Glutamate-positive neurons and axon terminals in cat sensory cortex: a correlative light and electron microscopic study

Immunocytochemical methods were used to perform a correlative light and electron microscopic study of neurons and axon terminals immunoreactive to the antiglutamate (Glu) serum of Hepler et al. ('88) in the visual and somatic sensory areas of cats. At the light microscopic level, numerous Glu-positive neurons were found in all layers except layer I of both cortical areas. On the basis of the dendritic staining of Glu-positive cells, two major morphological categories were found: pyramidal cells, which were the most frequent type of immunostained neuron, and multipolar neurons, which were more numerous in layer IV of area 17 than in any other layer. A large number of Glu-positive neurons, however, did not display dendritic labelling and were considered unidentified neurons. Counts of labelled neurons were performed in the striate cortex; approximately 40% were Glu-positive. Numerous lightly stained punctate structures were observed in all cortical layers: the majority of these Glu-positive puncta were in the neuropil. After resectioning the plastic sections for electron microscopy it was observed that: 1) the majority of neurons unidentifiable at light microscopic level were indeed pyramidal neurons except in layer IV of area 17, where many stained cells were probably spiny stellate neurons. Some Glu-positive neurons, however, exhibited clear ultrastructural features of nonspiny nonpyramidal cells; 2) all synaptic contacts made by Glu-positive axon terminals were of the asymmetric type, but not all asymmetric synaptic contacts were labelled. The vast majority of postsynaptic targets of Glu-positive axons were unlabelled dendritic spines and shafts. The present results provide further evidence that Glu (or a closely related compound) is probably the neurotransmitter of numerous excitatory neurons in the neocortex.     

Serotonin-containing axon terminals in the hypoglossal nucleus of the rat. An immuno-electronmicroscopic study

The morphology and distribution of serotonin-containing axon terminals in the rat hypoglossal nucleus (XII) was investigated immunocytochemically at the electron microscopic level. Serotonin-positive profiles were found throughout all regions of XII and included unmyelinated axons, varicosities and axon terminals. Most labeled profiles (68.1%) were nonsynaptic unmyelinated axons and varicosities, while synaptic profiles, ending on dendrites and somata, were seen less frequently (28.7%). The majority of labeled axon terminals (76.9%) ended on small-to-medium-sized dendrites. Most axodendritic terminals contained small, round agranular vesicles (20-55 microns), several large (60-100 microns) dense core vesicles, and were associated with a pronounced asymmetric postsynaptic specialization. By contrast, labeled axosomatic terminals were seen less often than those ending on dendrites (23.0%). Axosomatic terminals typically contained small, round, agranular and large dense core vesicles and were associated with a symmetric or no postsynaptic specialization. These results provide the structural substrates for elucidating the functional role of serotonin in tongue control.     

Serotonin-containing projections to the thalamus in the rat revealed by a horseradish peroxidase and peroxidase antiperoxidase double-staining technique

The retrograde transport of horseradish peroxidase (HRP) has been used in combination with peroxidase antiperoxidase (PAP) immunocytochemistry in order to investigate serotonin-containing projections to the thalamus of the rat. Sections were histochemically stained to reveal retrogradely transported HRP and then PAP immunostained using a monoclonal anti-serotonin (5-HT) antibody. Following HRP injections into the ventral thalamus, retrogradely labelled cells were observed in a number of sites in the brainstem and including areas known to be rich in 5-HT-containing neurons. At rostral levels of the dorsal raphe nucleus, retrogradely labelled cells were observed both on the midline and in a distinct lateral group extending diffusely into the periaqueductal gray (PAG). In both of these areas many 5-HT-immunoreactive HRP retrogradely labelled neurons were observed. However, except for the most rostral levels of the dorsal raphe nucleus, such double-labelled cells represented only a small proportion of the total population of 5-HT-immunoreactive neurons. In the lateral group, the retrograde labelling was mainly unilateral to the injection site but some contralateral labelling was also seen. At caudal levels of the dorsal raphe nucleus, retrogradely labelled cells were observed predominantly in the lateral group. At the level of the dorsolateral tegmental nucleus, few 5-HT of 5-HT/HRP labelled cells were observed in the lateral group, although HRP retrogradely labelled neurons were present. Double-stained cells were detected also in the medial raphe nucleus (corresponding to the B8 cell group according to the nomenclature of Dahlström and Fuxe¹³), among the fibres of the medial lemniscus (B9), and in nucleus raphe pontis (B5).     

