Level of p75 receptor expression in sensory ganglia is modulated by NGF level in the target tissue

Neurotrophins play an essential role in sensory development by providing trophic support to neurons that innervate peripheral targets. Nerve growth factor (NGF), neurotrophin-3, neurotrophin-4, and brain-derived neurotrophin exert their survival effect by binding to two transmembrane receptor types: trk receptors, which exhibit binding specificity, and the p75^NTR receptor, which binds all neurotrophins. To determine how target-derived neurotrophins affect sensory neuron development and function, we used transgenic mice that overexpress NGF in the skin to examine the impact of NGF overexpression on receptor expression. Previous studies of trk expression in trigeminal ganglia of adult NGF transgenics showed that the percentage of trkA neurons doubled and their number increased fivefold. The present study focused on the p75 receptor and shows that the percentage of neurons expressing p75^NTR also increase in NGF ganglia, but only by 10%. This increase did not encompass the small, BS-IB-4 isolectin-positive cells as they remained p75 negative in transgenic ganglia. Interestingly, levels of trkA protein were not increased on a per-cell level, whereas levels of p75^NTR increased nearly threefold. These results show that in sensory systems, target-derived NGF modulates the level of p75^NTR receptor expression, and in so doing, may act to regulate the formation of functional receptor complexes and subsequent trophic action. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 258–270, 1998     

Assembly of trigeminal sensory ganglia by chemokine signaling

Sensory neurons with related functions form ganglia, but how these precisely positioned clusters are assembled has been unclear. Here, we use the zebrafish trigeminal sensory ganglion as a model to address this question. We find that some trigeminal sensory neurons are born at the position where the ganglion is assembled, whereas others are born at a distance and have to migrate against opposing morphogenetic movements to reach the site of ganglion assembly. Loss of Cxcr4b-mediated chemokine signaling results in the formation of mispositioned ganglia. Conversely, ectopic sources of the chemokine SDF1a can attract sensory neurons. Transplantation experiments reveal that neuron-neuron interaction and the adhesion molecules E- and N-Cadherin also contribute to ganglion assembly. These results indicate that ganglion formation depends on the interplay of birthplace, chemokine attraction, cell-cell interaction, and cadherin-mediated adhesion.     

Novel roles for neurotrophins are suggested by BDNF and NT-3 mRNA expression in developing neurons.

The results of our in situ hybridization experiments demonstrate that sensory neurons, sympathetic neurons, and motoneurons express brain-derived neurotrophic factor and/or neurotrophin-3 mRNAs during development in mouse. In accordance with previous data, we also find neurotrophins in the targets of sensory neurons (skin) and motoneurons (muscle) and the neurotrophin receptors p75, trkA, and trkB in sensory and sympathetic ganglia. These results suggest that neurotrophins have roles other than being target-derived factors that support neuron survival during developmental cell death (neurotrophic hypothesis), but may be transported in an orthograde fashion in neurons and released from axon terminals. We discuss several novel roles for neurotrophins, including autocrine/paracrine regulation of neuron survival, regulation of Schwann cell activity, and neuron to target signaling.     

Bone morphogenetic protein down-regulation of neuronal pituitary adenylate cyclase-activating polypeptide and reciprocal effects on vasoactive intestinal peptide expression

Among bone morphogenetic proteins (BMPs), the decapentaplegic (Dpp; BMP2, BMP4) and glass bottom boat (Gbb/60A; BMP5, BMP6, BMP7) subgroups have well-described functions guiding autonomic and sensory neuronal development, fiber formation and neurophenotypic identities. Evaluation of rat superior cervical ganglia (SCG) post-ganglionic sympathetic neuron developmental regulators identified that selected BMPs of the transforming growth factor beta superfamily have reciprocal effects on neuronal pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) expression. Dpp and Gbb/60A BMPs rapidly down-regulated PACAP expression, while up-regulating other sympathetic neuropeptides, including PACAP-related VIP. The suppressive effects of BMP on PACAP mRNA and peptide expression were potent, efficacious and phosphorylated mothers against decapentaplegic homolog (Smad) signaling-dependent. Axotomy of SCG dramatically increases PACAP expression, and the possibility that abrogation of inhibitory retrograde target tissue BMP signaling may contribute to this up-regulation of sympathetic neuron PACAP was investigated. Replacement of BMP6 to SCG explant preparations significantly blunted the injury-induced elevated PACAP expression, with a concomitant decrease in sympathetic PACAP-immunoreactive neuron numbers. These studies suggested that BMPs modulate neuropeptide identity and diversity by stimulating or restricting the expression of specific peptidergic systems. Furthermore, the liberation of SCG neurons from target-derived BMP inhibition following axotomy may be one participating mechanism associated with injury-induced neuropeptidergic plasticity.     

