FMRFamide signaling promotes stress-induced sleep in Drosophila
Introduction
An increase in sleep following an immune challenge is common to many species (reviewed in (Imeri and Opp, 2009)), including humans (Mullington et al., 2000) and Drosophila (Kuo et al., 2010). Similar to mammals, the Drosophila acute sleep response to infection is dependent on the time of day of inoculation, and requires an NFκB transcription factor, Relish (Kuo et al., 2010). A recent study shows that bacterial toxins, as well as other stressors, such as osmotic shock or heat shock, induce sleep-like quiescence in adult Caenorhabditis elegans nematodes (Hill et al., 2014). Together, these findings indicate that sleep is a conserved response to stressful stimuli. Importantly, the sleep response promotes survival (Hill et al., 2014, Kuo and Williams, 2014a), indicating that it is adaptive and beneficial to the animal.
Many components of the mammalian innate immune response, particularly pro-inflammatory cytokines, exert sleep-promoting effects likely through actions in hypothalamic nuclei (Obal and Krueger, 2003). In flies, expression of Relish in the fat body, which is a major site of immune response signaling, is necessary for the sleep promoting effects of aseptic injury and immune challenge (Kuo et al., 2010) and has a role in daily night-time sleep regulation (Williams et al., 2007). We have recently demonstrated that altering neuronal excitability in the mushroom body to manipulate sleep (Joiner et al., 2006) influences survival outcome during bacterial infections (Kuo and Williams, 2014b). Thus, communication from peripheral tissue to the brain is likely a key mechanism that underlies the injury/infection-induced sleep response in Drosophila. However, the signaling molecules and neuronal circuitry that underlie these processes are unknown.
In C. elegans, the neurosecretory ALA neuron promotes the sleep-like quiescence in response to cellular stress (Hill et al., 2014). In response to heat stress, the ALA neuron depolarizes and releases neuropeptides encoded for by the gene flp-13 (Nelson et al., 2014). FLP-13 peptides are similar to Drosophila Phe-Met-Arg-Phe-amide (FMRFa) peptides. FMRFa is a member of the FMRFa related peptide family (FaRP), which in addition to FMRFa, include dromyosuppressin, drosulfakinin, neuropeptide F (NPF), and short neuropeptide F (sNPF) (Nassel and Winther, 2010). Both NPF and sNPF have sleep-regulating roles (Chen et al., 2013, He et al., 2013, Shang et al., 2013). Each of these peptide genes encodes for one or more peptides with Fmrfa encoding for eight (Schneider and Taghert, 1990). Fmrfa is expressed in the central nervous system (CNS) as well as in thoracic neurosecretory cells (Nassel and Winther, 2010). The function of fmrfa is not fully understood, but has been reported to be important for escape responses in larval (Klose et al., 2010) and adult flies (Kiss et al., 2013). The FMRFa receptor (FR; CG2114) has varying affinity for the different FMRFa peptides and is also capable of binding Dromysosuppressin (Cazzamali and Grimmelikhuijzen, 2002, Johnson et al., 2003, Meeusen et al., 2002). FR is related to the neurotensin/thyrotropin-releasing factor receptor family in mammals (Johnson et al., 2003). Based on the fact that FLP-13 peptides promote sleep-like quiescence in response to stress in C. elegans, we tested the hypothesis that FMRFa and its corresponding receptor promote the stress-induced sleep response in Drosophila.
Section snippets
Fly stocks
Flies were grown on standard dextrose-cornmeal media. Control strains included Canton-Special (CS), w1118, and yellow, white (y,w). Other strains were obtained from the Bloomington Drosophila Stock Center (Indiana University), and include Mi{ET1}FMRFaRMB04659, FMRFaRJF01879, Df(3L)BSC428, and P{SUPor-P}FMRFaKG01300. RelishE20 mutants (Hedengren et al., 1999) were isogenized to CS as previously described (Williams et al., 2007).
Behavioral assays
Locomotor activity and sleep were measured as described previously (
Heat stress induces sleep
We first determined the effects of heat stress (HS) on sleep in wild-type animals. Canton-S and w1118 wild-type flies were placed at 37 °C for 1 h (see Methods) at zeitgeber time (ZT) 18, which is 6 h into the dark phase of a 12 h:12 h light:dark cycle. This time point was chosen based on findings that infection and aseptic injury at ZT18 induce robust effects on sleep (Kuo et al., 2010). The HS flies exhibited increases in sleep relative to handled controls (HC) in the morning hours after HS, a
Discussion
Sleep is influenced by multiple environmental and behavioral factors, such as social experience (Ganguly-Fitzgerald et al., 2006), light stimuli (Harbison and Sehgal, 2009, Shang et al., 2011), copulation (Isaac et al., 2010), diet (Catterson et al., 2010, Keene et al., 2010, Linford et al., 2012, Thimgan et al., 2010, Yamazaki et al., 2012), and septic injury (Kuo et al., 2010, Shirasu-Hiza et al., 2007). Here we show a conserved response to heat stress, where similar to nematodes (Hill et
Acknowledgments
This work was supported by NIH Grants #R21NS078582 to J.A.W. and #R01NS064030 to D.M.R. We thank Ewa Strus for technical support.
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These authors contributed equally to this work.
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Present address: Department of Neurology, Changzheng Hospital of Second Military Medical University, Shanghai 200433, China.