Immunocytochemical study of GABAergic neurons containing the calcium-binding protein parvalbumin in the rat hippocampus

Summary The structural features of PV-immunoreactive (PV-I) neurons, a particular subpopulation of GABAergic neurons, in the hippocampus were studied by immunocytochemistry. The PV-I cell bodies were concentrated within the stratum pyramidale (SP) and stratum oriens (SO) in the hippocampus. PV-I puncta were frequent in SP, while they were rarely seen in other layers. The dendritic arborization of PV-I neurons resembled that of some of the nonpyramidal cells observed after Golgi-impregnation. The most commonly observed PV-I neurons had their perikarya located in SP with dendrites extending into SO and the stratum radiatum (SR). Most of the dendrites in SR had typical beaded or varicose segments. The dendrites extending into SO had few beaded parts. There were many bipolar and multipolar neurons with smooth dendrites in SO, but only a small number of such multipolar neurons in SR. An electron microscopic analysis revealed that PV-I products were located to perikarya, dendrites, myelinated axons and synaptic boutons. The perikarya of PV-I neurons exhibited several ultrastructural features of nonpyramidal cells, e.g., abundant cisternae of endoplasmic reticulum, mitochondria and other perikaryal organelles, an infolded nuclear envelope and intranuclear inclusions. They received many asymmetric synapses with round presynaptic vesicles. There were numerous PV-I boutons, presumably axonal endings, covering the pyramidal cell bodies. The PV-I boutons also contacted the axon initial segments and proximal dendrites of the pyramidal cells. In addition PV-I terminals were found on somata and dendrites of both PV-I or PV-negative nonpyramidal cells. The results suggest that PV-containing neurons include basket and axo-axonic cells.     

The projection of the lateral geniculate nucleus to area 17 of the rat cerebral cortex. V. Degenerating axon terminals synapsing with Golgi impregnated neurons

Summary The sites of termination of afferents from the lateral geniculate nucleus to layer IV and lower layer III in area 17 of the rat visual cortex have been determined by use of a combined degeneration—Golgi/EM technique. Degeneration of geniculocortical axon terminals was produced by making lesions in the lateral geniculate body. After the animals had been allowed to survive for two days, the ipsilateral visual cortex was removed and impregnated by the Golgi technique. Suitably impregnated neurons and their processes in layer IV and lower layer III were then gold-toned and deimpregnated for examination in the electron microscope. A search was made for synapses between degenerating axon terminals and the gold-labelled postsynaptic neurons.Geniculocortical synapses were found to involve: (1) the spines of basal dendrites, as well as those of proximal shafts and collaterals of apical dendrites of layer III pyramidal neurons; (2) the spines of the apical dendritic shafts and collaterals of layer V pyramidal neurons; (3) the perikaryon and dendritic spines of a sparsely-spined stellate cell; and (4) the perikaryon and dendrites of a smooth, bitufted stellate cell. In view of this variety of postsynaptic elements it is suggested that all parts of the perikarya and dendrites of neurons contained in layer IV and lower layer III which are capable of forming asymmetric synapses can be postsynaptic to the thalamic input.Finally, an analysis of the known neuronal interrelations within the rat visual cortex is presented.     

Immunogold demonstration of GABA in synaptic terminals of intracellularly recorded, horseradish peroxidase-filled basket cells and clutch cells in the cat's visual cortex

To identify the putative transmitter of large basket and clutch cells in the cat's visual cortex, an antiserum raised against GABA coupled to bovine serum albumen by glutaraldehyde and a postembedding, electron microscopic immunogold procedure were used. Two basket and four clutch cells were revealed by intracellular injection of horseradish peroxidase. They were identified on the basis of the distribution of their processes and their synaptic connections. Large basket cells terminate mainly in layer III, while clutch cells which have a more restricted axon, terminate mainly in layer IV. Both types of neuron have a small radial projection. They establish type II synaptic contacts and about 20-30% of their synapses are made with the somata of other neurons, the rest with dendrites and dendritic spines. Altogether 112 identified, HRP-filled boutons, the dendrites of three clutch cells and myelinated axons of both basket and clutch cells were tested for the presence of GABA. They were all immunopositive. The postsynaptic neurons received synapses from numerous other GABA-positive boutons in addition to the horseradish peroxidase-filled ones. Dendritic spines that received a synapse from a GABA-positive basket or clutch cell bouton also received a type I synaptic contact from a GABA-negative bouton. A few of the postsynaptic dendrites, but none of the postsynaptic somata, were immunoreactive for GABA. The fine structural characteristics of the majority of postsynaptic targets suggested that they were pyramidal and spiny stellate cells. These results provide direct evidence for the presence of immunoreactive GABA in identified basket and clutch cells and strongly suggest that GABA is a neurotransmitter at their synapses. The laminar distribution of the synaptic terminals of basket and clutch cells demonstrates that some GABAergic neurons with similar target specificity segregate into different laminae, and that the same GABAergic cells can take part in both horizontal and radial interactions.     

