Collateral axonal projections from hypothalamic hypocretin neurons to cardiovascular sites in nucleus ambiguus and nucleus tractus solitarius

Hypocretin-1 (hcrt-1)-containing axons have been shown to have an extensive distribution within the central nervous system, although the total number of hypothalamic hcrt-1 neurons has been shown to be small. This suggests that hcrt-1 neurons may innervate central structures with similar function through collateral axonal projections. Retrograde tract-tracing techniques combined with immunohistochemistry were used in this study to investigate whether hypothalamic hcrt-1-containing neurons send collateral axonal projections to cardiovascular sites in the nucleus of the solitary tract (NTS) and in the nucleus ambiguus (Amb) in the rat. Fluorogold- (FG) and/or rhodamine (Rd)-labeled latex microspheres were microinjected into either the NTS or Amb at sites that elicited bardycardia responses (L-glutamate; 0.25 M; 10 nl). After a survival period of 10-15 days, the rats were sacrificed and tissue sections of the hypothalamus were processed immunohistochemically for the identification of hcrt-1-containing cell bodies. After injection of the tract-tracers into the NTS or Amb, retrogradely labeled neurons were observed within several hypothalamic regions; the paraventricular hypothalamic nucleus, lateral hypothalamic area, perifornical hypothalamic area, and posterior hypothalamus, bilaterally, but with an ipsilateral predominance. In addition, after NTS injections, retrogradely labeled neurons were found within the ipsilateral caudal arcuate nucleus. Of the total number (1107+/-97) of hcrt-1-immunoreactive neurons found bilaterally within the lateral and perifornical hypothalamic nuclei, 7.9+/-1.4% were found to be retrogradely labeled from the NTS, 16.4+/-1.8% from the Amb, and 3.1+/-0.5% from both medullary sites. Hcrt-1 neurons projecting to the NTS were found mainly in and around the perifornical hypothalamic region, with a smaller number in the caudal lateral hypothalamic area. On the other hand, those innervating the Amb were primarily observed within the caudal lateral hypothalamic area, with a smaller number in the perifornical hypothalamic area. Neurons with collateral axonal projections to NTS and Amb were observed within two specific hypothalamic areas: one group of neurons was found in the perifornical hypothalamic area, and the other was observed in the lateral hypothalamic region just dorsal to the retrochiasmatic component of the supraoptic nucleus. These data indicate that axons from hcrt-1 neurons bifurcate to innervate functionally similar cardiovascular-responsive sites in the NTS and Amb. Although the function of these hcrt-1-containing hypothalamic-medullary pathways is not known, they likely represent the anatomical substrate by which the lateral hypothalamic hcrt-1 neurons simultaneously coordinate autonomic-cardiovascular responses to different behaviors.     

Ionotropic glutamate receptor subunit immunoreactivity of vagal preganglionic neurones projecting to the rat heart

The ionotropic glutamate receptor subunits expressed by vagal preganglionic neurones in the rat medulla oblongata were examined by using fluorescence immunolabelling combined with retrograde neuronal tracing. The general population of these neurones in the medulla was identified by intraperitoneal injections of Fluorogold and also with choline acetyltransferase antibodies. Cardiac projecting neurones were specifically identified by applying the fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine (DiI) to the heart or by injecting cholera toxin B-subunit into the pericardium. Both tracers labelled populations of neurones lying in the dorsal vagal nucleus, intermediate reticular formation and nucleus ambiguus, and when both tracers were applied simultaneously, approximately 50% of cells were dual-labelled. Control experiments established that the labelling was specific for neurones projecting to the heart. Most vagal preganglionic neurones, including those projecting to the heart, irrespective of their location in the medulla, had a similar profile of glutamate receptor immunoreactivity. Labelling of somata for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) subunit GluR1 was weak or absent, while labelling with antibodies directed to GluR2, a common sequence of GluR2 and GluR3, and GluR4 was moderate or intense. All neurones studied appeared to express the N-methyl-D-aspartate (NMDA) receptor subunit NR1, and while antibodies recognising the NR2A and NR2B splice variants gave strong labelling, immunoreactivity with a NR2B specific antibody was weaker. Weak to moderate labelling was seen in some neurones using antibodies to the kainate receptor subunits KA2 and GluR5-7. These results are consistent with neurophysiological data indicating the presence of AMPA, NMDA and kainate responses in cardiac vagal preganglionic neurones, and suggest that these neurones are similar to other vagal parasympathetic preganglionic neurones in expressing mainly AMPA receptor subunits GluR2-4.     

