Synaptic interaction of vagal afferents and catecholaminergic neurons in the rat nucleus tractus solitarius

Combined radioautography and immunocytochemistry were used to define the ultrastructure and synaptic relations between vagal sensory afferents and catecholaminergic (CA) neurons of the A2 group located within the nucleus tractus solitarius (NTS) of rat brain. The vagal afferents were radioautographically labeled by tritiated amino acids anterogradely transported from the nodose ganglion. Immunocytochemical labeling for tyrosine hydroxylase (TH) served for the identification of catecholaminergic neurons. The radiographically labeled axons seen by light microscopy were widely distributed throughout the more caudal NTS. The reduced silver grains were more densely distributed within the NTS located homolateral to the injected nodose ganglion. The radioautographically labeled processes were localized in regions containing catecholaminergic neurons as indicated by immunoreactivity for TH. Electron microscopic analysis of the medial NTS at the level of the obex demonstrated that the reduced silver grains were localized within axon terminals. The radioautographically labeled terminals were 2-3 microns in diameter, contained numerous small, clear and a few large, dense vesicles, and formed predominately axodendritic synapses. Many of the recipient dendrites contained immunoreactivity for TH. In rare instances, vagal afferents formed synaptic appositions with both TH-labeled and unlabeled axon terminals and neuronal soma. This study provides the first ultrastructural evidence that the catecholaminergic neurons within the NTS receive direct synapses from sensory neurons in the nodose ganglion.     

Relationship of Met-enkephalin-like immunoreactivity to vagal afferents and motor dendrites in the nucleus of the solitary tract: a light and electron microscopic dual labeling study

Methionine (Met⁵)-enkephalin has been implicated in autonomic functions involving vagal reflexes within the nucleus of the solitary tract (NTS). We examined the light and electron microscopic relationships between neurons containing methionine (Met⁵)-enkephalin-like immunoreactivity (MELI) and vagal afferents and motor dendrites in the rat NTS. A polyclonal antibody raised against Met⁵-enkephalin and showing maximal cross-reactivity with this peptide was localized by immunoautoradiography. In the same sections, vagal afferents and motor neurons were identified by histochemical detection of anterogradely and retrogradely transported horseradish peroxidase (HRP). By light microscopy, the MELI was detected in perikarya distributed principally in the dorsomedial, intermediate and parasolitary subdivisions of the NTS. These subnuclei as well as medial and commissural divisions of the NTS also showed: (1) aggregates of silver grains thought to overlie terminals containing MELI, and (2) anterogradely transported HRP in varicose processes. Electron microscopic analysis of the dorsomedial NTS at the level of the area postrema established that MELI was detectable in perikarya, dendrites, and axon terminals. Most of the MELI was associated with large dense core vesicles (dcvs). These opioid terminals formed primarily symmetric synapses on proximal and asymmetric synapses on distal dendrites. Analysis of the dendritic targets of terminals containing MELI revealed that 13/222 were in synaptic contact with dendrites also containing MELI. The remainder of the terminals containing MELI either lacked recognized junctions or formed synapses with unlabeled dendrites. In comparison to the terminals containing MELI in the same series of sections, anterogradely labeled vagal terminals extensively formed asymmetric junctions with distal dendrites and spines. Of the observed anterogradely labeled terminals 6/84 formed synapses with dendrites containing MELI and 3/84 with dendrites containing retrogradely transported HRP. The remainder of the junctions were with dendrites lacking detectable immunoautoradiographic or HRP-labeling. The majority of the recognized synapses on labeled dendrites were at more proximal sites possibly reflecting more limited detection of both MELI and retrogradely transported HRP in smaller dendrites. However, the presence of even a few junctions at proximal sites on dendrites where synaptic transmission is known to be more effective suggests a potentially strong modulation of both opioid and vagal motor neurons by visceral afferents in the NTS. In addition to forming synapses on dendrites, both vagal afferents and terminals containing MELI showed frequent synaptic associations with unlabeled terminals, but not with each other. This finding suggests that the previously demonstrated opiate binding sites on vagal afferents is most likely attributed to other endogenous opiates.     

