Intense Isolectin-B4 Binding in Rat Dorsal Root Ganglion Neurons Distinguishes C-Fiber Nociceptors with Broad Action Potentials and High Nav1.9 Expression

Abstract

Binding to isolectin-B4 (IB4) and expression of tyrosine kinase A (trkA) (the high-affinity NGF receptor) have been used to define two different subgroups of nociceptive small dorsal root ganglion (DRG) neurons. We previously showed that only nociceptors have high trkA levels. However, information about sensory and electrophysiological properties in vivo of single identified IB4-binding neurons, and about their trkA expression levels, is lacking. IB4-positive (IB4ϩ) and small dark neurons had similar size distributions. We examined IB4-binding levels in Ͼ120 dye-injected DRG neurons with sensory and electrophysiological properties recorded in vivo. Relative immunointensities for trkA and two TTX-resistant sodium channels (Nav1.8 and Nav1.9) were also measured in these neurons. IB4ϩ neurons were classified as strongly or weakly IB4ϩ.All strongly IB4ϩ neurons were C-nociceptor type (C-fiber nociceptive or unresponsive). Of 32 C-nociceptor-type neurons examined, ϳ50% were strongly IB4ϩ, ϳ20% were weakly IB4ϩ and ϳ30% were IB4Ϫ. A␦ low-threshold mechanoreceptive (LTM) neurons were weakly IB4ϩ or IB4Ϫ. All 33 A-fiber nociceptors and all 44 A␣/␤-LTM neurons examined were IB4Ϫ. IB4ϩ compared with IB4Ϫ C-nociceptor-type neurons had longer somatic action potential durations and rise times, slower conduction velocities, more negative membrane potentials, and greater immunointensities for Nav1.9 but not Nav1.8. Immunointensities of IB4 binding in C-neurons were positively correlated with those of Nav1.9 but not Nav1.8. Of 23 C-neurons tested for both trkA and IB4, ϳ35% were trkAϩ/IB4ϩ but with negatively correlated immunointensities; 26% were IB4ϩ/trkAϪ, and 35% were IB4Ϫ/trkAϩ. We conclude that strongly IB4ϩ DRG neurons are exclusively C-nociceptor type and that high Nav1.9 expression may contribute to their distinct membrane properties.

Differential Slow Inactivation and Use-Dependent Inhibition of Nav1.8 Channels Contribute to Distinct Firing Properties in IB4+ and IB4− DRG Neurons

“…This finding is surprising, given that Na v 1.9, a channel that is predominantly expressed in IB 4 ϩ neurons (Cummins et al 2000;Fjell et al 1999), produces a persistent inward current at rest that can acutely shift resting potential in a depolarizing direction (Baker et al 2003;Herzog et al 2001). Our data in this regard are similar to results recently reported by Fang et al (2006). One possible explanation is that, without added GTP, the Na v 1.9 persistent current is too low to effect membrane potential (Baker et al 2003).…”

“…For our characterization of the voltage-dependent and kinetic properties of Na v 1.8 current, cells were held at Ϫ70 mV for 10 min using fluoride-based pipette solution to inactivate Na v 1.9 current (Choi et al 2006;Coste et al 2004). Because Na v 1.9 channels produce persistent currents during the depolarization pulses of 100 ms and are more heavily expressed in IB 4 ϩ than in IB 4 Ϫ cells (Fang et al 2006;Fjell et al 1999;Rush et al 2005) we estimated the extent of possible contamination of Na v 1.9 current using the ratio of maximum late current amplitude at the end of 100-ms pulses to peak current amplitude. The maximal percentage of late current amplitudes to peak current for IB 4 ϩ and IB 4 Ϫ neurons was 5.0 Ϯ 0.5 and 3.0 Ϯ 0.5%, respectively, and were significantly different (P Ͻ 0.01).…”

“…Stucky and Lewin (1999) reported that IB 4 ϩ neurons have longer-duration action potentials (APs), higher AP threshold, and produce larger tetrodotoxin-resistant (TTX-R) currents than IB 4 Ϫ neurons in response to a long step current injection stimulus. Fang et al (2006), studying functionally identified C-nociceptor DRG neurons, found not only that strongly IB 4 ϩ cells have longer AP durations and rise times, but also that these cells have slower conduction velocities and more negative resting membrane potentials than those of IB 4 Ϫ neurons. Taken together, these neurochemical, neuroanatomical, and electrophysiological differences suggest that IB 4 ϩ and IB 4 Ϫ neurons may be functionally distinct.…”

Role of neurotrophin signalling in the differentiation of neurons from dorsal root ganglia and sympathetic ganglia

“…The TTXR sodium channel Nav1.9/SNS2/NaN is selectively expressed in small DRG neurons (Tate et al 1998) and is detected in nociceptive A and C fibre neurons as analysed by electrophysiology and IHC of labelled lumbar neuron in rats (Fang et al 2002). In C fibre nociceptors of lumbar DRG, Nav1.9 immunoreactivity is correlated with isolectin-B4 (IB4)-binding levels (Fang et al 2006). Expression is detected in rat L4/5 DRG by IHC at E15 for Nav1.8 and not before E17 for Nav1.9 (Benn et al 2001).…”

The role of glial cells in influencing neurite extension by dorsal root ganglion cells

“…The IB4 2ve cell fraction includes large diameter proprioceptive neurons and small diameter peptidergic nociceptors (Julius and Basbaum, 2001;Fang et al, 2006). Cell size analysis showed that the large diameter IB4 2ve cells were the principal cell types responsive to PTX.…”