Placode and neural crest-derived sensory neurons are responsive at early developmental stages to brain-derived neurotrophic factor

The response of embryonic chick nodose ganglion (neural placode-derived) and dorsal root ganglion (neural crest-derived) sensory neurons to the survival and neurite-promoting activity of brain-derived neurotrophic factor (BDNF) was studied in culture. In dissociated, neuron-enriched cultures established from chick embryos between Day 6 (E6) and Day 12 (E12) of development, both nodose ganglion (NG) and dorsal root ganglion (DRG) neurons were responsive on laminin-coated culture dishes to BDNF. In the case of NG, BDNF elicited neurite outgrowth from 40 to 50% of the neurons plated at three embryonic ages; E6, E9, and E12. At the same ages, nerve growth factor (NGF) alone or in combination with BDNF, had little or no effect upon neurite outgrowth from NG neurons. The response of NG neurons to BDNF was dose dependent and was sustainable for at least 7 days in culture. Surprisingly, in view of a previous study carried out using polyornithine as a substrate for neuronal cell attachment, on laminin-coated dishes BDNF also sustained survival and neurite outgrowth from a high percentage (60-70%) of DRG neurons taken from E6 embryos. In marked contrast to NG neurons, the combined effect of saturating levels of BDNF and NGF activity on DRG neurons was greater than the effect of either agent alone at all embryonic ages studied. Under similar culture conditions, BDNF did not elicit survival and neurite outgrowth from paravertebral chain sympathetic neurons or parasympathetic ciliary ganglion neurons. We propose that primary sensory neurons, regardless of their embryological origin, are responsive to a "central-target" (CNS) derived neurotrophic factor--BDNF, while they are differentially responsive to "peripheral-target"-derived growth factors, such as NGF, depending on whether the neurons are of neural crest or placodal origin.     

High K+-mediated survival of spinal sensory neurons depends on developmental stage

Elevated concentrations of K+ (35 mM) have previously been shown to support the survival of most embryonic chick sympathetic neurons in vitro (Wakade et al., Exp cell res 144 (1983) 377, [23]) and to be interchangeable with nerve growth factor (NGF) as a survival-promoting agent for these cells (Wakade & Thoenen, Neurosci lett 45 (1984) 71 [21]). In the present study, we show that dorsal root ganglion (DRG) neurons from embryonic day 6 do not survive in the presence of high K+, although both NGF and brain-derived neurotrophic factor (BDNF) each support the survival of more than 50% of the cells at this developmental stage. At E6, high K+ appears to have a cytotoxic effect on BDNF-dependent neurons, and there is also considerable inhibition of neurite outgrowth. At a later developmental stage (E12), high K+ supports the survival of about 40% of DRG cells. This subpopulation of neurons is distinct from that supported by NGF (as evidenced by the additivity of these two agents), but partially overlaps with that supported by BDNF (i.e., the two agents are less than additive). At E12, only approx. 20% of the cells can be supported by either NGF or BDNF, with the rest depending exclusively on one or the other of these factors. This is in contrast to the situation at E6, where there is considerable overlap between NGF- and BDNF-dependent populations.     

The survival of early chick sympathetic neurons in vitro is dependent on a suitable substrate but independent of NGF

The neuronal cell population of lumbosacral sympathetic ganglia from 7-day-old chick embryos is characterized by a high proportion of cells with the ability to proliferate in culture (Rohrer and Thoenen, 1987). It is now demonstrated that neither proliferation nor survival of these neurons depend on the presence of nerve growth factor (NGF). However, neuronal survival did depend on the culture substrate used: on laminin, E7 neurons survived and their number increased due to proliferation, whereas on fibronectin (FN) or a substrate of molecules from heart cell-conditioned medium (HCM) a significant number of the cells died during early culture periods. Less than 70 and 50% of the number of neurons surviving on a laminin substrate were found on FN and HCM, respectively, after 3 days in culture. Although NGF did not affect neuronal survival, a small increase in neurite extension on these substrates was observed in the presence of NGF. Furthermore, although NGF did not prevent neuronal death after extended culture periods, this could be prevented by elevated extracellular potassium concentrations. Sympathetic neurons of E8 chick embryos however showed a strikingly different response to NGF compared with those of E7: whereas neuronal survival on laminin was not influenced by NGF, a significant effect of NGF on survival and on neurite extension was observed for E8 neurons on a HCM substrate. In contrast to cells from E7 and E8 embryos, the majority of neurons from E11 chick embryos required NGF for survival even on a laminin substrate as described previously (D. Edgar, R. Timpl, and H. Thoenen, 1984, EMBO J. 3, 1463-1468). These results demonstrate that while sympathetic neurons from E7 chick embryos do not depend on the soluble neurotrophic factor NGF for survival in vitro, they are dependent on molecules of the extracellular matrix. With increasing age, the survival requirements demonstrated in vitro change toward the classical pattern of NGF dependency. Low amounts of laminin-like immunoreactivity were shown to be present in sympathetic ganglia of E7 chick embryos which were then shown to increase as development proceeded. These data indicate that laminin may play a role in the survival and development of chick sympathetic neurons not only in vitro, but also in vivo.     

