SUMMARY

GABAergic signalling is important for normal sleep in humans and flies. Here we advance the current understanding of GABAergic modulation of daily sleep patterns by focusing on the role of slow metabotropic GABAB receptors in the fruit fly Drosophila melanogaster. We asked whether GABAB-R2 receptors are regulatory elements in sleep regulation in addition to the already identified fast ionotropic Rdl GABAA receptors. By immunocytochemical and reporter-based techniques we show that the pigment dispersing factor (PDF)-positive ventrolateral clock neurons (LNv) express GABAB-R2 receptors. Downregulation of GABAB-R2 receptors in the large PDF neurons (l-LNv) by RNAi reduced sleep maintenance in the second half of the night, whereas sleep latency at the beginning of the night that was previously shown to depend on ionotropic Rdl GABAA receptors remained unaltered. Our results confirm the role of the l-LNv neurons as an important part of the sleep circuit in D. melanogaster and also identify the GABAB-R2 receptors as the thus far missing component in GABA-signalling that is essential for sleep maintenance. Despite the significant effects on sleep, we did not observe any changes in circadian behaviour in flies with downregulated GABAB-R2 receptors, indicating that the regulation of sleep maintenance via l-LNv neurons is independent of their function in the circadian clock circuit.

INTRODUCTION

The fruit fly Drosophila melanogaster has become a well-accepted model for sleep research (reviewed by Cirelli, 2009; Minot et al., 2011). As in mammals, it has been shown that the sleep-like state of Drosophila is associated with reduced sensory responsiveness and reduced brain activity (Nitz et al., 2002; van Swinderen et al., 2004), and is subject to both circadian and homeostatic regulation (Hendricks et al., 2000; Shaw et al., 2000). Similarly to in humans, monaminergic neurons (specifically dopaminergic and octopaminergic neurons) enhance arousal in fruit flies (Andretic et al., 2005; Kume et al., 2005; Lebestky et al., 2009; Crocker et al., 2010), whereas GABAergic neurons promote sleep (Agosto et al., 2008). As in humans, GABA advances sleep onset (reduces sleep latency) and prolongs total sleep (increases sleep maintenance) (Agosto et al., 2008). Brain regions possibly implicated in the regulation of sleep in D. melanogaster are the pars intercerebralis (Foltenyi et al., 2007; Crocker et al., 2010), the mushroom bodies (Joiner et al., 2006; Pitman et al., 2006; Yuan et al., 2006) and a subgroup of the pigment dispersing factor (PDF)-positive neurons called the l-LNv neurons (Parisky et al., 2008; Sheeba et al., 2008a; Chung et al., 2009; Lebestky et al., 2009; Shang et al., 2011). The l-LNv belong to the circadian clock neurons, indicating that in flies, as in mammals, the sleep circuit is intimately linked to the circadian clock and that the mechanisms employed to govern sleep in the brain are evolutionarily ancient.

The l-LNv are conspicuous clock neurons with wide arborisations in the optic lobe, fibres in the accessory medulla – the insect clock centre – and connections between the brain hemispheres (Helfrich-Förster et al., 2007a). Thus, the l-LNv neurons are anatomically well suited to modulate the activity of many neurons. In addition, their arborisations overlap with those of monaminergic neurons (Hamasaka and Nässel, 2006). Several studies show that they indeed receive dopaminergic, octopaminergic and GABAergic input and that they control the flies' arousal and sleep (Agosto et al., 2008; Parisky et al., 2008; Kula-Eversole et al., 2010; Shang et al., 2011). Furthermore, the l-LNv are directly light sensitive and promote arousal and activity in response to light, especially in the morning (Shang et al., 2008; Sheeba et al., 2008a; Sheeba et al., 2008b; Fogle et al., 2011).

A part of the sleep-promoting effect of GABA on the l-LNv has been shown to be mediated via the fast ionotropic GABAA receptor Rdl (Resistance to dieldrin) (Agosto et al., 2008). Rdl Cl channels are expressed in the l-LNv (Agosto et al., 2008) and, similar to mammalian GABAA receptors, they mediate fast inhibitory neurotransmission (Lee et al., 2003). As expected, GABA application reduced the action potential firing rate in the l-LNv, whereas application of picrotoxin, a GABAA receptor antagonist, increased it (McCarthy et al., 2011). Furthermore, an Rdl receptor mutant with prolonged channel opening and consequently increased channel current significantly decreased sleep latency of the flies after lights-off, whereas the downregulation of the Rdl receptor via RNAi increased it (Agosto et al., 2008).

Nevertheless, the manipulation of the Rdl receptor had no effect on sleep maintenance. Because the latter is significantly reduced after silencing the GABAergic neurons (Parisky et al., 2008), other GABA receptors must be responsible for maintaining sleep. Suitable candidates are slow metabotropic GABAB receptors that are often co-localised with ionotropic GABAA receptors (Enell et al., 2007). In Drosophila, like in mammals, the metabotropic GABAB receptors are G-protein-coupled seven-transmembrane proteins composed of two subunits, GABAB-R1 and GABAB-R2 (Kaupmann et al., 1998; Mezler et al., 2001). The GABAB-R1 is the ligand binding unit and GABAB-R2 is required for translocation to the cell membrane and for stronger coupling to the G-protein (Kaupmann et al., 1998; Galvez et al., 2001). In this study we show that the l-LNv do express metabotropic GABAB-R2 receptors and that these receptors are relevant for sleep maintenance but not for sleep latency. Thus, metabotropic and ionotropic GABA receptors are cooperating in sleep regulation.

