Trophic regulation of nodose ganglion cell development: evidence for an expanded role of nerve growth factor during embryogenesis in the rat

Peripheral sensory neurons are derived from two distinct embryonic tissues, the neural crest and epibranchial placodes. Studies in the chick suggest that embryonic lineage and trophic dependence are interrelated, such that many crest-derived cells depend on NGF for survival during development, whereas placodal derivates, including nodose ganglion neurons, do not (30). It remains controversial, however, whether or not a similar dichotomy exists in mammalian species, in which trophic requirements during early development of placodal ganglia have not been defined. To approach this issue, the present study examined the effects of nerve growth factor (NGF) on neuronal survival in embryonic rat nodose ganglion cultures. Treatment of E13.5-14.5 nodose explants with 20 ng/ml NGF resulted in a four-fold increase in neuronal survival that was blocked by anti-NGF antiserum. Increased neuronal survival and neurite outgrowth were also observed in neuron-enriched dissociated cell cultures; these effects were seen within 12 h of plating, indicating that NGF-responsive neurons or neuroblasts were already present in the ganglion at the time of explantation. This was further supported by immunocytochemical staining of nodose cell bodies in situ with the monoclonal antibody 192-IgG against the NGF receptor (12). These findings indicate that NGF may be important in regulating nodose development during early gangliogenesis in mammals and suggest that NGF plays a more widespread role in peripheral nervous system ontogeny than previously recognized.     

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.     

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.     

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.     

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.     

Expression of Ciliary Neurotrophic Factor and the Neurotrophins-Nerve Growth Factor,Brain-Derived Neurotrophic Factor and Neurotrophin 3-in Cultured Rat Hippocampal Astrocytes

Cultured astrocytes are known to possess a range of neurotrophic activities in culture. In order to examine which factors may be responsible for these activities, we have examined the expression of the genes for four known neurotrophic factors – ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) – in purified astrocyte cultures derived from neonatal rat hippocampus. Hippocampal astrocytes were found to express mRNA for three neurotrophic factors – CNTF, NGF and NT3 – at significantly higher levels than other cultured cell types or cell lines examined. BDNF messenger RNA (mRNA), however, was undetectable in these astrocytes. The levels of CNTF, NGF and NT3 mRNA in astrocytes were largely unaffected by their degree of confluency, while serum removal caused only a transient decrease in mRNA levels, which returned to basal levels within 48 h. Astrocyte-derived CNTF was found to comigrate with recombinant rat CNTF at 23 kD on a Western blot. Immunocytochemical analysis revealed strong CNTF immunoreactivity in the cytoplasm of astrocytes, weak staining in the nucleus, but no CNTF at the cell surface. NGF and NT3 were undetectable immunocytochemically. CNTF-like activity, as assessed by bioassay on ciliary ganglion neurons, was found in the extract of cultured astrocytes but not in conditioned medium, whereas astrocyte-conditioned medium supported survival of dorsal root ganglion neurons but not ciliary or nodose ganglion neurons. This conditioned medium activity was neutralized with antibodies to NGF. Astrocyte extract also supported survival of dorsal root ganglion and nodose ganglion neurons, but these activities were not blocked by anti-NGF. Part, but not all, of the activity in astrocyte extracts which sustained nodose ganglion neurons could be attributed to CNTF.     

The effects of nerve growth factor (NGF) and antiserum to NGF on the development of embryonic sympathetic neurons in vivo

The role of nerve growth factor (NGF) in the development of embryonic sympathetic neurons was examined in vivo. Individual mouse embryos received transuterine injections of NGF or antiserum to NGF (anti-NGF), and the effects on the superior cervical ganglion (SCG) were studied. Treatment with NGF at any gestational stage, from the time of ganglion aggregation to birth, increased ganglion tyrosine hydroxylase (T-OH) activity. Both the number of catecholaminergic neurons and T-OH activity per neutron were increased. Choline acetyltransferase (ChAc) activity was increased by NGF at early gestational stages, but not at later stages. These observations suggest that perikarya containing ChAc are responsive to NGF, whereas preganglionic nerve terminals are not. Treatment with anti-NGF rapidly and permanently decreased ganglion T-OH activity. The effects of anti-NGF were more pronounced at later gestational stages, suggesting that ganglia become increasingly dependent on NGF during development. Alteration of maternal levels of NGF had no effect on development of the embryonic SCG, suggesting that local embryonic concentrations of NGF are responsible for modulating sympathetic ontogeny.     

NGF/BDNF chimeric proteins: analysis of neurotrophin specificity by homolog-scanning mutagenesis.