Synaptic organization of immunocytochemically identified GABA neurons in the monkey sensory-motor cortex

Neurons in the monkey somatic sensory and motor cortex were labelled immunocytochemically for the GABA synthesizing enzyme, glutamic acid decarboxylase (GAD), and examined with the electron microscope. The somata and dendrites of many large GAD-positive neurons of layers III-VI receive numerous asymmetric synapses from unlabelled terminals and symmetric synapses from GAD-positive terminals. Comparisons with light and electron microscopic studies of Golgi-impregnated neurons suggest that the large labelled neurons are basket cells. Small GAD-positive neurons generally receive few synapses on their somata and dendrites, and probably conform to several morphological types. GAD-positive axons from symmetric synapses on many neuronal elements including the somata, dendrites and initial segments of pyramidal cells, and the somata and dendrites of non-pyramidal cells. Synapses between GAD-positive terminals and GAD-positive cell bodies and dendrites are common in all layers. Many GAD-positive terminals in layers III-VI arise from myelinated axons. Some of the axons form pericellular terminal nests on pyramidal cell somata and are interpreted as originating from basket cells while other GAD-positive myelinated axons synapse with the somata and dendrites of non-pyramidal cells. These findings suggest either that the sites of basket cell terminations are more heterogeneous than previously believed or that there are other classes of GAD-positive neurons with myelinated axons. Unmyelinated GAD-positive axons synapse with the initial segments of pyramidal cell axons or form en passant synapses with dendritic spines and small dendritic shafts and are interpreted as arising from the population of small GAD-positive neurons which appears to include several morphological types.     

Parvalbumin-immunoreactive neurons in the entorhinal cortex of the rat: localization, morphology, connectivity and ultrastructure

Summary We studied the distribution, morphology, ultrastructure and connectivity of parvalbumin-immunoreactive neurons in the entorhinal cortex of the rat. Immunoreactive cell bodies were found in all layers of the entorhinal cortex except layer I. The highest numbers were observed in layers II and III of the dorsal division of the lateral entorhinal area whereas the lowest numbers occurred in the ventral division of the lateral entorhinal area, Most such neurons displayed multipolar configurations with smooth dendrites. We distinguished a type with long dendrites and a type with short dendrites. We also observed pyramidal immunoreactive neurons. A dense plexus of immunoreactive dendrites and axons was prominent in layers II and III of the dorsal division of the lateral entorhinal area and the medial entorhinal area. None of the parvalbuminimmunoreactive cells became retrogradely labelled after injection of horseradish peroxidase into the hippocampal formation. By electron microscopy, immunoreactivity was observed in cell bodies, dendrites, myelinated and unmyelinated axons and axon terminals. Immunoreactive dendrites and axons occurred in all cortical layers. We noted many myelinated immunoreactive axons. Immunoreactive axon terminals were medium sized, contained pleomorphic synaptic vesicles, and established symmetrical synapses. Both horseradish peroxidase labelled and unlabelled immunonegative cell bodies often received synapses from immunopositive axon terminals arranged in baskets. Synapses between immunoreactive axon terminals and unlabelled dendritic shafts and spines were abundant. Synapses with initial axon segments occurred less frequently. In addition, synaptic contacts were present between immunopositive axon terminals and cell bodies and dendrites. Thus, the several types of parvalbumin-containing neuron in the entorhinal cortex are interneurons, connected to one another and to immunonegative neurons through a network of synaptic contacts. Immunonegative cells projecting to the hippocampal formation receive axo-somatic basket synapses from immunopositive terminals. This connectivity may form the morphological substrate underlying the reported strong inhibition of cells in layers II and III of the entorhinal cortex projecting to the hippocampal formation.     

