Tyrosine hydroxylase-immunoreactive boutons in synaptic contact with identified striatonigral neurons, with particular reference to dendritic spines

Tyrosine hydroxylase-immunoreactive fibres in the rat neostriatum were studied in the electron microscope in order to determine the nature of the contacts they make with other neural elements. The larger varicose parts of such fibres contained relatively few vesicles and rarely displayed synaptic membrane specializations; however, thinner parts of axons (0.1-0.4 micron) contained many vesicles and had symmetrical membrane specializations, indicative of en passant type synapses. By far the most common postsynaptic targets of tyrosine hydroxylase-immunoreactive boutons were dendritic spines and shafts, although neuronal cell bodies and axon initial segments also received such input. Six striatonigral neurons in the ventral striatum were identified by retrograde labelling with horseradish peroxidase and their dendritic processes were revealed by Golgi impregnation using the section-Golgi procedure. The same sections were also developed to reveal tyrosine hydroxylase immunoreactivity and so we were able to study immunoreactive boutons in contact with the Golgi-impregnated striatonigral neurons. Each of the 280 immunoreactive boutons examined in the electron microscope displayed symmetrical synaptic membrane specializations: 59% of the boutons were in synaptic contact with the dendritic spines, 35% with the dendritic shafts and 6% with the cell bodies of striatonigral neurons. The dendritic spines of striatonigral neurons that received input from immunoreactive boutons invariably also received input, usually more distally, from unstained boutons that formed asymmetrical synaptic specializations. A study of 87 spines along the dendrites of an identified striatonigral neuron showed that the most common type of synaptic input was from an individual unstained bouton making asymmetrical synaptic contact (53%), while 39% of the spines received one asymmetrical synapse and one symmetrical immunoreactive synapse. It is proposed that the spatial distribution of presumed dopaminergic terminals in synaptic contact with different parts of striatonigral neurons has important functional implications. Those synapses on the cell body and proximal dendritic shafts might mediate a relatively non-selective inhibition. In contrast, the major dopaminergic input that occurs on the necks of dendritic spines is likely to be highly selective since it could prevent the excitatory input to the same spines from reaching the dendritic shaft. One of the main functions of dopamine released from nigrostriatal fibres might thus be to alter the pattern of firing of striatal output neurons by regulating their input.     

Glutamate decarboxylase immunoreactivity in the rat interpeduncular nucleus: A light and electron microscope investigation

The distribution of immunohistochemically demonstrable glutamate decarboxylase, the synthetic enzyme for GABA, was examined in the rat interpeduncular nucleus at the light and electron microscope levels. Immunoreactive perikarya were distributed in a characteristic pattern among the subnuclear divisions. The rostral, ventral and caudal portions of the nucleus possessed numerous immunoreactive perikarya, while few immunoreactive somata were observed in the subnuclei of the dorsal aspect. A dense field of immunostained axons and terminals was also present throughout. Ultrastructural examination of glutamate decarboxylase immunoreactivity revealed numerous labelled somata, dendritic processes, axons and boutons. Axodendritic and axosomatic synapses with immunoreactive postsynaptic profiles were numerous throughout those subnuclei with large numbers of immunoreactive somata. Immunostained terminals in contact with both immunoreactive and non-immunoreactive somatic and dendritic profiles were also present. An abundance of immunostained terminals was observed in the subnuclei that possessed a sparse population of immunoreactive somata. Immunoreactive myelinated axons of unknown origin were also present.

This investigation demonstrates that the rat interpeduncular nucleus possesses a large population of glutamate decarboxylase-immunoreactive neurons coextensive with a plexus of immunostained axons and terminals. The results suggest that the immunoreactive neurons give rise to axons which contribute to an intrinsic circuit interconnecting the different subnuclear divisions. These immunoreactive neurons are in receipt of non-immunoreactive afferent inputs of variable morphology, as well as projections from intrinsic immunoreactive neurons.     

