Distribution and synaptic relations of NOS neurons in the dorsal raphe nucleus: A comparison to 5-HT neurons

Anti-nitric oxide synthase antibody was used to study the distribution, cytowchhecture, and synaptic relations of nitric oxide synthase-like immunoreactive neurons in the whole rostral-caudel length of the dorsal raphe nucleus of the rat and compared them with serotonergic neurons. Results showed that the distribution of the nitric oxide synthase in the dorsal raphe nucleus was similar to that of the serotonergic neurons at the rostral part of the dorsal raphe nucleus, including the mediodorsal and the medioventral cell groups, and changed at the middle and caudal parts of the dorsal raphe nucleus. The cytoarchitecture of the nitric oxide synthase-like immunoreactive neurons in the medioventrai cell group of the dorsal raphe nucleus was similar to that of the serotonergic neurons. Similar to the serotonergic neurons there, nitric oxide synthase-like immunoroactive neurons also received synapses from axon terminals that contained round, or flattened vesicles, or both kinds. Different to the serotonergic neurons, the few nitric oxide synthase-like immunoroactive axon terminals that were in this area formed synapses.     

Electron microscopic study of GABAergic synaptic innervation of neurotensin-immunoreactive neurons in the dorsal raphe nucleus

A double immunocytochemical method combining the preembedding avidin-biotin-peroxidase-complex technique and the postembedding immunogold technique was used to examine synaptic interactions between GABAergic and neurotensin-containing neurons in the same tissue sections of the dorsal raphe nucleus of the rat. Whereas the neurotensin-like immunoreactive perikarya rarely received synapses from GABA-like immunostaining axon terminals, the neurotensin-like immunoreactive dendrites frequently received synapses from GABA-like immunoreactive neurons. These results suggest that GABAergic neurons could modulate neurotensinergic neurons in the dorsal raphe nucleus through synaptic relations. The immunocytochemically identified local synaptic circuit in the dorsal raphe was discussed.     

GABAergic innervation of serotonergic neurons in the dorsal raphe nucleus of the rat studied by electron microscopy double immunostaining.

A double immunocytochemical method combining the preembedding PAP technique and the postembedding immunogold technique was used to examine interactions between GABAergic and serotonergic neurons in the same tissue sections of the dorsal raphe nucleus of the rat. A large number of immunogold stained GABAergic axon terminals were found to be presynaptic to strongly PAP immunostained serotonergic perikarya and dendrites. The types of synapses were mostly symmetrical although a few asymmetrical ones were also found. No axo-axonic synapse between the GABAergic axon terminals and the serotonergic neuronal profiles was found. These results suggest that GABAergic neurons could modulate serotonergic neurons in the dorsal raphe nucleus through synaptic relations.     

Immunoelectron microscopic study of beta-endorphinergic synaptic innervation of GABAergic neurons in the dorsal raphe nucleus.

Using a preembedding double immunoreactive technique by immunostaining with antirat beta-endorphin and antisynthetic glutamic acid decarboxylase antisera sequentially, the synaptic relationships between beta-endorphinergic neuronal fibers and GABAergic neurons in the dorsal raphe nucleus of the rat were examined at the ultrastructural level. Although both beta-endorphin-like immunoreactive fibers and glutamic acid decarboxylase-like immunoreactive neurons can be found in the mediodorsal and medioventral parts of the dorsal raphe nucleus, the synapses between them were found only in the mediodorsal part. Most of the beta-endorphin-like immunoreactive neuronal fibers contained many dense-cored vesicles. The synapses made by beta-endorphin-like immunoreactive neuronal axon terminals on glutamic acid decarboxylase-like immunoreactive neurons were both symmetrical and asymmetrical, with the latter predominant, especially in the axo-dendritic synapses. Perikarya with beta-endorphin-like immunoreactivity were found only in the ventrobasal hypothalamus. These findings suggest the possibility that the beta-endorphin-producing neurons in the ventrobasal hypothalamus could influence GABAergic neurons in the dorsal raphe nucleus directly by synaptic relationships.     

