Basket Cell Terminals Research Articles

Colocalization of glycine-like and GABA-like immunoreactivities in Golgi cell terminals in the rat cerebellum: a postembedding light and electron microscopic study

Consecutive sections of rat cerebella were incubated with antisera raised against glycine or γ-aminobutyric acid (GABA) conjugated to protein by glutaraldehyde. The sections were subsequently processed according to the peroxidase-antiperoxidase technique (semithin sections) or treated with secondary antibody coupled to colloidal gold particles (ultrathin sections). Corroborating previous light microscopic observations based on pre-embedding immunocytochemistry 20, a major proportion (about 70%) of the Golgi cell bodies showed immunoreactivity for both glycine and GABA. Analyses of semithin sections further suggested that the two immunoreactivities were colocalized in the same glomeruli and even in the same Golgi cell terminals. This was confirmed by electron microscopy. Quantification of the immunogold labelling for glycine (which is assumed to play metabolic roles in addition to its presumed role as a transmitter) showed that the net gold particle density was an order of magnitude higher over Golgi cell terminals than over the other constituents of the cerebellar glomeruli (mossy fibre terminals and granule cell dendrites). The total particle density over the latter was only slightly higher than the background level (over empty resin), suggesting that the concentration of ‘metabolic’ glycine is generally low compared to the concentration of glycine in Golgi cells. The stellate and basket cell terminals (which similarly to the Golgi cell are thought to release GABA as transmitter) were immunoreactive for GABA, but (with very few exceptions) virtually unlabelled for glycine, suggesting that our results were not confounded by any crossreactivity of the glycine antiserum with fixed GABA. Direct evidence that the sera reacted selectively with fixed glycine or GABA under the conditions used was obtained by incubating the tissue sections together with test sections containing a series of different amino acid-glutaraldehyde-brain macromolecule conjugates. Adsorption tests with soluble amino acid-glutaraldehyde complexes similarly suggested that the double-labelling of the Golgi terminals indeed reflected a colocalization of glycine and GABA. The results show that two ‘classical’ transmitters, both being inhibitory and acting on Cl − channels, may coexist in the same nerve terminals.

Redistribution of transmitter amino acids in rat hippocampus and cerebellum during seizures induced by l-allylglycine and bicuculline: An immunocytochemical study with antisera against conjugated gaba, glutamate and aspartate

The effects of the convulsants l-allylglycine and bicuculline on the distribution of γ-aminobutyric acid (GABA), glutamate and aspartate in rat brains were assessed immunocytochemically, using antisera raised against glutaraldehyde-protein conjugates of the respective amino acids. In accord with previous biochemical studies of GABA content, l-allylglycine treatment was followed by a decreased immunoreactivity for GABA in the hippocampus and cerebellum, whereas treatment with bicuculline led to an increased immunoreactivity in the hippocampus, but not in the cerebellum. Different cells and zones were affected differentially. With both convulsants the hippocampus showed the most pronounced changes in the neuropil of the pyramidal and granular cell layers. l-Allylglycine treatment led to a substantial decrease in the concentration of detectable GABA-immunoreactive bouton-like dots in the stratum oriens, radiatum and lacunosum-moleculare and in the deep hilar region, but did not produce statistically significant changes in this parameter in the outer and intermediate zones of the dentate molecular layer. In the cerebellum, the decrease in GABA immunoreactivity after l-allylglycine treatment was less in the basket cell terminals than in other GABA-containing elements. Neither convulsant altered the average staining intensity for aspartate or glutamate in the two regions studied, but l-allylglycine reduced the level of aspartate-like immunoreactivity in hippocampal hilar cells. All the changes described were evident after 20 min of seizure activity and were qualitatively similar after 60 min of seizure (animals paralysed and ventilated). Our results indicate that l-allylglycine or bicuculline given intravenously exerts specific effects on cerebral amino acid metabolism. The nature and magnitude of these effects show inter-regional variations and also differ among cellular compartments within each region. Amino acid immunocytochemistry may prove to be a valuable tool for the investigation of metabolic changes associated with epileptic seizures and should be particularly useful in regions showing heterogeneous changes that would tend to cancel each other in biochemical analyses.

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 ihe 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 Ihe distribution of their processes and their synaptic connections. Large basket cells terminale mainly in layer III, while clulch 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 slructural 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 dislribulion of the synaplic 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 lake part in both horizontal and radial interactions.

