Glial Cells Physiologically Modulate Clock Neurons and Circadian Behavior in a Calcium-Dependent Manner

Current Biology 21, 625–634, April 26, 2011 ª2011 Elsevier Ltd All rights reserved

DOI 10.1016/j.cub.2011.03.027

Article Glial Cells Physiologically Modulate Clock Neurons and Circadian Behavior in a Calcium-Dependent Manner Fanny S. Ng,1 Michelle M. Tangredi,1 and F. Rob Jackson1,* 1Department of Neuroscience, Center for Neuroscience Research, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA

Summary Background: An important goal of contemporary neuroscience research is to define the neural circuits and synaptic interactions that mediate behavior. In both mammals and Drosophila, the neuronal circuitry controlling circadian behavior has been the subject of intensive investigation, but roles for glial cells in the networks controlling rhythmic behavior have only begun to be defined in recent studies. Results: Here, we show that conditional, glial-specific genetic manipulations affecting membrane (vesicle) trafficking, the membrane ionic gradient, or calcium signaling lead to circadian arrhythmicity in adult behaving Drosophila. Correlated and reversible effects on a clock neuron peptide transmitter (PDF) and behavior demonstrate the capacity for glia-toneuron signaling in the circadian circuitry. These studies also reveal the importance of a single type of glial cell—the astrocyte—and glial internal calcium stores in the regulation of circadian rhythms. Conclusions: This is the first demonstration in any system that adult glial cells can physiologically modulate circadian neuronal circuitry and behavior. A role for astrocytes and glial calcium signaling in the regulation of Drosophila circadian rhythms emphasizes the conservation of cellular and molecular mechanisms that regulate behavior in mammals and insects. Introduction Understanding how the nervous system drives behavior requires knowledge of the relevant neural circuits and synaptic interactions. The neuronal circuitry regulating circadian behavior has been analyzed in Drosophila melanogaster via elegant genetic approaches. Previous work in this system has identified the neurons comprising the pacemaker and yielded an explicit biochemical model for the molecular oscillator within them that drives behavioral rhythms [1]. Recent work on the Drosophila circadian circuitry has focused on defining the biophysical properties of clock neurons [2–6] and implementing network approaches to understanding pacemaker circuitry. The latter studies, for example, have demonstrated that distinct groups of communicating clock neurons regulate the timing of the two daily activity bouts (evening and morning) as well as responses to environmental light and temperature signals [7–11]. Additional studies have identified a peptide neurotransmitter (PDF) and its receptor that mediate interactions among neuronal components of the network [12–15]. However, none of this work has addressed the role(s) of glial cells in the circuitry regulating circadian behavior.

*Correspondence: [email protected]

Astrocyte glial cells of the adult mammalian nervous system are known to interact with neurons to modulate physiological functions including water and ionic balance [16], energy metabolism [17], neurovascular interactions [18], and synaptic transmission [19]. Astrocytes produce and release transmitters of their own (‘‘gliotransmitters’’ such as glutamate, D-serine, and ATP) that act on neighboring neurons and glia [20–23], a process thought to be mediated by Ca2+ and SNAREdependent vesicular mechanisms, similar to mechanisms underlying neuronal transmitter release [24]. There has been controversy, however, about whether glial Ca2+ signaling is important for the regulation of neuronal excitability and plasticity [19, 25–27]. Recent studies have described the glial cell classes of the adult Drosophila nervous system, highlighting similarities to their mammalian counterparts [28, 29]. The adult fly central nervous system contains five main classes of glial cells: two neuropil classes (known as ensheathing and astrocytes), two types of surface glia (known as perineurial and subperineurial, forming the blood brain barrier), and one cortex (cell body) class of glia. These five classes include many subtypes, which have been particularly well studied in the insect visual system. Astrocytes and ensheathing glia extend into or wrap neuronal projections within the neuropil whereas cortex glia encompass neuronal cell bodies. Based on their positions and potential to regulate neurons, the neuropil and cortex glia are well suited to modulate neuronal excitability and behavior. Glia of both classes are present in many regions of the Drosophila brain, in close proximity to clock neurons and other neuronal cell types [30]; similar to mammalian astrocytes, fly astrocytes are associated with neuronal synapses [28]. Given our previous description of a glial-specific factor (Ebony) essential for normal circadian behavior [30], we were intrigued by the idea that glial cells might actively modulate adult circadian pacemaker neurons. Although recent studies suggest that neuron-to-astrocyte communication might occur in the mammalian circadian pacemaker [31, 32], there has not been an in vivo analysis in any system that documents modulation of circadian circuitry by glial cells. In the studies reported here, we have employed genetic approaches in Drosophila to determine whether glial cells can physiologically regulate the neuronal circuitry driving circadian behavior. We made use of promoter-enhancer (Gal4) drivers, which are available for all major classes of adult glia [28, 29], to perform genetic manipulations in a cell type-specific manner. Gal4-regulated (UAS) transgenes were employed to alter glial vesicle trafficking (exocytosis/endocytosis), the membrane electrochemical gradient, or internal calcium stores. In most studies, we utilized the Drosophila TARGET system [33], which makes use of a temperature-sensitive inhibitor of Gal4 called Gal80ts, to effect conditional perturbations in adult behaving animals, thus circumventing possible developmental effects. Our results indicate that glial cells of the adult brain physiologically regulate circadian neurons and behavior, and they demonstrate the importance of glial internal calcium stores for the modulation of behavior. Furthermore, these studies identify a specific class of glial cells—the astrocyte—that participates in the regulation of circadian behavior.

