Axotomy alters putative neurotransmitters in visceral sensory neurons of the nodose and petrosal ganglia.

Acute peripheral axotomy of the visceral sensory neurons of the vagus and glossopharyngeal nerves removes peripheral depolarizing and trophic influences to their sensory ganglia. To study axotomy-induced changes in the putative neurotransmitters of visceral sensory neurons, rats were sacrificed 1, 3, 7 or 14 days after transection of either the cervical vagus and superior laryngeal nerves (to affect peripheral axotomy of the nodose ganglion) or the glossopharyngeal and carotid sinus nerves (to affect peripheral axotomy of the petrosal ganglion). The numbers of tyrosine hydroxylase (TH)-immunoreactive (ir), vasoactive intestinal peptide (VIP)-ir, calcitonin-gene-related peptide (CGRP)-ir, and substance P (SP)-ir neurons in the respective ganglia were analyzed in axotomized and control ganglia. In the nodose ganglion, axotomy of the cervical vagus resulted in a rapid (by 1 day) reduction in the number of TH-ir cells, whereas VIP-ir neurons were dramatically increased in number by 3 days. CGRP- and SP-ir cells in the nodose ganglion were relatively unaffected by axotomy. In the petrosal ganglion, axotomy of the glossopharyngeal and carotid sinus nerves greatly reduced the number of TH-ir cells but did not alter the number VIP-ir neurons. CGRP- and SP-ir neurons in the petrosal ganglion were reduced in number by axotomy. Thus, axotomy of visceral sensory neurons differentially changed the content and perhaps the expression of putative transmitters. Differential changes were seen among transmitters in a single ganglia and between ganglia. These data demonstrate the plasticity of putative neurotransmitter systems in visceral afferent systems of adult rats.     

Studies on the coexistence of substance P with other putative transmitters in the nodose and petrosal ganglia.

Visceral afferent neurons of the nodose and petrosal ganglia are immunoreactive (ir) for many neurotransmitters [e.g., substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), and dopamine (tyrosine hydroxylase-ir; TH)]. Coexistence of SP-ir with NKA-, CGRP-, or TH-ir was studied in individual neurons of the rat ganglia using fluorescence immunocytochemistry. SP- and NKA-ir were present in equal numbers of cells and were consistently colocalized. SP- and CGRP-ir were found to be similarly distributed in scattered cells, concentrated mostly in the rostral pole of the nodose ganglion and in the petrosal ganglion. SP-ir completely coexisted with CGRP-ir. However, there was at least twice the number of CGRP-ir neurons as SP-ir neurons, and thus CGRP-ir neurons that did not contain SP-ir were also present. In contrast, SP- and TH-ir had different distributions in both the nodose and the petrosal ganglia. SP-ir was located in the more rostral regions of both the nodose and petrosal ganglia, whereas TH-ir was detected throughout the entire nodose ganglion and only in the most caudal region of the petrosal ganglion. There was no coexistence of SP- and TH-ir. These data demonstrate the differential localization and coexistence of putative transmitters in visceral sensory neurons in the nodose and petrosal ganglia.     

Streptozotocin-induced diabetes reduces retrograde axonal transport in the afferent and efferent vagus nerve

Diabetes-induced alterations in nerve function include reductions in the retrograde axonal transport of neurotrophins. A decreased axonal accumulation of endogenous nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the vagus nerve of streptozotocin (STZ)-induced diabetic rats was previously shown. In the current study, no changes in the NGF and NT-3 protein or mRNA levels in the stomach or atrium, two vagally innervated organs, were noted after 16 or 24 weeks of diabetes. Moreover, the amounts of neurotrophin receptor (p75, TrkA, TrkC) mRNAs in the vagus nerve and vagal afferent nodose ganglion were not reduced in diabetic rats. These data suggest that neither diminished access to target-derived neurotrophins nor the loss of relevant neurotrophin receptors accounts for the diabetes-induced alteration in the retrograde axonal transport of neurotrophins. To assess whether diabetes causes a defect in axonal transport that may not be specific to neurotrophin transport, we studied the ability of a neuronal tracer (FluoroGold, FG) to be retrogradely transported by vagal neurons of control and diabetic rats. After vagal target tissue (stomach) injections of FG, the numbers of FG-labeled afferent and efferent vagal neurons were counted in the nodose ganglion and in the dorsal motor nucleus of the vagus, respectively. After 24 weeks of diabetes, FG was retrogradely transported to more than 50% fewer afferent and efferent vagal neurons in the STZ-diabetic compared to control rats. The diabetes-induced deficit in retrograde axonal transport of FG is likely to reflect alterations in basic axonal transport mechanisms in both the afferent and efferent vagus nerve that contribute to the previously observed reductions in neurotrophin transport.     