Enkephalinergic innervation of GABAergic neurons in the dorsal raphe nucleus of the rat

The preembedding double immunoreaction method was used to study interrelations of enkephalinergic and GABAergic neuronal elements in the dorsal raphe nucleus of the Wistar albino rat. The enkephalin-like neuronal elements were immunoreacted by the peroxidase-antiperoxidase method and silver-gold intensified, which showed strongly and was specific. The GABA-like immunoreactive neurons were immunoreacted by the peroxidase-antiperoxidase method only. GABA-like neural somata were postsynaptic to both the enkephalin-like immunoreactive and the non-immunoreactive axon terminals. The enkephalin-like immunoreactive axon terminals were also found to synapse GABA-like immunoreactive dendrites. The GABA-like immunoreactive neuronal elements were also found to receive synapses from other non-immunoreactive as well as GABA-like immunoreactive axon terminals. Almost all of the synapses appeared to be asymmetrical. Possible functional activity of interactions among the enkephalinergic, GABAergic, and serotonergic neuronal elements in the dorsal raphe nucleus are discussed.     

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.     

Catecholaminergic, GABAergic, and hippocamposeptal innervation of GABAergic "somatospiny" neurons in the rat lateral septal area

This study deals with the neurochemical characterization of the rat lateral septal area (LSA) somatospiny neurons and their innervation by hippocamposeptal, catecholaminergic, and GABAergic fibers. Electron microscopic single and double immunostaining methods were used to label catecholaminergic fibers and GABAergic cells and boutons. Axon terminals originating in the hippocampus were labeled by acute anterograde axon degeneration induced by fimbria-fornix transection 36 hours before sacrifice. Three types of experiments were performed. The convergent catecholaminergic and hippocamposeptal innervation of LSA somatospiny neurons was studied by combining immunostaining for tyrosine hydroxylase (TH) with fimbria-fornix transection. GABAergic neurons and their hippocamposeptal afferents were identified and characterized in colchicine pretreated animals immunostained for glutamic acid decarboxylase (GAD) combined with fimbria-fornix transection. The third experiment aimed at simultaneously visualizing the relationships between catecholaminergic boutons, hippocamposeptal excitatory amino acid containing axon terminals and GABAergic profiles by double immunostaining for TH (the PAP technique) and GAD (the immunogold method) combined with fimbria-fornix transection. The results are summarized as follows: 1) The same LSA somatospiny neurons receive synaptic inputs from the hippocampus and TH immunoreactive fibers which form pericellular baskets around these cells. 2) LSA somatospiny neurons are GABAergic and are postsynaptic targets of GABAergic boutons with unknown origin and hippocamposeptal axon terminals. 3) The double immunostaining experiment, finally, provided direct evidence that the same GABAergic somatospiny neurons are postsynaptic targets of both catecholaminergic and hippocamposeptal afferents. The synaptic interconnections described in this study provide anatomical basis for a better understanding of the action of catecholamines, excitatory amino acids, and GABA on the activity of LSA neurons.     

Contribution of brainstem GABAergic circuitry to descending antinociceptive controls: II. Electron microscopic immunocytochemical evidence of GABAergic control over the projection from the periaqueductal gray to the nucleus raphe magnus in the rat

Pharmacological, physiological, and behavioral studies suggest that inhibitory GABAergic neurons influence the projection from the midbrain periaqueductal gray matter to the medullary nucleus raphe magnus. The present study used electron microscopic immunocytochemical techniques to examine the morphology and synaptic relationships of GABA-immunoreactive terminals in the ventrolateral periaqueductal gray. These putative GABAergic terminals comprise almost 40% of all axon terminals in the periaqueductal gray. GABA-immunoreactive terminals contain small, clear, pleomorphic or round, vesicles, and 46% also contain some dense-cored vesicles. In some experiments we also used a colloidal gold-conjugated retrograde tracer to label periaqueductal gray neurons that project to the nucleus raphe magnus. About half of the synaptic inputs onto the cell bodies and proximal dendrites of retrogradely labeled neurons are GABA-immunoreactive; these putative GABAergic synapses, which directly control activity in neurons projecting from the periaqueductal gray to the nucleus raphe magnus, might mediate the antinociception-related effects of exogenous GABAA receptor ligands.     