Cardiotopic organization of the nucleus ambiguus? An anatomical and physiological analysis of neurons regulating atrioventricular conduction

Previous data indicate that there are anatomically segregated and physiologically independent parasympathetic postganglionic vagal motoneurons on the surface of the heart which are capable of selective control of sinoatrial rate, atrioventricular conduction and atrial contractility. We have injected a retrograde tracer into the cardiac ganglion which selectively regulates atrioventricular conduction (the AV ganglion). Medullary tissues were processed for the histochemical detection of retrogradely labeled neurons by light and electron microscopic methods. Negative dromotropic retrogradely labeled cells were found in a long column in the ventrolateral nucleus ambiguus (NA-VL), which enlarged somewhat at the level of the area postrema, but reached its largest size rostral to the area postrema in an area termed the rostral ventrolateral nucleus ambiguus (rNA-VL). Three times as many cells were observed in the left rNA-VL as compared to the right (P < 0.025). Retrogradely labeled cells were also consistantly observed in the dorsal motor nucleus of the vagus (DMV). The DMV contained one third as many cells as the NA-VL. The right DMV contained twice as many cells as the left (P < 0.05). These data are consistent with physiological evidence that suggests that the left vagus nerve is dominant in the regulation of AV conduction, but that the right vagus nerve is also influential. While recording the electrocardiogram in paced and non-paced hearts,l-glutamate (GLU) was microinjected into the rNA-VL. Microinjections of GLU caused a 76% decrease in the rate of atrioventricular (AV) conduction (P < 0.05) and occasional second degree heart block, without changing heart rate. The effects of GLU were abolished by ipsilateral cervical vagotomy. These physiological data therefore support the anatomical inference that CNS neurons that are retrogradely labeled from the AV ganglion selectively exhibit negative dromotropic properties. Retrogradely labeled negative dromotropic neurons displayed a round nucleus with ample cytoplasm, abundant rough endoplasmic reticulum and the presence of distinctive somatic and dendritic spines. These neurons received synapses from afferent terminals containing small pleomorphic vesicles and large dense core vesicles. These terminals made both asymmetric and symmetric contacts with negative dromotropic dendrites and perikarya, respectively. In conclusion, the data presented indicate that there is a cardiotopic organization of ultrastructurally distinctive negative dromotropic neurons in the NA-VL. This central organization of parasympathetic preganglionic vagal motoneurons mirrors the functional organization of cardioinhibitory postganglionic neurons of the peripheral vagus nerve. These data are further discussed in comparison to a recent report on the light microscopic distribution and ultrastructural characteristics of negative chronotropic neurons in the NA-VL⁴². The data support the hypothesis that anatomically separated and functionally selective parasympathetic preganglionic vagal motoneurons in the NA may independently control AV conduction and cardiac rate.     

Control of negative inotropic vagal preganglionic neurons in the dog: synaptic interactions with substance P afferent terminals in the nucleus ambiguus?

Previous research from this laboratory has shown that substance P-immunoreactive (SP) terminals synapse upon negative chronotropic vagal preganglionic neurons (VPNs), but not upon negative dromotropic VPNs, of the ventrolateral nucleus ambiguus (NA-VL). Moreover, SP agonists injected into NA-VL cause bradycardia without decreasing AV conduction. In the current study, we have: (1) defined the electron microscopic characteristics of the SP neurons of NA-VL in dog; and (2) tested the hypothesis that SP nerve terminals synapse upon negative inotropic VPNs of NA-VL, retrogradely labeled from the cranial medial ventricular (CMV) ganglion. Numerous SP terminals and a few SP neurons were observed in the vicinity of retrogradely labeled neurons. SP terminals were observed forming synapses with unlabeled dendrites and with SP dendrites, but never with the retrogradely labeled neurons. Together, these results and earlier findings suggest that SP agonists may be able to induce bradycardia without decreasing AV conduction or ventricular contractility.     

