The higher level of PER (and REPO) in EnGl may indicate the need for stronger circadian regulation of the functions carried out by EnGl, e.g., the ensheathment of R-cells axon bundles. Being the robust circadian oscillators, the EnGl could modulate their capacity to process information in a circadian manner. As they wrap and insulate neuronal axons (Cameron et al., 2016), they increase the conduction velocity of electrical impulses (in both vertebrate and invertebrate nervous systems), and consequently, enhance the neuronal capacity to process information at a faster pace.

Studies on development of the nervous system in Drosophila indicate that this type of glia are able to undergo complex morphological changes to accommodate neuronal axons (Banerjee and Bhat, 2008; Subramanian et al., 2017). In the adult brain, on the other hand, the ensheathing glia (but not astrocytes) express the engulfment receptor Draper, which enables glial membranes to contact degenerating axons and proceed with engulfment of axonal debris. Blocking of endocytosis specifically in the EnGl cells inhibits the process of severed axons clearance (Doherty et al., 2009).

Interestingly, as we showed the EnGl of Drosophila express the DmMANF, a novel evolutionary conserved (in ∼50% identical to human MANF) neurotrophic factor (Petrova et al., 2003) that not only protects neurons against apoptosis and supports their survival (like other neurotrophic factors), but also plays a conserved immune modulatory function (Neves et al., 2016). It has been shown that the silencing of DmMANF in glial cells induces the appearance of so called MANF immunoreactive cells (MiCs) during metamorphosis. These are the migratory cells that closely resemble macrophages/hemocytes and vertebrate microglia. They express RELISH and DRAPER suggestive of their immune response activation (Stratoulias and Heino, 2015b).

FIGURE 6 | (A) Horizontal section of the medulla of NP6520-Gal4/UASmCD8-GFP flies immunolabeled with anti-PER antibody. The nuclei of GFP-expressing EnGl (frames 1–3) are PER-positive, which is well visible in higher magnification (panels 1–3). The processes of EnGl (arrowheads in panels 1–3) span the medula neuropil (Mn). R, retina; Lc, Lamina cortex; Ln, lamina neuropil; oCh, outer chiasm; Mc, medulla cortex; iCh, inner chiasm. Scale bars: 20µm for (A) and 10µm for panel 1, 2, and 3. (B) The nuclei of GFP-positive EnGl show high level of REPO-specific immunofluorescence (arrow). Arrowhead-the EnGl processes. Scale bar: 10 µm. (C) The distal part of the medulla in horizontal section of NP6520-Gal4/UAS-mCD8-GFP flies immunolabeled with anti-DmMANF antibody. The processes of EnGl are marked with arrows. R, retina; Lc, lamina cortex; Ln, lamina neuropil; Mc, medulla cortex; Mn, medulla neuropil. Scale bar: 20µm. (D-D") Higher magnification of EnGl marked in (C). The GFP-positive processes of EnGl (D, arrows) are marked with DmMANF (D',D"). DmMANF is also visible in the perinuclear space of cell bodies in the medulla cortex (D', arrowheads). Scale bar: 10µm.

Thus, the ensheathing glia appear to play the similar role as the highly rhythmic microglia in the mammalian brain (for review see Salter and Stevens, 2017). For example, the hippocampal

microglia have been shown to rhythmically expresses core clock genes: Per1, Per2, Rev-erb, Bmal1, as well as several proinflammatory cytokines. They also display profound differences in immune stimulation throughout the day (Hayashi et al., 2013; Fonken et al., 2015). The strong molecular rhythm of PER oscillations in the Drosophila EnGl definitely adds to its similarity to microglia.

In the Drosophila embryonic nervous system DmMANF can be found only in glia (Palgi et al., 2009), while it is present in both the glia and the neurons of the adult brain (Stratoulias and Heino, 2015a). In glia it is present in both cell somata and processes. In neurons, on the other hand, it can be found only in the somata (Stratoulias and Heino, 2015a). In our recent study (Walkowicz et al., 2017) we found that DmMANF is strongly expressed in the epithelial glial cells (EGl) of the first visual neuropil (the lamina). Here we show, that it is also clearly present in the cell bodies and, most importantly, the processes of EnGl (**Figures 6C,D"**).

The processes of EnGl in the distal medulla are enlarged and form a broad meshwork (Kremer et al., 2017). Since they are closely associated with R7 and R8 photoreceptor terminals, just like the processes of the lamina ensheathing glia (also called marginal glia) with the terminals of R1-R6 photoreceptors (Edwards et al., 2012; Kremer et al., 2017), one might put forth a hypothesis that the EnGl may be involved in the regulation of daily morphological changes in the photoreceptor terminals in the distal medulla. In the Drosophila medulla various types of axons innervate the neuropil unit (column), but they must target distinct layers while using the same tract. Even the subtle structural changes provided by the EnGl should thus enable modulation of the information flow during the day and night, affecting functions of the visual system. In the mammalian retina renewal of the photoreceptor outer segments is regulated in a circadian manner and this process is based on engulfing and phagocytosis of photoreceptors by the neighboring pigmented epithelium (reviewed in Kevany and Palczewski, 2010).

We found that the EnGl express REPO at higher level than the ALGl. It seems important in view of the recent findings (Matsuno et al., 2015) that the expression of REPO (regulated by neuronal protein KLINGON, KLG), just like per expression (Sakai et al., 2004; Chen et al., 2012), is vital for the formation of the long term memory (LTM). Experiments showed that repo mutants are defective for LTM and conditional knockdown of repo under control of ALGl-specific driver line results in reduction of 24 h memory, while it is not affected by similar inhibition in EnGl (Matsuno et al., 2015). It appears, therefore, that only the astrocyte glia is involved in this process. One should take into consideration, however, that this may be also caused by the fact that the EnGl, even without REPO, should still be able to partake in LTM formation, owing to the high level of PER, exceeding that in the ALGl.

The capability of changing morphology and governing the phagocytosis, as well as for the high expression of PER and daily fluctuations in REPO abundance suggest that both morphology and functioning of the EnGl of distal medulla has the capacity to undergo robust daily/circadian changes. The occurrence of daily alterations in glial morphology is known from the study on the epithelial glia of the lamina in the housefly (Pyza and Górska-Andrzejak, 2004). They are associated with oscillations of the axon diameter and the size of dendritic tree in L2 monopolar cells during the day and night (Pyza and Meinertzhagen, 1999; Weber et al., 2009). Moreover, the contribution of glial clocks to the circadian structural plasticity of the pacemaker neurons (the small LNvs) have just been reported by Herrero et al. (2017). The impaired glial clocks abolish circadian structural remodeling without affecting other clock-controlled outputs (Herrero et al., 2017).

# The T1 Interneurons Express PER and REPO at Very Low Levels

Interestingly, the so called T1 interneurons (Hamanaka and Meinertzhagen, 2010) first identified by Fischbach and Dittrich (1989) occurred to display low levels of PER and REPO. Because of the very presence of PER, we cannot rule out the possibility that the T1 interneurons are part of the medulla circadian network. Interestingly, however, these cells are perceived as neuronal anomaly (Takemura et al., 2008) as they fail to make presynaptic sites at chemical synapses in both the lamina and the medulla neuropils (that they innervate). Since their outputs have not been found in detailed EM study, the cells appear to be entirely postsynaptic (Hamanaka and Meinertzhagen, 2010). What we have found about T1 makes this cell even more peculiar (but also more interesting) as it expresses a very low level of the glial marker, REPO (**Figure 5**), in addition to expression of EAAT1 transporter (also often expressed by glia) that mediates the high affinity uptake of glutamate or aspartate into cells. Unveiling its precise function, however, requires further study.

# CONCLUSIONS

Our results revealed that the two types of dMnGl, the astrocyte like glia (ALGl) and the ensheathing glia (EnGl), differ among each other also with respect to the level of expression of the clock protein PER and the glial marker REPO. Since the ALGl glia

#### REFERENCES

controlling circadian behavior show little or no PER expression we conclude that the high levels of PER may not be necessary for the circadian functions in the peripheral oscillators, such as the ALGl. This makes the functioning of the circadian mechanism in the ALGl and other peripheral oscillators even more intriguing. On the other hand, the high level of PER and REPO in the EnGl, as well as the presence of daily changes in the number of cells that express REPO at relatively high levels, may indicate that the EnGl are highly rhythmic, just like their vertebrate counterparts, the microglia. They may constitute the particularly important and/or influential glial component of the Drosophila circadian network and deserve more consideration in future study on the role of glia in the circadian rhythms.

Our study reveal the heterogeneity of glial oscillators and the complexity of the distal medulla circadian network, thus establishing an essential basis for the study of its functioning. We believe that further study should be focused on the typedependent properties of glia in the circadian network and clarification of the EnGl circadian function.

## AUTHOR CONTRIBUTIONS

WK and LW: performed experiments, collected and analyzed data, and prepared the manuscript. AP: performed some experiments, collected, and analyzed data. JG-A: designed the study, performed experiments, collected and analyzed data, and prepared the manuscript.

# FUNDING

The study was supported by the grant K/ZDS/007356 and the Laboratory of Confocal Microscopy in the Institute of Zoology and Biomedical Research, as well as the National Science Centre in Poland (grant number 2014/15/B/NZ3/ 04754).

# ACKNOWLEDGMENTS

The authors express their gratitude for the fly strains that the Department of Cell Biology and Imaging received: to C. Förster (University of Würzburg) for Pdf 0 strain, F. Rouyer (Paris Saclay Institute of Neuroscience) for tim-Gal4 strain, C. Klämbt (University of Münster) for alrm-Gal4 strain, and Bloomington Drosophila Stock Center (USA) for EAAT1-Gal4 (8849), UASmcD8-GFP (5137), UAS-S65T-GFP (1521) lines. The authors also wish to express their gratitude to M. Andrzejak for his assistance in preparation of this manuscript.

Baylies, M. K., Bargiello, T. A., Jackson, F. R., and Young, M. W. (1987). Changes in abundance or structure of the per gene product can alter periodicity of the Drosophila clock. Nature 326, 390–392. doi: 10.1038/326390a0

Beaver, L. M., Rush, B. L., Gvakharia, B. O., and Giebultowicz, J. M. (2003). Noncircadian regulation and function of clock genes period and timeless in oogenesis of Drosophila melanogaster. J. Biol. Rhythms 18, 463–472. doi: 10.1177/0748730403259108

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Krzeptowski, Walkowicz, Płonczynska and Górska-Andrzejak. ´ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Age-Related Changes in the Expression of the Circadian Clock Protein PERIOD in Drosophila Glial Cells

#### Dani M. Long\* and Jadwiga M. Giebultowicz

Department of Integrative Biology, Oregon State University, Corvallis, OR, United States

Circadian clocks consist of molecular negative feedback loops that coordinate physiological, neurological, and behavioral variables into "circa" 24-h rhythms. Rhythms in behavioral and other circadian outputs tend to weaken during aging, as evident in progressive disruptions of sleep-wake cycles in aging organisms. However, less is known about the molecular changes in the expression of clock genes and proteins that may lead to the weakening of circadian outputs. Western blot studies have demonstrated that the expression of the core clock protein PERIOD (PER) declines in the heads of aged Drosophila melanogaster flies. This age-related decline in PER does not occur in the central pacemaker neurons but has been demonstrated so far in retinal photoreceptors. Besides photoreceptors, clock proteins are also expressed in fly glia, which play important roles in neuronal homeostasis and are further categorized into subtypes based on morphology and function. While previous studies of mammalian glial cells have demonstrated the presence of functional clocks in astrocytes and microglia, it is not known which glial cell types in Drosophila express clock proteins and how their expression may change in aged individuals. Here, we conducted immunocytochemistry experiments to identify which glial subtypes express PER protein suggestive of functional circadian clocks. Glial cell subtypes that showed night-time accumulation and day-time absence in PER consistent with oscillations reported in the pacemaker neurons were selected to compare the level of PER protein between young and old flies. Our data demonstrate that some glial subtypes show rhythmic PER expression and the relative PER levels become dampened with advanced age. Identification of glial cell types that display age-related dampening of PER levels may help to understand the cellular changes that contribute to the loss of homeostasis in the aging brain.

