Calbindin-containing interneurons are a target for VIP-immunoreactive synapses in rat primary somatosensory cortex

Inhibitory interneurons in cerebral cortex are morphologically and physiologically extremely heterogeneous. This greatly interferes with an understanding of their functions. Progress has been made by classifying these neurons with the aid of molecular markers, e.g., neuropeptides or calcium-binding proteins, which are reliably expressed by certain subpopulations. We have used this approach to demonstrate an output of a subpopulation of cortical interneurons which express vasoactive intestinal polypeptide (VIP). By double immunostaining and correlated light and electron microscopy, we show that calbindin (CB)-containing interneurons located in layers II-VI of rat barrel cortex are targets of symmetric VIP-immunoreactive synapses. All CB-immunoreactive interneurons showed numerous contacts of VIP boutons on proximal and distal dendritic segments. A great majority of CB-immunoreactive interneurons (214/222) displayed such close appositions with VIP boutons on their soma as well. Quantification revealed that the number of VIP-immunoreactive boutons on CB-immunoreactive somata and dendrites of specified order is comparable for the different cortical layers. In conclusion, all calbindin-containing cortical interneurons seem to be under direct influence of other GABAergic interneurons expressing the peptide VIP. An indirect functional consequence of this may be disinhibition of pyramidal cells, which are considered the major target of calbindin interneurons. However, since the examined types of interneurons are intricately embedded in networks of yet different interneurons, the outcome of these multiple inhibitory interactions is likely to be less simplistic. It may be related to the timing of pyramidal cell discharge within and across layers of cortical columns.     

Entorhinal cortical innervation of parvalbumin-containing neurons (basket and chandelier cells) in the rat ammon's horn

Physiological data suggest that in the CA1–CA3 hippocampal areas of rats, entorhinal cortical efferents directly influence the activity of interneurons, in addition to pyramidal cells. To verify this hypothesis, the following experiments were performed: 1) light microscopic double-immunostaining for parvalbumin and the anterograde tracer Phaseolus vulgaris-leucoagglutinin injected into the entorhinal cortex; 2) light and electron microscopic analysis of cleaved spectrin-immunostained (i.e., degenerating axons and boutons) hippocampal sections following entorhinal cortex lesion; and 3) an electron microscopic study of parvalbumin-immunostained hippocampal sections after entorhinal cortex lesion. The results demonstrate that in the stratum lacunosum-moleculare of the CA1 and CA3 regions, entorhinal cortical axons form asymmetric synaptic contacts on parvalbumin-containing dendritic shafts. In the stratum lacunosum-moleculare, parvalbumin-immunoreactive dendrites represent processes of GABAergic, inhibitory basket and chandelier cells; these interneurons innervate the perisomatic area and axon initial segments of pyramidal cells, respectively. A feed-forward activation of these neurons by the entorhinal input may explain the strong, short-latency inhibition of pyramidal cells. © 1996 Wiley-Liss, Inc.     

Innervation of VIP-immunoreactive neurons by the ventroposteromedial thalamic nucleus in the barrel cortex of the rat

We investigated the synaptic terminals of fibers originating in the ventroposteromedial thalamic nucleus (VPM) and projecting to the main input layers (IV/III) of the rat posteromedial barrel subfield. It was our aim to determine whether or not the subpopulation of vasoactive intestinal polypeptide (VIP)-immunoreactive neurons in these layers are directly innervated by the sensory thalamus. Anterograde tracing with Phaseolus vulgaris leucoagglutinin (PHA-L) and immunohistochemistry for VIP were combined for correlated light and electron microscopic examination. Columns of cortical tissue were well defined by barrel-like patches of PHA-L-labeled fibers and boutons in layers IV and III. Within these columns VIP-immunoreactive perikarya were located mainly in supragranular layers. Marked perikarya were also seen in infragranular layers, but their immunoreactivity was often weaker. Granular layer IV, which is the main terminal field for thalamic fibers, contained fewer VIP neurons than supragranular layers. In the light microscope, however, PHA-L-labeled fibers appeared to contact the somata or proximal dendrites of 60–86% of the layer IV VIP neurons. By contrast, only 18–35% of the VIP neurons in the supragranular layers, which receive a moderately dense projection from the VPM, appeared to be contacted. PHA-L-labeled boutons were seen close to 13–25% of infragranular VIP-positive cells. Electron microscopy showed that thalamic fibers formed at most four asymmetric synapses on a single layer IV, VIP-positive neuron. Although the proportion of VIP-positive neurons with labeled synapses was lower in supragranular layers, most of them shared multiple asymmetric synapses with labeled thalamic fibers. Up to six labeled synapses were seen on individual VIP neurons in layer III. We conclude that subpopulations of VIP-immunoreactive neurons, located in layers IV, III, and II are directly innervated by the VPM. These neurons may be involved in the initial stages of cortical processing of sensory information from the large, mystacial vibrissae. Since VIP is known to be colocalized with the inhibitory transmitter GABA, it is likely that VIP neurons participate in the shaping of the receptive fields in the barrel cortex. © 1996 Wiley-Liss, Inc.     