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Figure 1. Conditional Adult Expression of NaChBac in Glial Cells Induces Arrhythmic Circadian Behavior (A) Schematic drawing of experimental paradigm. The white and black bars represent the light/dark schedule in this and other figures. After 3–4 days entrainment at 23 C, flies were maintained in free-running (DD) conditions for at least 7 days at 30 C. (B) Representative actograms for each genotype during DD. Grey and black bars indicate subjective day and night, respectively. Only the experimental strain (tubG80ts; repoGal4 > UAS-NaChBac) exhibited significant arrhythmicity. (C) Representative actograms for tubG80ts; repoGal4 > UAS-NaChBac fly at 23 C in DD; in these conditions, all flies showed rhythmic locomotor activity. (D) Histograms summarizing percent rhythmicity and average RI value for different genotypes. As indicated by asterisks (***), the RI value for the experimental strain is significantly reduced compared to either control strain (p < 0.001, nonparametric one-way ANOVA with Dunn post-hoc test). Data are from at least two independent experiments. Sample sizes (n) for each genotype are shown at the bottom of the panel. For this figure and all others, error bars are standard error of the mean (SEM).

Results Glial Cell Function Is Essential in Adults for Normal Behavioral Rhythmicity To determine the importance of glial cells in the circadian circuitry, we assayed adult locomotor activity rhythms in transgenic Drosophila with altered glial cell functions. In a first set of experiments, a bacterial sodium channel (NaChBac) was expressed in all adult glial cells. This prokaryotic channel is selective for Na+ in mammalian cells [34]; its expression is predicted to alter glial ionic gradients, perhaps affecting transporter functions as certain mammalian glial transporters exhibit Na+-dependent activity [35]. It had previously been shown that expression of NaChBac in Drosophila pacemaker neurons perturbs behavioral rhythms [6]. Our studies utilized the TARGET system [33] and a temperature-sensitive tubulinGal80 (tubG80ts) transgene to limit expression of NaChBac to adult animals (in tubG80ts; repoGal4 > UAS-NaChBac flies); tubGal80ts is inactivated at 30 C, permitting expression of UAS transgenes at the high temperature. As shown in Figure 1A, an experimental protocol was employed that resulted in upregulation of Gal4 activity (and subsequent expression of NaChBac) at the beginning of subjective daytime on day 1 of constant darkness. During light/dark (LD) entrainment (at 23 C), both the control (repoGal4 and tubG80ts UAS-NaChBac) and tubG80ts; repoGal4 > UAS-NaChBac experimental flies displayed normal bimodal locomotor activity rhythms (Figure S1A and Table S1A

available online). Thus, all monitored flies were normally entrained by LD at the permissive temperature. However, when temperature was increased to 30 C in DD, the majority of NaChBac-expressing flies (tubG80ts; repoGal4 > UAS-NaChBac) became arrhythmic within 1 day whereas most control individuals (repoGal4 or tubG80ts; UAS-NaChBac) retained strong, persistent rhythmicity with a phase predicted from the previous LD cycle (Figure 1; Table S1A, Figure S1B). Representative actograms for all three types of flies are shown in Figure 1B, while population statistics summarizing percent rhythmicity and rhythmicity index (RI, a measure of robustness) are included in Figure 1D. Despite an increase in mean activity for experimental flies, they were arrhythmic and exhibited severely reduced RI values (RI z 0.1) compared to either control strain (for which RI z 0.5; p < 0.001). Importantly, experimental and control flies showed robust behavioral rhythms at 23 C, indicating that arrhythmicity is caused by activation of NaChBac expression (Figure 1C; Table S1B). These observations indicate that normal glial function in adult animals is crucial for rhythmic locomotor activity and suggest that glial cells can physiologically modulate circadian pacemaker neurons. Conditional Manipulation of Glial Vesicle Trafficking Results in Arrhythmic Locomotor Activity Previous studies demonstrate that mammalian astrocytes and insect glial cells can modulate synaptic transmission [19, 36], and in the case of astrocytes, this occurs via the release of gliotransmitters [21]. Thus, we performed genetic manipulations to conditionally disrupt glial vesicle trafficking to determine whether gliotransmission might be important for the regulation of circadian behavior. To conduct this study, a temperature-sensitive dynamin molecule (Shibirets or shits) was employed which has been well characterized [37]. At high temperature, shits mutants show rapid and reversible defects in synaptic vesicle trafficking and neurotransmitter release as well as altered endocytosis [38].

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Figure 2. Conditional Glial Cell-Specific Perturbation of Exocytosis/Endocytosis Has Reversible Effects on Rhythmic Behavior (A) Schematic drawing of experimental paradigm. All flies were entrained to LD 12:12 for 3–4 days before entering DD at 30 C for 3 days. After the 30 C treatment, flies were returned to 23 C (still in DD) for 8 days. (B) Representative actograms for different genotypes. The different actogram panels (from top to bottom) illustrate the three phases of the experiment: (1) top panels represent the LD behavioral record, (2) middle panels represent the high-temperature period (30 C, DD), (3) bottom panels represent the recovery period (23 C, DD). Only tubG80ts; repoGal4 > UAS-shits flies exhibited arrhythmic behavior during the 30 C treatment. We note that flies which experience the temperature shift paradigm have reduced activity levels relative to flies kept at 23 C the entire experiment. In addition, shits-expressing flies have slightly reduced activity at 30 C and during the subsequent 23 C interval relative to controls. (C) Representative actograms for tubG80ts; repoGal4 > UAS-shits flies that did not receive a heat treatment (23 C, DD); these flies were all rhythmic. (D) Histograms summarizing percent rhythmicity during high and low temperature conditions (30 C and the 23 C recovery period). There was a dramatic reduction in rhythmicity for tubG80ts; repoGal4 > UAS-shits flies at 30 C, but such flies became rhythmic again when temperature was reduced to 23 C. Results represent at least two independent experiments for each strain. Sample sizes (n) are indicated next to the genotypes.

