Patterns of glutamate decarboxylase immunostaining in the feline cochlear nuclear complex studied with silver enhancement and electron microscopy

The cochlear nuclear complex of the cat was immunostained with an antiserum to glutamate decarboxylase (GAD), the biosynthetic enzyme for the inhibitory neurotransmitter GABA, and studied with different procedures, including silver intensification, topical colchicine injections, semithin sections, and immunoelectron microscopy. Immunostaining was found in all portions of the nucleus. Relatively few immunostained cell bodies were observed: most of these were in the dorsal cochlear nucleus and included stellate cells, cartwheel cells, Golgi cells, and unidentified cells in the deep layers. An accumulation of immunoreactive cells was also found within the small cell cap and along the medial border of the ventral cochlear nucleus. Immunostained cells were sparse in magnocellular portions of the ventral nucleus. Most staining within the nucleus was of nerve terminals. These included small boutons that were prominent in the neuropil of the dorsal cochlear nucleus, the granule cell domain, in a region beneath the superficial granule cell layer within the small cell cap region, and along the medial border of the ventral nucleus. Octopus cells showed small, GAD-positive terminals distributed at moderate density on both cell bodies and dendrites. Larger, more distinctive terminals were identified on the large cells in the ventral nucleus, in particular on spherical cells and globular cells. There was a striking positive correlation of the size, location, and complexity of GAD-positive terminals with the size, location, and complexity of primary fiber endings on the same cells. This correlation did not hold in the dorsal nucleus, where pyramidal cells receive many large GAD-positive somatic terminals despite the paucity of primary endings on their cell bodies. The GAD-positive terminals contained pleomorphic synaptic vesicles and formed symmetric synaptic junctions that occupied a substantial portion of the appositional surface to cell bodies, dendrites, axon hillocks, and the beginning portion of the initial axon segments. Thus, the cells provided with large terminals can be subjected to considerable inhibition that may be activated indirectly through primary fibers and interneurons or by descending inputs from the auditory brainstem.     

Three classes of inhibitory amino acid terminals in the cochlear nucleus of the guinea pig

Electron microscopic postembedding immunocytochemistry was used to analyze and assess the synaptic distribution of glycine (GLY) and γ-amino butyric acid (GABA) immunoreactivities in the guinea pig cochlear nucleus (CN). Three classes of endings were identified containing immunolabeling for glycine, GABA, or both glycine and GABA (GLY/GABA). All classes were similar in that the terminals contained pleomorphic vesicles and formed symmetric synapses with their postsynaptic targets. A fourth class, which labeled with neither antibody, contained round vesicles and formed asymmetric synapses. Glycine endings predominated in the ventral CN, while GLY/GABA endings were prevalent in the dorsal CN. GABA endings were the least common and smallest in size. Glycine, GLY/GABA, and GABA endings differed in their proportions and patterns of distribution on the different classes of projection neurons in the CN, including spherical bushy, type I stellate/multipolar, and octopus cells in the ventral CN and fusiform cells in the dorsal CN. The vast majority of anatomically-defined, putative inhibitory endings contain GLY, GABA, or both, suggesting that most of the inhibition in the cochlear nucleus is mediated by these three cytochemically and, probably, functionally distinct classes of endings. The results of this study also suggest that a large proportion of the GABA available for inhibition in the CN coexists in terminals with glycine. © 1996 Wiley-Liss, Inc.     

Evidence for GABAergic interneurons in the red nucleus of the painted turtle.

