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.     

Immunocytochemical localization of gamma-aminobutyric acid in the hypoglossal nucleus of the macaque monkey, Macaca fuscata: a light and electron microscopic study

The hypoglossal nucleus of the macaque monkey Macaca fuscata was investigated with light and electron microscopic immunocytochemistry with an antibody directed against gamma-aminobutyric acid (GABA). At the light microscopic level, GABA immunoreactivity was present in small neurons, punctate structures, and thin, fiberlike structures. These GABA-positive elements were distributed throughout the hypoglossal nucleus at rostrocaudal levels. There was no immunoreactivity in the hypoglossal motoneurons. The GABA-positive small neurons were fusiform or ovoid (15 X 9 micron) and extended a few proximal dendrites from both poles. At the ultrastructural level, these small neurons were characterized by a markedly invaginated nucleus and a scanty cytoplasm in which cisternae of rough endoplasmic reticulum were not organized into extensive lamellar arrays as seen in the motorneurons. The GABA-positive punctate structures at the light microscopic level were identified as vesicle-containing axon boutons at the electron microscopic level. These GABA-positive axon terminals made synaptic contacts mainly with the dendrites of the motoneurons and infrequently with the somata. The majority of them made symmetric synapses and they contained pleomorphic synaptic vesicles. However, a small number of GABA-positive terminals (7%) formed asymmetric synapses with the dendrites of motoneurons, and these contacts exhibited postsynaptic dense bars or Taxi bodies lying beneath the postsynaptic membranes. There were no GABA-positive boutons that contacted the cell bodies of the small neurons. Although GABA-positive myelinated and unmyelinated axons were seen as thin, fiberlike structures, these myelinated and unmyelinated axons rarely gave rise to boutons on the motoneurons. The present study suggests that GABAergic inhibition in the monkey hypoglossal nucleus occurs mainly on the dendrites of the motoneurons and to some extent on the somata.     

Synaptic organization of cortico-cortical connections from the primary visual cortex to the posteromedial lateral suprasylvian visual area in the cat

The synaptic organization of the projection from the cat striate visual cortex to the posteromedial lateral suprasylvian cortical area (PMLS) was examined. The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was iontophorectically delivered into area 17, and anterogradely labeled fibers were revealed in PMLS by means of an immunocytochemical detection method. Most axons and presumptive terminal swellings were found in layers III and IV. The neuronal elements (n = 190) that were postsynaptic to anterogradely labeled boutons were quantitatively analyzed. All anterogradely labeled cortico-cortical boutons (n = 182) established type 1 synapses. The results show that 83% of the postsynaptic targets were dendritic spines, probably belonging to pyramidal cells. Dendritic shafts constituted 17% of the targets. The dendritic shafts postsynaptic to cortico-cortical boutons were studied for the presence of gamma-aminobutyric acid (GABA) with a postembedding immunogold method. Most dendritic shafts (85%) that were tested were found to be GABA-positive, demonstrating that they originate from local inhibitory neurons. Taking into account that most postsynaptic targets were spines and extending the results of the immunocytochemical testing to the total population of postsynaptic dendrites, it was calculated that at least 14% of targets originated from GABA-positive cells. Thus cortico-cortical axons establish direct monosynpatic connections mainly with pyramidal and to a lesser extent with GABAergic nonpyramidal neurons in area PMLS, providing both feedforward excitation and feedforward inhibition to a visual associational area known to be involved in the processing of motion information. The results are consistent with previously demonstrated deficits in physiological properties of neurons in PMLS following removal of cortico-cortical afferents.     

