Nicotinic acetylcholine receptor subtypes in nociceptive dorsal root ganglion neurons of the adult rat
Introduction
Peripheral nicotinic acetylcholine receptors (nAChR) participate in the modulation of pain perception. Stimulation of neuronal nAChR excites or sensitizes peripheral sensory nerve fibres but has also been reported to mediate cholinergic antinociception Steen and Reeh, 1993, Bernardini et al., 2001, Rueter et al., 2003. In a rat model of facial pain, for example, stimulation of peripheral nicotinic receptors lead to an antihyperalgesic effect (Gilbert et al., 2001) and application of the general nAChR antagonist mecamylamine decreased acute pain but increased the tonic pain after formalin application (Hama and Menzaghi, 2001).
The signalling pathways that underlies the nAChR-mediated antinociceptive effects remains to be described. Nicotinic receptor-dependent stimulation of nitric oxide synthase (NOS) has been demonstrated in DRG neurons (Haberberger et al., 2000). The synthesised NO is able to block dorsal root ganglion (DRG) ion channels Renganathan et al., 2002, Yoshimura et al., 2001, and the inhibition of the NOS–cGMP system has been shown to be involved in diabetic hyperalgesia (Kim et al., 2003).
These differences in nAChR-mediated effects could be related to the stimulation of different nAChR subtypes located on nociceptive neurons.
Subpopulations of primary afferent neurons in the DRG have been defined on the basis of cell size, ultrastructure, neurochemical and electrophysiological characteristics, peptide content and receptive properties Rambourg et al., 1983, Harper and Lawson, 1985, Villiere and McLachlan, 1996. The subgroup of nociceptive neurons is anatomically and functionally distinct from other neuron subpopulations and can be characterised by the sensitivity to the pungent principle of the plant family capsicum, capsaicin, and by the expression of the vanilloid receptor 1 (TRPV-1). TRPV-1 is a non-selective cation channel with high Ca2+ permeability that is activated, e.g., by temperatures above 43 °C, by acidification and by capsaicin the pungent ingredient of hot chilli peppers (Tominaga et al., 1998).
Nociceptive neurons express, in addition to TRPV-1, a vast array of receptors that modulate or mediate their activity (Julius and Basbaum, 2001). Nicotine for example evokes a pain sensation when injected into skin, induces heat hyperalgesia and excites and sensitises nociceptive neurons Magerl et al., 1990, Steen and Reeh, 1993, Bernardini et al., 2001, Lang et al., 2003. Binding assays, RT-PCR, immunohistochemistry and electrophysiological studies showed the presence of neuronal nAChR in sensory neurons Polz-Tejera et al., 1980, Sucher et al., 1990, Boyd et al., 1991, Puttfarcken et al., 1997, Genzen et al., 2001, Lips et al., 2002. Mammalian nAChR are ligand-gated ion channels that consist of either heteropentamers (α2–6 with β2–4 subunits or α7β2 and α9/α10 subunits) or α7-homopentamers Lindstrom et al., 1996, Elgoyhen et al., 2001, Le Novere et al., 2002, Khiroug et al., 2002. The subunit composition of nAChR determines the Ca2+ permeability Albuquerque et al., 1997, Le Novere et al., 2002, Michelmore et al., 2002. Nicotinic AChR subtypes with high calcium permeability consist of α7-subunit homopentamers (α7-nAChR; Roth et al., 2000, Conti-Fine et al., 2000, Drisdel and Green, 2000) or heteropentamers with α9 and α10 or β2 Elgoyhen et al., 2001, Khiroug et al., 2002. Another possible pathway for nAChR-dependent rises in [Ca2+]i might involve depolarization followed by Ca2+ influx through voltage gated Ca2+ channels (Cav), as has been suggested for α3β4, α4β2 or α6β3 heteromers Le Novere et al., 2002, Michelmore et al., 2002.
Although α2–7 and β2–4 nAChR subunits have been found in newborn sensory neurons (Genzen et al., 2001), little is known regarding the expression of nAChR subunits in adult nociceptive neurons. In the present study, we investigated the nAChR α4 and α7 subunits Le Novere et al., 2002, Michelmore et al., 2002, and the recently described α10 subunit in thoracic and lumbar nociceptive neurons from adult rat dorsal root ganglia (DRG) at the transcriptional (RT-PCR) translational (immunohistochemistry) and functional level (current recordings and calcium measurements).
Section snippets
Cell culture preparation
Lumbar and thoracic DRGs were excised from adult (140–160 g) Wistar rats of either sex from an in house colony that were killed in pure CO2 atmosphere. DRG neurons were then cultured as published previously Kress and Guenther, 1999, Haberberger et al., 2000. Briefly, ganglia from segmental levels L1 to L5 and from T1–T12 were harvested after sagittal sectioning of the spine and incubated with liberase (0.28 U/ml in DMEM, 75 min; Roche Biochemicals, Mannheim, Germany) and trypsin (25,000 U/ml in
Expression of nAChR mRNA in rat DRG cultures
RT-PCR analysis of mRNA isolated from rat thoracic and from lumbar DRG after 1 day in culture revealed expression of several nAChR α-subunits. Neuronal cultures contained products corresponding to the mRNAs of the subunits α2, α3, α4, α5, α6, α7, and α10. Products corresponding to the α9 subunit could not be detected in cultured thoracic DRG cells (Fig. 1). Interestingly, α9 mRNA was found in total RNA isolated from lumbar but not thoracic ganglia suggesting differential expression of this
Discussion
Here, we demonstrate the presence of the mRNA for all (lumbar) and seven out of eight (thoracic) neuronal nAChR α-subunits in cultures of adult DRG neurons by using RT-PCR. Indirect immunofluorescence revealed the presence of α-subunits 4, 7 and 10 in thoracic as well as in lumbar nociceptive neurons. The α4, α7 and α10 subunits were colocalised in small to medium-sized nociceptive TRPV-1-immunoreactive neurons in situ and in vitro. Nociceptive neurons responded to nicotinic stimulation with
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (HA 3140/1-1 to RVH and SFB353, A10 to MK).
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