Ca2+/calmodulin-dependent protein kinase II in the rat cranial sensory ganglia.

Immunohistochemistry for Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) was performed on the rat cranial sensory ganglia. More than one half of neurons was immunoreactive for the enzyme in the trigeminal (60%), jugular (70%), petrosal (55%) and nodose ganglia (63%). These neurons were mainly small to medium-sized. The co-expression study demonstrated that one half of CaMKII-immunoreactive (ir) neurons was also immunoreactive for calcitonin gene-related peptide (CGRP) or the vanilloid receptor subtype 1 (VR1) in the trigeminal, jugular and petrosal ganglia. In the nodose ganglion, CaMKII-ir neurons were mostly devoid of CGRP-immunoreactivity (ir) (8.2%) whereas the co-expression with VR1-ir was common among such neurons (72%). In the facial skin, nasal mucosa and palate, the epithelium and taste bud were innervated by CaMKII-ir nerve fibers. In addition, the retrograde tracing study demonstrated that 39.6% and 44.8% of trigeminal neurons which were retrogradely traced with fluorogold from the facial skin and nasal mucosa exhibited CaMKII-ir. Forty-six percent of petrosal neurons which innervated the soft palate were immunoreactive for the enzyme.

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.

Streptozotocin-induced diabetes causes metabolic changes and alterations in neurotrophin content and retrograde transport in the cervical vagus nerve.

Abnormal availability of neurotrophins, such as nerve growth factor (NGF), has been implicated in diabetic somatosensory polyneuropathy. However, the involvement of neurotrophins in diabetic neuropathy of autonomic nerves, particularly the vagus nerve which plays a critical role in visceral afferent and in autonomic motor functions, is unknown. To assess the effects of hyperglycemia on the neurotrophin content and transport in this system, cervical vagus nerves of streptozotocin (STZ)-induced diabetic rats were studied at 8, 16, and 24 weeks after the induction of diabetes. Elevations in vagus nerve hexose (glucose and fructose) and polyol levels (sorbitol), and their normalization with insulin treatment, verified that the STZ treatment resulted in hyperglycemia-induced metabolic abnormalities in the nerve. Neurotrophin (NGF and neurotrophin-3; NT-3) content and axonal transport were assessed in the cervical vagus nerves from nondiabetic control rats, STZ-induced diabetic rats, and diabetic rats treated with insulin. The NGF, but not the NT-3, content of intact vagus nerves from diabetic rats was increased at 8 and 16 weeks (but not at 24 weeks). Using a double-ligation model to assess the transport of endogenous neurotrophins, the retrograde transport of both NGF and NT-3 was found to be significantly reduced in the cervical vagus nerve at later stages of diabetes (16 and 24 weeks). Anterograde transport of NGF or NT-3 was not apparent in the vagus nerve of diabetic or control rats. These data suggest that an increase in vagus nerve NGF is an early, but transient, response to the diabetic hyperglycemia and that a subsequent reduction in neuronal access to NGF and NT-3 secondary to decreased retrograde axonal transport may play a role in diabetes-induced damage to the vagus nerve.

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.

Neurotrophins alter the numbers of neurotransmitter-ir mature vagal/glossopharyngeal visceral afferent neurons in vitro.

Mature nodose and petrosal ganglia neurons (placodally derived afferent neurons of the vagal and glossopharyngeal nerves) contain TrkA and TrkC, and transport specific neurotrophins [nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4)]. This study evaluated neurotrophin influences on the presence of neuropeptides and/or neurotransmitter enzymes in these visceral sensory neurons. NGF, NT-3 and NT-4 (10-100 ng/ml) were applied (5 days) to dissociated, enriched, cultures of mature nodose/petrosal ganglia neurons, and the neurons processed for tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and neurofilament (NF-200) immunocytochemistry. Addition of NGF to nodose/petrosal ganglia neuron-enriched cultures significantly increased the number of TH-immunoreactive (ir) neurons, decreased the number of VIP-ir neurons in the cultures, and did not affect the numbers of CGRP-ir neurons. The addition of an NGF neutralizing antibody attenuated the effects of NGF on TH and VIP-ir neurons. NT-3 increased the number of VIP-ir neurons in the nodose/petrosal ganglia cultures and did not alter the numbers of TH-, or CGRP-ir neurons. The addition of an NT-3 neutralizing antibody attenuated the effects of NT-3 on VIP-ir neurons. NT-4 had no significant effects on the numbers of TH, VIP and CGRP-ir neurons. The absence of neurotrophin-induced changes in the numbers of NF-200-ir neurons in culture showed the lack of neurotrophin-mediated changes in survival of mature vagal afferent neurons. These data demonstrate that specific neurotrophins influence the numbers of neurons labeled for specific neurochemicals in nodose/petrosal ganglia cultures. These data, coupled with previous evidence for the presence of TrkA and TrkC mRNA and of the retrograde transport of NGF and NT-3, suggest important roles for NGF and NT-3 in the maintenance of transmitter phenotype of these mature visceral afferent neurons.