Basket cells in the monkey fascia dentata: A Golgi/electron microscopic study

Summary This study describes non-granule cells in the fascia dentata of rhesus monkeys and baboons. Their cell bodies are located in the molecular layer and at the hilar border of the granular layer. They are called basket cells since their axons give rise to collaterals that branch in the close vicinity of the parent cell body and form symmetric synapses with dendrites and cell bodies of granule cells. These neurons are further classified with regard to the shape and location of their cell bodies and the orientation of their dendrites. Basket cells in the molecular layer are mainly bipolar with dendrites oriented perpendicular to the granular layer. These dendrites are densely innervated by presynaptic boutons forming asymmetric synapses. We have rarely observed molecular layer basket cells with dendrites traversing the granular layer and invading the hilus. We thus conclude that these cells are mainly activated by extrinsic afferents terminating in the molecular layer. Basket cells at the hilar border display pyramidal, fusiform or multipolar cell bodies that give rise to apical dendrites traversing the molecular layer and basal dendrites invading the hilar region. Large boutons establish asymmetric synapses with identified basal dendrites of these neurons. The dendrites of all types of basket cell are smooth, i.e. they had few or no spines. Many of them display varicosities. Cell counts in Cresyl Violet-stained sections revealed a ratio of basket cells to granule cells of 1:500. Essentially, the types of basket cell in the monkey fascia dentata are similar to those described previously for the rat. This contrasts sharply to our recent findings for pyramidal neurons and granule cells of the monkey hippocampus which showed an increased complexity and variability when compared with rodents. These data do not support the hypothesis that only local circuit neurons evolve in phylogeny.     

The axo-axonic interneuron in the cerebral cortex of the rat,cat and monkey

The synaptic connections of a specific type of identified cortical interneuron, the axo-axonic cell, were studied using Golgi methods. In the light-microscope axo-axonic cells were demonstrated in certain layers of the primary and secondary visual cortex of rat, cat and monkey, in the motor cortex of cat and in the subiculum and pyriform cortex of rat. The dendrites originating from the oval soma were oriented radially in a lower and upper spray within a cylinder about 100–150 μm wide. Electronmicroscopy of Golgi impregnated, gold-toned axo-axonic cells showed predominantly but not exclusively asymmetrical synaptic contacts on their dendrites and spines, few synaptic contacts on the perikarya some of which were asymmetrical, and no synaptic contacts on the axon initial segment. The axon usually arborized within the vicinity of the cell's own dendritic field in an area 100–200 μm in diameter. In the kitten motor cortex the axon of a neuron in layer III descended to layer VI, providing a columnar arborization.The axon formed specialized, 10–50 μm long terminal segments invariably oriented parallel with the axon initial segment of pyramidal cells. All 85 identified symmetrical-type synaptic contacts, deriving from 31 specialized terminal segments, were found exclusively on the axon initial segment of pyramidal neurons. Rare, lone boutons of axo-axonic cells also made synaptic contact only with axon initial segments, confirming the exclusive target specificity of these cells. In identified gold-toned boutons, flattened pleomorphic vesicles were present. Electron-microscopy showed that axons ending in specialized terminal segments may originate from myelinated fibres, indicating that Golgi impregnation has revealed only part of the axon. Counting of axon terminal segments, each of which was in contact with the axon initial segment of a pyramidal neuron, revealed 166 pyramidal neurons receiving input from a partially reconstructed axo-axonic cell in the motor cortex of the kitten, and 67 from another cell in the visual cortex of the cat. The convergence of five axo-axonic cells onto one pyramidal cell was demonstrated in the striate cortex of the cat by counting all synaptic contacts on three initial segments. Cells from a one-month-old kitten were compared with those of the adult. The axon of the developing neurons was more diverse, having many growth cones and filopodia which made no specialized membrane contacts. However, the developing specific terminal segments formed synapses only with axon initial segments.It is concluded that the presence of axo-axonic cells in all the species and cortical areas we have examined suggests their association with the structural design of pyramidal cells, wherever the latter occur, and with their participation in the information processing of pyramidal cells. Axo-axonic cells are uniquely endowed with the means of simultaneously influencing the action potential at the site of origin in groups of pyramidal cells. This strategic location may enable them to synchronise the activity of pyramidal neurons, either through inhibitory gating or through changing the threshold of pyramidal cells to certain inputs.     