In Vivo Regulation of Brain-Derived Neurotrophic Factor in Dorsal Root Ganglia Is Mediated by Nerve Growth Factor-Triggered Akt Activation during Cystitis

The role of brain-derived neurotrophic factor (BDNF) in sensory hypersensitivity has been suggested; however the molecular mechanisms and signal transduction that regulate BDNF expression in primary afferent neurons during visceral inflammation are not clear. Here we used a rat model of cystitis and found that the mRNA and protein levels of BDNF were increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. BDNF up-regulation in the L6 DRG was triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reduced BDNF levels during cystitis. The neutralizing NGF antibody also subsequently reduced cystitis-induced up-regulation of the serine/threonine kinase Akt activity in L6 DRG. To examine whether the NGF-induced Akt activation led to BDNF up-regulation in DRG in cystitis, we found that in cystitis the phospho-Akt immunoreactivity was co-localized with BDNF in L6 DRG, and prevention of the endogenous Akt activity in the L6 DRG by inhibition of phosphoinositide 3-kinase (PI3K) with a potent inhibitor LY294002 reversed cystitis-induced BDNF up-regulation. Further study showed that application of NGF to the nerve terminals of the ganglion-nerve two-compartmented preparation enhanced BDNF expression in the DRG neuronal soma; which was reduced by pre-treatment of the ganglia with the PI3K inhibitor LY294002 and wortmannin. These in vivo and in vitro experiments indicated that NGF played an important role in the activation of Akt and subsequent up-regulation of BDNF in the sensory neurons in visceral inflammation such as cystitis.     

Effects of the neurotrophins nerve growth factor,neurotrophin-3, and brain-derived neurotrophic factor (BDNF) on neurite growth from adult sensory neurons in compartmented cultures

We used compartmented cultures to study the regulation of adult sensory neurite growth by neurotrophins. We examined the effects of the neurotrophins nerve growth factor (NGF), neurotrophin-3 (NT3), and BDNF on distal neurite elongation from adult rat dorsal root ganglion (DRG) neurons. Neurons were plated in the center compartments of three-chambered dishes in the absence of neurotrophin, and neurite extension into the distal (side) compartments containing NGF, BDNF, or NT3 was quantitated. Initial proximal neurite growth did not require any of the neurotrophins, while subsequent elongation into distal compartments required NGF. After neurites had extended into NGF-containing distal compartments, removal of NGF by treatment with anti-NGF resulted in the cessation of growth with minimal neurite retraction. In contrast to the effects of NGF, no distal neurite elongation was observed into compartments with BDNF or NT3. To examine possible additive influences, neurite extension into compartments containing BDNF plus NGF or NT3 plus NGF was quantitated. There was no increased neurite extension into NGF plus NT3 compartments, while the combination of BDNF plus NGF resulted in an inhibition of neurite extension compared with NGF alone. We then investigated whether the regrowth of neurites that had originally grown into NGF subsequent to in vitro axotomy still required NGF. The results demonstrated that unlike adult sensory nerve regeneration in vivo, the in vitro regrowth did require NGF, and neither BDNF nor NT3 was able to substitute for NGF. Since the initial growth from neurons after dissociation (which is also a regenerative response) did not require NGF, it would appear that neuritic growth and regrowth of adult DRG neurons in vitro includes both NGF-independent and NGF-dependent components. The compartmented culture system provides a unique model to further study aspects of this differential regulation of neurite growth. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 395–410, 1997     

The response of cultured trigeminal and spinal cord motoneurons to nerve growth factor

Dissociated neurons from the trigeminal (V) region of the metencephalic basal plate or the ventral spinal cord from chick embryos of Day 4 (V basal plate) or Day 5 (spinal cord) were cultured on a laminin substratum either in the presence of nerve growth factor (NGF) or in control medium. Assessment was made of neuronal survival, the amount of neurite elaborated, and the percentage of neurons initiating neurites. The presence of motoneurons was verified by retrograde labeling with the fluorescent dye diI. NGF was found to significantly increase the quantity of neuritic processes produced by the spinal cord dissociates at both 24 and 48 hr in vitro. The percentage of neurons initiating neuritic processes was significantly increased by NGF in the trigeminal population at 48 hr in vitro. Neuronal survival was not enhanced by NGF in either group. Both trigeminal and spinal cord neurons were also found to specifically bind 125I-NGF in culture. These results provide direct evidence for an influence of NGF on process formation of early embryonic motoneurons in culture.     

