Electron microscopic study of GABAergic synaptic innervation of nitric oxide synthase immunoreactive neurons in the dorsal raphe nucleus in the rat

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 nitric oxide synthase containing neurons in the same tissue sections of the dorsal raphe nucleus of the Wistar white rat. Although a large number of immunogold stained GABAergic axon terminals were found to be presynaptic to dendrites containing nitric oxide synthase-like immunoreaction product, synapses between GABA-like immunoreactive axon terminals and nitric oxide synthase-like immunoreactive perikarya were rare. The labeled boutons were found to make symmetrical and asymmetrical synapses. No axo-axonic synapse was found. These results suggest that GABAergic neurons could modulate nitric oxide producing neurons in the dorsal raphe nucleus through direct synaptic relations. Synapse 25:24–29, 1997. © 1997 Wiley-Liss, Inc.     

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.     

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.     

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.     

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.     

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.     

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.     

Neurotoxic effects of p-chloroamphetamine on the serotoninergic innervation of the trigeminal motor nucleus: a retrograde transport study

The rat forebrain receives projections from both dorsal and median raphe nuclei. It has recently been shown that serotoninergic axons arising from the dorsal raphe nucleus, but not those from the median raphe nucleus, degenerate following systemic administration of p-chloroamphetamine (PCA). The present study was conducted to determine (i) whether the motor nucleus of the trigeminal nerve is innervated by overlapping projections from multiple serotonin cell groups and (ii) whether a particular subset of serotoninergic axon terminals in the trigeminal motor nucleus are sensitive to the neurotoxic effects of PCA. Retrograde transport was used in combination with immunofluorescence to identify the serotonin-positive cells that project to the trigeminal motor nucleus both in control rats and in rats previously treated with PCA. In untreated rats, an average of 95 retrogradely labeled serotonin-positive neurons were found in the dorsal raphe nucleus, 135 in the nucleus raphe obscurus, 132 in the nucleus raphe pallidus and 63 in the ventrolateral medulla. After treatment with PCA, there was a marked decrease (-77%) in the number of retrogradely labeled serotoninergic neurons in the dorsal raphe nucleus, whereas the number of labeled neurons was unchanged in the raphe obscurus and raphe pallidus. These results demonstrate that PCA selectively lesions serotonin axon terminals arising from the dorsal raphe nucleus, while sparing projections from the raphe obscurus and raphe pallidus to the trigeminal motor nucleus. This conclusion is in agreement with previous findings that in the forebrain only axons from the dorsal raphe are vulnerable to PCA. The data provide further evidence that serotoninergic axons originating in the dorsal raphe nucleus differ from other serotoninergic axons in their pharmacological properties and that the dorsal raphe may contain a functionally unique subset of serotonin neurons.     

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.     

Serotonergic retinopetal projections from the dorsal raphe nucleus in the mouse demonstrated by combined [(3)H] 5-HT retrograde tracing and immunolabeling of endogenous 5-HT

The present study demonstrated a direct serotonergic retinopetal projection in the mouse stemming from the lateral portion of the dorsal raphe nucleus bilaterally. A double-labeling technique was employed combining: (1) radioautography and retrograde axonal tracing following intraocular injection of [(3)H] 5-HT and (2) immunocytochemical identification of endogenous 5-HT. Radiolabeled neurons were only observed within the dorsal raphe nucleus and were always double-labeled with the 5-HT antibody. The radiolabeling appeared to be specific resulting from the retrograde transport of a radioactive 5-HT derivative product following uptake of the neurotransmitter by intraretinal terminals.     

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.     