A combined Golgi-electron microscopic study of the synapses made by the proximal axon and recurrent collaterals of a pyramidal cell in the somatic sensory cortex of the monkey

The synapses made within the cortex by the proximal axon and recurrent collateral branches of a pyramidal cell, the soma of which was at the boundary of layers II and III of the somatic sensory cortex of the monkey, have been studied by the combined Golgi-electron microscopic technique. In a large number of serial sections 62 synapses were found, all of which were asymmetric, 49 being formed by the main axon and 13 by the collaterals. Sixty per cent of the synapses were upon the shafts of dendrites, approximately half of which were identified as being of the large or aspinous stellate cell, and the remainder were upon dendritic spines.If the large stellate cell is inhibitory, then these findings provide a morphological basis for recurrent collateral inhibition.     

The distribution of intrinsic cortical axons in area 3b of cat primary somatosensory cortex

Summary The morphology of single neurons in area 3b of cat primary somatosensory (SI) cortex was examined after horseradish peroxidase (HRP) injections. Neurons were labeled either by intracellular injection of HRP following intracellular recording or by small extracellular iontophoretic HRP injections. Both pyramidal and nonpyramidal neurons were labeled and reconstructed from serial sections. Their axons had local, interlaminar and interareal patterns of termination. Most neurons formed local axonal fields around their cell bodies and dendrites. Pyramidal neurons in cortical layer IV sent axons up into layers II and III, neurons in layers II and III sent axons down to layer V, and layer V neurons sent axons to layer VI as well as back to the upper layers. Layer VI neurons sent axons back to the upper cortical layers in a unique bowl-shaped pattern. The horizontal distribution of axons of pyramidal cells in layer III was extremely widespread. Axons of layer III neurons in area 3b terminated within 3b and area 1, but not in other areas of SI. Layer III neurons in area 1 distributed axon collaterals to all fields of SI as well as projecting a main axon to motor cortex. In general, the axon collaterals of area 3b pyramidal cells outside layer III remained confined to area 3b. Most of the nonpyramidal neurons labeled were basket cells in layers III and VI. These neurons formed dense axonal fields around their cell bodies, and none of their axons could be followed into the underlying white matter. The results of the present study demonstrate that area 3b somatosensory cortical neurons and their axons are vertically organized in a manner similar to that reported for other sensory cortical areas. They also show that widespread horizontal connections are formed by pyramidal neurons of layer III, and that these horizontal axons can travel for great distances in the cortical grey matter.     

A correlative electron microscopic study of basket cells and large GABAergic neurons in the monkey sensory-motor cortex

Large basket cells were identified in Golgi and horseradish peroxidase labeled material from the sensory-motor cortex of adult monkeys. Their morphology was correlated at the light and electron microscopic level with large comparable cells stained immunocytochemically for glutamate decarboxylase. In Golgi-impregnated material these cells have a very large cell body and dendrites that extend through several layers of the cortex with a predominant vertical orientation. The axon is only stained for a few micrometers. The same cells studied electron microscopically in serial sections after gold-toning show very distinctive ultrastructural characteristics: the cell bodies contain a large number of organelles, the nuclei are rounded with homogeneously dispersed chromatin and synapsing onto the somata are many axon terminals, both symmetrical and asymmetrical but the symmetrical type forms 70-80% of the total; dendrites also receive a large number of both symmetrical and asymmetrical synaptic contacts. All the axons of basket cells become myelinated and the Golgi labeling of the initial segments is interrupted at the commencement of the first myelin internode. The axon initial segments receive several symmetrical synaptic contacts in the proximal one-third of their length. The axonal arborization of a basket cell retrogradely labeled in the somatosensory cortex after intracortical injection of horseradish peroxidase was analyzed in detail. The mainly horizontal axonal collaterals of this cell are myelinated for most of their trajectory and have a preferred orientation in the anteroposterior dimension. These axonal collaterals, although very long (more than 1.8 mm), at intervals give rise to a small number of short unmyelinated terminal branches that bear a series of boutons terminaux forming a multi-terminal ending. The multi-terminal endings surround somata and proximal dendrites of pyramidal and non-pyramidal cells. Dense pericellular terminations (baskets or nests) like those drawn by Ramón y Cajal and Marin-Padilla are not formed by the axon of a single basket cell. Thus, basket formations are presumably formed by converging axons from several basket cells. Immunocytochemical material was stained for glutamate decarboxylase, the enzyme involved in the synthesis of gamma-aminobutyrate (GABA). This shows that large glutamate decarboxylase-positive neurons of the same size as those positively identified as basket cells in the Golgi and horseradish peroxidase material have virtually the same morphological characteristics, at both the light and electron microscope levels, as the basket cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The termination of callosal fibres in the auditory cortex of the rat. A combined Golgi--electron microscope and degeneration study