BDNF and NT4/5 promote survival and neurite outgrowth of pontocerebellar mossy fiber neurons.

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and NT4/5 are all found in the developing cerebellum. Granule cells, the major target neurons of mossy fibers, express BDNF during mossy fiber synaptogenesis. To determine whether neurotrophins contribute to the development of cerebellar afferent axons, we characterized the effects of neurotrophins on the growth of mossy fiber neurons from mice and rats in vitro. For a mossy fiber source, we used the basilar pontine nuclei (BPN), the major source of cerebellar mossy fibers in mammals. BDNF and NT4/5 increased BPN neuron survival, neurite outgrowth, growth cone size, and elongation rate, while neither NT3 nor NGF increased survival or outgrowth. In addition, BDNF and NT4/5 reduced the size of neurite bundles. Consistent with these effects, in situ hybridization on cultured basilar pontine neurons revealed the presence of mRNA encoding the TrkB receptor which binds both BDNF and NT4/5 with high affinity. We detected little or no message encoding the TrkC receptor which preferentially binds NT3. BDNF and NT4/5 also increased TrkB mRNA levels in BPN neurons. In addition to previously established functions as an autocrine/paracrine trophic factor for granule cells, the present results indicate that cerebellar BDNF may also act as a target-derived trophic factor for basilar pontine mossy fibers.     

Neurotoxicity caused by didanosine on cultured dorsal root ganglion neurons

The purpose of the present study was to investigate whether didanosine (ddI) directly causes morphological and ultrastructural abnormalities of dorsal root ganglion (DRG) neurons in vitro. Dissociated DRG cells and organotypic DRG explants from embryonic 15-day-old Wistar rats were cultured for 3 days and then exposed to ddI (1 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) for another 3 days and 6 days, respectively. Neurons cultured continuously in medium served as normal controls. The diameter of the neuronal cell body and neurite length were measured in dissociated DRG cell cultures. Neuronal ultrastructural changes were observed in both culture models. ddI induced dose-dependent decreases in neurite number, length of the longest neurite in each neuron, and total neurite length per neuron in dissociated DRG cell cultures with 3 days treatment. There were no morphological changes seen in organotypic DRG cultures even with longer exposure time (6 days). But ddI induced ultrastructural changes in both culture models. Ultrastructural abnormalities included loss of cristae in mitochondria, clustering of microtubules and neurofilaments, accumulation of glycogen-like granules, and emergence of large dense particles between neurites or microtubules. Lysosome-like large particles emerged inconstantly in neurites. ddI induced a neurite retraction or neurite loss in a dose-dependent manner in dissociated DRG neurons, suggesting that ddI may partially contribute to developing peripheral neuropathy. Cytoskeletal rearrangement and ultrastructural abnormalities caused by ddI in both culture models may have a key role in neurite degeneration.     

Brain-derived neurotrophic factor increases survival and differentiated functions of rat septal cholinergic neurons in culture

Brain-derived neurotrophic factor (BDNF) was found to promote the survival of E17 rat embryo septal cholinergic neurons in culture, as assessed by a histochemical stain for acetylcholinesterase (AChE). A 2.4-fold increase in neuronal survival was achieved with 10 ng/ml BDNF. After initial deprivation of growth factor for 7 days, BDNF failed to bring about this increase, strongly suggesting that BDNF promotes cell survival and not just induction of AChE. BDNF was also found to increase the levels of cholinergic enzymes; choline acetyltransferase (ChAT) and AChE activities were increased by approximately 2-fold in the presence of 50 ng/ml BDNF. BDNF produced a 3-fold increase in the number of cells bearing the NGF receptor, as detected by the monoclonal antibody IgG-192. Although NGF had no additive effect with BDNF in terms of neuronal survival, suggesting that both act on a similar neuronal population, the combination of both produced an additive response, approximately a 6-fold increase, in ChAT activity.     