Combined golgi and electron microscopic study on the synapses formed by double bouquet cells in the visual cortex of the cat and monkey

The morphology of certain Golgi-stained cells was examined in the striate and peristriate cortex of the cat and in the striate cortex of the rhesus monkey. Neurons in layer III were selected on the basis of their characteristic vertical axon bundles, which are 20–150 μ in diameter and traverse layers II–V Selected neurons were examined under the electron microscope to characterize their synapses and to establish their postsynaptic targets. It was found that double bouquet cells form symmetrical or type II synapses. In the cat the postsynaptic membrane specialization was more extensive than in the monkey. After removing the Golgi precipitate from boutons of two cells in the cat, small pleomorphic and flattened vesicles were found in the boutons Earlier suggestions that double bouquet cells make synapses preferentially with spines of apical dendrites could not be confirmed. Out of 66 boutons in area 17 of the cat, 86.4% formed synapses with dendritic shafts, many of them belonging to nonpyramidal cells, 9% with perikarya of nonpyramidal cells, and only 4.6% with spines. Out of 19 synapses examined in area 18, 74% were contacting dendritic shafts and the rest contacted spines. In the monkey 60% of a total of 35 double bouquet cell synapses made synapses with dendritic shafts. A different type of double bouquet cell with densely spiny dendrites is also described in layer IV of the monkey striate cortex. This neuron formed asymmetrical synapses It is suggested that layer III double bouquet cells with vertical axon bundles are probably inhibitory and act on other nonpyramidal cells and certain parts of pyramidal cells.     

Fine structure and synaptic connections of the common spiny neuron of the rat neostriatum: A study employing intracellular injection of horseradish peroxidase

Medium-sized spiny neurons of the rat neostriatum, identified by intracellular injection of horseradish peroxidase, were examined at both light and electron microscopic levels. These neurons were characterized by their heavy investment of dendritic spines, beginning about 20 μm from the soma and continuing to the tips of the dendrites. Their axons arose from the soma or from a large dendritic trunk very near the soma, and tapered rapidly to form a main axonal branch from which arose several smaller initial collaterals. These arborized extensively throughout an area of about the same size as, and highly overlapping with, the dendritic field of the cell, while the main axon could be followed for distances of up to 1 mm in the direction of the globus pallidus. Three major synaptic types were seen in contact with spiny neurons. Boutons containing small round synaptic vesicles formed synapses exclusively with spiny regions of the dendrites, and most of these were axo-spinous. Small, very pleomorphic synaptic vesicles characterized a second bouton type of unknown origin, which made contacts with somata, initial segments, and dendrites, but not dendritic spines. Boutons containing large pleomorphic synaptic vesicles had the most widespread distribution, contacting all regions including dendritic spines. Spines receiving these contacts also were postsynaptic to boutons containing small round vesicles. Axon collaterals of spiny cells formed synapses with large pleomorphic vesicles and made synapses with somata, initial segments of axons, dendrites, and dendritic spines of striatal neurons, including other spiny cells.     

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.     

Synaptic relationships between axon terminals from the mediodorsal thalamic nucleus and gamma-aminobutyric acidergic cortical cells in the prelimbic cortex of the rat

Although the reciprocal interconnections between the prefrontal cortex and the mediodorsal nucleus of the thalamus (MD) are well known, the involvement of inhibitory cortical interneurons in the neural circuit has not been fully defined. To address this issue, we conducted three combined neuroanatomical studies on the rat brain. First, the frequency and the spatial distribution of synapses made by reconstructed dendrites of nonpyramidal neurons were identified by impregnation of cortical cells with the Golgi method and identification of thalamocortical terminals by degeneration following thalamic lesions. Terminals from MD were found to make synaptic contacts with small dendritic shafts or spines of Golgi-impregnated nonpyramidal cells with very sparse dendritic spines. Second, a combined study that used anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and postembedding gamma-aminobutyric acid (GABA) immunocytochemistry indicated that PHA-L-labeled terminals from MD made synaptic junctions with GABA-immunoreactive dendritic shafts and spines. Nonlabeled dendritic spines were found to receive both axonal inputs from MD with PHA-L labelings and from GABAergic cells. In addition, synapses were found between dendritic shafts and axon terminals that were both immunoreactive for GABA. Third, synaptic connections between corticothalamic neurons that project to MD and GABAergic terminals were investigated by using wheat germ agglutinin conjugated to horseradish peroxidase and postembedding GABA immunocytochemistry. GABAergic terminals in the prelimbic cortex made symmetrical synaptic contacts with retrogradely labeled corticothalamic neurons to MD. All of the synapses were found on cell somata and thick dendritic trunks. These results provide the first demonstration of synaptic contacts in the prelimbic cortex not only between thalamocortical terminals from MD and GABAergic interneurons but also between GABAergic terminals and corticothalamic neurons that project to MD. The anatomical findings indicate that GABAergic interneurons have a modulatory influence on excitatory reverberation between MD and the prefrontal cortex.     