Cholinergic synaptic circuitry in the macaque prefrontal cortex

Surprisingly little is known about the synaptic architecture of the cholinergic innervation in the primate cerebral cortex in spite of its acknowledged relevance to cognitive processing and Alzheimer's disease. To address this knowledge gap, we examined serially sectioned cholinergic axons in supra- and infragranular layers of the macaque prefrontal cortex by using an antibody against the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT). The tissue bound antibody was visualized with both immunoperoxidase and silver-enhanced diaminobenzidine sulfide (SEDS) techniques. Both methods revealed that cholinergic axons make synapses in all cortical layers and that these synapses are exclusively symmetric. Cholinergic axons formed synapses primarily on dendritic shafts (70.5%), dendritic spines (25%), and, to a lesser extent, cell bodies (4.5%). Both pyramidal neurons and cells exhibiting the morphological features of GABAergic cells were targets of the cholinergic innervation. Some spiny dendritic shafts received multiple, closely spaced synapses, suggesting that a subset of pyramidal neurons may be subject to a particularly strong cholinergic influence. Analysis of synaptic incidence of cholinergic profiles in the supragranular layers of the prefrontal cortex by the SEDS technique revealed that definitive synaptic junctions were formed by 44% of the cholinergic boutons. An unexpected finding was that chohnergic boutons were frequently apposed to spines and small dendrites without making any visible synaptic specializations. These same spines and dendrites often received asymmetric synapses, presumably of thalamocortical or corticocortical origin. Present ultrastructural findings suggest that acetylcholine may have a dual modulatory effect in the neocortex: one through classical synaptic junctions on dendritic shafts and spines, and the other through nonsynaptic appositions in close vicinity to asymmetric synapses. Further physiological studies are necessary to test the hypothesis of the nonsynaptic release of acetylcholine in the Cortex. © 1995 Wiley-Liss, Inc.     

Cholecystokinin-immunoreactive cells form symmetrical synaptic contacts with pyramidal and nonpyramidal neurons in the hippocampus

The ultrastructural features and synaptic relationships of cholecystokinin (CCK)-immunoreactive cells of rat and cat hippocampus were studied using the unlabeled antibody immunoperoxidase technique and correlated light and electron microscopy. CCK-positive perikarya of variable shape and size were distributed in all layers and were particularly concentrated in stratum pyramidale and radiatum: the CCK-immunoreactive neurons were nonpyramidal in shape and the three most common types had the morphological features of tufted, bipolar, and multipolar cells. Electron microscopic examination revealed that all the CCK-positive boutons established symmetrical (Gray's type II) synaptic contacts with perikarya and dendrites of pyramidal and nonpyramidal neurons. The origin of some of the boutons was established by tracing fine collaterals that arose from the main axon of two CCK-immunostained cells and terminated in the stratum pyramidale; these collaterals were then examined in the electron microscope. The axon of one such neuron exhibited a course parallel to the pyramidal layer and formed pericellular nets of synaptic boutons upon the perikarya of pyramidal neurons. This pattern of axonal arborization is very similar to that of some of the basket cells, previously suggested to be the anatomical correlate for pyramidal cell inhibition. Typical dendrites of pyramidal cells also received symmetrical synaptic contacts from CCK-immunoreactive boutons, and some of these boutons could be shown to originate from a local neuron in stratum radiatum. Many CCK-immunoreactive cells received CCK-labeled boutons upon their soma and dendritic shafts. Synaptic relationship, established by multiple "en passant" boutons, was observed between CCK-positive interneurons of the stratum lacunosum-moleculare and radiatum. The soma and dendrites of the CCK-immunostained neurons also received symmetrical and asymmetrical synapses from nonimmunoreactive boutons. These results indicate that the CCK-immunoreactive neurons participate in complex local synaptic interactions in the hippocampus.     