When the corpus callosum of the rat is sectioned, the callosal fibres in the cerebral cortex undergo degeneration. In the auditory cortex (area 41) the degenerating axon terminals form asymmetric synapses, and the vast majority of them synapse with dendritic spines. Some other synapse with the shafts of both spiny and smooth dendrites, and a few with the perikarya of non-pyramidal cells. The degenerating axon terminals are contained principally within layer II/III, in which they aggregate in patches. Using a technique in which neurons within the cortex are Golgi-impregnated, then gold-toned and examined in the electron microscope, it has been shown that the dendritic spines of pyramidal neurons with cell bodies in different layers receive the degenerating callosal afferents. The spines arise from the main apical dendritic shafts and their branches, from the dendrites of the apical tufts, and in some cases from the basal dendrites of the pyramidal neurons. The shafts of some pyramidal cell apical dendrites also form asymmetric synapses with callosal afferents. Since we have encountered no spiny non-pyramidal neurons in Golgi preparations of rat auditory cortex, and because other types of non-pyramidal cells have few dendritic spines, it is concluded that practically all of the dendritic spines synapsing with callosal afferents originate from pyramidal neurons.     

Synaptic organization of intracellularly stained CA3 pyramidal neurons in slice cultures of rat hippocampus

Pyramidal cells of regio inferior in slice cultures of the rat hippocampus were impaled and intracellularly stained with horseradish peroxidase. A correlated light- and electron-microscopic analysis was then performed to study the properties of these neurons under culture conditions with particular emphasis on input synapses onto these cells. Like pyramidal cells in situ, CA3 pyramidal neurons in slice cultures had a triangular cell body with an apical stem dendrite emerging from it. Several basal dendrites and the axon arose from the basal pole of the cell body. The peripheral thin branches of both apical and basal dendrites were covered with small spines, whereas proximal thick dendritic segments and portions of the cell body exhibited large spines or excrescences. The axon gave off numerous fine varicose collaterals which projected to stratum radiatum of CA1 (Schaffer collaterals), to the alveus and to the hilar region. In one case a collateral could be followed to stratum moleculare of the fascia dentata. Electron-microscopic analysis of the injected pyramidal neurons revealed that their cell bodies, dendritic shafts and spines formed synaptic contacts with presynaptic terminals. Mossy fiber endings were identified by their large size and their numerous clear synaptic vesicles with some dense-core vesicles intermingled, and were observed to form synaptic contacts on the large spines or excrescences. Since extrinsic afferents degenerate in slice cultures, the numerous synaptic boutons on the identified pyramidal neurons probably arise from axons of intrinsic neurons that have sprouted in response to deafferentation. This assumption is supported by the finding that collaterals of the injected neurons formed abundant synaptic contacts on dendritic shafts and spines of other cells. These results suggest that, although pyramidal cells under culture conditions retain a remarkable number of their normal characteristics, considerable synaptic reorganization does take place.     

Aspiny neurons and their local axons in the neostriatum of the rat: a correlated light and electron microscopic study of Golgi-impregnated material

Summary Three types of neuron with smooth (aspiny) dendrites could be distinguished in the Golgi-impregnated rat neostriatum. Examples of each type of aspiny neuron were found with local axon collaterals within the neostriatum and these were selected for gold-toning and examination in the electron microscope. One type of aspiny neuron had an elongated, usually spindle-shaped, medium-size soma with two, or rarely three, primary dendrites originating from opposite poles of the cell; one example of this type of neuron had two separate axons. The second type of aspiny neuron had a nearly round, medium-size soma with four primary dendrites that branched profusely quite close to the cell body. A third type of aspiny neuron had a very large polygonal-shaped cell body. Afferent axon terminals were found in synaptic contact with the dendrites and cell bodies of all three types of aspiny neuron.Axon collaterals of each type of neuron displayed varicosities which, when examined in the electron microscope, were frequently found to be boutons making synaptic contact. All such synaptic contacts had symmetrical membrane specializations and the most common postsynaptic targets were dendritic shafts, sometimes spine-bearing. Dendritic spines themselves also received synapses from each type of neuron. No axosomatic synapses involving boutons of identified axons were found. One example of a synapse between an axon collateral of an aspiny neuron and one of the same neuron's dendrites (an autapse) was demonstrated by electron microscopy.It is concluded that the synaptic terminals of at least four types of neuron, the three aspiny types described here and the medium-size densely spiny neuron, participate in local circuit interactions in the neostriatum.     

