The sensory mechanotransduction ion channel ASIC2 (acid sensitive ion channel 2) is regulated by neurotrophin availability.

Almost all sensory neurons of the dorsal root ganglia have a mechanosensitive receptive field in the periphery. We have shown that the sensitivity to mechanical stimuli of a subset of sensory neurons that are slowly adapting mechanoreceptors (SAM) is strongly dependent on the availability of brain-derived neurotrophic factor (BDNF). Here we have investigated whether the ASIC2 sodium channel, recently shown by us to be necessary for normal SAM sensitivity, might be regulated by BDNF and thus partially account for the down-regulation of SAM sensitivity seen in BDNF deficient mice. We show that the mRNA for ASIC2 channels is reduced in the DRG of BDNF deficient mice indicating that BDNF might maintain its expression in vivo. We also made short-term cultures of sensory neurons from adult BDNF deficient mice and used a specific antibody to detect the presence of ASIC2 channels in different classes of sensory neurons. We observed that the channel protein was dramatically down-regulated selectively in medium and large diameter neurons and this expression could be rescued in a dose and time dependent manner by addition of BDNF to the culture (10-100 ng/ml). Drugs that block new transcription or protein synthesis also prevented the rescue effects of BDNF. We observed that ASIC2 channels were down-regulated in sensory neurons taken from neurotrophin-4 and neurotrophin-3 deficient mice; these effects might be due to a selective loss of neurons that normally express large amounts of ASIC2 channels. In summary, our data identify the ASIC2 channel as a target of BDNF signaling in vivo and suggest that the functional down-regulation of sensory mechanotransduction in BDNF deficient mice is in part due to loss of ASIC2 expression.

Identification of caveolae-like structures on the surface of intact cells using scanning force microscopy.

Caveolae are small, functionally important membrane invaginations found on the surface of many different cell types. Using electron microscopy, caveolae can be unequivocally identified in cell membranes by virtue of their size and the presence of caveolin/VIP22 proteins in the caveolar coat. In this study we have applied for the first time scanning force microscopy (SFM), to visualize caveolae on the surface of living and fixed cells. By scanning the membranes of Chinese hamster ovary cells (CHO), using the tapping mode of the SFM in fluid, we could visualize small membrane pits on the cell membranes of living and fixed cells. Two populations of pits with mean diameters of around 100 nm and 200 nm were present. In addition, the location of many pits visualized with the SFM was coincident with membrane spots fluorescently labeled with a green fluorescent protein-caveolin-1 fusion protein. Scanning force microscopy on cells treated with methyl-beta-cyclodextrin, an agent that sequesters cholesterol and disrupts caveolae, abolished pits with a measured diameter of 100 nm but left pits of around 200 nm diameter intact. Thus, the smallest membrane pits measured with the SFM in CHO cells were indeed very likely to be identical to caveolae. These experiments show for the first time that SFM can be used to visualize caveolae in intact cells.

Selective activation of nociceptors by P2X receptor agonists in normal and inflamed rat skin.

1. ATP can elicit pain in humans and, together with other P2X channel agonists, can produce nocifensive responses in rodents. We used the rat in vitro skin-nerve preparation to quantify primary afferent responses to ATP and its stable analogue alpha,beta-methylene ATP in normal and carrageenan-inflamed skin. 2. Both ATP and alpha,beta-methylene ATP were found to specifically activate the peripheral terminals of Adelta and C-fibre nociceptors in the skin. Thirty-nine per cent of the nociceptors tested responded to the maximal dose of alpha,beta-methylene ATP (5 mM). In contrast, non-nociceptive, low-threshold mechano-sensitive fibres were never activated by the same agonist concentrations. 3. Amongst the nociceptor population, C-mechanoheat fibres (C-MH or polymodal nociceptors) were markedly more responsive to P2X agonists than mechanonociceptors (C-M nociceptors) with Adelta- or C-fibre axons. Both C-mechanoheat and C-mechanonociceptors were activated by alpha,beta-methylene ATP doses as low as 50 microM. 4. In skin inflamed with carrageenan 3-4 h before recording both the number of responsive C-fibre nociceptors and their response magnitude increased. The increased neural response under inflammatory conditions was largely observed in C-mechanoheat or polymodal nociceptors. After low doses of P2X agonists C-MH fibres but not C-M fibres developed elevated ongoing activity and this effect was only seen after carrageenan inflammation. The time course of alpha,beta-methylene ATP-evoked discharges in nociceptors was found to correlate well with the time course of behavioural nocifensive responses in rats to the same agonist described in a previous study (Hamilton et al. 1999). 5. We conclude that the rapid increase in the number of alpha,beta-methylene ATP responsive nociceptors and the increased magnitude of the neural response following carrageenan inflammation explains why very low concentrations of such agonists can cause pain in inflammatory states.