A UAS-shits transgene was utilized—which blocks vesicle trafficking in a temperature-dependent, dominant manner [37]—in conjunction with repoGal4 and tubG80ts to conditionally perturb adult glia at high temperature (30 C). tubG80ts was included because others have documented effects of shits even at seemingly permissive temperatures [39]. Initially, tubG80ts; repoGal4 > UAS-shits and control flies were examined at 23 C to ensure that rhythmicity was normal in the two types of flies at low temperature. As indicated in Figure 2C and Table S2B, all flies entrained well to LD and exhibited robust rhythms in DD when maintained at 23 C. In contrast, when temperature was increased to 30 C, all shitsexpressing flies became arrhythmic within 2 days, and prolonged exposure (4–5 days) to high temperature caused lethality (data not shown), indicating the importance of glial functions in the adult brain. To circumvent lethality, a paradigm was employed (Figure 2A) in which temperature is reversibly raised to 30 C for 3 days. With this paradigm, nearly all experimental flies (141/143; total of three independent experiments) became arrhythmic within 1–2 days whereas most control flies continued to exhibit robust rhythms throughout the high-temperature period (Figures 2B and 2D; Table S2A). Note that blind scoring of records was employed in these experiments to determine rhythmicity and arrhythmicity during the high-temperature period,

because short (3 day) activity records are not amenable to statistical analysis. In addition to arrhythmicity, there was a w54% reduction in activity level for experimental flies maintained at 30 C, relative to controls (Table S2A; p < 0.001), supporting the idea that glial cells can modulate neurons that determine activity levels. Note, however, that activity level is not correlated with rhythmicity. For example, deficits for the fly Dopamine Transporter or the Dopamine 2 Receptor (D2R) significantly increase or decrease activity, respectively, but neither causes arrhythmicity [40, 41]. As shown in the previous section, glial expression of NaChBac increases locomotor activity but nonetheless causes arrhythmicity. Notwithstanding the dramatic effect on rhythmicity, most of the shits-expressing flies (80%) survived the 30 C heat treatment and remained viable at the end of the 8 day 23 C (DD) recovery period. Equally important, clock neurons and glial cells of the adult brain appeared normal on days 2–3 of the heat treatment (Figures S2, S3A, and S4B); i.e., there was no apparent cellular degeneration. Consistent with that result, the shits-induced arrhythmicity was completely reversible: when temperature was decreased to 23 C after 3 days of high temperature treatment, most (91%) of the shits-expressing flies recovered strong, persistent rhythms (RI = 0.34 compared to 0.32 and 0.47 for controls; Figures 2B and 2D; Table S2A) with a phase similar to that of control flies and predicted by the previous LD entrainment (i.e., there was not a detectable phase shift of the clock). Thus, blockade of glial vesicle trafficking eliminated rhythmicity but in a completely reversible manner.

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Figure 3. Conditional Glial Expresson of shits Reduces PDF Immunoreactivity in Projections of the LNv Neurons (A) Schematic drawing of experimental paradigm. All flies were entrained to LD 12:12 at 23 C for 4 days, released into DD at 30 C for 3 days, then maintained in DD at 23 C for the last 3 days. Triangles indicate when flies were collected for examination of PDF immunoreactivity. (B and C) Representative data for red and blue triangles. Images acquired at time points represented by the black triangles are not shown. (B) Representative confocal images of PDF immunoreactivity in control (UAS-shits) and experimental (tubG80ts; repoGal4 > UAS-shits) flies maintained at 30 C. CT20 is a time point during subjective night, whereas CT0 and CT4 are times at the beginning of subjective day. PDF immunostaining in neuronal projections is significantly reduced in experimental flies at all time points examined. (C) Representative confocal images of PDF immunoreactivity at CT0 during the recovery period (23 C, DD). Signal intensity is comparable in tubG80ts; repoGal4 > UAS-shits and control flies. Four or more individual brains were examined per genotype at each circadian time point. Similar results were obtained in two independent experiments. All confocal images shown in this figure are w70 to 80 mm Z stacks of 3 mm optical sections from the LNv cell body to the dorsal end of the projections.

Glial Cell Perturbation Alters a Circadian Peptide Transmitter without Affecting Clock Protein Abundance Drosophila circadian locomotor activity is known to be regulated by neuronal transcriptional/translational oscillators. Given our finding that glial perturbation can cause behavioral arrhythmicity, we wondered whether alteration of the neuronal clock was responsible for the effect. To address this issue, PERIOD (PER) and PDP13 clock protein abundance were examined in experimental (tubG80ts; repoGal4 > UAS-shits) and control (UAS-shits) flies subjected to a light/temperature protocol identical to that utilized for behavioral experiments (Figure S2A). Those studies indicated that clock protein cycling was similar in experimental and control flies at high temperature (Figure S2C, data not shown). As shown in Figures S2B and S2C, PER was rhythmically expressed in all clock neurons of experimental and control flies, with a peak at CT0 and a trough at CT12. Similarly, the PER nuclear entry rhythm was normal in experimental flies with complete nuclear localization observed by CT0. As expected,