Nucleus ambiguus and bulbospinal ventral respiratory group neurons in the neonatal rat

The in vitro brainstem-spinal cord preparation of the neonatal rat is an important model system for studies of the respiratory control system, yet there have not been studies to anatomically characterize respiratory neuron populations in the neonate. Fluorescent retrograde tracers were used to identify bulbospinal neurons of the ventral respiratory group and motoneurons of nucleus ambiguus in neonatal rats. Fluoro-Gold injections into the C₄ ventral horn labeled bulbospinal neurons within a densely packed column within the ventrolateral intermediate reticular nucleus from the level of the pyramidal decussation to the facial nucleus. This cell column corresponded closely to the location of the ventral respiratory group of the adult rat. In particular, neurons were labeled in regions corresponding to the rostral ventral respiratory group and the Bötzinger complex. Unlike adult rats, the preBötzinger complex also contained many bulbospinal neurons. Fluoro-Gold–labeled neurons were also located in the medial reticular nuclei, raphe pallidus, and obscurus and spinal vestibular nucleus. As in adult rats, bulbospinal ventral respiratory group neurons overlapped with cervical vagal motoneurons in the external formation, and partially with those in the loose formation, but not with those in the semicompact or compact formation of nucleus ambiguus. These results indicate that the distribution of bulbospinal ventral respiratory group neurons corresponds with that observed in physiological studies of neonatal rats.     

Negative inotropic vagal preganglionic neurons in the nucleus ambiguus of the cat: neuroanatomical comparison with negative chronotropic neurons utilizing dual retrograde tracers

The vagal postganglionic controls of cardiac rate and left ventricular contractility are mediated by separate intracardiac ganglia, the sino-atrial (SA) and cranio-ventricular (CV) ganglia, respectively. We injected a different retrograde tracer into each of these ganglia (in the same animal) and subsequently examined the brain for the presence of single labeled or double labeled vagal preganglionic neurons. Retrogradely labeled cells from either ganglion were found exclusively in the ventrolateral nucleus ambiguus (NA-VL). There was considerable overlap in the distribution of labeled cells from either ganglion, however fewer than 3% of labeled neurons were double labeled. The data are consistent with the hypothesis that the preganglionic controls of cardiac rate and left ventricular contractility are mediated by largely separate but overlapping groups of cardioinhibitory neurons originating from the NA-VL. These neurons have parallel but morphologically independent pathways projecting to the SA and CV ganglia. Physiological experiments are needed to support this hypothesis.     

Stimulation of NTS activates NMDA and non-NMDA receptors in rat cardiac vagal neurons in the nucleus ambiguus

While it is widely accepted that tonic and reflex changes in cardiac vagal activity play significant roles in cardiovascular function, little is known about the synaptic pathways in the brainstem responsible for the control of cardiac vagal neurons in the nucleus ambiguus (NA). In this study, we identified the principal post-synaptic receptors activated in cardiac vagal neurons upon stimulation of the nucleus tractus solitarius (NTS). Cardiac vagal neurons were identified by the presence of a retrograde fluorescent tracer and were visualized in rat brainstem slices. Perforated patch clamp techniques were used to record post-synaptic currents. NTS stimulation activated glutamatergic currents in cardiac vagal neurons with a typical delay of 8–18 ms. Post-synaptic responses were separated into NMDA and non-NMDA components using

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-2-amino-5-phophonovalerate (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. In conclusion, this study characterizes a monosynaptic glutamatergic pathway from NTS that activates NMDA and kainate/AMPA post-synaptic receptors in cardiac vagal neurons.     

Substance P innervation of neurons projecting to the paraventricular hypothalamic nucleus in the rat nucleus tractus solitarius

After injection of WGA-HRP-colloidal gold in the rat paraventricular nucleus (PVN), retrogradely labeled neurons were found mainly in the medial and commissural subnuclei of the nucleus tractus solitarius (NTS) around 0.5 mm caudal to the obex which is closely related to cardiovascular function. Electron microscopic immunohistochemistry in these areas demonstrated synaptic contacts between retrogradely labeled neurons and substance P-immunoreactive terminals. Innervation of NTS-PVN projection systems by substance P is suggested.     

