LNv and Morning Anticipation Figure 5. Morning behavioral anticipation and high amplitude morning startle response behavior persists in flies lacking functional small LNv. (A) Mean locomotor activity counts of flies living in standard 12 h:12 h light:dark cycles with pdfGAL4 driven expression of control Q0-Htt (black squares), or Q128-Htt (red dots) and yw controls (grey triangles) are shown. Yellow shaded areas denote mean locomotor activity binned in 15 min intervals during the day while blue shaded areas denote mean locomotor activity at night. Average activity is plotted for age day 1–10 (top panel), age day 11–20 (middle panel) and age day 21–30 (lowest panel) to distinguish age dependent effects. (B) Morning and evening anticipation indices were estimated for individual flies at same 3 life-stages as panel A using the fraction of activity during the 3 hours before the transition states of dawn or dusk compared to the activity level through the six hours before transition (Harrisingh et al 2007). One-way ANOVA on the morning anticipation showed no significant differences between the genetic background controls or yw controls and the experimental lines pdf/Q0-Htt and pdf/Q128-Htt flies through all three life stages. Similar ANOVA was performed on the individual evening anticipation indices at all three lifestages. pdf/Q128-Htt flies exhibited significantly lower evening anticipation compared to pdf/Q0-Htt during the early life stages up to age day 10 (* indicates p,0.01), but during days 21–30 the levels are not different between the two genotypes. But we cannot rule out the non-specific effect of Q128 since yw/q128-Htt flies (genetic background controls) did not show significantly higher anticipation than pdf/Q128-Htt. (C) Group anticipation indices of morning (left panel) and evening activity (right panel), estimate the positive slope of activity for the 4 hours before dawn ZT0 or dusk ZT12 (Stoleru et al 2004; methods of this paper) for both control pdf/Q0-Htt and pdf/Q128-Htt. Relative anticipation indices are plotted for both control pdf/ Q0-Htt and pdf/Q128-Htt by normalizing over values of AI for their respective genetic background controls (yw/Q0-Htt and yw/Q128-Htt). Morning anticipation in flies expressing HTT-Q128 in the LNv appears to be not different from controls up to the age of 20 days and during the advanced age group of 21–30 days they show an enhanced level of morning anticipation suggesting that the network of neurons that regulate anticipatory behavior is plastic and undergoes modifications that allow flies to compensate for the lack of functional sLNv. No statistical comparisons were made as single values for anticipation index is obtained by this method from the average activity profile of each genotype. Similarly evening anticipation also appears unaffected by the loss of functional sLNv (right panel). doi:10.1371/journal.pone.0011628.g005 behavior for days 11–20 and 21–30 for both control pdf/Q0-Htt and experimental pdf/Q128-Htt flies (Figure 5C). The highest Relative AI value is seen for the morning behavior of experimental pdf/Q128-Htt flies analyzed between 21–30 days, which is consistent with visual inspection of the average locomotor actograms (Figure 5A,B). Relative AI for morning anticipation using this method was also calculated for other control lines, yw/ pdf-GAL4, and again using this method, defects in morning anticipation can be seen (Supplementary Figure S1), AI values not shown. Group/Relative AI values are positive for evening anticipatory behavior for both control pdf/Q0-Htt and experimen- tal pdf/Q128-Htt flies at all life stages measured with a noteworthy high value for control pdf/Q0-Htt flies between days 1–10, again, consistent with visual inspection of average locomotor activity profile actograms (Figure 5A). Based on low Group/Relative AI morning anticipation values for some of the time intervals analyzed for two control lines and the lack of robustness of this method for measuring AI, we also analyzed morning and evening anticipation using an AI described in [51] which calculates the amount of locomotor activity in the 3 hours preceding LD transition divided by the locomotor activity during 6 hours preceding LD transition. Unlike the Group/Relative AI, the individual AI method provides a statistical comparison [51]; see also [47]. Using the individual AI method, we calculate positive AI values for both control pdf/Q0-Htt and pdf/Q128-Htt flies, and see no significant difference in morning anticipation between these two genotypes and their respective genetic background controls (Figure 5B, Figure S1). The individual evening AI also do not show significant differences between genotypes at any life stage (Figure 5A,B; One way ANOVA comparing genetic background controls pdf/QO and pdf/Q128). Thus, while AI values calculated using either the Relative/Group or Individual methods show a few differences, both analyses yield the same fundamental conclusion that small LNv functionally ablated pdf/Q128-Htt flies exhibit measurable morning anticipation similar to controls as well as a high amplitude morning startle response coinciding with lights-on in standard 12 h:12 h LD cycles when examined for 30 days or between days 1–10, days 11–20, or days 21–30 (Figure 5A). The small LNv functionally ablated pdf/Q128-Htt flies do show one interesting consistent difference from pdf/Q0-Htt control lines: significantly higher overall night time behavioral activity for all time windows tested (Supplementary Figure S1B), indicating that the small LNv may inhibit night time activity, and may act as sleep promoting neurons. Significant increases in overall night time behavioral activity in pdf/Q128-Htt flies is robust and has been independently replicated in our laboratory where the genotype of the fly was coded to enable unbiased estimation of activity levels. The pdf/Q128-Htt flies with small LNv functionally ablated also show a small but significant increase in total day time behavioral activity (Supplementary Figure S1B) These results are consistent with recently published findings from our group and others [31,33]. Increased day time activity cannot be attributed to phase advanced evening anticipation as UAS control yw/UAS- Q128-Htt flies exhibit similar levels of evening anticipation as pdf/ Q128-Htt flies (Figure 5A,B). Morning anticipation is retained in DD but the high amplitude circadian morning peak is selectively lost in flies lacking functional small LNv Previous imaging and functional studies show that upon introduction to constant darkness molecular oscillations are robust in the small LNv while molecular oscillation quickly dampens in the large LNv; and that the small LNv, but not the large LNv are essential for circadian behavior [5,7,11–13,41,42]. This assertion is also supported by the comparative anatomy of the large and small LNvs [49]. However, circadian molecular oscillations in the large LNv appear to recover after long term exposure to constant darkness (9–14 days), indicating network plasticity [17,46]. We examined the behavior of control versus small LNv functionally ablated pdf/Q128-Htt flies during the transition between standard 12 h:12 h LD cycles to constant darkness. As seen in earlier experiments under 12 h:12 h LD conditions, control pdf/Q0-Htt and pdf/Q128-Htt exhibit both morning anticipatory increase in behavior preceding lights-on and the CT 0 transition from LD to DD (the first ‘‘cycle’’ of darkness). In contrast to control flies, the high amplitude circadian morning peak behavior is entirely absent in small LNv functionally ablated pdf/Q128-Htt flies and is thus similar to negative control pdf01 flies (Figure 6A third panel; pdfGAL/pdf01). One robust evening peak around CT 12 on day 1 in DD followed by the next weak evening peak (also around CT12) can be discerned approximately 24 hours later on day 2 DD in pdf/Q128-Htt flies, after which time, no discernable circadian behavioral rhythms can be observed (Figure 6A second panel). In contrast, control pdf/Q0-Htt flies exhibit a robust morning peak around CT 0 for days 1 and 2 in DD, and robust evening peaks around CT 12 for days 1–3 in DD (Figure 6A). In an attempt to assign a quantitative value to this behavior we adopted two methods both of which involved averaging of activity across flies PLoS ONE | www.plosone.org 8 July 2010 | Volume 5 | Issue 7 | e11628