4.4. Light Stimulation and Calcium Imaging

To visualize calcium activity of light-driven olfactory stimulation in this transgenic system, we used transgenic zebrafish Tg(huc:GAL4-VP16) [33] and Tg (uas:GCaMP7a) [34]. These transgenic lines were obtained from the Koichi Kawakami lab through the central facility of NBRP Japan (RIKEN). The Tg(omp:ChR2-Kaede) fish were crossed with Tg(huc:GAL4;uas:GCaMP7a). The triple transgenic zebrafish of 4-6 dpf were embedded in 5% methylcellulose (Sigma Chemical Co., St. Louis, MO, USA, M0512) in embryo medium (sea salt 0.006% Sigma, S9883). Each embryo was irradiated using a Leica DM 5000B upright compound microscope and 20×/0.5 NA objective equipped with a mercury lamp using a GFP filter (480nm). To target olfactory epithelium region, diaphragm was used. Odorant Citrus paradisi aroma oil was used in this experiment to monitor habenular activity. Calcium activity was observed using a fluorescence microscope (DM 5000B, Leica, Wetzlar, Germany) equipped with a CCD camera (DC300 FX, Leica, Wetzlar, Germany). Due to spectral overlap and subsequent cross-talk, the use of ChR2 specific blue light and GCaMP-aided imaging represents a problem. To minimize ChR2 activation during calcium imaging, we irradiated the olfactory epithelium first, and subsequently we adjusted the position of larvae to avoid direct olfactory region irradiation, which allowed us to focus our imaging on the other brain regions. To calculate dF/F0 we first measured the average intensity of GCaMP7a in the ROI 1 (left habenula) and ROI 2 (right habenula), before ChR2-light or odor activation as baseline using ImageJ (National Institute of Health, Bethesda, Maryland, MD, USA). After activation, we measured the average intensity and then we calculated dF/F0 by dividing intensity change (activation-before) by baseline intensity.