PDP13 protein was predominantly nuclear at all time points in experimental (Figure S2B, bottom) and control (Figure S2B, top) brains and exhibited peak abundance between CT16 and CT20 in both types of brains. Thus, the phasing and average abundances of neuronal clock proteins were normal after conditional glial cell manipulation, although slightly more variable PER and PDP signal intensities were observed among experimental flies compared to controls at several times of day. A peptide transmitter called pigment-dispersing factor (PDF) is localized to ventral lateral (LNv) pacemaker neurons of the fly brain, and its release from dorsal projections of the small LNv (s-LNv) neurons is essential for rhythmicity [14, 42]. PDF acts on a G protein-coupled receptor, localized to many clock cells [12, 14, 15, 43], to synchronize the clock neuronal network [13] and drive rhythmic behavior. To determine whether PDF synthesis, transport, or release might be regulated by glia, we assessed immunoreactivity for the peptide in s-LNv dorsal projections after glial cell perturbation. Whereas controls (UAS-shits) exhibited obvious and rhythmic PDF immunostaining in the LNv dorsal projections after 2 days at 30 C, tubG80ts; repoGal4 > UAS-shits brains had significantly reduced PDF at CT20, CT0, and CT4 (Figure 3B; Figure S3B). Importantly, the glial cell-mediated alteration of PDF was reversible, and the peptide could be detected at high levels in shits-expressing and control flies after the return to low (23 C) temperature (Figure 3C; Figure S3C). Reversibility of the effect demonstrates that the dorsal

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Figure 4. Glial PER Is Not Required for Normal Free-Running Behavioral Rhythmicity (A) PER staining at different times of the diurnal cycle in wild-type flies. PER exhibits rhythms in abundance in glia of the optic lobe, with a peak at ZT21–23. (B) PER (red) staining in control and repoGal4 > per RNAi flies at ZT1 (the high point of neuronal PER abundance). Green signal represents PDF. Glial PER is nearly eliminated in repoGal4 > per RNAi flies whereas abundance of the clock protein is normal in the LNv and DN neurons of such flies. Arrowheads indicate ocellar photoreceptors that were not detached from the brain. The dashed line in the right-hand panel delimits the brain. The magnified images for control and repoGal4 > per RNAi flies illustrate normal abundance of PER in clock neurons (LNv and LNd); glia are not apparent in these images because they are 10 mm optical sections whereas the adjacent low-magnification images are w100 mm Z stacks showing an entire hemisphere of the brain. (C) Intensity of PER signal in a CRY/PERexpressing population of glia at the junction of the optic medulla and lobula. PER signal was analyzed at ZT1 and ZT12 and compared statistically with a 2-tailed paired t test. *p < 0.05; ***p < 0.001. (D) Representative actograms of LD and DD behavior for repoGal4 > per RNAi and control flies. (E) Population statistics for per RNAi and control flies. A one-way ANOVA or nonparametric ANOVA was performed to determine statistical differences. Two asterisks (**) and one asterisk (*) represent significant differences from both control strains (UAS-per RNAi and repoGal4) at p < 0.01 and p < 0.05, respectively.

projections remain intact in such flies and suggests a physiological regulation of neuronal PDF. Although there are apparent differences in cell body PDF staining in the images shown in Figure 3, we note that a thorough analysis found no consistent differences between genotypes at several times of day (see Figure S3A). Based on these observations, we infer that PDF transport and/or release rather than synthesis is preferentially affected by glial cell perturbation. Altered PDF release is predicted to result in arrhythmic circadian behavior [14]. Glial Oscillators Are Not Required for Circadian Behavior in Constant Conditions Because glial cell manipulations might alter PER-based oscillator function, we wondered whether the glial molecular oscillator contributed to free-running behavior. Several studies demonstrate that clock proteins including PERIOD (PER), TIMELESS (TIM), and CRYPTOCHROME (CRY) are expressed in adult fly glial cells and these cells exhibit robust rhythms in PER abundance [30, 44, 45] (Figure 4A). A previous study suggested that PER-containing glia might be important for circadian locomotor activity rhythms [46], but it has not been

determined directly whether a glialbased molecular oscillator contributes to behavioral rhythmicity. To ask whether glial PER expression is required for normal circadian behavior, the clock protein was knocked down specifically in glial cells (using a per RNAi transgene [47]), leaving neuronal expression intact (Figures 4B and 4C); locomotor activity rhythms were then assayed in light/dark (LD) and constant dark (DD) conditions. Flies with a severe glial PER deficiency (repoGal4; UAS-per RNAi) nonetheless exhibited normal rhythmicity (Figures 4D and 4E); there were no significant differences between control (repoGal4 or UAS-per RNAi) and knockdown flies in activity level, average circadian period (Tau), robustness of rhythms (RI), or percent rhythmicity (%R). Similar results were obtained with the TARGET [33] system to limit per RNAi expression to adult glial cells (data not shown). Furthermore, a knockdown of CRY in glial cells did not affect free-running behavior (data not shown). In contrast, tim-gal4-driven expression of the same RNAi construct in all clock cells, including neurons, resulted in altered circadian period and some arrhythmicity (Figure 4E), similar to published studies [47]. This long-period phenotype is identical to that observed with perturbations that affect PER only in the LNv neurons [39] and thus it is probably due to neuronal-specific effects. These results indicate that the glial PER-based oscillator is not essential for free-running