Immunocytochemical and electrophysiological evidence supporting the presence of GABAergic interneurons in the turtle red nucleus is presented. Injections of HRP into the spinal cord produced labeling of large neurons in the contralateral red nucleus. The peroxidase-antiperoxidase (PAP) method revealed smaller cells immunoreactive to an antibody against glutamate decarboxylase (GAD), the synthetic enzyme for the inhibitory neurotransmitter GABA, that were interspersed among larger immunonegative neurons. Similar small neurons were densely immunostained by antibodies to GABA-glutaraldehyde conjugates obtained from different sources and applied according to pre-embedding and postembedding protocols. Rubrospinal neurons retrogradely labeled with HRP measured 16 and 27 microns in mean minor and major cell body diameters, while GABA-like immunopositive neurons situated within the red nucleus measured 7 and 13 microns. There was very little overlap in soma size between the two cell populations. Therefore, we suggest that the GAD- and GABA-positive neurons may be local inhibitory interneurons. This notion is further supported by observations of pre-embedding immunostaining for GAD and postembedding immunostaining for GABA showing that the turtle red nucleus is amply innervated by immunoreactive axon terminals. These puncta are closely apposed to cell bodies and dendrites of both immunonegative large neurons and immunopositive small neurons. Moreover, immunogold staining at the electron microscopic level demonstrated that GABA-like immunoreactive axon terminals with pleomorphic synaptic vesicles formed symmetric synapses with cell bodies and dendrites of the two types of red nucleus cells. These ultrastructural features are commonly assumed to indicate inhibitory synapses. A moderately labeled bouton with round vesicles and asymmetric synapses was also observed. In addition, the two types of red nucleus neurons received asymmetric axosomatic and axodendritic synapses with GABA-negative boutons provided with round vesicles, features usually associated with excitatory functions. To obtain electrophysiological evidence for inhibition, intracellular recordings from red nucleus neurons were conducted using an in vitro brainstem-cerebellum preparation from the turtle. Small, spontaneous IPSPs were recorded from 7 out of 14 red nucleus cells studied. These morphological and physiological results provide strong support for concluding that the turtle red nucleus, like its mammalian counterpart, contains GABAergic inhibitory interneurons. While we have not identified the main source of input to these interneurons, in view of the scarce development of the reptilian cerebral cortex, this input is unlikely to come from the motor cortex as it does in mammals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential distribution of synaptic endings containing glutamate, glycine, and GABA in the rat dorsal cochlear nucleus

The dorsal cochlear nucleus (DCN) integrates the synaptic information depending on the organization of the excitatory and inhibitory connections. This study provides, qualitatively and quantitatively, analyses of the organization and distribution of excitatory and inhibitory input on projection neurons (fusiform cells), and inhibitory interneurons (vertical and cartwheel cells) in the DCN, using a combination of high-resolution ultrastructural techniques together with postembedding immunogold labeling. The combination of ultrastructural morphometry together with immunogold labeling enables the identification and quantification of four major synaptic inputs according to their neurotransmitter content. Only one category of synaptic ending was immunoreactive for glutamate and three for glycine and/or gamma-aminobutyric-acid (GABA). Among those, nine subtypes of synaptic endings were identified. These differed in their ultrastructural characteristics and distribution in the nucleus and on three cell types analyzed. Four of the subtypes were immunoreactive for glutamate and contained round synaptic vesicles, whereas five were immunoreactive for glycine and/or GABA and contained flattened or pleomorphic synaptic vesicles. The analysis of the distribution of the nine synaptic endings on the cell types revealed that eight distributed on fusiform cells, six on vertical cells and five on cartwheel cells. In addition, postembedding immunogold labeling of the glycine receptor alpha1 subunit showed that it was present at postsynaptic membranes in apposition to synaptic endings containing flattened or pleomorphic synaptic vesicles and immunoreactive for glycine and/or GABA on the three cells analyzed. This information is valuable to our understanding of the response properties of DCN neurons.     

Enkephalin-like immunoreactivity in the cat superior colliculus: distribution, ultrastructure, and colocalization with GABA

