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.     

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.     

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. J. Comp. Neurol. 393:102–117, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US government work and, as such, is in the public domain in the United States of America.

     

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.     

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.     

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.     

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.     

In vivo survival requirement of a subset of nodose ganglion neurons for nerve growth factor

The sensory neurons of the nodose ganglion are the classic example of a population of peripheral nervous system neurons that do not require nerve growth factor (NGF) for survival during development but are dependent on other neurotrophins. We have re-examined this assertion by studying the development of the nodose ganglion of mice that have a null mutation in the NGF gene. Compared with wild-type embryos, the number of neurons undergoing apoptosis was elevated in NGF -/- mice, resulting in a significant reduction in the total number of neurons in the ganglion by the end of embryonic development. TrkA, the NGF receptor tyrosine kinase, was expressed in the nodose ganglion throughout development and there was a marked decrease in TrkA mRNA expression in the nodose ganglion of NGF -/- embryos. Although the in vitro survival of the majority of nodose neurons was promoted by brain-derived neurotrophic factor (BDNF), a minor proportion was supported by NGF in cultures established over a range of embryonic stages. These results clearly demonstrate that a subset of nodose ganglion neurons depends on NGF for survival during development. The finding that the expression of tyrosine hydroxylase (TH) mRNA was unaffected in the nodose ganglia of NGF-deficient embryos indicates that this NGF-dependent subset is distinct from the subset of catacholaminergic neurons in the nodose ganglion.     

Expression of cannabinoid receptors and neurotrophins in human gliomas

Recent studies have shown an anti-tumour activity of cannabinoid receptors CB1 and CB2 in gliomas. This effect was mediated by neurotrophins in breast and prostate carcinoma, while in gliomas this relationship has not yet been considered. The aim of this study was to investigate the expression of cannabinoid receptors CB1 and CB2, neurotrophin NGF and NT-3 and their receptors TrkA and TrkC in glioma and endothelial cells. The analysis was performed in 14 gliomas and 2 non-tumour brain specimens by immunohistochemistry and real-time quantitative-polymerase chain reaction (RTQ-PCR). Gliomas showed a weak immunoreactivity for CB1 and CB2 in tumour and in endothelial cells, and for NGF/TrkA mainly in tumour cells, while a moderate/diffuse immunoreactivity was found for NT-3/TrkC. CB2 was expressed on 3 out of 6 low-grade gliomas and in all high-grade gliomas. Non-tumour brain tissues were weakly positive in astrocytes and endothelium for CB1, CB2, NT-3 and TrkC and negative for NGF and TrkA. By RTQ-PCR, gliomas showed low mRNA levels of NGF/TrkA and moderate levels of CB1, NT-3 and TrkC. CB2 mRNA expression was low or absent. A potential role of cannabinoids, particularly of CB2 agonists devoid of psychotropic side effects, in glioma therapy could have a basis in glioblastomas, because they were all positive, though weakly, to CB2. The presence of neurotrophins and their receptors, mainly NT-3 and TrkC, suggests a possible role of these pathways in glioma growth/invasion, but further investigations are required to verify this hypothesis and a potential relationship between cannabinoids and neurotrophins.     

Distinct usages of phospholipase C gamma and Shc in intracellular signaling stimulated by neurotrophins

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), members of the neurotrophin family, bind to and activate TrkA, TrkB and TrkC, respectively, members of the Trk receptor tyrosine kinase family, to exert various effects including promotion of differentiation and survival, and regulation of synaptic plasticity in neuronal cells. Many reports have suggested that different neurotrophins show distinct biological functions, although molecular mechanisms by which neurotrophins exert their different functions remain unclear. In the present study, we found distinct usages of phospholipase Cgamma (PLCgamma) and Shc in intracellular signaling stimulated by neurotrophins. BDNF stimulated much stronger interactions of PLCgamma with Trk than NGF and NT-3 in PC12 cells stably expressing TrkB and cultured cerebral cortical neurons, respectively, although BDNF, NGF and NT-3 induced similar levels of tyrosine phosphorylation of Trk. Furthermore, the cultured cortical neurons showed large PLCgamma-dependent increases in intracellular Ca(2+) levels in response to BDNF compared with NT-3. In Shc signaling, NGF, but not BDNF, displayed interactions between Trk and Shc in a phenylarsine oxide (PAO; an inhibitor of tyrosine phosphatase)-dependent manner in TrkB-expressing PC12 cells. These results indicated that neurotrophins stimulate distinct kinds of interactions between Trk and PLCgamma and between Trk and Shc. These differences may lead to the distinct biological functions of neurotrophins.     

