大鼠舌咽神经中对氧气敏感神经元全细胞电流的生物物理特性

Campanucci V A, Nurse C A

Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1.

Neuroscience. 2005;132(2):437-51. doi: 10.1016/j.neuroscience.2004.11.058.

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 GOmega, 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 (IC50 approximately 0.2 microM), prolonged and transient Ca2+ 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 Ca2+ currents consisted of nifedipine-sensitive L-type, omega-agatoxin IVA-sensitive P/Q-type, omega-conotoxin GVIA-sensitive N-type, SNX-482-sensitive R-type, and Ni2+-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 (PO2 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 Ca2+-entry, activation of nNOS, and release of NO.

在本研究中，我们使用制霉菌素穿孔膜片钳和传统全细胞记录技术，来表征大鼠舌咽神经（GPN）中表达神经元型一氧化氮合酶（nNOS）的副神经节神经元的生物物理特性，这些神经元被认为可提供一氧化氮介导的对颈动脉体化学感受器的传出抑制。这些GPN神经元分为两类，一类靠近GPN与颈动脉窦神经的分叉处，另一类位于GPN更远端的位置。通过用苯乙烯基吡啶染料4-Di-2-ASP（D289）进行活体染色，在整装标本中可观察到这两类神经元。在体外分离后，近端和远端神经元具有相似的输入电阻（平均值分别为1.5和1.6 GΩ）、输入电容（平均值分别为25.0和27.4 pF）以及静息电位（平均值分别为-53.9和-53.3 mV）。所有神经元都具有相似的电压依赖性电流，包括：河豚毒素（TTX）敏感的Na⁺电流（IC50约为0.2 μM）、持续和瞬时的Ca²⁺电流以及延迟整流型K⁺电流。Na⁺电流的阈值激活约为-30 mV，并且在10 ms内失活。内向Ca²⁺电流由硝苯地平敏感的L型、ω-芋螺毒素IVA敏感的P/Q型、ω-芋螺毒素GVIA敏感的N型、SNX-482敏感的R型以及Ni²⁺敏感但SNX-482不敏感的T型通道组成。电压依赖性外向K⁺电流对四乙铵（TEA；10 mM）和4-氨基吡啶（4-AP；2 mM）敏感。暴露于化学感受刺激物低氧（PO₂范围：80 - 5 Torr）会导致K⁺电流呈剂量依赖性降低，在存在TEA和4-AP的情况下这种降低仍然持续，这与背景K⁺通道的参与一致。在电流钳模式下，GPN神经元产生TTX敏感的动作电位，并且在自发活动的神经元中，低氧会导致膜去极化并增加放电频率。这些特性使GPN神经元具有在低氧期间通过电压门控Ca²⁺内流、nNOS激活和NO释放来精确调节颈动脉体化学感受器功能的能力。