Despite their extensive sequence identities at the amino acid level (approximately 55%), NGF and brain-derived neurotrophic factor (BDNF) display distinct neuronal specificity toward neurons of both the PNS and CNS. To explore which region(s) within these neurotrophic factors might determine their differential actions on various subpopulations of peripheral neurons, a systematic series (homolog-scanning mutagenesis) of chimeric NGF/BDNF molecules was prepared using PCR overlap-extension techniques. After expression in COS-7 cells, the chimeric proteins were tested for their biological activities in neurite outgrowth and neuronal survival assays. This approach led to the functional expression of 12 NGF/BDNF chimeras. Surprisingly, despite replacing successive amino acid segments throughout the entire length of NGF with the corresponding parts of BDNF, all chimeras displayed full NGF-like activity in bioassays carried out with PC12 cells, embryonic chick dorsal root ganglion explants, sympathetic ganglion explants, and dissociated cultures of dorsal root ganglion neurons. Most of the chimeras additionally showed BDNF-like activity as defined by neurite outgrowth on chick nodose ganglion explants. However, none of the chimeras supported the survival of dissociated nodose ganglion neurons. Our results suggest that NGF and BDNF must share very similar higher-order protein structures, and we propose that the overall structure or conformation of NGF, in contrast to short amino acid "active-site" segments, may determine its exact neuronal specificity.     

Effects of nerve growth factor on sensory neurons in the chick embryo: a stereological study

Chicken embryos on days 6-13 of incubation received injections of purified beta NGF (80 micrograms/day) for 3 or 4 days and were then killed. Sensory ganglia were fixed and taken for embedding and sectioning. A stereological method based on unfolding of cell-diameter frequencies was used to determine the number of neurons of different size in the spinal, trigeminal and nodose ganglia. The total volume of the ganglia was also determined. NGF induced increases in diameter of the neural crest-derived dorsomedial (DM) neurons in spinal and trigeminal ganglia. Injected NGF did not influence ventrolateral (VL) neurons of neural crest origin in the spinal ganglia nor the ventrolateral neurons of placodal origin in the trigeminal ganglion. The volumes of spinal and trigeminal ganglia increased by 50 and 100%, respectively. The volume of the nodose ganglion and the total number and size of the placodal nodose neurons were unaffected by NGF. The results demonstrate a clear difference in the response to NGF in vivo between smaller and larger sensory neurons.     

BDNF activated TrkB/IRR receptor chimera promotes survival of sympathetic neurons through Ras and PI-3 kinase signaling

Insulin receptor-related receptor (IRR) expression is tightly coupled to the nerve growth factor (NGF) receptor, TrkA, throughout development. Expression of both receptors is primarily localized to neural crest derived sensory and sympathetic neurons. In contrast to TrkA, however, the physiological ligand for IRR is unknown. To analyze the intracellular signaling and potential function of the orphan IRR in neurons, an adenovirus expressing a TrkB/IRR chimeric receptor was used to infect cultured mouse superior cervical ganglion neurons that normally require NGF for survival. Brain derived neurotrophic factor (BDNF)-activated TrkB/IRR induced neuronal survival. We utilized numerous receptor mutants in order to identify the intracellular domains of IRR necessary for signaling and neuron survival. Finally, we employed adenovirus encoding dominant negative forms of the extracellular signal-regulated kinase (ERK) signaling cascade to demonstrate that IRR, like TrkA, requires ras activation to promote neuron survival. Therefore, by use of the chimeric TrkB/IRR receptor, we have demonstrated the ability of IRR to elicit activation of signaling cascades resulting in a biological response in superior cervical ganglion (SCG) neurons.     

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.     

The response of chick sensory neurons to brain-derived neurotrophic factor

To determine the spectrum of activity of brain-derived neurotrophic factor (BDNF) among first-order sensory neurons, explants of the nine distinct populations of sensory neurons from embryonic chicks of 3-14 d incubation (E3-E14) were grown in collagen gels with and without BDNF in the culture medium. The explants responded to BDNF with profuse neurite outgrowth and comprised those in which neurons are derived from neural crest (the dorsomedial part of the trigeminal ganglion, rostromedial part of the trigeminal mesencephalic nucleus, jugular ganglion, and lumbosacral dorsal root ganglia) and from epibranchial placodes (the ventrolateral part of the trigeminal ganglion and the geniculate, petrosal, and nodose ganglia). This response was first clearly observed in the ventrolateral trigeminal and nodose ganglia as early as E4, but did not appear until later in other explants. The dorsomedial trigeminal, jugular, and geniculate ganglia were the latest to develop a response, which was not apparent until E8. In all explants the response was maximal between E10 and E12, and there was a decline in the magnitude of the response from E12 to E14. Although explants of the vestibular ganglion failed to respond to BDNF, the survival and growth of vestibular neurons in dissociated neuron-enriched cultures were promoted by BDNF. To investigate whether all first-order sensory neurons respond to BDNF or whether BDNF-responsive neurons comprise a distinct subset, we studied the influence of BDNF and NGF on the survival and growth of the placode-derived and the neural crest-derived neurons of the trigeminal ganglion in dissociated neuron-enriched culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences and Developmental Changes in the Responsiveness of PNS Neurons to GDNF and Neurturin