BDNF but not NT-4 is required for normal flexion reflex plasticity and function.

Neurotrophins can directly modulate the function of diverse types of central nervous system synapses. Brain-derived neurotrophic factor (BDNF) might be released by nociceptors onto spinal neurons and mediate central sensitization associated with chronic pain. We have studied the role of BDNF and neurotrophin-4 (NT-4), both ligands of the trkB tyrosine kinase receptor, in synaptic transmission and reflex plasticity in the mouse spinal cord. We used an in vitro spinal cord preparation to measure monosynaptic and polysynaptic reflexes evoked by primary afferents in BDNF- and NT-4-deficient mice. In situ hybridization studies show that both these neurotrophins are synthesized by sensory neurons, and NT-4, but not BDNF, also is expressed by spinal neurons. BDNF null mutants display selective deficits in the ventral root potential (VRP) evoked by stimulating nociceptive primary afferents whereas the non-nociceptive portion of the VRP remained unaltered. In addition, activity-dependent plasticity of the VRP evoked by repetitive (1 Hz) stimulation of nociceptive primary afferents (termed wind-up) was substantially reduced in BDNF-deficient mice. This plasticity also was reduced in a reversible manner by the protein kinase inhibitor K252a. Although the trkB ligand NT-4 is normally present, reflex properties in NT-4 null mutant mice were normal. Pharmacological studies also indicated that spinal N-methyl-d-aspartate receptor function was unaltered in BDNF-deficient mice. Using immunocytochemistry for markers of nociceptive neurons we found no evidence that their number or connectivity was substantially altered in BDNF-deficient mice. Our data therefore are consistent with a direct role for presynaptic BDNF release from sensory neurons in the modulation of pain-related neurotransmission.

Lack of neurotrophin-4 causes selective structural and chemical deficits in sympathetic ganglia and their preganglionic innervation.

Neurotrophin-4 (NT-4) is perhaps the still most enigmatic member of the neurotrophin family. We show here that NT-4 is expressed in neurons of paravertebral and prevertebral sympathetic ganglia, i.e., the superior cervical (SCG), stellate (SG), and celiac (CG) ganglion. Mice deficient for NT-4 showed a significant reduction (20-30%) of preganglionic sympathetic neurons in the intermediolateral column (IML) of the thoracic spinal cord. In contrast, neuron numbers in the SCG, SG, and CG were unchanged. Numbers of axons in the thoracic sympathetic trunk (TST) connecting the SG with lower paravertebral ganglia were also reduced, whereas axon numbers in the cervical sympathetic trunk (CST) were unaltered. Axon losses in the TST were paralleled by losses of synaptic terminals on SG neurons visualized by electron microscopy. Furthermore, immunoreactivity for the synaptic vesicle antigen SV2 was clearly reduced in the SG and CG. Levels of catecholamines and tyrosine hydroxylase immunoreactivity were dramatically reduced in the SG and the CG but not in the SCG. Despite this severe phenotype in the sympathetic system, blood pressure levels were not reduced and displayed a pattern more typical of deficits in baroreceptor afferents. Numbers of IML neurons were unaltered at postnatal day 4, suggesting a postnatal requirement for their maintenance. In light of these and previous data, we hypothesize that NT-4 provided by postganglionic sympathetic neurons is required for establishing and/or maintaining synapses of IML neurons on postganglionic cells. Impairment of synaptic connectivity may consequently reduce impulse flow, causing a reduction in transmitter synthesis in postganglionic neurons.

