LNv and Morning Anticipation Figure 6. Functional loss of small LNv causes retention of morning anticipation in light-dark cycles, but rapid loss of the circadian- dependent morning peak behavioral activity in constant darkness. (A) Mean activity of flies during the transition from LD12 h:12 h (day 4 and 5; yellow shaded area represents light phase and blue shaded area represents dark phase) to DD (beginning on day 6; grey shaded area) is shown for control flies (pdf/Q0-Htt top panel; averaged across all flies that were rhythmic in DD and smoothed by a moving average of 45 mins - i.e., three consecutive 15 min bins) flies lacking sLNv (pdf/Q128-Htt second panel; averaged across all flies that were arrhythmic in DD and smoothed) and pdfGAL/pdf01controls (bottom panel). Under LD12 h:12 h both genotypes show robust anticipation of morning (yellow arrow) and evening (black arrow). In control flies the morning peak is clear for at least the first two days in DD along with a persistent robust evening peak. (B) Quantification of morning anticipation activity in LD and first 3 days of DD on data averaged across flies and smoothed across 45 mins. Left panel shows morning anticipation activity index measured as the ratio between activity levels 3 hrs compared to 6 hours preceding lights ON (Harrisingh et al 2007) while right panel shows normalized or baseline-corrected index of morning activity estimated as ratio between the activity levels in the 1 hour preceding lights ON normalized by activity levels at midnight preceding that transition. doi:10.1371/journal.pone.0011628.g006 assayed for each genotype. In the case of pdf/Q0-Htt only those flies that were confirmed to be rhythmic in DD were used for the analysis (n = 25; 78% of total flies). In the case of pdf/Q128-Htt only those flies that were confirmed as arrhythmic in DD (n = 29; 81% of total flies) were used for further analysis. For both the above genotypes and for negative control pdfGAL/pdf 01 flies we obtained a measure of morning activity (henceforth morning index Fig. 6B left panel) by estimating the ratio of activity immediately preceding the transition from lights ON to OFF or vice versa (similar to that of [51], except that this was done on activity averaged across flies for each day, see methods). In order to normalize for differences in baseline activity among the three genotypes we also estimated another morning index (normalized morning index) where the activity immediately preceding the lights ON and OFF transitions is normalized by the level of activity at midnight and midday respectively. Both methods clearly show that sLNv ablated flies exhibit morning anticipatory activity which is higher than that of pdf 01 flies. These results indicate that the behavioral morning peak can be readily dissected into two components: a low amplitude anticipa- tory component that shows small but steady increase for the several hours before lights-on, and a much larger high amplitude morning startle response component that peaks at lights-on. In the absence of functional small LNv, the high amplitude morning startle response component persists in LD indicating that it can be acutely light driven, but disappears immediately upon transition into DD and is thus clock- and small LNv dependent in the absence of light. Discussion Individual pacemaker neurons are capable of autonomously generating circadian rhythms over a large range of phases [52,53]. This raises the question as to how multiple pacemaker neurons coordinate their activity to generate a single coherent rhythm in overt behavior. Answering this difficult question is potentially more tractable for Drosophila than for mammals due to the relatively small number of pacemaker neurons in the fly circadian circuit and the available tools for fly genetics [50,54]. Several models have been proposed to account for the general organiza- tion of circadian circuits. A dual oscillator model posits that individual oscillators are anatomically restricted to two function- ally distinct groups of neurons that control ‘‘morning’’ versus ‘‘evening’’ behavior. The dual oscillator model can be contrasted with a distributed network model which suggests that many cell- autonomous oscillators are coordinated in a more complex, but ultimately flexible fashion (recent reviews of the experimental evidence supporting these respective models can be found in [23–25]). While the results above along with several recently published papers [8,16,17] supports the idea that there is considerable circuit plasticity in terms of which cells contribute to diurnal and circadian behavioral morning and evening bouts, there is strong consensus that PDF coordinates the entire fly circadian circuit [6,8,12,13,16,17,23,31,44,46,47,55,56]. The small LNv have received considerable attention as being critical for controlling multiple aspects of diurnal and circadian behavior [41,42,57], including control of anticipatory behavior immediately preceding the peak in morning behavior [6,7]. More recent work indicates that the large LNv function as light-driven arousal neurons that modulate sleep [30,31,33], see also [32]. To further clarify the relative contributions of the small and large LNv to regulating behavior in light/dark cycles, we made use of the selective functional ablation of the small LNv subset by expression of the neurotoxic protein Q128-Htt [31]. PolyQ protein expression has been shown previously to cause neurode- generation and functional loss in photoreceptors and other Drosophila neurons [37,39,58]. In contrast to the Q128-Htt- induced loss of function of the small LNv, large LNv appear to be spared following expression of Q128-Htt, as shown both by physiological and morphological assays. Such selective vulnerabil- ity of certain neuronal populations, while not well understood, is a hallmark feature of neurodegenerative diseases [59]. While pdfGAL4 drives expression in both the small and large LNv neurons, the PDF-positive small LNv precursor neurons appear much earlier in development relative to the PDF-positive large LNv [50].This developmental difference may account in part for the selective functional vulnerability of small LNv to Q128-Htt. PolyQ protein expression in circadian neurons has also been shown to cause abnormalities in courtship behavior [60]. In contrast to the loss of morning anticipation behavior in pdf 01 flies, pdf/Q128-Htt flies lacking functional small LNv retain morning anticipation along with the high amplitude morning startle response that occurs at the onset of morning at ZT0. Previous work shows that directed expression of PER to the small LNv in a per0 genetic background is sufficient to drive morning anticipation activity [7]. But this interpretation is complicated by the fact that the Mai179 driver line used for driving expression in the small LNv also drives expression in the three out of six LNd pairs and a smaller subset of the large LNv [7,20,61]. Furthermore, the interpretation that PER cycling in the small LNv is sufficient to rescue morning anticipation in a per0 genetic background relies on a rather complicated subtractive analysis: the comparison of PER expression rescue in both PDF-positive LNv subsets for which only morning anticipation (but not evening) is rescued versus lack of rescue of morning or evening anticipation behavior in flies with PER expression directed to the large LNv (and a large number of non-clock peptidergic neurons). The present results indicated that in the absence of small LNv, the morning anticipation behavior persists. This shows clearly that the large LNv operating within the circadian network along with the LNd and dorsal pacemaker neurons are capable of mediating morning anticipation behavior. This is consistent with earlier findings which show that flies retain both morning and evening anticipatory behavior when all their neurons express PLoS ONE | www.plosone.org 10 July 2010 | Volume 5 | Issue 7 | e11628