Nerve growth factor-independent development of embryonic mouse sympathetic neurons in dissociated cell culture

Although ganglia from neonatal mouse sympathetic ganglia require nerve growth factor (NGF) for survival in culture, explanted sympathetic ganglia from early embryonic stages do not require added NGF for survival and growth. To determine whether the change in growth factor requirement is due to changes in the neurons themselves, to variations in neuronal populations, or to changes in nonneuronal cells, we examined the response to growth factors by dissociated sympathetic neurons at various stages of development. Results indicate that neurons from the 14-day gestational (E14) superior cervical ganglion (SCG) do not require NGF for initial survival and neurite extension, but do require the conditioned medium neurite extension factor, CMF. By 2 to 3 days thereafter, whether in vivo or in culture, most neurons have developed a requirement for NGF for survival in culture. During the same period, there is a concomitant increase in responsiveness to NGF alone as a trophic agent. Changes in response to NGF are not due to changes in NGF content of ganglia, to interactions in culture with nonneuronal cells, or to age-related differences in NGF requirements for maximum survival. The changes in growth factor requirements may be related to mechanisms regulating specificity of nerve-target connections.     

Characterization of the Responses of Purkinje Cells to Neurotrophin Treatment

Abstract: The ability of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) to promote neuronal survival and phenotypic differentiation was examined in dissociated cultures from embryonic day 16 rat cerebellum. BDNF treatment increased the survival of neuron-specific enolase-immunopositive cells by 250 and 400% after 8 and 10 days in culture, respectively. A subpopulation of these neurons, the Purkinje cells, identified by calbindin staining, was increased to an equivalent extent, ∼200%, following BDNF, NT-4/5, or NT-3 treatment. The number of GABAergic neurons, identified by GABA immunoreactivity, was greatly increased by treatment with BDNF (470%) and moderately by NT-4/5 (46%), whereas NT-3 was without effect. NGF failed to increase the number of either Purkinje cells or GABAergic neurons. Addition of BDNF within 48 h of cell plating was required to obtain a maximal increase in Purkinje cell number after 8 days. In contrast, the NT-3 responses were nearly equivalent even if treatment was delayed for 96 h after plating. BDNF, NT-4/5, and NT-3, but not NGF, induced the rapid expression of the immediate early gene c- fos . Immunocytochemical double-labeling with antibodies to c-fos and calbindin was used to identify Purkinje cells that responded to neurotrophin treatment by induction of c-fos. After 4 days in vitro, both BDNF and NT-3 induced the formation of c-fos protein in calbindin-immunopositive neurons, whereas NT-4/5 did not. The latter results suggest that although BDNF and NT-4/5 have been shown to act through a common receptor, TrkB, it appears that the effects of BDNF and NT-4/5 are not identical.     

BDNF and NT4/5 promote survival and neurite outgrowth of pontocerebellar mossy fiber neurons

The neurotrophins nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT3), and NT4/5 are all found in the developing cerebellum. Granule cells, the major target neurons of mossy fibers, express BDNF during mossy fiber synaptogenesis. To determine whether neurotrophins contribute to the development of cerebellar afferent axons, we characterized the effects of neurotrophins on the growth of mossy fiber neurons from mice and rats in vitro. For a mossy fiber source, we used the basilar pontine nuclei (BPN), the major source of cerebellar mossy fibers in mammals. BDNF and NT4/5 increased BPN neuron survival, neurite outgrowth, growth cone size, and elongation rate, while neither NT3 nor NGF increased survival or outgrowth. In addition, BDNF and NT4/5 reduced the size of neurite bundles. Consistent with these effects, in situ hybridization on cultured basilar pontine neurons revealed the presence of mRNA encoding the TrkB receptor which binds both BDNF and NT4/5 with high affinity. We detected little or no message encoding the TrkC receptor which preferentially binds NT3. BDNF and NT4/5 also increased TrkB mRNA levels in BPN neurons. In addition to previously established functions as an autocrine/paracrine trophic factor for granule cells, the present results indicate that cerebellar BDNF may also act as a target‐derived trophic factor for basilar pontine mossy fibers. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 254–269, 1999     

Sympathetic neuronal survival induced by retinal trophic factors.