The distribution of enkephalin (ENK) immunoreactivity has been examined in the cat superior colliculus (SC) by means of light and electron microscope immunocytochemistry. The antisera were directed against leucine enkephalin but also recognized methionine enkephalin. Colocalization of ENK with gamma aminobutyric acid (GABA) was studied with a two-chromagen double-labeling technique. Enkephalin antiserum labeling was highly specific. Dense neuropil labeling was found only in a thin band 75-100 microns wide within the upper superficial gray layer of SC. Negligible neuropil labeling was seen deeper, except for patches of label within the intermediate gray layer. Intensely labeled neurons also had a specific distribution. Forty-seven percent were located within the upper 200 microns of SC, 40% within the deep superficial gray layer, 11% in the optic layer, and only 2% below that layer. Almost all ENK-labeled cells were small (mean area of 117 microns2). Some of these had horizontal fusiform cell bodies and horizontally oriented dendrites. Others had small round somata and thin, obliquely oriented dendrites. In double-labeling experiments, 18% of anti-ENK-labeled cells were also immunoreactive for GABA. Four distinct types of ENK-labeled profile were identified with the electron microscope. Presynaptic dendrites (PSD) with loose accumulations of synaptic vesicles were densely labeled with the antiserum. Conventional dendrites were also labeled. Both types of labeled profile received input from unlabeled synaptic terminals, including those from the retina that contained pale mitochondria and round synaptic vesicles and formed asymmetric synaptic contacts. Retinal terminals were never labeled with the antisera. However, some axon terminals with round synaptic vesicles, dark mitochondria, and symmetric synaptic densities were labeled by the antisera, as were some thinly myelinated axons. These results show that there is a small population of enkephalinergic neurons in the cat SC, some of which also contain GABA. Because not all cells with identical morphologies were double labeled, it appears that neurons of like morphology are chemically heterogeneous.     

Fine structure, synaptology and immunocytochemistry of large neurons in the rat dorsal cochlear nucleus connected to the inferior colliculus

Neurons in the rat dorsal cochlear nucleus that project to the inferior colliculus (pyramidal and giant) were retrograde labelled with wheat germ agglutinin conjugated to horseradish peroxydase. Both cell types showed a similar ultrastructural feature, particularly the rough endoplasmic reticulum was well developed and sometimes surrounded the nucleus. The synaptological profile was similar in pyramidal and giant cells. Axo-somatic terminals covered 40-70% of the perimeter of pyramidal cells and 35-60% of the perimeter of giant neurons. Giant neurons featured bipolar or multipolar shape and different orientation but they possessed a similar synaptic profile. Most axo-somatic terminals contained flat and pleomorphic synaptic vesicles, some pleomorphic vesicles. Few terminals contained round vesicles. These cells were consistently immuno-negative for both glycine and GABA and variably positive for glutamate. The immunoelectron microcopic study of thin sections showed that glycine immunoreactivity was constantly present in terminals enriched with flat vesicles, which often did not show GABA immunoreactivity. Few anterograde labelled boutons containing flat vesicles were in contact with the proximal dendrites and the cell bodies of pyramidal and giant neurons. The origin of these terminals is discussed. No other cells of the dorsal cochlear nucleus, in particular cartwheel and tuberculo-ventral neurons, were in contact with labelled boutons. The present results suggest that descending inhibitory collicular projections are essentially directed to the large excitatory neurons of the dorsal cochlear nucleus.     

GABA-immunoreactive neurons and terminals in the lateral cervical nucleus of the cynomolgus monkey

An antiserum against the inhibitory transmitter substance gamma-aminobutyric acid (GABA) was used to investigate the distribution of GABAergic nerve terminals and cell bodies in the lateral cervical nucleus (LCN) of the cynomolgus monkey. Light microscopic immunohistochemistry demonstrated GABA-immunoreactive puncta, suggestive of nerve terminals, scattered throughout the LCN. The terminal-like profiles are often present along the somata of unlabeled neurons, but most are located in the neuropil. GABA-immunoreactive neurons are present in the LCN, but constitute a very small number of the LCN neurons. Electron microscopy showed that the GABA-positive neurons are small with a relatively large nucleus. They are contacted by few somatic boutons. Numerous GABA-immunoreactive terminals containing densely packed round to oval synaptic vesicles were also found. Most GABA-positive terminals make synaptic contact with dendrites, but synapses with cell bodies are also present. Synaptic contacts between labeled and unlabeled terminals were not observed. Some GABA-positive terminals make contact with GABA-positive neurons. The present findings suggest that GABA is a major inhibitory transmitter substance in the LCN of the monkey. However, in comparison with other somatosensory relay nuclei, there are few GABA-immunoreactive neurons in the LCN. This may imply that the GABA-positive neurons branch extensively in the LCN or that an extrinsic source of GABAergic input exists.     

