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.     

Threshold-free population analysis identifies larger DRG neurons to respond stronger to NGF stimulation

Sensory neurons in dorsal root ganglia (DRG) are highly heterogeneous in terms of cell size, protein expression, and signaling activity. To analyze their heterogeneity, threshold-based methods are commonly used, which often yield highly variable results due to the subjectivity of the individual investigator. In this work, we introduce a threshold-free analysis approach for sparse and highly heterogeneous datasets obtained from cultures of sensory neurons. This approach is based on population estimates and completely free of investigator-set parameters. With a quantitative automated microscope we measured the signaling state of single DRG neurons by immunofluorescently labeling phosphorylated, i.e., activated Erk1/2. The population density of sensory neurons with and without pain-sensitizing nerve growth factor (NGF) treatment was estimated using a kernel density estimator (KDE). By subtraction of both densities and integration of the positive part, a robust estimate for the size of the responsive subpopulations was obtained. To assure sufficiently large datasets, we determined the number of cells required for reliable estimates using a bootstrapping approach. The proposed methods were employed to analyze response kinetics and response amplitude of DRG neurons after NGF stimulation. We thereby determined the portion of NGF responsive cells on a true population basis. The analysis of the dose dependent NGF response unraveled a biphasic behavior, while the study of its time dependence showed a rapid response, which approached a steady state after less than five minutes. Analyzing two parameter correlations, we found that not only the number of responsive small-sized neurons exceeds the number of responsive large-sized neurons--which is commonly reported and could be explained by the excess of small-sized cells--but also the probability that small-sized cells respond to NGF is higher. In contrast, medium-sized and large-sized neurons showed a larger response amplitude in their mean Erk1/2 activity.     

Sympathetic axons surround nerve growth factor-immunoreactive trigeminal neurons: Observations in mice overexpressing nerve growth factor

It has been postulated that the aberrant projection of sympathetic axons to individual primary sensory neurons may provide the morphological basis for pain-related behaviors in rat models of chronic pain syndrome. Since nerve growth factor (NGF) can elicit the collateral sprouting of noradrenergic sympathetic terminals, it might be predicted that NGF plays a role in mediating the sprouting of sympathetic axons into sensory ganglia. Using a line of transgenic mice overexpressing NGF among glial cells, it was first found that trigeminal ganglia from adult transgenic mice possessed significantly higher levels of NGF protein in comparison to age-matched wild-type mice; as well, detectable levels of NGF mRNA transgene expression were present in both the ganglia and brain stem. Within the trigeminal ganglia, a small proportion of the sensory neuronal population stained immunohistochemically for NGF; a higher percentage of NGF-positive neurons was evident in transgenic mice. New sympathetic axons extended into the trigeminal ganglia of transgenic mice only and formed perineuronal plexuses surrounding only those neurons immunostained for NGF. In addition, such plexuses were accompanied by glial processes from nonmyelinating Schwann cells. From these data, we propose that accumulation of glial-derived NGF by adult sensory neurons and its putative release into the ganglionic environment induce the directional growth of sympathetic axons to the source of NGF, namely, the cell bodies of primary sensory neurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 347–360, 1998     

Role of nerve growth factor in modulation of gastric afferent neurons in the rat

Recent studies demonstrated that experimental ulcers are associated with changes in the properties of voltage-sensitive sodium currents in sensory neurons. We hypothesized that nerve growth factor (NGF) contributes to these changes. Gastric ulcers were induced by acetic acid injection into the wall of the rat stomach. NGF expression was determined by ELISA and immunohistochemically. Sensory neurons were labeled by injection of a retrograde tracer into the gastric wall. Sodium currents were recorded in gastric sensory neurons from nodose and dorsal root ganglia cultured for 24 h in the presence of NGF or a neutralizing NGF antibody, respectively. Gastric ulcer formation caused a rise in NGF concentration within the gastric wall and an increase in NGF immunoreactivity. Exposure to NGF caused a significant increase in the TTX-resistant sodium current, whereas the TTX-sensitive sodium current remained unchanged. This was associated with an acceleration of the recovery from inactivation in spinal sensory neurons. Production and release of NGF in the gastric wall may contribute to sensitization of primary afferent neurons during gastric inflammation.     