Immunoelectron microscopic study of glutamate inputs from the retrosplenial granular cortex to identified thalamocortical projection neurons in the anterior thalamus of the rat

We have carried out an ultrastructural study to determine the characteristics and distribution of glutamate-containing constituents of the anterodorsal (AD) and anteroventral (AV) thalamic nuclei in adult rats. We used a polyclonal antibody to glutamate and a postembedding immunogold detection method in animals in which the neurons of AD/AV projecting to the cortex had been retrogradely labelled and the terminals of corticothalamic afferents anterogradely labelled by injection of cholera toxin-horseradish peroxidase (HRP) into the retrosplenial granular cortex. The heaviest immunogold labelling was over axon terminals 0.42 to 2.2 microm in diameter containing round synaptic vesicles and establishing Gray type 1 (asymmetric) synaptic contact (type 1 terminals) on HRP-labelled or non-labelled dendrites. Mean gold particle densities over such terminals were 3-4 times higher than the densities over the dendrites to which they were presynaptic and 5-6 times higher than over terminals establishing Gray type 2 (symmetric) synaptic contacts (type 2 terminals). Gold particle densities over neuronal cell bodies and dendrites and over a subpopulation of myelinated axons were intermediate between the densities over type 1 and type 2 terminals. In adjacent serial sections immunoreacted for gamma aminobutyric acid, type 2 terminals were heavily immunolabelled whereas type 1 terminals and other profiles with moderate gold particle densities after glutamate immunoreaction displayed very low labelling. A subpopulation of small type 1 axon terminals (up to 1 microm diameter) contained HRP reaction product identifying them as cortical in origin; they contacted small dendritic profiles (most <1 microm diameter) many of which also contained HRP reaction product. We conclude that terminals of the corticothalamic projection from retrosplenial granular cortex to AD/AV are glutamatergic and innervate predominantly distal dendrites of thalamocortical projection neurons.     

Ultrastructure and synaptology of the nucleus ambiguus in the rat: The semicompact and loose formations

The fine structure of the pharyngomotor semicompact and laryngomotor loose format formations of the rat nucleus ambiguus was studied in single and serial sections by means of light and electron microscopy. Motoneurons and their dendrites were identified after retrograde labelling by injections of neuroanatomical tracers into pharyngeal and laryngeal muscles or nerves. Pharyngeal motoneurons measured 39 × 29 μm and had 2–25 axosomatic synapses per somatic profile, representing an estimated average of 1S2 synapses per soma. Laryngeal motoneurons measured 42 × 30 μm with 6–33 synapses per profile, or an average of 339 synapses per soma. In both subdivisions, axon terminals that contained round vesicles and formed asymmetric junctions and terminals that contained pleomorphic vesicles and formed symmetric junctions were distributed in approximately equal proportions on somata and dendrites, forming over 90% of the synapse population. A small percentage (2–8%) of synapses had a subsurface cistern situated below the axon terminal (C type). Small, atypical motoneurons measuring 15 × 5 μm with an invaginated nucleus were also present in both subdivisions. The ultrastructure and synaptology of pharyngeal and laryngeal motoneurons are characterized by similarities to those of spinal motoneurons and by their relatively large numbers of axosomatic synapses in comparison to esophageal motoneurons of the compact formation of the nucleus ambiguus. © 1996 Wiley-Liss, Inc.     

Time course and distribution of motoneuronal loss in the dorsal motor vagal nucleus of guinea pig after cervical vagotomy