The coexistence of TrkA with putative transmitter agents and calcium-binding proteins in the vagal and glossopharyngeal sensory neurons of the adult rat.

The presence of the neurotrophin receptor, TrkA, in neurochemically identified vagal and glossopharyngeal sensory neurons of the adult rat was examined. TrkA was colocalized with calcitonin gene-related peptide (CGRP), parvalbumin, or calbindin D-28k in neurons of the nodose, petrosal and/or jugular ganglia. In contrast, no TrkA-immunoreactive (ir) neurons in these ganglia colocalized tyrosine hydroxylase-ir. About one-half of the TrkA-ir neurons in the jugular and petrosal ganglia contained CGRP-ir, whereas only a few of the numerous TrkA-ir neurons in the nodose ganglion contained CGRP-ir. Although 43% of the TrkA-ir neurons in the nodose ganglion contained calbindin D-28k-ir, few or no TrkA-ir neurons in the petrosal or jugular ganglia were also labeled for either calcium-binding protein. These data show distinct colocalizations of TrkA with specific neurochemicals in vagal and glossopharyngeal sensory neurons, and suggest that nerve growth factor (NGF), the neurotrophin ligand for TrkA, plays a role in functions of specific neurochemically defined subpopulations of mature vagal and glossopharyngeal sensory neurons.

Coexistence of s100beta and putative transmitter agents in vagal and glossopharyngeal sensory neurons of the rat.

The coexistence of S100beta 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. S100beta immunoreactive (-ir) neurons in the jugular and petrosal ganglia frequently colocalized CGRP- or SP-ir, whereas S100beta-ir neurons in the nodose ganglion infrequently contained CGRP- or SP-ir. No S100beta-ir neurons in the jugular and petrosal ganglia showed SOM-ir while the small number of SOM-ir neurons in the nodose ganglion colocalized S100beta-ir. Many neurons in the nodose ganglion colocalized S100beta-ir and NADPH-d activity, whereas S100beta-ir neurons in the jugular and nodose ganglia infrequently contained NADPH-d activity. S100beta- and TH-ir were frequently colocalized in nodose ganglion but not in petrosal or jugular ganglion neurons. These findings suggest relationships between S100beta and specific putative transmitters in functions of subpopulations of vagal and glossopharyngeal sensory neurons.

Axonal transport of neurotrophins by visceral afferent and efferent neurons of the vagus nerve of the rat.

The receptor-mediated axonal transport of [125I]-labeled neurotrophins by afferent and efferent neurons of the vagus nerve was determined to predict the responsiveness of these neurons to neurotrophins in vivo. [125I]-labeled neurotrophins were administered to the proximal stump of the transected cervical vagus nerve of adult rats. Vagal afferent neurons retrogradely transported [125I]neurotrophin-3 (NT-3), [125I]nerve growth factor (NGF), and [125I]neurotrophin-4 (NT-4) to perikarya in the ipsilateral nodose ganglion, and transganglionically transported [125I]NT-3, [125I]NGF, and [125I]NT-4 to the central terminal field, the nucleus tractus solitarius (NTS). Vagal afferent neurons showed minimal accumulation of [125I]brain-derived neurotrophic factor (BDNF). In contrast, efferent (parasympathetic and motor) neurons located in the dorsal motor nucleus of the vagus and nucleus ambiguus retrogradely transported [125I]BDNF, [125I]NT-3, and [125I]NT-4, but not [125I]NGF. The receptor specificity of neurotrophin transport was examined by applying [125I]-labeled neurotrophins with an excess of unlabeled neurotrophins. The retrograde transport of [125I]NT-3 to the nodose ganglion was reduced by NT-3 and by NGF, and the transport of [125I]NGF was reduced only by NGF, whereas the transport of [125I]NT-4 was significantly reduced by each of the neurotrophins. The competition profiles for the transport of NT-3 and NGF are consistent with the presence of TrkA and TrkC and the absence of TrkB in the nodose ganglion, whereas the profile for NT-4 suggests a p75 receptor-mediated transport mechanism. The transport profiles of neurotrophins by efferent vagal neurons in the dorsal motor nucleus of the vagus and nucleus ambiguus are consistent with the presence of TrkB and TrkC, but not TrkA, in these nuclei. These observations describe the unique receptor-mediated axonal transport of neurotrophins in adult vagal afferent and efferent neurons and thus serve as a template to discern the role of specific neurotrophins in the functions of these visceral sensory and motor neurons in vivo.