Cutaneous overexpression of NT-3 increases sensory and sympathetic neuron number and enhances touch dome and hair follicle innervation

Target-derived influences of nerve growth factor on neuronal survival and differentiation are well documented, though effects of other neurotrophins are less clear. To examine the influence of NT-3 neurotrophin overexpression in a target tissue of sensory and sympathetic neurons, transgenic mice were isolated that overexpress NT- 3 in the epidermis. Overexpression of NT-3 led to a 42% increase in the number of dorsal root ganglia sensory neurons, a 70% increase in the number of trigeminal sensory neurons, and a 32% increase in sympathetic neurons. Elevated NT-3 also caused enlargement of touch dome mechanoreceptor units, sensory end organs innervated by slowly adapting type 1 (SA1) neurons. The enlarged touch dome units of the transgenics had an increased number of associated Merkel cells, cells at which SA1s terminate. An additional alteration of skin innervation in NT-3 transgenics was an increased density of myelinated circular endings associated with the piloneural complex. The enhancement of innervation to the skin was accompanied by a doubling in the number of sensory neurons expressing trkC. In addition, measures of nerve fibers in cross- sectional profiles of cutaneous saphenous nerves of transgenics showed a 60% increase in myelinated fibers. These results indicate that in vivo overexpression of NT-3 by the epidermis enhances the number of sensory and sympathetic neurons and the development of selected sensory endings of the skin.     

Cranial sensory neuron development in the absence of brain-derived neurotrophic factor in BDNF/Bax double null mice

To investigate the role of brain-derived neurotrophic factor (BDNF) in differentiation of cranial sensory neurons in vivo, we analyzed development of nodose (NG), petrosal (PG), and vestibular (VG) ganglion cells in genetically engineered mice carrying null mutations in the genes encoding BDNF and the proapoptotic Bcl-2 homolog Bax. In bax(-/-) mutants, ganglion cell numbers were increased significantly compared to wild-type animals, indicating that naturally occurring cell death in these ganglia is regulated by Bax signaling. Analysis of bdnf(-/-)bax(-/-) mutants revealed that, although the Bax null mutation completely rescued cell loss in the absence of BDNF, it did not rescue the lethality of the BDNF null phenotype. Moreover, despite rescue of BDNF-dependent neurons by the bax null mutation, sensory target innervation was abnormal in double null mutants. Vagal sensory innervation to baroreceptor regions of the cardiac outflow tract was completely absent, and the density of vestibular sensory innervation to the cristae organs was markedly decreased, compared to wild-type controls. Moreover, vestibular afferents failed to selectively innervate their hair cell targets within the cristae organs in the double mutants. These innervation failures occurred despite successful navigation of sensory fibers to the peripheral field, demonstrating that BDNF is required locally for afferent ingrowth into target tissues. In addition, the bax null mutation failed to rescue expression of the dopaminergic phenotype in a subset of NG and PG neurons. These data demonstrate that BDNF signaling is required not only to support survival of cranial sensory neurons, but also to regulate local growth of afferent fibers into target tissues and, in some cells, transmitter phenotypic expression is required.     