The origin of catecholaminergic nerve fibers in the subdiaphragmatic vagus nerve of rat.

It is known that the vagus nerve contains catecholaminergic fibers. However, the origin of these fibers has not been systematically examined. In this study, we addressed this issue using retrograde tracing from the subdiaphragmatic vagus nerve combined with immunocytochemistry. The cervical and thoracic sympathetic trunk ganglia, the nodose ganglia and the dorsal motor nucleus of the vagus nerve were examined following injection of Fluoro-Gold or cholera toxin horseradish peroxidase conjugate into the trunks of the subdiaphragmatic vagus nerve of rats. Numerous retrogradely labeled neurons were seen in the nodose ganglion and the dorsal motor nucleus of the vagus nerve. Very few labeled neurons were found in the sympathetic ganglia (less than 0.06% of the neurons in either superior cervical ganglion or cervicothoracic ganglion were retrogradely labeled). Double labeling with immunofluoresence for catecholamine synthesizing enzymes revealed that: (1) 92% of all Fluoro-Gold retrogradely labeled tyrosine hydroxylase immunoreactive neurons were found in parasympathetic sources (75% in the dorsal motor nucleus of the vagus nerve and 17% in the nodose ganglia), and only 8% in the cervicothoracic sympathetic ganglia; (2) 12% of the retrogradely labeled catecholaminergic neurons in the dorsal motor nucleus of the vagus nerve were also dopamine-beta-hydroxylase immunopositive neurons; (3) 70% of the retrogradely labeled neurons in the sympathetic ganglia were tyrosine hydroxylase immunopositive and 54% of these catecholaminergic neurons contained dopamine-beta-hydroxylase, while 30% of the retrogradely labeled neurons were non-catecholaminergic neurons. These results indicate that catecholaminergic fibers in the abdominal vagus nerve are primarily dopaminergic and of parasympathetic origin, and that only an extremely small number of these fibers, mostly noradrenergic in nature, arise from postganglionic sympathetic neurons.     

Coexistence of S100β and putative transmitter agents in vagal and glossopharyngeal sensory neurons of the rat

The coexistence of S100β with calcitonin gene-related peptide (CGRP), substance P (SP), somatostatin (SOM), nicotinamide adenosine dinucleotide phosphate-diaphorase (NADPH-d), and tyrosine hydroxylase (TH) was examined in the glossopharyngeal and vagal sensory ganglia. S100β immunoreactive (-ir) neurons in the jugular and petrosal ganglia frequently colocalized CGRP- or SP-ir, whereas S100β-ir neurons in the nodose ganglion infrequently contained CGRP- or SP-ir. No S100β-ir neurons in the jugular and petrosal ganglia showed SOM-ir while the small number of SOM-ir neurons in the nodose ganglion colocalized S100β-ir. Many neurons in the nodose ganglion colocalized S100β-ir and NADPH-d activity, whereas S100β-ir neurons in the jugular and nodose ganglia infrequently contained NADPH-d activity. S100β- and TH-ir were frequently colocalized in nodose ganglion but not in petrosal or jugular ganglion neurons. These findings suggest relationships between S100β and specific putative transmitters in functions of subpopulations of vagal and glossopharyngeal sensory neurons.     

Presence of cholinergic neurons in the vagal afferent system: biochemical and immunohistochemical approaches

The presence of cholinergic fibers in the afferent vagal system of various species was shown using biochemical and immunohistochemical methods. Biochemical activity of choline acetyl transferase, the synthesizing enzyme for acetylcholine, was detected in the nodose ganglion of cat, rabbit, dog and sheep. Immunohistochemistry, using a monoclonal antibody raised against choline acetyl transferase, revealed labelled cell bodies in the nodose ganglion of the rabbit. Acetylcholine endogenous content, measured in nodose ganglia devoid of efferent fibers, was twice as high in the right ganglion as compared to the left. Enzyme transport and choline acetyl transferase activity analysis were each determined on separate peripheral vagus nerves. These results are discussed in terms of functional properties of the vagal afferent neurons, including the modulation of vagal afferent messages at the level of the nodose ganglion and the eventual control of peripheral intrinsic neurons by sensory vagal terminals.     

