TRH regulation of tracheal tension through vagal preganglionic motoneurons

TRH-immunoreactive nerve terminals innervate the ambiguous nucleus in the rabbit. Vagal preganglionic motoneurons that innervate the trachea, were revealed by HRP histochemistry in the rostral part of the ambiguous nucleus and the dorsal motor nucleus of the vagus. HRP histochemistry plus TRH immunocytochemistry revealed that TRH-immunoreactive axon terminals synapsed on ambiguous nucleus neurons retrogradely labeled by HRP injection into tracheal smooth muscle and the superior laryngeal nerve. Microinjection of 50 ng TRH into the rostral ambiguous nucleus caused slight dilation followed by constriction, which was inhibited by atropine and vagotomy. Results show that central TRH-containing neurons regulate tracheal tension through synapses on vagal preganglionic motoneurons that innervate tracheal smooth muscle.     

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.     

Medullary origin of vagal preganglionic axons to the heart of the cat

It is apparent from the literature that a controversy exists concerning the site of origin of cardiac vagal preganglionic axons. Physiological studies have suggested that the location of these neurons may be different in different species and there has been disagreement between physiological and anatomical findings in the same species. We now present anatomical and neurophysiological studies suggesting that in the cat cardiac vagal preganglionic neurons are located in two medullary regions: the areas of the dorsal motor nucleus of the vagus (DMV) and of the nucleus ambiguus (AMB). This suggestion is based on the following observations. Firstly, after application of horseradish peroxidase to the right cardiac branches of the vagus nerve, labeled neurons were found primarily in the regions of te DMV and AMB. Additional scattered neurons were found in the reticular formation between these two nuclei. Secondly, following injections of tritiated amino acids into either the DMV or AMB, labeled vagal fibers were found in the atrial myocardium. Finally, electrical stimulation of the right cardiac branches of the vagus nerve antidromically activated DMV or AMB, labeled vagal fibers were found in the atrial myocardium. Finally, neurons in the DMV and AMB regions with latencies corresponding to conduction velocities of B-fibers. In addition, these neurons were orthodromically excited by electrical stimulation of the carotid sinus and aortic depressor nerves.     

Distribution of motoneurons in the brain stem of monkeys, innervating the larynx

Following HRP (Horseradish Peroxidase) injections to cricothyroid muscle, recurrent laryngeal nerve and the vagal nerve at the level of nodose ganglion, labeled motoneurons were found to show a characteristic distribution in the brain stem of the monkey. Cricothyroid motoneurons extended from a level caudal to the facial nucleus to a level caudal to the middle part of the inferior olivary nucleus (IO) and were scattered around the outer area of nucleus ambiguus (Amb). Motoneurons supplying the recurrent laryngeal nerve were found between a level rostral to the middle of IO and its caudal end. Distribution was compact in the lateral part, but was scattered in the dorsomedial part of Amb. On injection of HRP into the nodose ganglion of the vagal nerve, labeled motoneurons were seen in two cell columns: In the Amb and in the dorsal motor nucleus of the vagus. The former extended from the rostral level of IO to the caudal end of IO, also showing connections with the retroambigual nucleus.     

Spinal origins of preganglionic B and C neurons that innervate paravertebral sympathetic ganglia nine and ten of the bullfrog

These experiments were designed to characterize the distribution, morphology, and number of spinal preganglionic neurons that selectively innervate the B- and C-type sympathetic neurons in paravertebral ganglia 9 and 10 of the bullfrog. For this purpose, horseradish peroxidase (HRP) was applied to the anterior end of the sectioned sympathetic chain between ganglia 8 and 9. Subsequent retrograde axonal transport of the HRP labeled ipsilateral spinal neurons whose cell bodies form a column having rostral and caudal boundaries that are, respectively, just caudal to the level of spinal nerve 4 and midway between the entry zones of spinal nerves 7 and 8. In all segments, the labeled preganglionic somata were found in the lateral half of the spinal gray and slightly dorsal to the central canal; a position analogous to that of the intermediolateral cell column in mammals. Most labeled preganglionic neurons were multipolar in shape, and the cell bodies lying between spinal nerves 4 and 5 were, on average, larger than those found between spinal nerves 7 and 8. In transverse sections that were cut near spinal nerve 5, the axons of preganglionic neurons could be seen to exit the cord through ventral roots. Counts of labeled preganglionic neurons indicate that an average +/- S.D. of 338 +/- 89 cells innervate ganglia 9 and 10. Selective labeling of preganglionic B neurons, by cutting spinal nerves 7 and 8 central to their rami communicantes at the time of HRP application, revealed an average +/- S.D. of 137 +/- 31 cells that lie exclusively between spinal nerves 4 and 6. By contrast, selective labeling of preganglionic C neurons, by cutting the sympathetic chain rostral to ganglion 7 at the time of HRP application, revealed an average +/- S.D. of 187 +/- 77 cells in an adjacent portion of the preganglionic column that is bounded by spinal nerve 6 and by a point midway between spinal nerves 7 and 8. These results thus demonstrate a clear segmental segregation between the preganglionic B and C neurons that innervate ganglia 9 and 10.     