Coexistence of calcium-binding proteins in vagal and glossopharyngeal sensory neurons of the rat.

The presence and coexistence of the calcium-binding proteins (CaBPs), calbindin D-28k, parvalbumin and S100 protein, were immunohistochemically examined in the glossopharyngeal and vagal sensory ganglia, the carotid body and taste buds. The CaBPs were found in each ganglion with the nodose ganglion containing the largest number of CaBP-immunoreactive (ir) cells (calbindin D-28k > or = S100 >> parvalbumin). The coexistence of CaBPs was found in neurons of the nodose, petrosal, and jugular ganglia. Calbindin D-28k-ir neurons in the nodose and petrosal ganglia frequently colocalized S100-ir whereas calbindin D-28k-ir neurons in the jugular ganglion less frequently contained S100-ir. Only small percentages of calbindin D-28k-ir neurons in each ganglion colocalized parvalbumin. Similarly, S100-ir neurons in the nodose and petrosal ganglia frequently colocalized calbindin D-28k-ir whereas S100-ir neurons in the jugular ganglion less frequently contained calbindin D-28k-ir. Moderate to small percentages of S100-ir neurons in each ganglion colocalized parvalbumin. Parvalbumin-ir neurons nearly always colocalized S100-ir in the nodose, petrosal and jugular ganglia. Moderate to small percentages of parvalbumin-ir neurons in each ganglion colocalized calbindin D-28k. Whereas calbindin D-28k- and S100-ir were colocalized in nerve fibers and cells within taste buds of circumvallate papilla of the tongue, the coexistence of these CaBPs could not be determined in the carotid body. These findings suggest a co-operative role for CaBPs in the functions of subpopulations of nodose and petrosal ganglia neurons.

Neurochemistry of the nodose ganglion.

Placode-derived general visceral afferent neurons of the nodose ganglion transmit visceral sensory information from specialized sensory endings of the vagus nerve and its branches to the nucleus of the solitary tract. These neurons are critical in relaying information such as elevations in blood pressure, changes in blood oxygenation, passage of contents through the esophagus and intestines, and distention of the heart, stomach, and lungs to the CNS for reflex maintenance of visceral functions. Multiple neurotransmitters, neuropeptides, calcium binding proteins, and other neuroactive substances are associated with neurons of the nodose ganglion. Many neurons colocalize 2 or more neuroactive substances creating the potential for complex interactions of neurochemical signals in the NTS. Neurons of the nodose ganglion also contain a variety of receptors which respond to transmitters, inflammatory mediators, and neurotrophic factors. The contents of these neurochemicals and receptors are not static as alterations in their expression are noted in response to epigenetic influences. Although not yet well understood, potential factors and mechanisms regulating neurochemical events in the nodose ganglion neurons are discussed.

Immunocytochemical studies of the 5-HT(1A) receptor in ventral medullaryneurons that project to the intermediolateral cell column and contain serotonin or tyrosine hydroxylase immunoreactivity.