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

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

Pituitary adenylate cyclase activating polypeptide-immunoreactive sensory neurons innervate rat adrenal medulla

Rat adrenal chromaffin cells were invested by a dense network of nerve fibers immunoreactive to pituitary adenylate cyclase activating polypeptide-38 (PACAP-IR). Immunohistochemical studies demonstrated the presence of PACAP-IR in nodose and dorsal root ganglion cells, but not in neurons of the intermediolateral cell column and other autonomic nuclei of the thoracic and upper lumbar spinal cord. Somata of the T7 to T12 paravertebral ganglia were PACAP-negative. A few lightly labeled neurons were occasionally noted in the dorsal motor nucleus of the vagus. Injection of the retrograde tracer Fluorogold into the left adrenal medulla 3 days prior to sacrifice resulted in the labeling of a population of neurons in the ipsilateral spinal cord intermediolateral cell column (T1 to L1), ipsilateral and contralateral nodose ganglia and ipsilateral dorsal root ganglia from T7 to T10 inclusive. A small number of lightly labeled somata was occasionally noted in the dorsal motor nucleus of the vagus. Combined retrograde tracing and PACAP immunohistochemistry showed that a population of Fluorogold-containing nodose and dorsal root ganglion cells were also PACAP-positive. Pre-treatment of the rats with capsaicin caused a marked reduction of the PACAP-IR in the adrenal gland as well as in the superficial layers of the dorsal horn and caudal spinal trigeminal nucleus. These findings, in conjunction with the apparent absence of PACAP-IR in spinal sympathetic preganglionic neurons, sympathetic postganglionic neurons, and dorsal motor nucleus of the vagus, raise the possibility that PACAP-IR fibers observed in the adrenal medulla are primarily sensory in origin. As a corollary, catecholamine secretion from chromaffin cells may be modulated by the peptidergic sensory afferents in addition to the cholinergic sympathetic preganglionic nerve fibers.     

Diabetic autonomic neuropathy: evidence for apoptosis in situ in the rat

We examined the hypothesis that activation of the apoptosis cascade occurs relatively early in diabetes mellitus affecting three distinct neuronal populations that are involved in regulating gut function: (i) dorsal root ganglion (DRG), (ii) vagus nodose ganglion and (iii) colon myenteric plexus. A validated streptozotocin-induced diabetic rat model and age-matched healthy controls were studied. After 4-8 weeks of diabetes the animals were anaesthetized, fixed in situ and the relevant tissues removed. After 1 month of diabetes some animals were treated with insulin for 2 weeks to restore euglycaemia. Apoptosis was measured using immunohistochemical detection of activated caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL)-positive cells in adjacent sections in neurones (PGP 9.5-positive cells). The level of apoptosis was confirmed using double-label assessment of caspase-3 and TUNEL in DRG preparations. Caspase-3 immunoreactive neurones demonstrated a range in staining intensity. When all grades of staining were included, 6-8% of the DRG, nodose ganglia and myenteric neurones were immunoreactive in the preparations from diabetic rats compared with 0.2-0.5% in controls. Neurones staining positive for both caspase-3 and TUNEL accounted for 1-2% of the total neuronal population in all three preparations in diabetic rats compared with 0.1-0.2% in controls (P < 0.05). Insulin treatment reversed the percentage of TUNEL-positive neurones in diabetic rats to control levels. Activation of the apoptosis cascade occurs relatively early in diabetic autonomic neuropathy and may contribute to the pathophysiology of this disorder.     

De novo synthesis and axoplasmic transport of [S]methionine-substance P in explants of nodose ganglion/vagus nerve

The synthesis and transport of substance P, the widely distributed undecapeptide, was studied in the vagus nerve of the guinea pig. In preliminary in vivo studies, the cervical vagus nerve was ligated 2 cm distal to the nodose ganglion. Twenty-four hours later, the content of immunoreactive substance P (IR-SP) in the 3-mm nerve segment proximal to ligature was2147 ± 207pg(mean±S.E.M.)vs133 ± 31pg in an equal segment of unligated nerver or243 ± 55pg in the nodose ganglion. When the vagus nerve was crushed above the ganglion and simultaneously ligated 2 cm distally, the IR-SP content proximal to the ligature was reduced 50% to1131 ± 99pg (P < 0.01), while nodose ganglion content increased to420 ± 140pg(n.s.).

