Tetrodotoxin-sensitive sodium channels mediate action potential firing and excitability in menthol-sensitive Vglut3-lineage sensory neurons

Abstract

Small-diameter vesicular glutamate transporter 3-lineage (Vglut3 lineage ) dorsal root ganglion (DRG) neurons play an important role in mechanosensation and thermal hypersensitivity; however, little is known about their intrinsic electrical properties. We therefore set out to investigate mechanisms of excitability within this population. Calcium microfluorimetry analysis of male and female mouse DRG neurons demonstrated that the cooling compound menthol selectively activates a subset of Vglut3 lineage neurons. Whole-cell recordings showed that smalldiameter Vglut3 lineage DRG neurons fire menthol-evoked action potentials and exhibited robust, transient receptor potential melastatin 8 (TRPM8)-dependent discharges at room temperature.This heightened excitability was confirmed by current-clamp and action potential phase-plot analyses, which showed menthol-sensitive Vglut3 lineage neurons to have more depolarized membrane potentials, lower firing thresholds, and higher evoked firing frequencies compared with menthol-insensitive Vglut3 lineage neurons. A biophysical analysis revealed voltage-gated sodium channel (Na V ) currents in menthol-sensitive Vglut3 lineage neurons were resistant to entry into slow inactivation compared with menthol-insensitive neurons. Multiplex in situ hybridization showed similar distributions of tetrodotoxin (TTX)-sensitive Na V s transcripts between TRPM8positive and -negative Vglut3 lineage neurons; however, Na V 1.8 transcripts, which encode TTXresistant channels, were more prevalent in TRPM8-negative neurons. Conversely, pharmacological analyses identified distinct functional contributions of Na V subunits, with Na V 1.1 driving firing in menthol-sensitive neurons, whereas other small-diameter Vglut3 lineage neurons rely primarily on TTX-resistant Na V channels. Additionally, when Na V 1.1 channels were blocked, the remaining Na V currents readily entered into slow inactivation in menthol-sensitive Vglut3 lineage neurons. Thus, these data demonstrate that TTX-sensitive Na V s drive action potential firing in menthol-sensitive sensory neurons and contribute to their heightened excitability. Significance StatementSomatosensensory neurons encode various sensory modalities including thermoreception, mechanoreception, nociception and itch. This report identifies a previously unknown requirement for tetrodotoxin-sensitive sodium channels in action potential firing in a discrete subpopulation of small-diameter sensory neurons that are activated by the cooling agent menthol. Together, our results provide a mechanistic understanding of factors that control intrinsic excitability in functionally distinct subsets of peripheral neurons. Furthermore, as menthol has been used for centuries as an analgesic and anti-pruritic, these findings support the viability of Na V 1.1 as a therapeutic target for sensory disorders.

The Sensory Coding of Warm Perception

“…Intriguingly, we observed a new population of C-fibers with ongoing spike activity in the absence of externally applied thermal stimuli ( Figure 3B). These fibers are reminiscent of low-threshold cold receptors in the cornea (Belmonte et al, 2009) and may correspond to recently described mentholsensitive Vglut3 lineage sensory neurons described in vitro with ongoing activity (Griffith et al, 2019). The physiological properties of these fibers closely resembled thermally responsive units recorded in humans (Campero and Bostock, 2010;Campero et al, 2001), monkeys (Dubner et al, 1975), and rabbits (Shea and Perl, 1985).…”

“…The loss of warm sensation in trpm8 À/À mice could be an indirect consequence of the early developmental loss of cool information reaching the brain. We addressed this issue by acutely inactivating TRPM8 in the forepaw of WT mice using PBMC (1-Phenylethyl-(2-aminoethyl)[4-(benzyloxy)-3-methoxybenzyl]carbamate), a selective antagonist of TRPM8 that has been shown to suppress cooling-responsive cells and reduce cooling-evoked behavioral responses in mice (Gonzá lez et al, 2017;Griffith et al, 2019;Knowlton et al, 2013;Yudin et al, 2016). We first trained WT animals to report warm stimuli and then we pharmacologically inactivated TRPM8 by performing a transdermal injection in the plantar side of the right forepaw ( Figures 6D-6F).…”

Molecular correlates of muscle spindle and Golgi tendon organ afferents

“…RNAscope analysis was performed using the ACDbio RNAscope kit for nonfixed tissue (ACD #320851) based on ref. 71 . In short, DRG extracted from adult mice were fixed for 15 min in 4% PFA, washed in PBS, and allowed to equilibrate in 30% sucrose for 2 h before embedding in OCT (Tissue Tek; Sakura Finetek).…”

In vivo calcium imaging identifies functionally and molecularly distinct subsets of tongue-innervating mechanosensory neurons

“…Cre-driver lines were crossed with mouse reporter lines that express membrane-bound GFP (Hippenmeyer et al, 2005; Muzumdar et al, 2007). We found that trigeminal ganglion neurons were labeled with three different Cre markers ( Figure 3A) : the GDNF receptor Ret (Ret CreERT2 tamoxifen-induced at P21–30), vesicular glutamate transporter 3 ( Vglut3 Cre ) and the NGF receptor Ntrk3 CreERT2 (tamoxifen-induced at P21–30; (Bai et al, 2015; Griffith et al, 2019; Grimes et al, 2011; Lou et al, 2013; Luo et al, 2009). We quantified expression of each marker in the trigeminal ganglion and found that Ret CreERT2 labeled 31% of trigeminal neurons, Vglut3 Cre labeled 29% of trigeminal neurons, and Ntrk3 CreERT2 labeled 10% of trigeminal neurons.…”