Medullary cells of origin of physiologically identified cardiac nerves in the dog

Individual cardiac nerves from which stimulation elicited cardioinhibition (bradycardia and negative inotropism) were identified in 24 of 38 dogs. Subsequently, 3-25 microliters of 30% horseradish peroxidase (HRP) were injected into an identified cardiac nerve. After a 3-day survival period, the medulla oblongata was processed for HRP histochemistry. Retrograde labeling was observed to be concentrated primarily in the ipsilateral nucleus ambiguus (NA) and in medium-sized neurons located ventral and lateral to the larger neurons of the principal NA cell column. This latter location was so characteristic that it has been designated the ventrolateral nucleus ambiguus (VLNA). Labeled neurons were found at all levels of the NA and VLNA and their distribution was similar irrespective of the cardiac nerve injected. Relatively few labeled neurons were observed in the dorsal motor nucleus of the vagus nerve (DMV) except after injections into the left and right recurrent cardiac nerves and the left cranial vagal nerve. In some dogs labeled cells were present only in and ventrolateral to the NA and not in the DMV, even though stimulation of the injected nerve elicited both bradycardia and negative inotropism. These results demonstrate that ventrolateral regions of the NA represent the major site of cardioinhibitory motor neurons in the dog that they can regulate both rate and force.     

Immunohistochemical characterization of cardiac vagal preganglionic neurons in the rat

Cardiac vagal preganglionic neurons (CVN) control cardiac activity by negative chronotropic, dromotropic and inotropic effects. We attempted to characterize the distribution and neuronal properties of the CVN by using double labeling with the retrograde tracer cholera toxin B subunit (CTb) and immunohistochemistry for choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP) or nitric oxide synthase (NOS). Injection of CTb into the sinoatrial ganglia resulted in many retrogradely labeled of neurons in the dorsal motor nucleus of the vagus (DMV), the compact (AmC), semicompact (AmS), loose (AmL), external (AmE) formations of the nucleus ambiguus, and the intermediate zone (IZ) between DMV and the nucleus ambiguus. Almost all CTb-labeled neurons showed ChAT immunoreactivity in the DMV, AmC, AmS, AmL and IZ, but most of the CTb-labeled neurons showed no ChAT immunoreactivity in the AmE. Most of the CTb-labeled neurons were double-labeled with CGRP immunoreactivity in the AmC, AmS and AmL, but a few double-labeled neurons were found in the DMV, IZ and AmE. A few CTb-labeled neurons were double-labeled with NOS immunoreactivity only in the DMV. No TH-immunoreactive neurons were found among the CVN. These results indicate that there are four kinds of neurons among the CVN: non-cholinergic CVN in the AmE, cholinergic and CGRP-containing CVN in the AmC, AmS and AmL, and cholinergic or cholinergic and NOS-containing CVN in the DMV.     

Characteristics of spontaneous and evoked GABAergic synaptic currents in cardiac vagal neurons in rats

Despite the importance of GABAergic input to cardiac vagal neurons the electrophysiological properties and possible origins of this innervation have not yet been studied. Individual cardiac vagal neurons were identified by a retrograde fluorescent tracer and were studied in an in vitro slice preparation using patch-clamp electrophysiology. Cardiac vagal neurons received spontaneous GABAergic inhibitory post-synaptic currents (IPSCs) that were blocked by the GABA(A) receptor antagonist bicuculline. The spontaneous presynaptic GABAergic input to cardiac vagal neurons in the nucleus ambiguus occurred at a significantly lower frequency than that recorded in cardiac vagal neurons in the dorsal motor nucleus of the vagus. To identify a possible source of the GABAergic innervation to cardiac vagal neurons the nucleus tractus solitarius was electrically stimulated. GABAergic synaptic currents in cardiac vagal neurons, in both the dorsal motor nucleus of the vagus (DMNX) and the nucleus ambiguus (NA), were consistently evoked upon stimulation of the nucleus tractus solitarius and these responses were also blocked by bicuculline.     

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.     