Localization of the M2 muscarinic cholinergic receptor in dendrites,cholinergic terminals,and noncholinergic terminals in the rat basolateral amygdala: An ultrastructural analysis

Activation of M2 muscarinic receptors (M2Rs) in the rat anterior basolateral nucleus (BLa) is critical for the consolidation of memories of emotionally arousing events. The present investigation used immunocytochemistry at the electron microscopic level to determine which structures in the BLa express M2Rs. In addition, dual localization of M2R and the vesicular acetylcholine transporter protein (VAChT), a marker for cholinergic axons, was performed to determine whether M2R is an autoreceptor in cholinergic axons innervating the BLa. M2R immunoreactivity (M2R‐ir) was absent from the perikarya of pyramidal neurons, with the exception of the Golgi complex, but was dense in the proximal dendrites and axon initial segments emanating from these neurons. Most perikarya of nonpyramidal neurons were also M2R–negative. About 95% of dendritic shafts and 60% of dendritic spines were M2 immunoreactive (M2R⁺). Some M2R⁺ dendrites had spines, suggesting that they belonged to pyramidal cells, whereas others had morphological features typical of nonpyramidal neurons. M2R‐ir was also seen in axon terminals, most of which formed asymmetrical synapses. The main targets of M2R⁺ terminals forming asymmetrical (putative excitatory) synapses were dendritic spines, most of which were M2R⁺. The main targets of M2R⁺ terminals forming symmetrical (putative inhibitory or neuromodulatory) synapses were unlabeled perikarya and M2R⁺ dendritic shafts. M2R‐ir was also seen in VAChT⁺ cholinergic terminals, indicating a possible autoreceptor role. These findings suggest that M2R‐mediated mechanisms in the BLa are very complex, involving postsynaptic effects in dendrites as well as regulating release of glutamate, γ‐aminobutyric acid, and acetylcholine from presynaptic axon terminals. J. Comp. Neurol. 524:2400–2417, 2016. © 2016 Wiley Periodicals, Inc.     

Organisation of the cerebellar nucleus of the dogfish,Scyliorhinus canicula L.: A light microscopic,immunocytochemical, and ultrastructural study

Elasmobranchs possess a well-developed cerebellum with an associated cerebellar nucleus. To determine whether the organization of this nucleus is comparable with that of the deep cerebellar nuclei of mammals, we studied the dogfish cerebellar nucleus with light microscopic methods (Nissl stain, Golgi method, reduced silver stain, NADPH-diaphorase histochemistry and immunocytochemistry) and with electron microscopy. We found the dogfish cerebellar nucleus to consist of about 1,050 large neurons, the ratio of Purkinje cells to cerebellar nucleus neurons being about 17:1. Immunocytochemistry showed large glutamatergic neurons in the main portions of the nucleus and small glutamate- and/or α-aminobutyric acid (GABA)-immunoreactive cells in the subventricular region of the nucleus. Large glutamatergic neurons corresponded to bipolar or triangular cells revealed by Golgi methods. Application of horseradish peroxidase to the cerebellar cortex produced the labelling of beaded fibres of Purkinje cells in the cerebellar nucleus. Unlike in mammals, GABAergic innervation of the cerebellar nucleus was scarce: Purkinje cell axon terminals in the cerebellar nucleus did not appear to be GABA-immunoreactive, most GABAergic fibres being found in the subventricular neuropile. Some fibres immunoreactive to serotonin and somatostatin were also observed in the subventricular neuropile of the cerebellar nucleus. Three neuron types were distinguished with electron microscopy (types A to C). Type A cells were abundant and smooth-surfaced, and appeared to correspond to Golgi-impregnated neurons and large glutamate-immunoreactive cells. Type B neurons were scarce and possessed dendrites covered by sessile or stalked spines. Type C neurons were small cells located mainly in the medialmost region of the nucleus and corresponded to subventricular glutamate- and GABA-immunoreactive cells. Six types of synaptic bouton were observed (types I to VI). The most abundant (type I boutons) made symmetrical contacts and appeared to correspond to Purkinje cell axons. Type I boutons were the only type observed on perikarya and initial axon segments of type A cells. Type IV and type V boutons made complex glomerular-like asymmetrical contacts with spines of type B cells. Type VI boutons appeared to correspond to peptidergic and/or monoaminergic axons. The functional significance of these results is discussed. © 1996 Wiley-Liss, Inc.     