Keywords: glia, circadian biology, aging, Drosophila, glial clocks

## INTRODUCTION

Many molecular, cellular, and physiological processes in most organisms are coordinated with the predictable changes of the 24-h solar day. The circadian clock provides the mechanism of time keeping that is based on a negative feedback loop of transcriptional activators and repressors that generate endogenous molecular oscillations of circa 24 h (Hardin, 2011). A core repressor in the clock mechanism is encoded by the gene period (per) and the translocation of the PER

#### Edited by:

Jolanta Górska-Andrzejak, Jagiellonian University, Poland

#### Reviewed by:

Colleen J. Doherty, North Carolina State University, United States Gustavo Bueno Rivas, University of Florida, United States Iris Salecker, Francis Crick Institute, United Kingdom

\*Correspondence: Dani M. Long<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology

Received: 30 September 2017 Accepted: 21 December 2017 Published: 09 January 2018

#### Citation:

Long DM and Giebultowicz JM (2018) Age-Related Changes in the Expression of the Circadian Clock Protein PERIOD in Drosophila Glial Cells. Front. Physiol. 8:1131. doi: 10.3389/fphys.2017.01131

**77**

protein into the cell nuclei followed by its degradation is the fundamental feature of clock function (Hardin, 2011). In both mammals and Drosophila, the circadian system consists of central and peripheral clocks (Hardin and Panda, 2013). Central pacemaker neurons in mammals are located in the suprachiasmatic nuclei (SCN). The central pacemaker neurons driving behavioral rest/activity rhythms consist of a network of about 150 neurons in the Drosophila brain. In addition to these central pacemaker neurons, mammals have intrinsic peripheral clocks in cells of fat tissues, kidneys, liver, and most other organs. Many of these peripheral tissues that express autonomous oscillators coordinate local tissue-specific processes. Similarly to mammals, peripheral clocks are widespread in fly tissues and function independently of the central pacemaker coordinating tissue-specific physiological processes (Giebultowicz, 2001). Within the nervous system, peripheral clocks are present in retinal photoreceptor cells and in other sensory neurons. In addition to neurons, some glial cells rhythmically express circadian clock genes in both mammals (Prolo et al., 2005; Marpegan et al., 2011; Hayashi et al., 2013) and in Drosophila (Ng et al., 2011). Early studies showed that PER protein is expressed in non-neuronal cells (Zerr et al., 1990; Ewer et al., 1992) and suggested that PER expression in these presumed glia is sufficient for manifestation of behavioral rhythmicity (Ewer et al., 1992). However, which glial cell subtypes express the PERbased oscillator and what their roles may be in the timekeeping processes remain poorly understood.

As organisms age, circadian rhythms tend to dampen as demonstrated in behavioral and molecular experiments both in mammals (Reddy and O'Neill, 2010) and Drosophila (Giebultowicz and Long, 2015). This phenomenon is implicated in declining cellular homeostasis and in various pathologies of aging, including altered inflammatory responses (Fonken et al., 2016), neurodegenerative diseases (Musiek et al., 2013) and impaired memory formation (Kondratova et al., 2010). In addition, physiological aging and late life diseases are accelerated by chronic disruption of clock functions in mammals (Kondratov et al., 2006; Antoch et al., 2008; Yu and Weaver, 2011; Hastings and Goedert, 2013). Similar to mammals, mutations in core clock genes accelerate aging phenotypes in Drosophila (Krishnan et al., 2009). Disruptions of the circadian clock in flies predispose them to neurodegeneration, although it is not known which clocks are involved (Krishnan et al., 2009, 2012). It was shown that Drosophila per01 mutants have increased levels of oxidative damage and neurodegeneration compared to agematched controls (Krishnan et al., 2009). However, it is not known to what extent per mRNA is expressed in the glia, and consequently, whether loss of per in these cells could contribute to neurodegeneration and aging in general.

Glial cells play important roles in such processes as neuronal guidance during development, neuronal homeostasis, clearance of damaged tissues, and neurotransmitter recycling (Freeman and Doherty, 2006; Edwards and Meinertzhagen, 2010; Stork et al., 2012). Recent studies implicate mammalian astrocytes in neuroprotection via involvement in toxin clearance from the brain during sleep (Xie et al., 2013) and removal of damaged mitochondria from neurons in the process of transmitophagy (Davis et al., 2014). Glial cells were first classified based upon their location within the brain as surface, cortex, and neuropil glia. Recent classifications in mammals include astrocytes, microglia, oligodendrocytes, and Schwann cells. The Drosophila adult central nervous system (CNS) has five glial subtypes divided into three main categories, namely surface, cortex or neuropil glia.

Surface glia consist of two distinct subtypes, the perineurial and subperineurial glia. Perineurial glia are narrow, oblong cells that make up the outermost covering of the adult CNS (Awasaki et al., 2008). During development, these cells increase their cell division to maintain complete coverage of the adult Drosophila nervous system (Avet-Rochex et al., 2012). The function of this glial cell subtype in the adult fly brain remains largely unknown (Edwards and Meinertzhagen, 2010), but a recent study suggests that perineurial glia may be important in the transport of trehalose (the main energy supplying carbohydrate in insects) across the blood-brain barrier (Volkenhoff et al., 2015). Subperineurial glia are large, flat polyploidal cells (Unhavaithaya and Orr-Weaver, 2012) that reside just underneath the perineurial glia layer. Unlike perineurial glia, subperineurial glia undergo endoreplication during larval development to increase their cell size to maintain coverage of the brain through metamorphosis (Unhavaithaya and Orr-Weaver, 2012). These cells contain several tight septate junctions, form the blood-brain barrier of Drosophila, and separate the CNS from pathogens, xenobiotics, and the high electrolyte content of the hemolymph ultimately protecting neuronal function (Limmer et al., 2014; Weiler et al., 2017). Consistent with these functions, the transcriptome of surface glia is enriched for gene categories associated with drug metabolism, cell adhesion, and various transporters (DeSalvo et al., 2014).

Cortex glia make contact with the subperineurial glia through adherens junctions and envelope neuronal cell bodies that reside in the cortex providing metabolic support to them (Edwards and Meinertzhagen, 2010). One cortex glial cell can cover many neuronal bodies, which gives these cells a mesh-like appearance (Awasaki et al., 2008).

Located below the cortex are two types of neuropil glia, astrocytes and ensheathing glia. Ensheathing glia have a fibrous lamellar morphology (Awasaki et al., 2008) and act as phagocytes of the brain, similar to mammalian microglia. These glia respond to axonal injury through the Draper receptor signaling pathway (Doherty et al., 2009). Astrocyte glial cell bodies are located at the cortex/neuropil border and have projections that are closely associated with neuronal synapses and contain multiple neurotransmitter recycling pathways (Stork et al., 2012). A recent study of the transcriptome of fly astrocytes showed enriched expression of genes involved in metabolism, redox reactions, neurotransmitter synthesis and transport (Ng et al., 2016). RNAimediated knockdown of some of these genes revealed alterations in behavior including changes in activity level, activity onset, and mechanical stress induced paralysis (Ng et al., 2016).

It has been established that some glial subtypes express circadian clock genes in a rhythmic manner. In mammals, both astrocytes (Prolo et al., 2005; Marpegan et al., 2011) and microglia (Hayashi et al., 2013; Fonken et al., 2015) rhythmically express Per1 and Per2 proteins. Cultured astrocytes from Per1::luciferase transgenic rats and knock-in mice are capable of maintaining modest rhythms in circadian clock gene expression that can be entrained by physiologically relevant temperature changes (Prolo et al., 2005). Rhythmic expression of several clock genes was also shown in cortical microglia by qRT-PCR (Hayashi et al., 2013; Fonken et al., 2015). Expression of the circadian clock gene per in glia have been also suggested in flies (Ewer et al., 1992) and this was confirmed more recently although, it is not clear which glial subtypes express PER protein (Ng et al., 2011).

Although impairments of the circadian system are believed to be involved in accelerated aging, little is known about how the circadian clock in different tissues is altered across the lifespan. In Drosophila, PER expression remains robust in central pacemaker neurons (Luo et al., 2012) but is significantly reduced in retinal photoreceptors (Luo et al., 2012; Rakshit et al., 2012). While glia have many important roles in maintaining nervous system homeostasis, it is not known which glial subtypes express the core clock protein PER and whether PER levels remain similar across lifespan or decline with age. To address these questions, we took advantage of the fact that glial subtypes of Drosophila have unique expression patterns and can be labeled separately by GFP via cell-type specific drivers. We performed 2-timepoint immunocytochemical experiments to identify Drosophila glia subtypes that express PER protein and determined that the PER level in these cells is reduced in old fly brains compared to young.

## METHODS

#### Fly Rearing and Genetics

Drosophila melanogaster were maintained on diet containing yeast (35 g/l), cornmeal (50 g/l), and molasses (5%). Temperature was maintained at 25 ± 1 ◦C with a 12:12 h light/dark cycle with fluorescent light of luminous energy of 8 ± 2 µmol m−2 s −1 . We used mated males in all experiments to minimize differences in lifespan, which may vary with sex and mating status. Males were aged in groups of 50 in polypropylene cages (Genesee Scientific, San Diego, CA) inverted over 35 mm petri dish (BD Falcon, San Jose, CA) containing 15 mL of diet. Diet dishes were replaced every 2–3 days. Young (day 5) and old (day 55) males expressing nuclear GFP in specific glial cell subtypes were obtained by crossing w;UAS-GFP with nuclear localization signal (Bloomington Drosophila Stock Center stock 4775) males with females carrying GAL4 drivers expressing in the following glia types: perineurial glia, NP6293-GAL4 (Awasaki et al., 2008); subperineurial glia moody-GAL4 (Schwabe et al., 2005); cortex glia NP577-GAL4 and NP2222-GAL4; ensheathing glia NP6520- GAL4 (Awasaki et al., 2008) and mz0709-GAL4 (Ito et al., 1995); astrocytes alrm-GAL4 (Doherty et al., 2009). UAS-GFP with the nuclear localization signal was chosen to clearly discern nuclear overlap between GFP and PER protein; however, some GFP was also visible in the cytoplasm of glial cells.

#### Immunocytochemistry (ICC)

Flies for brain dissection were collected at Zeitgeber time (ZT) 22 and ZT10 which correspond to high and low levels of PER protein in wild-type flies, respectively (Long et al., 2014). Whole brain mounts were made using established protocol (Long et al., 2014). Brains were incubated for 48 h in primary antibodies 1:500 chicken monoclonal anti-GFP (Aves Laboratories) and 1:10,000 pre-absorbed rabbit anti-PER, rinsed 6 times in phosphate buffered saline with 0.5% Triton-X (Fisher Scientific, Pittsburgh, PA) and incubated overnight in secondary antibodies Alexa Fluor 488 anti-chicken (1:500) and Alexa Fluor 555 anti-rabbit (1:1,000) (Life Technologies). After the final rinse, brains were mounted on microscope slides in Vectashield mounting media with DAPI (Vector Laboratories, Burlingame, California). Images were taken with a Zeiss LSM 780 NLO scanning confocal microscope (Zeiss) with all laser parameters set for optimal signal in young fly brains at ZT22 for each genotype and then held constant while imaging young ZT10 as well as old ZT22 and ZT10 flies of the same genotype. Young and old per01 mutant flies were dissected and stained along with each genotype using the same protocol. Lack of PER staining signal in per01 mutants was used as a negative control.

## Image Analysis and PER Quantification

In order to quantify the relative fluorescence of PER signal at ZT10 and ZT22 in each glia subtype, images were reviewed and maximum intensity projections were created using ZEN 2012 software (Zeiss). Due to their location on the outer surface of the adult Drosophila brain, in order to capture a sufficient number of surface glial cells for measurement, multiple images of nonoverlapping 6µm stacks were captured in several regions of each brain. The area of focus for perineurial glia was the dorsal brain while the surface of the optic lobes was used for subperineurial glial cells. For all other glial subtypes, a single 11µm thick stack from each brain was used for PER signal quantification. PER levels were evaluated by measuring the fluorescence intensity in an average of 15 GFP-positive cell nuclei located in the region of interest specified below. After converting the mean level of fluorescence to the Mean Gray Value the intensity was quantified using Fiji ImageJ software (Schindelin et al., 2012). For each stack, measurements of non-specific background fluorescence were taken from the adjacent areas of similar size as glial cell nuclei (avoiding non-specific red speckles). The background values were averaged and subtracted from the averaged PER measurements obtained from that stack. Five to seven brains were used to measure PER at given time point and age. Statistical significance for average intensity of PER staining between young and old brains at ZT22 was calculated by unpaired t-test with Welch's correction using GraphPad Prism 6 (GraphPad Prism v6.0;GraphPad Software Inc. San Diego, CA). The p- and t-values and the degrees of freedom (df) from these measurements are provided in the results section and in the figure captions.