Cholinergic neurons of the nucleus basalis of Meynert receive cholinergic, catecholaminergic and GABAergic synapses: an electron microscopic investigation in the monkey

An electron microscopic analysis of the nucleus basalis in the macaque monkey was carried out following the immunohistochemical labeling of choline acetyltransferase, either by itself or in conjunction with glutamate decarboxylase or tyrosine hydroxylase. Cholinergic axon varicosities were frequently encountered, and formed large, usually asymmetric, synapses on both choline acetyltransferase-immunopositive and -immunonegative dendrites of nucleus basalis neurons. Catecholaminergic (tyrosine hydroxylase-immunoreactive) axon varicosities formed synapses which in most cases were classified as asymmetric, and glutamate decarboxylase-immunoreactive (GABAergic) axons formed clearly symmetric synapses, each on to choline acetyltransferase-immunopositive or -immunonegative dendrites. These findings indicate that cholinergic cells in the nucleus basalis of the monkey, also known as Ch4 neurons, receive numerous synaptic inputs from cholinergic, catecholaminergic and GABAergic axons.     

Ultrastructural immunocytochemistry of gamma-aminobutyrate in the cerebral and cerebellar cortex of the rat

gamma-Aminobutyrate containing structures in the cerebral and cerebellar cortex of the rat were visualized by an immunocytochemical method using glutaraldehyde fixation and an antiserum developed against a gamma-aminobutyrate-glutaraldehyde-protein conjugate. Labelled elements (perikarya and cell processes) were observed to be distributed throughout the layers of the cerebral cortex in a pattern similar to that described using glutamate decarboxylase immunocytochemistry. The morphological features of many immunoreactive cell bodies were typical of stellate neurons. In the cerebellar cortex, Purkinje, basket, Golgi and stellate, cell bodies were found to be immunoreactive along with numerous labelled neuronal processes. At the ultrastructural level, the labelled processes in both areas corresponded to immunoreactive dendrites and fibres. Labelled synaptic boutons, generally of the symmetrical type, could also be seen in contact with positive or negative cell bodies and dendrites. In the cerebellum, glomeruli could be clearly identified including mossy fibres surrounded by unlabelled dendrites in contact with immunoreactive terminals. At the subcellular level in both brain regions, the areas occupied by the Golgi apparatus were never labelled, although the nuclei had varied reactions. The strong glutaraldehyde fixation that limits the diffusion of gamma-aminobutyrate limits also antibody diffusion. However, this fixation is compatible with a good morphological preservation and should enable immunocytochemistry studies to be compared to other methods such as autoradiography.     

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.     

Substance P immunoreactivity in the rat interpeduncular nucleus: synaptic interactions between substance P-positive profiles and choline acetyltransferase- or glutamate decarboxylase-immunoreactive structures.

The subnuclear and synaptic distribution of substance P immunoreactivity was examined in the rat interpeduncular nucleus at the light and electron microscope level. The nucleus possessed a prominent substance P-immunoreactive axonal plexus in the lateral and dorsomedial subnuclei, and in the dorsal cap of the rostral subnucleus. The density of substance P-immunoreactive axons in the remaining subnuclear divisions was sparse to moderate. Terminals of immunoreactive axons contained spherical vesicles and formed asymmetric contacts on dendritic processes exclusively. Immunoreactive neurons, restricted to the rostral subnucleus, possessed long, sparsely branched dendrites. Unlabelled terminals containing either spherical or pleomorphic vesicles contacted substance P-immunoreactive dendritic profiles. Axodendritic and axosomatic synapses containing substance P immunoreactivity pre- and postsynaptically were not observed. Ultrastructural evidence for synaptic relationships between substance P-containing profiles and those containing either choline acetyltransferase or glutamate decarboxylase was obtained by means of double antigen immunohistochemistry. Terminals of fasciculus retroflexus axons stained for choline acetyltransferase immunoreactivity formed asymmetric synaptic contacts with substance P-immunoreactive dendritic profiles. Few substance P-positive dendrites in the rostral subnucleus received terminals possessing glutamate decarboxylase activity. Unlabelled terminals containing either spherical or pleomorphic vesicles contacted substance P- and glutamate decarboxylase-immunoreactive dendritic profiles simultaneously. Terminals possessing either substance P or glutamate decarboxylase immunoreactivity formed synaptic contacts with dendritic processes of neurons in the lateral subnucleus. Many of the neurons within this subnuclear division contained glutamate decarboxylase. This study provides direct evidence of synaptic relationships between choline acetyltransferase-immunoreactive axons and substance P-immunoreactive dendritic profiles, and between substance P-positive axons and glutamate decarboxylase-immunoreactive dendrites. These findings reveal that two types of transmitter-specific axons of the fasciculus retroflexus innervate neuronal populations of the interpeduncular nucleus stained immunohistochemically for either substance P or glutamate decarboxylase.     