Ultrastructural analysis of synaptic relationships of intracellularly stained pyramidal cell axons in piriform cortex

Axons of pyramidal cells in piriform cortex stained by intracellular injection of horseradish peroxidase (HRP) have been analyzed by light and electron microscopy. Myelinated primary axons give rise to extensive, very fine caliber (0.2 micron) unmyelinated collaterals with stereotyped radiating branching patterns. Serial section electron microscopic analysis of the stained portions of the collateral systems (initial 1-2 mm) revealed that they give rise to synaptic contacts on dendritic spines and shafts. These synapses typically contain compact clusters of large, predominantly spherical synaptic vesicles subjacent to asymmetrical contacts with heavy postsynaptic densities. On the basis of comparisons with Golgi material and intracellularly stained dendrites, it was concluded that dendritic spines receiving synapses from the proximal portions of pyramidal cell axon collaterals originate primarily from pyramidal cell basal dendrites. Postsynaptic dendritic shafts contacted closely resemble dendrites of probable GABAergic neurons identified in antibody and [3H]-GABA uptake studies. Electron microscopic examination of pyramidal cell axon initial segments revealed a high density of symmetrical synaptic contacts on their surfaces. Synaptic vesicles in the presynaptic boutons were small and flattened. It is concluded that pyramidal cells synaptically interact over short distances with other pyramidal cells via basal dendrites and with deep nonpyramidal cells that probably include GABAergic cells mediating a feedback inhibition. This contrasts with long associational projections of pyramidal cells that terminate predominantly on apical dendrites of other pyramidal cells.     

Enrichment of cholinergic synaptic terminals on GABAergic neurons and coexistence of immunoreactive GABA and choline acetyltransferase in the same synaptic terminals in the striate cortex of the cat

The synaptic circuits underlying cholinergic activation of the cortex were studied by establishing the quantitative distribution of cholinergic terminals on GABAergic inhibitory interneurons and on non-GABAergic neurons in the striate cortex of the cat. Antibodies to choline acetyltransferase and GABA were used in combined electron microscopic immunocytochemical experiments. Most of the cholinergic boutons formed synapses with dendritic shafts (87.3%), much fewer with dendritic spines (11.5%), and only occasional synapses were made on neuronal somata (1.2%). Overall, 27.5% of the postsynaptic elements, all of them dendritic shafts, were immunoreactive for GABA, thus demonstrating that they originate from inhibitory neurons. This is the highest value for the proportion of GABAergic postsynaptic targets obtained so far for any intra- or subcortical afferents in cortex. There were marked variations in the laminar distribution of targets. Spines received synapses most frequently in layer IV (23%) and least frequently in layers V-VI (3%); most of these spines also received an additional synapse from a choline acetyltransferase-negative bouton. The proportion of GABA-positive postsynaptic elements was highest in layer IV (49%, two-thirds of all postsynaptic dendritic shafts), and lowest in layers V-VI (14%). The supragranular layers showed a distribution similar to that of the average of all layers. The quantitative distribution of targets postsynaptic to choline acetyltransferase-positive terminals is very different from the postsynaptic targets of GABAergic boutons, or from the targets of all boutons in layer IV reported previously. In both cases the proportion of GABA-positive dendrites was only 8-9% of the postsynaptic elements. At least 8% of the total population of choline acetyltransferase-positive boutons, presumably originating from the basal forebrain, were also immunoreactive for GABA. This raises the possibility of cotransmission at a significant proportion of cholinergic synapses in the cortex. The present results demonstrate that cortical GABAergic neurons receive a richer cholinergic synaptic input than non-GABAergic cells. The activation of GABAergic neurons by cholinergic afferents may increase the response specificity of cortical cells during cortical arousal thought to be mediated by the basal forebrain. The laminar differences indicate that in layer IV, at the first stage of the processing of thalamic input, the cholinergic afferents exert substantial inhibitory influence in order to raise the threshold and specificity of cortical neuronal responses. Once the correct level of activity has been set at the level of layer IV, the influence can be mainly facilitatory in the other layers.     

