3142 • The Journal of Neuroscience, February 26, 2014 • 34(9):3142–3160 Behavioral/Cognitive Neuropeptidergic Signaling Partitions Arousal Behaviors in Zebrafish Ian G. Woods,1,2 David Schoppik,2 Veronica J. Shi,2 Steven Zimmerman,2 Haley A. Coleman,1 Joel Greenwood,3 Edward R. Soucy,3 and Alexander F. Schier2,3 1Department of Biology, Ithaca College, Ithaca, New York 14850, and 2Department of Molecular and Cellular Biology and 3Center for Brain Science, Harvard University, Cambridge, Massachusetts 02138 Animals modulate their arousal state to ensure that their sensory responsiveness and locomotor activity match environmental demands. Neuropeptides can regulate arousal, but studies of their roles in vertebrates have been constrained by the vast array of neuropeptides and their pleiotropic effects. To overcome these limitations, we systematically dissected the neuropeptidergic modulation of arousal in larval zebrafish. We quantified spontaneous locomotor activity and responsiveness to sensory stimuli after genetically induced expression of seven evolutionarily conserved neuropeptides, including adenylate cyclase activating polypeptide 1b (adcyap1b), cocaine-related and amphetamine-related transcript (cart), cholecystokinin (cck), calcitonin gene-related peptide (cgrp), galanin, hypocretin, and nocicep- tin. Our study reveals that arousal behaviors are dissociable: neuropeptide expression uncoupled spontaneous activity from sensory responsiveness, and uncovered modality-specific effects upon sensory responsiveness. Principal components analysis and phenotypic clustering revealed both shared and divergent features of neuropeptidergic functions: hypocretin and cgrp stimulated spontaneous locomotor activity, whereas galanin and nociceptin attenuated these behaviors. In contrast, cart and adcyap1b enhanced sensory respon- siveness yet had minimal impacts on spontaneous activity, and cck expression induced the opposite effects. Furthermore, hypocretin and nociceptin induced modality-specific differences in responsiveness to changes in illumination. Our study provides the first systematic and high-throughput analysis of neuropeptidergic modulation of arousal, demonstrates that arousal can be partitioned into independent behavioral components, and reveals novel and conserved functions of neuropeptides in regulating arousal. Introduction Magoun, 1949; Saper et al., 2005, 2010; Pfaff and Banavar, 2007; Arousal is fundamental to life, from the vigilance required to Fuller et al., 2011). hunt prey or avoid predators, to the drive needed to obtain sus- Despite extensive studies of the arousal systems, several im- tenance and mates. Defects in arousal can be debilitating. For the portant questions remain. For example, the partitioning of ϳ15–20% of Americans with sleep disorders, approximately half arousal into individual behavioral components, including spon- exhibit insomnia, which is associated with an inability to regulate taneous locomotor activity and sensory responsiveness, has stim- physiological arousal (Mahowald and Schenk, 2005; Colten and ulated debate regarding the independence of different arousal Altevogt, 2006; Saper et al., 2010). Inappropriately elevated behaviors (Robbins, 1997; Garey et al., 2003; Pfaff, 2006; Jing et arousal is associated with stress, anxiety, and hyperactivity, al., 2009; Lebestky et al., 2009; Agmo, 2011; Van Swinderen and whereas abnormally low arousal can cause inattention, excessive Andretic, 2011; Yokogawa et al., 2012). In addition, relatively sleepiness, chronic fatigue, and vegetative states (Pfaff and Bana- little is known about how external and internal inputs interact to var, 2007; Pfaff et al., 2008; Berridge et al., 2010). Nearly a century set and maintain appropriate levels of arousal. Neuropeptides are of research has elucidated the primary arousal-promoting neu- attractive candidates to modulate these inputs (Pfaff et al., 2008; roanatomy: ascending projections from brainstem nuclei stimu- Bargmann, 2012), yet systematic and comparative interrogations late wakefulness in the brain (von Economo, 1930; Moruzzi and of neuropeptide function have been constrained by behavioral variability across experimental conditions. Received Aug. 16, 2013; revised Jan. 1, 2014; accepted Jan. 7, 2014. Larval zebrafish are especially useful for studying