Ultrastructure and synaptic connections of vasoactive intestinal polypeptide-like immunoreactive non-pyramidal neurons and axon terminals in the rat hippocampus

In the hippocampus, antibody raised against vasoactive intestinal polypeptide (VIP) labeled perikarya and processes of non-pyramidal neurons whereas these structures remained unlabeled in pyramidal cells and granule cells. In the present study, VIP-immunostaining was used to investigate the fine structure and synaptic connections of identified non-pyramidal neurons and of imrnunoreactive axon terminals in the CA1 region of the rat hippocampus by means of electron microscopic immunocytochemistry.From a number of cells studied, two VIP-like imrnunoreactive non-pyramidal neurons in the regio superior were selected for an electron microscopic analysis of serial thin sections. These cells were different with regard to the location of their cell bodies and the orientation of their dendrites. One cell was located in the stratum lacunosum-moleculare with dendritic processes oriented parallel to the hippocampal fissure. The second neuron was found in the inner one-third of the stratum radiatum. The dendrites of this cell ran nearly parallel to the ascending apical dendrites of the pyramidal cells. Both cells had a round or ovoid perikaryon and an infolded nucleus. The aspinous dendrites of both neurons were densely covered with synaptic boutons. These terminals were small, filled with spherical vesicles and established asymmetric synaptic contacts. No variations in the fine structure of the presynaptic boutons were found along the course of the labeled dendrites through the various hippocampal layers, although different afferents are known to terminate in these layers.Vasoactive intestinal polypeptide-like immunopositive axon terminals course through all layers of the hippocampus. In the stratum pyramidale they established symmetric synaptic contacts with the perikarya of pyramidal cells. In the stratum radiatum they made symmetric contacts with the shafts of apical dendrites of pyramidal cells but never contacted dendritic spines.The symmetric contacts with pyramidal cell perikarya suggest an involvement of the VIP-like immunoreactive axon terminals in pyramidal cell inhibition.     

The recurrent collaterals of Purkinje cell axons: A correlated study of the rat's cerebellar cortex with electron microscopy and the Golgi method

Summary Each Purkinje cell axon with its recurrent collaterals occupies a roughly triangular space in the folium, apex pointed towards the white matter and base against the Purkinje cell layer. The axon is smooth initially but develops distensions that become more obvious at twists and turns and at points where collaterals originate. These thin, finely beaded collaterals make characteristic acute angles with the axon from which they issue. The collaterals bifurcate further, their terminal branches becoming more varicose, intertwining with each other to form plexuses in the molecular and granular layers. These fiber plexuses are found in three locations: (1) the recurrent collateral plexus in the granular layer which synapses with dendrites and somata of deep Golgi II neurons; (2) the profuse infraganglionic plexus, boutons of which terminate in relation with the somata and dendrites of Purkinje cells and Lugaro cells, in addition to participating in other complex synaptic arrangements in the neuropil; (3) the sparse supraganglionic plexus which forms synapses with dendrites of Purkinje cells and occasionally with basket cells.In electron micrographs, terminals belonging to recurrent collaterals contain a mixture of neurofilaments, microtubules, and slender mitochondria with a loose array of flat, elliptical, and round synaptic vesicles embedded in a dark filamentous matrix. It is usual to find a cluster of boutons on the postsynaptic surface. Each synapse consists of several separate macular junctional complexes. The synaptic cleft is widened and contains a dense fibrous material while both pre- and postsynaptic components have very shallow, symmetrical filamentous densities adherent to the cytoplasmic surfaces of the membranes.It is suggested that recurrent collaterals from axons of Purkinje cells may provide a rapid monosynaptic feed-back mechanism for inhibitory control of Purkinje cell responses. These collaterals may also participate in a slower positive feed-forward circuit or resetting mechanism involving at least two synapses. The existence of this circuit is indicated by synapses on deep Golgi II neurons. The inhibition of Golgi II cells may depress their inhibitory activity on surrounding granule cells, thus resetting the mechanism for the subsequent responses to excitatory afferent input. Recurrent collateral inhibition also may aid in the disinhibition of Purkinje cells through the depression of basket cell activity.Supported by U.S. Public Health Service Research Grant NS03659 and Training Grant NS05591 from the National Institute of Neurological Diseases and Stroke.     