Synaptic connections of enkephalin-immunoreactive nerve terminals in the neostriatum: a correlated light and electron microscopic study

Summary Two different antisera to leucine-enkephalin were used to study the localization of enkephalin-like immunoreactive material in the neostriatum and globus pallidus of the rat, by means of the unlabelled antibody-enzyme method. Thin immunoreactive varicose fibres are scattered throughout the neostriatum. In the ventral striatum, fibres come together and follow a relatively straight course for several micrometers, forming tube-like structures which can be traced to cell bodies; these cell bodies are completely surrounded by immunoreactive fibres. Occasional immunoreactive varicose fibres are also found close to another type of neuron throughout the whole neostriatum.Examination by electron microscopy of immunoreactive structures that had been identified first in the light microscope, showed that each of the nearly 200 varicosities examined was a vesicle-containing bouton that formed a synaptic contact. Rarely were asymmetrical synaptic contacts found between immunoreactive boutons and dendritic spines. All other synapses formed by enkephalin-immunoreactive boutons were symmetrical. Two types of postsynaptic neuron were identified; the first type was a medium-sized neuron with the ultrastructural features of a typical striatal spiny neuron. The second type had a larger perikaryon surrounded by numerous immunoreactive varicosities that were found to be boutons forming symmetrical synapses. The long dendrites of this second type of neuron likewise received a dense input of immunoreactive boutons forming symmetrical synapses; such ensheathed dendrites were found to be the tube-like structures seen in the light microscope. The ultrastructural features of these neurons, notably a highly indented nucleus, were those of a rare type of striatonigral neuron. In the globus pallidus, all the enkaphalin-immunoreactive boutons studied formed symmetrical synapses with ensheathed dendrites and perikarya that were similar to the latter type of postsynaptic neuron in the neostriatum. Axo-axonic synapses involving immunoreactive boutons were not seen in our material.The results are consistent with the view that enkephalin-like substances may be synaptic transmitters in the neostriatum and that they may have different actions according to the nature of the postsynaptic target. The finding that one type of neostriatal neuron, and a very similar neuron in the globus pallidus, receives multiple enkephalin-immunoreactive boutons all over its perikaryon and along its dendrites indicates a potentially important role of enkephalin in the convergence of information within the neostriatum and pallidum on to output neurons.     

Characterization of cholinergic neurons in the rat neostriatum. A combination of choline acetyltransferase immunocytochemistry, Golgi-impregnation and electron microscopy

Immunocytochemistry with a monoclonal antibody against choline acetyltransferase has been used to characterise cholinergic neurons in the rat neostriatum. The light microscopic morphology, ultrastructure and synaptic input of these neurons was compared to that of the three types of large neuron found in Golgi preparations of the striatum. The cholinergic neurons are large and have long infrequently branching dendrites. Two of the immunoreactive neurons were also Golgi-impregnated and showed characteristics of the "classical" large neurons of the striatum. Examination in the electron microscope revealed that the synaptic input to perikarya and proximal dendrites is sparse, thus distinguishing them from another large type of neuron, found in the ventral regions of the striatum, whose dendrites and perikarya are ensheathed in synaptic boutons. It is concluded that one of the three morphologically distinguishable classes of large neuron in the striatum is a cholinergic neuron.     

A GABA immunocytochemical study of rat motor thalamus: light and electron microscopic observations.