Projection of olfactory bulb efferents to layer I GABAergic neurons in the entorhinal area. Combination of anterograde degeneration and immunoelectron microscopy in rat

Glutamic acid decarboxylase (GAD) immunoelectron microscopy in combination with anterograde degeneration was applied in rats to study the synaptic targets of olfactory bulb afferents to the lateral subdivision (LEA) of the entorhinal area (EA). Immunoreactive neurons and terminals are scattered throughout all layers of LEA. After olfactory bulb resection, terminal degeneration occurs in layer Ia of EA. Using the electron microscope we examined serial thin sections of 12 and 14 immunoreactive neurons sampled from layer Ia of the dorsal (DLEA) and ventral (VLEA) subdivisions of LEA, respectively. The morphology of all these neurons is similar: they are small (short axis 5-9 micron, long axis 7-12 micron) and possess eccentrically located, indented nuclei provided with filamentous nuclear rodlets. The immunoreactive neurons have thin, smooth dendrites which usually emerge abruptly from the somata. We observed a single cilium on 5 of the immunoreactive neurons. In layer Ia of both DLEA and VLEA, the somata of the immunoreactive neurons are contacted by degenerating, non-immunoreactive boutons showing asymmetric synaptic junctions. In addition to these boutons, 4 other categories of axo-somatic terminals can be distinguished: normal, non-immunoreactive boutons forming asymmetric synapses and containing spherical synaptic vesicles; normal, non-immunoreactive boutons with symmetric synapses and pleomorphic synaptic vesicles; normal, non-immunoreactive boutons with asymmetric synapses, containing dense-cored vesicles in addition to spherical synaptic vesicles; and normal, immunoreactive boutons with symmetric synapses and pleomorphic synaptic vesicles. It is suggested that the GAD-immunoreactive neurons which receive olfactory bulb input correspond to local circuit neurons with intralaminar axons which innervate each other as well as the distal segments of the apical dendrites of projection neurons with cell bodies in layers II and III. Thus, the olfactory input in EA seems to be wired not only for excitation of layers II and III pyramidal neurons but also for feed-forward inhibition using GABAergic intermediary neurons, strategically located in the area of termination of olfactory bulb fibers.     

GABA-immunoreactive boutons make synapses with inspiratory neurons of the dorsal respiratory group.

Intracellular labelling with horseradish peroxidase (HRP) combined with gamma-aminobutyric (GABA) immunocytochemistry was used to assess the GABAergic input to inspiratory bulbospinal neurons of the dorsal respiratory group in the cat. The relationship between GABA-immunoreactive (GABA-IR) boutons and intracellularly labelled neurons was examined at the light microscopic and ultrastructural levels. At the light microscopic level, GABA-IR boutons were frequently found in close apposition to dendrites and cell bodies of labelled neurons. The presence of synapses was confirmed with electron microscopy. In addition, synaptic specializations were observed between immunoreactive boutons and unlabelled terminals which in turn formed synaptic contacts with HRP-labelled dendrites, a finding consistent with presynaptic inhibition. These results demonstrate a direct GABAergic input to a functionally defined population of medullary respiratory neurons, and suggest involvement of this neurotransmitter in the control of these neurons.     

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.     

Innervation of interneurons immunoreactive for VIP by intrinsically bursting pyramidal cells and fast-spiking interneurons in infragranular layers of juvenile rat neocortex

