Mol. Cells 2014; 37(4): 295-301
Published online March 21, 2014
https://doi.org/10.14348/molcells.2014.2371
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: jchoe@kaist.ac.kr
SIFamide receptor (SIFR) is a
Keywords
There are many hypotheses as to why we sleep, but there are three major functions of sleep (Mignot, 2008). First, memory consolidation during sleep occurs by synaptic downscaling (Tononi and Cirelli, 2014). Second, sleep is needed for energy conservation; body temperature in warm-blooded animals goes down during sleep to save energy (Berger and Phillips, 1995), and metabolic activity during sleep is mainly anabolic rather than catabolic (Dworak et al., 2010). Feeding and sleep are two conflicting but interrelated behaviors important for homeostasis, as sleep is suppressed by starvation both in flies and mammals (Keene et al., 2010; Macfadye et al., 1973), whereas sleep deprivation promotes feeding behaviors (Rechtschaffen and Bergmann, 2002). Lastly, vital cellular components, such as those in immune system, are recovered during sleep (Besedovsky et al., 2012). Despite essential roles of sleep in daily physiology, it remains unclear how sleep processes are maintained and regulated at the levels of genes and neural pathways.
In the course of our behavioral screens to identify novel sleep-regulatory genes and neurons, we found evidence that SIFamide (SIFa)-expressing neurons promote sleep. The SIFa family was initially isolated as a myotropic peptide from the grey fleshfly
All flies were maintained on a 12-h light-dark cycle on standard cornmeal-yeast-agar medium at 25°C and 60% humidity.
Total RNA was isolated using TRIzol (Invitrogen) from 50 adult fly heads per genotype. After DNase I treatment (Promega), 2 μg of total RNA was reverse-transcribed using TOPscript™ RT DryMIX (Enzynomics) and oligo-(dT)18 primer. Relative transcript levels of
We placed 2?5 day old female flies in 65 mm × 5 mm glass vials containing 5% sucrose and 1% agar. Sleep behavior was monitored using
For anti-SIFa antibody labeling, adult fly brains were dissected in phosphate-buffered saline (PBS) and then fixed in PBS containing 4% formaldehyde. Brains were blocked with 3% normal goat serum in PBS containing 0.3% Triton X-100 (PBS-T) and then incubated overnight at 4°C with anti-SIFa serum (a kind gift from Dr. J. A. Veenstra) diluted in the blocking solution at 1:1,000. After washing with PBS-T, brains were incubated with fluorescein isothiocyanate (FITC)- or rhodamine-conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch) diluted in PBS-T at 1:1,000. Brains were mounted in VECTASHIELD Mounting Medium (Vector Laboratories, USA) and examined by confocal microscopy (LSM710).
To test if SIFa-expressing neurons regulate sleep behaviors, we genetically ablated SIFa-expressing neurons in transgenic flies using the Gal4/UAS system (Southall et al., 2008) and recorded their daily baseline sleep in light-dark (LD) conditions. SIFa is a neuropeptide expressed in four neurons within the PI and has been shown to modulate sexual behaviors in flies (Terhzaz et al., 2007). We confirmed that SIFa peptide was not detectable in SIFa-expressing PI neurons when the apoptosis-inducing gene,
To determine whether general hyperactivity led to the short sleep phenotype in SIFa-ablated flies, we examined locomotor activity during their wake state. As shown in Fig. 1D, wake activity was decreased by the ablation of SIFa-expressing PI neurons compared to the controls (Fig. 1D,
As our genetic ablation strategy revealed a sleep-promoting role of SIFa-expressing neurons, we next investigated whether signaling through the neuropeptide SIFa is directly involved in sleep regulation. To validate this hypothesis, we depleted endogenous expression of SIFa or the SIFa receptor, SIFR, by RNA interference (RNAi) in transgenic flies and examined sleep behaviors.
An SIFa RNAi transgene has been shown to efficiently knock down SIFa expression in PI neurons (Terhzaz et al., 2007). Thus, we expressed the same SIFa RNAi by
When SIFR expression was knocked down using the panneuronal
To further localize brain regions important for SIFR function in sleep regulation, we screened various Gal4 drivers to express transgenic SIFR RNAi in different groups of neurons, including the MB, FB, antennal lobe (AL), olfactory lobe (OL), PI, and circadian-clock neurons (Fig. 4A). SIFR depletion by most of our Gal4 drivers had no significant effects on total sleep amount when compared to both Gal4/+ and UAS-SIFR RNAi/+ control flies. However, daily sleep amount was reduced by ∼3 h when we expressed SIFR RNAi using
Our study provides new evidence that the neuropeptide SIFa and its G protein-coupled receptor SIFR are novel mediators for promoting sleep in
The PI in adult fly brain is homologous to the mammalian hypothalamus, the control center for neurotransmitter regulation. Several therapeutic targets for human sleep disorders are concentrated in the hypothalamus (Mignot et al., 2002). For instance, dopaminergic neurons blocked by amphetamine-like drugs induce wake-promoting signals to cure narcolepsy (Wisor et al., 2001). Benzodiazepine compounds increase gamma-aminobutryic acid (GABA)ergic neuronal transmission to enhance sleep-promoting signals to treat insomnia (Smith, 2001). Octopamine, which is similar to mammalian norepinephrine (Roeder, 1999), has been identified as a wake-promoting molecule in
Additional genes have been identified as sleep regulators in the PI region of adult fly brain, including members of the
Mol. Cells 2014; 37(4): 295-301
Published online April 30, 2014 https://doi.org/10.14348/molcells.2014.2371
Copyright © The Korean Society for Molecular and Cellular Biology.
