GABAergic Interneurons are the Major Postsynaptic Targets of Median Raphe Afferents in the Rat Dentate Gyrus

The termination pattern of median raphe axons was studied in the rat dentate gyrus using Phaseolus vulgaris leucoagglutinin as an anterograde tracer, in combination with postembedding immunostaining for γ-amino-butyric acid (GABA), and pre-embedding immunostaining for calbindin D28k, parvalbumin and GABA. Postembedding immunogold staining for GABA revealed that the majority (73.7%) of anterogradely labelled median raphe boutons make synaptic contacts with GABA-immunoreactive postsynaptic targets, mainly with dendritic shafts and perikarya. Pre-embedding immunocytochemical double staining for the anterograde tracer and GABA confirmed the electron microscopic results and showed that varicose median raphe axons establish multiple contacts with fusiform interneurons in the hilus and different types of basket cells in the granule cell layer. Some of the innervated cells were shown to contain calbindin D28k, whereas GABAergic interneurons containing another calcium-binding protein, parvalbumin, were never seen to receive multiple contacts from axons of raphe origin. Our results suggest that serotonergic median raphe fibres influence the firing of dentate granule cells via local inhibitory interneurons. The mechanism of using these interneurons with extensive local connections as monosynaptic targets may explain the great efficacy of this pathway in the control of hippocampal electrical activity.     

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.     

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.     

Synaptic connections of intracellularly filled clutch cells: a type of small basket cell in the visual cortex of the cat

Light and electron microscopic quantitative analysis was carried out on a type of neuron intracellularly filled with horseradish peroxidase. Two cells were studied in area 17, one of which was injected intra-axonally, and its soma was not recovered. One cell was studied in area 18. The two somata were on the border of layers IVa/b; they were radially elongated and received synapses from numerous large boutons with round synaptic vesicles. The dendrites were smooth and remained largely in layer IV. The cells can be recognised on the basis of their axonal arbor, which was restricted to layer IV (90-95% of boutons) with minor projections to layers III, V, and VI. Many of the large, bulbous boutons contacted neuronal somata, short collaterals often forming "claw"-like configurations around cells. The name "clutch cell" is suggested to delineate this type of neuron from other aspiny multipolar cells. Computer-assisted reconstruction of the axon showed that in layer IV the axons occupied a rectangular area about 300 X 500 microns, elongated anteroposteriorly in area 17 and mediolaterally in area 18. The distributions of synaptic boutons and postsynaptic cells were patchy within this area. A total of 321 boutons were serially sectioned in area 17. The boutons formed type II synaptic contacts. The postsynaptic targets were somata (20-30%), dendritic shafts (35-50%), spines (30%), and rarely axon initial segments. Most of the postsynaptic somata tested were not immunoreactive for GABA and their fine structural features suggest that they are spiny stellate, star pyramidal, and pyramidal neurons. The characteristics of most of the postsynaptic dendrites and spines also suggest that they belong to these spiny neurons. A few of the postsynaptic dendrites and somata exhibited characteristics of cells with smooth dendrites and these somata were immunoreactive for GABA. It is suggested that clutch cells are inhibitory interneurons exerting their effect mainly on layer IV spiny neurons in an area localised perhaps to a single ocular dominance column. The specific laminar location of the axons of clutch cell also suggests that they may be associated with the afferent terminals of lateral geniculate nucleus cells, and could thus be responsible for generating some of the selective properties of neurons of the first stage of cortical processing.     

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.     

Projections from basal forebrain to prefrontal cortex comprise cholinergic, GABAergic and glutamatergic inputs to pyramidal cells or interneurons