Cortical columns contain specific neuronal populations with characteristic sets of connections. This wiring forms the structural basis of dynamic information processing. However, at the single-cell level little is known about specific connectivity patterns. We performed experiments in infragranular layers (V and VI) of rat somatosensory cortex, to clarify further the input patterns of inhibitory interneurons immunoreactive (ir) for vasoactive intestinal polypeptide (VIP). Neurons in acute slices were electrophysiologically characterized using whole-cell recordings and filled with biocytin. This allowed us to determine their firing pattern as regular-spiking, intrinsically bursting and fast-spiking, respectively. Biocytin was revealed histochemically and VIP immunohistochemically. Sections were examined for contacts between the axons of the filled neurons and the VIP-ir targets. Twenty pyramidal cells and five nonpyramidal (inter)neurons were recovered and sufficiently stained for further analysis. Regular-spiking pyramidal cells displayed no axonal boutons in contact with VIP-ir targets. In contrast, intrinsically bursting layer V pyramidal cells showed four putative single contacts with a proximal dendrite of VIP neurons. Fast-spiking interneurons formed contacts with two to six VIP neurons, preferentially at their somata. Single as well as multiple contacts on individual target cells were found. Electron microscopic examinations showed that light-microscopically determined contacts represent sites of synaptic interactions. Our results suggest that, within infragranular local cortical circuits, (i) fast-spiking interneurons are more likely to influence VIP cells than are pyramidal cells and (ii) pyramidal cell input probably needs to be highly convergent to fire VIP target cells.     

Morphological evidence for altered synaptic organization and structure in the hippocampal formation of seizure-sensitive gerbils.

Seizure-sensitive (SS) and seizure-resistant (SR) Mongolian gerbils were used for three experiments. In the first experiment, GABAergic neurons and terminals in the dentate gyrus were localized with GAD immunocytochemistry. GAD-positive puncta adjacent to cell bodies of GABAergic pyramidal basket cells were counted in light microscopic preparations. The pyramidal basket cells of SS gerbils displayed a significant threefold increase in the number of GAD-positive puncta associated with their cell bodies as compared to those from SR gerbils. These data indicate that the number of GABAergic synapses with pyramidal basket cell bodies in the dentate gyrus was greater in SS gerbils. An electron microscopic (EM) analysis of GAD immunocytochemical preparations showed GAD-positive axon terminals forming symmetric synapses with GAD-positive basket cell bodies. However, numerous terminals forming symmetric axosomatic synapses with basket cells were not immunopositive, and other synapses formed by terminals were not classified because reaction product in the cell bodies obscured postsynaptic densities. Therefore, routine EM preparations were analyzed for symmetric and asymmetric axosomatic synapses on pyramidal basket cells and granule cells of SS and SR gerbils. The data obtained from these preparations showed that the pyramidal basket cells of SS gerbils had a selective increase in the number of symmetric synapses per 10 microns of soma as compared to those of the SR gerbils. In contrast, the granule cells did not show any significant difference in the number of either symmetric or asymmetric axosomatic synapses between SS and SR gerbils. These results indicate that pyramidal basket cell bodies of SS gerbils have more inhibitory synapses than do those of SR gerbils. The third experiment used SS gerbils with lesions of the perforant pathway that stopped seizure activity (Ribak, C. E., and S. U. Khan (1987) The effects of knife cuts of hippocampal pathways on epileptic activity in the seizure-sensitive gerbil. Brain Res. 418:251-260). The percentage of axon terminal area occupied by synaptic vesicles and their packing density was determined in CA3 mossy fiber boutons and compared for lesioned and nonlesioned SS gerbils. The mossy fibers of nonlesioned SS gerbils showed a depletion of synaptic vesicles consistent with the previous results of Peterson et al. (Peterson, G. M., C. E. Ribak, and W. H. Oertel (1985) A regional increase in the number of hippocampal GABAergic neurons and terminals in the seizure-sensitive gerbil. Brain Res. 340:384-389).(ABSTRACT TRUNCATED AT 400 WORDS)     

Synaptic output of physiologically identified spiny stellate neurons in cat visual cortex

Spiny stellate neurons of area 17 of the cat's visual cortex were physiologically characterised and injected intracellularly with horseradish peroxidase. Six neurons from sublamina 4A were selected. Five had the S-type of simple receptive fields; one had a complex receptive field. Their axons formed boutons mainly in layers 3 and 4. An electron microscopic examination of 45 boutons showed that each bouton formed one asymmetric synapse on average. Spines were the most frequent synaptic target (74%); dendritic shafts formed the remainder (26%). On the basis of ultrastructural characteristics, 8% of the target dendrites were characterised as originating from smooth γ-aminobutyrate-ergic (GABAergic) neurons. Thus the major output of spiny stellate neurons is to other spiny neurons, probably pyramidal neurons in layer 3 and spiny stellates in layer 4.     