Dose and age-dependent axonal responses of embryonic trigeminal neurons to localized NGF via p75NTR receptor

Nerve growth factor (NGF) and related neurotrophins are target-derived survival factors for sensory neurons. In addition, these peptides modulate neuronal differentiation, axon guidance, and synaptic plasticity. We tested axonal behavior of embryonic trigeminal neurons towards localized sources of NGF in collagen gel assays. Trigeminal axons preferentially grow towards lower doses of localized NGF and grow away from higher concentrations at earlier stages of development, but do not show this response later. Dorsal root ganglion axons also show similar responses to NGF, but NGF-dependent superior cervical ganglion axons do not. Such axonal responses to localized NGF sources were also observed in Bax-/- mice, suggesting that the axonal effects are largely independent of cell survival. Immunocytochemical studies indicated that axons, which grow towards or away from localized NGF are TrkA-positive, and TrkA-/- TG axons do not respond to any dose of NGF. We further show that axonal responses to NGF are absent in TG derived from mice that lack the p75 neurotrophin receptor (p75NTR). Collectively, our results suggest that localized sources of NGF can direct axon outgrowth from trigeminal ganglion in a dose- and age-dependent fashion, mediated by p75NTR signaling through TrkA expressing axons.     

Skin-derived nerve growth factor blocks programmed cell death in the trigeminal ganglia but does not enhance neuron proliferation.

Development of the cutaneous sensory nervous system is dependent on the production of neurotrophic factors, such as nerve growth factor (NGF), by the skin. Limited synthesis of NGF in developing skin is thought to underlie programmed cell death and cause a 50% neuronal loss. This loss does not occur in transgenic mice that overexpress NGF in the skin, which have double the number of neurons (J. Neurosci. 14 (1994) 1422). To determine whether increased NGF blocks neuronal death and/or increases neuronal precursor replication, we analyzed the trigeminal ganglia at embryonic days E12.5, E14.5 and E16.5 using transferase-mediated dUTP nick-end labeling (TUNEL) and bromodeoxyuridine labeling. Results show that excess target-derived NGF causes a major decrease in the percent of TUNEL-labeled neurons without affecting the percent of replicating neurons. Analysis of RNA and protein expression suggests this block in cell death is mediated via the anti-apoptotic protein bcl-2.     

Neurotrophins and their receptors in rat peripheral trigeminal system during maxillary nerve growth

We examined the expression of the neurotrophins (NTFs) and their receptor mRNAs in the rat trigeminal ganglion and the first branchial arch before and at the time of maxillary nerve growth. The maxillary nerve appears first at embryonic day (E)10 and reaches the epithelium of the first branchial arch at E12, as revealed by anti-L1 immunohistochemistry. In situ hybridization demonstrates, that at E10- E11, neurotrophin-3 (NT-3) mRNA is expressed mainly in the mesenchyme, but neurotrophin-4 (NT-4) mRNA in the epithelium of the first branchial arch. NGF and brain-derived neurotrophic factor (BDNF) mRNAs start to be expressed in the distal part of the first brachial arch shortly before its innervation by the maxillary nerve. Trigeminal ganglia strongly express the mRNA of trkA at E10 and thereafter. The expression of mRNAs for low-affinity neurotrophin receptor (LANR), trkB, and trkC in trigeminal ganglia is weak at E10, but increases by E11-E12. NT-3, NT-4, and more prominently BDNF, induce neurite outgrowth from explant cultures of the E10 trigeminal ganglia but no neurites are induced by NGF, despite the expression of trkA. By E12, the neuritogenic potency of NGF also appears. The expression of NT-3 and NT-4 and their receptors in the trigeminal system prior to target field innervation suggests that these NTFs have also other functions than being the target-derived trophic factors.     

BDNF-overexpression regulates the reactivity of small pulmonary arteries to neurokinin A

Brain-derived neurotrophic factor (BDNF) is a key modulator during the development of jugular and nodose ganglia neurons, which represent the origin of a large proportion of the sensory innervation of the lung. It belongs to the family of neurotrophins, which have been shown to induce the expression of tachykinins. To assess the interactions of BDNF and the tachykinin neurokinin A (NKA) in small pulmonary vessels, BDNF-transgenic mice were examined for tachykinin contents in the airways, heart, trigeminal ganglion and jugular and nodose ganglion complex (JNC) using reverse phase HPLC (rpHPLC) and radioimmunoassay. BDNF-overexpression led to increased NKA levels in the heart and the JNC, whereas only slightly enhanced levels in the trigeminal ganglion were detected. Lower NKA levels were found in the lung. To assess vasoreactivity in small arteries, precision cut lung slices were subjected to videomorphometry and the response to NKA was examined, which showed significantly stronger effects in the BDNF-transgenic mice, while NK-2 receptor mRNA expression, assayed by real-time RT-PCR, was reduced. In conclusion, BDNF-overexpression results in decreased levels of NKA in the lung with subsequently increased NKA-sensitivity of small arteries. These findings point to a modulatory role of neurotrophins in small respiratory vessel tone regulation.     