Reciprocal synaptic relations between enkephalinergic and GABAergic neurons in the area postrema of the rat

A preembedding, double immunostaining technique was used to study synaptic relations between enkephalinergic and GABAergic neurons in the area postrema of the rat. As a main result, the enkephalinergic dendrites received many synapses from GABAergic axon terminals, and most of the synapses were symmetrical. Enkephalinergic neuronal perikarya received a few synapses from GABAergic axon terminals, and a few enkephalinergic axon terminals were found presynaptic to GABAergic neurons. Synapses between enkephalinergic profiles were frequent, but no axo-axonic synapses were seen. These findings suggest that GABAergic innervation of enkephalinergic neurons is the main relation between the two kinds of neurons in the area postrema. The synapses between the ankephalinergic axon terminals and GABAergic neurons might be explained as being part of the local servo system of the area postrema.     

An electron microscopic observation of the vesicular acetylcholine transporter-immunoreactive fibers in the rat dorsal raphe nucleus

By using immunocytochemistry with an antibody directed against the vesicular acetylcholine transporter, many cholinergic neuronal processes were found to be immunopositive in the dorsal raphe nucleus. At the electron microscopic level, most of these processes were found to be axons. The immunopositive axon terminals made synapses on immunonegative dendrites and their spines whereas rare synapses were found between the immunopositive axon terminals and the immunonegative neuronal perikarya. Occasionally, the dendrites postsynaptic to an immunopositive axon terminal also received a synapse from an immunonegative axon terminal. The synapses made by the immunopositive axon terminals were usually symmetric and had a short active zone. Fewer immunostained dendrites were found, and they usually received asymmetric synapses from nonimmunostained axon terminals. The existence of cholinergic axon terminals and the synapses made by these terminals support the physiological data indicating that acetylcholine plays a role in the pain inhibition system in the dorsal raphe nucleus.     

Origin of the serotonergic innervation to the rat dorsolateral hypothalamus: retrograde transport of cholera toxin and upregulation of tryptophan hydroxylase mRNA expression following selective nerve terminals lesion.

The regulation of serotonin synthesis was investigated in the serotonergic neurons, which provide afferents to the dorsolateral hypothalamus (DLH). The origin of the DLH projection neurons within the raphe nucleus was identified by retrograde transport of Cholera toxin (CTb) and their serotonergic nature confirmed by tryptophan hydroxylase (TPH) immunocytochemistry. Disruption of serotonin synthesis steady-state was induced unilaterally by a selective and local destruction of serotonergic nerve terminals with 5,7-dihydroxytryptamine (5,7-DHT), stereotaxically injected in the right DLH. The results show that most of the serotonergic dorsal raphe neurons projecting to the DLH have an ipsilateral localization within the lateral aspects of the nucleus. In rats with unilateral DLH lesion, a population of serotonergic cells within the raphe nucleus exhibited a clear increase in TPH mRNA. These cells were about five times more numerous in the ipsilateral as compared to the contralateral dorsal raphe nucleus and they had, for the most part, a lateral localization within the raphe nucleus. Sham-operated rats did not exhibit any upregulation of TPH mRNA. Together, the present results provide the first demonstration that a discreet and selective destruction of serotonergic terminals induces a circumscribed and striking increase in TPH mRNA expression in a subset of brainstem serotonergic neurons projecting to and/or passing through the DLH. On the basis of these results and previous in vivo measurements of TPH activity (e.g., 5-HT synthesis), we suggest that this upregulation in TPH mRNA expression results from the loss of pre-synaptic and/or post-synaptic regulation of serotonin synthesis. These new findings raise important issues related to the repercussions of a local disruption in serotonergic neurotransmission on brain areas remote from the site of injury.     

Serotonergic retinopetal projections from the dorsal raphe nucleus in the mouse demonstrated by combined [H] 5-HT retrograde tracing and immunolabeling of endogenous 5-HT

The present study demonstrated a direct serotonergic retinopetal projection in the mouse stemming from the lateral portion of the dorsal raphe nucleus bilaterally. A double-labeling technique was employed combining: (1) radioautography and retrograde axonal tracing following intraocular injection of [³H] 5-HT and (2) immunocytochemical identification of endogenous 5-HT. Radiolabeled neurons were only observed within the dorsal raphe nucleus and were always double-labeled with the 5-HT antibody. The radiolabeling appeared to be specific resulting from the retrograde transport of a radioactive 5-HT derivative product following uptake of the neurotransmitter by intraretinal terminals.     