GABAergic neural elements in the rat basilar pons: electron microscopic immunochemistry

Previous light microscopic immunoperoxidase studies of glutamic acid decarboxylase (GAD)-immunoreactive neural elements in the rat basilar pontine nuclei revealed immunocytochemical reaction product in neuronal somata and axon terminals. In the present study, pre-embedding immunoperoxidase labeling of GAD or gamma-aminobutyric acid (GABA) and postembedding immunogold labeling of GABA allowed the ultrastructural visualization of these neural elements in the basilar pontine nuclei of colchicine-treated animals. At the electron microscopic level, immunolabeled neuronal somata exhibited smoothly contoured nuclei, whereas some dendrites also contained reaction product after immunocytochemical treatment and were postsynaptic to both immunoreactive and nonimmunoreactive axon terminals. Synaptic boutons immunoreactive for GAD or GABA exhibited cross-sectional areas that ranged from 0.1 to 3.8 microns 2 and generally appeared round or elongated in most sections. The majority (95%) of immunolabeled boutons contained pleomorphic synaptic vesicles and formed symmetric synapses at their postsynaptic loci; however, boutons exhibiting round vesicles and boutons forming asymmetric synapses (5%) were also immunopositive. Small (less than 1.5 microns 2) GAD- or GABA-labeled axon terminals formed synaptic contact mainly with small dendritic profiles, dendritic spines, and neuronal somata, whereas large labeled boutons (greater than 1.5 microns 2) formed synapses with all sizes of dendritic profiles. Occasionally, a single immunolabeled bouton formed synaptic contact with two separate postsynaptic dendrites. It is suggested that the immunolabeled neuronal somata and dendrites observed in the rat basilar pontine nuclei represent a population of pontine local circuit neurons; however, it is known that GABAergic cell groups extrinsic to the pontine gray provide afferent projections to the basilar pons, and therefore at least some immunoreactive axon terminals present in the pontine nuclei are derived from these extrinsic sources. The ultrastructural observation of GABAergic neural elements in the rat basilar pontine nuclei confirms previous light microscopic findings and provides an anatomical substrate through which GABAergic neurons, whether arising from an intrinsic or extrinsic source, might exert an inhibitory influence on target cells within the pontine nuclei.     

Axon terminals immunolabeled for dopamine or tyrosine hydroxylase synapse on GABA-immunoreactive dendrites in rat and monkey cortex

Dopamine afferents to the cortex regulate the excitability of pyramidal neurons via a direct synaptic input. However, it has not been established whether dopamine also modulates pyramidal cell activity indirectly through synapses on γ-aminobutyric acid (GABA) interneurons, and whether such inputs differ across cortical regions and species. We sought to address these issues by an immunocytochemical electron microscopic approach that combined peroxidase staining for dopamine or tyrosine hydroxylase (TH) with a pre-embedding gold-silver marker for GABA. In the deep layers of the rat prefrontal cortex and in the superficial layers of the monkey prefrontal and primary motor cortices, terminal varicosities immunoreactive for dopamine or TH formed primarily thin, symmetric synapses on distal dendrites. Both GABA-immunoreactive dendrites as well as unlabeled spines and dendrites were contacted by dopamine- or TH-immunoteactive terminals. Synaptic specializations were detected at some, but not all of these contacts. The relative frequency of these appositional and synaptic contacts did not appear to differ between the rat and monkey prefrontal cortex, or between the monkey prefrontal and motor cortices. Across regions and species, labeled and unlabeled targets of dopamine- or TH-positive terminals received additional synaptic input from unlabeled, and occasionally GABA-immunoreactive terminals. Close appositions between dopamine- or TH immunoreactive and GABA-positive terminals were observed only rarely. These findings indicate that dopamine afferents provide direct synaptic inputs to GABA local circuit neurons in a consistent fashion across cortical regions and species. Thus, dopamine's cellular actions involve direct as well as modulatory effects on both GABA interneurons and pyramidal projection neurons. © 1995 Wiley-Liss, Inc.     

