Astrocytic processes compensate for the apparent lack of GABA transporters in the axon terminals of cerebellar Purkinje cells

The aim of the present study was to evaluate the expression of two high affinity GABA transporters (GAT-1 and GAT-3) in the rat cerebellum using immunocytochemistry and affinity purified antibodies. GAT-1 immunoreactivity was prominent in punctate structures and axons in all layers of the cerebellar cortex, and was especially prominent around the somata of Purkinje cells. In contrast, the deep cerebellar nuclei showed few if any GAT-1 immunoreactive puncta. Weak GAT-3 immunoreactive processes were present in the cerebellar cortex, whereas GAT-3 immunostaining was prominent around the somata of neurons in the deep cerebellar nuclei. Electron microscopic preparations of the cerebellar cortex demonstrated that GAT-1 immunoreactive axon terminals formed symmetric synapses with somata, axon initial segments and dendrites of Purkinje cells and the dendrites of granule cells. Astrocytic processes in the cerebellar cortex were also immunolabeled for GAT-1. However, Purkinje cell axon terminals that formed symmetric synapses with neurons in the deep cerebellar nuclei lacked GAT-1 immunoreactivity. Instead, weak GAT 1 and strong GAT-3 immunoreactivities were expressed by astrocytic processes that enveloped the Purkinje cell axon terminals. In addition, GAT-3-immunoreactivity appeared in astrocytic processes in the cerebellar cortex. These observations demonstrate that GAT-1 is localized to axon terminals of three of the four neuronal types that were previously established as being GABAergic, i.e. basket, stellate and Golgi cells. GAT-1 and GAT-3 are expressed by astrocytes. The failure to identify a GABA transporter in Purkinje cells is consistent with previous data that indicated that Purkinje cells lacked terminal uptake mechanisms for GABA. The individual glial envelopment of Purkinje cell axon terminals in the deep cerebellar nuclei and the dense immunostaining of GAT-3, and to a lesser extent GAT-1, expressed by astrocytic processes provide a compensatory mechanism for the removal of GABA from the synaptic cleft of synapses formed by Purkinje cell axon terminals.     

An analysis of HRP-filled basket cell axons in the cat''s cerebellum

The axons of basket cells course horizontally within the lower molecular layer of the cerebellar cortex and give rise to three types of axon collaterals. One collateral distributes around the somata and axon hillocks of Purkinje cells, forming a complex pericellular basketlike formation or pinceau. The other two axonal specializations have the form of beaded tendrils, but differ in their origin and laminar distribution. In the previous paper, the morphometry and configuration of these axonal branches were described. In the present account, their synaptic relationships are analyzed based on serial section analysis and computer reconstructions. Our data indicate that: (1) Purkinje cells receive descending collaterals from more than one basket cell; (2) the contribution of the descending collaterals to individual pinceau is not uniform; Purkinje cells located close to the basket cell of origin receive a greater number of descending collaterals when compared to Purkinje cells located more distally; (3) few synaptic junctions are formed by the descending collaterals within the pinceaux; (4) beaded tendrils make synaptic contacts with the somata and dendrites of Purkinje cells, and (5) the horizontal axon forms synaptic junctions with the dendritic shafts and spiny branchlets of Purkinje cells. Functional considerations of these synaptic relationships are discussed in the light of the classic concept of off-beam inhibition mediated by basket cells.     

