LNv and Morning Anticipation Figure 3. Q128-Htt expression in both LNv subsets causes the selective loss of PDF immunoreactivity in the small LNv. (A) The average number of PDF-immunoreactive (PDF+) LNv (6 SEM) that can be detected in flies expressing Q0-Htt (black filled square) and Q128-Htt (red hollow circle) in the small and large LNv. The number of PDF+ large LNv remains constant for up to 34 days of age in Q0-Htt and Q128-Htt flies (left panels). The average number of PDF+ small LNv (right panel) remains constant in Q0 lines throughout the assay, while in Q128-Htt flies, PDF+ small LNv are almost never detected between 1–14 days of age, then not detected in any Q128-Htt flies thereafter. (B) Representative confocal maximum projections of anti-PDF staining in large and small LNv in adult flies expressing Q0-Htt or Q128-Htt at ages 12, 16, 18, 22 and 26 days. The PDF+ large LNv (denoted by .) are detectable in all the genotypes up to 26 days of age. The small LNv (thin arrow) are detectable in Q0-Htt flies up to 26 days of age, but in Q128-Htt flies, PDF+ small LNv are rarely detected between 1–18 days of age and never detected after 18 days of age. Scale bars = 20 mm in large LNv panels and 15 mm in small LNv panels. doi:10.1371/journal.pone.0011628.g003 inspection of averaged behavioral records of control yw/pdfGAL4- driver line flies (data not shown). Visual assessment of gradual increases in activity in anticipation of morning and evening is highly qualitative and imprecise. Several quantitative methods have been developed recently for measuring anticipation index (AI; [5,51], see Materials and Methods for details). Using the Stoleru method to measure Relative AI (henceforth Group/Relative AI method), control pdf/ Q0-Htt and pdf/Q128-Htt small LNv functionally impaired flies exhibit similar positive values for Relative AI for morning behavior and positive values for evening anticipation over 30 days of 12 h:12 h LD locomotor behavior (Figure 5C). Further analysis using the Relative AI method applied to 10-day successive intervals shows defects in morning anticipation for both control pdf/Q0-Htt and experimental pdf/Q128-Htt flies between days 1– 10, then increasing positive values for Relative AI for morning PLoS ONE | www.plosone.org 5 July 2010 | Volume 5 | Issue 7 | e11628