Neuronal survival in the vertebrate peripheral nervous system depends on neurotrophic factors available from target tissues. In an attempt to identify novel survival factors, we have studied the effect of secreted factors from retinal cells on the survival of chick sympathetic ganglion neurons. Embryonic day 10 sympathetic neurons undergo programmed cell death after 48 h without appropriate levels of nerve growth factor (NGF). Retina Conditioned Media (RCM) from explants of embryonic day 11 retinas maintained for 4 days in vitro supported 90% of E10 chick sympathetic neurons after 48 h. Conditioned medium from purified chick retinal Muller glial cells supported nearly 100% of E10 chick sympathetic neurons. Anti-NGF (1 microg/mL) blocked the survival effect of NGF, but did not block the trophic effect of RCM. Neither BDNF nor NT4 (0.1-50 ng/mL) supported E10 sympathetic neuron survival. Incubation of chimeric immunoglobulin-receptors TrkA, TrkB, or TrkC had no effect on RCM-induced sympathetic neuron survival. The survival effects were not blocked by anti-GDNF, anti-TGFbeta, and anti-CNTF and were not mimicked by FGFb (0.1-10 nM). LY294002 at 50 microM, but not PD098059 blocked sympathetic survival induced by RCM. Further, the combination of RCM and NGF did not result in an increase in neuronal survival compared with NGF alone (82% survival after 48 h). The secreted factor in RCM is retained in subfractions with a molecular weight above 100 kDa, binds to heparin, and is unaffected by dialysis, but is heat sensitive. Our results indicate the presence of a high-molecular weight retinal secreted factor that supports sympathetic neurons in culture.     

Fibroblast-like cells from rat plantar skin and neurotrophin-transfected 3T3 fibroblasts influence neurite growth from rat sensory neurons in vitro

Our previous finding that skin-derived and muscle-derived molecules can be used to sort regenerating rat sciatic nerve axons evoked questions concerning neuron-target interactions at the level of single cells, which prompted the present study. The results show that dorsal root ganglion (DRG) neurons co-cultured with fibroblast-like skin-derived cells emit many neurites. These have a proximal linear segment and a distal network of beaded branches in direct relation to skin-derived cells. Electron microscopic examination of such co-cultures showed bundles of neurites at some distance from the target cells and single profiles closely apposed to subjacent cells. RNase protection assay revealed that cultivated skin-derived cells express nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). In co-cultures of DRG neurons and 3T3 fibroblasts overexpressing either of the neurotrophins produced by skin-derived cells the picture varied. NT-3 transfected 3T3 fibroblasts gave a growth pattern similar to that seen with skin-derived cells. Neurons co-cultured with mock-transfected 3T3 fibroblasts were small and showed weak neurite growth. In co-cultures with a membrane insert between skin-derived cells or 3T3 fibroblasts and DRG neurons few neurons survived and neurite growth was very sparse. We conclude that skin-derived cells stimulate neurite growth from sensory neurons in vitro, that these cells produce NGF, BDNF, NT-3 and NT-4 and that 3T3 fibroblasts producing NT-3 mimic the effect of skin-derived cells on sensory neurons in co-culture. Finally the results suggest that cell surface molecules are important for neuritogenesis.     

Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF

mRNA coding for brain-derived neurotrophic factor (BDNF) has been detected in cultured L929 fibroblasts, rat dermal fibroblasts, and sciatic nerve Schwann cells, as well as in rat skin. Medium conditioned by cultured fibroblasts and Schwann cells also stimulates neurite growth from retinal explants and promotes the survival in culture of BDNF-responsive sensory neurons; biological activity is abolished by antibodies raised against NGF. These results suggest that molecules with BDNF-like activity may be produced by cells in the peripheral nervous system and that the BDNF-like activity in fibroblasts and Schwann cells is derived from molecules immunologically related to NGF. In support of this concept, antibodies against NGF have been found to reduce the biological activity of recombinant BDNF in culture and to cross-react with BDNF on Western blots.     

Differential Age-Dependent Trophic Responses of Nodose,Sensory, and Sympathetic Neurons to Neurotrophins and GDNF: Potencies for Neurite Extension in Explant Culture

The age-dependent trophic responses of sympathetic, sensory, and nodose neurons to the neuro-trophins NGF, BDNF, and NT-3 and to glial cell line-derived neurotrophic factor (GDNF) were examined by an explant culture system. Superior cervical ganglia (SCG), dorsal root ganglia (DRG), and nodose ganglia (NG) were removed from rat embryos (E18), neonatals ( 1 day old), young adults (3–6 months old), and aged adults (>24 months old). The ganglia were cultured with and without each neurotrophic factor; the neurite extension and neurite density were then assessed. The SCG from rats of all ages were significantly influenced by NGF, NT-3, and GDNF; the effects of NT-3 and GDNF were reduced after maturation. The DRG from embryos and neonates were influenced by all neurotrophic factors; however, the effects of BDNF and NT-3 disappeared after maturation. The GDNF showed little effect on adult DRG and no effect on aged DRG. The effect of NGF was preserved over all ages of DRG. The NG from embryonic rats were significantly responsive to BDNF and GDNF; their effects decreased in the neonatal NG, but a minimum effect remained in the aged NG. These results indicate that age-dependent profiles of trophic effects differ extensively among the lineages of the peripheral nervous system and also among the individual neurotrophic factors.     