# RESULTS

We investigated which glia subtypes express the circadian clock protein PER in a manner similar to that of the pacemaker neurons and whether the relative amounts of PER signal change with age. To label specific glial cells, we employed the GAL4/UAS system (Brand and Perriman, 1993) using GAL4 lines to drive GFP expression in subtypes of glia with specific location and function in combination with immunocytochemistry to measure PER levels. It has been reported that PER expression in lateral and dorsal pacemaker neurons are equally strong in young and old Drosophila brains (Luo et al., 2012); therefore, the presence of PER staining in these neurons was used as a positive control. These cells have rhythmic PER expression with high levels at ZT22 and lack PER at ZT10 in wild type flies (Long et al., 2014); therefore, we selected these time points to examine glial cells.

# PER Is Expressed in Both Layers of the Surface Glia

Surface glia consist of two distinct glial subtypes namely perineurial and subperineurial glia. Perineurial glial cells were labeled by crossing UAS-GFP to NP6293-GAL4 driver line, which specifically marks this layer of glia (Awasaki et al., 2008). All GFP-positive nuclei of cells on the dorsal surface of the brain showed PER signal at ZT22 but not at ZT10, suggesting rhythmic expression in the perineurial glia in young brains (**Figures 1A,B**). PER expression persisted in the brains of old flies but the average signal at ZT22 was significantly reduced (**Figure 1C**, p-value < 0.0001, t-value = 8.928, df = 9.171). Subperineurial glial cells were visualized via moody-GAL4 driver line (Schwabe et al., 2005) combined with UAS-GFP. We observed that GFP-labeled cells surrounded the entire brain in both young and old flies. PER signal was quantified in GFP positive subperineurial glia surrounding the optic lobe (**Figures 2A,B**). Although some GFP leaked and was observed in the cytoplasm of the subperineurial glia predominant signal came from their large nuclei. Subperineurial glia showed nuclear PER signal at ZT22 (**Figure 2A**, arrowheads) but not at ZT10 in brains of young flies suggesting rhythmic expression of this protein. PER was discernible from the background in old brains at ZT22 but the average PER signal was significantly reduced compared to young flies (**Figure 2C**, p-value ≤ 0.0031, t-value = 3.719, df =11.56).

## PER Is Weakly Expressed in Cortex Glia at ZT22

Cortex glia surround neuronal cell bodies that reside underneath the surface glia. To visualize cortex glia cells, we used two drivers, NP2222-GAL4 or NP577-GAL4 (Awasaki et al., 2008) combined with UAS-GFP. GFP-positive cells were abundant in both young and old flies having a mesh-like appearance as described previously (Awasaki et al., 2008). Given their large population and prolific distribution in the cortex, we focused on a small subset of GFP-positive cortex glia in the vicinity of the dorsal lateral pacemaker neurons (**Figures 3A,B**). PER signal was detected in both cortex glia lines at ZT22 but not at ZT10 similar to the circadian expression of PER in the lateral neurons (**Figure 3A**). The relative level of PER signal in cortex glia in brains of NP2222-GAL4>GFP flies was much lower than in the lateral neurons located nearby, but was present at ZT22 and not at ZT10 (**Figure 3A**). Analysis of PER signal in cortex glia in this region showed that the average PER levels were significantly reduced in old fly brains compared to young (**Figure 3C**, p-value ≤ 0.0099, t-value = 3.784, df = 5.743).

# Variable PER Expression in the Neuropil Glia

Neuropil glia consists of two morphologically distinct subtypes, the ensheathing glia and astrocytes. Ensheathing glia were visualized via mz0709-GAL4 (Ito et al., 1995) or NP6520- GAL4 (Awasaki et al., 2008) drivers combined with UAS-GFP. PER was detected in GFP-positive cells in both lines; however, mz0709-GAL4 has been reported to drive expression also in the subperineurial glia (Dutta et al., 2016); therefore, NP6520- GAL4>GFP flies were used for PER signal measurement. GFPpositive cells were observed at the border between cortex and several neuropil compartments in the central brain (**Figures 4A,B**). At ZT10, these ensheathing glial cells were negative for PER signal in both young and old brains (not shown); however, many of these cells were PER positive at ZT22 in both ages (**Figure 4A**). The intensity of PER signal was variable from brain to brain and cell to cell and there was no significant difference in average PER signal between young and old brains (**Figure 4C**, p-value = 0.8452, t-value = 0.2017, df =7.961).

Another group of glial cells marked with GFP via NP6520- GAL4 driver was observed in the medulla segment of the optic lobe (**Figures 5A,B**). Based on their position and large oblong nuclei, these cells appear to represent the giant glial cells of the medulla (Tix et al., 1997). Cell nuclei were PER negative at ZT10, but PER was detected at ZT22 albeit with somewhat variable intensity from cell to cell (**Figure 5A**). Nevertheless, the average intensity of PER signal was significantly reduced in these cells in the brains of old files compared to young (**Figure 5C**, p-value ≤ 0.0100, t-value = 3.056, df = 12) indicating that PER levels in these glial cells are reduced as the function of age similar to other glia types discussed above.

A second group of prominent neuropil glia are astrocytes which were visualized via alrm-GAL4 driver (Doherty et al., 2009) combined with UAS-GFP. Interestingly, it appears that alrm-GAL4 marked as GFP-positive the same giant glial cells in the medulla that were also labeled via ensheathing glia NP6520- GAL4 driver. PER was again detected in these cells at ZT22 (**Figures 6A,B**) with the average signal lower in old flies (not shown). This suggests that these cells share features of both ensheathing and astrocyte glial cells.

In mammals, astrocytes located among the central clock neurons in the SCN show robust oscillations in Per-reporter. Therefore, we investigated whether astrocytes located in the central brain neuropil are PER-positive in flies. These cells were marked with GFP via alrm-GAL4 driver and due to the leakiness of nuclear GFP also show some projections that extended into the neuropil (**Figure 6C**). GFP-positive astrocytes were examined in several areas of the central brain but PER protein staining was not detected in any of these cells at either ZT22 or ZT10 while nearby ventral lateral pacemaker neurons were PER-positive at ZT22 as expected (**Figures 6B,C**).

#### DISCUSSION

In this study, we show that most glial cell subtypes of the adult Drosophila CNS express PER in a manner suggesting

that circadian clock may function in these cells. These glial subtypes include perineurial glia, subperineurial glia, cortex glia, ensheathing glia of the central brain and the giant glia located in the medulla. While rhythmic PER expression in the medulla was reported previously (Suh and Jackson, 2007; Gorska-Andrzejak et al., 2009), our data suggest clock function in several other types of glial cells. However, the astrocyte glia appear to be an exception as we did not detect PER protein in these cells at either time point examined.

Our finding that perineurial and subperineurial glia express PER protein is consistent with a recent study of the surface glia transcriptome of adult Drosophila compared to the transcriptomes of all neurons, all glia, and to total brain lysates (DeSalvo et al., 2014). While not the focus of the study, their data do list per mRNA and other circadian clock genes as expressed in the surface glia (DeSalvo et al., 2014). Moreover, the core clock gene Clk was identified as one of the top 50 genes enriched in surface glia when compared to all glia (DeSalvo et al., 2014). The perineurial and subperineurial glia have distinct nonoverlapping roles in the formation and maintenance of the bloodbrain barrier that are not well understood (Awasaki et al., 2008; DeSalvo et al., 2014); the presence of the circadian clock in these cells may help to understand their functioning in the future.

Cortex glia constitute about 20% of the glia in the adult Drosophila brain (Kremer et al., 2017), but this subtype is relatively understudied in flies. Based on our results, cortex glia express PER protein similarly to the pacemaker neurons albeit at a much lower level even in young flies. However, we cannot exclude that cortex glia in other brain regions could show higher PER levels. Cortex glia are presumed to provide trophic

detectable in old brains (\*\*p = 0.0099, t = 3.784, df = 5.743). Number of brains analyzed are shown within each bar; error bars indicate SEM.

support to the neuronal cell bodies they envelop (Edwards and Meinertzhagen, 2010). A recent study supports this idea by demonstrating that genes involved with β-oxidation are expressed in cortex glia suggesting that these cells may generate and transport ketone bodies (Schulz et al., 2015). Cortex glia are known to produce Ca+2 oscillations and disruptions of

these oscillations by mutations in the glial-specific Na+/Ca2+, K + exchanger encoded by zydeco significantly decrease seizure threshold in flies (Melom and Littleton, 2013). While cortex glia are important for neuronal health and function, the role of potentially low amplitude PER oscillations in these cells has yet to be addressed.

Ensheathing glial cells are closely associated with neuronal arborizations and synaptic regions. We determined that these cells express PER in the central brain in Drosophila. It has been reported that the equivalent mammalian cells, the microglia express Per1 and Per2 in a circadian manner (Hayashi et al., 2013; Fonken et al., 2015). The ensheathing glia of Drosophila uniquely express key components of the glial phagocytic machinery such as the engulfment receptor Draper (Doherty et al., 2009). Interestingly, a recent study comparing circadian transcriptome in heads of young and old flies indicated that drpr mRNA show a rhythmic profile in young flies but the rhythm is dampened in old (Kuintzle et al., 2017).

We determined that the astrocyte glia in the central brain in the vicinity of the pacemaker neurons do not express PER. These results are consistent with a previous report that the astrocytes of the central brain are PER/TIM negative (Suh and Jackson, 2007). The absence of PER in fly astrocytes is somewhat unexpected given that mammalian astrocyte cultures from Per1::luciferase transgenic rats and knock-in mice are capable of maintaining modest rhythms in circadian clock genes expression (Prolo et al., 2005; Marpegan et al., 2011). In fact, several recent studies have demonstrated that mammalian astrocytes are important for controlling circadian timekeeping. Astrocytespecific loss of core clock gene BMAL1 (the mammalian ortholog of cycle) via two independent methods was shown to alter circadian locomotor activity whereas expression of clock-associated kinase CK1ε in astrocytes was sufficient to lengthen the period of PER oscillations (Tso et al., 2017). Other recent studies also demonstrated astrocytes roles in circadian timekeeping through glia-neuron communication involving different signaling molecules (Barca-Mayo et al., 2017; Brancaccio et al., 2017). Consistent with the lack of PER in the fly central brain astrocytes, a study of the astrocyte transcriptome did not identify any of the core circadian clock genes (Ng et al., 2016). While astrocytes in both flies and mammals associate with neuronal synapses and have similar star-shaped morphologies and molecular markers, it is conceivable that circadian clock function could have been acquired later in evolution aided by the substantial proliferation of glia in mammals (Kremer et al., 2017).

Little is known about clock-controlled output processes in Drosophila glia. In the lamina of the fly visual system, glial cells show rhythmic changes in volume coordinated with the volume changes in the photoreceptor-contacting interneurons (Gorska-Andrzejak, 2013). These rhythmic changes in structure coincide with the rhythmic expression of the α-subunit of the sodium pump, Na+/K+-ATPase, which is in high abundance in glia and its rhythmic expression is per-dependent, as per01 mutants lack rhythmic expression of this subunit (Gorska-Andrzejak et al., 2009). A recent study implicates glial cell oscillators in the control of Gclc, a rhythmically expressed component of the ratelimiting enzyme in glutathione synthesis (Chow et al., 2016). This study reported that pan glial knockdown of the circadian clock gene cycle via loco-GAL4>cycRNAi was sufficient to significantly decrease rhythmicity of Gclc expression (Chow et al., 2016).

The ubiquitous and rhythmic expression of PER in glia reported here opens more questions regarding the functional significance of glial clocks. The role of glia in locomotor activity rhythms is not clear. Pan-glial knockdown of the circadian clock genes per or cryptochrome via RNAi failed to alter the freerunning behavior of adult Drosophila (Ng et al., 2011). Yet, other studies have shown that Drosophila glial cell functions are required for behavioral rhythmicity (Jackson, 2011; Ng et al., 2011, 2016; Jackson et al., 2015). Finally, pan-glia knockdown of several astrocyte enriched genes can cause significant changes in activity level, sensitivity to mechanical stress, and/or alterations in circadian locomotor activity (Ng et al., 2016).

One of the important findings of our study is that, with the exception of the central brain neuropil glia, all PER expressing glial subtypes display significant age-related decline in PER

#### REFERENCES

protein levels. These data are consistent with the age-related decrease in PER protein reported by Western blot in whole heads (Luo et al., 2012; Rakshit et al., 2012; Kuintzle et al., 2017). Previous immunofluorescent studies detected PER decline in the retinal photoreceptors of old flies (Luo et al., 2012; Rakshit et al., 2012). Our data now show that similar decline occurs in the majority of glial cells. Although the reasons for age-related decrease in PER protein shown here are not known, it could be related to reported reduction of TIM protein in the heads of old flies (Luo et al., 2012; Rakshit et al., 2012). TIM protein is known to be required for PER stability (Hardin, 2011). It is also well known that PER is an essential repressor of CLK/CYC-activated circadian transcription of target genes (Hardin, 2011); therefore, our data suggest that the repressive arm of the circadian clock weakens in glia during aging due to decline of nuclear localized PER protein. Consistent with this hypothesis, a recent RNA-seq study showed that per mRNA expression is higher in the heads of old flies compared to young while protein levels are decreased in old (Kuintzle et al., 2017).