An ultrastructural analysis of afferent terminals to the anterior pretectal nucleus in the cat

The synaptic organization of three kinds of afferent projections in the feline anterior pretectal nucleus (PTA) was analyzed by a combination of the degeneration and retrograde transport of horseradish peroxidase (HRP) techniques, along with that of the degeneration and anterograde transport of HRP techniques. Retrograde labeling of PTA neurons was performed by injections of HRP in the dorsal accessory olivary nucleus (DAO). Three kinds of afferent sources of the PTA--the cerebral motor cortex, the anterior interpositus nucleus of the cerebellum, and the gracile nucleus--were subjected to electrolysis, suction, or injection of kainic acid or HRP for identification of axon terminals of each system. Axon terminals of these different afferent sources identified by degeneration or anterograde HRP transport techniques showed similar morphological features: They were relatively large (1-6.5 microns in diameter), contained round or ovoid synaptic vesicles, and made asymmetrical synaptic contacts. When the degeneration study was combined with the retrograde HRP transport technique, some degenerating terminals from the motor cortex, anterior interpositus, or gracile nuclei were found to synapse directly with HRP-labeled dendrites or somata of the PTA neurons projecting to the DAO. Each combination of the degeneration and anterograde HRP transport techniques revealed the fact that neither degenerating nor HRP-labeled terminals were found to synapse with the same neuronal structure. These observations indicate that the PTA neurons relay afferent inputs from three different sources directly to the DAO, and that there is a possibility of parallel processing rather than convergence of three different afferent systems via the PTA to the DAO.     

Morphology and frequency of axon terminals on the somata, proximal dendrites, and distal dendrites of dorsal neck motoneurons in the cat.

The purpose of the present study was to compare the frequency of different classes of axon terminals on selected regions of the somatodendritic surface of dorsal neck motoneurons. Single motoneurons supplying neck extensor muscles were antidromically identified and intracellularly stained with horseradish peroxidase. By using light microscopic reconstructions as a guide, axon terminals on the somata, proximal dendrites (within 250 microns of the soma), and distal dendrites (more than 540 microns from the soma) were examined at the electron microscopic level. Axon terminals were divided into several classes based on the shape, density, and distribution of their synaptic vesicles. The proportion of axon terminals belonging to each axon terminal class was similar on the somata and proximal dendrites. However, there were major shifts in the relative frequency of most classes of axon terminals on the distal dendrites. The most common classes of axon terminals on the somata and proximal dendrites contained clumps of either spherical or pleomorphic vesicles. These types of axon terminals accounted for more than 60% of the axon terminals on these regions. In contrast, only 11% of the axon terminals found on distal dendrites belonged to these types of axon terminals. The most commonly encountered axon terminal on distal dendrites contained a dense collection of uniformly distributed spherical vesicles. These types of axon terminals accounted for 40% of all terminals on the distal dendrites, but only 5-7% of the axon terminals on the somata and proximal dendrites. Total synaptic density on each of the three regions examined was similar. However, the percentage of membrane in contract with axon terminals was approximately four times smaller on distal dendrites than somata or proximal dendrites. Axon terminals (regardless of type) were usually larger on somata and proximal dendrites than distal dendrites. These results indicate that there are major differences in the types and arrangement of axon terminals on the proximal and distal regions of dorsal neck motoneurons and suggest that afferents from different sources may preferentially contact proximal or distal regions of the dendritic trees of these cells.     

