Figure 3.

Hcrt overexpression consolidates active states and reduces rest. A, Ventral views of 7 d postfertilization brains labeled with an Hcrt1-specific antibody from HS-Hcrt transgenic larva that either were not (top) or were (bottom) heat shocked 48 h earlier. The non-heat-shocked brain shows endogenous Hcrt protein, whereas the heat-shocked brain shows high Hcrt levels throughout much of the brain. Scale bars, 100 μm. B, Hcrt overexpression increases locomotor activity. Each data point represents the average seconds of locomotor activity every 10 min for 20 larvae of each genotype. Behavioral recording was initiated on day 4 of development. HS-Hcrt and WT larvae were heat shocked for 1 h on day 5 (arrow). HS-Hcrt and WT larvae had similar activity levels before heat shock. Hcrt-overexpressing larvae became significantly more active than WT larvae a few hours after heat shock and remained more active for over 48 h. Note that larvae of both genotypes became very active for several minutes after lights out. The spike in activity during the afternoon on days 6 and 7 resulted from the addition of water to offset evaporation. C, Activity plots of representative WT and Hcrt-overexpressing larvae during 1 h preceding and after lights out. Black squares represent 1 min periods during which any locomotor activity is recorded and are referred to as active bouts. Rest latency refers to the time between lights out and the first 1 min period with no activity. Rest bout refers to a period of at least 1 min with no activity. D–H, Combined results from 10 independent experiments are shown. D–G, Each bar represents the mean ± SEM of 302 HS-Hcrt or 219 WT larvae. Hcrt overexpression increases active bout length (D), decreases rest bout length at night (E), decreases total time at rest (F), and decreases the number of rest bouts (G) (**p < 0.01 by two-tailed Student's t test). H, Hcrt overexpression reduces rest in the entire larval population. The graph represents the distribution of total rest times for HS-Hcrt and WT larvae during the night after heat shock. I, Pie charts represent the percentage of time spent in each state, and arrows represent the frequencies of transitions between states during the night after the heat shock. HS-Hcrt larvae in the inactive and low-active states are more likely to transition to the high-active state than WT larvae, as represented by thicker arrows. Frequency values in bold are significantly different between HS-Hcrt and WT larvae (p < 0.05 by two-tailed Student's t test). For example, HS-Hcrt larvae spend 64 % of their time in the high-active state compared with 38% for WT, and the probability that WT larvae will transition from the inactive to the high-active state is only 13 versus 26 % for HS-Hcrt larvae. State transition frequencies do not add up to 1 because larvae often remain in the same state for > 1 min.