Multistepped optogenetics connects neurons and behavior

Figure 1. Multistepped optogenetics (MSO) and its application to neurons in a trigeminal ganglion (TGG). (A) Schematic outline of MSO. (B) Schematic diagram of the IR-LEGO microscopic system. An IR laser beam (1462 nm) was introduced into the IR-LEGO optical unit through an optical fiber. The irradiation duration was controlled by a shutter unit. (C) Dorsal view of the head of a 3 dpf embryo of Tg(hsp70:gal4; uas:chr2-eyfp) after heat shock by incubation at 37°C for 30 min at 10 and 36 hpf. Anterior, to the top. (D) Infrared (IR) laser-induced ChR2-EYFP. Lateral (D1; anterior, to the left) and dorsal (D2; anterior, to the top) views show that ChR2-EYFP was expressed in the TGG only on the left side of the embryo at 33 hpf. The central axons are indicated by the arrowhead and the peripheral axons by the arrow. (E) The escape response induced upon irradiation with blue light on the same embryo. (E1) Selected images of the 33 hpf embryo from a movie (Movie S1) taken with a high-speed camera starting with the C-escape during blue-light irradiation. (E2) Montage of images from 0 (red) to 28 ms (blue) at 4 ms interval. (F) The kinematics obtained from Movie S1. The vertical axis shows (H-T)/AL, the distance between the tips of the head and tail divided by the axial length. Blue bars show the periods of blue-light irradiation. (G) Percentage of experimental embryos with ChR2 expression in TGG on either side showing light-evoked escape behaviors. Contra, Ipsi: Embryos that showed a C-bend either towards the contralateral or ipsilateral side, respectively, to the ChR2-expressing TGG, initially after each blue-light irradiation. Control: wild-type (AB line) embryos that showed a C-bend towards either side. Data are means ± SEM; Experimental: 80.9% ± 5.3%, n = 51 events, four embryos; control: 5% ± 2.8%, n = 60 events, six embryos. Scale bars = 50 μm. Multistepped optogenetics connects neurons and behavior