Serotonergic synaptic input to cholinergic neurons in the rat mesopontine tegmentum

Serotonergic synaptic inputs to cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei were examined with pre-embedding dual-label immunoelectron microscopy. Numerous serotonin-immunoreactive axon terminals visualized with a silver-enhanced immunogold method were present in both of these tegmental nuclei. Serotonergic terminals occasionally made synaptic contacts with the soma and proximal dendrites of cholinergic tegmental neurons labelled with a choline acetyltransferase-immunoreactive peroxidase-anti-peroxidase diaminobenzidine reaction product. In the rostralmost region of the laterodorsal tegmental nucleus, a few serotonergic neurons of the dorsal raphe nucleus were interspersed among cholinergic neurons. Some dendrites of these serotonergic neurons appeared to contain synaptic vesicles. Both myelinated and unmyelinated serotonergic axons were present in the mesopontine tegmentum. The presence of serotonergic synapses onto tegmental cholinergic neurons is consistent with previous behavioral and electrophysiological findings suggesting an inhibitory role of serotonin in the induction of rapid eye movement sleep and its phenomenology through an action on cholinergic neurons in the mesopontine tegmentum.     

Synaptic relationship of the neurons containing a metabotropic glutamate receptor, MGluR5, with nociceptive primary afferent and GABAergic terminals in rat spinal superficial laminae

Recent pharmacological evidence showed that metabotropic glutamate receptors (mGluRs), particularly mGluRs1/5, had a potential role in spinal nociceptive processing. However, previous morphological studies on mGluRs have been limited mainly to their distribution in the spinal cord. In the present study, electron microscopic immunocytochemistry was employed to identify the synaptic relationship of the neurons containing mGluR5, with nociceptive primary afferent and gamma-aminobutyric acid-ergic (GABAergic) terminals in the superficial dorsal horn of the spinal cord. Nociceptive C- and A(delta)-primary afferent terminals selectively labeled with horseradish peroxidase conjugated to wheat-germ agglutinin were in asymmetric synaptic contacts with or in direct apposition to mGluR5 positive dendritic profiles. The double-labeling studies revealed that mGluR5 immunoreactive dendrites also received symmetric synaptic contacts from axon terminals labeled with immunogold particles indicating GABA. The present demonstration of mGluR5 neurons receiving inputs from both nociceptive primary afferents and GABAergic terminals of presumed interneurons further supports the involvement of mGluR5 in the transmission and modulation of nociceptive information in the spinal cord.     

Orexinergic innervation of POMC-containing neurons in the rat arcuate nucleus

A pre-embedding double immunostaining technique was used to study the synaptic relationships between orexin-like immunoreactive axon terminals and preopiomelanocortin (POMC)-like immunoreactive neurons in the rat arcuate nucleus. Most of the synapses were axo-dendritic, while some axo-somatic synapses were also found. Both the axo-somatic and axodendritic synapses were symmetrical. In some cases the presynaptic orexin-like immunoreactive axon terminals contained a few large dense-cored vesicles. The results suggest that the orexinergic axon terminals in the arcuate nucleus may play an important role in the regulation of food intake via synapses through POMC neurons.     

Synaptic interactions between GABAergic neurons and trigeminothalamic cells in the rat trigeminal nucleus caudalis.

Synaptic interactions between GABAergic neurons and thalamic projecting cells within the trigeminal nucleus caudalis were examined using a combined method of GABA immunohistochemistry and retrograde WGA-HRP labeling of the trigeminothalamic pathway. Results showed that GABA-positive neurons and projecting cells were separate but closely intermingled within the spinal trigeminal nucleus. GABAergic axon terminals formed symmetric synaptic connections with the cell bodies and dendrites of trigeminothalamic neurons. In turn, some WGA-HRP filled axon terminals, presumed to originate from axon collaterals of the projecting neurons, formed synaptic connections with GABA containing neurons. The results suggest that in the spinal trigeminal nucleus there is a reciprocal modulation between GABA neurons and trigeminothalamic cells.     