Ultrastructure and synaptic organization of axon terminals from brainstem structures to the mediodorsal thalamic nucleus of the rat.

The ultrastructural characteristics and synaptic organization of afferent terminals from the brainstem to the mediodorsal thalamic nucleus (MD) of the rat have been studied with the electron microscope, by means of anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Labeled fibers were seen predominantly in the lateral portion of MD after the injections of WGA-HRP into the substantia nigra pars reticulata (SNr), the superior colliculus (SC), and the dorsal tegmental region (DT). The boutons arising from the SC were relatively small (less than 1.5 microns in diameter), formed asymmetric synaptic contacts with small dendrites and dendritic spines, and contained round synaptic vesicles. The axon terminals from the DT were mostly large boutons (2-4.5 microns) with asymmetric synaptic specializations and round vesicles. These boutons and their postsynaptic targets formed synaptic glomeruli that were entirely or partially ensheathed by glial lamellae. The ultrastructural features are almost identical to those of boutons in the medial and central segments of MD that were previously shown to originate from the basal amygdaloid nucleus and the piriform cortex. The boutons from the SNr had a wide range in size, but the majority were medium-sized to large (1.5-4 microns). The nigral boutons established symmetric synaptic contacts with dendritic shafts and occasionally with somata, and contained pleomorphic vesicles. However, like the DT terminals, they participated in glomerular formations. The nigral terminals closely resemble previously described terminals in the medial part of MD from the ventral pallidum, except that the nigral terminals formed en passant and axosomatic synapses as well as axodendritic synapses. A combined immunohistochemistry and WGA-HRP tracing study revealed that the nigral inputs were immunoreactive for glutamic acid decarboxylase and the axon terminals from the DT were immunoreactive for choline acetyltransferase. In a separate study, the colliculothalamic fibers have been shown to take up and transport the transmitter specific tracer [3H]-D-aspartate, and are therefore putatively glutamatergic and/or aspartatergic. Taken together with this, the present results suggest that the collicular afferents are excitatory and glutamatergic and/or aspartatergic, that the inputs from the DT are also excitatory and cholinergic, while the nigral inputs are inhibitory and GABAergic.     

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)     

Mode of termination of pallidal afferents to the thalamus: A light and electron microscopic study with anterograde tracers and immunocytochemistry in Macaca mulatta

The mode of termination of individual pallidothalamic fibers in the densicellular subdivision of the ventral anterior thalamic nucleus (VAdc) of Macaca mulatta was analyzed with light and electron microscopy after injections of anterograde tracers in the medial globus pallidus. Three tracers were utilized: tritiated leucine, biotinylated dextran amine, and wheat germ agglutinin conjugated to horseradish peroxidase in combination with postembedding immunocytochemsitry for gamma-aminobutyric acid (GABA). Pallidothalamic fibers, upon entering the VAdc, gave off several collaterals that formed plexuses of varicose terminal branches within different cell clusters. The varicosities were aligned along somata and proximal dendrites of projection neurons providing dense input to each individual cell. At the electron microscopic level, labeled boutons displayed a predominantly flat and elongated shape. They contained a moderate number of pleomorphic synaptic vesicles and very large amounts of mitochondria, displayed symmetric synaptic contacts, and were immunoreactive for GABA. In the total sample of 128 autoradiographically labeled terminals, 64% were in synaptic contact with somata and primary dendrites of projection neurons, 14% formed synapses on proximal dendrites of undefined order, and only 7% established synaptic contacts on distal dendrites. Fifteen percent of the labeled boutons established synapses on distal dendrites of GABAergic local circuit neurons (LCN). Pallidal boutons were also found in complex synaptic arrangements: triads with three GABAergic synapses, and serial synapses with LCN dendrites that in turn established synaptic contacts on projection neuron somata or dendrites. These anatomical results suggest a dual effect of pallidal afferents to projection neurons: direct inhibition and disinhibition mediated by LCN. The findings indicate that the fine structure of pallidothalamic terminals in the monkey is similar to that described earlier in the cat. There are, however, interspecies differences in the distribution of pallidal input on postsynaptic targets and its participation in complex synaptic arrangements. J. Comp. Neurol. 386:601–612, 1997. © 1997 Wiley-Liss, Inc.     