Pressor response to electrical and chemical stimulation of nucleus raphe dorsalis in the cat

The dorsal raphe nucleus was stimulated electrically and chemically in 57 cats with blood pressure being measured by an arterial catheter. Blood pressure rose by a mean of 119.5 +/- 8.9% on stimulation of the dorsal raphe nucleus at a frequency of 200 sec-1. This pressor response was frequency-dependent over the range 0.2 to 200 sec-1 and the site of origin was localized to the dorsal raphe nucleus. The pressor response could also be obtained by the injection of D,L-homocysteic acid into the nucleus, and is therefore likely to be due to activation of cell bodies rather than axons of passage. The response was blocked by high spinal cord section but was not affected by bilateral clamping of the adrenal glands or supracollicular decerebration. The pressor response was significantly reduced by pretreatment of the cats with parachlorophenylalanine (PCPA) and abolished by the alpha receptor blocker phentolamine, suggesting that both serotonergic and noradrenergic synapses are involved.     

Distribution and quantification of 5-HT nerve cell bodies in the nucleus raphe dorsalis area of C57BL and BALBc mice. Relationship between anatomy and biochemistry

BALBc and C57BL mice have been shown to have a different 5-HT metabolism. The present study compares the number and the distribution of 5-HT cell bodies in the nucleus raphe dorsalis area (B7 + B6) of these strains. By using 5-HT immunohistochemistry, we found a higher number of 5-HT neurons in the most caudal part of NRD (B6) of BALBc mice compared to C57BL. This difference may be correlated with a higher level of endogenous 5-HT, a higher uptake capacity toward exogenous [³H]5-HT, and a lower release of the amine in this same area of BALBc mice compared to C57BL. It could also imply a significant participation of the nerve cell bodies in the regulation of 5-HT transmission inside 5-HT nuclei.     

Morphological diversity and glutamate immunoreactivity of retinal terminals in the suprachiasmatic nucleus of the cat

Although the cat visual system has been the subject of intensive investigation, little attention has been given to the morphological features of ganglion cell projections to the suprachiasmatic nucleus. The present study has utilized anterograde transport of horseradish peroxidase and wheat germ agglutinin–conjugated horseradish peroxidase to label ganglion cell terminals in the cat suprachiasmatic nucleus. Visualization of the reaction product was facilitated through the use of gold-substituted silver intensification. Ganglion cell terminals were found to be morphologically diverse, making both asymmetric and symmetric contacts with postsynaptic processes. Synaptic vesicles were either scattered or densely packed, sometimes forming paracrystalline arrays. In contrast to other retinorecipient areas in which ganglion cell terminals have, been characterized by the presence of lightly staining mitochondria, many of the retinal terminals in the suprachiasmatic nucleus were seen to contain darkly stained mitochondria. Postembedding antiglutamate immunocytochemistry was used to evaluate the level of endogenous glutamate in these ganglion cell terminals. Although morphologically diverse, all of the retinal terminals in the suprachiasmatic nucleus were glutamate positive, consistent with the postulated role of glutamate as the neurotransmitter of retinal ganglion cells. © 1995 Wiley-Liss, Inc.     

