Boccaccio Anna, Sagheddu Claudia, Menini Anna
SISSA, International School for Advanced Studies.
J Vis Exp. 2011 Oct 29(55):e3195. doi: 10.3791/3195.
Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP or caged Ca. We use a flash lamp to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration.
笼锁化合物的光解能够使各种生理活性化合物的浓度迅速且局部地升高。笼锁化合物是一类分子，其生理活性被化学笼锁所抑制，而这种笼锁可被一束紫外光破坏。在此，我们展示了如何在解离的小鼠嗅觉感觉神经元中，将膜片钳记录与笼锁化合物的光解相结合，用于嗅觉转导的研究。嗅觉转导过程（图1）发生在嗅觉感觉神经元的纤毛中，气味分子与受体结合会导致环磷酸腺苷（cAMP）增加，进而打开环核苷酸门控（CNG）通道。通过CNG通道进入的钙离子会激活钙激活氯离子通道。我们展示了如何从小鼠嗅觉上皮中解离出神经元，以及如何通过笼锁cAMP或笼锁钙离子的光解来激活CNG通道或钙激活氯离子通道。我们使用闪光灯向纤毛区域施加紫外闪光，以释放出cAMP或钙离子，同时进行膜片钳记录，在全细胞电压钳模式下测量电流。