NGF receptor-mediated reduction in axonal NGF uptake and retrograde transport following sciatic nerve injury and during regeneration.

Injury to the rat sciatic nerve leads to the induction of nerve growth factor (NGF) receptors on the denervated Schwann cells and their disappearance on the regenerating axons of the axotomized, normally NGF-sensitive sensory and sympathetic neurons. This disappearance in the axonal expression and retrograde transport of NGF receptors is associated with a similarly dramatic reduction in the axonal uptake and retrograde transport of NGF following axotomy and during regeneration. In view of the massive NGF synthesis occurring in the injured nerve, these results suggest that, while sensory and sympathetic neurons are the primary targets of NGF in the normal peripheral nervous system, the denervated Schwann cells may become its primary target in the aftermath of nerve injury.     

Raf and akt mediate distinct aspects of sensory axon growth

Nerve growth factor (NGF) induces dramatic axon growth from responsive embryonic peripheral neurons. However, the roles of the various NGF-triggered signaling cascades in determining specific axon morphological features remain unknown. Here, we transfected activated and inhibitory mutants of Trk effectors into sensory neurons lacking the proapoptotic protein Bax. This allowed axon growth to be studied in the absence of NGF, enabling us to observe the contributions of individual signaling mediators. While Ras was both necessary and sufficient for NGF-stimulated axon growth, the Ras effectors Raf and Akt induced distinct morphologies. Activated Raf-1 caused axon lengthening comparable to NGF, while active Akt increased axon caliber and branching. Our results suggest that the different Trk effector pathways mediate distinct morphological aspects of developing neurons.     

BDNF is essentially required for the early postnatal survival of nociceptors

Neurotrophins promote the survival of specific types of neurons during development and ensure proper maintenance and function of mature responsive neurons. Significant effects of BDNF (Brain-Derived Neurotrophic Factor) on pain physiology have been reported but the contribution of this neurotrophin to the development of nociceptors has not been investigated. We present evidence that BDNF is required for the survival of a significant fraction of peptidergic and non-peptidergic nociceptors in dorsal root ganglia (DRG) postnatally. Bdnf homozygous mutant mice lose approximately half of all nociceptive neurons during the first 2 weeks of life and adult heterozygotes exhibit hypoalgesia and a loss of 25% of all nociceptive neurons. Our in vitro analyses indicate that BDNF-dependent nociceptive neurons also respond to NGF and GDNF. Expression analyses at perinatal times indicate that BDNF is predominantly produced within sensory ganglia and is more abundant than skin-derived NGF or GDNF. Function-blocking studies with BDNF specific antibodies in vitro or cultures of BDNF-deficient sensory neurons suggest that BDNF acts in an autocrine/paracrine way to promote the early postnatal survival of nociceptors that are also responsive to NGF and GDNF. Altogether, the data demonstrate an essential requirement for BDNF in the early postnatal survival of nociceptive neurons.     

Activation of extracellular signal-regulated protein kinase 5 is essential for cystitis- and nerve growth factor-induced calcitonin gene-related peptide expression in sensory neurons

