Alfaxalone Causes Reduction of Glycinergic IPSCs, but Not Glutamatergic EPSCs, and Activates a Depolarizing Current in Rat Hypoglossal Motor Neurons
FRONTIERS IN CELLULAR NEUROSCIENCE
Authors: Lau, Cora; Thakre, Prajwal P.; Bellingham, Mark C.
We investigated effects of the neuroactive steroid anesthetic alfaxalone on intrinsic excitability, and on inhibitory and excitatory synaptic transmission to hypoglossal motor neurons (HMNs). Whole cell recordings were made from HMNs in brainstem slices from 7 to 14-day-old Wistar rats. Spontaneous, miniature, and evoked inhibitory post-synaptic currents (IPSCs), and spontaneous and evoked excitatory PSCs (EPSCs) were recorded at -60 mV. Alfaxalone did not alter spontaneous glycinergic IPSC peak amplitude, rise-time or half-width up to 10 mu M, but reduced IPSC frequency from 3 mu M. Evoked IPSC amplitude was reduced from 30 nM. Evoked IPSC rise-time was prolonged and evoked IPSC decay time was increased only by 10 mu M alfaxalone. Alfaxalone also decreased evoked IPSC paired pulse ratio (PPR). Spontaneous glutamatergic EPSC amplitude and frequency were not altered by alfaxalone, and evoked EPSC amplitude and PPR was also unchanged. Alfaxalone did not alter HMN repetitive firing or action potential amplitude. Baseline holding current at -60 mV with a CsCl-based pipette solution was increased in an inward direction; this effect was not seen when tetrodotoxin (TTX) was present. These results suggest that alfaxalone modulates glycine receptors (GlyRs), causing a delayed and prolonged channel opening, as well as causing presynaptic reduction of glycine release, and activates a membrane current, which remains to be identified. Alfaxalone selectively reduces glycinergic inhibitory transmission to rat HMNs via a combination of pre- and post-synaptic mechanisms. The net effect of these responses to alfaxalone is to increase HMN excitability and may therefore underlie neuro-motor excitation during neurosteroid anesthesia.
Tetrodotoxin-Sensitive Sodium Channels Mediate Action Potential Firing and Excitability in Menthol-Sensitive Vglut3-Lineage Sensory Neurons
JOURNAL OF NEUROSCIENCE
Authors: Griffith, Theanne N.; Docter, Trevor A.; Lumpkin, Ellen A.
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 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 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 Nay subunits, with Na-v 1.1 driving firing in menthol-sensitive neurons, whereas other small-diameter Vglut3(lineage) neurons rely primarily on TTX-resistant Nay channels. Additionally, when Na-v 1.1 channels were blocked, the remaining Nay 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.