Ponissery Saidu Samsudeen, Dibattista Michele, Matthews Hugh R, Reisert Johannes
Monell Chemical Senses Center.
J Vis Exp. 2012 Apr 5(62):e3862. doi: 10.3791/3862.
Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert & Zhao, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca(2+)-activated Cl(-) channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body. In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors. The suction pipette technique was later adapted to also record from ORNs. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.
动物通过位于鼻腔内的化学感应系统对周围有气味的环境进行采样。化学感应信号会影响诸如食物选择、对捕食者、同种个体及配偶的识别等复杂行为，以及其他与社交相关的线索。嗅觉受体神经元（ORN）位于鼻腔背侧，嵌于嗅觉上皮中。这些双极神经元向嗅球发送轴突（见图1，Reisert和Zhao，最初发表于《普通生理学杂志》），并从上皮边界延伸出一根单一的树突，纤毛从该树突处向覆盖嗅觉上皮的黏液中辐射伸展。纤毛含有信号转导机制，最终导致兴奋性电流通过纤毛转导通道流入，该通道包括一个环核苷酸门控（CNG）通道和一个Ca(2 +)激活的Cl(-)通道（图1）。随之而来的去极化触发细胞体处动作电位的产生。在本视频中，我们描述了使用“吸移管技术”来记录ORN对气味剂诱导的反应。该方法最初是为记录视杆光感受器的反应而开发的，并且在jove.com上可以找到该方法的一个变体，该变体经过修改后用于记录小鼠视锥光感受器的反应。吸移管技术后来也被应用于记录ORN的反应。简要地说，在嗅觉上皮解离和细胞分离之后，将一个ORN的整个细胞体吸进记录吸移管的尖端。树突和纤毛仍暴露于浴槽溶液中，因此可以通过改变溶液来接触，例如施加气味剂或药理学阻断剂。在这种配置下，无法进入细胞内环境（没有全细胞电压钳制），细胞内电压可以自由变化。这允许同时记录起源于纤毛的缓慢受体电流和细胞体激发的快速动作电位。这两种信号在动力学上的差异使得可以使用不同的滤波设置将它们分离。该技术可用于任何野生型或基因敲除小鼠，或者用于选择性地记录那些还表达绿色荧光蛋白（GFP）以标记ORN特定亚群的ORN，例如表达特定气味剂受体或离子通道 的ORN。