Serotonergic innervation of the isthmo-optic nucleus of the pigeon centrifugal visual system. An immunocytochemical electron microscopic study

The ultrastructural features of serotonergic fibers, terminals and synaptic contacts were studied with the pre-embedding immunocytochemical method in the isthmo-optic nucleus of the pigeon centrifugal visual system. The 5-HT immunoreactive (-ir) profiles were diffusely distributed and their density was low. The labeled axons were thin and unmyelinated (mean diameter=0.21+/-0.03 microm) though a few larger myelinated axons were observed (mean diameter=0.51+/-0.07 microm). The 5-HT-ir terminals or varicosities were small (diameter=0.71+/-0.54 microm) and contained small agranular synaptic vesicles (diameter=28.5+/-6.9 nm) and large granular vesicles (diameter=102.2+/-19.5 nm). The latter only constituted approximately 1% of the total profiles containing synaptic vesicles in the isthmo-optic nucleus. In single thin sections, only 5% of the 5-HT-ir varicosities exhibited an active asymmetrical zone synapsing upon dendritic profiles of centrifugal visual neurons. Calculations indicated that 17% of these 5-HT-ir varicosities were actually engaged in junctional synaptic relationships, whereas the remaining (83%) were nonjunctional. The data suggest that, within the isthmo-optic nucleus, 5-HT acts both at synaptic junctions (wiring transmission) and at a distance via the extracellular space (volume transmission). These 5-HT afferents could thus modulate the activity of the retinopetal neurons and visual information processing.     

Cellular effects of swim stress in the dorsal raphe nucleus

Swim stress regulates forebrain 5-hydroxytryptamine (5-HT) release in a complex manner and its effects are initiated in the serotonergic dorsal raphe nucleus (DRN). The purpose of this study was to examine the effects of swim stress on the physiology of DRN neurons in conjunction with 5-HT immunohistochemistry. Basic membrane properties, 5-HT(1A) and 5-HT(1B) receptor-mediated responses and glutamatergic excitatory postsynaptic currents (EPSCs) were measured using whole-cell patch clamp techniques. Rats were forced to swim for 15min and 24h later DRN brain slices were prepared for electrophysiology. Swim stress altered the resting membrane potential, input resistance and action potential duration of DRN neurons in a neurochemical-specific manner. Swim stress selectively elevated glutamate EPSC frequency in 5-HT DRN neurons. Swim stress non-selectively reduced EPSC amplitude in all DRN cells. Swim stress elevated the 5-HT(1B) receptor-mediated inhibition of glutamatergic synaptic activity that selectively targeted 5-HT cells. Non-5-HT DRN neurons appeared to be particularly responsive to the effects of a milder handling stress. Handling elevated EPSC frequency, reduced EPSC decay time and enhanced a 5-HT(1B) receptor-mediated inhibition of mEPSC frequency selectively in non-5-HT DRN cells. These results indicate that swim stress has both direct, i.e., changes in membrane characteristics, and indirect effects, i.e., via glutamatergic afferents, on DRN neurons. These results also indicate that there are distinct local glutamatergic afferents to neurochemically specific populations of DRN neurons, and furthermore that these distinct afferents are differentially regulated by swim stress. These cellular changes may contribute to the complex effects of swim stress on 5-HT neurotransmission and/or the behavioral changes underlying the forced swimming test model of depression.     

Morphometrical synaptology of Clarke cells and of distal dendrites in the nucleus dorsalis: An electron microscopic study in the cat

The fine structural synaptology of large Clarke cells in L3 has been investigated from a morphometrical point of view in both normal and adult cats which received horseradish peroxidase (HRP) injections in the cerebellum. This marking method made it possible to distinguish small or distal dendrites of large Clarke cells from those of interneurons and the marginal cells of Clarke's column.A total of 1036 boutons was observed on the perikarya of 21 large Clarke cells; 81.9% (848/1036) were small-sized boutons, the cross-sectional areas of which ranged between 0.3 and 2.9 sq. μm, while 18.1% (186/1036) were giant boutons ranging between 3.0 and 8.0 sq. μm. From 1075 boutons on 17 primary dendrites of Clarke cells, 72.4% (778/1075) were small-sized boutons and 27.6% (297/1075) were giant boutons. From 1679 boutons contacting 366 distal or small HRP-labeled dendrites, 89.9% (1507/1679) were small boutons and 10.1% were giant boutons. The giant boutons were more frequently located on the proximal dendrites than on the cell bodies or small distal dendrites of Clarke cells.The proportion of S-and F-type boutons was different in 3 parts of large Clarke cells. F-type boutons were more frequent on soma (55.0%, 570/1036) and primary dendrites (59.4%, 635/1075). S-type boutons outnumbered the F-type on small or distal dendrites (62.6%, 1052/1679). The S/F ratio seemed to increase from the cell body toward the distal dendrites. The results suggest that Clarke cells receive predominantly small S-type boutons since the total receptive area of the dendrites is supposed to exceed that of the cell body.     