Uptake of d-aspartate and l-glutamate in excitatory axon terminals in hippocampus: Autoradiographic and biochemical comparison with γ-aminobutyrate and other amino acids in normal rats and in rats with lesions

High affinity uptake sites for 3H-labelled amino acids were studied in synaptosome-containing homogenates processed biochemically or in surface autoradiograms of incubated slices of hippocampus. d-aspartate and l-glutamate had apparently identical distributions. In normal rat hippocampus the highest uptake was in the terminal fields of axons from the pyramidal cells of regio inferior and hilus fasciae dentatae, while there was a moderate uptake in the terminal fields of the medial and lateral perforant paths, slight uptake in the mossy fibre layer and no uptake in the terminal fields of the basket cells. Uptake sites for γ-aminobutyrate were concentrated in the latter fields, and in the most superficial cortical layers. The present method shows no uptake in cell bodies. The uptake activities were strongly inhibited by recognized blockers of (neuronal) high affinity uptake of glutamate or γ-aminobutyrate. Autoradiographically, several other amino acids showed negligible uptake. The uptake of d-aspartate was reduced by 80% in stratum oriens and stratum radiatum of regio superior 4–14 days (70% at 3 days) after transection of the afferent pyramidal cell axons from the ipsiand contralateral regio inferior. The reduction was in the number of uptake sites, not in their affinity. Uptake of γ-aminobutyrate was not reduced. Lesions affecting regio superior caused a loss of d-aspartate uptake in subiculum at a site known to receive hippocampal afferents. Autoradiographically, the uptake of d-aspartate was strongly reduced in the inner zone (i.e. the target zone), but increased in the middle zone of the dentate molecular layer after lesions of the hilus fasciae dentatae. At 4 days and longer after transection of the entorhinal afferents, there was a conspicuous reduction of d-aspartate and l-glutamate uptake in the target zones of both the medial and lateral contingent of these fibres. In the same animals, the terminal zone of afferents from hilus fasciae dentatae had an increased radioactivity and was slightly wider than normally. Concomitantly, the γ-aminobutyrate uptake was increased in the target zones of the degenerating perforant path fibres. The results demonstrate that uptake sites for d-aspartate and l-glutamate are highly localized in axon terminals of regio inferior pyramidal cells and in perforant path afferents. The latter category of terminals has a lower density of acidic amino acid uptake sites than the former. Uptake sites for γ-aminobutyrate are localized in terminals of intrinsic neurones, including the axosomatic terminals of basket cells. Lesion of one pathway induces reactive increase in the uptake sites in other pathways. The anatomy of the uptake sites matches the known anatomy of the fibre connections, normally and after denervation.

Glutamate decarboxylase-immunoreactive terminals of Golgi-impregnated axoaxonic cells and of presumed basket cells in synaptic contact with pyramidal neurons of the cat's visual cortex.

Glutamate decarboxylase (GAD)-immunoreactive varicosities were found around cell bodies of nonimmunoreactive and immunoreactive neurons in the cat's visual cortex; they also occurred along apical dendrites and axon initial segments of pyramidal neurons. By examination in the electron microscope of structures first identified in the light microscope, it was established that the GAD-immunoreactive varicosities were boutons in symmetrical synaptic contact with pyramidal cells in layers II-IV. More than 90% of 142 boutons surrounding the cell bodies of 20 pyramidal neurons were immunoreactive for GAD. Since such a high proportion of the axosomatic boutons are GAD-immunoreactive, it is likely that the terminals of basket cells are included in this population and so the basket cell probably uses gamma-aminobutyrate as a transmitter, as suggested by previous authors. Almost all the 68 boutons in symmetrical contact with the axon initial segments of six pyramidal neurons could be shown to be GAD-immunoreactive, which makes it very likely that the boutons of axoaxonic cells contain GAD-immunoreactivity. This was established unequivocally for an individual Golgi-impregnated axoaxonic cell by combining Golgi impregnation and immunocytochemistry in the same sections: A Golgi-impregnated axoaxonic cell whose cell body was in layer II gave rise to numerous terminal segments, some of which were examined in the electron microscope after gold-toning. These boutons were in synaptic contact with axon initial segments and not only contained the Golgi precipitate but were also immunoreactive for GAD. It is concluded that the axoaxonic cell in the visual cortex uses gamma-aminobutyrate as a transmitter. An individual axoaxonic cell in layer II/III was filled with horseradish peroxidase by intracellular iontophoresis. The very extensive local axonal field was composed of 330 terminal bouton rows in layer II/III and a sparse descending collateral projection to infragranular layers. A computer-assisted reconstruction of the axonal field in three dimensions revealed the following: The main output of the cell is to pyramidal neurons that lie deeper than the soma; the axonal arborization occupies an area of 400 micron in the anteroposterior axis and extends 200 micron along the mediolateral axis; the terminal bouton rows in layer II/III form clusters about 50 micron wide running approximately at right angles to the border between areas 17 and 18, with an intercluster interval of about 100 micron. These findings suggest that the terminals of an individual axoaxonic cell could be contained within one ocular dominance column but that there may be inhomogeneities in the weighting of the axoaxonic input to pyramidal cells in the supragranular layers.