Recurrent axon collaterals of corticothalamic projection neurons in rat primary somatosensory cortex contribute to excitatory and inhibitory feedback-loops

Intrinsic circuitry within the primary somatosensory cortex of the rat was examined in a combined light and electron microscope study. Corticothalamic projection neurons were retrogradely labeled by applying Phaseolus vulgaris leucoagglutinin (PHA-L) into the ventro-posteromedial thalamic nucleus (VPM). Most labeled neurons were pyramidal cells of layer VI. Postsynaptic targets of recurrent axon collaterals originating from these neurons were assessed in layers IV and V. Single labeled cells, complete with recurrent collaterals, could be isolated in barrels in which no anterograde transport had taken place. These findings were confirmed by first eliminating thalamocortical projections from the VPM with kainic acid and then applying PHA-L into the same nucleus. This procedure led to selective retrograde accumulation of tracer in layer VI pyramidal cells. Reconstructed portions of labeled axonal trees reached layer IV, bringing numerous boutons to layers IV, V and VI. The boutons had characteristic drumstick-like shapes. In order to identify postsynaptic targets, 4 sections of axons stemming from 3 neurons were reembedded and serially sectioned for electron microscopy. The ultrastructure of 72 asymmetric synapses, all belonging to identified collaterals, was analysed. Of the 72 terminals, 44 (59.5%) ended on dendritic spines and 30 on shafts of dendrites (40.5%). Perikarya were not among the targets. In a subset of the sample, the nature of the target neurons was examined by postembedding immunohistochemistry for -amino butyric acid (GABA) after staining for PHA-L. A total of 42 labeled terminals was found in layers IV and V; 23 (55%) were located on GABA-negative spines and 19 (45%) on dendritic shafts. Only 6 (32%) of the shafts were GABA-positive. The remaining ones were either clearly GABA-negative, or labeled only at background levels (n=13; 68%). The results show that most synapses of corticothalamic projection neurons found in layers IV and V terminate on spines and shafts of GABA-negative dendrites. This finding suggests that such recurrent collaterals are involved in both excitatory and inhibitory feedback mechanisms.     

Types and spatial distribution of vasoactive intestinal polypeptide (VIP)-containing synapses in the rat visual cortex

Summary In the rat visual cortex vasoactive intestinal polypeptide (VIP)-containing structures were studied by means of light and electron microscopy and image analysis. VIP-immunoreactive axon terminals were found to form symmetric synapses with small dendritic shafts, dendritic spines and somata of pyramidal cells and interneurons. VIP-terminals often occured in pairs with VIP-negative, asymmetric synapses on the same postsynaptic structure. VIP-immunostained dendrites and perikarya were contacted by a purely asymmetric and a mixed population of VIP-negative terminals, respectively. Synaptic connections between two VIP-neurons are seldom as compared to the other types of VIP-synapses. Quantitative studies obtained by the image analysis of VIP-stained boutons and dendritic particles in light microscopic preparations suggest a distinct laminar distribution. Dendritic particles are most frequent in layers I–II, whereas axonal boutons have three laminar accumulations: at the border of layers I–II, in layer IV and layer VI. Together with previous results, the present findings argue for a non-random spatial distribution of VIP-boutons.     

The stellate cells of the rat's cerebellar cortex

Summary Stellate cells were studied in rapid Golgi preparations and in electron micrographs. These small neurons can be classified on the basis of their position in the molecular layer and the patterns of their dendritic and axonal arborizations as follows: (1) superficial cells with short, contorted dendrites and a circumscribed axonal arbor (upper third of the molecular layer); (2) deep stellate cells with radiating, twisted dendrites and with long axons giving rise to thin, varicose collaterals (middle third of the molecular layer); (3) deep stellate cells with similar dendrites and long axons giving collaterals to the basket around the Purkinje cell bodies (middle third of the molecular layer). An important characteristic of the stellate cell axon is that it generates most of its collaterals close to its origin. Even in long axon cells, only a few collaterals issue from the more distant parts of the axon. These forms contrast with the basket cell, which sends out long, straighter dendrites, and an extended axon that first emits branches at some distance from its origin. Furthermore, basket cell axon collaterals are usually stout in contrast to the frail, beaded collaterals of the stellate cell axon. The two cell types are considered to be distinct.In electron micrographs stellate cells display folded nuclei and sparse cytoplasm with the characteristics usual for small neurons. Mitochondria are often the most conspicuous organelles because of their size and pleomorphism. The dendrites cannot be followed for long distances in thin sections because of their irregular caliber and course. Axons can be recognized on the basis of their appearance in Golgi preparations as short stretches of slender fibers distended at close intervals and running athwart the grid of the parallel fibers. These distensions, full of ovoid or flattened vesicles, synapse on the shafts of Purkinje cell dendrites and also on the dendrites of Golgi cells, basket cells, and other stellate cells. In all cases the synaptic complex occupies about a third of the junctional interface, the synaptic cleft is somewhat widened, and the pre- and postsynaptic dense plaques are thin and almost symmetrical.Varicosities in the parallel fibers synapse with the soma and dendrites of stellate cells. These junctions display a widened synaptic cleft and asymmetrical pre- and postsynaptic densities. Junctions with climbing fibers (Scheibel collaterals) have also been seen.The form of the stellate cell indicates that it plays a role in cerebellar circuitry different from that of the basket cell, although both cells are inhibitory. It is probably concerned with local effects on Purkinje cell dendrites within the field of its afferent parallel fibers.Supported by U. S. Public Health Service Research Grant NS03659 and Training Grant NS05591 from the National Institute of Neurological Diseases and Stroke.     