Subdivisions in the Multiple GABAergic Innervation of Granule Cells in the Dentate Gyrus of the Rat Hippocampus

The sources of GABAergic innervation to granule cells were studied to establish how the basic cortical circuit is implemented in the dentate gyrus. Five types of neuron having extensive local axons were recorded electrophysiologically in vitro and filled intracellularly with biocytin (Han et al., 1993). They were processed for electron microscopy in order to reveal their synaptic organization and postsynaptic targets, and to test whether their terminals contained GABA. (1) The hilar cell, with axon terminals in the commissural and association pathway termination field (HICAP cell), formed Gray's type 2 (symmetrical) synapses with large proximal dendritic shafts (n= 18), two-thirds of which could be shown to emit spines, and with small dendritic branches (n= 6). Other boutons of the HICAP neuron were found to make either Gray's type 1 (asymmetrical) synapses (n= 4) or type 2 synapses (n= 6) with dendritic spines. Using a highly sensitive silver-intensified immunogold method for the postembedding visualization of GABA immunoreactivity, both the terminals and the dendrites of the HICAP cell were found to be immunopositive, whereas its postsynaptic targets were GABA-immunonegative. The dendritic shafts of the HICAP cell received synapses from both GABA-negative and GABA-positive boutons; the dendritic spines which densely covered the main apical dendrite in the medial one-third of the molecular layer received synapses from GABA-negative boutons. (2) The hilar cell, with axon terminals distributed in conjunction with the perforant path termination field (HIPP cell), established type 2 synapses with distal dendritic shafts (n= 17), most of which could be shown to emit spines, small-calibre dendritic profiles (n= 2) and dendritic spines (n= 6), all showing characteristics of granule cell dendrites. The sparsely spiny dendrites of the HIPP cell were covered with many synaptic boutons on both their shafts and their spines. (3) The cell with soma in the molecular layer had an axon associated with the perforant path termination field (MOPP cell). This GABA-immunoreactive cell made type 2 synapses exclusively on dendritic shafts (n= 20), 60% of which could be shown to emit spines. The smooth dendrites of the MOPP cell were also restricted to the outer two-thirds of the molecular layer, where they received both GABA-negative and GABA-positive synaptic inputs. (4) The extensive axonal arborization of the dentate basket cell terminated mainly on somata (n= 26) and proximal dendrites (n= 9) in the granule cell layer, and some boutons made synapses on somatic spines (n= 6); all boutons established type 2 synapses. (5) The dentate axo-axonic cell established type 2 synapses (n= 14) exclusively on axon initial segments of granule cells in the granule cell layer, and on initial segments of presumed mossy cells in the hilus. The results demonstrate that granule cells receive inputs from the local circuit axons of at least five distinct types of dentate neuron terminating in mutually exclusive domains of the cell's surface in four out of five cases. Four of the cell types (HICAP cell, MOPP cell, basket cell, axo-axonic cell) contain GABA, and the HIPP cell may also be inhibitory. The specific local inhibitory neurons terminating in conjunction with particular excitatory amino acid inputs to the granule cells (types 1 – 3) are in a position to interact selectively with the specific inputs on the same dendritic segment. This arrangement provides a possibility for the independent regulation of the gain and long-term potentiation of separate excitatory inputs, through different sets of GABAergic local circuit neurons. The pairing of excitatory and inhibitory inputs may also provide a mechanism for the downward reseating of excitatory postsynaptic potentials, thereby extending their dynamic range.     

