Research ReportAcute and chronic effects of neurotrophic factors BDNF and GDNF on responses mediated by thermo-sensitive TRP channels in cultured rat dorsal root ganglion neurons
Introduction
Acute pain signaling is involved in detection or prevention of tissue damage and generates withdrawal responses which are essential for survival. However, chronic (inflammatory or neuropathic) pain serves no useful purpose and leads to a substantial decrease in life quality in many individuals. Pathological pain states are often associated with peripheral and central sensitization of neurons in the pain pathway (nociceptors), leading to both hyperalgesia (increased sensitivity to painful stimuli) and allodynia (pain produced by innocuous stimuli). The involvement of neurotrophic factors (NTFs) in pain signaling is well established, with particular emphasis on the prototypic neurotrophin NGF and its pro-algesic role (Pezet and McMahon, 2006, Nicol and Vasko, 2007). NTFs are important during development for supporting survival of neurons in both the central and the peripheral nervous system. They continue to play a role in the adult organism as key signaling molecules inducing phenotypic switches in neurons as a response to disease or injury of the nervous system (Sah et al., 2003). Neurotrophins (nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and neurotrophin 4/5 (NT-4/5)) activate specific tyrosine kinase receptors trkA (NGF), trkB (BDNF and NT-4/5) and trkC (NT-3), as well as the low affinity receptor p75NTR (Chao, 2003). Glial cell-derived neurotrophic factor (GDNF), together with the closely related molecules neurturin, artemin and persephin, belongs to the transforming growth factor-β superfamily. It activates a receptor complex consisting of the tyrosine kinase ret (involved in the signaling pathway of all GNDF family members) together with the GDNF family receptor α-1 (GFRα1), which confers specificity for GDNF itself (Takahashi, 2001, Bespalov and Saarma, 2007).
BDNF is known to be synthesized by a subpopulation of nociceptive neurons in the dorsal root ganglia (DRG) and transported anterogradely to their central and peripheral axon terminals, where it acts as a pain enhancing molecule (Pezet et al., 2002). While its central effect as a neuromodulator of pain signaling has been demonstrated (reviewed by Obata and Noguchi, 2006), a peripheral mode of action for this neurotrophin has also been proposed, as it was shown to induce behavioral heat hyperalgesia upon local injection in the hind paw, as well as nociceptor sensitization to heat in vitro (Shu et al., 1999). Inflammation and nerve injury increase the level of BDNF expression in the DRG, both in neurons and satellite cells, where it may act on trkB-expressing neurons in an autocrine or paracrine manner (Zhou et al., 2000, Price et al., 2005) leading to enhanced pain behavior. A variety of cell types may also release BDNF in inflammatory states: platelets, T-lymphocytes, macrophages, monocytes, and microglia (Batchelor et al., 1999, Braun et al., 1999, Kerschensteiner et al., 1999). The high affinity receptor for BDNF, trkB, is expressed in 27% of sciatic nerve afferents, mainly of intermediate to large size (McMahon et al., 1994). Chronic exposure to BDNF leads to altered gene expression in cultured sensory neurons: the mRNA for the delayed rectifier potassium channel Kv1.2 was down-regulated (Park et al., 2003), while the protein level of the acid-sensing ion channel ASIC2 was up-regulated (McIlwrath et al., 2005) in the presence of exogenous BDNF.