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Figure 5. Calcium-Dependent Pathways Are Important for the Glial Modulation of Circadian Behavior Flies were reared and treated according to the paradigm shown in Figure 1A; they were maintained in DD conditions for 11 days. (A–C) Representative actograms are shown for different genotypes. UAS-CaP60A RNAi control fly (A); tubG80ts; repoGal4 > UAS-Dicer2/UAS-CaP60A RNAi individuals (B and C) that became arrhythmic after 5–6 days in DD. The fly in (C) exhibits slightly more robust rhythms prior to becoming arrhythmic. (D) RI, Tau, and mean activity for CaP60A RNAi and control populations. Only 43% of knockdown flies were rhythmic at the end of experiments, even though 75% (68/91) of those flies survived the entire recording period. (R) and (AR) indicate the rhythmic and arrhythmic classes of CaP60A RNAi flies. RI is statistically decreased for the (AR) population relative to controls. Note that RI was calculated based on all days of DD including the several days of rhythmic behavior. N/A, sample sizes were too small for statistical analysis (n % 2).

activity rhythms, but this does not rule out a requirement for this oscillator in regulating circadian light (or temperature) sensitivity or other types of circadian rhythms. Glial Calcium Stores and SERCA Are Critical for Behavioral Rhythmicity As another method of showing that glial cells modulate circadian behavior, we conditionally altered an endogenous component of these cells in adult animals. In these experiments, intracellular calcium stores were altered due to evidence from certain studies that mammalian astrocytes modulate synaptic transmission in a calcium-dependent manner. The aforementioned TARGET system was employed to conditionally knock down the Drosophila Sarco-endoplasmic Reticulum (ER) Calcium ATPase (SERCA) in glia of the adult brain. Fly SERCA is encoded by the CaP60A gene, which is known to be expressed at high levels in neurons and glial cells of the adult central nervous system [48–50], similar to the mouse homolog [51, 52]. Analogous to mammalian SERCA, the fly ATPase regulates cellular calcium by pumping the ion from the cytosol into the ER [49]. Therefore, a knockdown of fly SERCA, via RNA interference (RNAi) methods, is predicted to increase cytosolic calcium levels. Using the paradigm shown in Figure 1A, adult tubG80ts; repoGal4 > UAS-Dicer2/UAS-CaP60A RNAi flies and controls (UAS-CaP60A RNAi and tubG80ts; repoGal4 > UAS-Dicer2) were subjected to high-temperature conditions after rearing and subsequent entrainment to LD at 23 C. The UAS-Dicer2

transgene was included in these experiments to enhance RNA interference effects [53]. As shown in Figure 5 and Table S4A, control and CaP60A RNAi flies exhibited normal bimodal locomotor activity patterns at 23 C in LD, indicative of normal light entrainment. Similarly, both types exhibited strong rhythmicity when maintained in DD at 23 C (Table S4B). However, behavioral rhythms dramatically declined in CaP60A RNAi flies beginning at about day 5 of the 30 C treatment (actograms and population statistics shown in Figure 5). Whereas control individuals were rhythmic for the duration of experiments, RNAi flies were weakly rhythmic or arrhythmic by the end of experiments (only 43% rhythmic). In addition, RI (calculated for the entire DD period) was significantly decreased for the arrhythmic class of RNAi flies (Figure 5D). Similar results were seen for such flies in three independent experiments. We attribute the delayed behavioral arrhythmicity of knockdown flies to a gradual decrease in CaP60A after induction of RNAi at high temperature (see Discussion). Drosophila Astrocytes Regulate Circadian Locomotor Activity As noted previously, adult Drosophila glial cells can be divided into multiple classes according to their morphologies and locations within the brain. To determine which glial classes are relevant for circadian behavior, we utilized 10 different class-specific Gal4 drivers that express in different glial populations [28, 29, 54]. Locomotor activity rhythms were monitored at 30 C for Gal4; UAS-shits experimental and UAS-shits

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Table 1. Astrocytes, but Not Other Glial Classes, Are Essential for Circadian Behavior

Glial Class Driver Perineurial Subperineurial Cortex

Ensheathing

Astrocyte

Genotype

N

% Rhythmic Flies during Induction of shits

Control- NP6293 NP6293 > UAS-shits Control- tubG80ts NP2276 tubG80ts NP2276 > UAS-shits Control- NP577 NP577 > UAS-shits Control- tubG80ts NP2222 tubG80ts NP2222 > UAS-shits Control- NP6520 NP6520 > UAS-shits Control- tubG80ts MZ0709 tubG80ts MZ0709 > UAS-shits Control- tubG80ts alrm Gal4 tubG80ts alrm Gal4 > UAS-shits Control- tubG80ts Eaat1 Gal4 tubG80ts Eaat1 Gal4 > UAS-shits

44 27 22 48 45 52 18 44 28 62 22 54 10 53 19 51

93 100 95 87 96 92 89 85 100 84 100 98 100 40 95 14

Behavioral results obtained from UAS-shits expression with Gal4 drivers representing five different glial cell classes. Percent rhythmicity is reduced (bold numbers) with two different astrocyte drivers, but it is normal for flies expressing UAS-shits in other types of glia. We experienced viability issue in certain genotypes (NP2276 > UAS-shits, NP2222 > UAS-shits, and MZ0709 > UAS-shits). Therefore, tubG80ts is included to eliminate any potential leaky effects from the shi mutant form during development. Detailed locomotor behavioral parameters are represented in Tables S3 and S4.