A light and electron microscopic study of GABA-immunoreactive neurons and profiles in the ventroanterior-ventrolateral and ventromedial nuclei of rat dorsal thalamus was conducted using antiserum raised against GABA. Less than 1% of the neurons in these motor-related nuclei exhibited GABA immunoreactivity, confirming previous reports that these nuclei are largely devoid of interneurons. Immunoreactive neurons in the ventral anterior-ventral lateral complex and ventromedial nucleus were bipolar or multipolar in shape, and tended to be smaller than non-immunoreactive neurons. GABA immunoreactivity in the neuropil consisted of labeled axon terminals and myelinated and unmyelinated axons, and was lower in the ventral anterior-ventral lateral complex and ventromedial nucleus than in neighboring thalamic nuclei. The density of neuropil immunolabeling was slightly higher in ventral anterior-ventral lateral complex than in ventromedial nucleus. GABA-immunoreactive axon terminals, collectively termed MP boutons for their medium size and pleomorphic vesicles (and corresponding to "F" profiles of some previous studies of thalamic ultrastructure), formed symmetric synapses and puncta adhaerentia contacts predominantly with large and medium-diameter (i.e. proximal) non-immunoreactive dendrites. Approximately 12 and 18% of boutons in the ventral anterior-ventral lateral complex and ventromedial nucleus, respectively, were GABA-immunopositive. Many of these immunoreactive profiles probably arose from GABAergic neurons in the thalamic reticular nucleus, substantia nigra pars reticulata and entopeduncular nucleus. Two types of non-immunoreactive axon terminals were distinguished based on differences in morphology and synaptic termination sites. Boutons with small ovoid profiles and round vesicles that formed prominent asymmetric synapses onto small-diameter dendrites were observed. Mitochondria were rarely observed within these boutons, which arose from thin unmyelinated axons. These boutons composed approximately 82 and 74% of boutons in the ventral anterior-ventral lateral complex and ventromedial nucleus, respectively, and were considered to arise predominantly from neurons in the cerebral cortex. In contrast, boutons with large terminals that contained round or plemorphic vesicles and formed multiple asymmetric synapses predominantly with large-diameter dendrites were also observed. Puncta adhaerentia contacts were also common. Mitochondria were numerous within large boutons with round vesicles, which arose from myelinated axons. Many of the large boutons were likely to have originated from neurons in the cerebellar nuclei. Approximately 6% of the boutons in the ventral anterior-ventral lateral complex and 8% in ventromedial nucleus were of the large type.(ABSTRACT TRUNCATED AT 400 WORDS)     

Light and electron microscopical studies of the normal nuclei ventralis lateralis and ventralis anterior thalami in the cat

Summary A light and electron microscopical investigation of the nucleus ventralis lateralis (VL) and nucleus ventralis anterior (VA) of the cat's thalamus was made. Light microscopical examination of Golgi impregnated material revealed the existence of two types of cells based on differences in their dendritic arborization and branching of the axon. One of the cells is considered to be the thalamocortical relay cell, whereas the other is tentatively considered to be a Golgi type II neuron. Electron microscopical investigations of the two nuclei revealed the existence of a high number of profiles containing pleomorphic vesicles, and which have been identified as dendrites. Based on correlation with the Golgi material as well as on cytological features of the parent cell bodies, the dendrites containing vesicles are believed to belong to Golgi type II neurons. In addition to the vesicle-filled dendritic profiles, five different types of boutons have been identified. Two of these boutons, type LR and type SR, contain ovoid vesicles and establish asymmetrical synaptic contacts with dendrites of both types of neurons. Type F1, F2 and F3 boutons contain pleomorphic vesicles, but can be distinguished from dendritic profiles containing pleomorphic vesicles. Type F2 and F3 boutons establish symmetrical contacts with dendrites of both thalamocortical relay cells and Golgi type II neurons. Type F1 boutons establish symmetrical synaptic contact with the proximal dendrites or soma of the thalamocortical relay neurons only.Dendrites of both thalamocortical relay cells and Golgi type II neurons, as well as type LR, SR, F2 and F3 boutons, are engaged in glomeruli. Dendro-dendritic synapses between Golgi type II dendrites and relay cell dendrites are frequently seen, whereas no evidence of axo-axonic synapses have been found.Differences and similarities in the ultrastructural organization of VL and VA are described in some detail.Some of the observations reported in this and the following two papers were presented at the 6th Symposium of the International Society for Research in Stereoencephalotomy, Tokyo, October 12th & 13th, 1973.     

GABAergic innervation of somatostatin-containing neurosecretory cells of the anterior periventricular hypothalamic area: a light and electron microscopy double immunolabelling study

Double immunolabelling on semithin sections revealed glutamate decarboxylase immunopositive dots surrounding somatostatin-containing cell sections in the rat periventricular hypothalamic area. Up to 12 appositions were observed per cell section with an average number of 2-3 and a unimodal distribution. At the electron microscopical level pre-embedding staining of glutamate decarboxylase showed that most immunoreactive elements consisted of immunolabelled axonal endings. Most of these glutamate decarboxylase immunopositive boutons were found within the neuropil where they frequently made synapses on unidentified dendrites. Some of them were apposed to somatostatin-containing cell bodies that were identified according to the presence of immunolabelled granules using combined immunogold post-embedding staining. In many instances glutamate decarboxylase immunoreactive endings were also found to be involved in synaptic contact with somatostatin-labelled perikarya, or neuronal processes. These contacts provide the morphological basis for a direct GABAergic control of the somatostatin-containing cells regulating the secretion of growth hormone.     