The DRASIC cation channel contributes to the detection of cutaneous touch and acid stimuli in mice.

Cation channels in the DEG/ENaC family are proposed to detect cutaneous stimuli in mammals. We localized one such channel, DRASIC, in several different specialized sensory nerve endings of skin, suggesting it might participate in mechanosensation and/or acid-evoked nociception. Disrupting the mouse DRASIC gene altered sensory transduction in specific and distinct ways. Loss of DRASIC increased the sensitivity of mechanoreceptors detecting light touch, but it reduced the sensitivity of a mechanoreceptor responding to noxious pinch and decreased the response of acid- and noxious heat-sensitive nociceptors. The data suggest that DRASIC subunits participate in heteromultimeric channel complexes in sensory neurons. Moreover, in different cellular contexts, DRASIC may respond to mechanical stimuli or to low pH to mediate normal touch and pain sensation.

The mammalian sodium channel BNC1 is required for normal touch sensation.

Of the vertebrate senses, touch is the least understood at the molecular level The ion channels that form the core of the mechanosensory complex and confer touch sensitivity remain unknown. However, the similarity of the brain sodium channel 1 (BNC1) to nematode proteins involved in mechanotransduction indicated that it might be a part of such a mechanosensor. Here we show that disrupting the mouse BNC1 gene markedly reduces the sensitivity of a specific component of mechanosensation: low-threshold rapidly adapting mechanoreceptors. In rodent hairy skin these mechanoreceptors are excited by hair movement. Consistent with this function, we found BNC1 in the lanceolate nerve endings that lie adjacent to and surround the hair follicle. Although BNC1 has been proposed to have a role in pH sensing, the acid-evoked current in cultured sensory neurons and the response of acid-stimulated nociceptors were normal in BNC1 null mice. These data identify the BNC1 channel as essential for the normal detection of light touch and indicate that BNC1 may be a central component of a mechanosensory complex.

Hypoalgesia and altered inflammatory responses in mice lacking kinin B1 receptors.

Kinins are important mediators in cardiovascular homeostasis, inflammation, and nociception. Two kinin receptors have been described, B1 and B2. The B2 receptor is constitutively expressed, and its targeted disruption leads to salt-sensitive hypertension and altered nociception. The B1 receptor is a heptahelical receptor distinct from the B2 receptor in that it is highly inducible by inflammatory mediators such as bacterial lipopolysaccharide and interleukins. To clarify its physiological function, we have generated mice with a targeted deletion of the gene for the B1 receptor. B1 receptor-deficient animals are healthy, fertile, and normotensive. In these mice, bacterial lipopolysaccharide-induced hypotension is blunted, and there is a reduced accumulation of polymorphonuclear leukocytes in inflamed tissue. Moreover, under normal noninflamed conditions, they are analgesic in behavioral tests of chemical and thermal nociception. Using whole-cell patch-clamp recordings, we show that the B1 receptor was not necessary for regulating the noxious heat sensitivity of isolated nociceptors. However, by using an in vitro preparation, we could show that functional B1 receptors are present in the spinal cord, and their activation can facilitate a nociceptive reflex. Furthermore, in B1 receptor-deficient mice, we observed a reduction in the activity-dependent facilitation (wind-up) of a nociceptive spinal reflex. Thus, the kinin B1 receptor plays an essential physiological role in the initiation of inflammatory responses and the modulation of spinal cord plasticity that underlies the central component of pain. The B1 receptor therefore represents a useful pharmacological target especially for the treatment of inflammatory disorders and pain.

Neurotrophins, nociceptors and pain.