Substance P in the rat nucleus accumbens: ultrastructural localization in axon terminals and their relation to dopaminergic afferents

Dual labeling electron microscopic immunocytochemistry was used to investigate the cellular substrate for functional interactions between substance P (SP) and dopamine in the rat nucleus accumbens. Coronal vibratome sections from acrolein-fixed brains were sequentially processed for the localization of: (1) a rat monoclonal antiserum against SP identified by the peroxidase--anti-peroxidase immunocytochemical method, and (2) a rabbit polyclonal antiserum against tyrosine hydroxylase (TH) identified by immunoautoradiography. The monoclonal rat antiserum recognized principally SP, but also exhibited cross-reactivity with certain other tachykinins such as substance K. Terminals showing SP-like immunoreactivity (SPLI) were 0.2-1.5 microns in diameter and contained numerous small (30-40 nm), round vesicles; one or more large (80-150 nm), dense-core vesicles; and an occasional membrane-bound multivesicular body. From a total of 114 SP-labeled terminals that were quantitatively analyzed, 30.1% formed symmetric synapses with dendrites; whereas only 8% formed asymmetric junctions with dendritic spines. Terminals showing SPLI also occasionally formed junctions with dendrites receiving synaptic input from other terminals that were similarly labeled for the peptide or from terminals immunoautoradiographically labeled for TH. In contrast to the low frequency of postsynaptic relationships, 39.8% of the terminals containing SPLI showed close associations with other unlabeled or TH-labeled terminal or preterminal axons. The axonic contacts were characterized by equally spaced membranes that were not separated by glial processes. Within the terminals containing SPLI, vesicles were located near the axonic contacts; whereas vesicles in unlabeled terminals were located more distally with respect to these appositions. We conclude that in the rat nucleus accumbens SP or a closely related tachykinin subserves principally inhibitory functions at postsynaptic sites as indicated by the prominence of symmetric junctions. The abundance of axonic associations and sparsity of convergent input from TH- and SP-labeled terminals at closely spaced sites on dendrites supports the concepts that a SP-like tachykinin also may modulate the release of dopamine through direct or indirect presynaptic mechanisms. The possibility that there may be more extensive postsynaptic associations through convergence at widely spaced sites on common neurons is discussed.     

Dendrodendritic synapses in the cat reticularis thalami nucleus: a structural basis for thalamic spindle synchronization

Dendrodentritic synapses have been found between the dendrites of reticularis thalami neurons. These synapses were symmetric and the presynaptic element contained pleomorphic vesicles. A few cases of reciprocal dendrodendritic synapses were also observed. Given the morphological features of the synapses and the well-established GABAergic nature of reticularis neurons it is concluded that reticularis cell dendrites form a local inhibitory network. The functional implications of this type of organization for the synchronization of spindle oscillations are discussed and a new hypothesis is proposed.     

