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 small-diameter 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 currentclamp 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 transcripts between TRPM8-positive and-negative Vglut3 lineage neurons; however, Na V 1.8 transcripts, which encode TTX-resistant 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 current 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.

Regulation of TRPM8 channels by PKCβ mediates morphine-induced cold hypersensitivity.

“…S1d, the response to cold or menthol did not differ in cultured sensory neurons exposed to morphine or in neurons isolated from morphine-treated mice, relative to control. In accordance with previous published results [12,21,25,26], we found that cold sensitive DRG neurons were a rare subset of small diameter neurons (< 20 µm in diameter). In control mice, 3.06% of total neurons (37/1200) responded to cold and 3.31% (37/1138) to menthol (100 µM).…”

Chronic morphine regulates TRPM8 channels via MOR-PKCβ signaling

“…S1d, the response to cold or menthol did not differ in cultured sensory neurons exposed to morphine or in neurons isolated from morphine-treated mice, relative to control. In accordance with previous published results [12,21,25,26], we found that cold sensitive DRG neurons were a rare subset of small diameter neurons (< 20 μm in diameter). In control mice, 3.06% of total neurons (37/1200) responded to cold and 3.31% (37/1138) to menthol (100 μM).…”

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).…”