Study on functional roles of a single neuron in animal behaviors by functional transplant of channel rhodopsin 2

本实验将衣藻分离的通道视紫红质2（channel rhodopsin 2, ChR2）移植到经鉴定的海兔颊神经节MA神经元中，并探讨了MA在摄食神经回路中的功能作用。将ChR2表达载体（pNEX-ChR2）注入MA细胞体后，于20℃孵育3 d。当亮蓝光（100W氙灯）照射MA的胞体区域时，大多数突触后跟随神经元都记录到了去极化反应。这一结果与电生理学数据不一致，其中MA除了电突触外，还通过抑制性化学突触连接许多跟随神经元。ChR2倾向于在整个细胞膜上表达，强光刺激会使整个细胞去极化。那么在胞体附近产生的动作电位的传导有可能在轴突区域受到抑制，我们可以单独观察到电偶联去极化。我们用蓝色激光束产生的细射光（直径10μm）刺激MA细胞体上的小区域，可以记录到大多数追随者的ipsp。此外，当MA细胞体受到长时间强光或细射灯照射时，MA或其他颊部神经元产生节律性爆裂反应，提示MA可能产生这种节律性反应。本研究将提出不适当的光刺激会在ChR2功能移植中产生不同于活体现象的反应。

In the present experiments we attempted functional transplant of a channel rhodopsin 2(ChR2) isolated from Chlamydomonas into the identified MA neuron in Aplysia buccal ganglia, and explored the functional roles of the MA in the feeding neural circuit. The preparations were incubated at 20 oC for 3 days after injection of the ChR2 expression vector(pNEX-ChR2) into the cell body of MA. When the cell body region of the MA was illuminated with bright blue light(100W Xenon lamp), the depolarizing responses were recorded in most of the postsynaptic follower neurons. This result was inconsistent with the electrophysiological data where the MA connects many follower neurons with inhibitory chemical synapses in addition to the electrical synapses. ChR2 tends to be expressed over the whole cell membrane and the bright light stimulation will depolarize the whole cell. Then there may be a possibility that the conduction of action potentials produced near the cell body might be suppressed in the axonal region and we can observe the electrically coupled depolarization alone. We stimulated the small region on the MA cell body with the fine spotlight(10μm diameter) produced by the blue laser beam and could record the IPSPs in most of the followers then. In addition, the rhythmic bursting responses were produced in the MA or the other buccal neurons when the MA cell body was illuminated with the long-lasting bright light or the fine spotlight, suggesting the potentiality of the MA to produce such rhythmic responses. The present study will propose that unsuitable light stimulation will produce the responses different from the living phenomena in the functional transplant of ChR2.