Nerve growth factor (NGF) is known to play a key role in the development of hyperalgesia after inflammatory injury. The increased levels of NGF that accompany injury lead to hyperalgesia via peripheral and central spinal mechanisms. New evidence reviewed here indicates that NGF can directly sensitize nociceptive neurones to noxious heat stimuli through rapid modulation of heat/vanilloid receptors or via de-novo increased expression of heat receptors. In addition, new data suggest that the central sensitization that can result from increased NGF may be mediated via central release of another neurotrophin, brain-derived neurotrophic factor.

Abundant production of brain-derived neurotrophic factor by adult visceral epithelia. Implications for paracrine and target-derived Neurotrophic functions.

Brain-derived neurotrophic factor (BDNF) plays a crucial role for the survival of visceral sensory neurons during development. However, the physiological sources and the function of BDNF in the adult viscera are poorly described. We have investigated the cellular sources and the potential role of BDNF in adult murine viscera. We found markedly different amounts of BDNF protein in different organs. Surprisingly, BDNF levels in the urinary bladder, lung, and colon were higher than those found in the brain or skin. In situ hybridization experiments revealed that BDNF mRNA was made by visceral epithelial cells, several types of smooth muscle, and neurons of the myenteric plexus. Epithelia that expressed BDNF lacked both the high- and low-affinity receptors for BDNF, trkB and p75(NTR). In contrast, both receptors were present on neurons of the peripheral nervous system. Studies with BDNF-/-mice demonstrated that epithelial and smooth muscle cells developed normally in the absence of BDNF. These data provide evidence that visceral epithelia are a major source, but not a target, of BDNF in the adult viscera. The abundance of BDNF protein in certain internal organs suggests that this neurotrophin may regulate the function of adult visceral sensory and motor neurons.

Cellular sources of enhanced brain-derived neurotrophic factor production in a mouse model of allergic inflammation.

The aim of this study was to investigate production and cellular sources of brain-derived neurotrophic factor (BDNF) production in allergic asthma. For this purpose a mouse model of chronic and severe ovalbumin (OVA)-induced airway inflammation was developed. Allergen-exposed mice developed elevated immunoglobulin E titers; airway inflammation with influx of lymphocytes, monocytes, and eosinophils; and airway hyperresponsiveness. In addition to an influx of inflammatory cells, interleukin (IL)-4 and IL-5 production were enhanced, macrophages showed morphologic signs of activation, and airway epithelium was thickened and displayed a goblet-cell hyperplasia with a marked mucus production. BDNF was detected using in situ hybridization and enzyme-linked immunosorbent assay. Constitutive expression of BDNF messenger RNA (mRNA) was observed in the respiratory epithelium of sensitized and nonsensitized mouse lungs. In addition, BDNF mRNA was detected in airway inflammatory infiltrations and bronchoalveolar lavage fluid (BALF) cells of OVA-sensitized and aerosol-challenged mice. Highest BDNF protein levels were detected in BALF after long-term allergen aerosol exposure. Analysis of BDNF production by isolated lymphocyte subsets revealed T but not B cells as a cellular source of BDNF. In addition, activated alveolar macrophages were identified as BDNF-positive cells. These data indicate that in allergic airway inflammation BDNF production is upregulated and immune cells serve as a source of BDNF.

Isolectin B(4)-positive and -negative nociceptors are functionally distinct.

Small-diameter sensory neurons that are primarily nociceptors can be divided neurochemically into two populations: isolectin B(4) (IB(4))-positive nonpeptidergic neurons, and IB(4)-negative peptidergic neurons. It has been shown that IB(4)-positive neurons depend on glial-derived neurotrophic factor (GDNF), whereas IB(4)-negative neurons depend on NGF for survival during postnatal development (Molliver et al., 1997). Furthermore, these two populations of nociceptors terminate in distinct regions of the superficial spinal cord. To date, however, no evidence exists that indicates whether these two groups of nociceptors have distinct functional roles in the process of nociception (Snider and McMahon, 1998). To search for functional differences, we performed whole-cell voltage and current-clamp recordings on acutely isolated adult mouse dorsal root ganglion neurons that were labeled with fluorescent IB(4). We found that IB(4)-positive neurons have longer-duration action potentials, higher densities of TTX-resistant sodium currents, and smaller noxious heat-activated currents than IB(4)-negative neurons. Furthermore, we show that NGF, but not GDNF, directly increases the number of neurons that respond to noxious heat. The different electrophysiological properties expressed by IB(4)-positive and -negative small neurons, including their different heat sensitivities, indicates that they may relay distinct aspects of noxious stimuli both acutely and after injury in vivo.