Activation of serotonin-1A receptors (5-HT(1A)R) in the medulla oblongata lowers sympathetic nerve discharge and blood pressure. Binding sites for 5-HT(1A)R ligands are present in ventral medullary nuclei [e.g., rostral ventrolateral medulla (RVLM), raphe pallidus (RPa), and parapyramidal region (PPR)] that project to sympathetic preganglionic neurons in the intermediolateral cell column (IML). However, the projections and the neurochemical contents of the ventral medullary neurons that are likely to be involved in the hypotensive actions of 5-HT(1A) agonists are unclear. Using a sheep antibody to a fragment of the third intracellular loop of the 5-HT(1A)R, we localized 5-HT(1A)R immunoreactivity (ir) to IML-projecting neurons that were retrogradely labeled with rhodamine beads injected into the IML of adult male rats. The percentages of IML-projecting neurons containing 5-HT(1A)R-ir were 49% in RPa, 34% in PPR, and 44% in RVLM. Using multiple-immunofluorescence labeling, we also demonstrated 5-HT(1A)R-ir in serotonergic (5-HT) and in catecholaminergic (tyrosine hydroxylase; TH-ir) neurons of the ventral medulla. The percentages of 5-HT-ir neurons containing 5-HT(1A)R-ir were 28% in RPa, 18% in PPR, and 31% in raphe obscurus. In addition, 5-HT(1A)R-ir was present in 14% of TH-ir neurons of the RVLM. Moreover, some IML-projecting neurons in the PPR and RPa were doubly immunolabeled for 5-HT(1A)R-ir and 5-HT, and some IML-projecting neurons in the RVLM were doubly immunolabeled for 5-HT(1A)R-ir and TH-ir. These data provide anatomical evidence for the presence of 5-HT(1A)R on serotonergic and catecholaminergic bulbospinal neurons and for their potential role in directly modifying the activity of these ventral medullary neurons.

A novel technique for producing antibody-coated microprobes using a thiol-terminal silane and a heterobifunctional crosslinker.

Antibody-coated microprobes are used to measure neuropeptide release in the central nervous system. Although they are not quantitative, they provide the most precise spatial resolution of the location of in vivo release of any currently available method. Previous methods of coating antibody microprobes are difficult and time-consuming. Moreover, using these methods we were unable to produce evenly coated antibody microprobes. This paper describes a novel method for the production of antibody microprobes using thiol-terminal silanes and the heterobifunctional crosslinker, 4-(4-N-maleimidophenyl)butyric acid hydrazide HCl 1/2 dioxane (MPBH). Following silation, glass micropipettes are incubated with antibody to substance P (SP) that has been conjugated to MPBH. This method results in a dense, even coating of antibody without decreasing the biological activity of the antibody. Additionally, this method takes considerably less time than previously described methods without sacrificing the use of antibody microprobes as micropipettes. The sensitivity of the microprobes for SP is in the picomolar range, and there is a linear correlation between the log of SP concentration (M) and B/B0 (r2 = 0.98). The microprobes are stable for up to 3 weeks when stored in 0.1 M sodium phosphate buffer with 50 mM NaCl (pH 7.4) at 5 degrees C. Finally, insertion into the exposed spinal cord of an anesthetized rat for 15 min produces no damage to the antibody coating.

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.

Characterization of substance P release from the intermediate area of rat thoracic spinal cord.

Substance P (SP) nerve terminals innervate the intermediolateral cell column (IML) of the thoracic spinal cord, where SP coexists with serotonin (5-HT), neurokinin A (NKA) and thyrotropin-releasing hormone (TRH). Neither the depolarization-induced release of SP nor the presence of other neurochemicals in the regulation of SP release has been directly studied in this system. In the present study, basal and K(+)-stimulated release of SP from the microdissected intermediate area (including the IML, intercalated nucleus and central autonomic nucleus) of the rat thoracic spinal cord, and the regulation of SP release by presynaptic autoreceptors and by coexisting neurochemicals (5-HT, NKA and TRH) were studied using an in vitro superfusion system. Potassium evoked a concentration- and extracellular Ca(2+)-dependent release of SP. In rats pretreated with the serotoninergic neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), both SP content and the absolute amount of SP released were decreased. However, the fraction of the remaining tissue content of SP released by K+ depolarization was not changed subsequent to 5,7-DHT treatment. Moreover, 5-HT, 5-HT1B agonists (CGS-12066B and RU 24969) and a 5-HT3 agonist (2-methyl-5-HT) did not alter the K(+)-evoked release of SP. These data demonstrate that SP is released from the intermediate area of the rat thoracic spinal cord and some of the SP released comes from serotoninergic nerve terminals. Although 5-HT coexists with SP in the IML, neither endogenous 5-HT nor 5-HT receptor ligands appear to regulate the release of SP. Other colocalized neuropeptides (NKA and TRH) are not involved in the regulation of SP release because neither NKA, a NK2 agonist (GR 64349) nor a TRH analog (MK-771) changed the K(+)-evoked release of SP. A neurokinin-1 (NK1) antagonist (GR 82334) dose-dependently (10(-9)-10(-7) M) increased the K(+)-stimulated release of SP. These data suggest the presence of presynaptic inhibitory NK1 autoreceptors. Whereas, NK1 agonists, [GR 73632 (10(-9)-10(-6) M) and [Sar9, Met (O2)11]SP (10(-8)-10(-6) M)], increased the basal and K(+)-stimulated release of SP, the excitatory effects of GR 73632 were not blocked by the NK1 antagonist. Moreover, GR 73632 increased the efflus of SP to a greater extent in the absence of peptidase inhibitors. Thus, the effect of NK1 agonists on the release of SP may be related to an inhibition of peptide degradation rather than activation of NK1 autoreceptors.

