Previous behavioral studies have revealed that CaV3.2 T-type calcium channels support peripheral nociceptive transmission and electrophysiological studies have established the presence of T-currents in putative nociceptive sensory neurons of dorsal root ganglion (DRG). To date, however, the localization pattern of this key nociceptive channel in the soma and peripheral axons of these cells has not been demonstrated due to lack of isoform-selective anti-CaV3.2 antibodies. In the present study a new polyclonal CaV3.2 antibody is used to localize CaV3.2 expression in rodent DRG neurons using different staining techniques including confocal and electron microscopy (EM). Confocal microscopy of both acutely dissociated cells and short-term cultures demonstrated strong immunofluorescence of anti-CaV3.2 antibody that was largely confined to smaller diameter DRG neurons where it co-localized with established immuno-markers of unmyelinated nociceptors, such as, CGRP, IB4 and peripherin. In contrast, a smaller proportion of these CaV3.2-labeled DRG cells also co-expressed neurofilament 200 (NF200), a marker of myelinated sensory neurons. In the rat sciatic nerve preparation, confocal microscopy demonstrated anti-CaV3.2 immunofluorescence which was co-localized with both peripherin and NF200. Further, EM revealed immuno-gold labeling of CaV3.2 preferentially in association with unmyelinated sensory fibers from mouse sciatic nerve. Finally, we demonstrated the expression of CaV3.2 channels in peripheral nerve endings of mouse hindpaw skin as shown by co-localization with Mrgpd-GFP-positive fibers. The CaV3.2 expression within the soma and peripheral axons of nociceptive sensory neurons further demonstrates the importance of this channel in peripheral pain transmission.
Keywords: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 4’, 6-diamidino-2-phenylindole; CGRP; CNS; Ca(2+); DAPI; DRG; DTNB; EM; GA; GFP; HEK; HEPES; IB(4); KI; KO; MYE; Mas-related G protein-coupled receptor D; Mrgprd; NF200; NONMYE; PB; PBS; PFA; T-channels; T-currents; T-type calcium channels; TBST; Tris buffer with 0.9% NaCl and Triton-X-100; WT; calcitonin-gene-related-peptide; central nervous system; di-thio nitro benzene; dorsal root ganglion; electron microscopy; glutaraldehyde; green fluorescent protein; human embryonic kidney; isolectin B4; knock-in; knock-out; low-voltage-activated; myelinated; neurofilament 200; nociceptors; paraformaldehyde; phosphate buffer; phosphate-buffered saline; unmyelinated; wild type.
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