Our detailed analysis of PER expression suggests that circadian clock may function in several glial subtypes. These data should facilitate future functional analysis of glial circadian clocks and their roles in homeostasis of the nervous system. The age-related decline in PER protein expression in various glial subtypes may provide new ways of investigating the physiological processes that decline with age.

#### AUTHOR CONTRIBUTIONS

This study was conceived, analyzed, and written by DL and JG. All experiments were performed by DL.

#### ACKNOWLEDGMENTS

This work was supported by the National Institute of Aging of NIH under award number R01AG045830 and R21AG052950 to JG. The authors also wish to acknowledge the Confocal Microscopy Facility of the Center for Genome Research and Biocomputing at Oregon State University. This publication was made possible in part by award number 1337774 from the National Science Foundation, MRI: Acquisition of Confocal and Two-Photon Excitation Microscope. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. We would like to thank Dr. R Jackson for all NP-GAL4 lines, mz0709-GAL4, and alrm-GAL4, Dr. R. Stanewsky for rabbit anti-PER primary antibody, and Eileen Chow for reading the manuscript.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Long and Giebultowicz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Neuronal and Glial Clocks Underlying Structural Remodeling of Pacemaker Neurons in Drosophila

#### Anastasia Herrero† , José M. Duhart † and Maria F. Ceriani\*

Laboratorio de Genética del Comportamiento, Fundación Instituto Leloir, IIB-BA CONICET, Buenos Aires, Argentina

A number of years ago we reported that ventral Lateral Neurons (LNvs), which are essential in the control of rest-activity cycles in Drosophila, undergo circadian remodeling of their axonal projections. This structural plasticity gives rise to changes in the degree of connectivity, which could provide a means of transmitting time of day information. Thus far, work from different laboratories has shown that circadian remodeling of adult projections relies on activity-dependent and -independent mechanisms. In terms of clock- dependent mechanisms, several neuronal types undergoing circadian remodeling hinted to a differential effect of clock genes; while per mutants exhibited poorly developed axonal terminals giving rise to low complexity arbors, tim mutants displayed a characteristic hyper branching phenotype, suggesting these genes could be playing additional roles to those ascribed to core clock function. To shed light onto this possibility we altered clock gene levels through RNAi- mediated downregulation and expression of a dominant negative form exclusively in the adult LNvs. These experiments confirmed that the LNv clock is necessary to drive the remodeling process. We next explored the contribution of glia to the structural plasticity of the small LNvs through acute disruption of their internal clock. Interestingly, impaired glial clocks also abolished circadian structural remodeling, without affecting other clock-controlled outputs. Taken together our data shows that both neuronal and glial clocks are recruited to define the architecture of the LNv projections along the day, thus enabling a precise reconfiguration of the circadian network.

Keywords: circadian remodeling, structural plasticity, LNvs, cell autonomous clocks

#### INTRODUCTION

Plasticity—the ability to make adaptive changes—is an integral property of the nervous system. There are numerous examples of functional and structural plasticity in invertebrates and vertebrates (Holtmaat and Svoboda, 2009; Bozorgmehr et al., 2013), highlighting its relevance. Plasticity occurs at different scales in time and structure, ranging from milliseconds to hours and from dendritic spines and axonal boutons to entire axonal and dendritic arbors. Structural plasticity contributes to synaptic and circuit function, and it is affected during aging (Barnes, 2001) and disease (Bernardinelli et al., 2014); despite their relevance, the mechanisms underlying structural plasticity, especially large-scale terminal remodeling in the adult brain remains elusive. Over the years, examples of structural remodeling of neuronal terminals taking place along the day have accumulated (Pyza and Meinertzhagen, 1999; Mehnert et al., 2007; Becquet et al., 2008; Fernández et al., 2008; Appelbaum et al., 2010), and have been shown to depend on an intact circadian clock (Fernández et al., 2008).

#### Edited by:

Jolanta Gorska-Andrzejak, Jagiellonian University, Poland

#### Reviewed by:

Ezio Rosato, University of Leicester, United Kingdom Monika Stengl, University of Kassel, Germany

#### \*Correspondence:

Maria F. Ceriani<EMAIL_ADDRESS>†Co-first authors.

#### Specialty section:

This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology

Received: 18 September 2017 Accepted: 30 October 2017 Published: 14 November 2017

#### Citation:

Herrero A, Duhart JM and Ceriani MF (2017) Neuronal and Glial Clocks Underlying Structural Remodeling of Pacemaker Neurons in Drosophila. Front. Physiol. 8:918. doi: 10.3389/fphys.2017.00918

**87**

The circadian clock is conserved throughout the animal kingdom. In Drosophila, 150 neurons in the adult brain support a circadian pacemaker. This molecular clock depends on the activity of the transcription factors CLOCK (CLK) and CYCLE (CYC), which drive circadian oscillations by promoting rhythmic transcription of several key genes, including period (per), timeless (tim), and clockwork orange (cwo), which repress CLK-CYC-mediated transcription (Ozkaya and Rosato, 2012). The coordinated operation of the circadian network is necessary for the adapted responses to synchronizing environmental stimuli. Clock neurons are anatomically clustered in distinct groups: small and large ventral-lateral (s-LNvs, l-LNvs, and the fifth s-LNv), the dorsal-lateral (LNds), the lateral posterior (LPNs) and three subgroups of dorsal neurons (DNs1-3). Only the LNvs express a neuropeptide called PIGMENT DISPERSING FACTOR (PDF), which plays a major role in the synchronization of the circadian network. PDF is essential for normal circadian activity patterns in light: dark cycles (LD) and for persistent circadian rhythms under constant free running conditions (DD). In fact, PDF synchronizes the phase of the sLNvs and DN1s, while slows down the pace and increases the amplitude of the LNds and the PDF negative 5th sLNv (Lin et al., 2004; Lear et al., 2009; Yoshii et al., 2009; Im et al., 2011).

PDF immunoreactivity changes throughout the day in the sLNv axonal termini, indicating that its regulation is under clock control. In addition, the sLNv axonal terminals exhibit a higher degree of arborization during the day and a reduced complexity at night, accompanying the changes in PDF levels. This phenomenon, called circadian structural plasticity, is lost in per01 and tim01 mutants, indicating that it depends on a functional clock, although substantial structural differences are observed (Fernández et al., 2008).

Glial cells have a critical role in plasticity and synaptic transmission.  Despite little is known about Drosophila circadian gliotransmitters, there is vast evidence of their relevance in circadian rhythmicity (Ng et al., 2011; Ng and Jackson, 2015). Several studies have reported rhythmic expression of clock proteins and other neural proteins (e.g., PER, TIM, Ebony) in glial cells of the adult brain (Suh and Jackson, 2007), although the importance of glial clocks in circadian outputs has not been extensively studied yet, prompting us to analyze its relevance in structural plasticity.

Given the implications to the circadian network of an active sLNv terminal remodeling, i.e., daily changes in connectivity (Gorostiza et al., 2014), we inquired whether affecting the molecular clock, particularly in the adult LNvs, would abolish circadian plasticity. To this end we deregulated specific clock genes and analyzed the impact of these genetic interventions on structural remodeling; interestingly, despite altering different clock genes similarly affected molecular oscillations, the terminals adopted a different configuration, suggesting that additional mechanisms are recruited. We next addressed the possibility that the clock in glial cells actively contributes to the structural plasticity of sLNv terminals, and uncovered that adult-specific impairment of their molecular clock also disrupts circadian remodeling. Thus, both, the molecular clock in LNvs and glia are necessary for sustaining this unusual form of plasticity.

# MATERIALS AND METHODS

### Fly Rearing and Strains

Flies were grown and maintained at 25◦C in vials containing standard cornmeal medium under 12:12 h LD cycles. For adult specific induction either the GeneSwitch or TARGET systems were employed (McGuire et al., 2004). GeneSwitch expression was induced transferring 2 day-old flies to vials containing food supplemented with RU486 (mifepristone, Sigma, USA) in 80% ethanol to a final concentration of 200µg/ml, or with the same amount of ethanol (vehicle) in control treatments. Adultspecific termosensitive Gal4 expression was induced transferring flies raised at 23◦C during development to 30◦C for 48 h. The pdf-GeneSwitch (pdf-GS) line was generated in our laboratory (Depetris-Chauvin et al., 2011); Stocks UAS-cycDN (#36317, Tanoue et al., 2004), UAS-timRNAi I (#29583), UAS-perRNAi (#40878 and #31285, I and II, respectively), pdf-Gal4 (#6900), repo-Gal4 (#7415) were obtained from the Bloomington Stock Center. The UAS-timRNAi II (#2886) stock was obtained from the Vienna RNAi Stock Center. pdf-dsRed was generously provided by J. Blau.

#### Adult Locomotor Activity

For locomotor activity experiments adult male flies were entrained for 3 days in 12:12 LD cycles at 25◦C and then transferred to constant darkness (DD) at 25◦C. Males were placed in glass tubes containing standard food and monitored for activity with infrared detectors and an automated data collection system (TriKinetics, Waltham, MA). Activity was monitored for 14 days (4 in LD and 9–10 in DD). Period, FFT and rhythmicity in DD were estimated using ClockLab software (Actimetrics, Evanston, IL) as previously described (Ceriani et al., 2002; Depetris-Chauvin et al., 2011).

# Immunohistochemistry and Image Acquisition

Adult fly heads were fixed with 4% p-formaldehyde (pH 7.5) for 30–40 min at room temperature. Brains were dissected and rinsed four times in PT buffer (PBS with 0.1% Triton X-100) for 30 min. Samples were blocked in 7% normal goat serum (in PT) for 1 h, and incubated with primary antibodies at room temperature for 2 days. The primary antibodies employed were chicken anti-GFP 1:500 (Aves Labs, Inc, USA), rabbit anti-DsRed 1:500 (Clontech, USA) and homemade rat anti-Drosophila-PDF 1:500 (Depetris-Chauvin et al., 2011). Samples were washed 4 x 15 min in PT, and incubated with secondary antibody at 1:250 for 2 h at room temperature. Secondary antibodies were washed 4 x 15 min in PT and mounted in Vectashield antifade mounting medium (Vector Laboratories, USA). The secondary antibodies used were Cy2-conjugated donkey anti-rabbit, Alexa Fluor 647-conjugated AffiniPure donkey anti-rat and Cy3-conjugated AffiniPure donkey anti-rabbit (Jackson ImmunoResearch, USA). Images were taken on a Zeiss LSM 710 confocal microscope.

# Structural Plasticity Analysis and PDF Immunoreactivity

Images were taken with a 40× objective and an optical zoom of 2×. CD8GFP signal was adjusted to threshold levels generating a selection that delimits the area of sLNv axonal terminals. This selection was then applied to the PDF channel and mean intensity was measured. For the analysis of PDF immunoreactivity all pictures were taken employing the same confocal settings and quantification was performed using Image J software (downloaded from http://rsbweb.nih.gov/ij/). Structural plasticity was analyzed by Scholl analysis, as reported (Fernández et al., 2008). In all cases the analysis was performed blind.

## Quantitative Real-Time PCR

Total RNA isolation from fly head extracts was performed using Trizol (Invitrogen, Carlsbad, CA). Superscript III was used for reverse transcription (ThermoFisher Scientific, USA) and FastStart Universal SYBR Green Master (Roche) was used for quantitative real-time PCR following manufacturer's instructions. The real-time assays were conducted in a Stratagene Mx3000P QPCR System (La Jolla, CA) using SYBR green as the detection system and ROX as the reference dye. The primers were designed using Primer3 (available online at http://frodo. wi.mit.edu/primer3/). mRNA levels were assessed from three independent RNA extractions and two technical replicates were performed on each sample. Only primer pairs with efficiency between 90 and 110% were used. The following primers were employed, to detect rpl49 (fw 5′GAACAAGAAGGCCCATCG TA3′ ; rev 5′AGTAACAGGCTTRGGCTTGC3′ ); per (for 5′GAC CGAATCCCTGCTCAATAA3′ ; rev 5′GGACTTCTTGCTCTT CTCACC3′ ); tim (fw 5′GGTAAACGGATCGCACTTCTCG3′ ; rev 5′AAGAGACATTGTCGCTGTTTAAT3′ ); dClk (fw 5′CAG AGTCAGTTGCAGGATCAA3′ ; rev 5′GCAGATATGTGTAGC GGGATAG3′ ); cyc (fw 5′TGGACAATCACCCGAACATAC3′ ; rev 5′CTGAGGCAGGAAACCAATCA3′ ).