MATERIALS AND METHODS

Fly strains and rearing

Oregon R was used as a wild-type strain for GABAB-R2, PDF and GAD1 immunohistochemistry. For visualizing GABAB-R2 receptors we also used a GABAB-R2-GAL4 line (Root et al., 2008) (kindly provided by Jing Wang, University of California, San Diego) to express green fluorescent protein (GFP) with the binary UAS-GAL4 system (using UAS-s65tGFP, stock 1522, Bloomington Stock Center, Bloomington, IN, USA). The specificity of the GABAB-R2-GAL4 line for GABAB-R2-expressing neurons has been demonstrated previously for the adult olfactory sensory neurons (OSNs) (Root et al., 2008) and seems to be correct also for most the other labelled neurons, judging from a wide overlap between immunostaining with a GABAB-R2 antiserum and GABAB-R2-GAL4-driven GFP (Hamasaka et al., 2005). In order to downregulate the GABAB-R2 receptor specifically in the PDF neurons (s-LNv and l-LNv), we used Pdf-GAL4 (Park et al., 2000) to either express UAS-GABAB-R2-RNAi (Root et al., 2008) (provided by Jing Wang) alone, or to simultaneously express UAS-GABAB-R2-RNAi and UAS-Dicer2 (no. 60012, Vienna Drosophila RNAi Center, Wien, Austria). In the first experiment, the Pdf-GAL4 driver and UAS-GABAB-R2-RNAi effector lines crossed to white1118 were taken as controls, and in the second experiment, the Pdf-GAL4 driver and UAS-GABAB-R2-RNAi effector lines crossed to UAS-Dicer2 were taken as controls. In a second set of experiments, we drove an independent UAS-Trip-GABAB-R2-RNAi line (no. 27699; Bloomington Stock Center) together with dicer2 under control of Pdf-GAL4 in order to downregulate GABAB-R2. All flies were raised on Drosophila food (0.8% agar, 2.2% sugar-beet syrup, 8.0% malt extract, 1.8% yeast, 1.0% soy flour, 8.0% corn flour and 0.3% hydroxybenzoic acid) at 25°C under a 12 h:12 h light:dark (LD) cycle and transferred to 20°C at an age of ~3 days.

Immunohistochemistry

For PDF and GFP co-labelling, whole-mount brains were dissected in PBS-TX (0.01 mol l−1 phosphate-buffered saline with 0.5% Triton X-100, pH 7.2) before fixing them in ice-cold 4% paraformaldehyde (in 0.1 mol l−1 phosphate buffer, pH 7.2) for 1 h. For GABAB-R2 immunolabelling, entire fly heads were fixed for 3 h prior to brain dissection. The fixed and dissected brains were washed in PBS-TX, blocked for 2 h in 5% normal goat serum (NGS, in PBS-TX) and then incubated with primary antibodies at 4°C overnight or for 48 h. Primary antibodies were used in the following concentrations in PBS-TX with 5% NGS: anti-PDF 1:2500 and anti-GABAB-R2 1:10,000 (for description of primary antibodies, see below). After washing several times in PBS-TX, the brains were incubated in secondary fluorochrome-conjugated antibodies in PBS-TX with 5% NGS either overnight at 4°C or for 2 h at room temperature. We used Alexa Fluor antibodies (Molecular Probes, Carlsbad, CA, USA) of 488 nm (goat anti-mouse) or 546 nm (goat anti-rabbit) and Cy2- or Cy3-tagged IgGs (goat anti-mouse or anti-rabbit; Jackson ImmunoResearch, West Grove, PA, USA) at a dilution of 1:1000. After incubation with the secondary antibodies, brains were washed in PB-TX and subsequently mounted in Vectashield medium (Vector Laboratories, Burlingame, CA, USA) on glass slides, all in the same orientation.

GABAB-R2 antiserum

Production and characterization of antiserum (code B7873/3) to GABAB-R2 was described previously (Hamasaka et al., 2005). In western blots of brain tissue from Drosophila the antiserum detected a band of appropriate size (Hamasaka et al., 2005), and in GABAB-R2 immunohistochemistry on Drosophila brains the antiserum labelled neurons that were in close proximity to neural elements immunopositive for a vesicular GABA transporter, GAD and a GABAA receptor (RDL), strongly indicating that the GABAB-R2 antiserum is reliably detecting a GABAB receptor (Enell et al., 2007). The anti-GABAB-R2 specificity was further established by diminished GABAB-R2 immunolabelling after GABAB-R2 RNAi in specific neurons (OSNs) in the antenna/antennal lobe (Root et al., 2008). Furthermore, physiological/pharmacological evidence (using Ca2+ imaging) for GABAB-R2 expression has been produced for the larval s-LNv and the adult OSN (Hamasaka et al., 2005; Root et al., 2008). Recently, physiological evidence for metabotropic GABA signalling was also provided for the adult s-LNv using cAMP imaging (Lelito and Shafer, 2012).

PDF antiserum

The mouse anti-PDF was provided by Justin Blau (obtained from the Developmental Studies Hybridoma Bank).

Microscopy and image analysis

We performed laser scanning confocal microscopy (Leica TCS SPE, Leica, Wetzlar, Germany) to analyse immunofluorescent brains. To avoid bleeding through, we used sequential scanning (laser lines 488 and 532 nm). Confocal stacks of 1.5 μm thickness were acquired. For quantifying intensity of GABAB-R2 immunostaining, the settings of the confocal microscope (laser power, gain and contrast) were kept the same for all preparations. The staining intensity of GABAB-R2 labelling was then quantified on single confocal images by two different methods. To compare labelling intensity between GABAB-R2 immunostaining and GABAB-R2 GFP reporter staining, we coded the images and judged by eye whether the intensity in each single PDF-positive neuron was strong, weak or completely absent. Subsequently, we calculated the percentage of l-LNv and s-LNv with strong, weak and no staining (Fig. 1J,K). To judge the degree of GABAB-R2 downregulation in the l-LNv and s-LNv by RNAi, entire confocal stacks were imported into ImageJ (Fiji distribution; http://fiji.sc/wiki/index.php/Fiji or http://rsb.info.nih.gov/ij/). Individual somata of l-LNv and s-LNv were selected using the magic wand selection tool on single confocal stacks. The average staining intensity of the selected area was calculated (grey value 0: no staining; grey value 255: maximal staining), and the same was done for the neighbouring area outside the neurons to obtain the staining intensity of the background. The background staining was subtracted from the value gained for the soma. An average staining intensity was calculated for all l-LNv and s-LNv of one brain hemisphere, and finally the values of all 11–13 brain hemispheres were averaged. After testing for normal distribution, a one-way ANOVA was used to test for significant staining differences between control flies and flies in which GABAB-R2 was downregulated.

Fig. 1.

The PDF-positive lateral neurons (l-LNv and s-LNv) express the GABAB receptor 2 (GABAB-R2). (A–I) All images are projections of ~10 confocal stacks of the anterior region of the right brain hemispheres of whole-mount brains. GABA-receptor-expressing neurons are depicted in green and the PDF-positive lateral neurons in magenta. The right column shows overlays of both labelling. Magnification is the same in all pictures (scale bar in C, 20 μm). (A–F) Neurons expressing GABAB-R2 visualized by green fluorescent protein (GFP) (in a GABAB-R2-GAL4;UAS-gfp line). Strong expression is found in the accessory medulla (aMe), in one to three l-LNv (A,D) and in few non-LNv neurons (asterisk in A). The s-LNv are only weakly marked or not at all. (G–I) Neurons expressing GABAB-R2 visualised by the GABAB-R2-antiserum. Both the l-LNv and the s-LNv show punctate staining, which is more pronounced in the s-LNv (G). (J,K) Judgement of the GABAB-R2 staining intensity (no, weak, strong) in the l-LNv and the s-LNv. Overall, GFP staining was rated in 22 brain hemispheres (85 l-LNv and 73 s-LNv) and GABAB-R2-antibody staining in 11 brain hemispheres (43 l-LNv and 44 s-LNv).