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)     

Differentiation of granule cells in relation to GABAergic neurons in the rat fascia dentata

Summary Golgi impregnation was used to study the dendritic differentiation of granule cells in the rat fascia dentata. The impregnated granule cells were gold-toned allowing for a fine structural study of the same identified neurons and of the input synapses onto their cell bodies and dendrites. Due to the long postnatal formation of these cells it was possible to describe a sequence of maturational stages coexisting on the same postnatal day (P5). Characteristic features of the dendritic development of granule cells were i) occurrence of varicose swellings along the dendrites, ii) growth cones on dendritic tips, iii) transient formation of basal dendrites, and iv) progressive development of dendritic spines. Incoming synapses on the differentiating granule cells were mainly found on dendritic shafts. Their membrane specializations were symmetric. At least some of these symmetric synapses were GABAergic because immunostaining of Vibratome sections from the same postnatal stage (P5) demonstrated a well-developed GABAergic axon plexus in the fascia dentata (antibodies against glutamate decarboxylase (GAD), the GABA synthesizing enzyme). Electron microscopy of the immunostained axon plexus revealed numerous GABAergic terminals that formed symmetric synaptic contacts, mainly on shafts of differentiating dendrites but also on cell bodies of granule cells. Our results thus indicate that the plexus of inhibitory GABAergic axons is already well developed at a stage when the target neurons, the granule cells, are still being formed.     

Gamma-aminobutyrate, gastrin releasing peptide, serotonin, somatostatin, and vasopressin: ultrastructural immunocytochemical localization in presynaptic axons in the suprachiasmatic nucleus

An ultrastructural immunocytochemical study was undertaken to identify neuroactive substances contained in presynaptic boutons in the hypothalamic suprachiasmatic nucleus. Axonal boutons containing immunoreactive gamma-aminobutyrate, glutamate decarboxylase, neurophysin/vasopressin, gastrin releasing peptide/bombesin, somatostatin and serotonin were localized within the hypothalamic suprachiasmatic nucleus with pre-embedding peroxidase immunostaining. Synaptic contacts were found between boutons containing each of these substances and postsynaptic structures. While some variation in synaptic morphology existed, most of the immunoreactive contacts were of the symmetrical type. Previous work has indicated that neuroactive peptides may be found in highest concentrations in dense-core vesicles, to examine the subcellular localization of the amino acid inhibitory transmitter gamma-aminobutyrate, ultrastructural immunocytochemistry with pre-embedding peroxidase was compared with post-embedding immunocytochemistry with colloidal gold. Ultracryothin sections were also used for ultrastructural localization of gamma-aminobutyrate and glutamate decarboxylase immunoreactivity. Both gamma-aminobutyrate and glutamate decarboxylase immunoreactivity were found throughout the cytoplasm of immunoreactive boutons when pre-embedding peroxidase was used; with post-embedding colloidal gold immunostaining, label was found over areas containing small clear vesicles, and over mitochondria of immunoreactive axons. At the dilutions used in this study, strongly immunoreactive gamma-aminobutyrate dendrites, boutons forming asymmetrical synapses, and cell bodies were not found. Differences between pre-embedding and post-embedding immunostaining may be due to antigen and label diffusion caused by mild fixation and membrane damage necessary for antisera penetration during pre-embedding immunostaining. These results suggest that gamma-aminobutyrate, gastrin releasing peptide, somatostatin and vasopressin are contained in axons making contact with neurons of the suprachiasmatic nucleus, and may function as neurotransmitters here. Since all of these substances can also be localized in perikarya within the suprachiasmatic nucleus, there is a strong possibility that at least some of the axons containing immunoreactivity for each of these substances may be involved in local circuit interactions between neurons within the suprachiasmatic nucleus.     