Substance P nerve terminals synapse upon negative chronotropic vagal motoneurons

Previous data indicate that there are anatomically segregated and physiologically independent parasympathetic ganglia on the surface of the heart which are capable of selective control of sino-atrial rate, atrio-ventricular conduction, and atrial contractility. We have injected a retrograde tracer into the cardiac ganglion which selectively regulates heart rate (the SA ganglion). Medullary tissues were processed for the histochemical visualization of retrogradely labeled neurons and for the immunohistochemical detection of the neurotransmitter substance P (SP) by dual labeling light and electron microscopic methods. Negative chronotropic retrogradely labeled cells were found in a long slender column in the ventrolateral nucleus ambiguus (NA-VL) which enlarged somewhat at the level of the area postrema. These cells were found bilaterally, but they were asymmetrically distributed. Half the animals showed a pronounced right side predominance in retrograde labeling, while the other half of the animals showed a lesser left side predominance. These observations may help to explain some of the controversy in the literature concerning the relative influence of the right and left vagus nerves on sinus rate. Ultrastructural examination demonstrated axo-somatic and axo-dendritic contacts between SP nerve terminals and retrogradely labeled negative chronotropic NA-VL neurons. SP immunoreactivity was often associated with large dense-core vesicles in terminals forming either symmetric or asymmetric synapses. These observations provide a potential anatomical substrate for the centrally mediated bradycardia elicited by microinjections of SP into the NA. SP immunoreactive terminals were also observed to make axo-somatic, axo-dendritic, and axo-axonic synapses with unlabeled neurons in NA-VL. These data suggest that SP may also modulate the activity of other vagal preganglionic neurons.     

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.     

Ultrastructural circuitry of cardiorespiratory reflexes: there is a monosynaptic path between the nucleus of the solitary tract and vagal preganglionic motoneurons controlling atrioventricular conduction in the cat

We have tested the hypothesis: (1) that presumptive negative dromotropic vagal preganglionic neurons in the ventrolateral nucleus ambiguus (NA-VL) can be selectively labelled from the heart, by injecting one of two fluorescent tracers into the two intracardiac ganglia which independently control sino–atrial (SA) rate or atrioventricular (AV) conduction; i.e., the SA and AV ganglia, respectively. The NA-VL was examined for the presence of single and/or double labelled cells. Over 91% of vagal preganglionic neurons in the NA-VL projecting to either intracardiac ganglion did not project to the second ganglion. Consequently, we also tested the hypothesis: (2) that there is a monosynaptic connection between neurons of the medial, and/or dorsolateral nucleus of the solitary tract (NTS), rostral to obex, and negative dromotropic neurons in the NA-VL. An anterograde tracer was injected into the NTS, and a retrograde tracer into the AV ganglion. The anterograde marker was found in both myelinated and unmyelinated axons in the NA-VL, as well as in nerve terminals. Axo–somatic and axo–dendritic synapses were detected between terminals labelled from the NTS, and retrogradely labelled negative dromotropic neurons in the NA-VL. This is the first ultrastructural demonstration of a monosynaptic pathway between neurons in the NTS and functionally associated (negative dromotropic) cardioinhibitory neurons. The data are consistent with the hypothesis that the neuroanatomical circuitry mediating the vagal baroreflex control of AV conduction may be composed of as few as four neurons in series, although interneurons may also be interposed within the NTS.     

Localization of motoneurons innervating the levator veli palatini muscle in the cat

Recent investigations of the nucleus ambiguus (NA) have attempted to identify motoneurons associated with the branchiomeric muscles of the larynx and pharynx. However, relatively little attention has been directed to the levator veli palatini muscle (LVP) which is critical in respiration, deglutition and eustachian tube function. Although the consensus is that cranial nerve X (vagus) innervates this muscle, some investigators have suggested that the LVP is innervated by either cranial nerve VII (facial) or IX (glossopharyngeal). The present study was designed to identify the specific location of LVP motoneurons within the brainstem. Horseradish peroxidase (HRP) was injected into the LVP of 18 cats. Following a survival period of 24–48 hours, animals were sacrificed and tissue processed according to Mesulam's TMB procedure. HRP labeled cells were located in the rostral NA both ipsilateral and contralateral to the side of injection and in the ipsilateral retrofacial nucleus (RFN). There were no labeled cells in the facial nucleus. Innervation of the LVP by cranial nerve VII would thus be excluded. This is the first report to definitively localize LVP motoneurons. Although the innervation of LVP by cranial nerve X is generally agreed upon in basic anatomy textbooks, identification of LVP motoneurons within the NA does not exclude innervation by cranial nerve IX.     

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.     

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.     