Immunocytochemical localization of glutamate decar☐ylase in rat cerebellum with a new antiserum

An antiserum prepared in sheep against rat brain glutamic acid decar☐ylase, the biosynthetic enzyme for γ-aminobutyric acid, has been employed to localize, at the light and electronmicroscopic levels, neurons containing glutamate decar☐ylase in the cerebellum of adult rats using the unlabeled antibody enzyme method of S ternberger (1979). With high dilutions of the primary antiserum (1:4000 and higher) glutamate decar☐ylase-immunoreactivity was localized in synaptic terminals of basket, stellate and Golgi cells and Purkinje cell axon recurrent collaterals in the cerebellar cortex, as well as in axosomatic and axodendritic synaptic terminals in the deep cerebellar nuclei, all of which contain pleomorphic synaptic vesicles. The reaction product was present in the axoplasm and at the cytoplasmic aspect of the plasmamembrane, the membrane of synaptic vesicles and axoplasmic reticulum, the outer mitochondrial membrane and also the microtubules. Lower dilutions of antiserum (1:1500) revealed light, specific immunoreactivity in somata of Purkinje, basket, stellate and Golgi-cells of the cerebellar cortex. We obtained specimens in which the vast majority of the members of these cells were stained. Staining of stellate cells was the least intense of all positive neurons in the cerebellar cortex. Very light labeling of some small cell bodies of the deep cerebellar nuclei was also observed. Granule cells, parallel fibers, mossy fibers and climbing fibers in the cerebellar cortex, the large neurons of the cerebellar nuclei, and glial cells were negative. When the antiserum was absorbed against acetone-dried rat liver powder, the same staining pattern was obtained, however, dilutions of 1:500 were necessary to obtain equally strong staining of neuronal cell bodies. These data indicate the specificity of the new anti-serum to rat brain glutamate decar☐ylase. They confirm and complement earlier immunocytochemical work with an antiserum to mouse brain glutamate decar☐ylase. The new antiserum not only shows that nearly all inhibitory axons in the cerebellum are glutamate decar☐ylase positive but also detects immunoreactivity in neuronal cell bodies in the cerebellar cortex without administration of blockers of axoplasmic transport.

Electrical inhibition of Purkinje cells in the cerebellum of the rat.

Monosynaptic activation of cerebellar Purkinje cells by a volley of parallel-fiber impulses is followed by a powerful disynaptic chemical inhibition mediated by molecular layer interneurons, including basket cells. Active zone established by basket preterminal axons on the body surface of the Purkinje cell account for this inhibition. However, morphological studies indicate that branches of the presynaptic fibers further descend along the initial segment of the Purkinje axon. Terminals from several basket cells converge and encapsulate each initial segment with a peculiar architectural structure that is reminiscent of that characterizing the axon cap of the teleost Mauthner cell. Because no function has yet been attributed to this pinceau, we have reanalyzed the successive Purkinje cell responses to activation of their presynaptic elements. Electrophysiological data provided by field-potential and single-unit measurements indicate that the classical phases of excitation and inhibition after a parallel-fiber volley are preceded by a brief inhibition of the Purkinje cells. Transmembrane hyperpolarizing potentials that exhibit the characteristics expected of electrically mediatd potentials underlie this early phase of inhibition; their properties are consistent with the hypothesis that they are generated by currents through terminals of nearby basket cells. Therefore, these hyperpolarizations, which are similar in their mechanism of generation to those described in the Mauthner cell system, represent a known case of electrical inhibition in the mammalian central nervous system.