Cells in the dorsal motor vagal nucleus (DMVN) of the adult guinea pig were counted at different times after unilateral cervical section of the vagus nerve. The counts were made from serial 30 μm coronal sections throughout the DMVN in normal and operated animals. There are three types of cells in the DMVN of guinea pig: medium-sized motoneurons that are retrogradely filled by HRP from the site of the vagotomy, small neurons, and glial cells. An interesting observation was a change in distribution of cells in the DMVN with age in unoperated guinea pigs. Following vagotomy degeneration was seen only in the motoneurons. Disappearance of motoneurons was slow and only 27% were present after 1 year. During that time the decrease in the total number of motoneurons was exponential with a time constant of 8.6 months, but degeneration in different parts of the nucleus was not uniform. Thirty-four percent of motoneurons in the caudal area of DMVN disappeared in the first month after vagotomy, while the rostral area was almost unchanged. The rostral area, however, showed rapid degeneration between 3 and 6 months after vagotomy. The central part of the nucleus degenerated at a constant rate between those of the rostral and caudal regions. At the end of 1 year, cell loss in all parts of the nucleus was approximately equal. Surviving motoneurons showed morphological changes: rounding of the soma, continuous reduction of the cell volume, and shrinkage of the nucleus. Occasional abnormal forms showing vacuolization or invaginated nuclei were seen. Calculations show that the process of degeneration lasts 25 days on the average. The marked degeneration found in dorsal vagal motoneurons, in contrast to recovery from axotomy in somatic motoneurons, is similar to that found in intrinsic neurons of the central nervous system. The slow and continuous time course of disappearance of motoneurons after vagotomy, however, is exceptional. It is reasonable to postulate that the increased vulnerability of these motoneurons may be sufficient to result in degeneration in response to what are normally nonpathological metabolic demands.     

Direct synaptic projections to esophageal motoneurons in the nucleus ambiguus from the nucleus of the solitary tract of the rat

Neurons of the nucleus of the solitary tract (NTS) serve as interneurons in swallowing. We investigated the synaptology of the terminals of these neurons and whether they project directly to the esophageal motoneurons in the compact formation of the nucleus ambiguus (AmC). Following wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) injection into the NTS, many anterogradely labeled axodendritic terminals were found in the neuropil of the AmC. The majority of labeled axodendritic terminals (89%) contained round vesicles and made asymmetric synaptic contacts (Gray's type I), but a few (11%) contained pleomorphic vesicles and made symmetric synaptic contacts (Gray's type II). More than half of the labeled terminals contacted intermediate dendrites (1-2 μm diameter). There were no retrogradely labeled medium-sized motoneurons, but there were many retrogradely labeled small neurons having anterogradely labeled axosomatic terminals. A combined retrograde and anterograde transport technique was developed to verify the direct projection from the NTS to the esophageal motoneurons. After the esophageal motoneurons were retrogradely labeled by cholera toxin subunit B conjugated HRP, the injection of WGA-HRP into the NTS permitted ultrastructural recognition of anterogradely labeled axosomatic terminals contacting directly labeled esophageal motoneurons. Serial sections showed that less than 20% of the axosomatic terminals were labeled in the esophageal motoneurons. They were mostly Gray's type I, but a few were Gray's type II. In the small neurons, more than 30% of axosomatic terminals were labeled, which were exclusively Gray's type I. These results indicate that NTS neurons project directly not only to the esophageal motoneurons, but also to the small neurons which have bidirectional connections with the NTS. J. Comp. Neurol. 381:18-30, 1997. © 1997 Wiley-Liss, Inc.     

White-matter dendrites in the upper cervical spinal cord of the adult cat: A light and electron microscopic study

The organization and structure of dendrites penetrating into the white matter of upper cervical spinal segments have been examined by means of Golgi staining techniques, intracellular horseradish peroxidase (HRP) injections, and ultrastructural studies. The Golgi studies established that several groups of neurons located in intermediate and ventral laminae of the upper cervical spinal cord have a substantial part of their dendritic tree extending into adjacent ventral and lateral funiculi. Most dendrites in white matter showed irregular varicosities along their length. They were devoid of spines and followed relatively direct paths. In contrast, grey matter dendrites were occasionally observed with spines and complex appendages and frequently followed tortuous paths. The size and location of some Golgi stained neurons suggested that white matter dendrites might originate from neck muscle motoneurons. This possibility was confirmed using intracellular HRP injections. These studies also showed that the distribution of white matter dendrites of neck muscle motoneurons depended on the location of the motoneuron soma. White matter dendrites of neck muscle motoneurons located deep in the ventral horn projected into all regions of white matter surrounding the ventral horn. Other neck muscle motoneurons, located in the spinal accessory nucleus, had white matter dendrites largely confined to the lateral funiculus. White matter dendrites of motoneurons in the commissural nucleus were found to project across the ventral commissure into the contralateral spinal cord. Light microscopic studies of semi-thin sections stained with toluidine blue and electron microscopic studies of thin sections revealed that white matter dendrites were confined to special regions of the white matter. These regions resembled the grey matter neuropil and contained dendrites and unmyelinated and small diameter myelinated axons. Axon terminals were also found in white matter. These terminals contained either flattened or spherical vesicles and formed synaptic contacts on white matter dendrites. White matter dendrites, by virtue of their frequency of occurrence, distribution, and type of synaptic contacts may represent a means by which descending or ascending spinal systems can influence spinal neurons without recourse to axon collaterals which terminate in grey matter.     

