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

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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.