The topographical organization of the vagal motor column in the elasmobranch fish, Scyliorhinus canicula L

The location within the brainstem of vagal preganglionic motoneurons has been determined in the dogfish Scyliorhinus canicula L. by means of the retrograde transport of horseradish peroxidase and cobalt applied to the vagus nerve and its component branches. Labelled vagal motoneurones were located in the ipsilateral caudal rhombencephalon from 2.1 mm caudal to 2.73 mm rostral to obex. The motoneurons of the vagal motor column are arranged as four distinct groups. Caudal to obex the column contains dorsomedial and ventromedial divisions, whilst rostrally it consists of a single rostromedial division and a short lateral division. The cells in the ventromedial division are approximately twice the size (mean area 1,094 microns 2) of the other vagal neurons. The dorsomedial division contains neurons that supply the heart and viscera; the ventromedial division supplies the viscera. The heart is also innervated by the neurons of the lateral division and the visceral nerve also receives axons from the rostromedial division. All neurons supplying axons to the gill arches are located in the rostromedial division. There is a sequential topographical representation of the vagus nerve in the vagal motor column. Neurons supplying the gastrointestinal tract are located caudally; those supplying the cardiac nerves lie in the midportion of the column, and the proximal supply to the gills is given by the most rostral neurons. There is some overlap between the pools of neurons supplying adjacent branches of the vagus.     

Brainstem origin of preganglionic cardiac motoneurons in the muskrat

The muskrat, an aquatic rodent with a brisk and reliable diving response, shows a remarkable bradycardia after nasal stimulation. However, the medullary origin of cardiac preganglionic motoneurons is unknown in this species. We injected fat pads near the base of the heart of muskrats with a WGA-HRP solution to label retrogradely preganglionic parasympathetic neurons that project to the cardiac plexi. Results showed that the preponderance of labeled neurons was in ventrolateral parts of the medulla from 1.5 mm caudal to the obex to 2.0 mm rostral. Eighty-nine percent of the labeled neurons were located bilaterally in the external formation of the nucleus ambiguus, 5.6% were in the lateral extreme of the dorsal motor nucleus of the vagus nerve and 5.3% were found in the intermediate area in between these two nuclei. Although controversy still exists concerning the medullary origin of preganglionic cardiac motoneurons, our results from muskrats agree with those from most other species where preganglionic cardiac motoneurons were located just ventral to the nucleus ambiguus.     

Localization and acetylcholinesterase content of vagal efferent neurons

The acetylcholinesterase (AChE) content of rat vagal efferent neurons was studied. Retrograde transport of horseradish peroxidase (HRP) by cut vagal axons provided a means for localizing efferent cell bodies; tissue sections were then processed for the simultaneous visualization of HRP and AChE. A dorsal vagal efferent column contained the dorsal motor nucleus of the vagus, as a primary component, and extended caudally into the upper cervical spinal cord. A ventral column contained neurons in the nucleus ambiguus and the surrounding reticular formation. Although most of the vagal efferent neurons stained with moderate to heave intensity for AChE there were some HRP-labeled cells that contained little AChE and a small percentage in which AChE was absent. In spite of the fact that AChE has been demonstrated in certain non-cholinergic neurons, it has also been found in all cholinergic neurons. Therefore, the presence of AChE has been regarded as a necessary (but not sufficient) component for identifying cholinergic neurons. The absence of AChE in a small percentage of the vagal efferent neurons indicates that some preganglionic parasympathetic fibers in the vagus nerve are not cholinergic.     