We have studied the ability of GDNF and neurturin to promote the in vitro survival of populations of embryonic chicken parasympathetic, sympathetic, and sensory neurons. We show that these neurons are more responsive to one or other of these factors at particular stages of development. Whereas the parasympathetic neurons are more sensitive to neurturin at late embryonic stages, sympathetic neurons are more sensitive to neurturin at early stages. In contrast, sensory neurons of the nodose ganglion are more sensitive to GDNF throughout embryonic development. Using competitive RT/PCR, we measured the levels of mRNAs encoding GDNF and neurturin receptors in purified neurons. All neurons expressed Ret mRNA, which encodes the common receptor tyrosine kinase for GDNF and neurturin. Neurons that were more sensitive to GDNF expressed higher levels of GFRalpha-1 mRNA than GFRalpha-2 mRNA and neurons that were more sensitive to neurturin expressed higher levels of GFRalpha-2 mRNA than GFRalpha-1 mRNA. These results show that populations of PNS neurons differ markedly in their responsiveness to GDNF and neurturin at certain stages of the development and suggest that these differences are governed in part by the relative levels of expression of members of the GFRalpha family of GPI-linked receptors.     

Selective regulation of nerve growth factor expression in developing cutaneous tissue by early sensory innervation

Background

In the developing vertebrate peripheral nervous system, the survival of sympathetic neurons and the majority of sensory neurons depends on a supply of nerve growth factor (NGF) from tissues they innervate. Although neurotrophic theory presupposes, and the available evidence suggests, that the level of NGF expression is completely independent of innervation, the possibility that innervation may regulate the timing or level of NGF expression has not been rigorously investigated in a sufficiently well-characterized developing system.

Results

To address this important question, we studied the influence of innervation on the regulation of NGF mRNA expression in the embryonic mouse maxillary process in vitro and in vivo. The maxillary process receives its innervation from predominantly NGF-dependent sensory neurons of the trigeminal ganglion and is the most densely innervated cutaneous territory with the highest levels of NGF in the embryo. When early, uninnervated maxillary processes were cultured alone, the level of NGF mRNA rose more slowly than in maxillary processes cultured with attached trigeminal ganglia. In contrast to the positive influence of early innervation on NGF mRNA expression, the levels of brain-derived neurotrophic factor (BDNF) mRNA and neurotrophin-3 (NT3) mRNA rose to the same extent in early maxillary processes grown with and without trigeminal ganglia. The level of NGF mRNA, but not BDNF mRNA or NT3 mRNA, was also significantly lower in the maxillary processes of erbB3 ^-/- mice, which have substantially fewer trigeminal neurons than wild-type mice.

Conclusions

This selective effect of initial innervation on target field NGF mRNA expression provokes a re-evaluation of a key assertion of neurotrophic theory that the level of NGF expression is independent of innervation.     

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.     

Overexpression of Nerve Growth Factor in Skin Increases Sensory Neuron Size and Modulates Trk Receptor Expression

Sensory neuron development and differentiation is dependent on a family of growth factors known as neurotrophins. Neurotrophins modulate neuron development via trk tyrosine kinase receptor proteins trkA, trkB and trkC. To determine how elevated levels of a target-derived neurotrophin might affect neuronal differentiation, we analysed trk expression in the trigeminal ganglion of transgenic mice that overexpressed nerve growth factor (NGF) in the skin. increased levels of NGF caused a five-fold increase in neurons expressing trkA mRNA and a two-fold increase in neurons expressing trkC. In control mice, cell size distributions of neuronal subpopulations expressing each trk mRNA showed the three subpopulations distributed over a narrow, overlapping range. In contrast, cell size distribution in NGF-transgenic mice was significantly divergent due in large part to hypertrophy of trkA neurons and, to a lesser extent, trkC neurons. In addition, we examined neurons that bound the isolectin B4 from Bandeiraea simplicifolia (BS-IB4) because most of these neurons do not express any trk receptor in the adult. There was a significant increase in the size of BS-IB4–positive neurons in transgenic mice; however, there was no increase in their number. These studies indicate that an increased level of target-derived NGF affects the development of sensory neurons that in the adult express trkA or trkC, as well as neurons that do not express trk receptors.