To confirm that residual transport following supranodose crush was derived from nodose ganglion-synthesized SP, SP synthesis and transport were studied in explants of nodose ganglion and attached distal vagus nerve removed from perfused animals and maintained in vitro for up to 24 h. At the time of resection, nerves were ligated 1.5 cm distal to the ganglion. Twenty-four hours following explantation, IR-SP content in proximal segments was1022 ± 142pg vs155 ± 22pg in unligated segments and560 ± 72pg in the nodose ganglion. Accumulation in the proximal segment was time dependent.

In separate experiments, [³⁵S]methionine was added to explant medium and the explants maintained for varying time intervals. nerve tissue was extracted and subjected to either serial reverse phase high performance liquid chromatography (HPLC), or immunoprecipitation with SP antiserum followed by a single HPLC separation. By 4 h, radiolabeled SP was present in nodose ganglia and lesser amounts in the proximal segments. By 12 h, [³⁵S]SP was present equally in ganglia and proximal segments whereas by 18 h, two-thirds or more of the newly synthesized peptide was present in proximal segments. At 18 h, the quantity of radiolabeled SP covaried with IR-SP content in the individual nerve segments. The addition of cycloheximide to explant medium reduced [³⁵S]SP synthesis by 90%.

These studies demonstrate that: (1) approximately 50% of immunoreactive SP transported efferently within the vagus nerve of the guinea pig is derived from the nodose ganglion, (2) de novo SP synthesis within and export from the nodose ganglion occurs within 4 h, (3) the changes in IR-SP content demonstrated in in vivo and in vitro ligation studies accurately reflect ongoing SP synthesis within the nodose ganglion. This transport model may provide one useful tool for studying the regulation of synthesis of SP, or other neuropeptides, within the sensory vagus nerve.     

Substance P content in cultured neonatal rat vagal sensory neurons: the effect of nerve growth factor

To begin to study the factors regulating the synthesis and release of substance P (SP) in the sensory vagus nerve, cultures of neonatal rat nodose ganglia were developed. In microexplant cultures, obtained from small fragments of nodose ganglia, SP was present in low amounts: after 3 weeks, 141 +/- 36 pg per well, 10 ganglia equivalents per well. To enhance neuron survival, nodose ganglia were enzymatically dissociated using neutral protease. Estimated survival at 5 days was 20-30%, with 800-1200 surviving neurons per plated ganglion, and decreased slowly thereafter. Specific SP immunostaining was present in 10-20% of neurons, mostly of small diameter (18-22 micron). SP content was low for 5 days then rose progressively after 14 days to 80-150 pg per plated ganglion. The addition of nerve growth factor (NGF, 100 ng/ml) to the culture medium did not alter neuron survival. However, SP content was doubled in the presence of NGF, or fell rapidly to one-half control levels following its withdrawal: e.g. following 12 days in culture with NGF 1185 +/- 176 pg/well vs NGF withdrawn day 8-12, 592 +/- 118 pg/well, mean +/- S.D., P less than 0.01. Somatostatin, present in one-sixth the amount of SP, was unaltered by NGF. In subsequent studies, plating of neurons onto previously dissociated rat atriacytes increased survival by 50% but did not alter SP content per surviving neurons. These studies demonstrate that SP is present in dissociated cultures of rat vagal sensory neurons; the quantities and estimated net synthesis rate correspond to previous observations in vivo. The studies also demonstrate that SP content but not neuron survival are regulated by NGF in nodose ganglion neurons. This model may prove valuable for the study of SP and other sensory neuropeptides in this important class of visceral afferent neurons.     