Fig. 1.

The PDF-positive lateral neurons (l-LNv and s-LNv) express the GABAB receptor 2 (GABAB-R2). (A–I) All images are projections of ~10 confocal stacks of the anterior region of the right brain hemispheres of whole-mount brains. GABA-receptor-expressing neurons are depicted in green and the PDF-positive lateral neurons in magenta. The right column shows overlays of both labelling. Magnification is the same in all pictures (scale bar in C, 20 μm). (A–F) Neurons expressing GABAB-R2 visualized by green fluorescent protein (GFP) (in a GABAB-R2-GAL4;UAS-gfp line). Strong expression is found in the accessory medulla (aMe), in one to three l-LNv (A,D) and in few non-LNv neurons (asterisk in A). The s-LNv are only weakly marked or not at all. (G–I) Neurons expressing GABAB-R2 visualised by the GABAB-R2-antiserum. Both the l-LNv and the s-LNv show punctate staining, which is more pronounced in the s-LNv (G). (J,K) Judgement of the GABAB-R2 staining intensity (no, weak, strong) in the l-LNv and the s-LNv. Overall, GFP staining was rated in 22 brain hemispheres (85 l-LNv and 73 s-LNv) and GABAB-R2-antibody staining in 11 brain hemispheres (43 l-LNv and 44 s-LNv).

Locomotor activity recording and activity analysis

The locomotor activity of single male control flies and flies with downregulated GABAB-R2 was recorded using the TriKinetics DAM2 System (TriKinetics, Waltham, MA, USA). Monitors were put in light-tight boxes (recording units) with white light LED illumination of 47.6 μW cm−2 (Lumitronix LED-Rechnik GmbH, Jungingen, Germany) during the light phase. These recording units were placed in a climate chamber with a constant temperature of 20±0.5°C and 60±1% relative humidity. Flies were exposed to 12 h:12 h LD cycles for 11 days and subsequently kept in constant darkness (DD) for another 10 days. Only male flies with an age of 3 to 6 days were taken for the experiments. Flies that died during the experiment were excluded from the calculations.

Raw data actograms were created using ActogramJ for ImageJ (Schmid et al., 2011). The average activity levels of all individual flies were calculated for the entire LD and DD period. To do so, the beam crosses per minute from days 2–11 (LD) and days 12–21 (DD) were first averaged for each single fly and then the activity values of all flies of one genotype were averaged. This calculation was possible during DD, because the periods of the individual flies were close to 24 h. During the LD period, average activity levels were additionally determined during the first and second halves of the day and the night, respectively, to be compared with the sleep values during the same time periods (see below). To see whether GABAB-R2 downregulation affects the speed of the circadian clock, we determined the free-running periods of all individual flies during days 12 to 21 in DD by periodogram analysis (Sokolove and Bushell, 1978) combined with a chi-square test with a 5% significance level (Schmid et al., 2011).

Sleep analysis

The sleep analysis was only performed during the LD cycles (from day 2 to day 11). Calculations of total sleep were performed for each hour of the day using a macro written in Microsoft Excel 2007. Average sleep bout durations were calculated for 6 h intervals using the very same macro. In previous studies sleep was defined as period of inactivity longer than 5 min (Hendricks et al., 2000; Shaw et al., 2000; Andretic and Shaw, 2005; Ho and Sehgal, 2005). At first we used the same criterion, but then we switched to a more stringent one (a period of inactivity longer than 10 min as definition of sleep) in order to make very sure that periods of inactivity that resemble rest rather than sleep are not included. An average sleeping curve was calculated for each genotype out of the hourly sleep duration determined for each individual fly. Furthermore, average total sleep and average sleep bout duration were calculated in 6-h intervals for each genotype for the first and second halves of the day and night, respectively. Sleep latency after lights-off was determined as the time in minutes after lights off until the first sleep bout of at least 10 min occurred. To do so, we imported the TriKinetics file into Excel and searched manually for the first occurrence of 10 zeros in a row after lights-off for each individual fly.

Fig. 2.

Downregulation of GABAB-R2 in the l-LNv increases the activity level in light:dark (LD) cycles and constant darkness (DD) but does not affect circadian rhythmicity. (A) GABAB-R2 immunolabelling is significantly reduced in the l-LNv (ANOVA: F1,21=7.409, P=0.0128), but not in the s-LNv (ANOVA: F1,21=0.292, P=0.5947) of UAS-Dicer2;Pdf-GAL4/UAS-GABAB-R2-RNAi flies (RNAi) as compared with UAS-Dicer2;UAS-GABAB-R2-RNAi flies (Control 1). (B) Flies with downregulated GABAB-R2 (RNAi) significantly increase their activity level in LD and DD as compared with Control 1 and Control 2 (UAS-Dicer2;Pdf-GAL4/+) flies. (C) Typical actograms of individual control flies and a fly with downregulated GABAB-R2 (RNAi). The black/white bars on top of the actograms indicate the light:dark cycle during LD. ANOVA followed by a post hoc test showed that period length (D) depended on the strain (ANOVA; power: F1,141=4.587, P=0.0118) and was significantly shorter in Control 2 flies than in Control 1 and RNAi flies. Nevertheless, the percentage of rhythmic flies and the power (E) of the free-running rhythm was independent of fly strain (ANOVA; power: F1,141=2.678, P=0.0722). Numbers in the columns indicate the number of tested flies; asterisks indicate significant differences. Error bars represent s.e.m.

Fig. 2.