Vagal and gastric connections to the central nervous system determined by the transport of horseradish peroxidase

Horseradish peroxidase (HRP, Sigma Type VI) crystals were encased in a parafilm envelope and applied to the transected central ends of the left and right cervical vagus nerves and the anterior and posterior esophageal vagus nerves of adult male hooded rats. Injections of 30% HRP were made into the muscle wall of the fundus and antrum regions of the stomach. After 48 hr survival time, animals were perfused intracardially with a phosphate buffer plus sucrose wash followed by glutaraldehyde and paraformaldehyde fixative. The brain stem, spinal cord and corresponding dorsal root ganglia, superior cervical sympathetic ganglion, and the nodose ganglion were removed and cut into 50 micron sections. All tissue was processed with tetramethylbenzidine (TMB) for the blue reaction according to Mesulum and counterstained with neutral red. Sequential sections were examined under a microscope. Labeled neurons and nerve terminals were identified using bright and dark field condensers and polarized light. In tissue from animals that had HRP applied to the cervical vagus nerves, retrogradely labeled neurons were identified ipsilaterally in the medulla located in the dorsal motor nucleus of the vagus (DMN) and the nucleus ambiguus (NA). Labeled cells extended from the DMN into the spinal cord in ventral-medial and laminae X regions C1 and C2 of cervical segments. Many neurons were labeled in the nodose ganglion. Anterogradely labeled terminals were observed throughout and adjacent to the solitary nucleus (NTS) dorsal to the DMN and intermixed among labeled neurons located in the DMN. In tissue from animals that had HRP applied to the esophageal vagus nerves, similar labeling was observed. However, fewer neurons were identified in the NA, the nodose ganglion, and only in laminae X of the cervical spinal cord segments C1 and C2. Also, very little terminal labeling was observed in and adjacent to the NTS. Labeled neurons in tissue from animals that had HRP injected into the stomach wall were observed bilaterally in the DMN, nodose ganglion, and only in laminae X at the C1 and C2 levels of the spinal cord. Labeled neurons also were observed in the dorsal root ganglia of the thoracic cord. These data indicate that cervical cord and NA neurons are important in the supradiaphragmatic motor innervation by the vagus. Also, many afferents to the NTS originate above the diaphragm. In addition, some afferents from the stomach enter the central nervous system via the thoracic spinal cord.     

Structures and connections of enkephalin- and γ-aminobutyric acid-immunoreactive profiles in the gustatory region of the nucleus tractus solitarius: a light and electron microscopic study

GABA and enkephalin (ENK) are principle inhibitory transmitters in the rostral portion of the nucleus tractus solitarius (rNTS) for regulating the gustatory information. Although the existence of GABA- and ENK-immunoreactive (ir) profiles in the rNTS has been demonstrated, there are no morphological data revealing the connections between them. In the present study, using immunofluorescent and electron microscopic methods, we examined their relationship in the rNTS of rat. Results showed the following: (1) dense ENK-ir fibers and terminals and GABA-ir cell bodies, fibers, and terminals were observed in the rNTS; (2) ENK-ir terminals mainly make symmetric synapses with GABA-ir and immunonegative somas and dendrites; (3) co-existence of ENK/GABA-ir axon terminals and convergence of ENK- and GABA-ir terminals on one immunonegative soma or dendrite can also be observed. These results suggest that ENK should inhibit directly or excite indirectly (by blocking the inhibition of the GABAergic neurons) the gustatory neurons in the rNTS.     