Axotomy alters neurotrophin and neurotrophin receptor mRNAs in the vagus nerve and nodose ganglion of the rat

Neurotrophins and neurotrophin receptors play an important role in survival and growth of injured peripheral nerves. To study the injury-mediated neurotrophic response in autonomic nerves, we investigated changes in mRNA expression of neurotrophins and their receptors in the transected vagus nerve and nodose ganglion. Studies using in situ hybridization histochemistry showed that axotomy of the cervical vagus nerve resulted in increased expression of mRNAs for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), and for TrkA, TrkB, and TrkC receptors in non-neuronal cells at both the proximal and distal segments of the transected cervical vagus nerve. Moreover, NGF protein was increased in the distal end, and NT-3 protein was increased in both the proximal and the distal ends of the transected nerve 3 days after axotomy. No change of p75(NTR) mRNA was detected in the transected vagus nerve. The induction of each neurotrophin and Trk receptor mRNA was apparent within 1 day after the axotomy and was sustained at least 14 days. By 45 days after the axotomy, a time when axonal reconnection with target tissue is made (integrity of the nerve-target connection was confirmed by the retrograde transport of FluoroGold from the stomach to vagal cell bodies), the levels of neurotrophin and Trk mRNAs in the vagus nerve declined to pre-axotomy levels. TrkA, TrkC, and p75(NTR) mRNA-containing vagal sensory neurons in the nodose ganglion were reduced in number after cervical vagotomy. Neurotrophin-mRNA-containing neurons were not found in the nodose ganglia from either intact or vagotomized rats. The axotomy-induced up-regulation of neurotrophins and Trk receptors mainly in the non-neuronal cells at or near the site of transection suggests that neurotrophins are involved in the survival and regeneration process of the vagus nerve after injury.     

Presence and coexistence of putative neurotransmitters in carotid sinus baro- and chemoreceptor afferent neurons

The presence and coexistence of tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP) and galanin (GAL) were studied in the petrosal and jugular neurons innervating the carotid body and carotid sinus of the rat. The retrograde labeling of the carotid sinus nerve with Fluoro-gold (FG) demonstrated that most (94.5%) FG-labeled ganglionic neurons were observed in the petrosal ganglion. Fewer (5.2%) FG-labeled neurons were seen in the jugular ganglion and very few (0.3%) were observed in the nodose ganglion. Immunohistochemistry revealed that subpopulations of TH-, VIP-, CGRP-, SP- and GAL-immunoreactive (-ir) neurons in the petrosal ganglion projected to the carotid sinus nerve. Approximately 4% of FG-labeled neurons contained TH-ir and were predominantly found in the caudal portion of the petrosal ganglion. Nearly 90% of total TH-ir neurons in the petrosal ganglion were labeled with FG. Less than 1% of FG-labeled neurons were immunoreactive for VIP in this ganglion. In the petrosal ganglion, 25% of FG-labeled neurons contained CGRP-ir, and 16.7% of FG-labeled neurons contained SP-ir. 30% of CGRP-ir or SP-ir neurons in the petrosal ganglion were labeled with FG. In the jugular ganglion, no TH- or VIP-ir neurons projected to the carotid sinus nerve and only small populations of CGRP- or SP-ir neurons projected to the carotid sinus nerve. Many FG-labeled and GAL-ir neurons were observed in the petrosal and jugular ganglia. The double-immunofluorescence method revealed the coexistence of CGRP- and SP-ir in carotid sinus nerve-projecting neurons in the petrosal and jugular ganglia. Likewise, GAL-ir coexisted with CGRP- and SP-ir in these ganglionic neurons. There was no coexistence of TH-ir and VIP-ir in carotid sinus nerve projections. The present study demonstrates the presence of multiple putative transmitters in baro- and chemoreceptor afferent neurons of the carotid sinus nerve. These neurochemicals are likely to contribute to transmission of signals from the carotid body and carotid sinus to neurons of the brainstem.     

Presence of galanin in rat vagal sensory neurons: evidence from immunohistochemistry and in situ hybridization.

Galanin (GAL), a 29 amino acid peptide originally isolated from the porcine upper small intestine, is widely distributed in the rat central nervous system, including the area postrema (AP) and nucleus of the solitary tract (NTS). Although vagal sensory neurons terminate in the AP/NTS, it is not known whether these neurons contain GAL in the rat. Therefore, we examined the presence and distribution of GAL in the rat nodose ganglia which contain the cell bodies of vagal sensory neurons. We used avidin-biotin-peroxidase immunohistochemistry and in situ hybridization histochemistry with a 35S-labeled oligonucleotide probe. Results with both techniques revealed the presence of GAL-containing cell bodies and fibers in the nodose ganglion. GAL-like immunoreactive cell bodies, mostly between 25 and 40 microns in diameter, were unevenly scattered throughout the nodose ganglia. The distribution and cell diameter range of GAL mRNA-labeled neurons appeared similar to those of GAL-like immunoreactive cells. These findings suggest a role for GAL in the transmission of visceral sensory information by the vagus nerve in rats.     