Downregulation of GABAB-R2 in the l-LNv increases the activity level in light:dark (LD) cycles and constant darkness (DD) but does not affect circadian rhythmicity. (A) GABAB-R2 immunolabelling is significantly reduced in the l-LNv (ANOVA: F1,21=7.409, P=0.0128), but not in the s-LNv (ANOVA: F1,21=0.292, P=0.5947) of UAS-Dicer2;Pdf-GAL4/UAS-GABAB-R2-RNAi flies (RNAi) as compared with UAS-Dicer2;UAS-GABAB-R2-RNAi flies (Control 1). (B) Flies with downregulated GABAB-R2 (RNAi) significantly increase their activity level in LD and DD as compared with Control 1 and Control 2 (UAS-Dicer2;Pdf-GAL4/+) flies. (C) Typical actograms of individual control flies and a fly with downregulated GABAB-R2 (RNAi). The black/white bars on top of the actograms indicate the light:dark cycle during LD. ANOVA followed by a post hoc test showed that period length (D) depended on the strain (ANOVA; power: F1,141=4.587, P=0.0118) and was significantly shorter in Control 2 flies than in Control 1 and RNAi flies. Nevertheless, the percentage of rhythmic flies and the power (E) of the free-running rhythm was independent of fly strain (ANOVA; power: F1,141=2.678, P=0.0722). Numbers in the columns indicate the number of tested flies; asterisks indicate significant differences. Error bars represent s.e.m.

RESULTS

Previous studies have shown that there are large numbers of GABA-producing neurons in the adult brain of Drosophila (Enell et al., 2007; Okada et al., 2009). The processes of some of these arborise in the region of the accessory medulla that contains presumed dendrites of the PDF neurons (Hamasaka et al., 2005). Thus GABAergic neurons may converge on PDF neurons.

GABAB-R2 receptors are located on the PDF cells

To identify whether PDF neurons express metabotropic GABAB receptors, we used two different markers: we expressed GFP with a GABAB-R2-specific GAL4 driver line (Root et al., 2008) and we used an antiserum raised against the GABAB-R2 protein (Hamasaka et al., 2005). In both cases we co-stained with anti-PDF to judge whether the two signals overlap (Fig. 1A–I). We found that the PDF-positive l-LNv neurons are reliably marked by both methods. GABAB-R2-GAL4-driven GFP was present in 98.8% of the l-LNv labelled by anti-PDF (Fig. 1J) and in 100% of the l-LNv that were labelled with the GABAB-R2 antiserum (Fig. 1K). Whereas the GABAB-R2-antibody labelling was similarly strong in all four l-LNv neurons (Fig. 1G), GFP was strongly expressed in only two to three of the four l-LNv neurons and weak in the remaining one to two cells (Fig. 1A,D). The PDF-positive s-LNv were not reliably stained by GABAB-R2-GAL4-driven GFP (Fig. 1A): only 39.7% of the s-LNv showed a GFP signal at all and just 2.7% of them revealed a strong signal (Fig. 1J). Nevertheless, the GABAB-R2 antibody revealed the s-LNv reliably (Fig. 1G,K): 100% of the s-LNv showed a prominent punctuate staining. We conclude that the GABAB-R2 receptor is expressed on all PDF neurons (the l-LNv and the s-LNv), but that GABAB-R2-GAL4 drives only weakly in the s-LNv.

Downregulation of GABAB-R2 receptors in PDF neurons increases the activity level but has little effect on free-running period

To investigate whether GABAB receptors on the PDF neurons might be relevant for locomotor activity rhythms and the general activity level, we downregulated the receptors by expressing two different constructs of UAS-RNAi for GABAB-R2 (with or without dicer2) in the PDF neurons (using Pdf-GAL4). First, we measured the intensity of immunolabelling in the PDF neurons in comparison to control flies (that carried only the RNAi construct). We observed a significant reduction in staining intensity only for one of the RNAi constructs that had previously been employed successfully (Root et al., 2008), and only if it was combined with dicer2. Furthermore, the knock-down was only significant in the l-LNv but not in the s-LNv neurons (Fig. 2A). The latter observation is in agreement with our findings that GABAB-R2 antibody staining revealed stronger signals in the s-LNv than in the l-LNv and that Pdf-GAL4 usually drives less strongly in the s-LNv as compared with the l-LNv (Renn et al., 1999).

After we had made sure that the GABAB-R2 downregulation is preferentially working in the l-LNv, we monitored locomotor activity of the flies for 11 days in 12 h:12 h LD followed by 10 days of DD, both at 20°C. We found that the flies with significantly downregulated GABAB-R2 in the l-LNv were significantly more active than the control flies, and this was true during both LD and DD (Fig. 2B,C). No significant differences in the activity level were present between controls and RNAi flies in the two lines, in which no GABAB-R2 knock-down was detectable by antibody staining (data not shown). The activity pattern of the flies was in principle similar for all strains, showing bimodal patterns with activity bouts in the morning and evening under LD conditions and more unimodal patterns under DD conditions. GABAB-R2 downregulation had no effect on the activity rhythms under DD conditions; the great majority of flies (86–100%) were rhythmic, free-ran with a period of approximately 24 h (Fig. 2D), which was of wild-type-like power (Fig. 2E).

Downregulation of GABAB-R2 receptors in PDF neurons reduces sleep maintenance

Next, we analysed the effects of GABAB-R2 downregulation on sleep. Again, we did not see any significant differences in sleep between controls and RNAi flies of the two lines, in which the GABAB-R2 knock-down appeared inefficient (data not shown). But, we found that the flies with significantly downregulated GABAB-R2 receptors in the l-LNv showed a largely reduced amount of night-time sleep during LD (Fig. 3). This difference was mainly due to a reduced amount of sleep in the second half of the night (Fig. 3A). During the 6-h interval in the late night, the experimental flies showed 30 min less sleep in total as compared with the controls (Fig. 3C). Simultaneously, they decreased their average sleep bout duration significantly (to 108 min in the 6-h interval, in contrast to 135 and 160 min, respectively, of the control strains) (Fig. 3D). Obviously, the flies with downregulated GABAB-R2 receptors were clearly unable to maintain sleep in the second half of the night. They woke up early and displayed a high amount of locomotor activity. This is also clearly visible in the actograms of the flies (Fig. 2C).

No effects on sleep were visible during the day or the first half of the night. Downregulation of GABAB-R2 receptors in the l-LNv did also not increase sleep latency. After lights-off, the experimental flies fell asleep as fast as flies of Control 2 (UAS-dicer2;Pdf-GAL4/+) (Fig. 3B). Just the flies of Control 1 (UAS-dicer2;UAS-GABAB-R2-RNAi) fell asleep faster.

As stated above, the expression of the GABAB-R2 RNAi in the PDF neurons also influenced the overall activity level of the flies, but in contrast to sleep, activity level was increased throughout the entire 24-h day (all four 6-h intervals; Fig. 3E). Thus the regulation of the overall activity level cannot be by the same mechanism as sleep regulation.