Collaterals of recurrent laryngeal nerve fibres innervate the thymus: a fluorescent tracer and HRP investigation of efferent vagal neurons in the rat brainstem

The origin and course of efferent vagal fibers, which innervate the rat thymus, were investigated by a fluorescent retrograde double labeling method, using Fast blue (FB) and Diamidino yellow dihydrochloride (DY) as tracers. In the same animal, one tracer was injected into the cranial portion of the right lobe of the thymus and the other dye was deposited around the cut end of the right recurrent laryngeal nerve. The neuronal population giving origin to the recurrent nerve was mapped by using retrograde labeling with HRP applied to the central stump of the nerve. The HRP retrograde axonal transport showed that most efferent vagal fibers of the recurrent nerve have their perikarya in the nucleus retroambigualis (NRA), nucleus ambiguus (NA), and to a lesser extent in the nucleus retrofacialis (NRF). In fluorescent retrograde double labeling of thymus and recurrent laryngeal nerve both single and double labeled cells were found. The cells labeled by the injections into the thymus were colocalized with the neurons labeled by the tracer deposited in the recurrent laryngeal nerve to the NRA, NA, and NRF. Moreover along the rostrocaudal extent of the NRF and NA double labeled cells were present, showing that some of the thymic efferents are collaterals of the recurrent nerve fibers. Our experiments shown that some thymic vagal fibres originate from neurons of nucleus dorsalis nervi vagi (NDV) as demonstrated both by HRP and FB injected thymuses. The possible role of these efferents in thymic function is briefly discussed.     

大鼠耳蜗核团向前庭终末器官的神经投射研究

目的 利用顺行荧光示踪剂研究Wistar大鼠耳蜗核团向前庭终末器官的神经投射.方法 Wistar大鼠20只随机分为实验组和对照组2组,每组10只动物.顺行示踪剂组Wistar大鼠10只,10只动物在脑干耳蜗核团注射绿色顺行荧光示踪剂PHA-L,10只动物在脑干耳蜗核团注射生理盐水作为对照.5d后处死动物,获取耳蜗和前庭外周器官,EDTA脱钙后行迷路连续冰冻切片,在荧光显微镜下观察荧光细胞在前庭终末器官的分布.结果 在Wistar大鼠脑干耳蜗核团注射绿色顺行荧光示踪剂PHA-L后在注射同侧听神经和前庭终末器官发现绿色示踪剂.结论 Wistar大鼠耳蜗核团存在向前庭终末器官的神经投射.     

Different astroglial reaction between the vagal dorsal motor nucleus and nucleus ambiguus following vagal–hypoglossal nerve anastomosis in cats

The dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (NA) were both traced with horseradish peroxidase (HRP) retrograde labelling technique after vagal–hypoglossal nerve anastomosis (VHA). By light microscopy, reinnervation of the new target, viz. tongue skeletal musculature, by DMV and NA was established at 22 days postoperation (dpo) as shown by the neuronal labelling with HRP. Ultrastructurally, signs of retrograde degeneration occurred in some DMV and NA neurons between 3 and 25 days after VHA. The incidence of darkened dendrites, an early sign of dendritic loss, was more common in the DMV compared to the NA. Accompanying the neuronal alteration were drastic astrocytic reactions in the DMV, but not in the NA. Between 3 and 7 dpo, the astrocytes in the DMV showed extensively hypertrophied processes and by 22 dpo, the somata and dendrites of HRP-labelled DMV neurons, but not NA’s, appeared to be delineated by the increased lamellar astrocytic processes. Such a feature was sustained throughout the remaining postoperative intervals up to 500 dpo. It is concluded that the DMV motoneurons being autonomic in nature are probably not conducive to the newly acquired target organ. Hence, the insulation of the regenerating DMV motoneurons by the astroglial ensheathment would be vital in the neuronal remodelling and reconstruction of the vagal–hypoglossal pathway.     

Ultrastructure and synaptology of the nucleus ambiguus in the rat: The compact formation

The fine structure of the esophagomotor compact formation of the nucleus ambiguus was studied. Esophageal motoneurons are atypical in that they have extensive direct somato somatic and somato-dendritic appositions without intervening glial processes. A unique feature is the presence of finger- and leaf-like somatic protrusions which partially wrap longitudinally oriented dendrites and occasionally, small groups of dendrites and axons. The neuropil contains many longitudinally oriented, small-diameter dendrites of relatively uniform size (1.1 ± 0.4 S. D. μm in diameter). Motoneuronal somatic profiles have 0–5 synapses per profile which represents a synaptic density of 10.6 synapses per soma. Axodendritic synapses measure 0.5 × 0.7 μm in the transverse plane and are up to 3.0 μm long in the sagittal plane. Many axon terminals contact both a soma and dendrite in close apposition. Most axon terminals (>90%) contain round vesicles and form asymmetric junctions with somata and dendrites. Axon terminal degeneration after electrolytic lesions and labelling after injections of wheat germ agglutinin-horseradish peroxidase in the nucleus of the tractus solitarius show that afferent connections to the compact formation form axodendritic synapses. The ultrastructure and synaptology of esophageal motoneurons is characterized by the close apposition of somata and dendrites (somatic-dendritic bundling), and the longitudinal orientation of dendrites (dendritic bundling), axons and axon terminals in the neuropil. These features may be important morphological substrates for synchronization and coordination of esophageal motoneuronal activity and esophageal peristalsis. © 1995 Wiley-Liss, Inc.     