Cytology and organization of rat cerebellar organ cultures

Roller tube cultures of parasagittal cerebellar slices were taken from young rats aged 9-11 days, and maintained in vitro for 1-2 weeks. Morphological aspects of cell types and synaptic relationships in such organ cultures were examined at light and electron microscopic levels. Some neurons were marked by intracellular injections of horseradish peroxidase for subsequent identification of their connection patterns. Cytoarchitecture of the cerebellar cortex was largely preserved in the organ cultures. Dendritic trees of Purkinje cells exhibited isoplanar organizations that often resembled their orientation at the time of explanation. Other cerebellar neurons, namely granule cells, Golgi cells, basket cells, stellate cells, all differentiated within the organ cultures. In addition, some neurons of the deep cerebellar nuclei remained viable during the period of culture. Mossy fibers most probably of cerebellar nuclear origin were found terminating on the dendrites of granule cells and Golgi cells. Quite unexpected were certain types of direct synapses of afferent fibers on short necked spines arising from Purkinje cell smooth dendrites and somata. Such terminals resembled climbing fibers. They were most likely modified mossy fiber afferents, since the organ cultures did not include neurons of the inferior olive which are well spearated from the cerebellar mass at postnatal stages. These "ascending" mossy fibers presumably occupied postsynaptic surfaces that were either vacated by deafferentation or induced by the afferent fibers themselves. Intracellularly labeled Purkinje cells had widely distributed axonal collateral branches. Labeled axons were distributed within the Purkinje cell layer. Several recurrent Purkinje cell axon collaterals stained with reaction products of horseradish peroxidase tracer were followed at the ultrastructural level. In one case, labeled terminals were examined in an area of approximately 2 mm2. Terminals of Purkinje cell collaterals formed symmetric synapses with somata of basket cells and dendrites of Golgi cells, but not Purkinje cell somata. Some large boutons of serially traced Purkinje cell axon collaterals formed asymmetric contacts with profiles interpreted as Golgi cell dendrites. In contrast to the apparent axonal sprouting in cerebellar organ cultures, maturation of dendritic processes remained static. Astroglia cells of diverse shapes were observed following immunocytochemical staining with antisera to glia filament proteins. The distribution patterns of immunoreactive astrocytes changed dramatically in cerebellar slice cultures maintained for 3-6 weeks in vitro.     

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.     

Identification of synapses formed in the cerebellar cortex by purkinje axon collaterals: an electron microscope study

Summary Purkinje axon collaterals and their synaptic terminals can be identified on the basis of three criteria: (1) They are the only myelinated axons of local elements, hence any myelinated axon persisting in chronically isolated folium is a Purkinje axon or its collateral; (2) They are the only known transfolial axons, so that axons and synapses found in the state of secondary degeneration after lesions placed into neighbouring folia of the cerebellar cortex are Purkinje axon collaterals and synapses; (3) The peculiar axonal tubular systems described by Andres (1965) are specific for Purkinje axons and their synaptic endings, which offers an additional clue for their identification. Using these three criteria numerous synapses of Purkinje axon collateral endings have been identified on the large Golgi neurons, both cell bodies and principal dendrites, and on the bodies of basket neurons. No evidence of the termination of Purkinje axon collaterals on other Purkinje cells could be detected.     

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)     

Synaptic connections of morphologically identified and physiologically characterized large basket cells in the striate cortex of cat

Neurons were studied in the striate cortex of the cat following intracellular recording and iontophoresis of horseradish peroxidase. The three selected neurons were identified as large basket cells on the basis that (i) the horizontal extent of their axonal arborization was three times or more than the extent of the dendritic arborization; (ii) some of their varicose terminal segments surrounded the perikarya of other neurons. The large elongated perikarya of the first two basket cells were located around the border of layers III and IV. The radially-elongated dendritic field, composed of beaded dendrites without spines, had a long axis of 300-350 microns, extending into layers III and IV, and a short axis of 200 microns. Only the axon, however, was recovered from the third basket cell. The lateral spread of the axons of the first two basket cells was 900 microns or more in layer III and, for the third cell, was over 1500 microns in the antero-posterior dimension, a value indicating that the latter neuron probably fulfills the first criterion above. The axon collaterals of all three cells often branched at approximately 90 degrees to the parent axon. The first two cells also had axon collaterals which descended to layers IV and V and had less extensive lateral spreads. The axons of all three cells formed clusters of boutons which could extend up a radial column of their target cells. Electron microscopic examination of the second basket cell showed a large lobulated nucleus and a high density of mitochondria in both the perikarya and dendrites. The soma and dendrites were densely covered by synaptic terminals. The axons of the second and third cells were myelinated up to the terminal segments. A total of 177 postsynaptic elements was analysed, involving 66 boutons of the second cell and 89 boutons of the third cell. The terminals contained pleomorphic vesicles and established symmetrical synapses with their postsynaptic targets. The basket cell axons formed synapses principally on pyramidal cell perikarya (approximately 33% of synapses), spines (20% of synapses) and the apical and basal dendrites of pyramidal cells (24% of synapses). Also contacted were the perikarya and dendrites of non-pyramidal cells, an axon, and an axon initial segment. A single pyramidal cell may receive input on its soma, apical and basal dendrites and spines from the same large basket cell.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification under the electron microscope of climbing fibers and their synaptic contacts