Evidence for interlaminar inhibitory circuits in the striate cortex of the cat

An interlaminar, ascending, and GABAergic projection is demonstrated in the striate cortex of the cat. We have examined a basket cell, with soma and smooth dendrites in layers V and VI, that was injected intracellularly with HRP in the kitten. Three-dimensional reconstruction of its axon revealed a horizontal plexus in layer V and upper VI, extending about 1.8 mm anteroposteriorly and 0.8 mm mediolaterally; a dense termination in the vicinity of the soma in layers V and VI; and an ascending tuft terminating in layers II and III in register above the soma and about 250 microns in diameter. Many boutons of this cell contacted neuronal somata and apical dendrites of pyramidal cells and subsequent electron microscopy showed that these boutons formed type II synaptic contacts with these structures. A random sample of postsynaptic targets (n = 199) in layers III, V, and VI showed that somata (20.1%), dendritic shafts (38.2%), and dendritic spines (41.2%) were contacted. The fine structural characteristics of postsynaptic elements indicated that the majority originated from pyramidal cells. Direct identification of postsynaptic neurons was achieved by Golgi impregnation of four large pyramidal cells in layer V, which were contacted on their somata and apical dendrites by between three and 34 boutons of the HRP-filled basket cell. Layer IV neurons were not contacted. Golgi-impregnated neurons similar to the HRP-filled basket cell were also found in the deep layers. The axonal boutons of one of them were studied; it also formed type II synapses with somata and apical dendrites of pyramidal cells. Boutons of the HRP-filled neuron were shown to be GABA-immunoreactive by the immunogold method. This is direct evidence in favour of the GABAergic nature of deep layer basket cells with ascending projections. The existence of an ascending GABAergic pathway was also demonstrated by injecting [3H]GABA into layers II and III. The labelled amino acid was transported retrogradely by a subpopulation of GABA-immunoreactive cells in layers V and VI, in addition to cells around the injection site. The axonal pattern and mode of termination of deep basket cells make them a candidate for producing or enhancing directional selectivity, a characteristic of layer V cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calretinin-immunoreactive local circuit neurons in area 17 of the cynomolgus monkey,Macaca fascicularis

The connections of local circuit neurons immunoreactive for calcium-binding protein calretinin (CR-ir) were studied in area 17 of the macaque monkey visual cortex. Most CR-ir neurons were located in layers 2 and 3A. They were polymorphic and included bitufted, multipolar, pyramid-shaped neurons with smooth dendrites and Cajal-Retzius cells. The majority of CR-ir neurons were γ-aminobutyric acid (GABA)-immunopositive (approximately 90%), and comprised about 14% of the total GABAergic neuron population. The axons of CR-ir cells had local arbors within layers 1–3, but the major trunks descended to deep layers 5 and 6 where they formed dense terminal fields within narrow columns (100–150 μm). This specific innervation of layers 5 and 6 appeared as a distinct feature of area 17 as it was not seen in the adjacent area 18. CR-ir boutons (n = 168) were GABA-ir (95%) and formed symmetric synapses. In layers 1–3, the majority of postsynaptic targets (n = 64) were GABAergic local circuit neurons [postsynaptic target distribution: GABA-positive dendrites (67%) and somata (14%), and GABA-negative dendrites (13%) and spines (6%)]. In deep layers, the most synapses (80%; n = 187) were formed with pyramidal cells where they provided a basket-type innervation [postsynaptic target distribution: GABA-positive dendrites (19%) and somata (1%), and GABA-negative dendrites (50%), spines (20%) and somata (10%)]. Unlike other GABAergic neurons, which innervate mainly pyramidal neurons, the CR-ir subpopulation only has pyramids as a preferred target in the deep layers (layers 5 and 6); however, in the superficial layers of the area 17, they selectively form synapses mainly with other GABAergic cells. Thus, the CR-ir neurons appear to have a dual function of disinhibiting superficial layer neurons and inhibiting pyramidal output neurons in the deep layers. J. Comp. Neurol. 379:113-132, 1997. © 1997 Wiley-Liss, Inc.     