DISCUSSION

Here we show that metabotropic GABAB-R2 receptors are expressed on the PDF-positive clock neurons (LNv neurons), and that their downregulation in the l-LNv by RNAi results in: (1) a higher activity level throughout the day and night and (2) reduced sleep maintenance in the second half of the night. Neither sleep onset nor circadian rhythm parameters were affected by the downregulation. We conclude that GABA signalling via metabotropic receptors on the l-LNv is essential for sustaining sleep throughout the night and for keeping activity at moderate levels throughout the 24-h day (preventing flies from hyperactivity). A major caveat of RNAi is off-target effects, particularly when Gal4 drivers are expressed in large numbers of non-target neurons. Though we have downregulated GABAB-R2 in only eight neurons per brain hemisphere and were careful to correlate the behavioural effects of our knockdown experiments with observation and measures of GABAB-R2 immunostaining in the s-LNv and l-LNv, it is still possible that some effects were due to off-target knockdown of other membrane proteins. Nevertheless, given the fact that no such effects have been reported in the previous paper that used the same GABAB-R2 RNAi line (Root et al., 2008), we think it is unlikely that the behavioural effects described here were due to off-target knockdown of other genes.

Fig. 3.

Downregulation of GABAB-R2 in the l-LNv decreases sleep maintenance in the second half of the night. (A) Average sleep curves during the 10 days of LD shown in Fig. 2C for flies with GABAB-R2 downregulated (RNAi) and the two controls (fly strains and number of flies are as in Fig. 2). The arrow points to the reduced sleep of the RNAi flies during the second half of the night. (B) Sleep latency after lights-off is unusually short in flies of Control 1, but not different between RNAi flies and flies of Control 2. (C–E) The 24-h day is divided in 6-h intervals (broken lines) to show mean total sleep (C), mean sleep bout duration (D) and mean activity (E) during these intervals. ANOVA followed by a post hoc test revealed that downregulation of GABAB-R2 affects the sleep parameters during the last 6-h interval (second half of the night), whereas it increases activity at all times. Significant differences are marked by asterisks (*P<0.05; **P<0.0005). Error bars represent s.e.m.

Fig. 3.

Downregulation of GABAB-R2 in the l-LNv decreases sleep maintenance in the second half of the night. (A) Average sleep curves during the 10 days of LD shown in Fig. 2C for flies with GABAB-R2 downregulated (RNAi) and the two controls (fly strains and number of flies are as in Fig. 2). The arrow points to the reduced sleep of the RNAi flies during the second half of the night. (B) Sleep latency after lights-off is unusually short in flies of Control 1, but not different between RNAi flies and flies of Control 2. (C–E) The 24-h day is divided in 6-h intervals (broken lines) to show mean total sleep (C), mean sleep bout duration (D) and mean activity (E) during these intervals. ANOVA followed by a post hoc test revealed that downregulation of GABAB-R2 affects the sleep parameters during the last 6-h interval (second half of the night), whereas it increases activity at all times. Significant differences are marked by asterisks (*P<0.05; **P<0.0005). Error bars represent s.e.m.

Our results are in line with a former study describing the location of GABAB receptors in D. melanogaster (Hamasaka et al., 2005). The ionotropic GABAA receptor Rdl has also been identified on the l-LNv neurons and has been shown to regulate sleep, but its downregulation delayed only sleep onset and did not perturb sleep maintenance (Parisky et al., 2008). In contrast, silencing GABAergic signalling influenced sleep onset and sleep maintenance, indicating that GABA works through the fast Rdl receptor, and also implying a longer-lasting signalling pathway. GABAB receptors are perfect candidates in mediating slow but longer-lasting effects of GABA. Often, GABAA and GABAB receptors cooperate in mediating such fast and slow effects. For example, in the olfactory system, GABAA receptors mediate the primary modulatory responses to odours whereas GABAB receptors are responsible for long-lasting effects (Wilson and Laurent, 2005).

In D. melanogaster, GABAB receptors consist of the two subunits GABAB-R1 and GABAB-R2, and only the two units together can efficiently activate the metabotropic GABA signalling cascade (Galvez et al., 2001; Mezler et al., 2001). In our experiments, we downregulated only GABAB-R2, but this manipulation should also have decreased the amount of functional GABAB-R1/GABAB-R2 heterodimers and, therefore, reduced GABAB signalling in general. Taking into account that sleep maintenance in the second half of the night was already significantly impaired by an ~46% reduction in detectable GABAB-R2 immunostaining intensity in the l-LNv clock neurons, it can be assumed that GABAB receptors account for an even larger portion of the sleep maintenance than detected in our experiments. Thus GABAB receptors play a crucial role in mediating GABAergic signals to the l-LNv neurons, which are needed to sustain sleep throughout the night. This is mainly due to the maintenance of extended sleep bout durations in the second half of the night. When signalling by the GABAB receptor is reduced, sleep bouts during this interval are significantly shortened, leading to less total sleep.

Most importantly, we confirm the l-LNv as important components in regulation of sleep and arousal (Agosto et al., 2008; Parisky et al., 2008; Kula-Eversole et al., 2010; Shang et al., 2011). In contrast, the s-LNv seem to be not involved in sleep–arousal regulation but are rather important for maintaining circadian rhythmicity under DD (reviewed by Helfrich-Förster et al., 2007b). One caveat in clearly distinguishing the function of s-LNv and l-LNv is the fact that both cell clusters express the neuropeptide PDF and, as a consequence, Pdf-GAL4 drives expression in both subsets of clock neurons. Though we did not see a significant GABAB-R2 knock-down in the s-LNv, we cannot completely exclude that GABAB-R2 was slightly downregulated in these clock neurons and that this knock-down contributed to the observed alterations in sleep. To restrict the knock-down to the s-LNv we used the R6-GAL4 line that is expressed in the s-LNv but not in the l-LNv (Helfrich-Förster et al., 2007a). We observed neither a reduction in GABAB-R2 staining intensity in the s-LNv nor any effects on sleep in the second half of the night (F.G. and C.H.-F., unpublished observations). The lack of any visible GABAB-R2 downregulation in the s-LNv with R6-GAL4 is in agreement with observations of Shafer and Taghert (Shafer and Taghert, 2009), who could completely downregulate PDF in the s-LNv using Pdf-GAL4 but not using R6-GAL4. Thus, R6-GAL4 is a weaker driver than Pdf-GAL4 and is obviously not able to influence GABAB-R2 in the s-LNv. Nevertheless, in our experiments the R6-Gal4-driven GABAB-R2 RNAi led to flies that had slightly higher diurnal activity levels and less diurnal rest than the control flies (F.G. and C.H.-F., unpublished observations; supplementary material Fig. S1). This suggests that GABAB-R2 was downregulated somewhere else. When checking the R6-GAL4 expression more carefully we found that it was not restricted to the brain, but was also present in many cells of the thoracic and especially the abdominal ganglia (F.G. and C.H.-F., unpublished observations). Given the broad expression of GABAB-R2, a putative knock-down in the ventral nervous system is likely to affect locomotor activity.