Substance P in the vagal sensory ganglia: Localization in cell bodies and pericellular arborizations

The distribution of Substance P-like immunoreactivity in the jugular and nodose ganglia of rabbits and pigeons has been studied using immunocytochemical staining techniques. Substance P-like immunoreactivity is localized to neuronal cell bodies and processes in the jugular and nodose ganglia, and to pericellular fiber plexi in the nodose ganglia of both species. The numbers and sizes of cells which exhibited Substance P-like immunoreactivity in each ganglion were determined using quantitative morphometric techniques. The distribution of Substance P-like immunoreactivity in the rabbit and pigeon vagal sensory ganglia is characterized by several general features. In most of the ganglia, immunoreactive neurons factor into discrete types which can be distinguished from one another, and from non-immunoreactive neurons, by size. In addition, immunoreactive nodose and jugular ganglion cells, respectively, are distinguishable on the basis of size. Finally, a considerably higher percentage of immunoreactive neurons is found in the jugular ganglion than in the nodose ganglion. Substance P-like immunoreactivity was also seen in pericellular fiber plexi which encircle individual neurons in the nodose ganglion of rabbits and pigeons. These plexi are composed of varicose fibers which appear to terminate as boutons on the surfaces of the cells which they encircle. The distribution of Substance P-like immunoreactivity within the vagal sensory ganglia is discussed with respect to the possible peripheral targets and functions of Substance P-containing vagal afferents. Our findings suggest that Substance P-containing vagal sensory neurons are involved in a variety of visceral and somatic afferent functions.     

Attenuation of the afferent limb of the baroreceptor reflex in streptozotocin-induced diabetic rats

Diabetic autonomic neuropathy is a common complication following prolonged diabetes. Alterations of cardiovascular reflexes contribute to the increased cardiovascular morbidity and mortality seen in diabetic patients. This study sought to better characterize these complications by investigating the afferent limb of the baroreceptor reflex in an experimental rat model of diabetes. Streptozotocin (STZ)-induced diabetic and euglycemic control rats were studied at 8- and 16-week time points after initiation of the experiment. Activation of the afferent limb of the baroreceptor reflex was assessed by measuring the numbers of c-Fos-immunoreactive (ir) neurons in the CNS site of termination of the baroreceptor afferent neurons, the nucleus of the solitary tract (NTS). Initial experiments established that baseline cardiovascular parameters and NTS expression of c-Fos-ir neurons were not different between diabetic and control rats at either time point. Phenylephrine (PE)-induced activation of baroreceptors resulted in a significant elevation in the numbers of c-Fos-ir neurons in the NTS of control rats. Although diabetic rats showed similar pressor responses to PE, the activation of c-Fos-ir neurons in the NTS of diabetic rats was significantly attenuated. At both 8 and 16 weeks, STZ-induced diabetic rats had significantly fewer c-Fos-ir neurons in the commissural NTS and in the caudal subpostrernal NTS when compared to the non-diabetic control animals receiving PE. These data suggest that STZ-induced diabetes, for a period of 8 and 16 weeks, results in reduced activity in the afferent baroreceptor input to the NTS, and are consistent with diabetes-induced damage to baroreceptor afferent nerves.     

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.     

The role of ischemic neurodegeneration of the nodose ganglia on cardiac arrest after subarachnoid hemorrhage: an experimental study

Background

The heart is innervated by several systems that contribute to the control of the heart's rhythm. The cardiac fibers of the vagus nerve have an important role in the regulation of heart rhythm under many emotional and physical conditions. Severe electrocardiographic disturbances have been reported following subarachnoid hemorrhage (SAH), but ischemic neuronal degeneration of the nodose ganglion of the vagus nerve has not been previously investigated. We examined if there is a relationship between ischemic injury of the nodose ganglion of the vagus nerve and the severity of heart rhythm disorders after subarachnoid hemorrhage.