ABSTRACT: BACKGROUND: Cystitis causes considerable neuronal plasticity in the primary afferent pathways. The molecular mechanism and signal transduction underlying cross talk between the inflamed urinary bladder and sensory sensitization has not been investigated. Results: In a rat cystitis model induced by cyclophosphamide (CYP) for 48 h, the mRNA and protein levels of the excitatory neurotransmitter calcitonin gene-related peptide (CGRP) are increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. Cystitis-induced CGRP expression in L6 DRG is triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reverses CGRP up-regulation during cystitis. CGRP expression in the L6 DRG neurons is also enhanced by retrograde NGF signaling when NGF is applied to the nerve terminals of the ganglion-nerve two-compartmented preparation. Characterization of the signaling pathways in cystitis- or NGF-induced CGRP expression reveals that the activation (phosphorylation) of extracellular signal-regulated protein kinase (ERK)5 but not Akt is involved. In L6 DRG during cystitis, CGRP is co-localized with phospho-ERK5 but not phospho-Akt. NGF-evoked CGRP up-regulation is also blocked by inhibition of the MEK/ERK pathway with specific MEK inhibitors U0126 and PD98059, but not by inhibition of the PI3K/Akt pathway with inhibitor LY294002. Further examination shows that cystitis-induced cAMP-responsive element binding protein (CREB) activity is expressed in CGRP bladder afferent neurons and is co-localized with phospho-ERK5 but not phospho-Akt. Blockade of NGF action in vivo reduces the number of DRG neurons co-expressing CGRP and p-CREB, and reverses cystitis-induced increases in micturition frequency. Conclusion: A specific pathway involving NGF-ERK5-CREB axis plays an essential role in cystitis-induced sensory activation.     

Merkel cells in vitro: production of nerve growth factor and selective interactions with sensory neurons

A method has been developed for obtaining mixed primary cultures of dissociated epidermis enriched in Merkel cells. Merkel cells obtained from embryonic rat buccal pads were grown in serum-free medium and identified in vitro using a variety of histological and immunohistochemical markers. Quinacrine, a fluorescent amine, which has been used to identify Merkel cells in situ, labeled a morphologically distinct population of cells in vitro. Cells labeled with quinacrine had a large, phase bright nucleus with prominent nucleoli, surrounded by a phase dark perinuclear ring. Antibodies directed against neuron-specific enolase, another marker for Merkel cells in situ, and antibodies against a well-characterized neuroendocrine vesicle antigen also labeled this population of quinacrine fluorescent cells. Electron microscopic examination of our cultures indicated that cells containing characteristic features of Merkel cells including cytoplasmic dense-cored granules were present. A small but significant increase in the number of Merkel cells was observed over time in culture. Merkel cells supported the survival and outgrowth of both trigeminal ganglion sensory neurons and sympathetic neurons from the superior cervical ganglion in serum-free medium in the absence of exogenous nerve growth factor (NGF). Immunoblots probed with antibodies directed against NGF demonstrated that NGF was present in the medium taken from these cultures. NGF-like immunoreactivity colocalized to cells containing quinacrine fluorescence in situ and in vitro. Addition of antibodies directed against NGF to cocultures of Merkel cells and neurons decreased survival of sympathetic neurons by 90% and decreased survival of sensory neurons by 60%. These results suggest that Merkel cells are capable of providing trophic support for their normal complement of sensory neurons by producing NGF. Selective recognition of these targets was studied in vitro by characterizing the interactions between Merkel cells and growth cones from sensory or sympathetic neurons using both time-lapse videomicroscopy and standard morphometry of fixed cocultures. The majority of trigeminal ganglion sensory neurons (approximately 60%) extended growth cones onto clusters of Merkel cells. Neurites which contacted clusters of Merkel cells were significantly more highly branched than those growing on collagen. In contrast, the majority of sympathetic neurons (greater than 90%) failed to grow onto Merkel cells. Growth cones of sympathetic neurons often "collapsed" and retracted when contact was made with a cluster of Merkel cells. Fixation of Merkel cells with paraformaldehyde prior to coculture did not affect this difference between sensory and sympathetic neurite extension onto the Merkel cells. However, prior fixation of Merkel cells eradicated the apparent Merkel ce-induced branching of sensory neurites.(ABSTRACT TRUNCATED AT 400 WORDS)     

p140trk mRNA marks NGF-responsive forebrain neurons: evidence that trk gene expression is induced by NGF.

