In spite of the claim that the M and E oscillators are distinct but coupled [5–7] this appealingly simple model must be considered in light of numerous previous studies that show a clear functional contribution by the so-called morning cells (LNv) to the evening bout of activity [11–13]. In flies lacking functional LNv or their output signal PDF, morning anticipatory activity is absent, and the phase of evening anticipation is advanced [11–13]. Ectopic PDF alters oscillator phase [14,15]. In attempts to reconcile these findings, it has been suggested that the ‘‘M’’ cells modulate the activity of the ‘‘E’’ cells [6,10]. However, additional work under different genetic and environmental conditions (constant light or constant darkness or alternating low light/darkness) on the Drosophila circadian circuit, along with comparison with mamma- lian circadian circuits, suggests a more complex model of the distribution and coordination of multiple oscillators in Drosophila beyond the simple two oscillator model [3,16–22]. We proposed recently that the circadian neuronal circuitry underlying the generation of morning and evening activity peaks is plastic rather than being composed of anatomically fixed oscillators to particular cell types. We posited that oscillator localization varies according to environmental conditions and the overall state of the circadian network (reviewed in [23]; see also [24] and [25]. Furthermore, the relative contribution of the small and large LNv as ‘‘morning’’ neurons is unclear. And while it has been recently demonstrated that non-PDF lateral neurons are modulated by the PDF+ large LNv [8], the question of whether non-PDF circadian neurons in the circuit can interact with the large LNv in a network fashion in the absence of the small LNv to modulate morning behavior remains unanswered. Also, in contrast to anatomically restricted dual oscillator models, large scale imaging and physiological studies of the SCN show that individual oscillators are organized in complex networks [26–28]. Electrophysiological analysis indicates that the large LNv exhibit preferential spontaneous firing in both circadian and actual morning [29,30] and that action potential firing rate of the large LNv is acutely sensitive to light [30]. Recent work further parses the LNv subsets, showing that the large LNv act as light- activated arousal neurons that modulate the circadian circuit for both the morning behavioral peak [30–32] (see also [33] and [34]) and the evening behavioral peak [8]. To clarify whether the small LNv are required for the morning behavioral anticipation and peak activity, and to distinguish between acute light versus circadian effects on the light-activated high amplitude morning startle response, we examined these questions using a method developed recently in our laboratory which functionally ablates the small LNv while the large LNv and the other circadian neurons remain functionally intact [31]. Results Peak spontaneous action potential firing rate of large LNv tends to be highest in the morning Spontaneous action potential firing of large LNv Drosophila pacemaker neurons can be measured by whole cell patch in current clamp mode [17,29–31,35], a recording method adapted from techniques devised for recording from olfactory neurons in adult Drosophila whole brain [36]. Flies expressing a membrane delimited GFP marker in the LNv (pdfGAL4/dORK-NC1-GFP; [13]) were maintained in standard 12 h:12 h light:dark cycles. Expression of dORK-NC1 in the LNv has no effect either on behavior [13] or membrane electrophysiological properties [30,35]. Individual flies (1–7 days old) were collected at time points throughout the 24 hr light: dark cycle and whole brains were dissected for whole cell patch clamp recording as described LNv and Morning Anticipation previously in detail [30,36]. Representative spontaneous action potential firing records are depicted for large LNv recorded under equal illumination conditions at all time points (7 klux, which corresponds to daylight illumination) in the early morning (ZT1) and late night (ZT22) (Figure 1, left and centre panel). Large LNv spontaneous action potential firing rate recorded under 7 klux light in 67 brains prepared at different phases throughout the entire 24 hour light:dark cycle show that large LNv spontaneous firing can occur throughout the entire light:dark cycle, but that higher firing tends to occur in the morning (Figure 1, right panel). Whole cell current clamp recordings of large LNv verify that spontaneous action potential firing peaks in the morning with firing rate gradually decreasing between ZT 0–12 (slope = 20.19) and increasing between ZT 13–24 (slope = 0.07, Figure 1, right panel). Although with this sample size of 67, which is approximately half of the sample size used in [29], they do not reach statistical significance. These results are in qualitative agreement with previous studies that show morning peak large LNv circadian-regulated firing [30] and large LNv peak firing in the morning under diurnal conditions [29], Again qualitatively similar to the results reported in [29], we tend to see more depolarized resting membrane potential values in the morning, however this is not statistically significant by regression analysis (data not shown), while this is reported as significant in [29]. This is probably due to sample size and the variance in large LNv firing rate and resting membrane potential as seen here and in [29]. Loss of rhythmic circadian behavior in flies expressing neurotoxic Huntingtin-Q128 in the LNv pacemaker neurons Expression of fragments of human Huntingtin (Htt) protein with poly-glutamine (poly-Q) sequences containing greater than 35 copies of Q results in functional neuronal loss and degeneration [31,37–40]. We expressed pathogenic UAS-Q128-Htt (henceforth Q128-Htt) or control non-pathogenic UAS-Q0-Htt (henceforth Q0-Htt) in the LNv using the pdfGAL4 (pdf) driver line and examined locomotor behavior of newly eclosed flies in standard light:dark (LD) conditions for 6 days followed by constant darkness (DD) starting at 7 days of age. For control comparison, the offspring of Q128-Htt and Q0-Htt crossed with yw and pdfGAL4 driver crossed with yw were assayed in parallel to test for potential genetic background effects of the UAS lines and the driver. In standard 12 h:12 h LD conditions, both pdf/Q0-Htt and pdf/Q128- Htt flies exhibit the typical profile of locomotor activity, with peak activity in the morning and evening along with low levels during mid-day and at night. Similar behavior is observed for both Q0- Htt and Q128-Htt UAS genetic background controls, except that pdf/Q128-Htt flies exhibit significantly greater locomotor activity at night at all ages tested (Supplementary Figure S1, S2). In contrast, while most control pdf/Q0-Htt and genetic background control flies exhibit robust circadian locomotor behavior in constant darkness, the locomotor behavior of most pdf/Q128-Htt flies is arrhythmic (Figure 2A,B) (** indicates significant differences at p,0.01.) The predominance of circadian arrhyth- mic behavior of pdf/Q128-Htt flies relative to control pdf/Q0-Htt flies is stable and seen for days 7–13, 14–20, and 21–27 in constant darkness. Thus, pdf/Q128-Htt flies do not later recover from the behavioral circadian arrhythmicity that occurs immediately when exposed to constant darkness. As the small LNv are required for circadian rhythmicity in constant darkness [41,42], the results indicate that all small LNv neurons are functionally impaired by Q128-Htt expression in young adult flies and that the small LNv contribute to restful behavior at night (Supplementary Figure S1). PLoS ONE | www.plosone.org 2 July 2010 | Volume 5 | Issue 7 | e11628