Localization of NADPH diaphorase activity in monoaminergic neurons of the rat brain

Nitric oxide has recently been implicated as a neurotransmitter, and may modulate synaptic transmission, cerebral blood flow, and neurotoxicity. NADPH diaphorase histochemistry has been shown to be a reliable marker for nitric oxide synthase, the enzyme that synthesizes nitric oxide, in the nervous system. Because monoaminergic neurons frequently contain co-transmitters, we examined whether these cells also exhibit NADPH diaphorase activity. Frozen sections from postnatal and adult rat brains were stained for NADPH diaphorase activity and either serotonin-like immunoreactivity or tyrosine hydroxylase-like immunoreactivity. Numerous neurons in the mesopontine serotoninergic cell groups (including the caudal linear, dorsal, median, supralemniscal, and pontine raphe nuclei) contained both serotonin-like immunoreactivity and NADPH diaphorase activity. Within the dorsal raphe nucleus, approximately 70% of the serotoninergic neurons in the medial subnuclei displayed NADPH diaphorase activity, while less than 10% of the serotoninergic neurons in the lateral subnuclei were doubly labeled. Retrograde labeling with fluorescent microspheres indicated that many raphe-cortical neurons contained NADPH diaphorase activity. No NADPH diaphorase activity was detected in serotoninergic neurons in the medullary nuclei (including the raphe magnus, raphe pallidum, and raphe obscurus). Only a small proportion of tyrosine hydroxylase-like immunoreactive neurons in the periaqueductal gray, rostral linear nucleus, and rostrtrodorsal ventral tegmental area contained NADPH diaphorase activity. Tyrosine hydroxylase-like immunoreactive neurons in the substantia nigra, locus coeruleus, hypothalamus, olfactory bulb, and dorsal raphe nucleus did not contain detectable NADPH diaphorase activity. The observation that many mesopontine (but not medullary) serotoninergic neurons contain NADPH diaphorase activity suggests that these neurons may release both serotonin and nitric oxide. © Wiley-Liss, Inc.     

Ultrastructural localization of delta-1 opioid receptor in the dorsal raphe nucleus of the rat

The ultrastructural localization of delta-1 opioid receptor in the rat dorsal raphe nucleus was studied by the preembedding avidin-biotin-peroxidase complex technique. With application of a low concentration of the first antiserum in incubation and control of short-time reaction to 3,3′-diaminobenzidine, the immunoreaction seemed to be faint at the light microscopic level. At the electron microscopic level, however, delta-1 opioid receptor immunoreaction products were found to be localized specifically on the postsynaptic membrane of dendrites, dense-cored vesicles, and the surface of the small, clear vesicles in axon terminals with strong immunoreactivity. Of the total 659 immunopositive profiles observed, up to 62.4% (411/659) were dendrites, whereas 33.8% (223/659) were axon terminals. The immunostained myelinated axons and perikarya were relatively rare, with the frequencies 0.8% (5/659) and 3.0% (20/659), respectively. Most of the immunopositive dendrites (338/411, 82.2%) were immunostained only at the postsynaptic membranes. Other immunoreactive dendrites showed their immunoreaction products also in some other contents besides the postsynaptic membranes (44/411, 10.7%) or only in those contents but not the postsynaptic membranes (25/411, 6.1%). Only four dendrites showed their immunoreactive results only at the membrane not related to synapse (4/267, 1.0%). No dendrite was found immunostained in all the contents. About half of the immunopositive axon terminals (125/223, 56.1%) were found to make synapse with nonimmunoreactive dendrites (76/223, 34.1%) or immunoreactive dendrites (49/223, 22.0%), while only one was found to make contact with immunoreactive perikarya. The present study showed that delta-1 opioid receptor in the dorsal raphe nucleus is mostly localized on postsynaptic membrane; the main function of the delta-1 receptor in the dorsal raphe nucleus is to receive signals from the opioid-containing axon terminals through synapses. Synapse 26:243–253, 1997. © 1997 Wiley-Liss Inc.     

Ultrastructural Relationships Between GABAergic Terminals and Cardiac Vagal Preganglionic Motoneurons and Vagal Afferents in the Cat: A Combined HRP Tracing and Immunogold Labelling Study