Nerve growth factor (NGF) appears to act as a neurotrophic factor for basal forebrain and caudate-putamen cholinergic neurons. The mechanism by which NGF transduces its signal in these neurons is yet to be defined. Recent data indicate that the product of the trk gene, p140trk, is a critical component of the NGF receptor. Herein, we show that p140trk mRNA is highly restricted in its distribution in the adult rat forebrain, that it is present in cholinergic neurons, and that most if not all cholinergic neurons contain p140trk mRNA. Furthermore, induction of trk expression by NGF suggests that neurotrophin-mediated up-regulation of their receptor tyrosine kinases is an important feature of their actions and that neurotrophins may regulate the activity of responsive neurons through increasing the level of their receptors.     

The neurotrophins and CNTF: specificity of action towards PNS and CNS neurons.

The availability of relatively large amounts of nerve growth factor (NGF) has allowed extensive in vitro and in vivo characterization of the neuronal specificity of this neurotrophic factor. The restricted neuronal specificity of NGF (sympathetic neurons, neural crest-derived sensory neurons, basal forebrain cholinergic neurons) has long predicted the existence of other neurotrophic factors possessing different neuronal specificities. Whereas there have been many reports of "activities" distinct from NGF, full characterization of such molecules has been hampered by their extremely low abundance. The recent molecular cloning of brain-derived neurotrophic factor (BDNF) revealed that this protein is closely related to NGF and suggested that these two factors might be members of an even larger gene family. A PCR cloning strategy based on homologies between NGF and BDNF has allowed us to identify and clone a third member of the NGF family which we have termed neurotrophin-3 (NT-3). The establishment of suitable expression systems has now made available sufficient quantities of these proteins to allow us to begin to establish the neuronal specificity of each member of the neurotrophin family, and the role of each in development, maintenance and repair of the PNS and CNS. Using primary cultures of various PNS and CNS regions of the developing chick and rat, and Northern blot analysis, we describe novel neuronal specificities of BDNF, NT-3 and an unrelated neurotrophic factor-ciliary neurotrophic factor (CNTF).     

Brain-Derived Neurotrophic Factor Is More Highly Conserved in Structure and Function than Nerve Growth Factor During Vertebrate Evolution

Mammalian nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are members of a protein family with perfectly conserved domains arranged around the cysteine residues thought to stabilize an invariant three-dimensional scaffold in addition to distinct sequence motifs that convey different neuronal functions. To study their structural and functional conservation during evolution, we have compared NGF and BDNF from a lower vertebrate, the teleost fish Xiphophorus, with the mammalian homologues. Genomic clones encoding fish NGF and BDNF were isolated by cross-hybridization using probes from the cloned mammalian factors. Fish NGF and BDNF were expressed by means of recombinant vaccinia viruses, purified, and their neuronal survival specificities for different classes of neurons were found to mirror those of the mammalian factors. The half-maximal survival concentration for chick sensory neurons was 60 pg/ml for both fish and mammalian purified recombinant BDNF. However, the activity of recombinant fish NGF on both chick sensory and sympathetic neurons was 6 ng/ml, 75-fold lower than that of mouse NGF. The different functional conservation of NGF and BDNF is also reflected in their structures. The DNA-deduced amino acid sequences of processed mature fish NGF and BDNF showed, compared to mouse, 63% and 90% identity, respectively, indicating that NGF had reached an optimized structure later than BDNF. The retrograde extrapolation of these data indicates that NGF and BDNF evolved at strikingly different rates from a common ancestral gene about 600 million years ago. By RNA gel blot analysis NGF mRNA was detected during late embryonic development; BDNF was present in adult brain.     

Binding of brain-derived neurotrophic factor to the nerve growth factor receptor

The neurotrophic proteins BDNF and NGF are related in their primary structures, and both have high- and low-affinity receptors on their responsive neurons. In this study, we investigate the extent to which these receptors can discriminate between BDNF and NGF. We found that a 1000-fold excess of the heterologous ligand is needed to reduce binding to the high-affinity receptor by 50%, but that the same concentrations of BDNF and NGF similarly reduce the binding of either ligand to the low-affinity receptor. Results obtained with cells transfected with the low-affinity NGF receptor gene indicate that these cells bind BDNF, in addition to NGF, whereas cells before transfection do not. These data indicate that the low-affinity NGF receptor is also a low-affinity BDNF receptor and that whatever is conferring high-affinity binding and biological response also considerably reinforces the ability of the low-affinity receptor to discriminate between NGF and BDNF.     