The present study was undertaken to characterize the pre- and postsynaptic constituents of the basal forebrain (BF) projection to the prefrontal cortex in the rat, and determine whether it includes glutamatergic in addition to established γ-aminobutyric acid (GABA)ergic and cholinergic elements. BF fibres were labelled by anterograde transport using biotin dextran amine (BDA) and dual-stained for the vesicular transporter proteins (VTPs) for glutamate (VGluT), GABA (VGAT) or acetylcholine (VAChT). Viewed by fluorescence microscopy and estimated by stereology, proportions of BDA-labelled varicosities were found to be stained for VGluT2 (and not VGluT1 or 3), VGAT or VAChT (representing, respectively, ∼15%, ∼52% and ∼19% within the infralimbic cortex). Each type was present in all, though commonly most densely in deep, cortical layers. Material was triple-stained for postsynaptic proteins to examine whether BDA+VTP+ varicosities might form excitatory or inhibitory synapses, respectively, labelled by postsynaptic density-95 kDA (PSD-95) or gephyrin (Geph). Viewed by confocal microscopy, a majority of BDA+/VGluT2+ varicosities were found to be apposed to PSD-95+ elements, and a majority of BDA+/VGAT+ varicosities to be apposed to Geph+ elements. Other series were triple-stained for cell marker proteins to assess whether the varicosities contacted interneurons or pyramidal cells. Viewed by confocal microscopy, BDA-labelled VGluT2+, VGAT+ and VAChT+ BF terminals were all found in contact with calbindin+ interneurons, whereas VGAT+ BF terminals were also seen in contact with parvalbumin+ interneurons and non-phosphorylated neurofilament+ pyramidal cells. Through distinct glutamatergic, GABAergic and cholinergic projections, the BF can thus influence cortical activity in a diverse manner.     

Arborisation pattern and postsynaptic targets of physiologically identified thalamocortical afferents in striate cortex of the macaque monkey

The monosynaptic targets of different functional types of geniculocortical axons were compared in the primary visual cortex of monkeys. Single thalamocortical axons were recorded extracellularly in the white matter by using horseradish-peroxidase-filled pipettes. Their receptive fields were mapped and classified as corresponding to those of parvi- or magnocellular neurons in the lateral geniculate nucleus. The axons were then impaled and injected intraaxonally with horseradish peroxidase. Two magnocellular (MA) and two parvicellular (PA) axons were successfully recovered and reconstructed in three dimensions. The two MA axons arborised mainly in layer 4C alpha, as did the two PA axons in layer 4C beta. Few collaterals formed varicosities in layer 6. Both MA axons had two large, elongated clumps of bouton (approx. 300-500 x 600-1,200 microns each) and a small clump. One PA axon had two clumps (each with a core appr. 200 microns in diameter); the other had only one (appr. 150-200 microns in axon had 1,380; one MA axon had 3,200 boutons; and those of the more extensive MA axon were not counted. The distribution of postsynaptic targets as well as the number of synapses per bouton has been established for a sample of 150 PA boutons and 173 MA boutons from serial ultrathin sections. The MA axons made on average 2.1 synapses per bouton compared to 1.79 for one PA axon and 2.6 for the other. The sample of boutons taken from the two physiological types of axons contacted similar proportions of dendritic spines (52-68%), shafts (33-47%), and somata (0-3%). The postsynaptic elements were further characterized by immunostaining for GABA. All postsynaptic perikarya and some of the dendrites (4.5-9.5% of all targets) were positive for the amino acid. Near the thalamic synapse GABA-negative dendritic shafts frequently contained lamellar bodies, an organelle identical in structure to spine apparatus. Dendritic shafts and spines postsynaptic to the thalamocortical boutons frequently received an adjacent synapse from GABA-immunoreactive boutons. The similarity between the magno-and parvicellular axons in their targeting of postsynaptic elements, including the GABAergic neurons, suggests that the structural basis of the physiological differences between 4C alpha and 4C beta neurons should be sought in other aspects of the circuitry of layer 4C, such as local cortical circuits, or in the far greater horizontal extent of the thalamocortical and GABAergic axons in layer 4C alpha compared to those in the beta subdivision.     

GABA-immunoreactive synaptic boutons in the rat basal forebrain: comparison of neurons that project to the neocortex with pallidosubthalamic neurons

Although the basal forebrain, including the globus pallidus, contains a high concentration of gamma-aminobutyric acid (GABA), it is not known whether all types of neuron in the globus pallidus receive GABAergic synaptic input. We have studied two types of neuron: typical pallidal neurons that project to the subthalamic nucleus and magnocellular neurons which are found in the medial and ventral borders of the globus and project to the sensorimotor cortex. The postembedding immunogold staining of endogenous GABA revealed many preterminal axons and synaptic boutons that contained GABA immunoreactivity. Neurons that projected to the neocortex were postsynaptic to some of the GABA-immunoreactive boutons, the majority of which formed symmetrical membrane specializations. From a series of random electron micrographs through the perikarya and proximal dendrites of such retrogradely labelled neurons the density of GABA-containing afferent synaptic boutons was estimated to be 0.58 GABA-containing boutons per 100 micron of neuronal membrane. The GABA-containing boutons accounted for 72% of the total afferent input in the proximal regions of the pallidocortical neurons examined. The pallidosubthalamic neurons received many more afferent boutons than did the cortically projecting neurons, a high proportion (80.4%) of which were immunoreactive for GABA. The density of GABA-containing boutons in contact with pallidosubthalamic neurons was 8.9 boutons per 100 micron. It is concluded that cortically projecting basal forebrain neurons, that are probably cholinergic, are innervated by GABA-containing afferent boutons. However, pallidosubthalamic neurons in the same part of the basal forebrain are much more densely innervated by GABA-containing boutons.     