Association of neurotensin binding sites with sensory and visceromotor components of the vagus nerve

Specific neurotensin (NT) binding sites were recently shown to be highly concentrated in the nucleus of the solitary tract (NTS), which receives primary vagal afferents, and in the dorsal motor nucleus of the vagus (DMN), which contains the cell bodies of origin of vagal preganglionic neurons. To investigate the relationship of these binding sites with sensory and visceromotor components of the vagus nerve, they were labeled here in vitro, using monoiodo[Tyr3]neurotensin (125I-NT) and visualized by light microscopic radioautography in the dorsomedial medulla of both intact and unilaterally vagotomized rats, in the nodose ganglia of intact animals, and in ligated vagus nerves. Unilateral vagotomy performed above the nodose ganglion resulted in a significant ipsilateral decrease in 125I-NT binding within both the NTS and the DMN, suggesting that NT binding sites were associated with both primary afferent fibers and preganglionic nerve cell bodies. The selective radioautographic labeling of a subpopulation (approximately 15%) of neuronal perikarya in the nodose ganglion confirmed that a proportion of vagal afferent neurons contained NT binding sites. Following vagus nerve ligation, a pile up of radiolabeled NT binding sites was observed on both sides of the nerve crush, indicating that NT receptor components were transported both anterogradely and retrogradely along fibers of the vagus nerve. We conclude that NT receptors are synthesized and transported within a subpopulation of afferent and efferent components of the vagus nerve and that NT may therefore act presynaptically upon vagal axon terminals in both central and peripheral nervous systems.     

Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla

Projections from the nucleus tractus solitarii (NTS) to autonomic control regions of the ventrolateral medulla, particularly the nucleus reticularis rostroventrolateralis (RVL), which serves as a tonic vasomotor center, were analyzed in rat by anterograde, retrograde, and combined axonal transport techniques. Autonomic portions of the NTS, including its commissural, dorsal, intermediate, interstitial, ventral, and ventrolateral subnuclei directly project to RVL as well as to other regions of the ventrolateral medulla. The projections are organized topographically. Rostrally, a small cluster of neurons in the intermediate third of NTS, the subnucleus centralis, and neurons in proximity to the solitary tract selectively innervate neurons in the retrofacial nucleus and nucleus ambiguus. Neurons generally located in more caudal and lateral sites in the NTS innervate the caudal ventrolateral medulla (CVL). The RVL, CVL, and nucleus retroambiguus are interconnected. A combined retrograde and anterograde transport technique was developed so as to prove that projections from the NTS to the ventrolateral medulla specifically innervate the region of RVL containing neurons projecting to the thoracic spinal cord or the region of the nucleus containing vagal preganglionic neurons. When the retrograde tracer, fast blue, was injected into the thoracic spinal cord, and wheat germ agglutinin-conjugate horseradish peroxidase (HRP) was injected into the NTS, anterogradely labeled terminals from the NTS surrounded the retrogradely labeled neurons in the RVL and in the nucleus retroambiguus in the caudal medulla. Among the bulbospinal neurons in the RVL innervated by the NTS were adrenaline-synthesizing neurons of the C1 group. When fast blue was applied to the cervical vagus, and HRP was injected into the NTS, anterogradely labeled terminals from the NTS surrounded retrogradely labeled neurons in the rostral dorsal motor nucleus of the vagus, the region of the nucleus ambiguus, the retrofacial nucleus, and the dorsal portion of the RVL, a region previously shown to contain cardiac vagal preganglionic neurons. This combined anterograde and retrograde transport technique provides a useful method for tracing disynaptic connections in the brain. These data suggest that the RVL is part of a complex of visceral output regions in the ventrolateral medulla, all of which receive afferent projections from autonomic portions of the NTS. Bulbospinal neurons in the RVL, in particular the C1 adrenaline neurons, may provide a portion of the anatomic substrate of the baroreceptor and other visceral reflexes.     