Viscerotopic representation of the subdiaphragmatic tracts of the digestive apparatus within the vagus complex in the sheep

The distribution in the brainstem and cervical spinal cord of neurons supplying the reticulum and the reticular groove, the rumen, the omasum, the abomasum, and the small and large intestine was investigated in the sheep using the fluorescent retrograde tracer technique. Only the reticulum and reticular groove were represented in the dorsal motor nucleus of the vagus nerve (DMNX), in the nucleus ambiguus (NA), and in the nucleus retroambigualis (NRA). The other forestomach, the abomasum and the small intestine were supplied by the DMNX only, with the exception of the rumen which was also innervated by the NRA. Some reticular formation neurons were found labeled after the injection of the tracer into the reticulum, the reticular groove, and the rumen. We present evidence that the reticular groove is the part of the forestomach having the widest representation, and also the richest innervation.     

Axotomy increases NADPH-diaphorase staining in rat vagal motor neurons

Left cervical vagotomy increased NADPH-diaphorase (NADPH-d) histochemical staining in neuronal perikarya of the ipsilateral dorsal motor nucleus of the vagus (dmnX) and the rostral part of the nucleus ambiguus (nAmb). This effect appeared by 2 days, was maximal around 10 days, and declined by 30 days after vagotomy. Light and dark stained perikarya occurred in dmn X ipsilateral to the vagotomy which could not be explained on the basis of the biochemical or transmitter content of these neurons. It is unlikely that the increases of NADPH-d activity resulted from changes in cholinergic transmission since vagotomy is known to decrease cholinergic enzyme function. Since vagotomy increased both the glucose metabolic rate and NADPH-d staining of dmnX and nAmb in these experiments, it is more likely that these effects represent regenerative metabolic responses to axotomy.     

Horseradish peroxidase localization of cardiac vagal preganglionic somata

Cardiac vagal preganglionic somata were labeled in cats by the horseradish peroxidase (HRP) technique. The anatomical characteristics of cell bodies with axons in the left and right cervicl vagi were compared. HRP was injected subepicardially in three groups of pentobarbital anesthetized animals. In two test groups, injections were made after a left and right cervical vagotomy, respectively. In a control group, peripheral cardiac parasympathectomies were performed prior to HRP injection. The controls served to determine the number of somata labeled by HRP uptake via vagal fibers innervating viscera closely approximating the myocardium. After a 48 h survival period the cats were reanesthetized, perfused and fixed. Brain stems were removed, cut in the transverse or sagittal plane and developed with 3,3′-diaminobenzidine.Control cats had 6.8% the number of labeled cell bodies identified in animals with an intact vagus. Thus, few labeled somata in test cats were associated with noncardia ti tissue.The number, distribution and sizes of labeled cell bodies in test cats were similar. Somata were located ipsilateral to the intact vagus in three regions: the nucleus ambiguus (NA), the dorsal motor nucleus of the vagus (DMN) and an intermediate zone (IZ) between the NA and DMN. The NA contained the maximum number of cell bodies while successively fewer somata were located in the DMN and IZ. Somata of the NA were heterogeneously distributed along the longitudinal neuraxis. The region of maximal cell body concentration was between 1.0 and 1.8 mm rostral to the obex. Somata of the DMN and IZ were homogeneously and sparsely distributed along the neuraxis. The long and short axes of NA somata were larger than corresponding dimensions of cell bodies in the DMN or IZ. However, the dimensions of somata in the DMN and IZ were similar. The identification of labeled cell bodies in three medullary regions and the size differences of the somata in these regions may reflect a central physiological organization of cardia vagal somata.     

Localization of cardiac vagal preganglionic motoneurones in the rat: Immunocytochemical evidence of synaptic inputs containing 5-hydroxytryptamine

The origin of cardiac vagal preganglionic motoneurones in the rat is still controversial and knowledge of the chemistry of synaptic inputs onto these neurones is limited. In this investigation vagal preganglionic motoneurones innervating the heart were identified by the retrograde transport of cholera toxin conjugated to horseradish peroxidase (CT-HRP) combined with the immunocytochemical localization of 5-hydroxytryptamine. Injection of CT-HRP into the myocardium resulted in the retrograde labelling of neurones primarily in the ventral regions of the nucleus ambiguus (75.1%). Labelled neurones were also distributed in a narrow band through the reticular formation extending between the dorsal motor nucleus of the vagus nerve and the nucleus ambiguus (17.3%) as well as in the dorsal motor nucleus itself (7.6%). A combination of retrograde labelling with immunocytochemistry for 5-hydroxytrypta-mine revealed that the neuronal perikarya and the dendrites of cardiac vagal motoneurones in the nucleus ambiguus were often ensheathed in 5-hydroxytryptamine-immunoreactive axonal boutons. Electron microscopic examination of this material confirmed that there were synaptic specializations between these boutons and the cardiac vagal motoneurones. The identification of 5-hydroxytryptamine-containing synaptic inputs to this population of vagal motoneurones provides further detail towards the understanding of the regulation of heart rate by the parasympathetic nervous system. © 1993 Wiley-Liss, Inc.     