Sangjin Park1,3, Jun Young Sonn1,3, Yangkyun Oh1, Chunghun Lim2, and Joonho Choe1,*
1Department of Biological Sciences, College of Life Science and Bioengineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea, 2Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan 689-798, Korea
Correspondence to:*Correspondence: jchoe@kaist.ac.kr
SIFamide receptor (SIFR) is a
Keywords:
There are many hypotheses as to why we sleep, but there are three major functions of sleep (Mignot, 2008). First, memory consolidation during sleep occurs by synaptic downscaling (Tononi and Cirelli, 2014). Second, sleep is needed for energy conservation; body temperature in warm-blooded animals goes down during sleep to save energy (Berger and Phillips, 1995), and metabolic activity during sleep is mainly anabolic rather than catabolic (Dworak et al., 2010). Feeding and sleep are two conflicting but interrelated behaviors important for homeostasis, as sleep is suppressed by starvation both in flies and mammals (Keene et al., 2010; Macfadye et al., 1973), whereas sleep deprivation promotes feeding behaviors (Rechtschaffen and Bergmann, 2002). Lastly, vital cellular components, such as those in immune system, are recovered during sleep (Besedovsky et al., 2012). Despite essential roles of sleep in daily physiology, it remains unclear how sleep processes are maintained and regulated at the levels of genes and neural pathways.
In the course of our behavioral screens to identify novel sleep-regulatory genes and neurons, we found evidence that SIFamide (SIFa)-expressing neurons promote sleep. The SIFa family was initially isolated as a myotropic peptide from the grey fleshfly
All flies were maintained on a 12-h light-dark cycle on standard cornmeal-yeast-agar medium at 25°C and 60% humidity.
Total RNA was isolated using TRIzol (Invitrogen) from 50 adult fly heads per genotype. After DNase I treatment (Promega), 2 μg of total RNA was reverse-transcribed using TOPscript™ RT DryMIX (Enzynomics) and oligo-(dT)18 primer. Relative transcript levels of
We placed 2?5 day old female flies in 65 mm × 5 mm glass vials containing 5% sucrose and 1% agar. Sleep behavior was monitored using
For anti-SIFa antibody labeling, adult fly brains were dissected in phosphate-buffered saline (PBS) and then fixed in PBS containing 4% formaldehyde. Brains were blocked with 3% normal goat serum in PBS containing 0.3% Triton X-100 (PBS-T) and then incubated overnight at 4°C with anti-SIFa serum (a kind gift from Dr. J. A. Veenstra) diluted in the blocking solution at 1:1,000. After washing with PBS-T, brains were incubated with fluorescein isothiocyanate (FITC)- or rhodamine-conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch) diluted in PBS-T at 1:1,000. Brains were mounted in VECTASHIELD Mounting Medium (Vector Laboratories, USA) and examined by confocal microscopy (LSM710).
To test if SIFa-expressing neurons regulate sleep behaviors, we genetically ablated SIFa-expressing neurons in transgenic flies using the Gal4/UAS system (Southall et al., 2008) and recorded their daily baseline sleep in light-dark (LD) conditions. SIFa is a neuropeptide expressed in four neurons within the PI and has been shown to modulate sexual behaviors in flies (Terhzaz et al., 2007). We confirmed that SIFa peptide was not detectable in SIFa-expressing PI neurons when the apoptosis-inducing gene,
To determine whether general hyperactivity led to the short sleep phenotype in SIFa-ablated flies, we examined locomotor activity during their wake state. As shown in Fig. 1D, wake activity was decreased by the ablation of SIFa-expressing PI neurons compared to the controls (Fig. 1D,
As our genetic ablation strategy revealed a sleep-promoting role of SIFa-expressing neurons, we next investigated whether signaling through the neuropeptide SIFa is directly involved in sleep regulation. To validate this hypothesis, we depleted endogenous expression of SIFa or the SIFa receptor, SIFR, by RNA interference (RNAi) in transgenic flies and examined sleep behaviors.
An SIFa RNAi transgene has been shown to efficiently knock down SIFa expression in PI neurons (Terhzaz et al., 2007). Thus, we expressed the same SIFa RNAi by
When SIFR expression was knocked down using the panneuronal
To further localize brain regions important for SIFR function in sleep regulation, we screened various Gal4 drivers to express transgenic SIFR RNAi in different groups of neurons, including the MB, FB, antennal lobe (AL), olfactory lobe (OL), PI, and circadian-clock neurons (Fig. 4A). SIFR depletion by most of our Gal4 drivers had no significant effects on total sleep amount when compared to both Gal4/+ and UAS-SIFR RNAi/+ control flies. However, daily sleep amount was reduced by ∼3 h when we expressed SIFR RNAi using
Our study provides new evidence that the neuropeptide SIFa and its G protein-coupled receptor SIFR are novel mediators for promoting sleep in
The PI in adult fly brain is homologous to the mammalian hypothalamus, the control center for neurotransmitter regulation. Several therapeutic targets for human sleep disorders are concentrated in the hypothalamus (Mignot et al., 2002). For instance, dopaminergic neurons blocked by amphetamine-like drugs induce wake-promoting signals to cure narcolepsy (Wisor et al., 2001). Benzodiazepine compounds increase gamma-aminobutryic acid (GABA)ergic neuronal transmission to enhance sleep-promoting signals to treat insomnia (Smith, 2001). Octopamine, which is similar to mammalian norepinephrine (Roeder, 1999), has been identified as a wake-promoting molecule in
Additional genes have been identified as sleep regulators in the PI region of adult fly brain, including members of the
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