Q128-Htt expression in both LNv subsets causes the selective loss of PDF immunoreactivity in the small LNv, but not in the large LNv Selective vulnerability of specific subsets of neurons is a common feature of neurodegenerative diseases [43]. The small LNv, but not the large LNv, are required for circadian behavioral rhythmicity [41,42], (see also [23] and [24] for detailed reviews of additional supporting evidence). Thus, the functional loss of circadian rhythmic behavior in pdf/Q128-Htt flies suggests that all of the small LNv neurons in the circadian arrhythmic flies are impaired by Q128-Htt expression. To verify this, we measured the levels of the functionally critical circadian neuropeptide pigment dispersing factor (PDF) [12,13,17,44–48] levels in both the small and large LNv subsets in flies expressing control Q0-Htt versus pathogenic Q128-Htt driven by pdf-GAL4 to both LNv neuronal subsets. Anti-PDF immunocyto- chemical analysis was performed on whole brains dissected from 1– 34 day old pdf/Q0-Htt and pdf/Q128-Htt flies that were maintained under standard LD conditions. There are typically 4–5 large LNv per brain hemisphere and 4–5 small LNv per brain hemisphere in wild type Drosophila [49,50]. Consistent with earlier observations, PDF- positive cell-counts per brain hemisphere in control pdf/Q0-Htt flies for both large and small LNv sub-groups ranged between 3–5 cells up to 34 days of age (Figure 3A). No consistent loss of PDF-positive large LNv occurs in pdf/Q0-Htt or pdf/Q128-Htt flies up to 34 days of age, so PDF expression appears to be unaffected by polyQ-Htt expression. In contrast, the numbers of PDF-positive small LNv were severely diminished relative to controls at all ages tested between ages 1–34 days in pdf/Q128-Htt flies (Figure 3A). No PDF-positive small LNv were observed between the ages of 18–34 days in pdf/Q128-Htt flies sampled and they were observed rarely in extremely low numbers in 6–16 day old pdf/Q128-Htt flies. Closer examination of the PDF-positive cell bodies shows robust healthy-appearing cells in Q0-Htt expressing flies while only small fragments of PDF-positive material is typically seen in the region of the small LNv in Q128-Htt expressing flies at all ages (Figure 3B, scale bar is 20 mM for the large LNv panels and 15 mM for the small LNv panels). Under the fixation conditions used, it was not possible to discern whether the small fragments of PDF-positive signal in the region of the small LNv of Q128-Htt expressing flies is cellular or non-cellular. Similarly, membrane delimited GFP expression is selectively lost in small LNv expressing Q128-Htt (pdfGAL4/dORK-NC1-GFP/Q128-Htt), but no loss of GFP signal is observed in large LNv expressing Q128-Htt (data not shown). While loss of PDF-immunoreactivity and GFP expression are clear indications of neural dysfunction, these markers are not equivalent to cell death. To test for polyQ-Htt induced cell death, we performed TUNEL assays using whole mount brains of control and polyQ-Htt expressing flies at various ages. We consistently observed small numbers of TUNEL-positive cells throughout the brain samples for all ages. However, we could detect no specific TUNEL signal in the region of the LNv (data not shown). Thus, there is no direct evidence for polyQ-Htt-induced cell death distinguishable from neuronal dysfunction of small LNv. However, these results indicate that Q128-Htt expression differentially affects the large and small LNv function and suggests that the function of the small LNv are selectively vulnerable to polyQ-Htt protein expression while the large LNv are potentially spared. Long-term persistence of spontaneous action potential firing and acute physiological light response in large LNv expressing Q128-Htt The results described above suggest that Q128-Htt expression in the PDF-expressing LNv causes selective functional disruption LNv and Morning Anticipation of the small LNv while sparing the large LNv. To test the functionality of the large LNv expressing Q128-Htt, we patch recorded spontaneous action potential firing and physiological light responsiveness of GFP-labelled large LNv from whole brains of 1–20 day old pdf/Q128-Htt flies in current clamp mode. Large LNv that express Q128-Htt show normal action potential firing rate and normal firing pattern as shown in recordings made up to 20 days of age. A representative tonic firing neuron is shown in Figure 4A, compare this with recording traces of wild type large LNv in Figure 1 above and in [30,35]. Large LNv acutely increase their spontaneous action potential firing rate on average by 50% when exposed to daytime levels of light (.2000 lux). This light-induced increase in firing rate is rapid and occurs typically between 1–2 seconds following light exposure and reverses between 1–2 seconds following lights-off [30]. Normal acute large LNv physiological light responses are seen in neurons expressing Q128-Htt up to 20 days of age (n = 12, 11/12 recordings made from flies 7 days old or less; one fly 20 days old), with a significantly higher firing rate during lights-on versus lights-off (Figure 4B, left panel). No differences in firing frequency or electrophysiological light response are observed between control pdfGAL4/NC1 flies and pdfGAL4/Q128/NC1 flies (Figure 4B). There are no apparent age dependent differences in firing rate or physiological light responsiveness in large LNv expressing Q128- Htt (small LNv expressing Q128-Htt cannot be identified for recording as they do not express GFP). From these results and the anti-PDF immunostaining results, we conclude that large LNv are selectively spared while small LNv are functionally impaired following Q128-Htt expression. Morning behavioral anticipation and high amplitude morning startle response persist in LD in flies lacking functional small LNv Loss of PDF or functional ablation of all PDF-expressing LNv in Drosophila leads to the loss of anticipatory behavior preceding the onset of morning, and in some cases that appears to depend on absolute light levels, attenuation of high amplitude light-induced morning startle response [5,12,13]. Similar losses of morning (and evening) anticipatory behavior are seen in per0 mutant flies [7]. Directed PER expression to both the small and large subset of LNv in per0 mutant flies rescues the morning peak defect (but not the evening peak defect), while expression directed to small LNv, some of the large LNv, and the majority of the LNd in per0 mutant flies rescues both the morning and evening peak defects. PER expression directed to the large LNv, but not the small LNv, is insufficient to rescue either the morning or evening peak defect under the environmental conditions tested for that study [7]. More recent studies using the Mai179 and R6 driver lines further suggest a specific small LNv contribution to morning anticipation [8]. However, other recent work shows that the Mai179 and R6 driver lines direct expression consistently to a subset of the large LNv as well as the small LNv [9]. To determine the contribution of the small LNv to the morning peak in flies that have functionally intact large LNv and all other circadian neurons, we examined LD behavior in control versus pdf/Q128-Htt flies that specifically lack functional PDF-positive small LNv but by all measures retain functional large LNv (Figures 2, 3 and 4). Visual inspection of averaged locomotor activity of control pdf/Q0-Htt and experimen- tal pdf/Q128-Htt flies shows similar gradual increases in activity in anticipation of morning and evening; and high amplitude morning and evening startle responses coinciding with lights-on and lights- off 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). Similar morning and evening anticipation is seen by visual PLoS ONE | www.plosone.org 4 July 2010 | Volume 5 | Issue 7 | e11628