Synaptic structure of the monoamine and peptide nerve terminals in the intermediolateral nucleus of the guinea pig thoracic spinal cord

Synaptic organization of the intermediolateral nucleus of the guinea pig thoracic spinal cord was examined with particular focus on monoamine- and peptide-containing nerve terminals. Axon varicosities having flat synaptic vesicles constituted 17% of all axons in the nucleus and formed exclusively symmetric synapses. Enkephalin-, substance P-, somatostatin-, 5-hydroxytryptamine-, and catecholamine-immunoreactive nerve terminals were densely distributed, while neurotensin, vasoactive intestinal polypeptide-, oxytocin-, and cholecystokinin-8-immunoreactive nerves were sparse in the nucleus. Coexistence of 5-hydroxytryptamine and enkephalin was demonstrated, and coexistence of somatostatin and enkephalin as well as somatostatin and 5-hydroxytryptamine in the same axons was also shown by serial semithin sections. Catecholamine axons labelled by 5-hydroxydopamine formed axodendritic and axosomatic synapses and made direct synaptic contacts on the preganglionic sympathetic neurons identified by retrograde transport of horseradish peroxidase. Direct synaptic contacts from enkephalin- and substance P-immunoreactive axons to preganglionic sympathetic neurons were also revealed. Enkephalin-, substance P-, and 5-hydroxytryptamine-immunoreactive axons formed axodendritic and axosomatic synapses. Catecholamine axon varicosities constituted 19% of all axon varicosities in the nucleus and 30% of them showed synaptic specializations in a sectional plane. Axon varicosities immunoreactive to enkephalin, 5-hydroxytryptamine, and substance P constituted approximately 35, 19, and 13% of all axon varicosities, respectively, while those with synaptic contacts made up 27, 30, and 26%, respectively, in a sectional plane. Enkephalin-, 5-hydroxytryptamine-, and noradrenaline-immunoreactive axons showed mainly symmetric synaptic contacts.     

The GABAergic neurons and axon terminals in the lateralis medialis-suprageniculate nuclear complex of the cat: GABA-immunocytochemical and WGA-HRP studies by light and electron microscopy

In order to get more detailed information on the neural circuit of the lateralis medialis-suprageniculate nuclear (LM-Sg) complex of the cat, the GABAergic innervation of this complex was studied by GABA immunohistochemical techniques. Small immunoreactive cells were found throughout the LM-Sg complex. On the basis of their ultrastructural features, these GABAergic cells were identified as Golgi type II interneurons. The neuropil of this nucleus displayed a conspicuous granular immunoreactivity. Ultrastructurally, the immunoreactive neural profiles in the neuropil were identified as the presynaptic dendrites of interneurons, myelinated axons, or axon terminals. The GABAergic dendritic profiles, containing pleomorphic synaptic vesicles, were involved in synaptic glomeruli. Additionally, GABAergic axon terminals containing pleomorphic synaptic vesicles formed symmetric axodendritic synaptic contacts mainly in the extraglomerular neuropil. They appeared to correspond to either axon terminals from the thalamic reticular nucleus (TRN) or the axon terminals of interneurons. The projections from the TRN to the LM-Sg complex were studied by using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Following injection of WGA-HRP into the LM-Sg complex, a number of retrogradely labeled cells were observed in the TRN. The connections between the TRN and the LM-Sg complex appeared to be topographically organized, the dorsal TRN being connected mainly with the dorsomedial portion of the LM-Sg complex, and the ventral TRN being connected chiefly with the ventrolateral portion of the LM-Sg complex. Following injection of the tracer into the TRN, ultrastructural examination of anterograde labeling in the LM-Sg complex revealed that labeled terminals contain pleomorphic vesicles and make symmetric synaptic contacts mainly with small to medium-sized dendrites. The labeled terminals were not involved in synaptic glomeruli. The present results provide anatomic support for the contention that the projection cells of the LM-Sg complex may be inhibited by both the TRN axons and interneurons, probably through the mediation of GABA.     

Ultrastructural morphology of axon terminals of an inhibitory spinal interneurone in the cat

Axon collateral terminals of an interneurone mediating Ia reciprocal inhibition of spinal α-motoneurones have been ultrastructurally investigated after intracellular staining with horseradish peroxidase. The size of bouton profiles, the size and shape of synaptic vesicles and the characteristics of synaptic contacts were analyzed in 38 terminals. The morphology of the terminals was compared with the morphology of previously investigated terminals of excitatory spinocervical tract cells. The flatness of synaptic vesicles was only marginally greater in the terminals of the inhibitory interneurone.     