Selective dependence of mammalian dorsal root ganglion neurons on nerve growth factor during embryonic development.

We have investigated the NGF dependence of dorsal root ganglion (DRG) neurons in mammals using a paradigm of multiple in utero injections of a high titer anti-NGF antiserum. We have determined the specificity of our antiserum in relation to other members of the NGF neurotrophin family and found no cross-reactivity with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). To identify various classes of DRG neurons, we have stained their characteristic central projections with Dil. We show here that the NGF dependence of DRG neurons is strikingly selective. Although a majority of DRG neurons are lost after NGF deprivation during embryonic life, these are almost exclusively small diameter neurons that project to laminae I and II of the dorsal horn and presumably subserve nociception and thermoreception. Larger neurons that project to more ventral spinal laminae and subserve other sensory modalities do not require NGF for survival. These NGF-independent DRG neurons likely require one of the more recently identified neurotrophins, BDNF or NT-3.     

The cranial sensory ganglia in culture: Differences in the response of placode-derived and neural crest-derived neurons to nerve growth factor

Explants of cranial sensory ganglia and dorsal root ganglia from embryonic chicks of 4 to 16 days incubation (E4 to E16) were grown for 24 hr in collagen gels with and without nerve growth factor (NGF) in the culture medium. NGF elicited marked neurite outgrowth from neural crest-derived explants, i.e., dorsal root ganglia, the dorsomedial part of the trigeminal ganglion, and the jugular ganglion. This response was first observed in ganglia taken from E6 embryos, reached a maximum between E8 and E11, and gradually declined through E16. Explants in which the neurons were of placodal origin varied in their response to NGF. There was negligible neurite outgrowth from explants of the ventrolateral part of the trigeminal ganglion and the vestibular ganglion grown in the presence of NGF. The geniculate, petrosal, and nodose ganglia exhibited an early moderate response to NGF. This was first evident in ganglia taken from E5 embryos, reached a maximum by E6, and declined through later ages, becoming negligible by E13. Dissociated neuron-enriched cultures of vestibular, petrosal, jugular, and dorsal root ganglia were established from embryos taken at E6 and E9. At both ages NGF elicited neurite outgrowth from a substantial proportion of neural crest-derived neurons (jugular and dorsal root ganglia) but did not promote the growth of placode-derived neurons (vestibular and petrosal ganglia). Our findings demonstrate a marked difference in the response of neural crest and placode-derived sensory neurones to NGF. The data from dissociated neuron-enriched cultures suggest that NGF promotes survival and growth of sensory ganglionic neurons of neural crest origin but not of placodal origin. The data from explant cultures suggest that NGF promotes neurite outgrowth from placodal neurons of the geniculate, petrosal, and nodose ganglia early in their ontogeny. However, we argue that this fibre outgrowth emanates not from the placodal neurons but from neural crest-derived cells which normally give rise only to satellite cells of these ganglia.     

Expression and histochemical localization of ciliary neurotrophic factor in cultured adult rat dorsal root ganglion neurons

Ciliary neurotrophic factor (CNTF) is abundantly expressed in Schwann cells in adult mammalian peripheral nerves, but not in neurons. After peripheral nerve injury, CNTF released from disrupted Schwann cells is likely to promote neuronal survival and axonal regeneration. In the present study, we examined the expression and histochemical localization of CNTF in adult rat DRG in vivo and in vitro. In contrast to the restricted expression in Schwann cells in vivo, we observed abundant CNTF mRNA and protein expression in DRG neurons after 3 h, 2, 7, and 15 days in dissociated cell culture. At later stages (7 and 15 days) of culture, CNTF immunoreactivity was detected in both neuronal cell bodies and regenerating neurites. These results suggest that CNTF is synthesized and transported to neurites in cultured DRG neurons. Since we failed to observe CNTF immunoreactivity in DRG neurons in explant culture, disruption of cell–cell interactions, rather than the culture itself, may be an inducible factor for localization of CNTF in the neurons.