The ultrastructural relationships between gamma-aminobutyric acid-immunoreactive (GABA-ir) neurons and other neurons of the nucleus tractus solitarius (NTS) and motoneurons of the nucleus ambiguus (NA) and dorsal motor vagal nucleus (DMVN), were examined by electron microscopic (EM) immunogold labelling with an anti-GABA antiserum on brain stem sections in which vagal motoneurons and vagal afferent fibres were labelled with horseradish peroxidase (HRP). HRP was applied to the cervical vagus or the cardiac vagal branch of anaesthetized cats. After 24 - 48 h survival, brains were glutaraldehyde-fixed and a stable HRP-tetramethylbenzidine reaction product compatible with EM processing was revealed on 250 microm vibratome sections. Following osmium postfixation, dehydration and resin embedding, GABA-ir was localized on ultrathin sections by an immunogold technique. GABA-ir axon terminals, heavily and specifically labelled with gold particles, were very numerous within NTS, DMVN and NA. All terminals contained small, clear, pleomorphic vesicles and a few also contained larger dense cored vesicles. The density of gold particles over clear vesicles, dense cored vesicles and mitochondria was significantly greater than over the cytoplasm of these terminals. GABA-ir synapses were found on the soma and dendrites of neurons, but rarely on other axon terminals within NTS, where GABA-ir cell bodies and dendrites were also seen. These received synaptic contacts from both GABA-ir terminals and from HRP-labelled vagal afferents. In both the DMVN and NA, similar GABA-ir synapses were present on both the soma and dendrites of HRP-labelled motoneurons. GABA synapses were also present on other cell types in DMVN. These observations provide an anatomical basis for a GABAergic inhibition of neurons forming the central pathways of cardiovascular and other autonomic reflexes.     

Serotonin-containing structures in the nucleus raphe dorsalis of the cat: an ultrastructural analysis of dendrites, presynaptic dendrites, and axon terminals

In the nucleus raphe dorsalis of the cat, an electron microscopic immunocytochemistry method was used to identify the fine structure of serotoninergic dendritic profiles and axon terminals analyzed in serial sections. Two classes of serotoninergic dendrites were distinguished in the nucleus. The first class was constituted by conventional serotonin (5-HT) dendrites that were contacted by unlabeled axon terminals containing differing populations of synaptic vesicles. The second class consisted of serotoninergic dendrites that contained vesicles in their dendritic shafts. Such 5-HT dendrites were further subdivided into two groups according to their synaptic contacts. In some 5-HT vesicle-containing dendrites, the vesicles were densely packed in small clusters and were associated with a well-defined synaptic specialization. These dendrites were classified as serotoninergic presynaptic dendrites and established synaptic contacts with unlabeled and labeled dendrites and were contacted by unlabeled axon terminals. In other 5-HT vesicle-containing dendrites, extensive serial section examination showed that the vesicles could be observed near the membrane but were never found to be associated with any synaptic membrane specialization. Serotoninergic axon terminals that were presumed to be recurrent collaterals of 5-HT neurons were present in the nucleus. Some of them were observed in synaptic contact with dendrites or dendritic protrusions whereas others did not exhibit synaptic specializations. The existence of serotoninergic dendrodendritic synaptic contacts and axon terminals suggests direct local interactions between serotoninergic neurons within the nucleus raphe dorsalis.     

Glycine-immunoreactive synaptic terminals in the nucleus tractus solitarii of the cat: ultrastructure and relationship to GABA-immunoreactive terminals.

Postembedding immunogold labeling methods applied to ultrathin and semithin sections of cat dorsomedial medulla showed that neuronal perikarya, dendrites, myelinated and nonmyelinated axons, and axon terminals in the nucleus tractus solitarii contain glycine immunoreactivity. Light microscopic observations on semithin sections revealed that these immunoreactive structures were unevenly distributed throughout the entire nucleus. At the electron microscopic level, synaptic terminals with high levels of glycine-immunoreactivity, assumed to represent those releasing glycine as a neurotransmitter, were discriminated from terminals containing low, probably metabolic levels of glycine-immunoreactivity, by a quantitative analysis method. This compared the immunolabeling of randomly sampled terminals with a reference level of labeling derived from sampling the perikarya of dorsal vagal neurones. The vast majority of these "glycinergic" terminals contained pleomorphic vesicles, formed symmetrical synaptic active zones, and targeted dendrites. They appeared to be more numerous in areas of the nucleus tractus solitarii adjoining the tractus solitarius, but rather scarce caudally, medially, ventrally, and in the dorsal motor vagal nucleus. In a random analysis of the entire nucleus tractus solitarii, 26.2% of sampled terminals were found to qualify as glycine-immunoreactive. In contrast, boutons immunoreactive for gamma-aminobutyric acid (GABA) were more evenly distributed throughout the dorsal vagal complex and accounted for 33.7% of the synaptic terminals sampled. A comparison of serial ultrathin sections suggested three subpopulations of synaptic terminals: one containing high levels of both GABA- and glycine-immunoreactivities (21% of all terminals sampled), one containing only GABA-immunoreactivity (12.7%), and relatively few terminals (5.2%) that were immunoreactive for glycine alone. These results were confirmed by dual labeling of sections using gold particles of different sizes. This study reports the first analysis of the ultrastructure of glycinergic nerve terminals in the cat dorsal vagal complex, and the pattern of coexistence of glycine and GABA observed provides an anatomical explanation for our previously reported inhibitory effects of glycine and GABA on neurones with cardiovascular and respiratory functions in the nucleus tractus solitarii.     

