Figure 4.
Parvalbumin expression in LNV circadian pacemaker neurons slows free-running behavioral rhythms. a, Representative actograms of individual flies expressing PV by combining pdf–GAL4 driver and indicated number of UAS–PV transgene insertions demonstrate dose-dependent slowing of free-running locomotor rhythms measured in DD. Nonexpressing 8× UAS–PV flies contain each of the independent transgene insertions used but no pdf–GAL4 driver. b, Flies expressing the highest doses of PV exhibit continuous lengthening of free-running period over time in DD. Flies expressing PV from four or six UAS–PV insertions exhibit longer periods than both nonexpressing flies and flies expressing PV from two UAS–PV insertions for all 4 weeks in DD (p < 0.01). Periods of flies expressing PV from six UAS–PV insertions increase over the 4 weeks in DD (p < 0.01) and are longer than those of flies expressing PV from four UAS–PV insertions during the fourth week in DD (p < 0.01). Line graph depicts mean ± SEM. c, Categorization of free-running rhythms during the fourth week in DD. Approximately 20% of flies expressing PV from six UAS–PV insertions suddenly shift to a short free-running period. (These individuals are excluded from the average periods shown in b.) The differences between control and experimental groups are statistically significant for “>24.5” (p < 0.001), “<24.5” (p < 0.001), and “<24.5 after being >24.5” (p < 0.05, χ2 test). d, Representative actogram of a pdf > 6× PV fly that shifts to a short-period rhythm. n > 30 flies per experimental condition. Multiple independent replicates reveal similar slowing and continuous lengthening of free-running period (data not shown). Statistical analysis is by repeated-measures ANOVA with Tukey–Kramer paired comparison test. e, Averaged normalized actograms of groups of flies of the indicated genotypes maintained for 3 weeks in LD, followed by release into DD for 1 week. f, Average free-running periods of flies maintained in DD for 4 weeks (measured for the 4th week; n > 12) or of flies maintained in LD for 3 weeks and then released into DD for 1 week (measured for that 1 week; n > 45). Mean ± SEM. There is no significant effect of environmental condition (i.e., maintenance in DD or maintenance in LD followed by shift to DD) on free-running period (p > 0.05; two-way ANOVA).