Cultured basal forebrain cholinergic neurons from postnatal rats show both overlapping and non-overlapping responses to the neurotrophins

Basal forebrain cholinergic neurons respond in vitro and in vivo to nerve growth factor (NGF) and to brain-derived neurotrophic factor (BDNF). It is not clear to what extent the neurons that respond to these two factors, or to neurotrophin-3 or−45 (NT-3;NT-45) are identical or only partially overlapping populations. We have addressed this issue in cultures of basal forebrain neurons derived from 2-week-old postnatal rats, using choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) as cholinergic markers. Cholinergic neuron survival was enhanced in the presence of NGF, BDNF andNT-45.NT-45 was as effective as BDNF. NT-3 was without effect at this age, although in cultures derived from embryonic forebrain, cholinergic differentiation was induced by NT-3. Cotreatment with NGF and BDNF resulted in small, but consistent, increases in the number of ChAT-positive neurons, compared with either factor alone.NT-45 was also found to be additive with NGF, whereas cotreatment with BDNF andNT-45 showed no addivity. NT-3 had no additive effects with any other neurotrophin on any cholinergic parameters in postnatal cultures. Taken together, the results indicate the existence in postnatal rat brain of a large overlapping population of cholinergic neurons that are responsive to ligands for the neurotrophin receptors TrkA (NGF) and TrkB (BDNF andNT-45), but not TrkC (NT-3), and small distinct populations that show specificity for NGF or BDNF but not both. We hypothesize that cholinergic neurons projecting into different regions of the hippocampus may derive trophic support from distinct neurotrophins.     

Differential effects of NGF and NT-3 on embryonic trigeminal axon growth patterns

We examined the effects of neurotrophins nerve growth factor (NGF) and neurotrophin-3 (NT-3) on trigeminal axon growth patterns. Embryonic (E13-15) wholemount explants of the rat trigeminal pathway including the whisker pads, trigeminal ganglia, and brainstem were cultured in serum-free medium (SFM) or SFM supplemented with NGF or NT-3 for 3 days. Trigeminal axon growth patterns were analyzed with the use of lipophilic tracer DiI. In wholemount cultures grown in SFM, trigeminal axon projections, growth patterns, and differentiation of peripheral and central targets are similar to in vivo conditions. We show that in the presence of NGF, central trigeminal axons leave the tract and grow into the surrounding brainstem regions in the elongation phase without any branching. On the other hand, NT-3 promotes precocious development of short axon collaterals endowed with focal arbors along the sides of the central trigeminal tract. These neurotrophins also affect trigeminal axon growth within the whisker pad. Additionally, we cultured dissociated trigeminal ganglion cells in the presence of NGF, NT-3, or NGF+NT-3. The number of trigeminal ganglion cells, their size distribution under each condition were charted, and axon growth was analyzed following immunohistochemical labeling with TrkA and parvalbumin antibodies. In these cultures too, NGF led to axon elongation and NT-3 to axon arborization. Our in vitro analyses suggest that aside from their survival promoting effects, NGF and NT-3 can differentially influence axon growth patterns of embryonic trigeminal 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.     

Reduction of NT-3 or TrkC results in fewer putative vagal mechanoreceptors in the mouse esophagus

Intraganglionic laminar endings (IGLEs) represent major vagal afferent structures throughout the gastrointestinal tract. Both morphological and functional data suggested a mechanosensory role. Elucidation of their functional significance in a particular organ would be facilitated by the availability of animal models with significantly altered numbers of IGLEs. The present study was aimed at searching for mouse strains fulfilling this criterion in the esophagus. Anterograde wheat germ agglutinin-horseradish peroxidase tracing (WGA-HRP) from nodose ganglion was used in order to label esophageal IGLEs in mice deficient for neurotrophin-3 (NT-3) or tyrosine kinase C-receptor (TrkC) and in control littermates. This approach was feasible only in heterozygous mutants which are viable. IGLEs were counted in tetramethylbenzidine (TMB) processed wholemounts using a standardised protocol. Quantification of myenteric neurons was done in cuprolinic blue-stained specimens. Nodose neuron counts were performed in cryostat sections stained with cresyl violet. Numbers of IGLEs in the esophagus were significantly reduced in both heterozygous NT-3 (NT-3+/-) and heterozygous TrkC (TrkC+/-) mutants (65% and 40% reduction, respectively). Numbers of nodose neurons were also significantly reduced in NT-3+/- mice (48% reduction), while their reduction in TrkC+/- mutants was insignificant (11% reduction). There was no reduction of myenteric neurons in the esophagus of either mutant strain. The numeric deficiency of IGLEs was unlikely to be secondary to reduction of myenteric neurons. Although only heterozygous mutants could be studied, these results suggest that esophageal IGLEs share neurotrophin dependence on NT-3/TrkC with spinal proprioceptors and some cutaneous mechanosensors. This concurs with their proposed function as vagal mechanosensors crucial for reflex peristalsis.     