Summary An attempt is made to identify, under the electron microscope, the climbing fibers of the cerebellum (in the cat) and their synaptic contacts with Purkinje cells and other cortical neurons. — Two kinds of axonal profiles, having synaptic contacts with primary and secondary dendrites of Purkinje neurons, can be recognized: One being terminal fibers densely packed with neurofilaments, having mainly contacts de passage with the dendrite surface, with small accumulations of synaptic vesicles at the presynaptic side of the contact. The others are rather knob-shaped contacts filled with synaptic vesicles and poor in neurofilaments. In chronically isolated folia, in which only local neurons and their processes have survived, all filamentous profiles have disappeared while vesicular ones are not appreciably reduced in number. It is inferred from this, that the neurofilamentous profiles correspond to climbing fibers, whereas the vesicular ones could be the endings of outer stellate axons, recurrent Purkinje axon collaterals, or ascending basket axon collaterals. — Similar two kinds of axon-terminal profiles are found in synaptic contact with Golgi and basket cell bodies. As in chronically isolated folia only the vesicular profiles survive, it is inferred that the climbing fiber has axo-somatic terminals on Golgi cells and basket cells as well. Previous information of this kind, gained with the light microscope and with degeneration studies, is thus substantiated with the aid of the electron microscope. The vesicular presynaptic profiles on Golgi and basket neurons are in the first case certainly and in the second with high probability endings of recurrent Purkinje axon collaterals. — The few axosomatic synapses found on outer stellate neurons may also be terminals of climbing fibers, but degeneration evidence for this is not conclusive. — The observations are summarized and evaluated from the functional point of view in a diagram, with consideration to recent physiological information on the function of climbing fibers.     

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.     

The cytology of neurons and their dendrites in the simple mammalian nucleus lateralis an electron microscope study

Summary Profiles of large neurons in the lateral nucleus range from 16 m to 35 m in diameter with dimpled nuclei, large Nissl bodies, and well developed Golgi apparatus. Two types of perikarya are distinguished, those that are smooth and those with irregular somatic and dendritic protuberances. About 86% of all large neuronal somata are covered with axosomatic synapses, predominantly with terminals of Purkinje axons and a few belonging to axons of the small neurons. The remaining 14% have no axosomatic synapses. The thick, fleshy dendrites of these cells are covered with terminals, the majority of which synapse directly upon the dendritic shaft. A few are present on spines. The initial segment of the large neuron is thick and robust and receives synapses upon its shaft or upon a spinous projection. The small neurons measure less than 12 m in diameter and have very lobulated nuclei in a sparse cytoplasm characterized by small Nissl bodies and a poorly elaborated Golgi apparatus. About 52% of all small neuronal somata bear no synapses whereas the remaining 48% are covered with axosomatic synapses, mainly from the axons of Purkinje cells and a few axons of other small cells. The slender long dendrites of both large and small cells bear synapses with six classes of axons in the neuropil. Synaptic protuberances of two varieties occur on the surfaces of both perikarya and dendrites, (a) dome-shaped ones capped with a pronounced asymmetrical synaptic junction and (b) ones with thin long necks and bulbous heads having synapses on both parts. Frond-like dendritic excrescences are borne on the processes of some small and large neurons and they are postsynaptic to many axon terminals clustered around them.Supported in part by U.S. Public Health Service Research grants NS10536, NS03659, Training grant NS05591 from the National Institute of Neurological Diseases and Stroke, and a William F. Milton Fund Award from Harvard University.     