Cellular and subcellular distribution of substance P receptor immunoreactivity in the dorsal vagal complex of the rat and cat: A light and electron microscope study

Immunoreactivity for the substance P receptor (NK1 receptor) has been investigated by light and electron microscopy in the dorsal vagal complexes of adult rats and cats. The general pattern of NK1 immunoreactivity was similar for both rat and cat. Numerous NK1-immunoreactive neurons were present in the area postrema, the nucleus of the solitary tract, and the dorsal motor nucleus of the vagus nerve. The density of labelled neurons differed between the subnuclei of the nucleus of the solitary tract. Overall, the efferent neurons of the dorsal motor nucleus of the vagus nerve highly expressed NK1 when compared to neurons in the nucleus of the solitary tract. The results are discussed with reference to the viscerotopic organisation of the dorsal vagal complex. Ultrastructural analysis demonstrated that NK1 immunoreactivity was present only at the membrane surface of somatic and dendritic profiles of neurons. No labelling was found in axon terminals, axons, or glial processes. NK1 immunoreactivity, as revealed by a preembedding immunogold technique in serial ultrathin sections, was preferentially located at nonsynaptic sites. A semiquantitative study suggested that the density of NK1 receptors is statistically higher at membrane sites free of any contact (synaptic or not) with axon terminals. The subcellular localisation of NK1 immunoreactivity was similar for neurons of both rat and cat. These results suggest that in the dorsal vagal complex, substance P might act on NK1 receptors through a process of volume transmission. J. Comp. Neurol. 402:181–196, 1998. © 1998 Wiley-Liss, Inc.     

大鼠腰骶髓后连合核内5—HT样阳性纤维和终末的来源

采用荧光金（ＦＧ）逆行追踪与免疫荧光组织化学染色相结合技术，对大鼠腰骶髓后连合核（ＤＣＮ）内５－羟色胺（５－ＨＴ）能纤维和终末的分布及其来源进行了观察。结果显示ＤＣＮ内分布有大量５－ＨＴ阳性纤维和终末。将ＦＧ注入到ＤＣＮ后，ＦＧ／５－ＨＴ双标细胞出现于延髓中缝核群中的中缝大核、中缝隐核和中缝苍白核以及外侧旁巨细胞核的腹侧部。结果提示５－ＨＴ对ＤＣＮ内盆内脏感觉信息的传递可能具有调控作用。     

Serotonergic dorsal raphe nucleus projections to the cholinergic and noncholinergic neurons of the pedunculopontine tegmental region: a light and electron microscopic anterograde tracing and immunohistochemical study

The serotonergic dorsal raphe nucleus is considered an important modulator of state-dependent neural activity via projections to cholinergic neurons of the pedunculopontine tegmental nucleus (PPT). Light and electron microscopic analysis of anterogradely transported biotinylated dextran, combined with choline acetyltransferase (ChAT) immunohistochemistry, were employed to describe the synaptic organization of mesopontine projections from the dorsal raphe to the PPT. In a separate set of experiments, we utilized immunohistochemistry for the serotonin transporter (SERT), combined with ChAT immunohistochemistry at the light and electron microscopic levels, to determine whether PPT neurons receive serotonergic innervation. The results of these studies indicate that: (1) anterogradely labeled and SERT-immunoreactive axons and presumptive boutons invest the PPT at the light microscopic level; (2) at the ultrastructural level, dorsal raphe terminals in the PPT pars compacta synapse mainly with dendrites and axosomatic contacts were not observed; (3) approximately 12% of dorsal raphe terminals synapse with ChAT-immunoreactive dendrites; and (4) at least 2-4% of the total synaptic input to ChAT-immunoreactive dendrites is of dorsal raphe and/or serotonergic origin. This serotonergic dorsal raphe innervation may modulate cholinergic PPT neurons during alterations in behavioral state. The role of these projections in the initiation of rapid eye movement (REM) sleep and the ponto-geniculo-occipital waves that precede and accompany REM sleep is discussed. J. Comp. Neurol. 382:302-322, 1997. © 1997 Wiley-Liss, Inc.     

