Fig. 5

Fig. 5

a Maximum-projection image showing regions of optogenetic stimulation (aSPa^y321 neurons) and calcium imaging (OXT neurons in PO or PT, or neuropil bundles in PT outlined in yellow). This experiment was repeated on 18 fish with similar results. b Mean Δf/f traces from either aSPa^y321 neurons or OXT cell bodies/neuropil bundles after stimulation with a 633 nm laser focused on the aSPa region at 60-s intervals. Black traces depict mean Δf/f of an example ReaChR-negative-control fish (top) or y321-negative-control fish (bottom). Red traces depict mean Δf/f from an example Tg(y321:Gal4, oxt:Gal4; UAS:GCaMP6s; UAS:ReaChR-RFP) fish of its aSPa^y321 neurons (top), OXT_PO/PT cell bodies (middle), or neuropil (bottom), subject to aSPa^y321 optogenetic stimulation. Orange lines indicate stimulus onset. Each box represents calcium traces from an individual representative fish. Shaded region indicates SEM. Blue arrows indicate dips in OXT neuropil activity in the presence or absence of optogenetic y321 activation. c Stimulus-triggered averages showing the mean Δf/f before and after the light stimulus. Responses to each stimulus were first averaged per unit (cell body or neuropil bundle) before being averaged across all units to obtain the Δf/f trace. Shaded region indicates SEM. Two-sided Wilcoxon rank-sum test was used for all statistical comparisons. Top panel: The calcium response (Δf/f) of aSPa^y321 neurons to 633 nm laser stimulation is significantly higher in the presence (red, n = 320 neurons from 15 fish) than absence (black, n = 118 neurons from 4 fish) of ReaChR (*p = 0.031). Middle panel: The calcium response of OXT_PO/PT neurons under aSPa^y321 optogenetic stimulation is significantly lower (***p = 5.4 × 10⁻⁶, n = 410 neurons from 18 fish) than for negative controls (Tg(oxt:Gal4; UAS:GCaMP6s; UAS:ReaChR-RFP) fish) exposed to the same light stimulus (n = 103 neurons from 5 fish). When we separated these OXT neurons according to their anterior–posterior position, we found that OXT_PO neurons showed significant suppression after aSPa^y321 optogenetic stimulation compared with negative controls (***p = 0.00028, n = 294/91 neurons), but no significant difference was observed for OXT_PT neurons (OXT_PT; p = 0.198, n = 116/12 neurons). Bottom panel: OXT neuropil calcium responses under y321 optogenetic stimulation are overall significantly lower than for negative controls exposed to the same light stimulus (***p = 2.7 × 10⁻⁵, n = 36/10 neuropil regions). The dip in neuropil activity under y321 stimulation is significantly less negative than the negative controls in the first 10-s epoch (**p = 0.0094), whereas neuropil activity is significantly lower than the negative controls in the next 30-s epoch (***p = 2.4 × 10⁻⁵). Gray bars indicate the 40-s period across which Δf/f was integrated for statistical analysis; asterisk color reflects the group with the more negative (i.e., lower) integrated value. Source data are provided as a Source Data file.