Presence and localization of neurotrophin receptor tyrosine kinase (TrkA, TrkB, TrkC) mRNAs in visceral afferent neurons of the nodose and petrosal ganglia.

The presence of mRNAs to the high affinity tyrosine kinase (Trk) receptors for neurotrophins was studied in visceral afferent neurons of the nodose and petrosal ganglia of adult and neonatal rats using in situ hybridization histochemistry. Neurons containing TrkA mRNA were found in the adult nodose and petrosal ganglia. About 10% of nodose ganglion neurons and 38% of petrosal ganglion neurons contained TrkA mRNA. The nodose and petrosal ganglia from 1 day old neonates also expressed TrkA mRNA. No TrkB mRNA-containing neurons were detected in the adult nodose and petrosal ganglia, whereas TrkB mRNA was detected in 1 day old neonatal nodose and petrosal ganglia. TrkC mRNA was found in about 9% of nodose ganglion neurons and 11% of petrosal ganglion neurons of adult rats. Likewise, low but detectable levels of TrkC mRNA were seen in 1 day old neonatal nodose and petrosal ganglia. These data demonstrate the presence of TrkA and TrkC in the adult nodose and petrosal ganglia and provide a substrate for the ongoing neurotrophin-induced regulation of these placodally derived visceral afferent neurons. The altered expression of Trk receptor mRNAs in the nodose and petrosal ganglia between the adult and neonatal rats may reflect developmentally regulated changes in neurotrophin responsiveness.

Inhibition of axoplasmic transport in the rat vagus nerve alters the numbers of neuropeptide and tyrosine hydroxylase messenger RNA-containing and immunoreactive visceral afferent neurons of the nodose ganglion.

Previous work showed that axotomy-induced deafferentation of the placode-derived visceral afferent neurons of the nodose ganglion altered their expression of some neuropeptides and tyrosine hydroxylase. The present studies were designed to selectively evaluate the loss of axonal transport on the numbers of vasoactive intestinal polypeptide, tyrosine hydroxylase, and calcitonin gene-related peptide mRNA-containing and immunoreactive neurons in the nodose ganglion of the adult rat. Vinblastine (0.15 mM) application to the cervical vagus nerve was used to block axonal transport between ganglionic perikarya and peripheral targets. In situ hybridization histochemistry with 35S-labeled oligonucleotide probes was used to both quantify the number of mRNA-containing neurons and to assess the density of mRNA expression per neuron, and immunocytochemistry was used to visualize the number of immunoreactive neurons. The efficacy of vinblastine to inhibit axonal transport was verified by evaluating the build-up of calcitonin gene-related peptide immunoreactive in the vagus nerve immediately rostral to the site of drug application. The absence of vinblastine-induced neuronal damage was verified by the relative absence of degenerating nerves in the vagus nerve caudal to the site of drug application. Vinblastine treatment of the vagus nerve increased the numbers of vasoactive intestinal peptide mRNA-containing neurons and vasoactive intestinal peptide-immunoreactive neurons in the nodose ganglion at three, seven and 14 days, and increased the numbers of calcitonin gene-related peptide mRNA-containing and calcitonin gene-related peptide-immunoreactive neurons in the nodose ganglion at one, three and seven days. The average labeling density of vasoactive intestinal peptide mRNA-containing neurons was also increased following vinblastine treatment. Vinblastine treatment of the cervical vagus nerve, however, led to the appearance of low-labeling density calcitonin gene-related peptide mRNA-neurons and resulted in reduction of the average labeling density for calcitonin gene-related peptide mRNA-containing neurons. In contrast, application of vinblastine to the cervical vagus nerve, decreased the number of tyrosine hydroxylase mRNA-containing and tyrosine hydroxylase-immunoreactive neurons in the nodose ganglion. In summary, inhibition of the axoplasmic transport between the periphery and the visceral sensory perikarya appeared to alter vasoactive intestinal peptide, calcitonin gene-related peptide, and tyrosine hydroxylase expression and content in visceral sensory neurons of the nodose ganglion. These data suggest the presence of an axonally transported influence on the regulation of neuropeptide and neurotransmitter enzyme synthesis in mature placode-derived visceral sensory neurons.