Postnatal development of the light and electron microscopic features of basket cells in the hippocampal dentate gyrus of the rat

Summary Light and electron microscopic preparations were used to analyze the postnatal development of the basket cells of the rat dentate gyrus. The basket cells, located at the hilar border, were recognized in 2-day-old rats in Golgi preparations, where they displayed immature dendrites and a small axon arbor in the granule cell layer. At 5 days, the basket cells were found to have a large perikaryal cytoplasm, a round nucleus, an axon that forms symmetric synapses with granule cells, and dendrites and somata that are contacted by other axon terminals. The 10-day basket cells display more mature features, such as Nissl bodies and well-developed Golgi complexes. The basket cells from 16-day-old rats are mature in terms of their ultrastructural features, in that the nuclei are highly indented and display intranuclear rods or sheets, the perikaryal cytoplasm is packed with organelles, and the axon has developed an extensive arborization with the somata and dendrites of granule cells at the border with the molecular layer. This arborization will continue to expand as more granule cells are generated and added to the hilar border. These data correlate well with the immunocytochemical and biochemical development of GABAergic neurons in the dentate gyrus. Furthermore, the maturation of the structure of basket cells appears to precede the appearance of adult-like electrical activity in the hippocampus.     

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.     

An analysis of HRP-filled basket cell axons in the cat's cerebellum

Basket cell axons terminate directly on the Purkinje cell body in the mammalian cerebellum and thus are in a position to have a direct influence on this neuron. In the present study, individual basket cells were intracellularly filled with horseradish peroxidase and the morphological characteristics and distribution of their axons were analyzed and quantified. The horizontally directed axon of basket cells preferentially distributes toward one side of the cell body, and is usually directed toward the base of the folium. In their course, the axons give rise to three types of axonal specializations including descending collaterals that form the complex endings around the axon hillocks and somata of Purkinje cells (i.e. the pinceau), beaded tendrils that arise from the descending collaterals and distribute to the Purkinje cell and upper granule cell layer, and beaded tendrils that arise directly from the horizontal axon to enter the molecular layer. In some respects, basket cells comprise a heterogeneous population of neurons on the basis of the length of their axon, as well as the number, distribution, and morphological characteristics of the axonal collaterals. The major finding of this study is that a single basket cell does not form a pinceau around the axon hillock of every Purkinje cell located along the course of its horizontal axon. Further, the pinceaux that are formed vary in their complexity. These findings suggest that the classic concept of off-beam inhibition mediated by basket cells is more complex than previously described.     

Quantitative assessment of taurine-like immunoreactivity in different cell types and processes in rat cerebellum: an electronmicroscopic study based on a postembedding immunogold labelling procedure

Summary Ultrathin sections of plastic-embedded rat cerebella were incubated with an antiserum against conjugated taurine and subsequently treated with a secondary antibody coupled to colloidal gold. The density of gold particles in various cellular profiles was calculated with the assistance of a computer. In the cerebellar cortex the highest density was found in the somata, dendrites, and dendritic spines of the Purkinje cells, supporting parallel light-microscopical observations in postembedding stained semithin sections from the same tissue blocks. The remaining profiles could be divided into three groups according to their immunolabelling intensity, in descending order: 1) somata and processes of granule and Golgi cells; 2) somata and processes of stellate, basket, and glial cells, and 3) mossy fiber terminals. In a representative experiment, the structures in the first and second groups showed gold particle densities in the range of 19–25%, and 4–11%, respectively, of that in the Purkinje cell somata (values corrected for background) whereas the particle density in the mossy fiber terminals was not significantly above background level. In the cerebellar nuclei, taurine-like immunoreactivity was concentrated in terminals that typically established symmetric or intermediate type contacts with weakly labelled dendrites and cell bodies. These terminals, which shared the ultrastructural features of Purkinje cell terminals, showed an average gold particle density that was about 60% higher than that of the Purkinje somata.For specificity control, ultrathin sections containing a series of different amino acid conjugates were incubated in the same drops of sera as the tissue sections. The highly selective labelling of the taurine conjugate indicated that the distribution of gold particles in the tissue was not confounded by crossreactivity with GABA, glutamate or other common amino acids but adequately reflected the distribution of fixed taurine. For additional control of specificity, the taurine antiserum was applied to the soluble fraction of a rat brain extract separated by thin layer chromatography. In this system the taurine antiserum stained a single spot that comigrated with free taurine.The present results suggest that all cell types and processes in the rat cerebellum (with the exception of the mossy fiber terminals) contain taurine. However, the concentration of taurine appears to vary considerably among the different cell types and may also differ between different parts of the same neuron.     