Brainstem cells of origin of the cervical vagus and cardiopulmonary nerves in the neonatal pig (Sus scrofa)

The distribution of the cells of origin of the cervical vagus and cardiopulmonary nerves has been studied in neonatal piglets (Sus scrofa) ranging in age from 1 to 60 days. Cardiopulmonary nerves were identified physiologically and anatomically prior to injection of horseradish peroxidase (HRP) into the nerves. Following injection of HRP into the cervical vagus nerve retrogradely labeled neurons were present in the dorsal motor nucleus of the vagus nerve (DMV), the nucleus of the solitary tract, the nucleus ambiguus (NA), ventrolateral to the NA and in an intermediate zone between the DMV and the NA. Two unique clusters of neurons were also retrogradely labeled after injections into the vagus nerve. One group was located lateral to the most caudal levels of the DMV and extended as far caudally as the C1 spinal segment. The second distinctive group was located ventrolateral to the nucleus ambiguus in a cell column identified as the ventrolateral nucleus ambiguus (VLNA). After injections of HRP into cardiopulmonary nerves, the majority of neurons were found in the VLNA and the distinct clusters of neurons in this cell column were particularly heavily labeled. Small numbers of cells were labeled in the DMV and NA and none were labeled in the solitary nucleus after cardiopulmonary nerve injections. There were no apparent age-related differences in the degree or distribution of retrograde labeling.The distribution of neurons in the medulla oblongata projecting into cardiopulmonary nerves in the piglet is similar to that described in other species, i.e., the nucleus ambiguus, particularly its ventrolateral cell column, is the primary site of cardiomotor neurons. In addition, in the piglet there is a morphologically distinct cluster of cells related to the heart, and possibly the lungs, which does not appear to be present in other species.     

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.     

Brain stem projections of sensory and motor components of the vagus complex in the cat: I. The cervical vagus and nodose ganglion

The motor and sensory connections of the cervical vagus nerve and of its inferior ganglion (nodose ganglion) have been traced in the medulla oblongata of 32 adult cats with the neuroanatomical methods of horseradish peroxidase (HRP) histochemistry and amino acid autoradiography (ARG). In 14 of these subjects, an aqueous solution of HRP was applied unilaterally to the central end of the severed cervical vagus nerve. In 13 other cases, HRP was injected directly into the nodose ganglion. Three of these 13 subjects had undergone infranodose vagotomy 6 weeks prior to the HRP injection. A mixture of tritiated amino acid was injected into the nodose ganglion in five additional cats. The retrograde transport of HRP yielded reaction product in nerve fibers and perikarya of parasympathetic and somatic motoneurons in the medulla oblongata. Furthermore, a tetramethyl benzidine (TMB) method for visualizing HRP enabled the demonstration of anterograde and transganglionic transport, so that central sensory connections of the nodose ganglion and of the vagus nerve could also be traced. The central distribution of silver grain following injections of tritiated amino acids in the nodose ganglion corresponded closely with the distribution of sensory projections demonstrated with HRP, thus confirming the validity of HRP histochemistry as a method for tracing these projections. The histochemical and autoradiographic experiments showed that the vagus nerve enters the medulla from its lateral aspect in multiple fascicles and that it contains three major components—axons of preganglionic parasympathetic neurones, axons of skeletal motoneurons, and central processes of the sensory neurons in the nodose ganglion. Retrogradely labeled neurons were seen in the dorsal motor nucleus of X(dmnX), the nucleus ambiguus (nA), the nucleus retroambigualis (nRA), the nucleus dorsomedialis (ndm) and the spinal nucleus of the accessory nerve (nspA). The axons arising from motoneurons in the nA did not traverse the medulla directly laterally; rather, all of these axons were initially directed dorsomedially toward the dmnX, where they formed a hairpin loop and then accompanied the axons of dmnX neurons to their points of exit. Afferent fibers in the vagus nerve reached most of the subnuclei of the nTS bilaterally, with the more intense labeling being found on the ipsilateral side. Labeling of sensory vagal projections was also found in the area postrema of both sides and around neurons of the dmnX. These direct sensory projections terminating within the dmnX may provide an anatomical substrate for vagally mediated monosynpatic reflexes. Following deefferentiation by infranodose vagotomy 6 weeks prior to HRP injections into the nodose ganglion, a number of neurons in the dmnX were still intensely labeled with the HRP reaction product. The axons of these HRP-labeled perikarya may constitute the bulbar component of the accessory nerve.     

