Subclassified Acutely Dissociated Cells of Rat DRG: Histochemistry and Patterns of Capsaicin-, Proton-, and ATP-Activated Currents

Abstract

We used a "current signature" method to subclassify acutely dissociated dorsal root ganglion (DRG) cells into nine subgroups. Cells subclassified by current signature had uniform properties. The type 1 cell had moderate capsaicin sensitivity (25.9 pA/pF), powerful, slowly desensitizing (tau = 2,300 ms), ATP-activated current (13.3 pA/pF), and small nondesensitizing responses to acidic solutions (5.6 pA/pF). Type 1 cells expressed calcitonin gene-related peptide immunoreactivity (CGRP-IR), manifested a wide action potential (7.3 ms), long duration afterhyperpolarization (57.0 ms), and were IB4 positive. The type 2 cell exhibited large capsaicin activated currents (134.9 pA/pF) but weak nondesensitizing responses to protons (15.3 pA/pF). Currents activated by ATP and alphabeta-m-ATP (51.7 and 44.6 pA/pF, respectively) had fast desensitization kinetics (tau = 214 ms) that were distinct from all other cell types. Type 2 cells were IB4 positive but did not contain either substance P (SP) or CGRP-IR. Similar to capsaicin-sensitive nociceptors in vivo, the afterhyperpolarization of the type 2 cell was prolonged (54.7 ms). The type 3 cell expressed, amiloride-sensitive, rapidly desensitizing (tau = 683 ms) proton-activated currents (127.0 pA/pF), and was insensitive to ATP or capsaicin. The type 3 cell was IB4 negative and contained neither CGRP nor SP-IR. The afterhyperpolarization (17.5 ms) suggested nonnociceptive function. The type 4 cell had powerful ATP-activated currents (17.4 pA/pF) with slow desensitization kinetics (tau = 2, 813 ms). The afterhyperpolarization was prolonged (46.5 ms), suggesting that this cell type might belong to a capsaicin-insensitive nociceptor population. The type 4 cell did not contain peptides. The type 7 cell manifested amiloride-sensitive, proton-activated currents (45.8 pA/pF) with very fast desensitization kinetics (tau = 255 ms) and was further distinct from the type 3 cell by virtue of a nondesensitizing amiloride-insensitive component (6.0 pA/pF). Capsaicin and ATP sensitivity were relatively weak (4.3 and 2.9 pA/pF, respectively). Type 7 cells were IB4 positive and contained both SP and CGRP-IR. They exhibited an exceptionally long afterhyperpolarization (110 ms) that was suggestive of a silent (mechanically insensitive) nociceptor. We concluded that presorting of DRG cells by current signatures separated them into internally homogenous subpopulations that were distinct from other subclassified cell types.

Roles of Tetrodotoxin (TTX)-Sensitive Na+Current, TTX-Resistant Na+Current, and Ca2+Current in the Action Potentials of Nociceptive Sensory Neurons

“…The correspondence of the characteristics of the cells we studied with type 1 and 2 cells, together with the presence of TTX-R current (in all but one cell), is consistent with their identification as nociceptors corresponding to C-type and A␦-type fibers (Cardenas et al, 1995(Cardenas et al, , 1997. It is possible that a few cells that we studied with small but detectable I h would fall into the type 7 classification in the scheme of Petruska et al (2000), also believed to correspond with a type of nociceptor.…”

“…The contribution of TTX-S sodium currents to threshold and upstroke of nociceptor action potentials is likely to be sensitive to small changes in resting potential, because the midpoint of inactivation for TTX-S current is near Ϫ70 mV, and reported resting potentials of nociceptors range from Ϫ50 to Ϫ80 mV (Cardenas et al, 1995;Safronov et al, 1996;Petruska et al, 2000;Sung et al, 2000). However, even in neurons with resting potentials negative to Ϫ80 mV, where there is little steady-state inactivation of TTX-S current (Fig.…”

“…C ells were selected for recording on the basis of size and then were tested for the magnitude of several ionic currents that have been proposed to divide DRG neurons into f unctional groups (C ardenas et al, 1995;Petruska et al, 2000). Images of cells were taken with a CCD camera (Hitachi, Woodbury, N Y), captured with a video acquisition card (Scion, Frederick, MD) with a resolution of 0.4 m, and stored on a computer.…”

“…The long-lasting AHPs probably reflect a combination of slowly decaying potassium conductances together with long membrane time constants. Cardenas et al (1995Cardenas et al ( , 1997 and Petruska et al (2000) have developed a system to classify acutely dissociated DRG neurons into distinct groups on the basis of a combination of properties, including cell diameter, action potential characteristics, and level of expression of ionic currents such as the hyperpolarizationactivated cation current I h , and the transient potassium current I A . Virtually all of the cells we studied corresponded well with the types of neurons designated types 1 and 2 in their systems in having long-duration action potentials with shoulders and longlasting AHPs as well as expressing little or no I h , with an average density of 1.4 Ϯ 1.5 pA/pF (n ϭ 52).…”

Morphological and functional diversity of first-order somatosensory neurons

“…More recently, the discovery of molecular mechanisms of sensory signal transduction have permitted classifications based on the selective expression of certain ion channels, especially TRP channels, ASIC channels, P2X receptors, certain isoforms of Na V channels, among others (McCleskey and Gold 1999;Petruska et al 2000; Le Pichon and Chesler 2014; Moraes et al 2014). Retrograde labeling allows characterization of the neurons that innervate specific organs.…”