The termination pattern and postsynaptic targets of rubrospinal fibers in the rat spinal cord: a light and electron microscopic study.

The spinal course, termination pattern, and postsynaptic targets of the rubrospinal tract, which is known to contribute to the initiation and execution of movements, were studied in the rat at the light and electron microscopic levels by using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) in combination with calbindin-D28k (CaBP), gamma-aminobutyric acid (GABA), and glycine immunocytochemistry. After injections of PHA-L unilaterally into the red nucleus, labelled fibers and terminals were detected at cervical, thoracic, and lumbar segments of the spinal cord. Most of the descending fibers were located in the dorsolateral funiculus contralateral to the injection site, but axons descending ipsilaterally were also revealed. Rubrospinal axon terminals were predominantly found in laminae V-VI and in the dorsal part of lamina VII at all levels and on both sides of the spinal cord, but stained collaterals were also seen in the ventrolateral aspect of Clark's column and in the ventral regions of lamina VII on both sides. The proportion of axonal varicosities revealed on the ipsilateral side varied at different segments and represented 10-28% of the total number of labelled boutons. Most of the labelled boutons were engaged in synaptic contacts with dendrites. Of the 137 rubrospinal boutons investigated, only 2 were found to establish axosomatic synaptic junctions in the lumbar spinal cord contralateral to the PHA-L injection. With the postembedding immunogold method, 80.8% of dendrites establishing synaptic contacts with rubrospinal terminals did not show immunoreactivity for either GABA or glycine, whereas 19.2% of them were immunoreactive for both amino acids. Rubrospinal axons made multiple contacts with CaBP-immunoreactive neurons in laminae V-VI. Synaptic contacts between rubrospinal terminals and CaBP-immunoreactive dendrites were identified at the electron microscopic level, and all CaBP-containing postsynaptic dendrites investigated were negative for both GABA and glycine. The results suggest that rubrospinal terminals establish synaptic contacts with both excitatory and inhibitory interneurons in the rat spinal cord, and a population of excitatory interneurons receiving monosynaptic rubrospinal input is located in laminae V-VI.     

Cholinergic innervation of pyramidal cells and parvalbumin-immunoreactive interneurons in the rat basolateral amygdala

The basolateral nucleus of the amygdala receives an extremely dense cholinergic innervation from the basal forebrain that is critical for memory consolidation. Although previous electron microscopic studies determined some of the postsynaptic targets of cholinergic afferents, the majority of postsynaptic structures were dendritic shafts whose neurons of origin were not identified. To make this determination, the present study analyzed the cholinergic innervation of the anterior subdivision of the basolateral amygdalar nucleus (BLa) of the rat using electron microscopic dual-labeling immunocytochemistry. The vesicular acetylcholine transporter (VAChT) was used as a marker for cholinergic terminals; calcium/calmodulin-dependent protein kinase II (CaMK) was used as a marker for pyramidal cells, the principal neurons of the BLa; and parvalbumin (PV) was used as a marker for the predominant interneuronal subpopulation in this nucleus. VAChT(+) terminals were visualized by using diaminobenzidine as a chromogen, whereas CAMK(+) or PV(+) neurons were visualized with Vector very intense purple (VIP) as a chromogen. Quantitative analyses revealed that the great majority of dendritic shafts receiving cholinergic inputs were CAMK(+) , indicating that they were of pyramidal cell origin. In fact, 89% of the postsynaptic targets of cholinergic terminals in the BLa were pyramidal cells, including perikarya (3%), dendritic shafts (47%), and dendritic spines (39%). PV(+) structures, including perikarya and dendrites, constituted 7% of the postsynaptic targets of cholinergic axon terminals. The cholinergic innervation of both pyramidal cells and PV(+) interneurons may constitute an anatomical substrate for the generation of oscillatory activity involved in memory consolidation by the BLa.     

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.     

Axoaxonic synapses on terminals of group II muscle spindle afferent axons in the spinal cord of the cat.