# Data Analysis and Statistics

Statistical analyses were performed with the InfoStat package version 2009 (Grupo InfoStat, FCA, Universidad Nacional de Córdoba, Argentina). In all graphs, experimental groups with different letters indicate statistically significant differences, with a p < 0.05. Validation of RNAi lines was tested with a Student's ttest. Clock gene oscillation under different clock modulations was analyzed by One-way ANOVA within each clock gene, followed by Tukey's post-hoc test. Effects on structural plasticity were analyzed by Two-way ANOVA, followed by Tukey's post-hoc test. Effects on PDF levels were analyzed by Kruskal–Wallis One way ANOVA, followed by Conover's post-hoc test. Number of flies or brains in each experiment is referred as n, and the number of experiments is referred as N, and was used for statistical analysis.

#### RESULTS

#### Disrupting the Molecular Clock in the Adult LNvs Impairs Locomotor Rhythmicity

To address the possibility that the circadian remodeling of sLNv terminals exclusively depends on their own molecular clock we took advantage of the spatially restricted Gal4/UAS system to alter clock protein levels exclusively in the LNvs. To monitor the efficiency of the different strategies and potentially uncover a differential effect, we first analyzed locomotor activity patterns in flies in which different clock genes were constitutively deregulated through RNAi-mediated silencing (per, tim), or through the expression of a dominant-negative CYC version (CYCDN, Tanoue et al., 2004).

Control (pdf-Gal4>+) flies showed a clear rhythmic pattern in the presence of synchronizing cues (LD cycles) as well as in DD (**Figure 1A**). In DD this rhythm has a period of around 24 h and flies consolidate their activity across the subjective day (**Table 1**). Downregulating per and tim mRNA levels in the LNvs (through pdf-Gal4) employing different RNAi lines triggered significant effects on the patterns of locomotor activity, mostly a ∼80% decrease in the percentage of rhythmicity (**Figure 1B**); surprisingly, period length was not different from the control (Supplementary Figure 1 and **Table 1**). We next measured steady state levels of per and tim mRNA by quantitative real-time PCR in total RNA extracts from wild type (tim-Gal4,dcr2>+) and flies expressing specific RNAis (perRNAi and timRNAi) at the peak of their endogenous levels (CT14, CT stands for circadian time, and refers to the hours passed since the last day to night transition). Significant differences were observed, with a decrease to about 30% compared to wild type levels both in the case of per and tim mRNA (**Figure 1C**). To overcome potential unspecific effects of the knockdown strategy, we employed a second RNAi line that showed similar results (Supplementary Figure 1). Thus, both RNAis are efficient to downregulate clock protein levels and affect clock outputs (i.e., behavioral rhythmicity).

In addition, we took advantage of a well-characterized dominant negative variant of CYC, called CYCDN (Tanoue et al., 2004) to block CLK/CYC activated transcription. As previously reported, expressing cycDN in the LNvs produced a drastic reduction of behavioral rhythmicity (**Figures 1A,B**; see **Table 1** for an in depth analysis of behavioral parameters) down to about 14%, suggesting this strategy is a very efficient one to impair clock function (Tanoue et al., 2004). Taken together, these experiments support the relevance of LNvs in the control of rhythmic restactivity cycles since deregulating different core clock genes in a cluster-specific fashion significantly impacts the consolidation of rhythmic locomotor behavior.

#### Modulation of Different Clock Components Leads to Dampened Oscillations

Rhythmicity of clock-controlled outputs depends on the precise regulation of the cell autonomous molecular clock. Since affecting different clock protein levels in the LNvs altered behavioral patterns to a different degree we assessed the state of the molecular clock after long term deregulation of each specific clock protein. To this end we examined per, tim, dClk, and cyc

cycles and then transferred to DD (gray area) for 9 additional days.  For every fly actogram, periodograms of the free-running rhythm in DD are shown (bottom). Rhythmic behavior of a typical male control is shown (orange). perRNAi (in blue), timRNAi (in pink), and cycDN (in green) expressing animals are shown. All genetic manipulations gave rise to a largely arrhythmic locomotor behavior. (B) Percentage of rhythmicity. Data represents at least three independent experiments; over 44 flies were analyzed. (C) Validation of the effectiveness of the RNAis employed. Both UAS-perRNAi and UAS-timRNAi were expressed under tim-Gal4. Levels are normalized to rpl49. Both tim and per mRNA levels are reduced compared to their respective control. Student's t-test showed a significant difference in levels of expression. Triple asterisks (\*\*\*) indicate significant differences with p < 0.001. Three independent experiments were performed.

mRNA levels at two timepoints during the day, CT2 and CT14, upon deregulation of the different genes using the pan-circadian driver tim-Gal4.

Interestingly, we found that the amplitude of the peak/trough oscillation of per or tim transcript levels was greatly reduced in the conditions tested regardless of the clock protein targeted, that is, as a result of impairing repression or activation (Supplementary Figure 2 and **Figure 2**). As expected (Lerner et al., 2015), dClk mRNA levels were low compared to those of per and tim in controls, which could partially account for the difficulty in determining a precise change in peak/trough amplitude (only two-fold in this set of measurements for the controls). Accordingly, a clear effect on the amplitude of dClk oscillations was detectable in timRNAi and cycDN. In addition, despite cyclevels were originally reported not to cycle by northern blot (Rutila et al., 1998), qPCR analysis showed a shallow (threefold) cycling in endogenous cyc levels in controls (higher at CT14 than CT2), that were not significantly affected in any of the combinations analyzed (Supplementary Figure 2).

In sum these results highlight that deregulation of different clock components results in a dampened molecular clock, regardless of the affected process (that is, impaired CLK/CYC mediated transcription or its repression), and confirm that any of these genetic interventions are useful to assess the relevance of the cell autonomous clock on a particular output, i.e., the control of the structural plasticity of the sLNv terminals.

# Circadian Structural Plasticity Is Differentially Altered by Clock Genes

A number of years ago we reported morphological changes of the axonal terminals of the sLNvs across the day and showed that this phenomenon is under the control of the circadian clock since it is abolished in per01 and tim01 mutants (Fernández et al., 2008). However, despite circadian changes in the complexity of the sLNv axonal arbor was abrogated in both null mutants, the overall morphology of the terminals was quite distinct, suggesting PER and TIM could be playing additional "non-circadian" roles. Thus, we set out to examine the impact of altering different molecular clock components specifically in the LNvs on the architecture of the dorsal terminals exclusively in the adult brain to avoid potential developmental effects.

We took advantage of an inducible Gal4 version termed GeneSwitch with restricted expression to the LNv neurons (pdf-GS, Depetris-Chauvin et al., 2011), combined with a membranetethered version of GFP (CD8GFP) to describe the complexity of the axonal arborizations, along with perRNAi , timRNAi, or cycDN to address the role of the LNv clock in structural plasticity. Flies transferred to RU486-containing food 2 days after eclosion were dissected at CT2 and CT14 on DD3 (**Figure 3A**). As previously reported, the overall structure of the dorsal terminals is more complex in the morning and less arborized at night time in controls (**Figures 3B,C**, shown in orange; Fernández et al., 2008; Gorostiza et al., 2014). Interestingly, affecting the negative elements of the molecular clock, per (shown in blue) and tim (in pink), the complexity was significantly reduced from that displayed by controls in the subjective morning (**Figures 3B,C**), and resembled the nighttime configuration of control terminals. On the other hand, expressing cycDN (in green) gave rise to maximally spread axonal termini throughout the day; in fact, the architecture of the termini (to the level described through confocal microscopy) was different from that of controls at any timepoint; specifically, the number of higher order neurites (ramifications of primary and secondary processes) was clearly increased compared to controls, suggesting that actively impairing CYC function in the adult triggers clear morphological defects, beyond those anticipated from affecting the endogenous molecular clock.

PDF immunoreactivity in the axonal terminals at the dorsal protocerebrum has been shown to oscillate in a circadian fashion both under LD and DD conditions (Park et al., 2000); remarkably, this cycling is blocked in mutants with impaired clock function (Park et al., 2000). Immunohistochemistry analysis on wholemount adult brains dissected at times when PDF levels peak and reach a trough was examined. PDF levels were assessed at CT2 and CT14 upon adult- specific expression of either

TABLE 1 | Detailed circadian parameters for all the behavioral experiments performed.

The analysis included the assessment of period (Tau) and different measurements of rhythm strength, such as FFT, power, and percentage of rhythmicity (%R). Average ± S.E.M. of different clock deregulating genotypes along with the control is shown. Different letters indicate statistically significant differences with a p < 0.05 (One-way ANOVA with a Tukey post-hoc test). N indicates the number of experiments, which was used for statistical analysis. n indicates the number of animals tested. perRNAi I (Bloomington Stock Center: #40878) and timRNAi I (Bloomington Stock Center: #29583) refers to the RNAis used in the main figures. The ones indicated in gray correspond to the RNAi lines shown in Supplementary Figure 1, as follows: perRNAi II (Bloomington Stock Center: #31285) and timRNAi II (VDRC Stock Center: #2886).

perRNAi , timRNAi, or cycDN along with controls. Control pdf-GS > CD8GFP flies (orange) in the presence of RU486 exhibited a significant difference in PDF immunoreactivity between these two time points (**Figure 3D**). In contrast, PDF immunoreactivity at the sLNv dorsal terminals in every experimental condition was significantly different to controls. Such difference was more pronounced when expressing perRNAi (blue) or timRNAi (pink), in which the amplitude of the oscillation is markedly reduced, even when compared to cycDN (green).

In sum, these experiments demonstrate that circadian remodeling of the sLNv terminals is driven by the LNv molecular clock. Furthermore, affecting the positive and negative elements of the feedback loop triggered a distinctive "architecture" of the axonal termini. Under these conditions, not only structural plasticity but also PDF levels are altered, indicating that both outputs are dependent on the correct operation of the LNv molecular oscillator.

#### Glial Clocks Also Contributes to Circadian Remodeling of the sLNv Terminals

Having demonstrated that the LNv clock is necessary for the remodeling of the sLNv termini, we wondered whether additional clocks could contribute to this phenomenon. One evident candidate is the one in glial cells, which plays a role in rhythmic locomotor behavior (Ng et al., 2011); in addition, it is well established that astrocytes modulate the activities of many different neuronal synapses, further strengthening this possibility. Flies expressing cycDN, in our hands the most effective means to block CLK/CYC activated transcription (**Figures 1**, **2**), was employed to acutely interrupt the glial clock in the adult brain.

At the restrictive temperature (23◦C, **Figure 4A**), where no expression of the dominant negative CYC is achieved, repo-Gal4;tub-Gal80TS >cycDN flies exhibited the expected remodeling of the sLNv terminals, more elaborated during the subjective day than at night (**Figures 4B,C**). In contrast, disrupting the clock in glia led to the absence of circadian remodeling, resulting in a minimally spread arbor, reminiscent of controls at night (**Figures 4B,C**). Controls shifted to the permissive temperature still displayed circadian remodeling (Supplementary Figure 3). These observations bring further support to the notion that glial clocks play an active role regulating circadian clock outputs, and more specifically, are essential for circadian structural plasticity.

We also explored PDF immunoreactivity in the dorsal protocerebrum, given its established relevance to this form of plasticity (Depetris-Chauvin et al., 2014). Surprisingly, we found that PDF levels still change between the subjective day and night when the clock in glia is impaired; in fact, under these conditions PDF levels were not different from controls (**Figure 4D**), providing further evidence that these two clock outputs (PDF levels and structural remodeling) can be uncoupled (Depetris-Chauvin et al., 2011).

Together these results indicate that circadian structural plasticity of the sLNvs depends not only on its own molecular clock, but also that glial clocks actively contribute to this form of plasticity.

#### DISCUSSION

A number of years ago we discovered that in wild type flies there are conspicuous structural changes in neurons that are key to the control of circadian locomotor activity (the sLNvs), which undergo remodeling of their axonal arborizations on daily basis (Fernández et al., 2008). Those initial observations led to additional discoveries, namely, that circadian remodeling involves changes in the number of synapses and connectivity, and concomitantly, that sLNvs neurons contact different postsynaptic targets across the day (Gorostiza et al., 2014); thus, structural plasticity results in changes in the strength of the communication between circadian clusters that could contribute to seasonal adaptation (Gorostiza et al., 2014; Petsakou et al., 2015). Circadian structural remodeling has been observed in clock brain structures and in other brain regions receiving input from the circadian clock (Bosler et al., 2015); interestingly, it has been shown to coexist with circadian changes in neuronal excitability and synaptic efficacy within and outside clock structures, but the precise relationship between these two forms of plasticity remains poorly understood (Frank and Cantera, 2014).