Our results on the l-LNv certainly do not exclude a role of GABA in the circadian clock controlling activity rhythms under DD conditions (here represented by the s-LNv). In mammals, GABA is the most abundant neurotransmitter in the circadian clock centre in the brain – the suprachiasmatic nucleus (van den Pol and Tsujimoto, 1985). GABA interacts with GABAA and GABAB receptors, producing primarily but not exclusively inhibitory responses through membrane hyperpolarisation (Wagner et al., 1997; Choi et al., 2008). GABA signalling is important for maintaining behavioural circadian rhythmicity, it affects the amplitude of molecular oscillations and might contribute to synchronisation of clock cells within the suprachiasmatic nucleus (Liu and Reppert, 2000; Albus et al., 2005; Aton et al., 2006; Ehlen et al., 2006). The same seems to be true for fruit flies. The s-LNv neurons of adults alter cAMP levels upon GABA application on isolated brains in vitro (Lelito and Shafer, 2012). Hyperexcitation of GABAergic neurons disrupts the molecular rhythms in the s-LNv and renders the flies arrhythmic (Dahdal et al., 2010). Thus, GABA signalling affects the circadian clock in the s-LNv. We found that flies with downregulated GABAB-R2 receptors had slightly longer free-running periods than the control flies, but this turned out to be only significant in comparison with Control 2 and not to Control 1 (Fig. 2D). Dahdal et al. (Dahdal et al., 2010) found similar small effects on period after downregulating GABAB-R2 receptors, but a significant period lengthening after downregulating GABAB-R3 receptors. This indicates that GABA signals via GABAB-R3 receptors to the s-LNv and was confirmed in vitro in the larval Drosophila brain by Ca2+ imaging (Dahdal et al., 2010). Nevertheless, the study of Dahdal et al. (Dahdal et al., 2010) does not rule out that GABA signals via GABAB-R3 plus GABAB-R2 receptors on the adult s-LNv. We found a rather strong expression of GABAB-R2 receptors in these clock neurons, and were not able to downregulate it significantly by RNAi, although we used dicer2 as amplification. Dahdal et al. (Dahdal et al., 2010) did not use dicer2, and they also did not measure the effectiveness of the downregulation of GABAB-R2 by RNAi immunocytochemically directly in the s-LNv. Thus, the exact GABAB receptors that mediate GABA responses in the adult s-LNv need still to be determined.

In summary, we conclude that the l-LNv subgroup of the PDF-positive clock neurons is a principal target of sleep-promoting and activity-repressing GABAergic neurons and sits at the heart of the sleep circuit in D. melanogaster. Thus, the sleep circuitry of flies is clearly more circumscribed and simpler than that of mammals. Mammals have many targets of sleep-promoting GABAergic neurons, and the circadian clock seems to have a mainly modulatory and less direct influence on sleep (Mistlberger, 2005). The fly sleep circuitry may therefore have condensed the mammalian arousal and sleep stimulating systems (e.g. monaminergic, cholinergic, peptidergic and GABAergic systems) into a simpler and more compact region, which seems to largely coincide with the eight PDF-positive l-LNv cells of the circadian circuit.

Acknowledgements

We thank Justin Blau, Jae Park and Jim Wang for supplying fly strains and antibodies.

FOOTNOTES

FUNDING

This research was supported by the German Research Foundation (DFG; SFB581, TP28; Fo207/14-1) and The Swedish Research Council (621-2007-6500).