control flies. At high temperature, drivers specific for perineurial, subperineurial, cortex, and ensheathing classes of glia did not affect percent rhythmicity when combined with UAS-shits (Table 1), although flies expressing UAS-shits in perineurial and subperineurial glia had slightly less robust rhythms (Table S3); however, population plots for these flies reveal normal bimodal rhythmicity (Figure S5 and data not shown). Not unexpectedly, certain of these drivers had effects on survival when expressed constitutively throughout development (data not shown). Importantly, when expressed constitutively, four different Gal4 drivers—dEaat1Gal4, alrmGal4, NP1243, and NP3233—were associated with arrhythmicity, similar to the phenotype of tubG80ts; repoGal4 > shits flies (data not shown). When expression of shits was restricted to adulthood for two drivers (dEaat1Gal4 and alrmGal4), population rhythmicity was, similarly, significantly reduced (Table 1). As expected, percent rhythmicity was further reduced in flies carrying two copies of the UAS-shits transgene, indicating a dose-dependent effect of shits on circadian behavior (data not shown). Notably, all four of the Gal4 drivers affecting rhythmicity express in astrocytes of the adult brain. Whereas dEaat1-Gal4 is also expressed in T1 lamina neurons of the visual system [55], alrm-Gal4 is exclusively expressed in astrocytes of the adult brain [28]. Thus, we conclude that fly astrocytes can physiologically regulate neuronal functions, similar to their mammalian counterparts. Discussion This study is the first to demonstrate a role for glial cells of the adult brain in regulating circadian behavior. Our results show that genetic manipulations of glial vesicle trafficking, the membrane ionic gradient, or internal calcium stores all lead to a similar phenotype: arrhythmic locomotor activity. Conditional alteration of glial membrane trafficking, via the shits

method, caused reversible effects on behavior (Figure 2) and neuronal function (Figure 3), strongly suggesting a physiological regulation of neuronal function rather than a neural degeneration-induced phenotype. Consistent with this interpretation, we found no evidence for glial or neuronal cell death after conditional perturbations of glia (Figure S4). Although expression of shits is expected to affect many different aspects of vesicle trafficking—and viability with prolonged exposure to high temperature—NaChBac channel expression may have more limited effects on glia. Flies expressing this channel in glia for long periods of time exhibit normal viability even though they are entirely arrhythmic (Figure 1). Thus, NaChBac expression may alter glia in a more subtle fashion; e.g., perhaps only affecting release of glial signaling molecules and communication with neurons. A question of interest is how the NaChBac voltage-gated channel affects glial function as mammalian astrocytes, for example, are known to have high input resistance and stable membrane potential. We note, however, that such electrical measurements have been made only at the astrocyte cell body and it is unknown how membrane potential changes in fine astrocytic processes (where gliotransmission may occur). In addition, membrane potential of other types of mammalian glia (OPCs) becomes depolarized in response to neurotransmitter actions [56]. Thus, if fly glia can similarly be depolarized by neurotransmitters, then NaChBac expression might have dramatic effects on glial membrane potential and gliotransmission. In addition to effects on rhythmicity, glial perturbations were associated with alterations of mean locomotor activity level. With shits activation (at 30 C) or SERCA knockdown, locomotor activity was decreased relative to control flies (i.e., such flies did not show the normal temperature-dependent increase in activity observed in controls; Figures 2 and 5; Tables S2 and S5). In contrast, expression of the NaChBac channel led to increased locomotor activity relative to controls (Table S1). Decreased or increased activity may result from opposite effects on glia-to-neuron signaling (i.e., gliotransmission). For example, shits activation is known to decrease transmitter release in neurons and the same may be true of glia. Similarly, neuronal SERCA deficits reduce Ca2+-activated potassium channel (slo) currents in Drosophila even though intracellular Ca2+ levels are elevated [50], consistent with a requirement for internal Ca2+ stores in the regulation of channel activity, action potential generation, and neurotransmitter release. With regard to NaChBac expression, it is known that astrocyte glutamate transporter activity is Na+ dependent, i.e., Na+ is cotransported with glutamate [35]. Thus, increased locomotor activity might occur because of an altered glial ionic gradient—caused by expression of NaChBac. Because glial cells are nonexcitable, such an alteration might result in reduced transporter activity or a reversal of glutamate transport, with either effect increasing extracellular glutamate levels and glia-to-neuron signaling. Alteration of PDF—a well-characterized circadian neuropeptide—by glial cell manipulations provides strong evidence for glia-to-neuron communication in the adult Drosophila brain, although the relevant signals remain unknown. Candidates include gliotransmitters such as those employed by mammalian astrocytes (adenosine, glutamate, D-serine, and others) or other types of protein signaling pathways (e.g., FGF, EGF, Notch, etc.). Completely reversible effects on PDF, with no obvious alteration of neuronal morphology or clock protein cycling, suggests that glial cells physiologically modulate

