Fig. 2-S2

c-fos expression correlates with overexpression of RFRP peptides that induce sleep.

One hour after heat-shock, sleep is significantly increased in Tg(hs:RFRP1-3) animals compared to their WT siblings (A, arrow) but there is no effect on sleep in Tg(hs:RFRPscr) animals compared to their WT siblings (D, arrow). We assayed neuronal activity at this time point in animals overexpressing different RFRP peptides by performing c-fos ISH. In animals overexpressing RFRP1-3, we observed prominent c-fos expression along the brain ventricular lining from the hypothalamus to the hindbrain (C), four blue arrowheads) that was absent in WT siblings (B, four white arrowheads). Overexpression of specific RFRP peptides affected c-fos expression in a manner that correlated with their effects on behavior (Figure 2E). No c-fos expression was observed in animals overexpressing RFRPscr, in which all 3 RFRP peptides are scrambled (F, four white arrowheads), weak c-fos expression was observed in animals overexpressing either RFRP1 or RFRP3 (G,I, 2 blue and two white arrowheads), and strong c-fos expression was observed in animals overexpressing RFRP2, or RFRP1 and RFRP3 (H,J, four blue arrowheads), similar to overexpression of RFRP1-3 (C). Yellow bars indicate heat shock. White and black bars under behavioral traces indicate day (14 h) and night, respectively. Scale: 50 μm.