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.     

Synaptic connections, axonal and dendritic patterns of neurons immunoreactive for cholecystokinin in the visual cortex of the cat

A subpopulation of gamma-aminobutyric acid (GABA) containing neurons was reported to contain cholecystokinin-immunoreactive material in the visual cortex of cat [Somogyi et al., J. Neurosci. (1984) 4, 2590-2603]. In the present study pre-embedding immunocytochemistry was used to identify which of the several types of presumed GABAergic nonpyramidal cells in areas 17 and 18 contain cholecystokinin immunoreactivity. Most of the cholecystokinin-immunoreactive somata were found in layers II-III, they were less frequent in layers I and VI, and relatively rare in layers IV and V. The distribution and density of the axon terminals resembled that of the cell bodies. Two well defined types of cholecystokinin-immunoreactive neuron were distinguished: (1) double bouquet cells in layers II-III with vertically projecting axons, and (2) small basket cells with local axons either restricted to layers II-III, or descending to layer V. Additional cholecystokinin-positive cells showed features of bitufted or multipolar neurons in layers II-VI and horizontal cells in layer I, but these cells could be defined less well due to partial staining. Cholecystokinin-immunoreactive dendrites were found to run horizontally in layer I for several hundred micrometers. Some of the cholecystokinin-immunoreactive cells in layer VI had very long dendrites ascending radially up to layer III, as did their axons. A few cholecystokinin-immunoreactive cells appeared to have two axons and this was confirmed by electron microscopy. All cholecystokinin-immunoreactive neurons and terminals were separated from the basal lamina of blood vessels by glial endfeet. Random samples of boutons from each layer as well as identified terminals traced to their origin from local neurons were examined in the electron microscope. All of the boutons established symmetrical (type II) synaptic contacts with dendritic shafts, spines or somata. Quantitative electron microscopy of the postsynaptic targets of double bouquet cells and small basket cells demonstrated clear differences between these two types of neuron; basket cells having a higher proportion of their terminals in synaptic contact with somata. The findings that several distinct types of cortical neurons, as defined by their synaptic connections, contain cholecystokinin-immunoreactive material and that identified axons of all examined neurons form type II synaptic contacts suggests that the majority, if not all cholecystokinin-positive boutons forming type II contacts originate from local cortical cells. The distribution of targets postsynaptic to cholecystokinin-positive neurons is compared to those of cells labelled by other methods.     

On the development of the cerebellum of the trout, Salmo gairdneri. IV. Development of the pattern of connectivity

Summary Synaptogenesis has been studied in the corpus cerebelli of the troutSalmo gairdneri, Richardson, 1836. The first synapses are observed in hatchlings and occur between parallel fibres and the shafts of Purkinje dendrites. Subsequently the axosomatic synapses of Purkinje axon collaterals on the neurons of the ganglionic layer appear, and finally the synapses made by climbing fibres and mossy fibres, and by stellate cell axons develop. Young synapses in the cerebellum of the trout resemble the mature structures so closely that the criteria for the identification of the latter can also be applied to the former. The number of parallel fibre synapses and of Purkinje axon collateral synapses increases considerably during development. Eurydendroid cells, the axons of which leave the cerebellum, receive an abundance of Purkinje axon collaterals on their somata and main dendritic trunks. Mossy fibre synapses are numerous in the granular layer. Climbing fibre contacts and synapses of stellate cell axons, both with Purkinje cells, are found occasionally. the following pattern of connectivity is proposed. The main input-output system is formed by the mossy fibres, the granule cells, the Purkinje cells and the eurydendroid cells. Additional pathways are formed by (1) the mossy fibres, granule cells and eurydendroid cells, and (2) the climbing fibres, Purkinje cells and eurydendroid cells. The afferent-efferent systems, mentioned above, are influenced by a number of internuncial elements: (1) The Golgi cells receive their input from the parallel fibres and contact with their axon collaterals the dendrites of granule cells. (2) Axon collaterals of Purkinje cells are in synaptic relation with Golgi cells. (3) Axon collaterals of Purkinje cells impinge upon the somata and main dendrites of other Purkinje cells. (4) Stellate cells, which derive their input from the parallel fibres, synapse with the dendrites and somata of Purkinje cells. The possible functional roles of all of these neuronal elements are discussed.     