The origins of innervation of the esophagus of the dog

This study defined the origins of extrinsic efferent and afferent innervation of the normal canine esophagus. When all the layers of the wall of the 3 esophageal regions (cervical, thoracic and abdominal) were injected with horseradish peroxidase (HRP), labeled nerve cells were found in the nucleus ambiguus (NA) and parasympathetic nucleus of X (PX) of the brainstem. Most labeled cells in the NA were located in the compact column (retrofacial nucleus) while labeled cells in the PX were located in separate rostral and caudal areas. There was no somatotopic organization in either the NA or PX. Labeled sympathetic postganglionic neurons were found in the cranial cervical, middle cervical, cervicothoracic, thoracic sympathetic trunk and celiacomesenteric ganglia. The HRP injection of the esophageal wall labeled sensory cell bodies in the glossopharyngeal, proximal and distal vagal, and C2-T6 spinal ganglia. There was no discernible pattern of distribution of labeled cells in the autonomic or sensory ganglia. When the HRP injections were confined to the mucosa-submucosa layers of the thoracic esophagus, a small number of labeled cells were identified in the NA; however, no labeled cells were found in the NA when injections were confined to the mucosa-submucosa of either the cervical or abdominal esophageal regions. With these confined injections, the labeled nerve cells appeared in the rostral part of the PX. Thus, it appeared that the internal tunics of the esophagus (i.e., the mucosa and submucosa) were innervated by neurons in the rostral PX while the muscular tunic was innervated by neurons in the caudal PX and the rostral NA. After mucosa-submucosa injections, labeled sympathetic neurons appeared in the same ganglia that were identified after whole wall injections and these had a similar random distribution. These injections also labeled neurons in the glossopharyngeal, proximal vagal, and distal vagal ganglia, but unlike the whole wall injections there was no labeling in the spinal ganglia. This suggested that the labeled cells of the spinal ganglia seen after whole wall injections conveyed impulses from the tunica muscularis and serosa.     

Monoaminergic and peptidergic axonal projections to the vagal motor cell column of a teleost, the filefish Stephanolepis cirrhifer

In an immunohistochemical study, the vagal motor nucleus of a teleost, the filefish Stephanolepis cirrhifer, could be divided into a rostral part and a caudal part, and the former into a dorsolateral group and a ventromedial group. The dorsolateral group consisted of neurons immunoreactive for calcitonin gene-related peptide, whereas the ventrolateral-caudal group was negative for calcitonin gene-related peptide. The latter group was retrogradely labeled after dextran amine injection to the visceral ramus of the vagus nerve, suggesting that it is a general visceral efferent column, made up of parasympathetic preganglionic neurons, whereas the dorsolateral rostral group is a special visceral efferent column. In the general visceral efferent column, a dense concentration of nerve fibers immunoreactive for serotonin, tyrosine hydroxylase, cholecystokinin-8, and substance P, and a small number of fibers immunoreactive for neuropeptide Y was observed. Perikarya in contact with varicose terminals immunoreactive for these substances were frequently seen. In contrast, in the special visceral efferent column, only a moderate concentration of neuropeptide Y-immunoreactive nerve fibers and a sparse distribution of fibers immunoreactive for tyrosine hydroxylase were observed. Perikarya in contact with varicose terminals immunoreactive for these substances were rare. These results suggest that the vagal parasympathetic preganglionic neurons might receive multiple inputs of monoaminergic and peptidergic fibers involved in the regulation of the visceral organs. On the other hand, monoaminergic and peptidergic afferent fibers might be of much less significance in the activity of the special visceral efferent component of the vagus nerve.     

Fibers supplying the laryngeal musculature in the cranial root of the rabbit accessory nerve: Nucleus of origin, peripheral course, and innervated muscles

The location of the rabbit laryngeal motoneurons whose axons traverse the cranial root of the accessory nerve was studied with injection of HRP or nuclear yellow into the laryngeal muscles in combination with the intracranial severing of either the rootlets of the vagus nerve or those of the cranial root. The motoneurons were located in the diffuse cell group that forms a subnucleus occupying the caudal two-thirds of the nucleus ambiguus and sending fibers to the inferior laryngeal nerve. The caudal one-third of the diffuse cell group supplying the lateral cricoarytenoid muscle, was occupied only by these motoneurons, whereas in its rostral two-thirds, they were intermingled with motoneurons having axons that traversed the vagal rootlets. The thyroarytenoid and posterior cricoarytenoid motoneurons are present in the rostral two-thirds of the diffuse cell group; axons of the former traversed the rootlets of the cranial root, and of the latter traversed the vagal rootlets. On the other hand, the medial scattered cell group, located in the rostral one-third of the nucleus ambiguus and sending fibers to the cricothyroid muscle via the superior laryngeal nerve, contained only motoneurons with axons traversing the vagal rootlets. The above findings clarified that fibers of the cranial root enter the inferior laryngeal nerve after joining the vagus, and then reach the adductor muscles for the vocal fold, with their neurons of origin in a caudal portion of the nucleus ambiguus. The vagal rootlet fibers, with their neurons of origin situated more rostrally in the nucleus, innervate the tensor and abductor muscles via the superior and inferior laryngeal nerve, respectively.     