Nerve Growth Factor Mediates a Switch in Intracellular Signaling for PGE2-Induced Sensitization of Sensory Neurons from Protein Kinase A to Epac

We examined whether nerve growth factor (NGF), an inflammatory mediator that contributes to chronic hypersensitivity, alters the intracellular signaling that mediates the sensitizing actions of PGE₂ from activation of protein kinase A (PKA) to exchange proteins directly activated by cAMP (Epacs). When isolated sensory neurons are grown in the absence of added NGF, but not in cultures grown with 30 ng/ml NGF, inhibiting protein kinase A (PKA) activity blocks the ability of PGE₂ to augment capsaicin-evoked release of the neuropeptide CGRP and to increase the number of action potentials (APs) evoked by a ramp of current. Growing sensory neurons in culture in the presence of increasing concentrations of NGF increases the expression of Epac2, but not Epac1. An intradermal injection of complete Freund''s adjuvant into the rat hindpaw also increases the expression of Epac2, but not Epac1 in the dorsal root ganglia and spinal cord: an effect blocked by intraplantar administration of NGF antibodies. Treating cultures grown in the presence of 30 ng/ml NGF with Epac1siRNA significantly reduced the expression of Epac1, but not Epac2, and did not block the ability of PGE₂ to augment capsaicin-evoked release of CGRP from sensory neurons. Exposing neuronal cultures grown in NGF to Epac2siRNAreduced the expression of Epac2, but not Epac1 and prevented the PGE₂-induced augmentation of capsaicin and potassium-evoked CGRP release in sensory neurons and the PGE₂-induced increase in the number of APs generated by a ramp of current. In neurons grown with no added NGF, Epac siRNAs did not attenuate PGE₂-induced sensitization. These results demonstrate that NGF, through increasing Epac2 expression, alters the signaling cascade that mediates PGE₂-induced sensitization of sensory neurons, thus providing a novel mechanism for maintaining PGE₂-induced hypersensitivity during inflammation.     

Development and regulation of substance P in sensory neurons in vitro

Substance P (SP), the putative neuropeptide mediator of pain sensation, is contained in small dorsomedial sensory neurons of the dorsal root ganglion. Using different culture techniques and a sensitive radioimmunoassay for SP, we studied the ontogeny and regulation of this functionally important neurotransmitter in these neurons, obtained from neonatal rats. In ganglion explants grown by two different techniques, SP increased two- to threefold during the first week in culture. This rise was predominantly due to mechanisms intrinsic to the ganglion since it occurred in a fully defined medium, in the absence of added nerve growth factor (NGF). Blockade of protein synthesis with cycloheximide prevented the increase in SP suggesting that ongoing protein synthesis was necessary. Furthermore, depolarization with veratridine blocked the increase in SP, an effect which was reversed by tetrodotoxin, suggesting that transmitter characteristics in sensory neurons may be regulated by depolarization and/or transmembrane sodium flux. After a week in culture on a collagen substratum, supplementary NGF was necessary for the continued rise in SP. However, raising the dose of the trophic factor had no incremental effect on SP content, suggesting that NGF was acting primarily on neuronal survival. To approach such questions at the cellular level, ganglia were dissociated and grown in cell culture. In all cultures, SP increased 1.5-fold during the first day. In the absence of NGF, however, SP and cell numbers fell progressively after the second day. NGF elicited parallel increases in cell survival and SP content, supporting the suggestion that NGF acts primarily through neuronal survival to increase SP. Veratridine blocked the increase in SP in a tetrodotoxin-reversible manner, without affecting neuronal survival, indicating that the effects of these agents do not depend on normal ganglionic cellular architecture. Consequently, depolarization probably affects ganglionic sensory neurons directly. Our studies suggest that the development of transmitter characteristics in primary sensory neurons may be regulated by multiple factors, including neuronal activity as well as trophic agents such as NGF.     

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.