A correlated light and electron microscopic immunocytochemical study of cholinergic terminals and neurons in the rat amygdaloid body with special emphasis on the basolateral amygdaloid nucleus

The cholinergic innervation of the rat basolateral amygdaloid nucleus (BL) was determined by the immunocytochemical localization of the acetylcholine biosynthetic enzyme, choline acetyltransferase (ChAT). ChAT-immunoreactive (ChAT-IR) elements were observed throughout the BL in the form of fine puncta and varicose fibers. Electron microscopy revealed that the immunoreactive puncta represented small terminals (0.3-1.2 micron), most of which formed synaptic contacts with unlabeled dendritic shafts or spines. Less frequently, ChAT-IR terminals established synaptic contacts with large neuronal cell bodies, which had all the characteristics of projection neurons as defined on the basis of axonal projections to the ventral striatum. ChAT-IR terminals were sometimes seen to form synaptic contacts with small neuronal cell bodies, including those of ChAT-IR neurons. The ChAT-IR boutons contained pleomorphic clear vesicles of varying size, and the large majority of the synapses were of the symmetric type. Small ChAT-IR neurons were observed in all parts of the BL. Although the ChAT-IR cell bodies varied widely in shape from typical fusiform to round, most had a more or less oval shape with a major diameter of 10-14 micron. Most of the ChAT-IR neurons seemed to display a radial bipolar dendritic pattern, but multipolar cells were also observed. The ChAT-IR neurons contained an indented nucleus, which was often eccentrically located and surrounded by a thin or moderately thin rim of cytoplasm. The results obtained are discussed in relation to a quasi-cortical organization of the BL.     

GABAergic neurons and axon terminals in the brainstem auditory nuclei of the gerbil

The anatomical localization of glutamic acid decarboxylase (GAD), the synthesizing enzyme for GABA, was analyzed in the brainstem auditory nuclei of the adult gerbil. GAD-positive terminals and somata were present in the cochlear nucleus, superior olivary complex, lateral lemniscus, and inferior colliculus in varying concentrations and patterns. One of the highest densities of GAD-positive terminals is found in the superficial layers of the dorsal cochlear nucleus (DCN), whereas the ventral cochlear nucleus (VCN) has somewhat fewer terminals that are arranged in pericellular plexuses. GAD-positive neurons occur mainly in the superficial and fusiform layers of the DCN and are scattered throughout the VCN. Within the superior olivary complex, the highest concentration of immunoreactive terminals and neurons occurs in the ventral and lateral nuclei of the trapezoid body. In contrast, the medial nucleus of the trapezoid body and the medial superior olive contain fewer GAD-positive puncta and probably no immunoreactive somata. The lateral superior olive and superior periolivary nucleus contain a few immunoreactive puncta but a large number of immunoreactive somata. In the midbrain, the nuclei of the lateral lemniscus contain a moderate number of GAD-positive puncta and a large number of different types of GAD-positive neurons. The inferior colliculus also contains a heterogeneous population of labeled somata, most of which are multipolar neurons. In addition, a high concentration of immunoreactive puncta occurs in this region. These data demonstrate a diverse distribution of GAD-positive neurons and puncta throughout the brainstem auditory nuclei and suggest that GABA might be an important neurotransmitter in the processing of auditory information.     

Observation of the ultrastructure and synaptic relationships of angiotensin II-like immunoreactive neurons in the rat area postrema

The ultrastructure and synaptic relationships of the angiotensin II-containing neurons in the area postrema of the rat were studied by immunocytochemistry using the avidin-biotin-complex-DAB method, and also using silver-gold intensification following the DAB reaction. At the light microscopic level, the angiotensin II-like immunoreactive neurons were observed within the area postrema, especially in the upper region. At the electron microscopic level, the angiotensin II-like immunoreactive cell bodies were observed as having a round, unindented nucleus. The nuclei of these neurons were not immunostained. The angiotensin II-like immunoreactive axon terminals often contained a few dense core vesicles in addition to many small clear synaptic vesicles. Numerous axon terminals were found to make synapses on immunonegative dendrites; they were also found to make synapses on angiotensin II-like immunoreactive dendrites. Many angiotensin II-like immunoreactive dendrites received synapses from immunonegative axon terminals. Although angiotensin II-like immunoreactive cell bodies were sometimes postsynaptic to immunoreactive axon terminals, they did not receive synapses from immunonegative axon terminals. These results provide solid morphological evidence of AP endogenous angiotensin II and confirm that in spite of circulating angiotensin II, the local neurons in the AP may also play an important role in angiotensin II-induced cardiovascular regulation.     