Ultrastructural features of synapse from dorsal parvocellular reticular formation neurons to hypoglossal motoneurons of the rat

The dorsal parvocellular reticular formation (PCRt) receives projection of the trigeminal mesencephalic nucleus neurons. It contains the dorsal group of interneurons that integrate and coordinate activity of the oral motor nuclei. Ultrastructural features of synaptic connection from the dorsal PCRt neurons to the motoneurons of the hypoglossal nucleus (XII) were examined at both the light and electron microscopic levels in rats. Biotinylated dextran amine (BDA) was initially iontophoresed into the dorsal part of PCRt unilaterally. Seven days later horseradish peroxidase (HRP) was injected into the body of the tongue. After histochemical reaction for visualization of HRP and BDA, the BDA-labeled fibers and terminals were seen distributing bilaterally in XII with ipsilateral predominance. BDA-labeled terminals were closely apposed upon HRP retrogradely labeled somata and dendrites of the XII motoneurons. A total of 1408 BDA-labeled boutons were examined ultrastructurally, which had mean size of 1.22+/-0.37 microm in diameter. Five hundred-ninety three of these boutons in both the ipsilateral (n=401) and contralateral (n=192) XII were seen to synapse on both the dendrites and somata of HRP-labeled motoneurons. The vast majorities of synapses were axodendritic (98%, 580/593), while 2% of them were axosomatic. Of the 1408 BDA-labeled boutons, 69.6% of them were S-type boutons containing small clear and spherical synaptic vesicles and 30.4% of them were PF-type boutons containing pleomorphic and flattened synaptic vesicles. Approximately 64% of synapses between BDA-labeled boutons and HRP-labeled motoneurons were asymmetric, and 33% of synapses were symmetric. No axoaxodendritic or axoaxosomatic synaptic triad was observed. The present study illustrated the anatomical pathway and synaptological characteristics of neuronal connection between the dorsal PCRt premotor neurons and the XII motoneurons. Its functional significance in coordinating activity of XII motoneurons during oral motor behaviors has been discussed.     

Ultrastructural characteristics of callosal neurons in deep layers of the primary auditory cortex (AI) in cat]

An electron microscope study of retrogradely labelled nonpyramidal neurons has been carried out in layers V-VI of the primary auditory cortex (AI) after HRP injections into the contralateral AI of cats. From 2 to 9 synapses were usually revealed on somatic profiles of these callosal neurons. Synapses occupied 15.8 +/- 1.7% (on the average) of the somatic surface of these neurons. All of the revealed synapses on the somata of these callosal neurons had symmetric contacts and were formed by axon terminals with small elongated synaptic vesicles. An average length of these synaptic contacts in sections was 1.6 +/- 0.1 mm. HRP-labelled axon terminals of callosal fibres in layers V-VI contained round synaptic vesicles and formed asymmetric synapses on spines and dendrites. Possible functional significance of axo-somatic synapses in formation of impulsation patterns of the callosal neurons is discussed.     

Cytoarchitecture, synaptic organization and Fiber Connections of the nucleus olfactoretinalis in a teleost (Navodon modestus)

Cytoarchitecture, synaptic organization and fiber connections of the nucleus olfactoretinalis (NOR) in a teleost, Navodon modestus, have been studied light- and electron-microscopically using an HRP or HRP-degeneration combined method. Following HRP injections into the optic nerve, most contralateral and a few ipsilateral neurons in the NOR were labeled. There are two types of neurons in NOR. Type I neurons have a medium-sized spindle-shaped soma with a round nucleus, and type II neurons have a large oval soma with an invaginated nucleus and contain cored vesicles (80-130 nm in diameter). Afferent terminals which form synaptic contacts with cell bodies of NOR neurons were classified into 3 types according to their morphological characteristics; S, F1 and F2 terminals. S terminals originated in ipsilateral area ventralis telencephali pars supracommissuralis (Vs). These terminals contain both spherical and cored vesicles, and make synaptic contacts with both type I and type II neurons. F1 terminals, which originated in ipsilateral area dorsalis telencephali pars posterior (Dp), are large in profile, and contain flat vesicles and mitochondria with irregularly arranged cristae. These terminals make synaptic contacts only with type I neurons. F2 terminals are small in profile, and contain flat vesicles, cored vesicles and small mitochondria with regularly arranged cristae. F2 terminals make synaptic contacts with both type I and type II neurons. The functional significance of NOR and the relationship between NOR and the ganglion of the nervus terminalis are discussed.     