The synaptic connections of basket cell axons in the developing rat hippocampal formation

Summary Recent studies have indicated that hippocampal basket cells in both the dentate gyrus and Ammon's horn develop their somal and dendritic features during the first two postnatal weeks in rats. Their axon terminals form exclusively symmetric synapses that are found as early as 5 postnatal days in both regions. The present study used Golgi-electron microscopic material from 10 and 16 day old rats to demonstrate that the axon terminals of basket cells form synapses not only with somata, dendrites, and dendritic spines as reported for adult material but also with axon initial segments. However, the terminals forming synapses with axon initial segments and dendritic spines represent only a minor portion of the total number of basket cell terminals. Quantitative results indicate that 36–62% of the total number of these terminals form axosomatic synapses and 32–50% form axodendritic synapses depending on the analyzed cell. These data indicate that hippocampal basket cells have an axonal distribution similar to that found for cortical basket cells.     

Immunocytochemical evidence suggests that taurine is colocalized with GABA in the Purkinje cell terminals,but that the stellate cell terminals predominantly contain GABA: a light- and electronmicroscopic study of the rat cerebellum

Summary The distributions of taurine-like and GABA-like immunoreactivities in the rat cerebellum were compared by analysis of consecutive semithin and ultrathin sections, postembedding labeled with the peroxidase-antiperoxidase technique or with an indirect immunogold procedure, respectively. Taurine-like immunoreactivity was selectively enriched in Purkinje cell bodies, dendrites and spines, and boutons in the cerebellar nuclei exhibiting ultrastructural features typical of Purkinje cell terminals. The stellate and basket cell bodies and terminals were very weakly labeled. A computer assisted quantitative assessment of the net immunogold labeling revealed that the mean gold particle density in the Purkinje cell terminals was about 70% higher than that in the Purkinje cell dendrites, and about 14 times higher than that in the stellate/basket cell terminals in the molecular layer. Stellate, basket and Purkinje cell terminals emerged as intensely immunoreactive in adjacent sections processed with an antiserum against conjugated GABA. These findings indicate, contrary to recent electrophysiological data, that GABA is a more likely transmitter candidate than taurine in the stellate cells. The apparent colocalization of GABA and taurine in the terminals of Purkinje cells raises the possibility that these terminals are capable of releasing two different inhibitory amino acids.     

大鼠三叉神经脊束核Ⅱ尾侧亚核层内CB样、PV样阳性结构的分布及其突触联系

本研究用免疫组织化学方法观察了 Calbindin D-2 8k( CB)样和 Parvalbumin ( PV)样胞体、纤维和终末在三叉神经脊束核尾侧亚核 ( Vc) 层内的分布及它们的突触联系。在光镜下观察到 CB样和 PV样阳性胞体、纤维和终末在 II层内侧带 ( IIi)最为密集 ,PV样阳性神经元的胞体稍大 ,但数量少于 CB样阳性神经元。在电镜下观察到 CB样或 PV样阳性结构主要形成下列 4种突触联系 :( 1)阳性轴突终末与阳性或阴性轴突终末形成对称性轴 -轴突触和少量非对称性轴 -轴突触 ;( 2 )阳性轴突终末与阳性树突形成非对称性和对称性轴 -树突触 ;( 3 ) CB样阳性轴突终末与阴性树突主要形成非对称性轴 -树突触 ,PV样阳性轴突终末与阴性树突主要形成对称性轴 -树突触 ;( 4 )阴性轴突终末与阳性树突形成非对称性和对称性轴 -树突触。另外还可见到 CB样或PV样阳性或阴性树突、轴突及终末与 CB样、PV样阳性或阴性的初级传入纤维终末形成 型和 II型突触小球。 型突触小球数量较多 ,有典型的扇贝样初级传入纤维终末和不均一的小泡 ,线粒体少 ;II型突触小球的初级传入纤维终末粗大而清亮 ,外观不规则 ,有均匀一致的小泡和丰富的线粒体。根据上述结果可以推知在面口部伤害性信息的传递和调控过程中 ,Vc II层神经元发挥着重要的作用