Patterns of glutamate, glycine, and GABA immunolabeling in four synaptic terminal classes in the lateral superior olive of the guinea pig.

The goal of this study was to correlate synaptic ultrastructure with transmitter specificity and function in the lateral superior olive (LSO), a nucleus that is thought to play a major role in sound localization. This was accomplished by means of postembedding immunogold immunocytochemistry. Four classes of synaptic terminals were identified in the LSO. They were distinguishable from one another both morphologically and on the basis of their different patterns of immunolabeling for glutamate, glycine, and gamma-aminobutyric acid (GABA). The highest level of glutamate immunoreactivity was found in terminals that contained round vesicles (R) and formed synaptic contacts with asymmetric synaptic junctions. Round-vesicle terminals predominated on small caliber dendrites by a ratio of at least 2:1 over the other classes combined. The thinnest dendrites were typically contacted by R terminals only. The ratio of R terminals to the other types decreased as the caliber of the dendritic profiles they apposed increased so that on the soma, R terminals were outnumbered by at least 2:1 by the other types. Terminals containing flattened vesicles (F) exhibited intense immunoreactivity for both glycine and glutamate, although the glutamate immunolabeling was not as high as that in the R terminals. Flattened-vesicle terminals formed symmetric synaptic contacts with their targets and their distribution was the reverse of that described for R terminals; i.e., they were most abundant on LSO perikarya and fewest on small caliber dendrites. Two terminal types, both containing pleomorphic vesicles and forming symmetric synaptic junctions, were found in far fewer numbers. One group contained large pleomorphic vesicles (LP) and was immunoreactive for both glycine and GABA. The other group contained small pleomorphic vesicles (SP) along with a few dense-core vesicles and labeled for GABA only. The LP terminals were preferentially distributed on somata and large-caliber dendrites, while the SP terminals most often contacted smaller dendrites. Previous work suggests that a large percentage of the R terminals arise from spherical cells in the ipsilateral cochlear nucleus and are excitatory in action. This pathway may use glutamate as a transmitter. Many of the F terminals are thought to originate from the ipsilateral medial nucleus of the trapezoid body and appear to be the inhibitory (glycinergic) terminals from a pathway that originates from the contralateral ear. The origins and functions of LP and SP terminals are unknown, but a few possibilities are discussed along with the significance of cocontainment of neuroactive substances in specific terminal types.     

Distribution and fine structure of neuronal elements containing glutamate decar☐ylase in the rat cochlear nucleus

Distribution and fine structure of γ-aminobutyric acid (GABA)-containing structures were examined in the rat cochlear nuclear complex by means of immunohistochemistry using glutamate decar☐ylase (GAD) as a marker. GAD-like immunoreactive (GADI) terminals were diffusely distributed in the dorsal cochlear nucleus, while in the ventral cochlear nucleus numerous immunoreactive fibers were situated around the cell bodies. These light-microscopic observations were confirmed by electron microscopy. Evidence suggesting that many of GADI boutons in the cochlear nucleus are of intrinsic origin was also shown.     

Red nucleus of Macaca fascicularis: an electron microscopic study of its synaptic organization