### Cell Autonomous Mechanisms Underlie Continuous Remodeling of Neuronal Terminals

Rhythmic changes in neuronal morphology reported thus far in Drosophila include those in axonal caliber, branching complexity, synaptic vesicles and synapse numbers. One of the structures undergoing daily and circadian remodeling is the visual system that shows changes in the number of synaptic contacts as well as in the morphology of neurons and glial cells (Pyza and Meinertzhagen, 1999; Weber et al., 2009; Damulewicz et al., 2013; Gorska-Andrzejak et al., 2013); likewise, cyclical changes in neuronal morphology are exhibited by motor neurons in the adult (Mehnert et al., 2007; Mehnert and Cantera, 2008; Ruiz et al., 2010, 2013), as well as by interneurons in the central brain (Fernández et al., 2008).

In terms of the underlying molecular processes that trigger structural changes not much is known; activity dependent and independent mechanisms appear to be recruited to drive terminal remodeling in the central brain (Depetris-Chauvin et al., 2011; Sivachenko et al., 2013), associated to changes in the degree of fasciculation (Sivachenko et al., 2013), pruning (Depetris-Chauvin et al., 2014), and actin cytoskeleton remodeling (Petsakou et al., 2015), although the timing of these events has not been explored in any length.

As it is the case for many clock-controlled outputs, it was expected that at least part of the molecules responsible for orchestrating active structural remodeling show circadian modulation of gene expression, protein stability and/or activity. Consequently, circadian remodeling would be directly controlled by a cell-autonomous circadian clock, i.e., the one operating in those specific neurons, a possibility that had not been specifically examined yet. Thus, to examine whether the LNv molecular clock is necessary to drive structural remodeling we resorted to different genetic strategies to obliterate molecular oscillations. Surprisingly, while adult-specific downregulation of the repressors of the molecular clock (through the expression of perRNAi and timRNAi) resulted in less complex arborization patterns, impairing CLK/CYC mediated transcriptional activation (through the expression of cycDN) correlated with maximally spread terminals, despite an overall similar effect on clock genes at the mRNA level. Closer inspection of the architecture of the arborizations suggests that additional phenomena are also affected; particularly in the case of CYCDN expression, not only the terminals are complex throughout the day as controls exhibit in the early (subjective) morning, but also membrane integrity appears severely disrupted unlike neuronal terminals in controls. Additional experiments are required to understand this process in full; one obvious candidate that could mediate the altered morphology is the circadian modulation of actin dynamics (Petsakou et al., 2015) that directly impacts on structural integrity and plasticity of neurons and their synapses. These results underscore that the circadian clock not only drives circadian remodeling but it could additionally play an active role in maintaining neuronal shape (Mehnert and Cantera, 2011).

## Distinct Long Term and Acute Effects Derived from Clock Disruption

While addressing the circadian nature of the remodeling phenomenon in different structures it became clear that loss-offunction mutations in per and tim not only abolish circadian remodeling but also trigger abnormal branching, which in turn would suggest that circadian plasticity plays a role in maintaining normal morphology (Mehnert and Cantera, 2011). Incidentally, per and tim null mutants showed quite distinct phenotypes in the architecture of the neuronal terminals (Mehnert et al., 2007; Fernández et al., 2008), suggesting that both proteins could play additional roles than those limited to the circadian clock. To delve further into this possibility,

we downregulated per or tim levels in the post-developmental brain, once the whole circadian network was established and well connected. Interestingly, adult-specific downregulation of either gene resulted in a similar structure, which at all times resembled the less complex arborization pattern exhibited by controls at night. These results reinforce the notion that the structural differences associated to loss of function mutations could uncover additional processes in which these proteins participate during early development or during establishment of the circadian circuitry.

# The Contribution of Glial Clocks to LNv Structural Plasticity

Over the years it was established that glia, and particularly neuron-glia communications, plays an active role in the control of rhythmic outputs, affecting PDF immunoreactivity at the dorsal terminals and concomitantly, rhythmic behavior (Suh and Jackson, 2007; Ng et al., 2011; Ng and Jackson, 2015). Surprisingly, affecting glial clocks per se did not impact on rhythmic patterns of locomotor activity, at least upon chronic downregulation of PER levels (Ng et al., 2011).

In the visual system circadian remodeling of neuronal terminals is likely driven by different circadian oscillators, and takes place in photoreceptor cells, the most abundant peripheral oscillator in the fly head, but also in non- clock cells such as the L1 and L2 monopolar neurons in the lamina (Weber et al., 2009). In the latter, circadian input driving remodeling likely derives from the photoreceptors, the PDF+ central clock neurons, as well as the surrounding glia (reviewed in Gorska-Andrzejak, 2013). Structural changes also correlate with changes in the abundance of a marker of presynaptic active zones (Bruchpilot, BRP; Gorska-Andrzejak et al., 2013). Interestingly, it was reported that blocking clock function in glia alters, though it does not obliterate, the daily changes in BRP accumulation in the lamina cartridges (Gorska-Andrzejak et al., 2013). In the house fly, glial cells change in size in the opposite phase compared to neurons, and remodeling is affected either when perturbing glial metabolism or, more dramatically, glial communication (Pyza and Gorska-Andrzejak, 2004).

To begin to assess whether clocks in glia would contribute to the structural remodeling of the LNv projections, panglial CYCDN expression was restricted to the adult. Interestingly, acute (for 2 days) disruption of glial clocks completely abolished circadian plasticity, underscoring their active contribution to the remodeling process. However, under those conditions, PDF immunoreactivity at the dorsal terminals exhibited no differences compared to controls, unexpectedly uncoupling both clock outputs. Despite a subtle effect derived from a short term blockage of CLK/CYC function cannot be ruled out, our results suggest that structural remodeling of the LNv terminals is even more sensitive to the alterations in glial physiology than PDF levels themselves.

Glia-to-neuron communication actively participates in the circadian regulation of terminal remodeling despite the mechanisms remain to be uncovered. One possible scenario would depend on circadian release of gliotransmitters, as it has been shown to take place in mammalian astrocytes (i.e., ATP; Burkeen et al., 2011; Marpegan et al., 2011), or other ligands known to mediate neuro-glial communication [obvious candidates to test belong to the Fibroblast growth factor (FGF) and Bone morphogenetic protein BMP signaling pathways; Awasaki et al., 2011; Fuentes-Medel et al., 2012; Stork et al., 2014]. These molecules could alter excitability of the neuronal terminals, ultimately affecting activity-dependent mechanisms known to be required for structural plasticity (Sivachenko et al., 2013), or be more directly involved in the remodeling process.

Circadian structural remodeling has also been described in the mammalian suprachiasmatic nucleus (SCN). Interestingly, antiphasic cyclical changes in glial coverage of VIP and AVP neurons were reported in the rat SCN and were proposed to contribute to synchronization of the clock to the light-dark cycle (Becquet et al., 2008; Girardet et al., 2010). Although, little is known about circadian structural remodeling in the mammalian brain the pervasive conservation of the mechanisms underlying the molecular clock as well as those underlying synaptic plasticity would predict conservation on this phenomenon as well.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

AH, JD, MFC designed experiments. AH, JD performed and analyzed experiments. AH, JD, MFC wrote the manuscript.

#### ACKNOWLEDGMENTS

We are indebted to members of the Ceriani lab for helpful discussion. We would like to specially thank Justin Blau (NYU. USA), the Bloomington (USA) Stock Center and the Vienna Drosophila Resource Center for fly stocks. MFC is a member of the Argentine Research Council (CONICET). AH and JD are supported by fellowships from CONICET. This work was supported by a grant from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Argentina, PICT2015-2041 (to MFC) and by a FIRCA-NIH grant (1R03TW008342) to MFC. The funders had no role in study design, 8 data collection and analysis, decision to publish, or preparation of the manuscript.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2017.00918/full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Herrero, Duhart and Ceriani. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Dynamic Neuron-Glia Interactions in an Oscillatory Network Controlling Behavioral Plasticity in the Weakly Electric Fish, Apteronotus leptorhynchus

#### Günther K. H. Zupanc\*

Laboratory of Neurobiology, Department of Biology, Northeastern University, Boston, MA, United States

The involvement of glial cells in the regulation of physiological functions is being increasingly recognized, yet their role in plasticity of neural oscillators has remained largely elusive. An excellent model system to address the latter function is the pacemaker nucleus of the weakly electric fish, Apteronotus leptorhynchus. This brainstem oscillator drives the fish's electric organ discharge in a one-to-one fashion, with median frequencies of 880 Hz in males and 740 Hz in females. Morphometric analysis of the pacemaker nucleus has shown that astrocytes outnumber mature neurons seven-fold, and oscillator neurons even 200-fold. A similar dominance of astrocytes occurs among the adult-born cells that differentiate into glia and neurons. The astrocytes form a dense meshwork of cells interconnected by gap junctions. The degree of association of astrocytic fibers with the neural oscillator cells, and the gap-junction coupling between individual astrocytes, exhibit a sexual dimorphism, which parallels the sexual dimorphisms in the output frequency of the pacemaker nucleus, and ultimately in the electric organ discharge of the fish. It is hypothesized that the dynamics in astroglial structure mediate differences in the capacity to buffer potassium, which increases during the generation of action potentials. These differences, in turn, affect the excitability of the neural oscillator cells, and thus the output frequency of the pacemaker nucleus. Comparison of the pacemaker nucleus with other brain oscillators suggests that modulation of the output activity is one of the chief functions of the interaction of glia with the neural oscillator cells.

Keywords: neural oscillator, neuron-glia interaction, plasticity, astrocytes, gliogenesis, structural reorganization, pacemaker nucleus, Apteronotus leptorhynchus

# INTRODUCTION

Since Maiken Nedergaard and Philip G. Haydon and associates reported in two independent papers in 1994 that stimulation of astrocytes results in elevated levels of Ca2+ in adjacent neurons (Nedergaard, 1994; Parpura et al., 1994), a plethora of studies have led to the notion that glial cells, particularly astrocytes, play an active role in the modulation of physiological functions of neural circuits. These studies have indicated that the modulatory actions of astrocytes can be grouped into two broad categories, according to the duration of their effects (Hatton, 1997; Theodosis et al., 2008; Haydon and Nedergaard, 2015). Transient changes, which operate on a second scale, are

#### Edited by:

Jolanta Górska-Andrzejak, Jagiellonian University, Poland

#### Reviewed by:

Piotr Bebas, University of Warsaw, Poland Tycho Hoogland, Erasmus Medical Center, Erasmus University Rotterdam, Netherlands

> \*Correspondence: Günther K. H. Zupanc

<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology

Received: 30 October 2017 Accepted: 11 December 2017 Published: 22 December 2017

#### Citation:

Zupanc GKH (2017) Dynamic Neuron-Glia Interactions in an Oscillatory Network Controlling Behavioral Plasticity in the Weakly Electric Fish, Apteronotus leptorhynchus. Front. Physiol. 8:1087. doi: 10.3389/fphys.2017.01087

**97**

based on molecular signals exchanged between astrocytes and neurons. Long-term changes, on the other hand, occur over minutes, hours, or days and involve restructuring of glial morphology, in addition to molecular-signaling.

Despite the recognition of astrocytes as key players in brain plasticity, their role in functional plasticity of neural oscillators has received surprisingly little attention. A neural oscillator well characterized in terms of its behavioral function and the plasticity associated with the sexually dimorphic output pattern is the pacemaker nucleus in the medulla oblongata of the weakly electric fish Apteronotus leptorhynchus (Dye and Meyer, 1986). Although the existence of glia in this nucleus has been known since its early morphological description (Elekes and Szabo, 1985), their potential role in the regulation of the oscillatory activity has been examined only in recent years (Sîrbulescu et al., 2014; Zupanc et al., 2014). In the following, I will summarize the key findings, discuss them in the broader context of glianeuron interactions in oscillatory networks, and propose a model to explain how astrocytes might modulate the activity of neurons in the pacemaker nucleus to produce a sexually dimorphic behavior.

## CELLULAR COMPOSITION OF THE PACEMAKER NUCLEUS

Like all apteronotids, A. leptorhynchus possesses an electric organ formed by modified axons of spinal motor neurons, which are commonly referred to as electromotor neurons. The synchronous depolarization of these electrocytes results in electric organ discharges (EODs) in a species-specific frequency range of 650– 1,000 Hz (de Oliveira-Castro, 1955; Bennett, 1971; Waxman et al., 1972). Within this frequency band, males discharge at higher frequencies than females (**Figure 1A**; Meyer et al., 1987; Zupanc et al., 2014).

