratio and obliquity; (3) directionality ratio, monocularity and vvariability; and (4) width to length ratio, variability and sideband ratio. We hypothesize that each of these components identifies a physiologically important feature of cortical organization. PC₁ and PC₄ describe those relationships among variables that have been used previously by other workers to classify cells in the visual cortex of the cat. Specifically, PC₁ is highly correlated with those variables associated with type of afferent input and PC₄ is highly correlated with those variables that differentiate simple cells from complex cells. PC₂ and PC₃, however, are highly correlated with variables that have not been used in classiying cells. Our results demonstrate that: (1) each of two major classification schemes (simple/complex and classification by afferent input) describe important differences among neurons in the cat's visual cortex and both must be incorporated into a comprehensive system for classifying cells in the cat's visual cortex; and (2) these two schemes when combined do not account for all the major differences among neurons in cat visual cortex, and at least two additional components are required: one related to orientation selectivity and dominance by the ipsilateral eye, and another related to directional selectivity and the degree of binocular interaction.     

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.