Brainstem distribution of neurons with efferent projections in the cervical vagus of the dog

The distribution within the brainstem of cell bodies and efferent fibers projecting in the cervical vagus was studied with retrograde transport of horseradish peroxidase (HRP). Five to eight days after multiple microinjections of HRP into either the cervical vagosympathetic trunk or the nodose ganglion the brainstems and nodose ganglia were perfused and processed by the tetramethyl benzidine method. HRP-positive neurons were found in three brainstem regions: a dorsal cell column comprising the dorsal motor nucleus of the vagus (dmnX), a ventrolateral group in the region of nucleus ambiguus (nA), and scattered cells along a line between these columns. The density of labeled neurons was greatest within dmnX. Axons from cells of the ventrolateral column projected dorsomedially; just ventral to dmnX they turned laterally to exit the medulla in multiple rootlets. Within nA labelled neurons were distributed according to size, with larger cells more medial and smaller ones more lateral. Caudal to nA in nucleus retroambigualis and nucleus dorsalis medialis cell bodies appeared segregated into clusters.     

Induction of Fos-like protein in neurons of the medulla oblongata after electrical stimulation of the vagus nerve in anesthetized rabbit

Antibodies against the c-fos protein product Fos were used to map the first- and higher-order neurons in the rabbit medulla oblongata after electrical stimulation of the vagus nerve. Fos immunoreactivity appeared bilaterally except in the nucleus tractus solitarii. Seven areas were labeled: the nucleus tractus solitarii, the area postrema, the subnucleus lateralis caudalis magnocellularis medullar oblongata, the lateral reticular nucleus, the ambiguus nucleus, the dorsal part of the spinal trigeminal nucleus, the nucleus reticularis lateralis, the lateral border of the external cuneatus nucleus, the medial part of the inferior olivary nucleus (subnucleus β). The last two areas have never been visualized with conventional tracing techniques and may represent higher-order neurons connected to visceral vagal pathways. No labeling was observed in the nodose ganglion.     

Serotonin-mediated excitation of recurrent laryngeal and phrenic motoneurons evoked by stimulation of the raphe obscurus

Short-latency averaged responses in the recurrent laryngeal nerve (RLN) and C₅ phrenic nerve to electrical stimulation (2.5–80 μA; 2.5–160 Hz; 150 μs pulse duration) of the medullary nucleus raphe obscurus (RO) were investigated in anaesthetized, paralyzed and artificially ventilated cats. The response evoked in RLN by stimulation within RO was excitatory and consisted of a single peak. Characteristics of this response in RLN were compared with those of the delayed excitatory response in C₅ phrenic nerve, which we previously showed to be elicited by stimulation within RO. Mean latency to onset for the excitatory response in RLN was5.7 ± 0.3ms, while the delayed excitatory response in C₅ phrenic nerve occurred at7.0 ± 0.3ms. The excitatory response in both could be evoked when stimulation was applied during inspiration as well as during expiration. The stimulus threshold varied between 2.5 and 5 μA for evoked the inspiratory-phase response in each nerve. The magnitude of this response in RLN and in C₅ phrenic nerve was directly related to current intensity and was dependent upon stimulus frequency. Intravenous administration of the serotonin receptor antagonist, methysergide (0.1–2.4 mg/kg) caused significant dose-related reductions in the response in each nerve. In summary, characteristics of the evoked response in RLN and phrenic nerve are similar in several ways. Both responses are: (1) excitatory in nature, (2) elicited at small stimulus currents, (3) affected similarly by increasing stimulus current and frequency, (4) elicited by stimulation during inspiration and expiration, and (5) mediated at least in part by activation of pathways using serotonin as a neurotransmitter.     