The role of GDNF in pain signaling appears to be more complex and it may have both pro-algesic and analgesic effects depending on the type of chronic pain (inflammatory vs. neuropathic; Boucher et al., 2000, Fang et al., 2003). The population of non-peptidergic, IB4-positive neurons down-regulates expression of trkA at late-embryonic stages and becomes GDNF-dependent in adult life, as most of these neurons express ret and GFRα1 and 2 (Molliver et al., 1997). GDNF production is increased in Schwann cells following nerve injury (Hammarberg et al., 1996), and inflammation leads to increased GDNF level in the DRG (Amaya et al., 2004). Skin injection of exogenous GDNF induces behavioral heat hyperalgesia (Malin et al., 2006). As in the case of BDNF, chronic exposure to GDNF induces changes in gene expression in the DRG: immunostaining for the vanilloid receptor subtype 1 (TRPV1) as well as capsaicin-induced cobalt uptake (a functional assay for TRPV1) is increased upon GDNF treatment for 3 days (Bron et al., 2003) and expression of the B1 bradykinin receptor is increased in cultured DRG neurons after exposure to GDNF (but not NGF) (Vellani et al., 2004). These results indicate that GDNF may act as a pain producing agent in the periphery and that some of the effects could be mediated by altered expression of pain transducing receptors.
Thermo-sensitive TRP channels are ion channels expressed by sensory nerve endings or skin keratinocytes and activated by various degrees of changes in ambient temperature (Dhaka et al., 2006). Among these are TRPM8 (activated by cooling and menthol (McKemy et al., 2002, Peier et al., 2002)), TRPA1 (activated by noxious cold (at least in the mouse; Sawada et al., 2007, Karashima et al., 2009) and pungent compounds, such as allyl isothiocyanate (AITC), the active compound in mustard and horseradish (Story et al., 2003, Bandell et al., 2004)) and TRPV1 (the capsaicin receptor (Caterina et al., 1997)). All these channels are proposed to be involved in pain signaling and are known to be regulated by a variety of pro-inflammatory agents, as well as following nerve injury (reviewed by Huang et al., 2006, Levine and Alessandri-Haber, 2007). Using a functional assay (calcium microfluorimetry), we show that long-term exposure (12–24 h) to BDNF or GDNF leads to profound changes in the expression pattern and level of activity of TRPA1 and TRPV1. In addition, acute treatment with NTFs leads to a sensitization of cultured DRG neurons to noxious stimuli (heat, in the case of both BDNF and GDNF, and AITC, in the case of GDNF only). We also show that recombinant rat TRPA1 is cold-sensitive.
Section snippets
The cold transducer in cold- and menthol-sensitive (CMS) dorsal root ganglion (DRG) neurons is most likely TRPM8
The protocol used for functional identification of thermoTRP channels is illustrated in Fig. 1: it consisted in a cold ramp (32 to 17 °C, 50 s), followed by application of (−)-menthol (100 μM, 70 s) and a second cold ramp in the presence of menthol (20 s into the menthol application), AITC (100 μM, 1 min), capsaicin (2 μM, 20 s) and KCl (50 mM, 20 s). The intervals between applications were: 5 min between the first cold ramp and menthol, 5 min between the second cold ramp and AITC, 10 min
Discussion
We investigated the effects of chronic and acute exposure of cultured rat DRG neurons to neurotrophic factors (NTFs) BDNF and GDNF. Following chronic treatment with NTFs we have monitored the functional expression of three thermo-sensitive TRP (thermoTRP) channels (TRPM8, TRPA1 and TRPV1), identified by sensitivity to thermal (cold) and chemical agonists of these channels (menthol, mustard oil (allyl isothiocyanate or AITC) and capsaicin) in a calcium microfluorimetric assay. The acute effects
Cell culture
Dorsal root ganglion (DRG) neurons were obtained from spinal levels L1–S1 from adult Wistar rats (150–180 g) as described elsewhere (Reid et al., 2002). Briefly, the animals were killed by 100% CO2 inhalation followed by decapitation, following a UK Home Office approved (Schedule 1) procedure. DRGs were removed and incubated in a mixture of 1.5 mg/ml Collagenase (type IX, Sigma) and 2.5 mg/ml Dispase (nonspecific protease, Sigma) in IncMix solution (see Section 4.3) for 1 h at 37 °C. After
Acknowledgments
This work was supported by grant PN2 Idei 164/2007 from the Romanian Council for Research (CNCSIS) (to AB) and Junior Fellowship Grant 0207 from the Physiological Society (to CC).
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