Uptake sites of horseradish peroxidase after injection into peritoneal structures: defining some pitfalls

Following injection of horseradish peroxidase (HRP) either into the jejunal wall or the peritoneal cavity, neurons in the dorsal motor nucleus of the vagus nerve, celiac, nodose and spinal ganglia, and ventral and lateral horns of the spinal cord from the mid-thoracic to lumbar segments were labeled. When HRP was injected into the wall of the exteriorized gut, neurons of the spinal cord were not labeled. Furthermore, there was a significant decrease in the number of labeled neurons in the dorsal motor nucleus and ganglia examined. These results indicate that HRP injected into the intestinal wall could leak into the peritoneal cavity and be taken up and transported to neuronal cell bodies by nerve fibers not terminating in the injection area. The leakage of HRP to nearby abdominal structures and its subsequent uptake by nerve fibers is attributed to the lack of a diffusion barrier across the surfaces of the intestinal wall and the abdominal structures. It is suggested that in applying the HRP techniques for the study of neuronal connections in the peripheral nervous system, it is essential that carefully planned control experiments be undertaken which can overcome the problem of mislabeling due to diffusion from the injection site.     

Localization of neurons in the rat superior cervical ganglion that project into different postganglionic trunks.

Horseradish peroxidase (HRP) was used to determine whether neurons in the rat superior cervical ganglion (SCG) are localized in regions of the ganglion as a function of the postganglionic trunk they utilize. In separate experiments, each of the two major postganglionic trunks was cut 1-3 mm from the SCG and solid HRP was applied to the cut end proximal to the ganglion. The results demonstrated that the cell bodies of neurons whose axons project out the internal carotid nerve (ICN) were located primarily in the rostral part of the ganglion. Cell bodies of neurons whose axons project out the external carotid nerve (ECN) were located primarily in the caudal part. The total percentages of neuronc with axons in the ICN and ECN were about 35% and 45%, respectively. When HRP was applied to both these trunks, 73% of the neurons in the SCG were labeled. In the caudal portion of the ganglion, an additional group of neurons was observed whose axons project into the cervical sympathetic trunk. Control studies indicated that the neuronal labeling observed in our experiments was due to retrograde axonal transport rather than the direct uptake of HRP by neuronal cell bodies. Thus, neuronal subpopulations exist in specific regions of the rat SCG. The significance of these results to biochemical and electrophysiological studies is discussed.     

Retrograde axonal transport of horseradish peroxidase in peripheral autonomic nerves.

An exogeneous marker protein, horseradish peroxidase (HRP) was used to race peripheral autonomic pathways in adult guinea pigs and cats. Small doses of HRP were injected into various organs and after a brief survival period, HRP activity appeared in the perikarya of autonomic neurons that supplied each injection site. After injection of HRP into the anterior chamber of the eye, reaction product was detected in the postganglionic sympathetic neurons of the superior cervical sympathetic ganglion. In another experiment, HRP reaction product was found in the cell bodies of the preganglionic sympathetic neurons that supply the adrenal medulla. These were located in the lateral gray column of the spinal cord at T6 and T7 segmental levels. Reaction product appeared in intramural postganglionic parasympathetic neurons close to an injection site in the wall of the urinary bladder and in a similiar situation in Meissner's ganglia of the ileum. Following injection into the walls of the stomach and ileum, HRP labelled cells were detected in the nodose ganglion of the vagus and in preganglionic parasympathetic neurons in the dorsal motor nucleus of this nerve. After injection into the subepicardial tissue of the heart, reaction product appeared in the stellate ganglion and also in an upper thoracic dorsal root ganglion. These data suggest that HRP is taken up by peripheral autonomic nerves of all types, and then undergoes rapid retrograde axonal transport to the perikaryon. It appears, therefore, that HRP may be useful in tracing both motor and sensory peripheral autonomic pathways.