Persistence of Morning Anticipation Behavior and High Amplitude Morning Startle Response Following Functional Loss of Small Ventral Lateral Neurons in Drosophila Vasu Sheeba¤, Keri J. Fogle, Todd C. Holmes* Department of Physiology and Biophysics, University of California Irvine, Irvine, California, United States of America Abstract Light-activated large ventral lateral clock neurons (large LNv) modulate behavioral arousal and sleep in Drosophila while their counterparts, the small LNv (s-LNv) are important for circadian behavior. Recently, it has been proposed that the pattern of day-night locomotor behavioral activity is mediated by two anatomically distinct oscillators composed of a morning oscillator in the small LNv and an evening oscillator in the lateral dorsal neurons and an undefined number of dorsal pacemaker neurons. This contrasts with a circuit described by network models which are not as anatomically constrained. By selectively ablating the small LNv while sparing the large LNv, we tested the relative importance of the small and large LNv for regulating morning behavior of animals living in standard light/dark cycles. Behavioral anticipation of the onset of morning and the high amplitude morning startle response which coincides with light onset are preserved in small LNv functionally-ablated animals. However, the amplitude of the morning behavioral peak is severely attenuated in these animals during the transition from regular light/dark cycles to constant darkness, providing further support that small LNv are necessary for circadian behavior. The large LNv, in combination with the network of other circadian neurons, in the absence of functional small LNv are sufficient for the morning anticipation and the high amplitude light-activated morning startle response. Citation: Sheeba V, Fogle KJ, Holmes TC (2010) Persistence of Morning Anticipation Behavior and High Amplitude Morning Startle Response Following Functional Loss of Small Ventral Lateral Neurons in Drosophila. PLoS ONE 5(7): e11628. doi:10.1371/journal.pone.0011628 Editor: Fabien Tell, The Research Center of Neurobiology-Neurophysiology of Marseille, France Received November 19, 2009; Accepted June 9, 2010; Published July 16, 2010 Copyright: 2010 Sheeba et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Science Foundation (NSF) grant IBN-0757242 (www.nsf.gov), and National Institutes of Health (NIH) grant NS046750 (www.nih.gov) to TCH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: tholmes@uci.edu ¤ Current address: Evolutionary and Organismal Biology Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India Introduction Pittendrigh and Daan proposed that circadian clocks simulta- neously ‘‘measure’’ daily and seasonal changes in day lengths using mutually coupled binary oscillators: the ’’Morning’’ (‘‘M’’) oscillator that tracks dawn and the ’’Evening’’ (‘‘E’’) oscillator that tracks dusk [1]. While the binary oscillator model was originally developed to explain the peculiar phenomenon of ‘‘splitting’’ and ‘‘re-fusion’’ of morning and evening activity bouts in mammals in response to constant light, this model has recently been adapted to account for the bimodal activity pattern in Drosophila melanogaster. Using several genetic and behavioral approaches, attempts have been made to identify the putative ‘‘M’’ and ‘‘E’’ oscillators in circadian neuronal circuit. The ‘‘M’’ and ‘‘E’’ oscillator model is particularly attractive for Drosophila as this insect exhibits two distinct bouts of locomotor behavior under 12:12 h LD cycles - at dawn (morning peak) and dusk (evening peak). Helfrich-Fo¨ rster [2] suggested that the morning peak in activity is governed by a per-independent clock and is entrained by light signals via photoreceptors, while the evening peak is regulated by the circadian clock involving per and entrained by CRY (see also [3]). Subsequently, several papers indicate that the ‘‘M’’ and ‘‘E’’ oscillators in Drosophila may have distinct anatomical locations as shown by the effects of eliminating different subgroups of clock neurons or by restoring clock gene expression in specific neurons in clock mutants [4–7]. These studies conclude that the small LNv function as the ‘‘M’’ oscillator in the Drosophila circadian pacemaker circuit, while the LNd and an unspecified number of dorsal neurons function as the ‘‘E’’ oscillator. PER-null flies lack both morning and evening anticipatory behavior. In an attempt to localize oscillator function between subsets of the LNvs, PER expression was directed in PER-null flies comparing the Mai179 and c929 driver lines. The Mai179 driver line putatively targets the small LNv, while the c929 driver line directs expression to the large LNv along with a large number of non-clock peptidergic neurons, but not the small LNv. Similar claims of small LNv specificity have been made also for the R6 driver line [8]. However, other reports state that the Mai179 and R6 lines drive expression in the both the small LNv and a subset of large LNv [9]. In spite of lacking a specific GAL4 driver for isolating the small LNv for PER expression rescue, the small LNv have been designated as the ‘‘morning’’ neurons. This interpre- tation of the small LNv as the locus of the morning oscillator appears to have gained some acceptance [6,10]. PLoS ONE | www.plosone.org 1 July 2010 | Volume 5 | Issue 7 | e11628