The parvicellular and magnocellular divisions of the red nucleus of the old world monkey, Macaca fascicularis, were analyzed at an electron microscopic level to examine the morphology of the synaptic profiles terminating on rubral neurons and to categorize them by their individual characteristics. The parvicellular division, or anterior two-thirds of the nucleus, is composed of small (10-15 microns) and medium-size (20-30 microns) cells, which are uniformly distributed with high packing density throughout this portion of the nucleus. These cells have invaginated nuclei and are often indented by blood vessels and glial cell somata (satellite cells) that lie in close proximity. The magnocellular portion, occupying the caudal one-third of the nucleus, is composed of an additional population of large cells, ranging from 50-90 microns in diameter, which often contain prominent lipofuscin granules and are frequently indented by blood vessels. Satellite glial cells are not a prominent feature in the magnocellularis portion of the nucleus. The large cells are separated one from the other by fields of myelinated axons either coursing through the nucleus or projecting to and from the nucleus itself. Although the divisions of the nucleus in the Macaca fascicularis are spatially distinct, each possesses a morphological similarity in regard to the categories of synaptic profiles seen at the electron microscopic level. These synaptic profiles are classified as follows: large terminals containing numerous, predominantly rounded vesicles (LR), which can often be seen to form the central profile in a synaptic glomerular arrangement; terminals of similar size with predominantly rounded vesicles but with a pale axoplasmic matrix (LRP); small profiles with rounded vesicles (SR); profiles containing granular dense-cored vesicles (DCV); profiles with numerous flattened vesicles (F); profiles containing pleomorphic vesicles (PL), some of which can be interpreted as presynaptic dendrites (PSD) because they are seen to be postsynaptic and contain ribosomes; and profiles with rounded synaptic vesicles, which are associated with subsynaptic Taxi bodies (T). Most of the various synaptic profile types were found to have similar distributions on the dendritic arbors of rubral neurons in both divisions of the nucleus. However, the LRP-type terminal predominates on the cell bodies and proximal dendrites of the large neurons in magnocellularis. Unlike other regions in the nervous system, F type terminals are rarely seen to contact neuronal somata. This study provides a basis for future experimental studies of afferents to the nucleus in this species.(ABSTRACT TRUNCATED AT 400 WORDS)     

Topography and synaptology of mamillary body projections to the mesencephalon and pons in the rat.

The anterograde and retrograde transport of horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) was used to study the anatomical organization of descending projections from the mamillary body (MB) to the mesencephalon and pons at light and electron microscopic levels. Injections of WGA-HRP into the medial mamillary nucleus resulted in dense anterograde and retrograde labeling in the ventral tegmental nucleus, while injections in the lateral mamillary nucleus resulted in dense anterograde labeling in the dorsal tegmental nucleus pars dorsalis and dense anterograde and retrograde labeling in the pars ventralis of the dorsal tegmental nucleus. Anterogradely labeled fibers in the mamillotegmental tract diverged from the principal mamillary tract in an extensive dorsocaudally oriented swath of axons which extended to the dorsal and ventral tegmental nuclei, and numerous axons turned sharply ventrally and rostrally to terminate topographically in the dorsomedial nucleus reticularis tegmenti pontis and rostromedial pontine nuclei. The anterograde labeling in these two precerebellar relay nuclei was distributed near the midline such that projections from the lateral mamillary nucleus terminated mainly dorsomedial to the terminal fields of projections from the medial mamillary nucleus. In the dorsal and ventral tegmental nuclei, labeled axon terminals contained round synaptic vesicles and formed asymmetric synaptic junctions primarily with small diameter dendrites and to a lesser extent with neuronal somata. A few labeled terminals contained pleomorphic vesicles and formed symmetric synaptic junctions with dendrites and neuronal somata. Labeled axon terminals were also frequently found in synaptic contact with retrogradely labeled dendrites and neuronal somata in the dorsal and ventral tegmental nuclei. These findings indicate that neurons in the dorsal and ventral tegmental nuclei are reciprocally connected with MB projection neurons. In the nucleus reticularis tegmenti pontis and medial pontine nuclei, labeled axon terminals contained round synaptic vesicles and formed asymmetric synaptic junctions primarily with small diameter dendrites. The present study demonstrates that projections from the medial and lateral nuclei of the MB are topographically organized in the mesencephalon and pons. The synaptic morphology of mamillotegmental projections suggests that they may have excitatory influences primarily on the distal dendrites of neurons in these brain regions.     