Nitric oxide-synthesising neurons in the central subnucleus of the nucleus tractus solitarius provide a major innervation of the rostral nucleus ambiguus in the rabbit

We describe an intramedullary nitric oxide synthase (NOS) neural pathway that projects from the nucleus tractus solitarius (NTS) to the rostral nucleus ambiguus (NA) in the rabbit. With the use of NADPH diaphorase histochemistry and NOS immunohistochemistry, a compact group of NOS-positive perikarya was identified in the central subnucleus of the NTS dorsomedial to the tractus solitarius and rostral to the obex. A dense network of NOS terminals was seen in the rostral NA. We investigated whether NOS terminals in the NA derive from NOS perikarya in the central NTS and whether the central NOS pathway links esophageal afferents and efferents. In some rabbits, the central NTS was unilaterally lesioned. In others, Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected into the central NTS, or cholera toxin-gold was injected into the NA, or cholera toxin-horseradish peroxidase (HRP) was injected into the wall of the esophagus. The medulla was subsequently processed to demonstrate PHA-L, cholera toxin-gold, HRP, and NOS reactivity. Seven days after the NTS lesion, we observed a marked decrease in the density of NOS terminals in the ipsilateral NA. After injection of PHA-L into the central NTS, a dense group of PHA-L fibres was seen in the rostral NA, principally ipsilaterally. Afferent fibres from the esophagus were found around the NOS cell bodies in the central NTS, and many of these NOS neurons were double labeled with cholera toxin-gold after injection of this tracer into the NA. NOS terminals were found around NA neurons that were retrogradely labelled from the esophagus. We conclude that the NOS neurons in the central NTS act as interneurons in a central pathway connecting esophageal afferents and efferents. © 1995 Wiley-Liss, Inc.     

Light and electron microscopic observations of a direct projection from mesencephalic trigeminal nucleus neurons to hypoglossal motoneurons in the rat

A direct projection from rat mesencephalic trigeminal nucleus (Vme) neurons to the hypoglossal nucleus (XII) motoneurons was studied using a double labeling method of anterogradely biotinylated dextran amine (BDA) tracing combined with retrogradely horseradish peroxidase (HRP) transport at both light and electron microscopic levels. BDA was iontophoresed unilaterally into the caudal Vme, and 7 days later HRP was injected into the ipsilateral tongue to label hypoglossal motoneurons. The BDA-labeled fibers were seen descended along Probst' tract and were traced to the caudal medulla. In this course, the fibers gave off axon collaterals bearing varicosities in the trigeminal motor nucleus (Vmo), the parvicellular reticular formation (PCRt), the dorsomedial portions of the subnuclei of oralis (Vodm) and interpolaris (Vidm) and in the XII ipsilaterally. The labeling of terminals was most dense in the PCRt at the levels of caudal pons and rostral medulla, which displayed a "dumbbell-shaped" form in the transverse planes. In the XII, labeled terminals were distributed mainly in the dorsal compartment of the nucleus. One hundred sixty-eight appositions made by BDA-labeled terminals on HRP-labeled motoneurons were seen in the dorsal compartment (71%) and in the lateral subcompartment (24%) of the ventral XII. Under electron microscopy BDA-labeled boutons containing clear, spherical synaptic vesicles were found to form synaptic contacts with the somata and dendrites of hypoglossal motoneurons with asymmetric specializations. The present study provides new evidence that the trigeminal proprioceptive afferent neurons terminate in the XII and make synaptic contacts with their motoneurons.     

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.     