Spatial relationships between the terminations of somatic sensory motor pathways in the rostral brainstem of cats and monkeys. II. Cerebellar projections compared with those of the ascending somatic sensory pathways in lateral diencephalon

In order to classify the presynaptic elements contacting the principle class of globus pallidus neurons, electron microscopic examination of serial sections made from a medially located large globus pallidus neuron, labeled with intracellular horseradish peroxidase, was undertaken. In addition, the use of labeled and light microscopically reconstructed material allowed us to quantitatively determine the distribution of each bouton type along the soma and dendrites. Six types of presynaptic terminals contacting the labeled cell have been recognized. Type 1 endings, the most numerous (84%), make symmetrical contacts on all portions of the cell, except spines, contain large pleomorphic, and a few large dense-core vesicles. Type 2 endings are filled with small spherical-to-ellipsoidal synaptic vesicles. They make asymmetrical contacts only with higher-order dendrites and account for 12% of synaptic contacts onto the labeled neuron. Type 3 endings are large, contain sparsely distributed large pleomorphic vesicles, and make two symmetrical synapses per bouton, one onto a spine head and the other onto the underlying dendritic shaft. They are infrequent (0.2%), being found only in association with dendritic spines. Type 4 endings contain large pleomorphic synaptic vesicles and no dense-core vesicles. They make symmetrical contacts with the short primary dendrites. Type 5 endings contain a mixture of small clear pleomorphic vesicles and numerous large dense-core vesicles. They contact only the cell body and the short primary dendrites, making up 20% of somatic synaptic contacts but less than 1% of contacts onto dendrites. Type 6 boutons contain oval and flattened synaptic vesicles and establish symmetrical contacts with higher-order dendritic branches and the cell body.     

GABAergic elements in the neuronal circuits of the monkey neostriatum: a light and electron microscopic immunocytochemical study

An antibody raised in rabbits against a GABA-BSA conjugate was used together with the PAP technique to label elements in the neostriatum of three Old World monkeys. Light microscopy revealed numerous immunoreactive medium-size neurons of various staining intensities, some of which had indented nuclei, as well as an occasional large cell. The neuropil showed a plexus of fine processes with frequent puncta. Ultrastructurally, the medium-size GABA-positive neurons were of two types: one with smooth nuclei and scanty cytoplasm, similar to spiny I cells, the other with invaginated nuclear envelopes and more abundant perikaryon, resembling the aspiny type. Correspondingly, labeled dendrites were either spiny or varicose. Some stained axons were myelinated, and the boutons had either large and ovoid, or small and pleomorphic vesicles. All of these boutons formed symmetric synapses, the former type with GABA-positive dendritic shafts but also with unlabeled dendrites; the latter type usually with GABA-negative elements, either dendrites, dendritic spines, or somata. Synapses were also observed between unreactive boutons and immunostained dendrites. Terminals with densely packed, small round vesicles that established asymmetric synapses with spines were always GABA-negative. Glial elements were consistently unlabeled, save for some astroglial endfeet. These findings provide positive evidence for the existence of two classes of GABAergic striatal neurons corresponding to a long-axoned spiny I type and an aspiny interneuron. Furthermore, the simultaneous labeling of GABA-immunoreactive presynaptic and postsynaptic profiles offers possible morphologic bases for the various kinds of intrastriatal inhibitory processes, including the feedforward, feedback, and "autaptic" types.     

Inhibitory contacts on dendritic spines of the dentate fascia

GABA-containing axon terminals were observed in the distal two-thirds of the dentate molecular layer to contact spines and dendrites of the granule cells. These contacts have the morphological characteristics of inhibitory synapses: they contain pleomorphic vesicles and have symmetrical junctional specializations. Convergence of an asymmetrical, non-GABAergic and a symmetrical, GABAergic synapse on one spine was often observed.     

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.     