mu-opioid receptors are present in vagal afferents and their dendritic targets in the medial nucleus tractus solitarius

Ligands of the mu-opiate receptor (MOR) are known to influence many functions that involve vagal afferent input to the nucleus tractus solitarius (NTS), including cardiopulmonary responses, gastrointestinal activity, and cortical arousal. The current study sought to determine whether a cellular substrate exists for direct modulation of vagal afferents and/or their neuronal targets in the NTS by ligands of the MOR. Anterograde tracing of vagal afferents arising from the nodose ganglion was achieved with biotinylated dextran amine (BDA), and the MOR was detected by using antipeptide MOR antiserum. The medial subdivision of the intermediate NTS was examined by electron microscopy for the presence of peroxidase-labeled, BDA-containing vagal afferents and immunogold MOR labeling. MOR was present in both presynaptic axon terminals and at postsynaptic sites, primarily dendrites. In dendrites, MOR immunogold particles usually were located along extrasynaptic portions of the plasma membrane. Of 173 observed BDA-labeled vagal afferent axon terminals, 33% contained immunogold labeling for MOR within the axon terminal. Many of these BDA-labeled terminals formed asymmetric, excitatory-type synapses with dendrites, some of which contained MOR immunogold labeling. MORs were present in 19% of the dendrites contacted by BDA-labeled terminals but were present rarely in both the vagal afferent and its dendritic target. Together, these results suggest that MOR ligands modulate either the presynaptic release from or the postsynaptic responses to largely separate populations of vagal afferents in the intermediate NTS. These results provide a cellular substrate for direct actions of MOR ligands on primary visceral afferents and their second-order neuronal targets in NTS.     

The central organization of the vagus nerve innervating the stomach of the rat

We employed the neural tracers cholera toxin-horseradish peroxidase and wheat germ agglutinin-horseradish peroxidase to examine the organization of the afferent and efferent connections of the stomach within the medulla oblongata of the rat. The major finding of this study is that gastric motoneurons of the dorsal motor nucleus (DMN) possess numerous dendrites penetrating discrete regions of the overlying nucleus of the solitary tract (NTS). In particular, dendritic labelling was present in areas of NTS which also received terminals of gastric vagal afferent fibers such as the subnucleus gelatinosus, nucleus commissuralis, and medial nucleus of NTS. This codistribution of afferent and efferent elements of the gastric vagus may provide loci for monosynaptic vagovagal interactions. A small number of dendrites of DMN neurons penetrated the ependyma of the fourth ventricle and a few others entered the ventral aspect of the area postrema, thus making possible the direct contact of preganglionic neurons with humoral input from the cerebrospinal fluid and/or the peripheral plasma. Nucleus ambiguus neurons projecting to the stomach predominantly innervate the forestomach. The dendrites of these cells, when labelled, were generally short, and extended beyond the compact cluster of ambiguus neurons in a ventrolateral direction, parallel to the fascicles of vagal efferent fibers traversing the medulla.     

Axonal projections and synapses from the supratrigeminal region to hypoglossal motoneurons in the rat

Neural circuits from the supratrigeminal region (Vsup) to the hypoglossal motor nucleus were studied in rats using anterograde and retrograde neuroanatomical tracing methodologies. Iontophoretic injection of 10% biotinylated dextran amine (BDA) unilaterally into the Vsup anterogradely labeled axons and axon terminals bilaterally in the hypoglossal nucleus (XII) as well as other regions of the brainstem. In the ipsilateral XII, the highest density of BDA labeling was found in the dorsal compartment and the ventromedial subcompartment of the ventral compartment, where BDA labeling formed a dense, patchy distribution. Microinjection of 20% horseradish peroxidase (HRP) ipsilaterally or bilaterally into the tongue resulted in retrograde labeling of XII motoneurons confined to the dorsal and ventral compartments of the hypoglossal motor nucleus. Under light microscopical examination, BDA-labeled terminals were observed closely apposing the somata and primary dendrites of HRP-labeled hypoglossal motoneurons. Two hundred and sixty-five of these BDA-labeled terminals were examined at the ultrastructural level. One hundred and twelve BDA-labeled axon terminals were observed synapsing with either the somata (39%, 44/112) or the large or medium-size dendrites (61%, 68/112) of retrogradely labeled hypoglossal motoneurons. Axon terminals containing spherical vesicles (S-type) formed asymmetric synapses with HRP-labeled hypoglossal motoneuron dendrites. In contrast to this, F_F-type axon terminals, containing flattened vesicles, formed symmetric synapses with both the somata and dendrites of HRP-labeled hypoglossal motoneurons with a preponderance of the contacts on their somata. Axon terminals containing pleomorphic vesicles (F_P-type) were noted forming both symmetric and asymmetric synapses with HRP-labeled hypoglossal motoneuron somata and dendrites. The present study provides anatomical evidence of neuronal projections and synaptic connections from the supratrigeminal region to hypoglossal motoneurons. These data suggest that the supratrigeminal region, as one of the premotor neuronal pools of the hypoglossal nucleus, may coordinate and modulate the activity of tongue muscles during oral motor behaviors.     