REFERENCES

Agosto
J.
,
Choi
J. C.
,
Parisky
K. M.
,
Stilwell
G.
,
Rosbash
M.
,
Griffith
L. C.
(
2008
).
Modulation of GABAA receptor desensitization uncouples sleep onset and maintenance in Drosophila
.
Nat. Neurosci.
11
,
354
-
359
.
Albus
H.
,
Vansteensel
M. J.
,
Michel
S.
,
Block
G. D.
,
Meijer
J. H.
(
2005
).
A GABAergic mechanism is necessary for coupling dissociable ventral and dorsal regional oscillators within the circadian clock
.
Curr. Biol.
15
,
886
-
893
.
Andretic
R.
,
Shaw
P. J.
(
2005
).
Essentials of sleep recordings in Drosophila: moving beyond sleep time
.
Methods Enzymol.
393
,
759
-
772
.
Andretic
R.
,
van Swinderen
B.
,
Greenspan
R. J.
(
2005
).
Dopaminergic modulation of arousal in Drosophila
.
Curr. Biol.
15
,
1165
-
1175
.
Aton
S. J.
,
Huettner
J. E.
,
Straume
M.
,
Herzog
E. D.
(
2006
).
GABA and Gi/o differentially control circadian rhythms and synchrony in clock neurons
.
Proc. Natl. Acad. Sci. USA
103
,
19188
-
19193
.
Choi
H. J.
,
Lee
C. J.
,
Schroeder
A.
,
Kim
Y. S.
,
Jung
S. H.
,
Kim
J. S.
,
Kim
Y.
,
Son
E. J.
,
Han
H. C.
,
Hong
S. K.
, et al. 
. (
2008
).
Excitatory actions of GABA in the suprachiasmatic nucleus
.
J. Neurosci.
28
,
5450
-
5459
.
Chung
B. Y.
,
Kilman
V. L.
,
Keath
J. R.
,
Pitman
J. L.
,
Allada
R.
(
2009
).
The GABAA receptor RDL acts in peptidergic PDF neurons to promote sleep in Drosophila
.
Curr. Biol.
19
,
386
-
390
.
Cirelli
C.
(
2009
).
The genetic and molecular regulation of sleep: from fruit flies to humans
.
Nat. Rev. Neurosci.
10
,
549
-
560
.
Crocker
A.
,
Shahidullah
M.
,
Levitan
I. B.
,
Sehgal
A.
(
2010
).
Identification of a neural circuit that underlies the effects of octopamine on sleep:wake behavior
.
Neuron
65
,
670
-
681
.
Dahdal
D.
,
Reeves
D. C.
,
Ruben
M.
,
Akabas
M. H.
,
Blau
J.
(
2010
).
Drosophila pacemaker neurons require g protein signaling and GABAergic inputs to generate twenty-four hour behavioral rhythms
.
Neuron
68
,
964
-
977
.
Ehlen
J. C.
,
Novak
C. M.
,
Karom
M. C.
,
Gamble
K. L.
,
Paul
K. N.
,
Albers
H. E.
(
2006
).
GABAA receptor activation suppresses Period 1 mRNA and Period 2 mRNA in the suprachiasmatic nucleus during the mid-subjective day
.
Eur. J. Neurosci.
23
,
3328
-
3336
.
Enell
L.
,
Hamasaka
Y.
,
Kolodziejczyk
A.
,
Nässel
D. R.
(
2007
).
γ-Aminobutyric acid (GABA) signaling components in Drosophila: immunocytochemical localization of GABAB receptors in relation to the GABAA receptor subunit RDL and a vesicular GABA transporter
.
J. Comp. Neurol.
505
,
18
-
31
.
Fogle
K. J.
,
Parson
K. G.
,
Dahm
N. A.
,
Holmes
T. C.
(
2011
).
CRYPTOCHROME is a blue-light sensor that regulates neuronal firing rate
.
Science
331
,
1409
-
1413
.
Foltenyi
K.
,
Greenspan
R. J.
,
Newport
J. W.
(
2007
).
Activation of EGFR and ERK by rhomboid signaling regulates the consolidation and maintenance of sleep in Drosophila
.
Nat. Neurosci.
10
,
1160
-
1167
.
Galvez
T.
,
Duthey
B.
,
Kniazeff
J.
,
Blahos
J.
,
Rovelli
G.
,
Bettler
B.
,
Prézeau
L.
,
Pin
J. P.
(
2001
).
Allosteric interactions between GB1 and GB2 subunits are required for optimal GABAB receptor function
.
EMBO J.
20
,
2152
-
2159
.
Hamasaka
Y.
,
Nässel
D. R.
(
2006
).
Mapping of serotonin, dopamine, and histamine in relation to different clock neurons in the brain of Drosophila
.
J. Comp. Neurol.
494
,
314
-
330
.
Hamasaka
Y.
,
Wegener
C.
,
Nässel
D. R.
(
2005
).
GABA modulates Drosophila circadian clock neurons via GABAB receptors and decreases in calcium
.
J. Neurobiol.
65
,
225
-
240
.
Helfrich-Förster
C.
,
Shafer
O. T.
,
Wülbeck
C.
,
Grieshaber
E.
,
Rieger
D.
,
Taghert
P.
(
2007a
).
Development and morphology of the clock-gene-expressing lateral neurons of Drosophila melanogaster
.
J. Comp. Neurol.
500
,
47
-
70
.
Helfrich-Förster
C.
,
Yoshii
T.
,
Wülbeck
C.
,
Grieshaber
E.
,
Rieger
D.
,
Bachleitner
W.
,
Cusamano
P.
,
Rouyer
F.
(
2007b
).
The lateral and dorsal neurons of Drosophila melanogaster: new insights about their morphology and function
.
Cold Spring Harb. Symp. Quant. Biol.
72
,
517
-
525
.
Hendricks
J. C.
,
Finn
S. M.
,
Panckeri
K. A.
,
Chavkin
J.
,
Williams
J. A.
,
Sehgal
A.
,
Pack
A. I.
(
2000
).
Rest in Drosophila is a sleep-like state
.
Neuron
25
,
129
-
138
.
Ho
K. S.
,
Sehgal
A.
(
2005
).
Drosophila melanogaster: an insect model for fundamental studies of sleep
.
Methods Enzymol.
393
,
772
-
793
.
Joiner
W. J.
,
Crocker
A.
,
White
B. H.
,
Sehgal
A.
(
2006
).
Sleep in Drosophila is regulated by adult mushroom bodies
.
Nature
441
,
757
-
760
.
Kaupmann
K.
,
Malitschek
B.
,
Schuler
V.
,
Heid
J.
,
Froestl
W.
,
Beck
P.
,
Mosbacher
J.
,
Bischoff
S.
,
Kulik
A.
,
Shigemoto
R.
, et al. 
. (
1998
).
GABAB-receptor subtypes assemble into functional heteromeric complexes
.
Nature
396
,
683
-
687
.
Kula-Eversole
E.
,
Nagoshi
E.
,
Shang
Y.
,
Rodriguez
J.
,
Allada
R.
,
Rosbash
M.
(
2010
).
Surprising gene expression patterns within and between PDF-containing circadian neurons in Drosophila
.
Proc. Natl. Acad. Sci. USA
107
,
13497
-
13502
.
Kume
K.
,
Kume
S.
,
Park
S. K.
,
Hirsh
J.
,
Jackson
F. R.
(
2005
).
Dopamine is a regulator of arousal in the fruit fly
.
J. Neurosci.
25
,
7377
-
7384
.
Lebestky
T.
,
Chang
J. S.
,
Dankert
H.
,
Zelnik
L.
,
Kim
Y. C.
,
Han
K. A.
,
Wolf
F. W.
,
Perona
P.
,
Anderson
D. J.
(
2009
).