Stomatin, a MEC-2 like protein, is expressed by mammalian sensory neurons.

The molecular mechanism whereby vertebrate primary sensory neurons convert mechanical energy at their receptive fields into action potentials is unknown. In recent years, genetic screens for touch insensitive mutants of the nematode worm Caenorhabditis elegans have led to the identification of several genes required for mechanical sensitivity. A model has been proposed in which a mechanically gated ion channel is connected both to the extracellular matrix and to the cytoskeleton. Displacement of the membrane is proposed to produce a shearing force that pulls the channel open. MEC-2 is thought to play an important role in this complex by linking the ion channel to the cytoskeleton. MEC-2 is highly homologous to a vertebrate protein called stomatin. Stomatin was first isolated from erythrocytes where it is a major integral membrane protein. To date, however, no data on neuronal expression of stomatin in the peripheral nervous system (PNS) or central nervous system (CNS) is available. Here, we have used RT-PCR, in situ hybridization, Northern blotting, and immunocytochemistry to demonstrate that stomatin is expressed by all sensory neurons in mouse dorsal root ganglia. Indirect immunofluorescence together with transfection of cultured adult sensory neurons with epitope-tagged stomatin show that stomatin is localized in spots on somatic and axonal membranes. During development, stomatin begins to be expressed by sensory neurons only as target innervation occurs. The onset of expression of stomatin thus coincides with the onset of functional mechanical sensitivity. Together, our data suggest that stomatin, like the C. elegans MEC-2 gene, is expressed in an appropriate temporal and spatial manner to participate in a putative vertebrate mechanotransduction complex.

Neurotrophins: a link between airway inflammation and airway smooth muscle contractility in asthma?

BACKGROUND: Bronchial asthma (BA) is characterized by a unique type of airway inflammation, epithelial cell damage and increased airway smooth muscle (ASM) contractility. The regulatory network between the immunological events and the neuronal control of ASM contractility remains to be defined. METHODS: Utilizing a well-characterized mouse model of airway inflammation and BA, we analyzed the production and function of neurotrophins in allergic asthma. To confirm these data in humans, segmental allergen provocation was performed in mild asthmatics. RESULTS: Allergen-induced airway inflammation was associated with increased local production of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in mice as well as in humans. In bronchoalveolar lavage fluid (BALF), NGF levels were increased 4- to 5-fold in men and mice 1 day after allergen provocation. The increase in BDNF was about 2-fold in both models. Treatment of mice with anti-NGF prevented development of airway hyperresponsiveness (AHR). In the human study group, NGF levels in BALF after allergen provocation were correlated significantly with baseline FEV1 levels. CONCLUSION: These data strongly suggest that neurotrophins serve as a link between airway inflammation and neuronal control of ASM constriction in BA.

Stability and plasticity of primary afferent projections following nerve regeneration and central degeneration.