On the identification of the afferent axon terminals in the nucleus lateralis of the cerebellum an electron microscope study

Summary Axon terminals in the neuropil of the lateral nucleus can be divided into six classes, each with a specific constellation of characteristics that consistently occur together. Two of these classes have synaptic varicosities with elliptical synaptic vesicles, one in a dense, the other in a sparse matrix, and both make axosomatic and axodendritic synapses. The remaining four classes all have round synaptic vesicles and do not make axosomatic synapses. In the first of these four, the vesicles are tightly packed in a dense matrix, in another they are loosely dispersed, and in the third they are clustered. In the fourth, large granular vesicles predominate. Of these six classes, the most numerous belong to the axons of the Purkinje cell terminal arborization. These boutons resemble their counterparts in the cerebellar cortex, the recurrent collaterals of the Purkinje axon. They have elliptical and flat synaptic vesicles in a dark matrix. The varicosities terminate on somata and dendrites of large and small neurons and constitute the majority of their input. Purkinje axons constitute 86% of the total population of terminals on large neuronal perikarya and 50% of those on their dendrites, but only 78% on the somata of small neurons and 31% on their dendrites. The terminals of climbing fiber collaterals are recognized by their resemblance in electron micrographs to the terminals of the climbing fiber arborization in the cerebellar cortex. They bear round synaptic vesicles packed into a dense axoplasmic matrix and make Gray's type 1 axodendritic synapses with large and small neurons. These axons are restricted to the lateral and ventral aspects of the nucleus and constitute 5% of the terminals on large cell dendrites and 6% of those on small neurons. The axons tentatively identified as collaterals of mossy fibers are myelinated fibers with a light axoplasm containing round synaptic vesicles, dispersed throughout their varicosities. They make Gray's type 1 synapses and constitute a fair percentage of the total axodendritic contacts in the neuropil, 22% on large neurons and 28% on small neurons. The bases for these tentative identifications are discussed in detail, as are the various synaptic relationships undertaken by each class of axon. The remaining 4 classes of axons of the neuropil will be described in subsequent papers.Supported in part by U.S. Public Health Service grants NS 10536 and NS 03659, Training grant NS 05591 from the National Institute of Neurological Diseases and Stroke, and a William F. Milton Fund Award from Harvard University.     

Agmatine containing axon terminals in rat hippocampus form synapses on pyramidal cells

We examined the cellular and subcellular localization of agmatine in the hippocampal CA1 region by immunocytochemistry. By light microscopy, agmatine-like immunoreactivity (agmatine-LI) was found primarily in the perikarya and dendritic profiles of pyramidal cells and in punctate processes preponderantly in stratum radiatum. Electron microscopy revealed that agmatine-LI was cytoplasmic and concentrated in ‘clusters' associated with mitochondria and tubular vesicles. In stratum radiatum, agmatine-LI was primarily in axons and axon terminals associated with small, synaptic vesicles. The terminals almost exclusively formed asymmetric synapses on the spines of dendrites, many of which originated from pyramidal cells. Some agmatine-LI also was present in shafts and spines of pyramidal cell dendrites and in astrocytic processes. The results demonstrate that agmatine in the hippocampus is found primarily in terminals forming excitatory (asymmetric) synapses on pyramidal cells, some of which contain agmatine-LI. These findings further implicate agmatine as an endogenous neurotransmitter which may be co-stored with -glutamate and may act in part in the rat hippocampus as a blocker of the N-methyl- -aspartate receptor and nitric oxide synthase.