The ultrastructural identification of reticulo-hypoglossal axon terminals anterogradely labeled with horseradish peroxidase

Injections of horseradish peroxidase (HRP) or wheat-germ agglutinin-horseradish peroxidase (WGA-HRP) into the nucleus reticularis parvocellularis (RPc) produced anterograde labeling of axon terminals within the hypoglossal nucleus. Based on morphological parameters of vesicle population, membrane specializations, and postsynaptic articulations, two types of axon terminals derived from neurons in RPc end on hypoglossal neurons. More than half of the terminals contained spherical vesicles (S-type), established asymmetrical membrane specializations and contacted proximal and medium-sized dendrites. The remaining labeled terminals had flattened vesicles (F-type), symmetrical membrane densities and apposed medium and small dendrites. The morphological differences expressed in the two types of terminals may reflect physiological and/or pharmacological differences in the action of RPc neurons on motoneurons in the hypoglossal nucleus.     

A light and electron microscopic study of the dorsal motor nucleus of the vagus nerve in the cat

The morphology of the dorsal motor nucleus of the vagus nerve (DMV) in the cat was studied with the aid of light and electron microscopy. In frozen sections stained by the Kluver-Barrera method or stained to show retrograde labeling in the DMV following injections of horseradish peroxidase (HRP) in the cervical vagus nerve and the stomach wall a range of sizes of DMV neurons was observed but it was not possible to distinguish separate types. In contrast, two distinct types of neurons, one medium-sized and the other small, were identified with the light microscope in Golgi-Cox and 1-μm Epon sections and with the electron microscope in ultrathin sections. The medium-sized neurons had a range of sizes but generally measured 18 × 25 μm and possessed three to four proximal dendrites which branched two or three times. Spines were observed occasionally on the soma and on dendrites. These neurons contained a well-developed cytoplasm and a noninvaginated round to oval nucleus. The small neurons generally measured 9 × 14 μm and were round or slightly elongated in shape. Their dendritic processes were fewer and thinner than those of the medium-sized neurons and extended for shorter lengths. Their soma contained scanty cytoplasm and an invaginated nucleus. The medium-sized neurons outnumbered the small neurons by more than three to one but both neuronal types were distributed evenly throughout the nucleus. The medium-sized neurons seemed to correspond in size to the parasympathetic efferent neurons of the viscera as indicated by the HRP studies. Axosomatic synapses on both types of neurons and axodendritic synapses were observed in the DMV. Terminals containing mainly small clear round vesicles and making asymmetrical contact with the postsynaptic membrane were involved in the majority of synapses on both the soma and dendrites. Terminals containing predominantly pleomorphic vesicles and making symmetrical contact with the postsynaptic membrane were also common, comprising up to one-third of all synapses observed. Serial sections revealed that most synaptic terminals contained varying numbers of large (75–110 nm) dense-cored vesicles. Smaller dense-cored vesicles (45–55 nm) were sometimes observed, often close to the area of synaptic contact. Terminals 1–2 μm in diameter which contacted dendrites 1–3 μm in diameter formed the most common synaptic combination throughout the rostral to caudal extent of the DMV. No distinct regional differences were observed with respect to distribution of synaptic types.     

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.     