Immunocytochemical localization of choline acetyltransferase within the rat neostriatum: a correlated light and electron microscopic study of cholinergic neurons and synapses

Monoclonal antibodies to choline acetyltransferase (ChAT) were used in an immunocytochemical study to characterize putative cholinergic neurons and synaptic junctions in rat caudate-putamen. Light microscopy (LM) revealed that ChAT-positive neurons are distributed throughout the striatum. These cells have large oval or multipolar somata, and exhibit three to four primary dendrites that branch and extend long distances. Quantitative analysis of counterstained preparations indicated that ChAT-positive neurons constitute 1.7% of the total neuronal population. Electron microscopy (EM) of immunoreactive neurons initially studied by LM revealed somata characterized by deeply invaginated nuclei and by abundant amounts of organelle-rich cytoplasm. Surfaces of ChAT-positive neurons are frequently smooth, but occasional somatic protrusions and dendritic spines occur. Although infrequently observed, axons of ChAT-positive neurons branch, receive synapses, and become myelinated. Unlabeled boutons make both symmetrical and asymmetrical synapses with ChAT-positive somata and proximal dendrites, but are more numerous on distal dendrites. In addition, some unlabeled terminals form asymmetrical synapses with ChAT-positive somata and dendrites that are distinguished by prominent subsynaptic dense bodies. Light microscopy demonstrated a dense distribution of ChAT-positive fibers and punctate structures in the striatum, and these structures appear to correlate, respectively, with labeled preterminal axons and presynaptic boutons identified by EM. ChAT-positive boutons contain pleomorphic vesicles, and make symmetrical synapses primarily with unlabeled dendritic shafts. Furthermore, they establish synaptic contacts with somata, dendrites and axon initial segments of unlabeled neurons that ultrastructurally resemble medium spiny neurons. These observations, together with the results of other investigations, suggest that medium spiny GABAergic projection neurons receive a cholinergic innervation that is probably derived from ChAT-positive striatal cells. The results of this study also indicate that cholinergic neurons within caudate-putamen belong to a single population of cells that have large somata and extensive sparsely spined dendrites. Such neurons, in combination with dense concentrations of ChAT-positive fibers and terminals, are the likely basis for the large amounts of ChAT and acetylcholine detected biochemically within the neostriatum.     

Monosynaptic cortical input and local axon collaterals of identified striatonigral neurons. A light and electron microscopic study using the golgi-peroxidase transport-degeneration procedure

Following the injection of horseradish peroxidase into the ipsilaeral substantia nigra, 36 retrogradely labelled neurons in the striatum were characterized (in three rats) by Golgi staining and gold toning: each neuron was of the medium-size, densely spinous type. Prior to the injection of horseradish peroxidase, two of the rats had had lesions placed in the ipsilateral motor cortex, the third rat had had a lesion placed in the ipsilateral frontal and prefrontal cortex. In the electron microscope, degenerating boutons of cortical neurons were found in asymmetrical synaptic contact with the spines of proximal and distal dendrites of all six of the identified striatonigral neurons that were studied. Some of the degenerating boutons were small (diameter 0.1–0.3 μ), while others were larger (1–2 μ). An individual dendrite of a striatonigral neuron was in synaptic contact with very few degenerating boutons Local axon collaterals im the striatum could be traced from two of the identified striatonigral neurons that received degenerating cortical boutons. These were studied in the electron microscope; their boutons formed symmetrical synapses with spines or dendritic shafts of other striatal neurons. The synaptic boutons contained large, clear, round and pleomorphic vesicles. The postsynaptic targets of these boutons morphologically resemble the dendrites of medium-size spiny neurons It is concluded that afferents from the cortex make monosynaptic contact with the dendritic spines of medium-size spiny striatonigral neurons and that such neurons have local axon collaterals in the striatum that form synapses with other spiny neurons.     