Methods

This study was conducted on 20 rabbits. Four rabbits were used as a baseline group. Experimental subarachnoid hemorrhage was applied to half of the remaining animals (n = 8) by injecting homologous blood into the cisterna magna, and the others (SHAM group, n = 8) were injected with isotonic saline solution in the same manner. For 20 days after the injection, heart rhythm changes were observed daily. After the experiment, normal and ischemic neuron densities in the nodose ganglia of the vagus nerves were examined stereologically. The number of heart rhythm irregularities and the number of degenerated neurons in the nodose ganglia were compared statistically.

Results

The normal heart rhythm rate was 280 ± 45/min. At the beginning of the SAH, the average heart rate was 220 ± 30/min; about 10 hours later, it decreased to 189 ± 30/min, indicating severe bradycardia. However, after 7 days, the average heart rate had increased to 350 ± 30/min. Six animals died due to irregularities in cardiac function and respiration. Histopathological examinations showed that the average density of normal neurons in the nodose ganglion was 10,500 ± 2500 in the baseline animals and the SHAM group, but the normal neuron density was 8250 ± 1500 in survivors and 6450 ± 1330 in dead animals. The ischemic neuronal degeneration in the nodose ganglia was more severe in the dead animals than in the survivors (p < 0.0001).

Conclusion

Afferent vagus nerves originating from the nodose ganglia have an important role in regulating heart rhythm via their afferent fibers and efferent connections. If neurons of the nodose ganglia are lesioned due to ischemic insult during subarachnoid hemorrhage, heart rhythm regulation by vagus afferent reflexes is disturbed. Vagus pathway paralysis may result in indirect sympathetic overactivity. The development of tachycardia causes depletion of the heart's reserves, and cardiac arrest may be inevitable following extensive subarachnoid hemorrhage.     

Coexistence of calbindin D-28k and NADPH-diaphorase in vagal and glossopharyngeal sensory neurons of the rat

The presence and coexistence of calbindin D-28k-immunoreactivity (ir) and nicotinamide adenosine dinucleotide phosphate (NADPH)-diaphorase activity (a marker of neurons that are presumed to convert L-arginine to L-citrulline and nitric oxide) were examined in the glossopharyngeal and vagal sensory ganglia (jugular, petrosal and nodose ganglia) of the rat. Calbindin D-28k-ir nerve cells were found in moderate and large numbers in the petrosal and nodose ganglia, respectively. Some calbindin D-28k-ir nerve cells were also observed in the jugular ganglion. NADPH-diaphorase positive nerve cells were localized to the jugular and nodose ganglia and were rare in the petrosal ganglion. A considerable portion (33–51%) of the NADPH-diaphorase positive neurons in these ganglia colocalized calbindin D-28k-ir. The presence and colocalization of calbindin D-28k-ir and NADPH-diaphorase activity in neurotransmitter-identified subpopulations of visceral sensory neurons were also studied. In all three ganglia, calcitonin gene-related peptide (CGRP)-ir was present in many NADPH-diaphorase positive neurons, a subset of which also contained calbindin D-28k-ir. In the nodose ganglion, many (42%) of tyrosine hydroxylase (TH)-ir neurons also contained NADPH diaphorase activity but did not contain calbindin D-28k-ir. These data are consistent with a potential co-operative role for calbindin D-28k and NADPH-diaphorase in the functions of a subpopulation of vagal and glossopharyngeal sensory neurons.     

The ovarian innervation in the dog: a preliminary study for the base for electro-acupuncture.

The origin of the canine ovarian sensory and sympathetic nerves was studied by applying horseradish peroxidase (HRP) or wheat germ agglutinin conjugated to HRP (WGA-HRP) to the ovarian stroma and into the ovarian bursa. HRP/WGA-HRP positive neurons were found bilaterally in the dorsal root ganglia of T10 to L4 segment with the majority located in T13 to L2. In sympathetic paravertebral ganglia, labeled neurons were distributed bilaterally in ganglia from T11 to L4 with the majorities located in segments T13 to L2. Both distributions show ipsilateral predominance. Labeled prevertebral neurons were mainly located in the aorticorenal ganglion, ovarian ganglia and caudal mesenteric ganglion. No labeled neurons were found in the dorsal motor nucleus of vagus, nodose ganglia or sacral segment from S1 to S3. This study provides the possible morphological basis of electro-acupuncture concerning the somato-visceral reflex of the ovary.