Organization of the ambiguus nucleus in the frog (Rana esculenta)

The common root of the glossopharyngeal, vagal, and accessory nerves and the individual branches of the vagus complex were labeled with cobalt, and the organization of the ambiguus nucleus was studied. The cell column labeled through the common root extended from the upper part of the medulla to the rostral spinal cord over a distance of about 3,500 μm. The labeling of individual branches revealed four subdivisions. 1) The pharyngomotor subdivision occupied the rostral 800 μm of the cell column. It gave origin to the innervation of the pharyngeal muscles. 2) The visceromotor subdivision, consisting of small and medium-sized cells labeled by way of the visceral branches of the vagus, was found in the rostrocaudal extent of the medulla. 3) The laryngomotor subdivision extended in the obex region over a distance of more than 1,000 μm. It supplied the sphincter muscles of the larynx. The dilator laryngeal muscle was represented in the rostral part of the visceromotor subdivision. 4) The accessory nerve subdivision was located in the lower medulla and the rostral spinal cord. From the results, the following conclusions are drawn. 1) The basic organization of the frog ambiguus nucleus is comparable to that of the rat, differences in nuclear organization reflecting differences in peripheral structures. 2) The cytoarchitectonic structure of the four subdivisions innervating different peripheral targets characteristically differ from each other. 3) On the basis of its characteristic neuronal morphology, the accessory nerve nucleus is regarded as an independent structure. © 1996 Wiley-Liss, Inc.     

Synaptology of the direct projections from the nucleus of the solitary tract to pharyngeal motoneurons in the nucleus ambiguus of the rat

During the pharyngeal phase of the swallowing reflex, the nucleus of the solitary tract (NTS) receives peripheral inputs from the pharynx by means of the glossopharyngeal ganglion and is the location of premotor neurons for the pharyngeal (PH) motoneurons. The semicompact formation of the nucleus ambiguus (AmS) is composed of small and medium-sized neurons that do not project to the pharynx, and large PH motoneurons. We investigated whether the neurons in the NTS projected directly to the PH motoneurons or to the other kinds of neurons in the AmS by using the electron microscope. When wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the NTS after cholera toxin subunit B-conjugated HRP (CT-HRP) injections into the pharyngeal muscles of male Sprague-Dawley rats, many nerve terminals anterogradely labeled with WGA-HRP were found to contact PH motoneurons retrogradely labeled with CT-HRP. Most of the labeled axodendritic terminals (63%) contained pleomorphic vesicles with symmetric synaptic contacts (Gray's type II), and the remaining ones contained round vesicles with asymmetric synaptic contacts (Gray's type I). About 14% of the axosomatic terminals on PH motoneuron in a sectional plane were anterogradely labeled, and about 70% of the labeled axosomatic terminals were Gray's type II. Observations of serial ultrathin sections revealed that both the small and the medium-sized neurons received only a few labeled axosomatic terminals that were exclusively Gray's type I. These results indicate that the NTS neurons may send mainly inhibitory as well as a few excitatory inputs directly to the PH motoneurons in the AmS. J. Comp. Neurol. 393:391–401, 1998. © 1998 Wiley-Liss, Inc.     

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 of calcitonin gene-related peptide-like immunoreactivity in the nucleus ambiguus of the cat

The distribution of calcitonin gene-related peptide (CGRP) in the cat nucleus ambiguus was examined by means of a combination of horseradish peroxidase (HRP) tracing and immunohistochemical techniques. Vagal motoneurones in the nucleus ambiguus were identified by applying HRP to either the thoracic vagus or the superior laryngeal nerve or the cervical vagus. Motoneurones in the nucleus ambiguus labelled with HRP from the thoracic vagus did not contain CGRP-like immunoreactivity although CGRP-like immunoreactive cells were present in this nucleus on the same sections. In contrast, a large proportion of the motoneurones labelled from the superior laryngeal nerve and a smaller proportion of cells labelled from the cervical vagus did contain CGRP-like immunoreactivity. It is concluded that CGRP-like immunoreactivity is absent from vagal preganglionic motoneurones projecting to structures in the thorax and abdomen but is present in vagal motoneurones projecting to striated muscle of the larynx and pharynx.     

Evidence for a glycinergic pathway connecting the two cochlear nuclei: an immunocytochemical and retrograde transport study

A combined retrograde transport and immunocytochemical study was carried out to determine the projections of the large glycine-immunoreactive neurons in the ventral cochlear nucleus of the guinea pig. Horseradish peroxidase (HRP) was injected into the contralateral cochlear nucleus, and cochlear nucleus sections were developed for HRP activity followed by staining for glycine using affinity-purified anti-glycine antibodies. These studies show that glycine-immunoreactive neurons in the ventral cochlear nucleus project to the contralateral cochlear nucleus and suggest that this pathway may be glycinergic.