Recent Advances in the Morphological Localization of γ-Aminobutyric Acid Receptors in the Cerebellum by Means of [3H]Muscimol

Muscimol, a structural analogue and potent agonist of γ-aminobutyric acid (GABA), was used in its tritiated form for the autoradiographic localization of GABA receptors in the rat cerebellum. [ 3 H]Muscimol ([ 3 H]M) was incubated with brain slices or was injected intracortically or into intraocular brain transplants. As indicated by [ 3 H]M binding and autoradiographic silver grains, GABA receptors display a laminar distribution over the cerebellar cortex. Specificity was indicated in control experiments by inhibition of binding by [ 3 H]muscimol after pretreatment with GABA, GABA and aminooxyacetic acid or the GABA analogues, THIP (isoxazole 16) and isoguvacine, or with receptor antagonist bicuculline and picrotoxin. Tritiated muscimol binding and electron microscope autoradiography localized GABA receptor sites in the cerebellum of the rat. In the cerebellar cortex, silver grains were associated with somata and dendrites of basket and stellate cells in the molecular layer; with somata and primary and secondary dendritic shafts of Purkinje cells; with axons and terminals of basket cells in the pinceau or basket, initial axonal segments and myelinated axons of Purkinje cells; with dendrites of granule and Golgi cells in the granular layer; and with somata and dendritic shafts of large and small cells in the cerebellar nuclei.

Ultrastructural localization of γ-aminobutyric acid receptors in the mammalian central nervous system by means of [ 3 H]muscimol binding

This study utilizes tritiated muscimol binding and electron microscope autoradiography (Ilford L4 emulsion and phenidone development) to localize gamma-aminobutyric acid (GABA) receptor sites in the cerebellum of the rat. In the cerebellar cortex, silver grains were associated with somata and dendrites of basket and stellate cells in the molecular layer, with somata and primary and secondary dendritic shafts of Purkinje cells, axons and terminals of basket cells in the pinceau or basket, initial axonal segments and myelinated axons of Purkinje cells, and dendrites of granule and Golgi cells in the granular layer, and with somata and dendritic shafts of large and small cells in the cerebellar nuclei. These data correspond well to the light-microscope-autoradiographic observations in the cerebellum previously reported [Chan-Palay, V. (1978) Proc. Natl. Acad. Sci. 75, 1024-1028]. Label over GABA receptor sites can be localized to the plasma membranes between pre- and postsynaptic elements at synaptic junctions, of which 88.9% of the samples are axodendritic and the remaining 11.1% are axosomatic. GABA receptor sites are also found along axonal membranes of the GABA-containing basket cell within the basket surrounding Purkinje cells, where true axo-axonal synapses are rare. It is speculated that GABA receptors in the basket may suggest a possibility of the basis for synchronization, either self-inhibition or facilitation within the basket formation, or presynaptic suppression of inhibitory action of basket cell on Purkinje cell.

Localization of putative transmitters in the hippocampal formation: with a note on the connections to septum and hypothalamus.

Biochemical assays on microdissected samples, denervation studies, subcellular fractionation, and light and electron microscopic autoradiography of high affinity uptake have been performed to study the cellular localization of transmitter candidates in the rat hippocampal formation. High affinity uptake of glutamate and aspartate is localized in the terminals of several excitatory systems, such as the entorhino-dentate fibres (perforant path), mossy fibres (from granular cells) and pyramidal cell axons. Thus, in stratum radiatum and oriens of CA1, 85% of glutamate and asparate uptake and 40% of glutamate and aspartate content are lost after lesions of ipsilateral plus commissural fibres from CA3/CA4. Hippocampal efferents also take up aspartate and glutamate, since these activities are heavily reduced in the lateral septum and mamillary bodies after transection of fimbria and the dorsal fornix. The synthesis (by glutamic acid decarboxylase), content and high affinity uptake of gamma-aminobutyrate (GABA) are not reduced after lesions of these or other projection fibre systems. A localization in intrinsic neurons is confirmed by a selective loss of glutamic acid decarboxylase after local injections of kainic acid. Peak concentrations of the enzyme occur near the pyramidal and granular cell bodies, corresponding to the site of the inhibitory basket cell terminals, and in the outer parts of the molecular layers. Some 85% of glutamic acid decarboxylase is situated in 'nerve ending particles'. Acetylcholine synthesis (by choline acetyltransferase) disappears after lesions of septo-hippocampal fibres. Since 80% of the hippocampal choline acetyltransferase is in 'nerve ending particles', the characteristic topographical distribution of this enzyme should reflect the distribution of cholinergic septo-hippocampal afferents. Serotonin, noradrenaline, dopamine and histamine are located/synthesized in afferent fibre systems. Some monoamine-containing afferents to the hippocampal formation pass via the septal area, others via the amygdala. The hippocampal formation also contains nerve elements reacting with antibodies against neuroactive peptides, such as enkephalin, substance P, somatostatin and gastrin/cholecystokinin.