Immunocytochemical localization of gamma-aminobutyric acid in the hypoglossal nucleus of the macaque monkey, Macaca fuscata: a light and electron microscopic study

The hypoglossal nucleus of the macaque monkey Macaca fuscata was investigated with light and electron microscopic immunocytochemistry with an antibody directed against gamma-aminobutyric acid (GABA). At the light microscopic level, GABA immunoreactivity was present in small neurons, punctate structures, and thin, fiberlike structures. These GABA-positive elements were distributed throughout the hypoglossal nucleus at rostrocaudal levels. There was no immunoreactivity in the hypoglossal motoneurons. The GABA-positive small neurons were fusiform or ovoid (15 X 9 micron) and extended a few proximal dendrites from both poles. At the ultrastructural level, these small neurons were characterized by a markedly invaginated nucleus and a scanty cytoplasm in which cisternae of rough endoplasmic reticulum were not organized into extensive lamellar arrays as seen in the motorneurons. The GABA-positive punctate structures at the light microscopic level were identified as vesicle-containing axon boutons at the electron microscopic level. These GABA-positive axon terminals made synaptic contacts mainly with the dendrites of the motoneurons and infrequently with the somata. The majority of them made symmetric synapses and they contained pleomorphic synaptic vesicles. However, a small number of GABA-positive terminals (7%) formed asymmetric synapses with the dendrites of motoneurons, and these contacts exhibited postsynaptic dense bars or Taxi bodies lying beneath the postsynaptic membranes. There were no GABA-positive boutons that contacted the cell bodies of the small neurons. Although GABA-positive myelinated and unmyelinated axons were seen as thin, fiberlike structures, these myelinated and unmyelinated axons rarely gave rise to boutons on the motoneurons. The present study suggests that GABAergic inhibition in the monkey hypoglossal nucleus occurs mainly on the dendrites of the motoneurons and to some extent on the somata.     

Monosynaptic input from Leu5-enkephalin-immunoreactive terminals to vagal motor neurons in the nucleus ambiguus: Comparison with the dorsal motor nucleus of the vagus

Vagal motor neurons in the rat dorsal motor nucleus of the vagus (DMN) are known to receive direct synaptic input from enkephalin-containing terminals. We examined (1) whether the vagal motor neurons within the nucleus ambiguus (NA) also received monosynaptic input from enkephalin-immunoreactive terminals and (2), if so, whether their ultrastructural relations differed from those in the DMN. In both regions, terminals containing Leu⁵-enkephalinlike immunoreactivity (LE-LI) were examined in relation to motor neurons identified by retrograde transport of wheat germ-agglutinated horseradish peroxidase (WGA-HRP) applied to the cut end of the cervical vagus nerve in single sections of the medulla oblongata of adult rats. By light microscopy, the most significant overlap between varicose processes with LE-LI and WGA-HRP-containing neurons was seen in the rostral compact portion of the NA and the DMN at the level of the obex. Thus, only these regions were examined by electron microscopy. The most distinguishing ultrastructural feature of WGA-HRP-labeled neurons in the NA compared to the DMN was their higher incidence of nonsynaptic appositions with other neurons. In both the NA and the DMN, terminals with LE-LI formed primarily symmetric synapses on smaller (presumably distal) dendrites; many of these dendrites, as well as most target perikarya, contained WGA-HRP. Additionally, in the compact portion of the NA compared to the DMN (1) multiple LE-labeled terminals more frequently contacted single perikarya or dendrites and (2) single terminals with LE-LI more commonly showed two contacts or active zones and contained more abundant LE-immunoreactive large (80–100 nm) densecore vesicles (dcvs). In contrast to small (40–50 nm), clear vesicles, which were usually aggregated near active zones, the immunoreactive dcvs were usually located near glial processes distal to these zones. These results indicate that enkephalin immunoreactivity is intensely localized to dcvs within terminals that may have direct inhibitory (symmetric synapses) actions on vagal motor neurons in both the compact portion of the NA and the DMN. Moreover, because numbers of dcvs and active zones have been equated with synaptic strength, our findings suggest enhanced potencies of enkephalin-immunoreactive terminals in the compact portion of the NA. Our findings support a prominent role for enkephalin in the coordinated activity of esophageal motor neurons located in the compact portion of the NA. © 1995 Wiley-Liss, Inc.