The pre-Bötzinger complex and phase-spanning neurons in the adult rat

To characterise respiratory neurons in the pre-Bötzinger complex of adult rats, extracellular recordings were made from 302 respiratory neurons in the ventral respiratory group of sodium pentobarbitone anaesthetised adult rats. Neurons were located 0 to 1.6 mm caudal to the facial nucleus, and ventral to the nucleus ambiguus. The pre-Bötzinger complex comprised expiratory neurons (22%, 22/100), inspiratory neurons (37%, 37/100) and phase-spanning neurons (41%, 41/100). In contrast, 80% (125/157) of Bötzinger neurons were expiratory, and 80% (36/45) of rostral ventral respiratory group neurons were inspiratory. Rostrocaudally, the pre-Bötzinger complex extended about 400 μm, starting at the caudal pole of the nucleus ambiguus compact formation. The pre-Bötzinger complex was also characterised by a predominance of propriobulbar neurons (81%, 13/16). Furthermore, 68% (33/48) of expiratory–inspiratory neurons found were located within the pre-Bötzinger complex. The variety of neuronal subtypes in the pre-Bötzinger complex, including many firing during the expiratory–inspiratory transition is consistent with the hypothesis that this nucleus plays a key role in respiratory rhythm generation in the adult rat.     

The central distribution of the cervical vagus nerve and gastric afferent and efferent projections in the rat

The medullary distribution of afferent fibers and cells of origin of the cervical vagal trunk and of the vagal innervation of the stomach have been studied using the anterograde and retrograde transport of horseradish peroxidase (HRP). Injections of HRP were made into the cervical vagus nerve, the stomach wall, the proximal small intestine, or the peritoneal cavity. Two to four days following the injections, the rats were perfused and the medullae oblongatae and nodose ganglia were processed using the tetramethyl benzidine method. Cervical vagus nerve injections of HRP resulted in heavy anterograde labeling in the ipsilateral nucleus of the tractus solitarius (NTS) and the commissural nucleus. Lighter labeling was seen in these regions on the contralateral side, but did not extend as far rostrally in the NTS. Labeling was also seen in the area postrema. Retrogade labeling of somata was present in the ipsilateral side in the nodose ganglion, throughout the whole extent of the dorsal motor nucleus of the vagus, much of the nucleus ambiguus and in rostral levels of the cervical spinal cord. After stomach injections, labeling indicative of afferent fibers was observed bilaterally in the dorsomedial and medial portions of the NTS and in the commissural nucleus. Labeled efferent fibres arose from neurons in the dorsal motor nucleus of the vagus, nucleus ambiguus and the cervical spinal cord. Retrogradely labeled somata were found bilaterally, throughout the rostrocaudal length of the dorsal motor nucleus in all cases with stomach injections. In some, but not all cases, labeled somata were seen bilaterally in compact areas within the nucleus ambiguus, particularly rostrally. Control injections of HRP into the intestinal wall and peritoneal cavity indicated that the stomach was the primary source of afferent and efferent labeling in the medulla following subdiaphragmatic injections.     

CNS cell groups projecting to pancreatic parasympathetic preganglionic neurons

The CNS cell groups that project to the pancreatic parasympathetic preganglionic neurons were identified by the viral retrograde transneuronal labeling method. Pseudorabies virus (PRV) was injected into the pancreas of C8 spinal rats and after 6 days survival, the animals were perfused and their brains processed for immunohistochemical detection of PRV. Parasympathetic preganglionic neurons of the dorsal vagal nucleus were retrogradely labeled with PRV. Several CNS cell groups consistently contained transneuronally labeled neurons. In the medulla oblongata, labeled neurons were found in the nucleus tractus solitarius, area postrema, paratrigeminal nucleus, lateral paragigantocellular reticular nucleus, raphe pallidus and obscurus nuclei, C3 region and scattered cells in the ventral medullary reticular formation. In the pons, the A5 cell group, Barrington's nucleus and the subcoeruleus region contained labeled neurons. Only an occasional labeled cell was identified in the parabrachial nucleus. In the midbrain, almost no labeling was found except for an occasional neuron in the central gray matter. In the diencephalon, labeling was found in the paraventricular hypothalamic nucleus (PVN) as well as in the lateral hypothalamic nucleus at two levels (one at the level of the PVN and the other at the level of the subthalamic nucleus). in addition, the perifornical and dorsal hypothalamic nuclei contained labeled neurons. A few cells were found in the peripheral part of the dorsomedial hypothalamic nucleus. No labeling was seen in the ventromedial hypothalamic nucleus. In the telencephalon, the central amygdaloid nucleus and the bed nucleus of the stria terminalis were labeled.