Ultrastructure of glutamate and GABA immunoreactive axon terminals of the rat nucleus tractus solitarius, with a note on infralimbic cortex afferents

The principal fast neurotransmitters in the CNS are glutamate and GABA. Our aim was to provide a baseline account on the ultrastructure of the axon terminals immunoreactive to glutamate or GABA present in the nucleus tractus solitarius (NTS) of the rat. In addition, we wanted to complete our study of cortico-solitary afferents at the electron microscopic level, by analyzing the inputs from the infralimbic cortex. Using post-embedding immunogold, we found that nearly 61% of the axon terminals were glutamatergic, and 36% were GABAergic in the rat visceral NTS. In general, axons making asymmetric synaptic contacts were enriched in glutamate, compared to axons involved in symmetric synapses. In contrast, the vast majority of the GABAergic axon terminals made symmetric synaptic contacts. We could discern five types of glutamatergic and two types of GABAergic axon terminals that differed in their fine structure. Afferents from the infralimbic cortex were small, with clear synaptic vesicles and no dense core vesicles; they made asymmetric contacts with fine dendrites, and were glutamatergic. We conclude that most axon terminals in the NTS use glutamate or GABA as fast transmitters, in addition to being a heterogeneous population of morphological types.     

TRH axon terminals in synapsis with GRF neurons in the arcuate nucleus of the rat hypothalamus as revealed by double labeling immunocytochemistry

Synaptic input to neurons containing growth hormone-releasing factor (GRF) by axon terminals containing thyrotropin-releasing hormone (TRH) in the arcuate nucleus (AN) of the rat hypothalamus was examined using a method combining pre-embedding peroxidase-anti-peroxidase for GRF with postembedding immunocolloidal gold staining for TRH. The TRH-like immunoreactive axon terminals were found to make synaptic contact with GRF-like immunoreactive neurons with unlabeled axon terminals. From these findings, TRH-containing neurons in the hypothalamic AN of the rat may be considered to innervate GRF neurons, to regulate GRF secretion or to have some other functions via synapses.     

Evidence for a GABAergic interface between cortical afferents and brainstem projection neurons in the rat central extended amygdala

The synaptic circuitry of the intrinsic GABAergic system of the central extended amygdala (CEA) in relation to efferent neurons and cortical afferents was examined in the present study. Neurons in the CEA projecting to the dorsal vagal complex and the parabrachial complex were identified by the retrograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Postembedding GABA-immunocytochemistry revealed that GABA-immunoreactive (GABA-IR) terminals formed largely symmetrical synaptic contacts with the perikarya and proximal dendritic processes of almost all WGA-HRP-labeled neurons in the CEA. To determine the relationship between cortical afferents and CEA GABAergic neurons, WGA-HRP was used to anterogradely label afferents from the insular cortex in combination with postembedding immunogold detection of GABA. Cortical afferents formed asymmetrical synaptic contacts predominantly on small dendrites and dendritic spines. Many of the dendrites postsynaptic to cortical terminals in the central nucleus were immunoreactive for GABA although only relatively few spines were GABA-IR. Combining pre-embedding GAD-immunocytochemistry with cortical lesions resulted in approximately 40% of degenerating terminals of insular cortical origin in the central nucleus in contact with small, GAD-IR dendrites and spines. The present results demonstrate that the neurons providing the major CEA outputs to the brainstem receive an extensive GABAergic innervation, strongly supporting our proposal that CEA efferent neurons are under strong tonic inhibition by intrinsic GABAergic neurons. Further, our finding that the major cortical input to the central nucleus preferentially innervates intrinsic GABAergic neurons suggests that these neurons in the CEA may serve as an interface between the principal inputs and outputs of this forebrain region. © Wiley-Liss, Inc.