We describe a functional profiling strategy to identify and characterize subtypes of neurons present in a peripheral ganglion, which should be extendable to neurons in the CNS. In this study, dissociated dorsal-root ganglion neurons from mice were exposed to various pharmacological agents (challenge compounds), while at the same time the individual responses of >100 neurons were simultaneously monitored by calcium imaging. Each challenge compound elicited responses in only a subset of dorsal-root ganglion neurons. Two general types of challenge compounds were used: agonists of receptors (ionotropic and metabotropic) that alter cytoplasmic calcium concentration (receptor-agonist challenges) and compounds that affect voltage-gated ion channels (membrane-potential challenges). Notably, among the latter are K-channel antagonists, which elicited unexpectedly diverse types of calcium responses in different cells (i.e., phenotypes). We used various challenge compounds to identify several putative neuronal subtypes on the basis of their shared and/or divergent functional, phenotypic profiles. Our results indicate that multiple receptor-agonist and membrane-potential challenges may be applied to a neuronal population to identify, characterize, and discriminate among neuronal subtypes. This experimental approach can uncover constellations of plasma membrane macromolecules that are functionally coupled to confer a specific phenotypic profile on each neuronal subtype. This experimental platform has the potential to bridge a gap between systems and molecular neuroscience with a cellular-focused neuropharmacology, ultimately leading to the identification and functional characterization of all neuronal subtypes at a given locus in the nervous system.

译文

:我们描述了一种功能分析策略,以识别和表征周围神经节中存在的神经元亚型,该亚型应可扩展至中枢神经系统中的神经元。在这项研究中,将来自小鼠的离解的背根神经节神经元暴露于各种药理剂(挑战化合物),同时通过钙成像同时监测> 100个神经元的单个反应。每个挑战化合物仅在背根神经节神经元的一个子集中引起反应。使用了两种一般类型的挑战化合物:改变细胞质钙浓度的受体激动剂(离子型和代谢型)(影响受体激动剂的挑战)和影响电压门控离子通道的化合物(膜电位挑战)。值得注意的是,后者是K通道拮抗剂,它在不同细胞中引起意想不到的不同类型的钙反应(即表型)。我们使用了各种挑战性化合物,以根据其共有和/或不同的功能性,表型特征来鉴定几种推定的神经元亚型。我们的结果表明,多种受体激动剂和膜电位挑战可应用于神经元群体,以识别,表征和区分神经元亚型。这种实验方法可以揭示质膜大分子的星座,这些星座在功能上耦合以赋予每种神经元亚型特定的表型特征。该实验平台具有弥合以细胞为中心的神经药理学的系统与分子神经科学之间的鸿沟的潜力,最终导致了神经系统中给定基因座上所有神经元亚型的鉴定和功能表征。

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