Postembedding immunocytochemistry demonstrates directly that both retinal and cortical terminals in the cat superior colliculus are glutamate immunoreactive

Although the excitatory neurotransmitter glutamate is known to be present in the cat superior colliculus (SC), the types of synapses that contain glutamate have not been examined. We, therefore, studied the ultrastructure of synaptic profiles labeled by a glutamate antibody by using electron microscopic postembedding immunocytochemistry. In addition, unilateral aspiration lesions of areas 17–18 were made at 5–28 days before death in order to determine whether degenerating terminals from visual cortex were glutamate immunoreactive (Glu-ir). Three types of axon terminal were glu-ir: 1) those containing large, round synaptic vesicles and pale mitochondria, characteristic of retinal terminals (RT profiles); 2) those containing small, round synaptic vesicles and dark mitochondria (RSD profiles); and 3) those containing large, round synaptic vesicles and dark mitochondria (RLD profiles). Measures of mean gold particle density revealed that RT, RSD, and RLD profiles had similar average grain densities (11.3–12.7 particles/unit area). Other labeled profile types included cell bodies, large-calibre dendrites, and myelinated axons. Axon terminals containing flattened synaptic vesicles and vesicle-containing presynaptic dendrites, both of which contain γ-aminobutyric acid (GABA), had many fewer gold particles (3.6 and 4.8 mean particles/unit area, respectively). Following unilateral removal of visual cortex, normal RSD terminals were observed infrequently in the SC ipsilateral to the lesion. Synaptic terminals in the initial stages of degeneration were heavily labeled by the glutamate antibody, as were axon terminals and myelinated axons undergoing hypertrophied or neurofilamentous degeneration. These results show that both major sensory afferents to the superficial layers of cat SC contain glutamate—RT terminals from the retina and RSD terminals from visual cortex. The origin of RLD terminals is unknown. © 1996 Wiley-Liss, Inc.     

Distribution of GABAergic neurons and terminals in the auditory system of the barn owl

Antisera to GAD (glutamic acid decarboxylase) and GABA were used to determine the distribution of GABAergic cells and terminals in the brainstem and midbrain auditory nuclei of the barn owl. The owl processes time and intensity components of the auditory signal in separate pathways, and each pathway has a distinctive pattern of GAD- and GABA-like immunoreactivity. In the time pathway, all the cells of the cochlear nucleus magnocellularis and nucleus laminaris receive perisomatic GABAergic terminals, and small numbers of GABAergic neurons surround both nuclei. The ventral nucleus of the lateral lemniscus (anterior division) contains both immunoreactive terminals and some GABAergic neurons. In the intensity pathway, dense immunoreactive terminals are distributed throughout the cochlear nucleus angularis, which also contains a small number of GABAergic neurons. The superior olive contains two GABAergic cell types and immunoreactive terminals distributed throughout the neuropil. All the neurons of the nucleus of the lateral lemniscus (ventral part) appear to be GABAergic, and this nucleus also contains a moderate number of immunoreactive terminals. Immunoreactive terminals are distributed throughout the neuropil of the ventral nucleus of the lateral lemniscus (posterior division), whereas multipolar and small fusiform GABAergic neurons predominate in the dorsal regions of the nucleus. The time and intensity pathways combine in the inferior colliculus. The central nucleus of the inferior colliculus contains a larger number of fusiform and stellate GABAergic neurons and a dense plexus of immunoreactive terminals, whereas the external nucleus contains slightly fewer immunoreactive cells and terminals. The superficial nucleus contains dense, fine immunoreactive terminals and a small number of GABAergic neurons.     

Ultrastructure and synaptic organization of axon terminals from brainstem structures to the mediodorsal thalamic nucleus of the rat.