Parvalbumin and calbindin D-28k in vagal and glossopharyngeal sensory neurons of the rat.

Parvalbumin- and calbindin D-28k-immunoreactivities (ir) were examined in the glossopharyngeal and vagal sensory ganglia (petrosal, nodose and jugular ganglia), the carotid sinus nerve and the carotid body. Parvalbumin-ir nerve cells were mostly localized in the petrosal and nodose ganglia and were rare in the jugular ganglion. Calbindin D-28k-ir nerve cells were found in moderate and large numbers in the petrosal and nodose ganglia, respectively. Only a few calbindin D-28k-ir nerve cells were observed in the jugular ganglion. The carotid sinus nerve and carotid body contained numerous calbindin D-28k-ir nerve fibers but few parvalbumin-ir nerve fibers. Studies of the coexistence of these calcium-binding proteins with calcitonin gene-related peptide (CGRP)- and tyrosine hydroxylase (TH)-ir showed that CGRP-ir was rarely colocalized in parvalbumin- or calbindin D-28k-ir nerve cells in the petrosal or nodose ganglion. Moreover, TH-ir was not generally contained in parvalbumin-ir nerve cells in the petrosal, nodose and jugular ganglia while a portion (15-19%) of calbindin D-28k-ir neurons in the petrosal and nodose ganglia colocalized TH-ir. These findings are consistent with the involvement of calcium-binding proteins, particularly calbindin D-28k, in the function of visceral sensory neural systems of the glossopharyngeal and vagus nerves and, perhaps, in baro- and chemoreceptor neurotransmission.

Tachykinin receptors in the spinal cord.

In summary, all three tachykinin receptors appear to be important modulators of physiological systems in the spinal cord. However, although there is a good deal of data concerning binding characteristics in peripheral tissues, work done in the spinal cord is scanty, leading to a number of unanswered questions. Firstly, Lui et al. (1993) have suggested a discrepancy between the location of SP binding sites and SP containing terminals. This might explain the conflicting evidence on the role of NK1 receptors in the dorsal horn. Furthermore, evidence that NK2 receptors are involved in nociception is increasing, however binding sites for these receptors in the spinal cord have not been demonstrated. This appears to be due to the difficulty in locating an ideal receptor specific ligand. The role of NK2 receptors in autonomic function is also unclear, perhaps for the same reason. Finally, there is evidence indicating that NK3 binding sites are increased following transection of the LIV-VI dorsal roots, however, studies on the effects of inflammation have not been done, as they have with the NK1 and NK2 receptors. All of these and many more unanswered questions require further investigation.

Release of [3H]5-hydroxytryptamine from the intermediate area of rat thoracic spinal cord is modulated by presynaptic autoreceptors.

Serotonin (5-HT) nerve terminals innervate sympathetic preganglionic neurons of the intermediolateral cell column (IML); however, neither the depolarization-induced release of 5-HT nor the presence of presynaptic modulatory autoreceptors have been directly studied in this system. We used in vitro superfusion of the microdissected intermediate area (including the intermediolateral cell column, intercalated nucleus, and central autonomic nucleus) of the rat thoracic spinal cord to measure basal and stimulated release of preloaded [3H]5-HT. Elevated K+ evoked a concentration- and Ca(2+)-dependent release of [3H]5-HT. Exogenous 5-HT and the 5-HT1B agonist, CGS-12066B, both decreased the K(+)-stimulated release of [3H]5-HT. A 5-HT1B antagonist (methiothepin) blocked the 5-HT- and the CGS-12066B-induced inhibition of K(+)-evoked release of [3H]5-HT. A 5-HT1A antagonist (NAN-190) did not alter the inhibitory actions of exogenous 5-HT. Moreover, a 5-HT1A agonist (8-OH-DPAT), a 5-HT2A/2C agonist [(+/-)-DOI hydrochloride), and a 5-HT3 agonist (2-methyl-5-HT) did not alter the K(+)-evoked release of [3H]5-HT. These data demonstrate that 5-HT is released from the intermediate area of the rat thoracic spinal cord. The 5-HT receptor subtype involved in the inhibition of the evoked release of [3H]5-HT is of the 5-HT1B subtype. These findings may help clarify the complex role of 5-HT in spinal regulation of the sympathetic nervous system.