Expression of Trk receptors in the developing mouse trigeminal ganglion: in vivo evidence for NT-3 activation of TrkA and TrkB in addition to TrkC

Animals lacking neurotrophin-3 (NT-3) are born with deficits in almost all sensory ganglia. Among these, the trigeminal ganglion is missing 70% of the normal number of neurons, a deficit which develops during the major period of neurogenesis between embryonic stages (E) 10.5 and E13.5. In order to identify the mechanisms for this deficit, we used antisera specific for TrkA, TrkB, and TrkC to characterize and compare the expression patterns of each Trk receptor in trigeminal ganglia of wild type and NT-3 mutants between E10.5 and E15.5. Strikingly, TrkA, TrkB, and TrkC proteins appear to be exclusively associated with neurons, not precursors. While some neurons show limited co-expression of Trk receptors at E11.5, by E13. 5 each neuron expresses only one Trk receptor. Neuronal birth dating and cell counts show that in wild-type animals all TrkB- and TrkC-expressing neurons are generated before E11.5, while the majority of TrkA-expressing neurons are generated between E11.5 and E13.5. In mice lacking NT-3, the initial formation of the ganglion, as assessed at E10.5, is similar to that in wild-type animals. At E11.5, however, the number of TrkC-expressing neurons is dramatically reduced and the number of TrkC-immunopositive apoptotic profiles is markedly elevated. By E13.5, TrkC-expressing neurons are virtually eliminated. At E11.5, compared to wild type, the number of TrkB-expressing neurons is also reduced and the number of TrkB immunoreactive apoptotic profiles is increased. TrkA neurons are also reduced in the NT-3 mutants, but the major deficit develops between E12.5 and E13.5 when elevated numbers of TrkA-immunoreactive apoptotic profiles are detected. Normal numbers of TrkA- and TrkB-expressing neurons are seen in a TrkC-deficient mutant. Therefore, our data provide evidence that NT-3 supports the survival of TrkA-, TrkB- and TrkC-expressing neurons in the trigeminal ganglion by activating directly each of these receptors in vivo.     

Macrophage stimulating protein is a target-derived neurotrophic factor for developing sensory and sympathetic neurons

Macrophage stimulating protein (MSP) is a pleiotropic growth factor that signals via the Ron receptor tyrosine kinase. We report that Ron mRNA is expressed by NGF-dependent sensory and sympathetic neurons and that these neurons survive and grow with MSP at different stages of development. Whereas NGF-dependent sensory neurons become increasingly responsive to MSP with age, sympathetic neurons exhibit an early response to MSP that is lost by birth. MSP mRNA expression increases with age in sensory neuron targets and decreases in sympathetic targets. After the phase of naturally occurring neuronal death, significant numbers of NGF-dependent sensory neurons, but not sensory neurons, dependent on other neurotrophins, are lost in mice lacking a functional Ron receptor. These results show that MSP is a target-derived neurotrophic factor for subsets of sensory and sympathetic neurons at different times during their development.     

Differential Trk expression in explant and dissociated trigeminal ganglion cell cultures

During embryonic development, expression of neurotrophin receptor tyrosine kinases (Trks) by sensory ganglia is continuously and dynamically regulated. Neurotrophin signaling promotes selective survival and axonal differentiation of sensory neurons. In embryonic day (E) 15 rat trigeminal ganglion (TG), NGF receptor TrkA is expressed by small diameter neurons, NT-3 receptor TrkC and BDNF receptor TrkB are expressed by large diameter neurons. Organotypic explant and dissociated cell cultures of the TG (and dorsal root ganglia) are commonly used to assay neurotrophin effects on developing sensory neurons. In this study, we compared Trk expression in E15 rat TG explant and dissociated cell cultures with or without neurotrophin treatment. Only a subset of TG cells express each of the three Trk receptors in wholemount explant cultures as in vivo conditions. In contrast, all TG neurons co-express all three Trk receptors upon dissociation, regardless of neurotrophin treatment. Neurons cultured in low concentrations of one neurotrophin first, and switched to higher concentrations of another after 1 day, survive and display morphological characteristics of neurons cultured in a mixture of both neurotrophins for 3 days. Our results indicate that wholemount explant cultures of sensory ganglia represent in vivo conditions in terms of Trk expression patterns; whereas dissociation dramatically alters Trk expression by primary sensory neurons.     

Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system.

We have generated mice carrying a mutation of the gene encoding the low affinity NGF receptor p75NGFR by targeted mutation in embryonic stem cells. Mice homozygous for the mutation were viable and fertile. Immunohistochemical analyses of the footpad skin of mutant mice revealed markedly decreased sensory innervation by calcitonin gene-related peptide- and substance P-immunoreactive fibers. The defective innervation was correlated with loss of heat sensitivity and associated with the development of ulcers in the distal extremities. Complicated by secondary bacterial infection, the ulcers progressed to toenail and hair loss. Crossing a human transgene encoding p75NGFR into the mutant animals rescued the absent heat sensitivity and the occurrence of skin ulcers and increased the density of neuropeptide-immunoreactive sensory innervation of footpad skin. The mutation in the gene encoding p75NGFR did not decrease the size of sympathetic ganglia or the density of sympathetic innervation of the iris or salivary gland. Our results suggest that p75NGFR has an important role in the development and function of sensory neurons.     

Phenotypic alterations of neuropeptide Y and calcitonin gene-related peptide-containing neurons innervating the rat temporomandibular joint during carrageenan-induced arthritis

The aim of this study was to identify immunoreactive neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) neurons in the autonomic and sensory ganglia, specifically neurons that innervate the rat temporomandibular joint (TMJ). A possible variation between the percentages of these neurons in acute and chronic phases of carrageenan-induced arthritis was examined. Retrograde neuronal tracing was combined with indirect immunofluorescence to identify NPY-immunoreactive (NPY-IR) and CGRP- immunoreactive (CGRP-IR) neurons that send nerve fibers to the normal and arthritic temporomandibular joint. In normal joints, NPY-IR neurons constitute 78±3%, 77±6% and 10±4% of double-labeled nucleated neuronal profile originated from the superior cervical, stellate and otic ganglia, respectively. These percentages in the sympathetic ganglia were significantly decreased in acute (58±2% for superior cervical ganglion and 58±8% for stellate ganglion) and chronic (60±2% for superior cervical ganglion and 59±15% for stellate ganglion) phases of arthritis, while in the otic ganglion these percentages were significantly increased to 19±5% and 13±3%, respectively. In the trigeminal ganglion, CGRP-IR neurons innervating the joint significantly increased from 31±3% in normal animals to 54±2% and 49±3% in the acute and chronic phases of arthritis, respectively. It can be concluded that NPY neurons that send nerve fibers to the rat temporomandibular joint are located mainly in the superior cervical, stellate and otic ganglia. Acute and chronic phases of carrageenan-induced arthritis lead to an increase in the percentage of NPY-IR parasympathetic and CGRP-IR sensory neurons and to a decrease in the percentage of NPY-IR sympathetic neurons related to TMJ innervation.Key words: trigeminal ganglion, otic ganglion, superior cervical ganglion, arthritis, temporomandibular joint.     

Competition during innervation of embryonic amphibian head skin

We have examined the initial innervation of the head skin in Xenopus laevis embryos which is by two classes of trigeminal mechanoreceptor with beaded 'free' nerve-endings. By recording receptive areas electrophysiologically and staining peripheral sensory neurites with horseradish peroxidase, we have shown that 'movement detector' neurites from one trigeminal ganglion do not normally cross the dorsal midline of the head to innervate areas of skin on the opposite side. However, if one trigeminal ganglion is removed before peripheral innervation starts, movement detector neurites from the intact side will now cross the midline to innervate contralateral skin. These observations suggest a specific competitive interaction between movement detector neurites during their innervation of head skin. The second class of receptor, 'rapid transient' detectors, have a different pattern of innervation, crossing the midline in both normal and operated animals.