The purpose of the present study was to determine if terminals of identified group II muscle spindle afferents participate in axoaxonic synaptic arrangements and, if so, to investigate the transmitter content of presynaptic terminals in these arrangements. Group II muscle afferents supplying the gastrocnemius-soleus or semitendinosus muscles were identified in adult cats and stained intra-axonally with horseradish peroxidase. In total, three group II axons were labelled and processed for combined light and electron microscopy. Group II axons gave rise to collaterals which characteristically descended through the superficial dorsal horn and formed relatively sparse terminal arborizations in the dorsal horn (laminae IV and V) and more profuse arbors in the intermediate grey matter (laminae VI-VII). Forty boutons were examined through series of ultrathin sections and all but four were postsynaptic to other axon terminals. Occasionally, more than one axon was presynaptic to a single group II terminal. Immunogold studies showed that all axons in presynaptic apposition to group II boutons contained gamma-aminobutyric acid (GABA) and also that glycine was colocalized in the majority of these axons. This evidence suggests that transmission from group II muscle afferents is under strong presynaptic inhibitory control and that it is mainly the subgroup of GABAergic interneurons with colocalized glycine which mediate this inhibition. Seventeen group II boutons were components of synaptic triads where the presynaptic axoaxonic bouton formed a synapse with the same dendrite as the group II axon. Therefore, a proportion of the interneurons which form axoaxonic synapses with group II axons are also likely to have postsynaptic inhibitory actions on target neurons of group II afferents.     

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)     

Convergence and segregation of septal and median raphe inputs onto different subsets of hippocampal inhibitory interneurons

The convergence and segregation of medial septal and median raphe afferents in the innervation of different subpopulations of GABAergic interneurons was investigated in the rat hippocampal formation. Following local injections of 5,7-dihydroxytryptamine into the median raphe nucleus destroying all serotonergic neurons, iontophoretic injections of Phaseolus vulgaris leucoagglutinin (PHAL) into the medial septum resulted in anterograde labelling of axons in the hippocampus. The labelled varicose fibres made multiple contacts with calbindin D28K-, parvalbumin-, and cholecystokinin-immunoreactive interneurons. These results disproved the possibility that PHAL-labelled afferents innervating hippocampal interneurons following septal PHAL injections would have been raphe axons passing through the injection site. In the second set of experiments a double anterograde tracing technique (PHAL from the septum and biotinylated-PHAL from the median raphe) and a triple or double immunostaining procedure was used to determine the types of interneurons (calbindin D28K-, parvalbumin-, or cholecystokinin-immunoreactive) innervated by one, or the other, or both pathways. The results showed that parvalbumin-containing neurons were innervated by septal afferents but avoided by raphe axons, whereas calbindin D28K-containing cells, and to a smaller extent cholecystokinin-containing cells served as targets for both pathways. In some cases the same individual calbindin D28K- or cholecystokinin-containing neurons received multiple contacts from afferents of both septal and raphe origin. Thus, our results indicate that different subcortical nuclei modulate largely different inhibitory circuits in the hippocampal formation. However, considering the occasional convergence of the two subcortical nuclei not only onto the same type, but even onto the same individual calbindin D28K-containing interneurons, we propose that a particular inhibitory function, most probably feed-forward inhibition in the distal dendritic region, is under the control of both pathways.     

Enkephalin‐containing interneurons are specialized to innervate other interneurons in the hippocampal CA1 region of the rat and guinea‐pig

Enkephalins are known to have a profound effect on hippocampal inhibition, but the possible endogenous source of these neuropeptides, and their relationship to inhibitory interneurons is still to be identified. In the present study we analysed the morphological characteristics of met-enkephalin-immunoreactive cells in the CA1 region of the rat and guinea-pig hippocampus, their coexistence with other neuronal markers and their target selectivity at the light and electron microscopic levels. Several interneurons in all subfields of the hippocampus were found to be immunoreactive for met-enkephalin. In the guinea-pig, fibres arising from immunoreactive interneurons were seen to form a plexus in the stratum oriens/alveus border zone, and basket-like arrays of boutons on both enkephalin-immunoreactive and immunonegative cell bodies in all strata. Immunoreactive boutons always established symmetric synaptic contacts on somata and dendritic shafts. Enkephalin-immunoreactive cells co-localized GABA, vasoactive intestinal polypeptide and calretinin. Postembedding immunogold staining for GABA showed that all the analysed enkephalin-immunoreactive boutons contacted GABAergic postsynaptic structures. In double-immunostained sections, enkephalin-positive axons were seen to innervate calbindin D28k-, somatostatin-, calretinin- and vasoactive intestinal polypeptide-immunoreactive cells with multiple contacts. Based on these characteristics, enkephalin-containing cells in the hippocampus are classified as interneurons specialized to innervate other interneurons, and represent a subset of vasoactive intestinal polypeptide- and calretinin-containing cells. The striking match of ligand and receptor distribution in the case of enkephalin-mediated interneuronal communication suggests that this neuropeptide may play an important role in the synchronization and timing of inhibition involved in rhythmic network activities of the hippocampus.     