The EOD frequency is controlled, in a one-to-one fashion, by the frequency of the oscillatory network defined by two of the neuronal cell types of the pacemaker nucleus, pacemaker cells and relay cells (Elekes and Szabo, 1985; Dye and Heiligenberg, 1987). While the pacemaker cells are restricted to the pacemaker nucleus, the relay cells project down the spinal cord to form electrotonic junctions with the electromotor neurons.

Although pacemaker and relay cells are the only neuronal cells known to be involved in the generation of the oscillatory activity of the pacemaker nucleus, they are—with 87 and 20, respectively—a diminishing minority among neurons. The vast majority of neurons (all of which have been identified by their immunoreactivity against the neuron-specific marker protein Hu C/D) are small interneurons (**Figure 2A**; Elekes and Szabo, 1985; Turner and Moroz, 1995; Zupanc et al., 2014). They total ∼5,300 in mid-aged adult fish (Elekes and Szabo, 1985; Turner and Moroz, 1995; Sîrbulescu et al., 2014; Zupanc et al., 2014). Nevertheless, even when considering all three types of neurons combined, they comprise only a minute fraction of the roughly 80,000 cells whose cellular identity is known. (The pacemaker nucleus of a mid-aged fish is formed by an estimated 200,000 cells; the identity of ∼120,000 cells remains unknown). Besides neurons, stem/progenitor cells (∼35,000; see section "Development of Astrocytes and Neurons in the Adult Pacemaker Nucleus," below) and glia (∼40,000; Sîrbulescu et al., 2014) have been identified. The latter express a variety of glial marker proteins, including S100, glial fibrillary acidic protein (GFAP), glutamine synthetase, and vimentin, with a large degree of overlap among the individual expression patterns.

The above morphometric data indicate that, among identified cells, glia outnumber the three types of neurons more than 7-fold, and the pacemaker and relay cells even more than 200 fold. A glia-neuron ratio of 7:1, as found among identified cells in the pacemaker nucleus, is, by any means, extremely high. For comparison, in the spinal cord of A. leptorhynchus S100 expressing glia and Hu C/D-expressing neurons coexist in a 2:1 ratio (Sîrbulescu et al., 2017). Importantly, these two ratios are directly comparable, as they have been determined in the pacemaker nucleus and the spinal cord by employing identical immunostaining and image processing protocols. On the other hand, the 2:1 ratio in the spinal cord of A. leptorhynchus is in line with measures in various regions of rat and primate brains, which yield typical glia-to-neuron ratios between 0.5 and 2 (Hilgetag and Barbas, 2009; von Bartheld et al., 2016).

### DEVELOPMENT OF ASTROCYTES AND NEURONS IN THE ADULT PACEMAKER NUCLEUS

Morphometric analysis of the pacemaker nucleus of fish of various ages has demonstrated an age-related increase in the total number of cells during adulthood, from ∼100,000 cells in young adults to 300,000 in adults toward the end of their lives (Sîrbulescu et al., 2014). However, whereas the small interneuron population grows at the same rate as the total number of pacemaker nucleus cells, no age-dependent increase in cell numbers has been found among pacemaker and relay cells—their numbers are remarkably constant over adult life.

The continuous growth of the pacemaker nucleus during adulthood is the result of continuous generation of new neurons and glial cells from adult stem/progenitor cells, most of which express the stem-cell markers Sox2 and Meis 1/2/3 (Sîrbulescu et al., 2014). However, only a minor fraction (2–3%) of them are mitotically active, whereas the rest are quiescent at any given time point. Further characterization of the stem/progenitor cells has shown that the vast majority of them express S100 and assume morphological properties of radial glia. Thus, these observations suggest that radial glia-like precursors that express Sox2 and Meis 1/2/3 serve as the adult stem/progenitor cells, which give rise to the new neurons and glia in the pacemaker nucleus. Radial glia have also been identified as endogenous multipotent stem cells in the adult mammalian brain (Doetsch et al., 1999; Laywell et al., 2000; Seri et al., 2001; Bonaguidi et al., 2011).

Combination of labeling of S-phase cells with a single pulse of the thymidine analog 5-bromo-2′ -deoxyuridine, and immunostaining against the neuron-specific marker protein

FIGURE 1 | Sexual dimorphism of the electric organ discharge (EOD) of the weakly electric fish, Apteronotus leptorhynchus. (A) Box-and-whisker plot of the EOD frequencies of males (35) and females (48), adjusted to an ambient temperature of 26◦C. Whiskers mark the minimum and maximum values in each group, and boxes indicate the interquartile ranges. Middle lines denote medians. The difference in the EOD frequency between males and females is highly significant (\*\*\*). The inset shows a trace of the EOD signal. (B) Effect of β-estradiol on the EOD frequency of Apteronotus leptorhynchus. The EOD frequency was determined on 7 consecutive days before the operation (days −6 through 0) and on 8 days following the surgery (days 1 through 8). Fish received implants with (circles; n = 14) or without (triangles; n = 17) β-estradiol immediately following the measurement of the EOD frequency on day 0 (indicated by arrow). For each fish, the changes in EOD frequency were quantified relative to its average frequency over the 7-day pre-implantation period. The dashed line indicates no change from this baseline. Starting with day 4 after the implantation, the EOD frequencies of the β-estradiol-treated fish were significantly different from the discharge frequencies of the controls as well as from the pre-implantation frequencies (\*\*\*) (From: Zupanc et al., 2014. Copyright permission not required due to author's own work).

FIGURE 2 | Cellular structure of the pacemaker nucleus of Apteronotus leptorhynchus. (A) Three-dimensional model of the neural organization of the pacemaker nucleus, based on a statistical-mapping approach. Neurons form three classes—relay cells, pacemaker cells, and small interneurons—distinguished by the sizes of their cell bodies. The small interneurons can be further divided into subtypes 1 and 2. Glial cells are not shown because, due to their abundance, they would cover essentially the entire area of the nucleus, making it impossible to visualize the neuronal cells in this representation. (B) Association of glia with neurons in the pacemaker nucleus. The image is based on a three-dimensional shadow projection of a z-stack of confocal images. Neurons expressing Hu C/D (blue) are embedded in a dense meshwork of GFAP-expressing astrocytes (green). The glia express the gap-junction protein connexin-43 in a punctate pattern (red). In the center of the image, a pacemaker cell (P), surrounded by a dense astrocytic syncytium, is shown. Scale bar = 5µm (After: Sîrbulescu et al., 2014. Copyright permission not required due to author's own work).

Hu C/D and the glia-specific marker protein S100 after various chase times, has revealed some notable details of the developmental pattern of the adult-born cells in the pacemaker nucleus (Sîrbulescu et al., 2014). At early stages of the cellular development, about equal portions of the progeny acquire neuronal and astrocytic identities. However, starting at about 10 days, the number of young neurons decreases, while the number of cells that differentiate into astrocytes continuously increases, resulting, at 100 days of cellular age, in a glia-to-neuron ratio of 4:1. Such a ratio is in line with the dominance of astrocytes observed among the entire population of mature cells in the pacemaker nucleus.

# GLIAL DYNAMICS IN THE PACEMAKER NUCLEUS ASSOCIATED WITH BEHAVIORAL PLASTICITY

The abundance of astrocytes, relative to the number of neurons, and in particular to the number of pacemaker and relay cells, prompts the question of what role glia play in the pacemaker nucleus. Some important insights toward finding an answer to this question have been provided by analysis of the structural interconnection of pacemaker and relay cells with astrocytes, and the dynamics of this system. Three-dimensional reconstruction of pacemaker nucleus tissue based on confocal laser scanning micrographs has revealed a dense meshwork of astrocytic fibers closely associated with the pacemaker and relay cells (**Figure 2B**; Zupanc et al., 2014), reminiscent of the close apposition of glial processes to pacemaker and relay cells revealed by electron microscopy (Elekes and Szabo, 1985). These S100 immunopositive astrocytes express high levels of connexin-43, a member of the connexin family of transmembrane gap junction proteins that plays a critical role in the formation of the astrocytic syncytium (Giaume and Liu, 2012). Thus, the morphological data suggest that the pacemaker and relay cells are embedded in a dense meshwork of astrocytes interconnected by gap junctions to form an astrocytic syncytium.

The astrocytic syncytium undergoes dynamic changes in concert with alterations in the EOD frequency. As mentioned above, males and females occupy different frequency domains within the species-specific EOD range. At a water temperature of 26◦C, the mean EOD frequency of males is ∼880 Hz, whereas the mean frequency of females is ∼740 Hz (**Figure 1A**). This sexual dimorphism is controlled by steroid hormones. Chronic administration of β-estradiol results in both males and females in a gradual decrease of the EOD frequency (**Figure 1B**; Meyer et al., 1987; Schaefer and Zakon, 1996; Zupanc et al., 2014).

The sexual dimorphism in the fish's EOD frequency is paralleled by changes in the morphology and expression pattern of the astrocytes in the pacemaker nucleus (Zupanc et al., 2014). In females, the pacemaker nucleus is occupied by numerous, intensely labeled GFAP-immunoreactive fibers. In males, the number and intensity of labeling of such fibers is significantly lower. This overall difference extends to individual pacemaker and relay cells. Quantitative analysis has shown that the total GFAP labeling associated with the area covered by pacemaker and relay cells is significantly higher in females than in males. Similarly, the relative area covered by connexin-43 immunoreactivity in the region around pacemaker cells is almost 3-times larger in females than in males.

The changes in astrocytic architecture that occur as part of the development of the sexual dimorphism in the structure and function of the pacemaker nucleus could be due to the generation of new astrocytes, or the outgrowth of existing astrocytes, or both, and are probably controlled by estradiol. Evidence in support of both estradiol-induced gliogenesis and astrocytic remodeling has been obtained in mammals. In mice, sex differences have been demonstrated in cell-cycle regulation of hematopoietic stem cells. Such cells divide significantly more frequently in females than in males—an effect that can be mimicked by administration of estradiol in both males and females (Nakada et al., 2014). In the basal hypothalamus of mammals, the portion of neuronal somata covered by astrocytic processes fluctuates over the estrous cycle, being high when estrogen levels are elevated, and low when estrogen levels have decreased (García-Segura et al., 1994). The retraction and elongation of astrocytic processes over the surfaces of neurons can be mimicked by administration of estradiol to ovariectomized animals, an effect that occurs as rapidly as within 2 h after the steroid hormone application (Langle et al., 2003). It is likely that this modulatory effect of estradiol on glial structure is direct, as estrogen receptors have been found in astrocytic cells of hypothalamic areas (Langub and Watson, 1992).

# WHAT IS THE FUNCTION OF ASTROCYTES IN THE PACEMAKER NUCLEUS?

The relative abundance of astrocytes, the dominance of gliogenesis over neurogenesis, the close association of astrocytic processes with pacemaker and relay cells, the structural reorganization of the astrocytic syncytium occurring in concert with sexual dimorphism-related changes in the oscillatory output frequency of the pacemaker nucleus—taken together, these observations point to a role of astrocytes in mediating the function of the pacemaker nucleus as the master oscillator in the neural network that controls the EOD frequency. However, despite this compelling correlative evidence, the role of the astrocytic meshwork in the regulation of the pacemaker frequency, and thus the EOD frequency, has remained elusive.

Given the high-frequency firing of the pacemaker and relay cells and the resulting continuous release of potassium from these cells into the extracellular space, it is plausible to hypothesize that one of the chief functions of the astrocytic meshwork in the pacemaker nucleus is to buffer the increases in K+ concentrations. Potassium buffering by glia was first suggested over half a century ago (Hertz, 1965). Since then, a large body of experimental evidence has established astrocytic buffering of excess K+ as a key concept of glial function in the nervous system (Kofuji and Newman, 2004). It is thought that the uptake of extracellular K + is primarily achieved via Na+/K+-ATPase, but K + channels have also been implicated in this function. It is, therefore, worth noting that Kv1.2 potassium channels have been identified through immunohistochemical staining in thin fibers resembling the astrocytic processes that wrap around pacemaker and relay cells, but are absent from these neurons (Smith and Zakon, 2000). Following uptake of extracellular K+, the abundant coupling of astrocytes via gap junctions, as indicated by the intense connexin-43 immunoreactivity associated with astrocytic cells (Zupanc et al., 2014), might serve as a mechanism to aid redistribution of K + from sites of excessive levels to sites of lower K + concentrations within the pacemaker nucleus.

