摘要
目的建立急性分离成年犬结状神经节神经元(VGN)电生理学研究平台。方法通过急性分离和培养雌雄成年比格犬VGN,利用全细胞膜片钳技术分别记录动作电位,电压依赖性的外向钾电流(IK)和超极化激活的阳离子电流(Ih)。结果动作电位波形分析结果显示:①雌性比格犬VGN不仅可以记录到传统分类中的髓鞘化A型和非髓鞘化C型神经元,还可以记录到雌性特异分布的Ah型神经元;②步阶电流除极在诱发相同持续放电频率时,A型所需要的刺激强度最小,Ah型所需要的电流强度与A型近似或轻微增加,而C型需要的刺激强度最大;③河豚毒素显著降低Ah型神经元的除极速率和触及阈值;蝎毒素可显著增加A-型的神经兴奋性;④所有受试神经元均表达IK和Ih,且2种电流密度均呈现A型>Ah型>C型;⑤尽管雌雄VGN分布的传入纤维种类有别,已知的A和C型VGN放电差异性并未在本研究中探讨。结论成年比格犬VGN可被成功急性分离,并用于动作电位、IK和Ih的记录;分类和电生理学特性足以完成药理学试验的需要,持续至少30 min的紧密封接应该满足药理学观察的需要。
OBJECTIVE To establish a platform of electrophysiology using vagal ganglion neurons(VGNs)isolated from adult canines.METHODS The VGNs were enzymatically isolated from adult canines of either gender and cultured under experimental conditions.Action potential(AP),repetitive firing,voltage-gated outward K+currents(IK)and hyperpolarization-mediated inward currents(Ih)were recorded under current-and voltage-clamp configurations before and after treatment.RESULTS Analysis of AP waveform showed that①inaddition to traditionally classified myelinated A-and unmyelinated C-types,myelinated Ah-types could also be identified in females rather than in males;②step current depolarization evoked a stimulus intensity-dependent repetitive discharge,and to reach a similar firing frequency,the lowest stimulus intensity was required for A-types,a similar or slightly higher stimulus intensity was needed for Ah-types,and the highest stimulus intensity was required for C-types;③tetro⁃dotoxin significantly reduced the rate of depolarization and positively shifted the AP firing threshold of Ah-types,and iberiotoxin dramatically increased the neuroexcitability of Ah-types;④all tested neurons functionally expressed IK and Ih,and the current density for both channels on average was A-types>Ah-types>C-types;⑤although the distribution of afferent types of VGNs differed between males and females,the known difference in discharge profiles of A-and C-types isolated from male and female rats was not studied here.CONCLUSION The VGNs can be successfully isolated from adult canines,AP,IK and Ih can be recorded.The tight seal can be held for at least 30 min,which may be enough for pharmacological investigation.
作者
钱钊
乔国芬
李柏岩
QIAN Zhao;QIAO Guo-fen;LI Bai-yan(Department of Pharmacology,College of Pharmacy,Harbin 150081,China;State-Province Key Laboratories of Biomedicine-Pharmaceutics of China,Harbin 150081,China;Key Laboratory of Cardiovascular Medicine Research,Ministry of Education,Harbin 150081,China;Department of Pharmacy,the First Affiliated Hospital,Harbin Medical University,Harbin 150081,China)
出处
《中国药理学与毒理学杂志》
CAS
北大核心
2020年第7期490-501,共12页
Chinese Journal of Pharmacology and Toxicology
基金
国家自然科学基金(31171122)
国家自然科学基金(81573431)
国家自然科学基金(81971326)
哈尔滨医科大学创新科学研究基金。
关键词
结状神经节
电生理学
膜片钳技术
比格犬
nodose ganglion
electrophysiology
patch-clamp technique
canine