The W cell pathway to cat primary visual cortex

The thalamic input to area 17 in the cat can be divided into at least three parallel pathways, the W, X, and Y. Although the latter two are some of the best studied synaptic connections in the brain, the former remains poorly understood both in structure and in function. By combining light and electron microscopy, we have reconstructed in 3‐D single W axons and described quantitatively the synapses that they form. We have also made a structural comparison of reconstructed synapses from the three visual pathways. Thalamic axons were labeled in vivo by injections of biotinylated dextran amine into the dLGN. W axons originating from C laminae injections arborized in layers 1, 2/3, and 5. Axons that traversed layer 1 supplied a few descending collaterals to layer 2/3, but the most extensive innervation in layer 2/3 was provided by axons ascending from the white matter. Most W boutons formed a single synapse, dendritic spines being the most common target, with dendritic shafts forming the remaining targets. In layer 1, the area of the postsynaptic density of spine synapses (0.16 μm²) was significantly larger than that of layers 2/3 (0.11 μm²) and 5 (0.09 μm²). Synapses from X and Y axons in layer 4 were similar in size to synapses formed by W boutons in layer 1. In layer 1, the main targets of the W axons are likely the apical dendrites of pyramidal cells, so that both proximal and distal regions of pyramidal cell dendritic trees can be excited by the W pathway. J. Comp. Neurol. 516:20–35, 2009. © 2009 Wiley‐Liss, Inc.     

Synaptic connection from cortical area V4 to V2 in macaque monkey

The major target of the V4 projection in V2 is layer 1, where it forms a tangential spread of asymmetric (excitatory) synapses. This is characteristic of a "feedback" projection. Some axons formed discrete clusters of bouton terminaux between lengths of myelinated axon, while others were unbranched and formed a continuous distribution of en passant boutons with no intercalated myelin. Minor projections were found in layers 2/3 and 6. Dendritic spines were the most frequently encountered targets of the V4 projection (80% in layer 1 and layer 2/3, 94% in layer 6). The remaining targets were dendritic shafts. In layer 1, 69% of target dendrites (12% of all targets) had characteristics identifying them as smooth (GABAergic) cells. In layer 2/3 and layer 6 virtually all the shaft synapses were on smooth dendrites (86% and 100%, respectively). Multisynaptic boutons were rare (mean 1.1 synapses per bouton). Synapses formed in layer 6 were smaller than those of layer 1 (mean area 0.073 microm(2) vs. 0.117 microm(2)). Synapses formed with spines had a more complex postsynaptic density than those formed with dendritic shafts. With respect to targets and synaptic type and size and morphology of synapses, the feedback projection from V4 to V2 resembles those of feedforward projections. The principal difference between the feedforward and feedback projection is in the lamina location of their terminal boutons. The concentration of the V4 projection on layer 1, where it forms asymmetric synapses mainly with spines, suggests that it excites the distal apical dendrites of pyramidal cells.     

Simultaneous Demonstration of Serotonin-immunoreactive Terminals and GABAergic Neurons in the VPL Nucleus of the Cat Thalamus

Pre-embedding immunoperoxidase (for serotonin) and postembedding immunogold (for γ-aminobutyric acid; GABA) labelling were combined at light and electron microscopic levels to demonstrate the neuronal targets of serotonin (5-HT) afferents in the ventral posterior lateral nucleus (VPL) of the cat thalamus. 5-HT-immunoreactive fibres and terminal varicosities were found in close proximity to GABA-immunoreactive interneurons and non-GABAergic relay neurons. Ultrastructurally, the vast majority of 5-HT terminals made close membrane contacts without overt membrane specializations with GABAergic axon terminals, GABAergic presynaptic dendrites and GABAergic somata. A very small number of 5-HT terminals formed typical asymmetrical synapses with GABAergic presynaptic dendrites and with dendritic shafts of relay cells. Some 5-HT terminals participated with the presynaptic dendrites in triadic synaptic arrangements. These findings suggest a dual innervation pattern by 5-HT afferents in VPL and the release of 5-HT in large part at sites not associated with morphologically detectable synapses.     