Fine structure of the medullary lateral line area of Chelon labrosus (order perciformes), a nonelectroreceptive teleost

The ultrastructure and synaptic organization of the nucleus medialis and cerebellar crest of the teleost Chelon labrosus have been investigated. The nucleus medialis receives projections from the anterior and posterior lateral line nerves. This nucleus consists of oval neurons and large crest cells (“Purkinje-like” cells) whose apical dendrites branch in the overlying molecular layer, the cerebellar crest. In the dorsal region of the nucleus medialis, the perikarya and smooth primary dendrites of the crest cells are interspersed among myelinated fibers and nerve boutons. The ventral layer of the nucleus medialis contains crest cell perikarya and dendrites as well as oval neurons. The cerebellar crest lacks neuronal bodies, but the apical dendrites of crest cells receive synapses from unmyelinated and myelinated fibers. In the cerebellar crest, two types of terminals are presynaptic to the crest cell dendrites: boutons with spherical vesicles that from asymmetric synapses with dendritic spines and boutons containing pleomorphic vesicles that from symmetric synapses with dendritic spines and boutons containing pleomorphic vesicles that from symmetric synapses directly on the dendritic shaft. Most axon terminals found on the somata and primary dedrites of crest cells in the nucleus medialis have pleomorphic vesicles and form symmetric contacts, though asymmetric with spherical vesicles and mixed synapses can be observed; these mixed synapses exhibit gap junctions and contain spherical vesicles. Unlike crest cells, the oval neuron perikarya receive three types of contacts (symmetric, asymmetric, and mixed). The origins and functions of these different bouton types in the nucleus medialis are discussed. © 1995 Willy-Liss, Inc.     

Synaptic reorganisation in the rat striatum after dopaminergic deafferentation: An ultrastructural study using glutamate decarboxylase immunocytochemistry

The ultrastructure of GABAergic and non-GABAergic synapses in the adult rat neostriatum was examined 6-8 months after unilateral removal of the nigrostriatal dopaminergic pathway by 6-hydroxydopamine injection into the medial forebrain bundle. GABAergic profiles were identified by preembedding glutamate decarboxylase (GAD) immunocytochemistry performed on parasagittal vibratome sections. In three representative fields of the striatum, the nature and number of boutons and their postsynaptic partners were determined and the differences between the striata ipsi- and contralateral to the lesion analyzed. The percentage of GAD-immunoreactive boutons was increased from 23% on the intact side to 28% on the lesioned side. In addition, the GABAergic boutons underwent significantly more multiple contacts with several independent postsynaptic profiles, preferentially with dendritic spines. This could reflect a lesion-induced sprouting of local GABAergic axon terminals. On the other hand, although the vast majority of GABAergic boutons underwent synaptic contacts with dendrites (77% vs. 80%), the number of boutons per dendrite or per dendritic circumference remained unchanged. Thus, the higher frequency of GABAergic boutons may simply reflect the loss of the dopaminergic nerve endings, without a heterosynaptic replacement by GABAergic boutons. The deafferentation also induced structural changes of the postsynaptic profiles. Some dendritic spines had a shortened neck; others were completely integrated in the dendrite which now contained a spine apparatus and was contacted by boutons with the features of axospinous synapses. The spine retraction resulted in a quantitative decrease in the number of spines. Analysis of the synaptic curvature revealed that only spines with a flat contact zone were lost. Concurrently, the number of dendrites was increased, of the GAD-containing in particular, suggesting that the denrites of GABAergic interzeurons tend to elongate and/or arborize. Taken together, the results of the present study show that the dopaminergic denervation caused a remodeling of the postsynaptic neurons. The relative increase of the number of GABAergic boutons and their synaptic contacts suggests that an altered wiring of the intrinsic GABAergic system contributes to the changes in the striatal output activity. © 1995 Wiley-Liss, Inc.     

GABAergic synaptic boutons in the granule cell layer of rat dentate gyrus

GABAergic synapses in the granule cell layer of the rat dentate gyrus were examined light and electron microscopically with glutamate decarboxylase (GAD) immunocytochemistry. GAD-immunoreactive synaptic boutons formed synapses with axon initial segments and somatic spines as well as somata and dendritic shafts of the granule cell. Most of these synapses were symmetrical, while a few were asymmetrical.