Synaptic connections of GABA-containing boutons in the lateral cervical nucleus of the cat: an ultrastructural study employing pre- and post-embedding immunocytochemical methods

The lateral cervical nucleus receives input from the spinocervical tract and projects to the thalamus and mesencephalon. The organization of this nucleus was examined using two immunocytochemical methods. Pre-embedding immunolabelling was performed using an antibody against glutamate decarboxylase, and post-embedding immunogold-reaction was performed with an antibody to glutaraldehyde-fixed GABA. Light microscopic analysis of material reacted for glutamate decarboxylase revealed that punctate structures were present throughout the nucleus and were associated with large cells in the dorsolateral region of the nucleus. Electron microscopy demonstrated that the punctate structures were synaptic boutons which formed symmetrical synaptic junctions with dendrites and somata of cells in the nucleus. The ultrastructural preservation of material prepared for the post-embedding immunogold technique was superior to that prepared for pre-embedding immunostaining. Positively labelled synaptic boutons exhibited high colloidal gold density and, like those prepared for the pre-embedding method, formed symmetrical synaptic junctions with dendrites and somata of neurons. Labelled boutons were densely packed with irregularly-shaped synaptic vesicles. They displayed characteristics which were distinct from those unlabelled boutons. Boutons, revealed by both immunolabelling methods, were not observed to form synaptic associations with other axon terminals and were presynaptic to dendrites and somata only. Therefore, it is probable that such boutons are responsible for postsynaptic inhibition of cells in the nucleus. In view of this evidence, it is concluded that the lateral cervical nucleus is not simply a relay but is actively involved in processing sensory information.     

Ultrastructural features of non-commissural GABAergic neurons in the medial vestibular nucleus of the monkey.

The ultrastructural characteristics of non-degenerating GABAergic neurons in rostrolateral medial vestibular nucleus were identified in monkeys following midline transection of vestibular commissural fibers. In the previous papers, we reported that most degenerated cells and terminals in this tissue were located in rostrolateral medial vestibular nucleus, and that many of these neurons were GABA-immunoreactive. In the present study, we examined the ultrastructural features of the remaining neuronal elements in this medial vestibular nucleus region, in order to identify and characterize the GABAergic cells that are not directly involved in the vestibular commissural pathway related to the velocity storage mechanism. Such cells are primarily small, with centrally-placed nuclei. Axosomatic synapses are concentrated on polar regions of the somata. The proximal dendrites of GABAergic cells are surrounded by boutons, although distal dendrites receive only occasional synaptic contacts. Two types of non-degenerated GABAergic boutons are distinguished. Type A terminals are large, with very densely-packed spherical synaptic vesicles and clusters of large, irregularly-shaped mitochondria with wide matrix spaces. Such boutons form symmetric synapses, primarily with small GABAergic and non-GABAergic dendrites. Type B terminals are smaller and contain a moderate density of round/pleomorphic vesicles, numerous small round or tubular mitochondria, cisterns and vacuoles. These boutons serve both pre- and postsynaptic roles in symmetric contacts with non-GABAergic axon terminals. On the basis of ultrastructural observations of immunostained tissue, we conclude that at least two types of GABAergic neurons are present in the rostrolateral portion of the monkey medial vestibular nucleus: neurons related to the velocity storage pathway, and a class of vestibular interneurons. A multiplicity of GABAergic bouton types are also observed, and categorized on the basis of subcellular morphology. We hypothesize that "Type A" boutons correspond to Purkinje cell afferents in rostrolateral medial vestibular nucleus, "Type B" terminals represent the axons of GABAergic medial vestibular nucleus interneurons, and "Type C" boutons take origin from vestibular commissural neurons of the velocity storage pathway.     

Characterization of pallidonigral neurons in the rat by a combination of Golgi impregnation and retrograde transport of horseradish peroxidase: their monosynaptic input from the neostriatum