Neurotensin Excitation of Serotonergic Neurons in the Dorsal Raphe Nucleus of the Rat In Vitro

Neurotensin-containing terminals and radioligand binding sites are present in the dorsal raphe nucleus. The purpose of this study was to test, in brain slices containing this nucleus, the effect of neurotensin on the electrical activity of serotonergic neurons. In extracellular recordings, the cells were identified by the ability of the α₁-adrenoceptor agonist phenylephrine to induce firing, and serotonin to reduce this effect. After washout of phenylephrine, neurotensin (10 nM to 10 μM) induced a concentration-dependent increase in the firing rate of serotonergic neurons (EC₅₀= 142 nM; maximum effect ˜1 μM). The neurotensin excitation, which was mimicked by neurotensin fragments 8–13 but not neurotensin peptide fragment 1–8 and selectively blocked by SR 48692 (100 nM), was observed mainly in the ventral part of the nucleus. Most serotonergic neurons showed marked desensitization to neurotensin, even at low concentrations. The neurotensin response was occluded by supramaximal concentrations of phenylephrine. In intracellular recordings using KCl-containing electrodes, neurotensin induced an inward current associated in some cases with a decrease in apparent input conductance. In conclusion, neurotensin was found to have an excitatory action on serotonergic neurons in the ventral part of the dorsal raphe nucleus, an effect which could be subject to desensitization and was occluded by phenylephrine. This occlusion phenomenon may be important for the physiological role of neurotensin in the dorsal raphe nucleus.     

Phenylethanolamine N-methyltransferase-containing neurons in the rostral ventrolateral medulla. II. Synaptic relationships with GABAergic terminals

The ultrastructural morphology of terminals synthesizing gamma-aminobutyric acid (GABA), as indicated by peroxidase immunoreactivity for its synthetic enzyme L-glutamate decarboxylase (GAD), was examined in the rostral ventrolateral medulla (RVL) of the adult rat brain. The objective of the study was to determine the types of synaptic associations between the GABAergic terminals and other neurons in the RVL, particularly the C1-adrenergic neurons containing phenylethanolamine N-methyltransferase (PNMT). The brains were fixed by perfusion with 3.75% acrolein and 2.0% paraformaldehyde in phosphate buffer. Coronal Vibratome sections through the RVL were singly labeled with a sheep antiserum to GAD using the peroxidase-antiperoxidase (PAP) method. Additional sections were dually labeled using the PAP technique for the GAD antiserum and immunogold labeling for a rabbit antiserum against PNMT. Ultrastructural analysis revealed that peroxidase labeling for GAD was localized primarily to axons and axon terminals in both single and dual labeled material. The axons were small and unmyelinated. The GAD-labeled terminals were 0.5-2.0 microns in diameter and contained a large population of small clear vesicles usually associated with a few mitochondria. These terminals formed synapses with many dendrites, a few nerve cell bodies and axon terminals. The junctions were all symmetric and the postsynaptic structures failed to exhibit immunoreactivity when processed only for GAD labeling. In sections incubated with both GAD and PNMT antisera, the peroxidase-labeled GABAergic terminals formed symmetric synapses with nerve cell bodies and dendrites showing immunogold labeling for PNMT. In addition, the GAD-labeled terminals were presynaptic to other dendrites which appeared to have equal access to the antisera and gold markers, but failed to exhibit detectable immunoreactivity for PNMT. Both the PNMT-labeled and unlabeled somata and dendrites also received symmetric and asymmetric contacts from terminals containing neither GAD nor PNMT-immunoreactivity. We conclude that GABA is at least one of the inhibitory transmitters regulating adrenergic as well as non-adrenergic outflow from the RVL.