Nicotinic acetylcholine receptor distribution in relation to spinal neurotransmission pathways

Abstract

Neuronal nicotinic receptors (nAChR) are pentameric assemblies of subunits of a gene family where specified combinations of alpha and beta subunits form functional receptors. To extend our understanding of the role of spinal nAChR in the processing of sensory stimuli and regulation of autonomic and motor responses, we initiated investigations to localize nAChR subunit expression within discrete spinal regions and cell types. High-affinity epibatidine binding was present in the superficial dorsal and ventral horns, the mediolateral and central canal regions. RT-PCR identified transcripts for alpha3, alpha4, alpha5, beta2, and beta4 in both spinal cord parenchyma and dorsal root ganglia (DRG). Our affinity-purified antibodies against alpha3, alpha4, alpha5, beta2, and beta4 subunits identified specific protein bands of appropriate molecular mass (preadsorbed with the respective antigens) in specific tissues and cells that express nicotinic receptors, including the spinal cord and DRG neurons. Having established the absence of crossreactivity with related subunits, specific fluorescence labeling of nerve terminals and cell bodies was achieved and correlated with the distribution of defined marker proteins and nicotinic receptor binding sites determined autoradiographically. Our findings indicate that alpha3, alpha4, alpha5, beta2, and beta4 subunits are all expressed on primary afferents (IB4-positive terminals) in the spinal cord. The predominant presynaptic (synaptophysin colocalization) labeling is in the superficial layer of the dorsal horn. These receptor subunits, except for beta4, are also present in postsynaptic autonomic (anti-bNOS-positive) and somatic motor neurons (anti-VAChT-positive). The alpha3, alpha5, and beta2 subunits showed additional staining in glial (anti-GFAP-positive) cells. These studies reveal a dense and distinguishable distribution of nAChR subunits in the spinal cord and point toward future therapeutic targeting for specific spinal actions.

Intrathecal α-conotoxins Vc1.1, AuIB and MII acting on distinct nicotinic receptor subtypes reverse signs of neuropathic pain

“…It was therefore suggested that Vc1.1 might produce pain relief via inhibition of 3-and/or 5-containing nAChRs on sensory nerves. α3, α4, α5, β2 and β4 subunit transcripts are all found in spinal cord parenchyma and sensory ganglia (Khan et al 2003). Although the specificity of nAChR antibodies has been questioned (Moser et al 2007), the subunits all appear to be expressed on primary afferents that co-label immunohistochemically with IB4, and with synaptophysin in superficial dorsal horn (Khan et al 2003).…”

“…α3, α4, α5, β2 and β4 subunit transcripts are all found in spinal cord parenchyma and sensory ganglia (Khan et al 2003). Although the specificity of nAChR antibodies has been questioned (Moser et al 2007), the subunits all appear to be expressed on primary afferents that co-label immunohistochemically with IB4, and with synaptophysin in superficial dorsal horn (Khan et al 2003). It therefore remains possible that pain relief may be achieved after intrathecal administration by antagonism of nAChRs comprising complex combinations of α3 with α5, β4 and β2 subunits, perhaps in combination with other less common subunits.…”

Membrane Receptors Involved in Modulation of Responses of Spinal Dorsal Horn Interneurons Evoked by Feline Group II Muscle Afferents

“…Other complicating factors are that, in the presence of bicuculline, the depression was not only counteracted but replaced by excitation (possibly by unmasking an excitatory pathway) and that bicuculline might also reduce the test responses. The latter effect suggests that it might also interact with an excitatory receptor, such as the nicotinic acetylcholine receptor possibly found on primary afferents (Ninkovic and Hunt, 1983;Khan et al, 2003) and known to be blocked by bicuculline (Demuro et al, 2001). …”

Conus Venom Peptide Pharmacology

“…Bovine adrenal chromaffin cells express ␣3-, ␣5-, ␣7-, and ␤4-nAChR subunits (Sala et al, 2008). Accordingly, the main nAChR subtypes expressed on nociceptive peripheral fibers and their central terminations are composed of ␣3, ␣5, ␤4, and ␤2 subunits (Khan et al, 2003;Lang et al, 2003;Rau et al, 2005). Vc1.1 also inhibited peripheral nerve-mediated vascular responses in rats (Satkunanathan et al, 2005) and, at low concentrations (100 nM), inhibited excitatory responses of Sandall et al, 2003;Satkunanathan et al, 2005;Vincler et al, 2006;Klimis et al, 2011 CyclizedVc1.1 Partially reverses mechanical allodynia in several neuropathic models after oral dosing.…”