Quantitative ultrastructural study of the perikaryon of the Purkinje cell and of the adjacent area in normal and hypothyroid rats aged 21 days

The effects of neonatal hypothyroidism on synaptic organization, glial surrounding and cytoplasmic structures of the Purkinje cell perikaryon of the 21 days old rat were studied by electron microscopy. Hypothyroidism decreases the size of the perikaryon but does not change the nucleo-cytoplasmic ratio of the cell. At 21 days the axonal endings that make synapse on the Purkinje cell perikaryon, already show the adult morphological features and hypothyroidism does not change these. The general synaptic density on the Purkinje perikaryon is not significantly altered, but the cells of hypothyroid animals still recieve 1/3 of their axo-somatic synapses on somatic spines, whereas euthyroid animals have practically no synapses on somatic spines. Hypothyroidism leads the disappearance of the synchronism which normally exists between the translocation or resorption of the Purkinje cell somatic spines and the establishment of the basket cell synapses. The basket cell axons of the normal animals form synapses only on the smooth surfaces of the Purkinje cell perikaryon while they also establish it on the somatic spines of the hypothyroid animals. The density of the axon terminals of basket cells is decreased, while that of the endings of Purkinje cell axon collaterals and of climbing fibers are increased. Hypothyroidism produces an increase in the size of the glial sheath around the Purkinje perikaryon. It does not alter the proportion of the cytoplasmic area occupied by mitochondria, but in thyroid deficiency there is an increase in the numbers of mitochondria which are reduced in average size. The Golgi apparatus is only slightly affected. Thyroid defiency does not alter the density of the cytoplasmic agreggates of free ribosomes but there is a decrease in density of the ergastoplasm and a disorganization of the Nissl bodies.

Experimental reorganization of the cerebellar cortex. II. Effects of elimination of most microneurons with prolonged x-irradiation started at four days.

AbstractThe heads of Long‐Evans rats were irradiated from the fourth day after birth with a schedule of repeated doses of low‐level x‐ray which essentially prevented the formation of basket, stellate and granule cells in all except the earliest‐forming lobules (nodulus and uvula). The morphogenic and synaptogenic effects of this treatment were examined with light and electron microscopy in 30 day old animals, with particular attention paid to the pyramis. Although when irradiation was started the Purkinje cells formed a monolayer and had upward oriented apical poles, they became scattered later and had randomly oriented dendrites. This secondary disorientation was attributed to insufficient space available in the arrested cerebellum for the rapidly expanding Purkinje cells. Although basket cell terminals were scarce, basket cell‐like terminals were formed on the somata of Purkinje cells, apparently by recurrent axon collaterals of these cells. The most common synapses with the thorns of Purkinje dendrites were formed by climbing fibers but other elements, including glial processes, were also in contact with postsynaptic loci on the thorns. Many mossy fiber terminals reached the surface. Where parallel fibers were present they were often thicker than in unirradiated animals and contained neurofilaments. No pathological changes were seen in these cerebella, with the possible exception of excessive lobulation of the nuclei of many Purkinje cells.

SPECIALIZED MEMBRANE JUNCTIONS BETWEEN NEURONS IN THE VERTEBRATE CEREBELLAR CORTEX

"Gap" junctions, the morphological correlate for low-resistance junctions, are demonstrated between some mossy fiber terminals and granule cell dendrites in some lower vertebrate cerebella (gymnotid and frog). Most of the gap junctions (GJs) seen in the gymnotid-fish cerebellum exhibit an asymmetrical configuration, the electron-opaque cytoplasmic material underlying the junction being more extensive in the dendritic than in the axonal side. In the frog cerebellum, the GJs have a symmetrical distribution of such electron-opaque material. In both species the GJs are encountered at the same synaptic interface as the conventional synaptic zone (CSZ), constituting "mixed synapses" in a morphological sense. The axonal surface covered by CSZs is larger than that covered by GJs. In mammalian cerebellum, GJs are observed only in the molecular layer, between perikarya, dendrites, or perikarya and dendrites of the inhibitory interneurons. These GJs are intermixed with attachment plates and intermediary junctions interpreted as simply adhesive. In the mammalian cerebellum, a new type of junction which resembles the septate junctions (SJs) of invertebrate epithelia is observed between axonal branches forming the tip of the brush of basket fibers around the initial segment of the Purkinje cell axon. It is suggested that such junctions may be modified forms of septate junctions. The physiological implications of the possible existence of high-resistance cross-bridges between basket cell terminals, which may compartmentalize the extracellular space and thus regulate extracellular current flow, must be considered.