Sensory neurons of the dorsal root ganglia (DRG) regenerate their peripheral axons with relative ease following a nerve lesion. The capacity for central regeneration appears more limited. However, after nerve lesion, some DRG neurons gain a regenerative advantage to sprout centrally. We developed a lesion model in the rat to test whether, after prior lesion of their peripheral axons, subsets of cutaneous afferents benefit differently in their ability to sprout into adjacent spinal segments denervated by dorsal rhizotomy. We found that under identical circumstances, myelinated sensory neurons, small-diameter peptidergic sensory neurons containing calcitonin gene related peptide (CGRP), and small-diameter nonpeptidergic neurons that bind the lectin from the plant Griffonia simplificolia, isolectin B4 (IB4) differ dramatically in their ability to regenerate centrally. Myelinated afferent terminals labelled transganglionically with cholera-toxin beta-subunit gain a small advantage in collaterally sprouting into the adjacent denervated neuropil in lamina III after prior peripheral nerve lesion. This central regenerative response was not mimicked by experimentally induced inflammation of sensory neuron cell bodies. Intact and unlesioned sensory neurons positive for CGRP sprout vigorously into segments denervated by rhizotomy in a nonsomatotopic manner. In contrast, IB4-positive sensory neurons maintain a somatotopic distribution centrally, which is not altered by prior nerve lesion. These data reveal a remarkably heterogeneous response to regeneration-promoting stimuli amongst three different types of cutaneous sensory neurons. In particular, the divergent responses of peptidergic and nonpeptidergic sensory neurons suggests profound functional differences between these neurochemically distinct neurons.

A new role for neurotrophins: involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control.

Neurotrophins exert many biological effects not directly targeted at neurons, including modulation of keratinocyte proliferation and apoptosis in vitro. Here we exploit the cyclic growth and regression activity of the murine hair follicle to explore potential nonneuronal functions of neurotrophins in the skin, and analyze the follicular expression and hair growth-modulatory function of BDNF, NT-4, and their high-affinity receptor, TrkB. The cutaneous expression of BDNF and NT-4 mRNA was strikingly hair cycle dependent and peaked during the spontaneous, apoptosis-driven hair follicle regression (catagen). During catagen, BDNF mRNA and immunoreactivity, as well as NT-4-immunoreactivity, were expressed in the regressing hair follicle compartments, whereas TrkB mRNA and immunoreactivity were seen in dermal papilla fibroblasts, epithelial strand, and hair germ. BDNF or NT-4 knockout mice showed significant catagen retardation, whereas BDNF-overexpressing mice displayed acceleration of catagen and significant shortening of hair length. Finally, BDNF and NT-4 accelerated catagen development in murine skin organ culture. Together, our data suggest that BDNF and NT-4 play a previously unrecognized role in skin physiology as agents of hair growth control. Thus, TrkB agonists and antagonists deserve exploration as novel hair growth-modulatory drugs for the management of common hair growth disorders.

BDNF overexpression induces differential increases among subsets of sympathetic innervation in murine back skin.

Besides their recognized dependence on nerve growth factor (NGF) during development, the dependence of mature sympathetic ganglion neurons on other neurotrophins is still unclear. Here, we have investigated the sympathetic innervation of back skin in mice overexpressing brain-derived neurotrophic factor (BDNF) under the alpha-myosin heavy-chain promoter, as well as in BDNF knockout (-/-) mice. Compared with wild-type controls, the dorsal skin of BDNF overexpressing mice displayed a significantly enhanced number of adrenergic, tyrosine hydroxylase-immunoreactive (IR) nerve fibres, while cholinergic or peptidergic sensory nerve fibres appeared unaltered. The adrenergic hyperinnervation in dorsal skin of BDNF overexpressing mice was most pronounced in the arrector pili muscle of hair follicles, while no increase of tyrosine hydroxylase-or neuropeptide Y-IR fibres associated with subcutaneous blood vessels was found. Instead, back skin of BDNF knockout (-/-) mice contained significantly fewer tyrosine hydroxylase-IR dermal nerve fibres than wild-type animals. This suggests that BDNF plays an important role in the control of different subsets of adrenergic innervation in murine back skin, and indicates that paravertebral sympathetic ganglia display a previously unrecognized differential BDNF-dependence in vivo.

A new role for neurotrophin-3: involvement in the regulation of hair follicle regression (catagen).