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circadian output or communication among elements of the neuronal circuitry driving rhythmicity. As previously mentioned, glial perturbation may affect trafficking of PDF to the projections or release of the peptide from neurons—through yet-tobe defined mechanisms—as we observe no significant alteration of neuronal cell body peptide intensity. Any effect on PDF release is expected to produce arrhythmic behavior [14]. Previous studies demonstrate that a PER-based oscillator exists in fly glial cells [30, 45] and mammalian astrocytes [57], but the function of this glial oscillator has not previously been addressed. In the present study, we asked whether it is required for circadian rhythms in locomotor activity. Surprisingly, a strong RNAi-mediated knockdown of PER in all REPO-positive glia of the adult brain (>99% of adult glial cells) had no effect on free-running behavioral rhythms (rhythmic activity in constant darkness), although neuronal expression of the same RNAi transgene caused a lengthened circadian period (Figure 4). Thus, the glial oscillator is not essential for free-running behavioral rhythmicity. However, these results do not exclude the possibility that this oscillator is required for circadian photic (or temperature) sensitivity or the expression of a different rhythm (e.g., changes in dendrite morphology in the visual system [58] or abundance of Na+/ K+-ATPase subunits [59]). The results of our studies utilizing glial class Gal4 drivers indicate that astrocytes, but not other glial cell types, are relevant for the circadian modulation of behavior (Table 1). A function for fly astrocytes in behavioral rhythmicity is significant because this class of mammalian glia has a well-documented role in the modulation of neurotransmission via release of gliotransmitters [19]. In addition, recent studies suggest that astrocytes may be relevant for the function of the mammalian circadian system [60]. Astrocytes of the fly and mammalian brains are remarkably similar with regard to their morphology and molecular signatures (e.g., expression of similar transporters and enzymes that regulate neurotransmitter retrieval [28, 61]), further suggesting a conservation of function. However, little is known about invertebrate gliotransmission [60], and it will be of great interest to determine how Drosophila glia communicate with neurons. Although mammalian astrocytes can regulate neuronal function, there has been controversy concerning roles of astrocytic Ca2+ in neurotransmission and plasticity [19, 25]. To determine whether glial Ca2+ stores are essential for circadian rhythmicity, we examined CaP60A, the fly homolog of SERCA, an ATPase present in the ER that regulates cytosolic levels of the cation [49]. The SERCA-2b isoform is expressed in mammalian primary cultured astrocytes, and it functions in the regulation of astrocytic calcium waves and gliotransmission [24, 62]. In Drosophila, a single ubiquitously expressed SERCA isoform exists [49] with known roles in the regulation of neuronal membrane excitability [50] and ERK-mediated transcription [63]. Deficits for SERCA are expected to result in increased cytosolic Ca2+ levels because of reduced ionic pumping into the ER and enhanced store-operated Ca2+ entry (SOCE) through membrane channels that open in response to ER Ca2+ depletion [64, 65]. A glia-specific knockdown of SERCA was performed to assess whether internal glial Ca2+ stores were critical for rhythmicity. We found that SERCA deficits led to behavioral arrhythmicity (Figure 5) that appeared 4–5 days after conditional induction of RNAi; in addition, SERCA RNAi flies had decreased activity levels (Table S4). The delayed expression of the knockdown phenotype during behavioral experiments

may be due to a gradual increase in RNA interference (after inducible expression in adults) or a long half-life for CaP60A, a known membrane protein. Whatever the reason for the delayed expression of arrhythmicity, the results support the idea that glial Ca2+ signaling is critical for the modulation of the neuronal circadian circuitry. This finding together with a function for fly astrocytes in behavioral rhythmicity underscores the utility of Drosophila as a genetic model for further understanding neuron-glia communication in the adult nervous system. Experimental Procedures Fly Strains and Maintenance All Drosophila cultures were reared at 23 C on standard cornmeal/sugar/ wheat germ medium inside an environmentally controlled incubator on a 12 hr:12 hr light-dark (LD) schedule. To assay effects of NaChBac expression, three different fly strains were used: (1) tubG80ts; repoGal4 > UASNaChBac, (2) repoGal4, and (3) tubG80ts; UAS-NaChBac. The experimental strain tubG80ts; repoGal4 > UAS-NaChBac was generated by crossing w; Sp/SM1; repoGal4 (from M. Freeman, U. Massachusetts, Worcester, MA) females to w; tubG80ts;UAS-NaChBac males. Genetic background control strains (repoGal4 and tubG80ts; UAS-NaChBac) were generated by crossing w;Sp/SM1; repoGal4 females or w;tubG80ts;UAS-NaChBac males to white1118 (w1118) flies. For perturbation of glial vesicle exocytosis/endocytosis, three additional strains were generated: (1) tubG80ts; repoGal4 > UAS-shits, (2) tubG80ts; repoGal4, and (3) UAS-shits. The tubG80ts; repoGal4 > UAS-shits strain was generated by crossing UAS-shits;; UASshits females [37] to w; tubG80ts; repoGal4 males. Control strains (tubG80ts; repoGal4 and UAS-shits) were created by crossing UAS-shits;; UAS-shits females or w; tubG80ts; repoGal4 males to w1118 flies. repoGal4 or timGal4 males were crossed to UAS-perRNAi females [47] to generate flies with glial or clock cell PER deficits. Control flies carried either the Gal4 or UAS transgene in a w1118 genetic background. To examine SERCA deficits, we generated tubG80ts; repoGal4 > UAS-Dicer2/CaP60A RNAi flies by crossing tubG80ts UAS-Dicer2; repoGal4 males to CaP60A RNAi females. To examine roles of different glial cell classes on rhythms, we generated the following experimental genotypes: (1) NP6293 > UAS- shits, (2) NP2276 > UAS- shits, (3) NP2222 > UAS-shits, (4) NP577 > UAS-shits, (5) NP6520 > UAS-shits, (6) MZ709 > UAS-shits, (7) NP1234 > UAS-shits, (8) NP3233 > UAS-shits, (9) alrmGal4 > UAS-shits, and (10) dEaat1Gal4 > UAS-shits. To create fly strains with two copies of UAS-shits expressed in a specific glial cell class, UASshits, UAS-shits females were crossed to males carrying the different NP transgenes [29] or alrmGal4 [28] with or without tubG80ts. Females carrying different glial class Gal4 drivers were crossed to UAS-shits;; UAS-shits males to generate transgenic strains expressing one copy of UAS-shits in glia. Genetic control strains were generated by crossing flies carrying glial-Gal4 or UAS-shits transgenes to w1118 flies. Locomotor Activity Assay and Data Analysis Three- to four-day-old flies were placed in Trikinetics Drosophila Activity Monitors housed in a temperature-controlled incubator. In each experiment, flies were entrained under LD 12:12 at 23 C for at least 3 days, then monitored in free-running (DD) conditions at a specific temperature (see Results) for an additional 7 to 10 days. Behavioral data (beam breaks) were collected in 30 min bins and then analyzed with the MATLAB-based signal processing toolbox [66]. Percent rhythmicity for populations was determined via the rhythmicity index (RI, a measurement of robustness), the correlogram (a statistical measurement of rhythmicity), and the pattern of activity (assessed by examining the actograms of individual flies). A fly was considered rhythmic if it had (1) an RI value > 0.1, (2) a statistically significant correlogram (above the 95% confidence level), and (3) a clearly observed pattern of rhythmic activity in the actogram. To assess statistical significance of the circadian behavioral parameters, we used a Kruskal-Wallis test (nonparametric ANOVA) with Dunn’s Multiple Comparison test if the data did not pass a normality test (using the method of Kolmogorov and Smirnov). One-way ANOVA with Tukey-Kramer Multiple Comparisons test was used if the data passed a normality test (Instat, GraphPad). Immunohistochemistry Prior to immunostaining, six to ten individual males of experimental and control strains (3 to 4 days old) were maintained in vials for 4 days under