The ultrastructural characteristics and synaptic organization of afferent terminals from the brainstem to the mediodorsal thalamic nucleus (MD) of the rat have been studied with the electron microscope, by means of anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Labeled fibers were seen predominantly in the lateral portion of MD after the injections of WGA-HRP into the substantia nigra pars reticulata (SNr), the superior colliculus (SC), and the dorsal tegmental region (DT). The boutons arising from the SC were relatively small (less than 1.5 microns in diameter), formed asymmetric synaptic contacts with small dendrites and dendritic spines, and contained round synaptic vesicles. The axon terminals from the DT were mostly large boutons (2-4.5 microns) with asymmetric synaptic specializations and round vesicles. These boutons and their postsynaptic targets formed synaptic glomeruli that were entirely or partially ensheathed by glial lamellae. The ultrastructural features are almost identical to those of boutons in the medial and central segments of MD that were previously shown to originate from the basal amygdaloid nucleus and the piriform cortex. The boutons from the SNr had a wide range in size, but the majority were medium-sized to large (1.5-4 microns). The nigral boutons established symmetric synaptic contacts with dendritic shafts and occasionally with somata, and contained pleomorphic vesicles. However, like the DT terminals, they participated in glomerular formations. The nigral terminals closely resemble previously described terminals in the medial part of MD from the ventral pallidum, except that the nigral terminals formed en passant and axosomatic synapses as well as axodendritic synapses. A combined immunohistochemistry and WGA-HRP tracing study revealed that the nigral inputs were immunoreactive for glutamic acid decarboxylase and the axon terminals from the DT were immunoreactive for choline acetyltransferase. In a separate study, the colliculothalamic fibers have been shown to take up and transport the transmitter specific tracer [3H]-D-aspartate, and are therefore putatively glutamatergic and/or aspartatergic. Taken together with this, the present results suggest that the collicular afferents are excitatory and glutamatergic and/or aspartatergic, that the inputs from the DT are also excitatory and cholinergic, while the nigral inputs are inhibitory and GABAergic.     

Projections from auditory cortex to the cochlear nucleus in rats: Synapses on granule cell dendrites

Previous work has demonstrated that layer V pyramidal cells of primary auditory cortex project directly to the cochlear nucleus. The postsynaptic targets of these centrifugal projections, however, are not known. For the present study, biotinylated dextran amine, an anterograde tracer, was injected into the auditory cortex of rats, and labeled terminals were examined with light and electron microscopy. Labeled corticobulbar axons and terminals in the cochlear nucleus are found almost exclusively in the granule cell domain, and the terminals appear as boutons (1–2 μm in diameter) or as small mossy fiber endings (2–5 μm in diameter). These cortical endings contain round synaptic vesicles and form asymmetric synapses on hairy dendritic profiles, from which thin (0.1 μm in diameter), nonsynaptic “hairs” protrude deep into the labeled endings. These postsynaptic dendrites, which are typical of granule cells, surround and receive synapses from large, unlabeled mossy fiber endings containing round synaptic vesicles and are also postsynaptic to unlabeled axon terminals containing pleomorphic synaptic vesicles. No labeled fibers were observed synapsing on profiles that did not fit the characteristics of granule cell dendrites. We describe a circuit in the auditory system by which ascending information in the cochlear nucleus can be modified directly by descending cortical influences. © 1996 Wiley-Liss, Inc.     

Post-embedding immunogold labeling of gamma-aminobutyric acid in lamina II of the spinal trigeminal subnucleus pars caudalis: I. A qualitative study

This study examines the normal synaptic organization of the feline spinal trigeminal nucleus pars caudalis (PC). A primary goal of this study is to identify and characterize the synaptic complexes within PC based on their specific neurotransmitter content. Post-embedding immunogold techniques are utilized with electron microscopy to determine the ultrastructural localization of gamma-aminobutyric acid (GABA) immunoreactivity within lamina II of PC. The colloidal gold particles (10 nm) are randomly distributed over immunoreactive (IR) profiles without preference toward membranous or cytoplasmic regions. GABA immunoreactivity occurs on small unmyelinated axons, on terminals which form synaptic contacts, and on some vesicle-containing dendrites. The GABA-IR terminals form symmetric (type II) contacts onto unlabeled somata and dendrites of various sizes, and onto other unlabeled axon terminals. The GABA-IR terminal in axo-axonic complexes is presynaptic to a round vesicle-containing terminal, which itself may form a type I asymmetric contact onto an unlabeled dendrite or soma. A proportion of vesicle-containing dendrites show GABA-immunoreactivity and are postsynaptic to unlabeled terminals with round vesicles. Other, but far fewer, vesicle-containing dendrites are GABA negative and postsynaptic to GABA-IR terminals. In summary, the findings are consistent with the localization of GABA in intrinsic neurons, and may be associated with presynaptic and postsynaptic inhibition within nociceptive related pathways. © 1996 Wiley-Liss, Inc.