Nervous system and hair follicle epithelium share a common ectodermal origin, and some neurotrophins (NTs) can modulate keratinocyte proliferation and apoptosis. Therefore, it is reasonable to ask whether NTs are also involved in hair growth control. Here, we show that the expression of NT-3 and its high-affinity receptor, tyrosine kinase C, in the skin of C57BL/6 mice is strikingly hair cycle-dependent, with maximal transcript and protein expression seen during spontaneous hair follicle regression (catagen). During catagen, NT-3 and tyrosine kinase C are co-expressed by terminal deoxynucleotidyl transferase-mediated in situ nick end labeling-positive keratinocytes in the club hair and secondary germ. NT-3-overexpressing transgenic mice show precocious catagen development during the postnatal initiation of hair follicle cycling, whereas heterozygous NT-3 knockout (+/-) mice display a significant catagen retardation. Finally, NT-3 stimulates catagen development in organ culture of normal C57BL/6 mouse skin. These observations suggest that the hair follicle is both a source and target of NT-3 and that NT-3/tyrosine kinase C signaling is functionally important in the control of hair follicle regression. Therefore, tyrosine kinase C agonists and antagonists deserve systematic exploration for the management of hair growth disorders that are related to premature (alopecia/effluvium) or retarded catagen (hirsutism/hypertrichosis).

TrkB and neurotrophin-4 are important for development and maintenance of sympathetic preganglionic neurons innervating the adrenal medulla.

The adrenal medulla receives its major presynaptic input from sympathetic preganglionic neurons that are located in the intermediolateral (IML) column of the thoracic spinal cord. The neurotrophic factor concept would predict that these IML neurons receive trophic support from chromaffin cells in the adrenal medulla. We show here that adrenal chromaffin cells in the adult rat store neurotrophin (NT)-4, but do not synthesize or store detectable levels of BDNF or NT-3, respectively. Preganglionic neurons to the adrenal medulla identified by retrograde tracing with fast blue or Fluoro-Gold (FG) express TrkB mRNA. After unilateral destruction of the adrenal medulla, 24% of IML neurons, i.e., all neurons that are preganglionic to the adrenal medulla in spinal cord segments T7-T10, disappear. Administration of NT-4 in gelfoams (6 microgram) implanted into the medullectomized adrenal gland rescued all preganglionic neurons as evidenced by their presence after 4 weeks. NT-3 and cytochrome C were not effective. The action of NT-4 is accompanied by massive sprouting of axons in the vicinity of the NT-4 source as monitored by staining for acetylcholinesterase and synaptophysin immunoreactivity, suggesting that NT-4 may enlarge the terminal field of preganglionic nerves and enhance their access to trophic factors. Analysis of TrkB-deficient mice revealed degenerative changes in axon terminals on chromaffin cells. Furthermore, numbers of FG-labeled IML neurons in spinal cord segments T7-T10 of NT-4-deficient adult mice were significantly reduced. These data are consistent with the notion that NT-4 from chromaffin cells operates through TrkB receptors to regulate development and maintenance of the preganglionic innervation of the adrenal medulla.

Point mutation in trkB causes loss of NT4-dependent neurons without major effects on diverse BDNF responses.

Neurotrophins are a family of soluble ligands that promote the survival and differentiation of peripheral and central neurons and regulate synaptic function. The two neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4), bind and activate a single high-affinity receptor, TrkB. Experiments in cell culture have revealed that an intact Shc adaptor binding site on TrkB and subsequent activation of the Ras/MAPK pathway are important for neuronal survival and neurite outgrowth. To elucidate the intracellular signaling pathways that mediate the diverse effects of BDNF and NT4 in vivo, we have mutated in the mouse germline the Shc binding site in the trkB gene. This trkB(shc) mutation revealed distinctive responses to BDNF and NT4. While nearly all NT4-dependent sensory neurons were lost in trkB(shc/shc) mutant mice, BDNF-dependent neurons were only modestly affected. Activation of MAP kinases and in vitro survival of cultured trkB(shc/shc) neurons were reduced in response to both neurotrophins, with NT4 being less potent than BDNF, suggesting differential activation of TrkB by the two ligands. Moreover, while the Ras/MAPK pathway is required for in vitro differentiation of neuronal cells, trkB(shc/shc) mutant mice do not show any defects in BDNF-dependent differentiation of CNS neurons or in the function of sensory neurons that mediate innocuous touch.