Biophysical characterization of whole-cell currents in O 2-sensitive neurons from the rat glossopharyngeal nerve

Abstract

In this study we use nystatin perforated-patch and conventional whole-cell recording to characterize the biophysical properties of neuronal nitric oxide synthase (nNOS)-expressing paraganglion neurons from the rat glossopharyngeal nerve (GPN), that are thought to provide NO-mediated efferent inhibition of carotid body chemoreceptors. These GPN neurons occur in two populations, a proximal one near the bifurcation of the GPN and the carotid sinus nerve, and a more distal one located further along the GPN. Both populations were visualized in whole mounts by vital staining with the styryl pyridinium dye, 4-Di-2-ASP (D289). Following isolation in vitro, proximal and distal neurons had similar input resistances (mean: 1.5 and 1.6 GΩ, respectively), input capacitances (mean: 25.0 and 27.4 pF, respectively), and resting potentials (mean: −53.9 and −53.3 mV, respectively). All neurons had similar voltage-dependent currents composed of: tetrodotoxin (TTX)-sensitive Na + currents (IC 50 approximately 0.2 μM), prolonged and transient Ca 2+ currents, and delayed rectifier-type K + currents. Threshold activation for the Na + currents was approximately −30 mV and they were inactivated within 10 ms. Inward Ca 2+ currents consisted of nifedipine-sensitive L-type, ω-agatoxin IVA-sensitive P/Q-type, ω-conotoxin GVIA-sensitive N-type, SNX-482-sensitive R-type, and Ni 2+-sensitive, but SNX-482-insensitive, T-type channels. The voltage-dependent outward K + currents were sensitive to tetraethylammonium (TEA; 10 mM) and 4-aminopyridine (4-AP; 2 mM). Exposure to a chemosensory stimulus, hypoxia (PO 2 range: 80–5 Torr), caused a dose-dependent decrease in K + current which persisted in the presence of TEA and 4-AP, consistent with the involvement of background K + channels. Under current clamp, GPN neurons generated TTX-sensitive action potentials, and in spontaneously active neurons, hypoxia caused membrane depolarization and an increase in firing frequency. These properties endow GPN neurons with an exquisite ability to regulate carotid body chemoreceptor function during hypoxia, via voltage-gated Ca 2+-entry, activation of nNOS, and release of NO.