Two different forms of arousal in Drosophila are oppositely regulated by the dopamine D1 receptor ortholog DopR via distinct neural circuits
.
Neuron
64
,
522
-
536
.
Lee
D.
,
Su
H.
,
O'Dowd
D. K.
(
2003
).
GABA receptors containing Rdl subunits mediate fast inhibitory synaptic transmission in Drosophila neurons
.
J. Neurosci.
23
,
4625
-
4634
.
Lelito
K. R.
,
Shafer
O. T.
(
2012
).
Reciprocal cholinergic and GABAergic modulation of the small ventrolateral pacemaker neurons of Drosophila's circadian clock neuron network
.
J. Neurophysiol.
107
,
2096
-
2108
.
Liu
C.
,
Reppert
S. M.
(
2000
).
GABA synchronizes clock cells within the suprachiasmatic circadian clock
.
Neuron
25
,
123
-
128
.
McCarthy
E. V.
,
Wu
Y.
,
Decarvalho
T.
,
Brandt
C.
,
Cao
G.
,
Nitabach
M. N.
(
2011
).
Synchronized bilateral synaptic inputs to Drosophila melanogaster neuropeptidergic rest/arousal neurons
.
J. Neurosci.
31
,
8181
-
8193
.
Mezler
M.
,
Müller
T.
,
Raming
K.
(
2001
).
Cloning and functional expression of GABAB receptors from Drosophila
.
Eur. J. Neurosci.
13
,
477
-
486
.
Minot
S.
,
Sinha
R.
,
Chen
J.
,
Li
H.
,
Keilbaugh
S. A.
,
Wu
G. D.
,
Lewis
J. D.
,
Bushman
F. D.
(
2011
).
The human gut virome: inter-individual variation and dynamic response to diet
.
Genome Res.
21
,
1616
-
1625
.
Mistlberger
R. E.
(
2005
).
Circadian regulation of sleep in mammals: role of the suprachiasmatic nucleus
.
Brain Res. Brain Res. Rev.
49
,
429
-
454
.
Nitz
D. A.
,
van Swinderen
B.
,
Tononi
G.
,
Greenspan
R. J.
(
2002
).
Electrophysiological correlates of rest and activity in Drosophila melanogaster
.
Curr. Biol.
12
,
1934
-
1940
.
Okada
R.
,
Awasaki
T.
,
Ito
K.
(
2009
).
Gamma-aminobutyric acid (GABA)-mediated neural connections in the Drosophila antennal lobe
.
J. Comp. Neurol.
514
,
74
-
91
.
Parisky
K. M.
,
Agosto
J.
,
Pulver
S. R.
,
Shang
Y.
,
Kuklin
E.
,
Hodge
J. J.
,
Kang
K.
,
Liu
X.
,
Garrity
P. A.
,
Rosbash
M.
, et al. 
. (
2008
).
PDF cells are a GABA-responsive wake-promoting component of the Drosophila sleep circuit
.
Neuron
60
,
672
-
682
.
Park
J. H.
,
Helfrich-Förster
C.
,
Lee
G.
,
Liu
L.
,
Rosbash
M.
,
Hall
J. C.
(
2000
).
Differential regulation of circadian pacemaker output by separate clock genes in Drosophila
.
Proc. Natl. Acad. Sci. USA
97
,
3608
-
3613
.
Pitman
J. L.
,
McGill
J. J.
,
Keegan
K. P.
,
Allada
R.
(
2006
).
A dynamic role for the mushroom bodies in promoting sleep in Drosophila
.
Nature
441
,
753
-
756
.
Renn
S. C.
,
Park
J. H.
,
Rosbash
M.
,
Hall
J. C.
,
Taghert
P. H.
(
1999
).
A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila
.
Cell
99
,
791
-
802
.
Root
C. M.
,
Masuyama
K.
,
Green
D. S.
,
Enell
L. E.
,
Nässel
D. R.
,
Lee
C. H.
,
Wang
J. W.
(
2008
).
A presynaptic gain control mechanism fine-tunes olfactory behavior
.
Neuron
59
,
311
-
321
.
Schmid
B.
,
Helfrich-Förster
C.
,
Yoshii
T.
(
2011
).
A new ImageJ plug-in ‘ActogramJ’ for chronobiological analyses
.
J. Biol. Rhythms
26
,
464
-
467
.
Shafer
O. T.
,
Taghert
P. H.
(
2009
).
RNA-interference knockdown of Drosophila pigment dispersing factor in neuronal subsets: the anatomical basis of a neuropeptide's circadian functions
.
PLoS ONE
4
,
e8298
.
Shang
Y.
,
Griffith
L. C.
,
Rosbash
M.
(
2008
).
Light-arousal and circadian photoreception circuits intersect at the large PDF cells of the Drosophila brain
.
Proc. Natl. Acad. Sci. USA
105
,
19587
-
19594
.
Shang
Y.
,
Haynes
P.
,
Pírez
N.
,
Harrington
K. I.
,
Guo
F.
,
Pollack
J.
,
Hong
P.
,
Griffith
L. C.
,
Rosbash
M.
(
2011
).
Imaging analysis of clock neurons reveals light buffers the wake-promoting effect of dopamine
.
Nat. Neurosci.
14
,
889
-
895
.
Shaw
P. J.
,
Cirelli
C.
,
Greenspan
R. J.
,
Tononi
G.
(
2000
).
Correlates of sleep and waking in Drosophila melanogaster
.
Science
287
,
1834
-
1837
.
Sheeba
V.
,
Fogle
K. J.
,
Kaneko
M.
,
Rashid
S.
,
Chou
Y. T.
,
Sharma
V. K.
,
Holmes
T. C.
(
2008a
).
Large ventral lateral neurons modulate arousal and sleep in Drosophila
.
Curr. Biol.
18
,
1537
-
1545
.
Sheeba
V.
,
Gu
H.
,
Sharma
V. K.
,
O'Dowd
D. K.
,
Holmes
T. C.
(
2008b
).
Circadian- and light-dependent regulation of resting membrane potential and spontaneous action potential firing of Drosophila circadian pacemaker neurons
.
J. Neurophysiol.
99
,
976
-
988
.
Sokolove
P. G.
,
Bushell
W. N.
(
1978
).
The chi square periodogram: its utility for analysis of circadian rhythms
.
J. Theor. Biol.
72
,
131
-
160
.
van den Pol
A. N.
,
Tsujimoto
K. L.
(
1985
).
Neurotransmitters of the hypothalamic suprachiasmatic nucleus: immunocytochemical analysis of 25 neuronal antigens
.
Neuroscience
15
,
1049
-
1086
.
van Swinderen
B.
,
Nitz
D. A.
,
Greenspan
R. J.
(
2004
).
Uncoupling of brain activity from movement defines arousal states in Drosophila
.
Curr. Biol.
14
,
81
-
87
.
Wagner
S.
,
Castel
M.
,
Gainer
H.
,
Yarom
Y.
(
1997
).
GABA in the mammalian suprachiasmatic nucleus and its role in diurnal rhythmicity
.
Nature
387
,
598
-
603
.
Wilson
R. I.
,
Laurent
G.
(
2005
).
Role of GABAergic inhibition in shaping odor-evoked spatiotemporal patterns in the Drosophila antennal lobe
.
J. Neurosci.
25
,
9069
-
9079
.
Yuan
Q.
,
Joiner
W. J.
,
Sehgal
A.
(
2006
).
A sleep-promoting role for the Drosophila serotonin receptor 1A
.
Curr. Biol.
16
,
1051
-
1062
.

COMPETING INTERESTS

No competing interests declared.

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