the molecu- Author contributions: I.G.W., D.S., and A.F.S. designed research; I.G.W., V.J.S., and H.A.C. performed research; lar and cellular control of arousal, as they possess a conserved yet I.G.W., D.S., S.Z., J.G., and E.R.S. contributed unpublished reagents/analytic tools; I.G.W. analyzed data; I.G.W. and relatively simple nervous system and display arousal-associated A.F.S. wrote the paper. behaviors similar to mammals (Prober et al., 2006; Burgess and This work was supported by a postdoctoral fellowship from the American Cancer Society (PF-07-262-01-DDC to I.G.W.), by a Helen Hay Whitney postdoctoral fellowship (to D.S.), by the McKnight Endowment Fund for Neurosci- Granato, 2007a,b; Wolman and Granato, 2012; Chiu and Prober, ence (to A.F.S.), and by the National Institutes of Health (R01HL109525 to A.F.S.). We thank Constance Richter for 2013). Moreover, their small size facilitates uniformly controlled critical reading of the manuscript. analyses of behavior across experimental manipulations. The di- The authors declare no competing financial interests. vergence between fish and other vertebrates, separated by 450 Correspondence should be addressed to either of the following: Ian G. Woods at the above address. E-mail: [email protected]; or Alexander F. Schier at the above address. E-mail: [email protected]. million years of evolution, may also be used to infer the ancestral DOI:10.1523/JNEUROSCI.3529-13.2014 regulation of arousal states (Kumar and Hedges, 1998; Garrison Copyright © 2014 the authors 0270-6474/14/333142-19$15.00/0 et al., 2012). Woods et al. • Neuropeptides and Arousal in Zebrafish J. Neurosci., February 26, 2014 • 34(9):3142–3160 • 3143 Table 1. Locomotor behavior analysis of wild-type larvae p values Day 5 Night 5 Day 6 Day 5 vs Night 5 Day 5 vs Day 6 Night 5 vs Day 6 Normal light–dark (n ϭ 80) Light Dark Light Waking activity (s/waking min) 7.78 Ϯ 0.303 1.41 Ϯ 0.048 4.28 Ϯ 0.168 Ͻ1.0E-16 2.2E-16 Ͻ1.0E-16 Rest (min/10 min) 0.19 Ϯ 0.041 1.75 Ϯ 0.131 0.51 Ϯ 0.069 Ͻ1.0E-16 4.7E-07 1.6E-14 Maximum amplitude/movement (pixels) 9.07 Ϯ 0.098 7.13 Ϯ 0.082 8.01 Ϯ 0.108 Ͻ1.0E-16 2.3E-11 1.4E-09 Movement duration (s) 0.19 Ϯ 0.002 0.20 Ϯ 0.002 0.17 Ϯ 0.002 6.0E-04 2.7E-09 6.5E-14 Movement frequency (Hz) 0.67 Ϯ 0.024 0.10 Ϯ 0.004 0.37 Ϯ 0.014 Ͻ1.0E-16 Ͻ1.0E-16 Ͻ1.0E-16 Bout duration (s) 1.17 Ϯ 0.056 0.36 Ϯ 0.004 0.89 Ϯ 0.032 Ͻ1.0E-16 1.8E-05 Ͻ1.0E-16 Bout frequency (s) 0.26 Ϯ 0.005 0.08 Ϯ 0.003 0.18 Ϯ 0.005 Ͻ1.0E-16 3.3E-16 Ͻ1.0E-16 Constant dark (n ϭ 79) Dark Dark Dark Waking activity (s/waking min) 2.18 Ϯ 0.075 1.29 Ϯ 0.050 1.69 Ϯ 0.051 2.2E-16 2.7E-07 4.7E-08 Rest (min/10 min) 1.65 Ϯ 0.129 3.01 Ϯ 0.160 1.80 Ϯ 0.125 2.7E-09 3.0E-01 9.9E-08 Maximum amplitude/movement (pixels) 9.43 Ϯ 0.209 8.03 Ϯ 0.183 9.29 Ϯ 0.209 1.1E-06 4.9E-01 1.6E-05 Movement duration (s) 0.23 Ϯ 0.003 0.20 Ϯ 0.003 0.21 Ϯ 0.003 1.6E-08 8.2E-03 4.1E-04 Movement frequency (Hz) 0.12 Ϯ 0.005 0.08 Ϯ 0.003 0.11 Ϯ 0.004 1.8E-11 2.4E-01 1.7E-09 Bout duration (s) 0.42 Ϯ 0.010 0.36 Ϯ 0.008 0.46 Ϯ 0.010 6.2E-07 1.9E-03 1.7E-13 Bout frequency (s) 0.09 Ϯ 0.003 0.06 Ϯ 0.003 0.08 Ϯ 0.003 1.1E-10 9.0E-03 1.4E-06 Constant light (n ϭ 79) Light Light Light Waking activity (s/waking min) 5.85 Ϯ 0.224 2.85 Ϯ 0.125 3.55 Ϯ 0.138 Ͻ1.0E-16 2.1E-14 7.1E-04 Rest (min/10 min) 0.35 Ϯ 0.094 0.59 Ϯ 0.127 0.39 Ϯ 0.049 1.7E-03 1.2E-05 2.1E-02 Maximum amplitude/movement (pixels) 7.94 Ϯ 0.133 6.67 Ϯ 0.119 7.11 Ϯ 0.120 5.3E-13 1.3E-07 2.0E-03 Movement duration (s) 0.18 Ϯ 0.002 0.16 Ϯ 0.002 0.17 Ϯ 0.002 6.7E-13 3.1E-05 2.3E-05 Movement frequency (Hz) 0.52 Ϯ 0.018 0.28 Ϯ 0.012 0.34 Ϯ 0.013 Ͻ1.0E-16 6.7E-13 2.4E-03 Bout duration (s) 0.91 Ϯ 0.044 0.50 Ϯ 0.018 0.72 Ϯ 0.031 Ͻ1.0E-16 3.9E-06 4.1E-14 Bout frequency (s) 0.24 Ϯ 0.007 0.17 Ϯ 0.007 0.17 Ϯ 0.005 1.4E-09 1.8E-11 5.0E-01 Values are mean Ϯ SEM p values from Kruskall–Wallis one-way ANOVA. Here we establish quantitative assays of behavioral arousal by peptide; stable transgenic lines were derived from the founders that decomposing multidimensional and complex locomotor behav- produced the strongest and most widespread expression upon heat- iors into simple parameters (Wolman and Granato, 2012). Via shock. Transgenic larvae in the behavioral analyses were distin- genetic expression of seven evolutionarily conserved neuropep- guished from their wild-type siblings by PCR.