Serotoninergic afferents preferentially innervate distinct subclasses of peptidergic interneurons in the rat visual cortex

Although it is well documented that the non-pyramidal neurons of the cerebral cortex are under the influence of the vast serotoninergic input, the ultrastructural substrate for such functional interactions appears largely obscure. We sought to address this issue by dual immunoelectron microscopy, combining antibodies against serotonin (5-HT) and three neurochemical markers for peptidergic interneurons, namely somatostatin (SRIF), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP). The gold-substituted silver-peroxidase method was employed to intensify and differentiate the end-product of the peptide-immunoreaction from the non-intensified 5-HT fibers. Mainly the SRIF but also the NPY neurons were encountered among the postsynaptic targets of the 5-HT boutons. Recipients of synapses were perikarya and proximal dendrites of SRIF and NPY cells but also distal dendrites of the SRIF neurons. Neither synaptic relationships nor close appositions were ever identified between 5-HT boutons and VIP-immunoreactive elements. This remarkable synaptic preference/avoidance of 5-HT afferents for specific peptidergic subpopulations reveals a 'wired' component of cortical serotonin neurotransmission, which should be carefully interpreted within the frame of the available literature for extrasynaptic serotonin release.     

Targets and Quantitative Distribution of GABAergic Synapses in the Visual Cortex of the Cat

The morphology and postsynaptic targets of GABA-containing boutons were determined in the striate cortex of cat, using a postembedding immunocytochemical technique at the electron microscopic level. Two types of terminals, both making symmetrical synaptic contacts, were GABA-positive. The first type (95% of all GABA-positive boutons) contained small pleomorphic vesicles, the second type (5%) contained larger ovoid vesicles. Furthermore, 99% of all cortical boutons containing pleomorphic vesicles were GABA positive, and all boutons with pleomorphic vesicles made symmetrical synaptic contacts. These results together with previously published stereological data (Beaulieu and Colonnier, 1985, 1987) were used to estimate the density of GABA-containing synapses, which is about 48 million/mm3 in the striate cortex. The postsynaptic targets of GABA positive boutons were also identified and the distribution was calculated to be as follows: 58% dendritic shafts, 26.4% dendritic spines, 13.1% somata and 2.5% axon initial segments. A total of 11% of the postsynaptic targets were GABA immunoreactive and therefore originated from GABAergic neurons. The results demonstrate that the majority of GABAergic synapses exert their action on the membrane of dendrites and spines rather than on the somata and axons of neurons.     

High level of mGluR7 in the presynaptic active zones of select populations of GABAergic terminals innervating interneurons in the rat hippocampus

The release of neurotransmitters is modulated by presynaptic metabotropic glutamate receptors (mGluRs), which show a highly selective expression and subcellular location in glutamatergic terminals in the hippocampus. Using immunocytochemistry, we investigated whether one of the receptors, mGluR7, whose level of expression is governed by the postsynaptic target, was present in GABAergic terminals and whether such terminals targeted particular cells. A total of 165 interneuron dendritic profiles receiving 466 synapses (82% mGluR7a-positive) were analysed. The presynaptic active zones of most GAD-(77%) or GABA-positive (94%) synaptic boutons on interneurons innervated by mGluR7a-enriched glutamatergic terminals (mGluR7a-decorated) were immunopositive for mGluR7a. GABAergic terminals on pyramidal cells and most other interneurons in str. oriens were mGluR7a-immunonegative. The mGluR7a-decorated cells were mostly somatostatin- and mGluR1alpha-immunopositive neurons in str. oriens and the alveus. Their GABAergic input mainly originated from VIP-positive terminals, 90% of which expressed high levels of mGluR7a in the presynaptic active zone. Parvalbumin-positive synaptic terminals were rare on mGluR7a-decorated cells, but on these neurons 73% of them were mGluR7a-immunopositive. Some type II synapses innervating interneurons were immunopositive for mGluR7b, as were some type I synapses. Because not all target cells of VIP-positive neurons are known it has not been possible to determine whether mGluR7 is expressed in a target-cell-specific manner in the terminals of single GABAergic cells. The activation of mGluR7 may decrease GABA release to mGluR7-decorated cells at times of high pyramidal cell activity, which elevates extracellular glutamate levels. Alternatively, the presynaptic receptor may be activated by as yet unidentified endogenous ligands released by the GABAergic terminals or the postsynaptic dendrites.