Distribution of GABAergic Elements Postsynaptic to Ventroposteromedial Thalamic Projections in Layer IV of Rat Barrel Cortex

The spatial synaptic pattern formed by boutons, originating in the ventroposteromedial thalamic nucleus, with GABAergic neurons in the rat barrel cortex was mapped. The aim was to shed light on the structural basis by which inhibitory circuits may be activated at the first stage of cortical information processing. The thalamic afferent projection was labelled by anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L), whereas the GABAergic targets in layer IV of the rat barrel cortex were visualized by postembedding GABA immunogold-labelling or by pre-embedding parvalbumin immunocytochemistry. In the first set of experiments, we mapped barrels, contained in single ultrathin sections, by means of a computer-controlled electron microscope stage in their entire layer IV representation. From a total of 1199 asymmetric PHA-L-labelled synapses, only 98 were on GABAergic elements, mainly on dendritic shafts. This corresponded to 8.2% of all synapses counted. These synapses on GABAergic targets were essentially homogeneously distributed without a reliable relationship to barrel subdivisions, i.e. hollow versus wall; or layer IVa versus layer IVb. In the second part of the study, we demonstrated that parvalbumin-containing neurons represent the major GABAergic cell type targetted by thalamic afferents in layer IV of the barrel cortex, since all parvalbumin-positive cells investigated received multiple synaptic contacts (up to eight synapses per neuron) from the ventroposteromedial thalamic nucleus. These results imply that interneurons responsible for perisomatic inhibition (basket and chandelier cells known to contain parvalbumin) are likely to be strongly excited by thalamic afferents, despite the relatively low proportion of thalamic synapses on GABAergic elements compared to spines of principal cells, and participate in the early stages of cortical sensory information processing.     

Organization,Development and Enucleation-induced Alterations in the Visual Callosal Projection of the Hamster: Single Axon Tracing with Phaseolus vulgaris leucoagglutinin and Di-I

The distribution of callosal axons interconnecting lateral area 17 and medial area 18 of the rodent's occipital cortex is dramatically altered by neonatal enucleation, but it is not known how this manipulation affects the morphology of individual callosal axons or whether the enucleation-induced changes in this pathway reflect maintenance of a transient developmental state by these fibres. In the present study, these questions were addressed by tracing the individual callosal axons in normal adult and neonatally enucleated adult hamsters with Phaseolus vulgaris leucoagglutinin (PHAL) and by anterograde labelling of developing callosal axons with the carbocyanine dye, Di-I. In normal adults, injections of PHAL into the region of the 17 - 18a border produced dense labelling in all layers in the region of the contralateral 17 - 18a border. Larger injections resulted in callosal labelling that extended across the lateral one-half of area 17, primarily in layers I and V. Thirty-four callosal axons from normal adult hamsters were reconstructed through all the cortical laminae. Most of these had very simple terminal arbors. They gave off short collaterals in the infragranular layers and branched more extensively in the uppermost part of layer II - III and in lamina I. Small injections of PHAL into the occipital cortex of neonatally enucleated adult hamsters resulted in labelled axons throughout most of areas 17 and 18a in the contralateral hemisphere. The terminal arbors of most individual callosal axons in eyeless hamsters were not appreciably different from those in sighted animals. However, 26.8% of 28 fibres reconstructed through all cortical laminae in the neonatally enucleated hamsters had much more widespread branches than any of the axons recovered from normal hamsters. As a result, the average total length of the callosal axons from the blinded hamsters was significantly greater than that for such fibres from the sighted animals. Anterograde labelling with Di-I demonstrated axons in the anterior commissure and anterior part of the corpus callosum on P-0. Labelled fibres extended into the white matter underlying the occipital cortex on P-1 and entered the cortical plate on P-2. Some of these axons reached into the marginal layer. Many developing callosal axons had short branches in the white matter, but generally extended only a single collateral into the cortical grey matter. Callosal axons in perinatal animals branched very little within the cortex and, in this respect, resembled fibres labelled with PHAL in adult hamsters. These results support the conclusion that the expanded tangential distribution of the occipital callosal projection in neonatally enucleated adult hamsters results, at least in part, from individual axons with abnormally widespread terminal arbors which are not present in large numbers at any time during normal development.