Cells Expressing mRNA for Neurotrophins and their Receptors During Embryonic Rat Development

In situ hybridization analysis of cells expressing messenger RNAs (mRNAs) for the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and their high-affinity receptors (trk, trkB and trkC) in the rat embryo revealed a complex but specific expression pattern for each of these mRNAs. For all mRNAs a developmentally regulated expression was seen in many different tissues. BDNF and NT-3 mRNAs were expressed in the sensory epithelia of the cochlea and vestibule macula of the sacculus and utricle, and both trkB and trkC mRNA were expressed in the spiral and vestibule ganglia innervating these sensory structures. NGF and NT-3 mRNA were found in the iris, innervated by the sympathetic neurons of the superior cervical ganglion and sensory neurons from the trigeminal ganglion, which expressed both trk and trkC mRNAs. Both NGF and NT-3 mRNAs were also expressed in other target fields of the trigeminal ganglion, the epithelium of the whisker follicles (NT-3 mRNA) and in the epithelium of the nose, tongue and jaw. NT-3 mRNA was found in the cerebellar external granule layer and trkC mRNA in the Purkinje layer of the cerebellar primordia. These sites of synthesis are consistent with a target-derived neurotrophic interaction for NGF, BDNF and NT-3. However, in some cases mRNAs for both the neurotrophins and their high-affinity receptors were detected in the same tissue, including the dorsal root, geniculate, superior, jugular, petrose and nodose ganglia, as well as in the hippocampus, frontal cortical plate and pineal recess, implying a local mode of action. Combined, these data suggest a broad function for the neurotrophins and their receptors in supporting neural innervation during embryonic development. The results also identify several novel neuronal systems that are likely to depend on the neurotrophins in vivo.     

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.     

Expression of neurotrophins and Trk receptors in the avian retina

Using the RNase protection assay, we have found that nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are expressed in the avian retina during development. The expression peaks around embryonic days 12–15, with decreasing levels at later stages of development. Abundant levels of NGF and BDNF but low levels of NT-3 mRNA were found in the adult retina. We also found that light/darkness regulated the levels of NGF and BDNF mRNAs but not the levels of NT-3 mRNA in the 5-day-old chicken retina. It was demonstrated that NGF and BDNF mRNA levels were up-regulated by light exposure. The cellular localization of mRNA expression for the neurotrophins and neurotrophin receptors TrkA, TrkB, and TrkC in the retina was studied using in situ hybridization. The patterns of NGF and trkA mRNA expression were very similar and were localized to the external part of the inner nuclear layer on the border with the outer plexiform layer and corresponded to the localization of horizontal cells. NT-3 labeling was also found over the external part of the inner nuclear layer, whereas trkC mRNA was found over all layers in the retina. BDNF labeling was found over all layers in the retina, whereas TrkB labeling was intense over cells in the ganglion cell layer, which is in agreement with the response of ganglion cells to BDNF stimulation. Functional neurotrophin receptors were suggested by the response of retinal explants to neurotrophin stimulation. These data indicate that the neurotrophins play local roles in the retina that involve interactions between specific neuronal populations, which were identified by the localization of the Trk receptor expression. The data also suggest that NGF and BDNF expression is regulated by normal neuron usage in the retina. © 1996 Wiley-Liss, Inc.     

Co-localization of μ-opioid receptor-like immunoreactivity with substance P-LI, calcitonin gene-related peptide-LI and nitric oxide synthase-LI in vagal and glossopharyngeal afferent neurons of the rat

Co-localization of μ-opioid receptor (MOR)-like immunoreactivity (-LI) with substance P (SP)-LI, calcitonin gene-related peptide (CGRP)-LI and nitric oxide synthase (NOS)-LI in the nodose, petrosal and jugular ganglia was examined in the rat by a double immunofluorescence histochemical method. About 0.6%, 41% and 95% of neurons with MOR-LI, respectively, in the nodose, petrosal and jugular ganglia showed SP-LI; about 2%, 51% and 66% of MOR-like immunoreactive neurons displayed CGRP-LI in the nodose, petrosal and jugular ganglia, respectively. In addition, about 59% of MOR-like immunoreactive neurons in the nodose ganglia displayed NOS-LI, whereas no NOS-LI was detected in the petrosal or jugular ganglion. These data provide evidence for co-localization of MOR-LI with SP-LI, CGRP-LI and NOS-LI in the vagal and glossopharyngeal afferent neurons, and suggest that MOR may regulate the release of SP, CGRP and nitric oxide from the visceral primary afferent terminals in the nucleus of the solitary tract of the rat.