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LD 12:12 (similar to conditions during locomotor activity experiments). Flies were then housed in constant darkness (DD) at 30 C after the entrainment period before collection for PERIOD, PDP13, or PDF immunostaining. Starting at CT0 of DD day 2 (DD2), brains were hand dissected and fixed with 4% paraformaldehyde (PFA) every 4 hr until CT4 of DD3. Experimental and control brains were collected, fixed, and stained at the same time within experiments so as to minimize variability due to methodological differences (see Supplemental Experimental Procedures). To detect specific circadian clock proteins, the following antibodies were used: rabbit anti-PER (1:7500, a gift of R. Stanewsky); guinea pig anti-PDP1 (1:30,000) [67]; and anti-PDF (1:100, Developmental Studies Hybridoma Bank, DSHB). The PER antibody was preabsorbed against per01 embryos to eliminate nonspecific signal. Relevant secondary antibodies were employed at 1:500. These include Alexa Fluor 488, goat anti-rabbit; Alexa Fluor 488, goat anti-mouse; Alexa Fluor 488, goat anti-guinea pig; Cy3, goat anti-rabbit; and Alexa Fluor 647, goat anti-mouse. All Alexa Fluor secondary antibodies were purchased from Invitrogen; the Cy3, goat anti-rabbit secondary was obtained from Jackson ImmunoResearch Laboratories. Brain images were acquired with a Leica TCS SP2 AOBS confocal microscope. For all experiments, control and experimental brains were examined at the same time with similar acquisition parameters. Laser power was adjusted in each experiment such that the high point of clock protein or PDF staining intensity was not saturated in control brains. Experimental brains were examined with the same conditions. The Leica LCS ‘‘Lite’’ software was used to generate 2- or 3-dimensional projected images from optical sections of the circadian clock cells or the whole brain. Two or three independent experiments were performed for each study, and image intensities were quantified with NIH Image-J 1.33. Images were further processed with Adobe Photoshop (see Supplemental Experimental Procedures for details).

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Supplemental Information Supplemental Information includes Supplemental Experimental Procedures, five figures, and four tables and can be found with this article online at doi:10.1016/j.cub.2011.03.027. Acknowledgments We wish to thank Alenka Lovy-Wheeler for use of the Center for Neuroscience Research (CNR) Imaging Core, Lax Iyer for data mining of glial expression results, Takeshi Awasaki for NP Gal4 lines, Marc Freeman and Ozge Tasdemir for repoGal4 and alrmGal4 strains, Serge Birman for dEAAT1Gal4, Mike Young for UAS-per RNAi, Paul Hardin for anti-PDP1, and Ralf Stanewsky for anti-PER. We are grateful to the Bloomington Drosophila Stock Center, the Vienna Drosophila RNAi Center, and the Harvard TRiP Project (NIH/NIGMS R01-GM084947) for transgenic flies. This work was supported by NIH R01 HL59873 (F.R.J.), NIH R01 NS065900 (F.R.J.), NIH P30 NS047243 (F.R.J.), and a Russo Award from Tufts School of Medicine. F.S.N. is supported by training grant NIH T32 HD049341. Received: November 27, 2010 Revised: February 9, 2011 Accepted: March 9, 2011 Published online: April 14, 2011 References 1. Nitabach, M.N., and Taghert, P.H. (2008). Organization of the Drosophila circadian control circuit. Curr. Biol. 18, R84–R93. 2. Park, D., and Griffith, L.C. (2006). Electrophysiological and anatomical characterization of PDF-positive clock neurons in the intact adult Drosophila brain. J. Neurophysiol. 95, 3955–3960. 3. Harrisingh, M.C., Wu, Y., Lnenicka, G.A., and Nitabach, M.N. (2007). Intracellular Ca2+ regulates free-running circadian clock oscillation in vivo. J. Neurosci. 27, 12489–12499. 4. Cao, G., and Nitabach, M.N. (2008). Circadian control of membrane excitability in Ilateral ventral clock neurons. J. Neurosci. 28, 6493–6501. 5. Sheeba, V., Gu, H., Sharma, V.K., O’Dowd, D.K., and Holmes, T.C. (2008). Circadian- and light-dependent regulation of resting membrane potential and spontaneous action potential firing of Drosophila circadian pacemaker neurons. J. Neurophysiol. 99, 976–988. 6. Nitabach, M.N., Wu, Y., Sheeba, V., Lemon, W.C., Strumbos, J., Zelensky, P.K., White, B.H., and Holmes, T.C. (2006). Electrical

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