Summary After injection of horseradish peroxidase, or its conjugate with wheatgerm agglutinin, into the substantia nigra of rats, retrogradely labelled cells were found in the globus pallidus. Forty-six of these neurons were also impregnated by the Golgi procedure and then gold-toned: their somata ranged from 15 to 30 m in diameter and these pallidonigral neurons had from two to five primary dendrites that were long and smooth, that branched infrequently and that bore occasional spines on their distal regions. Most of the neurons studied came from the lateral part of the globus pallidus. At the ultrastructural level, the identified pallidonigral neurons were found to have deeply infolded nuclei and an abundant cytoplasm; their perikarya were richly innervated by two distinct types of bouton, both of which formed symmetrical synaptic contacts. The dendrites of pallidonigral neurons were ensheathed in boutons, the majority forming symmetrical synaptic contacts. After placement of electrolytic lesions in the rostro-dorsal neostriatum, degenerating boutons were found in symmetrical synaptic contact with the cell bodies and dendrites of six identified pallidonigral neurons.It is concluded that pallidonigral neurons belong to the Golgi category of large pallidal neurons with smooth dendrites and that they receive monosynaptic input from the neostriatum. Thus, in addition to the direct striatonigral pathway, the neostriatum can influence the substantia nigra by a monosynaptic relay through the globus pallidus, which might allow other pallidal afferents to influence the transfer of information from neostriatum to substantia nigra.     

Synaptic input and local output of dopaminergic neurons in grafts that functionally reinnervate the host neostriatum

Summary In adult rats with a unilateral 6-hydroxydopamine-induced lesion of the nigrostriatal dopamine pathway, grafts of embryonic ventral mesencephalon can establish extensive efferent connections with the previously denervated host neostriatum and can compensate for motor and sensorimotor asymmetries induced by the lesion. The object of this study was to examine the afferent synaptic inputs to grafted dopaminergic neurons, implanted into a cortical cavity overlying the previously denervated caudate-putamen, using electron microscopic immunocytochemistry. The dopaminergic neurons of the grafts in the same animals had previously been shown to re-innervate the host neostriatum, to form synaptic connections therein and to attenuate the lesion-induced motor asymmetry that occured in response to amphetamine (Freund et al. 1985). In the light microscope, the grafts were found to contain numerous tyrosine hydroxylase-immunoreactive perikarya, dendrites, axons and axonal swellings which had distinct distributions. In addition axons and axonal swellings that were immunoreactive for either substance P or glutamate decarboxylase were present. Electron microscopic analysis of the boutons contacting tyrosine hydroxylase-immunoreactive neurons in the grafts revealed the presence of at least five distinct types of afferent synaptic boutons based on their immunochemistry, morphology, or types of membrane specialization. One type was itself immunoreactive for tyrosine hydroxylase; such synapses are extremely rare in the intact substantia nigra, none were found in the contralateral substantia nigrae or the substantia nigra of a control rat. Three of the remaining types had ultrastructural features that were similar to synaptic terminals that were immunoreactive for substance P or glutamate decarboxylase. These synapses were similar to the types of synapses found contacting dopaminergic neurons in the substantia nigra contralateral to the graft or the substantia nigra of a control rat. The results demonstrate that, in the absence of the normal extrinsic afferent inputs, the intracortical mesencephalic grafts have a well-developed local synaptic circuitry. It is suggested that local circuit regulation of dopaminergic neurons within the graft may, at least in part, be responsible for the maintenance of a normal or close to normal functional activity.     

Quantitative analysis of the ultrastructural distribution of GABA-like immunoreactivity in the intermediate and medial part of hyperstriatum ventrale of chick

Summary The intermediate and medial part of the hyperstriatum ventrale of the chick telencephalon plays a crucial role in the learning processes of imprinting. The distribution within the intermediate and medial part of the hyperstriatum ventrale of the neurotransmitter -amino butyric acid was studied with light and electron microscopy using an antibody against this amino acid. The antibody labelled 18.4% of neuronal somata. GABA-labelled terminals made symmetrical synapses onto somata and dendrites of labelled and unlabelled neurons. Labelled somata received about three times as many synaptic boutons as unlabelled somata. Approximately 21% of synaptic terminals on labelled somata were themselves labelled; unlabelled somata received a higher proportion (37.6%) of such terminals. Most labelled terminals synapsing with dendrites were confined to the shafts; very few labelled terminals contributed to axospinous synapses. Synaptic contacts made on dendritic shafts by labelled boutons were intermingled with symmetrical and asymmetrical contacts from non-immunoreactive terminals. The proportion of labelled terminals received by labelled dendrites (33.1%) was approximately twice that received by unlabelled dendrites (15.9%). Labelled neurons therefore received a higher proportion of labelled terminals on their dendrites and a lower proportion on their somata compared with unlabelled neurons. No immunoreactivity was seen in glial cells or ependyma.