Coexistence of GABA and glutamate in mossy fiber terminals of the primate hippocampus: an ultrastructural study.

One of the links in the trisynaptic circuit of the hippocampus is the synapse between the mossy fibre terminals of dentate granule cells and CA3 pyramidal cells of Ammon's horn. This synapse has been physiologically characterized as excitatory, and there is pharmacological and immunohistochemical evidence that mossy fibre terminals utilize glutamate as a neurotransmitter. This study demonstrates the presence of GABA-immunoreactivity in mossy fibre axons and terminals of the monkey at the electron microscopic level. We combined Golgi impregnation to identify CA3 pyramidal neurones, with postembedding immunocytochemistry to characterize the inputs to the identified cells. GABA immunoreactivity was present in mossy fibre terminals that made synaptic contact with complex embedded spines of identified Golgi-impregnated CA3 pyramidal neurones. GABA immunoreactivity could be demonstrated in serial sections of the same mossy fibre terminals by using 3 different antisera raised against GABA. In serial sections, the mossy fibre terminals were shown to be immunoreactive for both glutamate and GABA. In contrast, glutamate immunoreactivity but not GABA immunoreactivity was found in other terminals that did not have the morphological characteristics of mossy fibre terminals. GABA immunoreactivity in mossy fibre terminals was also demonstrated in a human surgical specimen of hippocampus. The coexistence of an "excitatory" amino acid and of an "inhibitory" amino acid in the same "excitatory" nerve terminal raises the possibility of corelease of the two transmitters, suggesting that the control of hippocampal neural activity is more complex than hitherto suspected.     

Immunocytochemical evidence for the existence of GABAergic neurons in the nucleus raphe dorsalis. possible existence of neurons containing serotonin and GABA

It has been established that nerve cell bodies of the nucleus raphe dorsalis (NRD) belong to ascending 5-hydroxytryptamine systems. These neurons could be modulated by GABAergic interneurons or interposed GABA neurons. A high glutamate decar☐ylase (GAD) activity in the NRD and a specific high-affinity uptake mechanism for GABA suggest the presence of GABA synthesizing elements in the NRD. Anti-GAD antibodies were used by an immunocytochemical procedure to demonstrate the presence of GABAergic elements. Anti-GAD antibodies were previously tested in the cerebellum and substantia nigra. Large amounts of GAD-positive reaction product were observed in the cytoplasm of some neurons (fusiform, ovoid or multipolar) or appeared as punctate deposits apposed to dendrites, soma and dispersed in the neuropil of the NRD. At the electron microscopic level, GAD-positive reaction product was observed within the cytoplasm of numerous somata in sections from colchicine-treated rats. GAD-positive staining was observed in numerous fibers or axonal terminals and two types of morphologically different fibers could be distinguished. The first displays small clear vesicles and few large granular vesicles (LGV) (80–100 nm), the second displays only clear round vesicles (40–60 nm). After 5,7-dihydroxytryptamine treatment (a neurotoxic for 5-HT terminals), the immunocytochemical labeling is much decreased. Some reactive neurons are still dispersed in the nucleus but the fibers containing LGV are no longer observed. These results strongly suggest that some neuronal elements in the NRD are morphologically, pharmacologically and anatomically similar to 5-HT neurons described at this level. Such cell elements could possess a double GABA and 5-HT potentiality. If this is not the case, a population of GABA neurons could be sensitive to 5,7-DHT and so have the capacity to take up 5-HT. The other reactive elements, insensitive to 5,7-DHT, could represent the GABAergic interneurons postulated at this level. Numerous GAD positive fibers or axon terminals were observed in synaptic contact with dendrites, axons or soma of other neurons. The chemical nature of the neuronal postsynaptic elements remains unknown. These findings strongly support the hypothesis for GABA-mediated inhibition in the NRD.