The astrocytic syncytium surrounding the pacemaker and relay cells may be involved not only in elimination of excess K + ions from the extracellular space, but also in regulating the output frequency of the pacemaker because a major determinant of the membrane potential is the extracellular K+ concentration. An increase in the extracellular K+ concentration leads to a reduction in the K+ equilibrium potential, whereas a decrease in the extracellular K+ concentration results in an increase in the K+ equilibrium potential. In either case, these changes affect the excitability of neurons by altering the difference between the resting potential and the threshold potential at which voltage-gated sodium channels are activated (Wang et al., 2012).

Some important insights into the role of astrocytic buffering of K+ in the generation of high-frequency oscillations has been gained by studying epilepsy and experimental models of this condition. Epileptic seizures are characterized by high-frequency oscillations (Bragin et al., 1999; Jirsch et al., 2006; Pearce et al., 2013). Impairment of K+ buffering induced in astrocytes by altering the expression of inwardly rectifying K+ channels enhances the susceptibility for epileptic seizures (Steinhäuser et al., 2012). Similarly, seizures can also be triggered by increases in the extracellular K+ concentration (Traynelis and Dingledine, 1988; Rangroo Thrane et al., 2013).

Conversely, enhancement of the efficiency in K+ uptake and inter-astrocytic redistribution will reduce the excitability of neural networks by widening the gap between the resting membrane potential and the threshold potential at which action potentials are triggered. Such increase in astrocytic buffering capacity is predicted to result in a reduction in output frequency of neural oscillators. In the pacemaker nucleus, enhanced buffering capacity might be achieved by increasing the surface area of pacemaker and relay cells covered by astrocytic processes, and by strengthening gap junction coupling between individual astrocytes.

#### MODEL OF THE DEVELOPMENT OF THE SEXUALLY DIMORPHIC EOD FREQUENCY

Based on the above empirical evidence and theoretical considerations, the following working hypothesis emerges to mechanistically explain the development of the sexual dimorphism in EOD frequency of A. leptorhynchus: Differences in the association of the astrocytic syncytium with pacemaker and relay cells in the pacemaker nucleus develop as part of sexual differentiation under the direct control of steroid hormones, foremost estradiol. In females, the larger surface area of pacemaker and relay cells covered by astrocytic processes, accompanied by increases in specific types of K+ channel and/or in Na+/K+-ATPase expression, and the stronger gap-junction coupling of the individual astrocytes within the astrocytic syncytium, serve as mechanisms to increase astrocytic K+ buffering. As a result, the levels of extracellular K+ are reduced. This reduction leads to a decrease in the excitability of the pacemaker and relay cells, thereby lowering the oscillator frequency of the pacemaker nucleus, and ultimately the EOD frequency of the fish.

## CONCLUSIONS

Involvement of glial cells in neural oscillations has been suspected since circadian fluctuations in GFAP labeling in the suprachiasmatic nucleus of hamsters (Lavialle and Servière, 1993; Lavialle et al., 2001) and in the abundance of clock gene products in glial cells of Drosophila (Siwicki et al., 1988; Zerr et al., 1990; Ewer et al., 1992) were first reported. Despite accumulating (mostly circumstantial) evidence, the precise role of glia in the regulation of oscillatory activity has remained largely a matter of speculation. A key question is whether glia in different oscillatory networks share similar functions and mechanisms that mediate these functions, despite major differences in cellular organization and output patterns. Besides the pacemaker nucleus of A. leptorhynchus, two other oscillatory systems in which the role of glia has been studied are circadian clocks and olfactory glomeruli. While the period of the pacemaker nucleus oscillations is on the order of 1 ms, corresponding values of circadian rhythms approximate 1 day. Mitral cells in individual olfactory glomeruli of mice exhibit oscillations in membrane potential with period lengths somewhat in between these two extremes, namely ∼5 s (Roux et al., 2015).

Notwithstanding the diversity of these systems, functional studies support the notion that the role of glia in these oscillatory networks is similar. Manipulation of clock genes specifically in astrocytes within the suprachiasmatic nucleus of mice has indicated that the astrocytic circadian clock, although neither necessary nor sufficient for sustaining circadian rhythms, interacts with the neuronal clock system to modulate the period length, and thus the frequency, of the circadian rhythm (Tso et al., 2017). Similarly, disruption by toxins of the metabolism of glial cells in the visual clock system of dipterans does not abolish circadian rhythms but affects the amplitude of circadian fluctuations in the size of neurons within this system (Pyza and Górska-Andrzejak, 2004). Lastly, combination of genetic and pharmacological approaches in the olfactory bulb system has shown that the activity of gap-junction channels interconnecting astrocytes modulates the amplitude and the firing rate of mitral cells (Roux et al., 2015).

Taken together, structural and functional studies suggest that astrocytes, although not essential for the generation of neural activity in oscillatory networks, play a critical role in the modulation of the output pattern of such networks. They appear to exert this function by undergoing both short-term and longterm plastic changes in morphological structure, ion channel expression, and/or gap-junction coupling between individual astrocytic cells. Although many details of the modulatory action of glia remain to be elucidated, it becomes increasingly clear that biologically realistic models of plasticity of oscillatory neural networks will have to take the role of glia-neuron interactions in this phenomenon into account.

#### AUTHOR CONTRIBUTIONS

The author confirms being the sole contributor of this work and approved it for publication.

#### REFERENCES

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**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Zupanc. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The RNA Helicase BELLE Is Involved in Circadian Rhythmicity and in Transposons Regulation in Drosophila melanogaster

Paola Cusumano1† , Milena Damulewicz2† , Elena Carbognin1† , Laura Caccin1 , Antonietta Puricella3 , Valeria Specchia3 , Maria Pia Bozzetti3 , Rodolfo Costa1 and Gabriella M. Mazzotta1 \*

1 Department of Biology, University of Padua, Padua, Italy, 2 Department of Cell Biology and Imaging, Jagiellonian University, Kraków, Poland, 3 Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy

#### Edited by:

Geoffrey A. Head, Baker Heart and Diabetes Institute, Australia

#### Reviewed by:

Francesco Cappello, Università degli Studi di Palermo, Italy Joon (Kyungjoon) Lim, La Trobe University, Australia

\*Correspondence:

Gabriella M. Mazzotta<EMAIL_ADDRESS>†These authors have contributed

equally to this work

#### Specialty section:

This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology

Received: 31 October 2017 Accepted: 04 February 2019 Published: 20 February 2019

#### Citation:

Cusumano P, Damulewicz M, Carbognin E, Caccin L, Puricella A, Specchia V, Bozzetti MP, Costa R and Mazzotta GM (2019) The RNA Helicase BELLE Is Involved in Circadian Rhythmicity and in Transposons Regulation in Drosophila melanogaster. Front. Physiol. 10:133. doi: 10.3389/fphys.2019.00133 Circadian clocks control and synchronize biological rhythms of several behavioral and physiological phenomena in most, if not all, organisms. Rhythm generation relies on molecular auto-regulatory oscillations of interlocked transcriptional-translational feedback loops. Rhythmic clock-gene expression is at the base of rhythmic protein accumulation, though post-transcriptional and post-translational mechanisms have evolved to adjust and consolidate the proper pace of the clock. In Drosophila, BELLE, a conserved DEAD-box RNA helicase playing important roles in reproductive capacity, is involved in the small RNA-mediated regulation associated to the piRNA pathway. Here, we report that BELLE is implicated in the circadian rhythmicity and in the regulation of endogenous transposable elements (TEs) in both nervous system and gonads. We suggest that BELLE acts as important element in the piRNA-mediated regulation of the TEs and raise the hypothesis that this specific regulation could represent another level of post-transcriptional control adopted by the clock to ensure the proper rhythmicity.

Keywords: RNA helicase BELLE, Drosophila, post-transcriptional control, circadian clock, piRNA-mediated transposons regulation

#### INTRODUCTION

Circadian clocks have evolved in most, if not all, organisms to time their physiology and behavior to the most appropriate phases of the day–night cycles (Young and Kay, 2001; Dubruille and Emery, 2008; Peschel and Helfrich-Forster, 2011). They control and synchronize the biological rhythms of a vast array of behavioral and physiological phenomena, including visual sensitivity, hormone secretion, locomotion, hatching, eclosion, ecdysteroid synthesis, sperm release, gene expression, and sleep wake cycle (Sandrelli et al., 2008; Allada and Chung, 2010; Schiesari et al., 2011). Biological clocks are self-sustained in the absence of environmental cues. Rhythm generation occurs in single cells and is based on molecular auto-regulatory oscillations of interlocked transcriptionaltranslational feedback loops. In Drosophila, the transcription factors CLOCK (CLK) and CYCLE (CYC) activate, as heterodimer, the expression of the clock genes period (per) and timeless (tim). The correspondent proteins, PER and TIM, are post-translational regulated and eventually accumulate in the cytoplasm and enter the nucleus, where they ultimately inhibit their own transcription by

repressing CLK-CYC activity. Two other interlocked feedback loops regulating the expression of the Clock gene and CLK/CYC mediated transcription, respectively, contribute to the robustness of the circadian oscillations (reviewed in Ozkaya and Rosato, 2012).

Rhythmic clock-gene expression is important to generate rhythmic protein accumulation; however, cycling protein levels do not depend only on cycling mRNAs but post-transcriptional and post-translational mechanisms have also evolved to adjust and consolidate the proper pace of the clock (Kojima et al., 2011). Among these, alternative splicing, mRNA nuclear export, polyadenilation, regulation of translation and degradation, play fundamental roles in the post-transcriptional regulation of circadian gene expression (Majercak et al., 1999, 2004; Collins et al., 2004; Boothroyd et al., 2007; Kojima et al., 2012; Huang et al., 2013; MacGregor et al., 2013; Robles et al., 2014; Montelli et al., 2015).

In recent years, a role for small, non-coding RNAs (miRNA) as post-transcriptional regulators of circadian rhythmicity in Drosophila has been also established. They regulate the proper development and maintenance of circadian rhythms acting either directly on the expression of clock genes or indirectly on signaling outputs (reviewed in Xue and Zhang, 2018).

Another class of small RNA, the piRNAs, has recently been associated to the age-dependent rhythmicity (Kuintzle et al., 2017). In particular, putative primary piRNA transcripts corresponding to transposons exhibit a de novo oscillation in old flies and circadian clock components appear to regulate these late rhythmicity (Kuintzle et al., 2017). piRNAs are small molecules protecting animal cells from the de-regulation of transposons and other repetitive genetic elements, preserving genome stability (Li et al., 2009; Thomson and Lin, 2009; Le Thomas et al., 2014). The piRNA biogenesis starts with the transcription of genomic clusters dispersed in the genome, producing the primary piRNA precursor transcripts (Brennecke et al., 2007). The piRNA pathway was first discovered in the gonads (Vagin et al., 2006; Aravin et al., 2007; Brennecke et al., 2007; Specchia et al., 2008, 2010, 2017; Specchia and Bozzetti, 2009; Bozzetti et al., 2015; Iwasaki et al., 2015; Sahin et al., 2016), but later it was observed in the nervous system of both Drosophila and mice (Lee et al., 2011; Perrat et al., 2013).

belle emerged in a screen for genes involved in the RNAi pathway: it binds to Ago1 and Ago2, involved in the small RNAmediated regulation (Kim et al., 2005; Ulvila et al., 2006; Zhou et al., 2008). Very recently, a role of belle in the regulation on specific P-derived constructs in the ovary has been described (Lo et al., 2016), and therefore it is now considered a "bona fide" piRNA gene. belle encodes a DEAD-box RNA helicase: it is the closest paralog of vasa and plays roles in the reproductive system allowing fertility in Drosophila melanogaster. Like other members of the DEAD box family, Vasa and BELLE are supposed to have ATPase, RNA binding, and RNA unwinding activities (Liang et al., 1994; Sengoku et al., 2006). BELLE is essential for both male and female reproductive capacity and its function in gonads is conserved during evolution. It localizes at the perinuclear region, called "nuage," of the germ cells in male and female gonads (Johnstone et al., 2005; Kibanov et al., 2011), where the majority of key components regulating transposable elements (TEs) are located (Lim and Kai, 2007; Klattenhoff and Theurkauf, 2008; Kibanov et al., 2011; Nagao et al., 2011).

Here, we have identified for BELLE important roles as putative circadian clock component and regulator of TEs in brain and in gonads. These apparently unrelated functions suggest the hypothesis that BELLE might be a key element in small RNA (piRNA)-mediated regulation of the TEs and that this regulatory pathway is involved in circadian rhythmicity.

# MATERIALS AND METHODS

#### Fly Strains

The following strains of Drosophila melanogaster were used: w 1118 , CantonS, yw;tim-Gal4 (Emery et al., 1998), UAS-belleRNAi (VDRC #6299), GMR-Gal4 (BDSC #8440), ninaE-Gal4 (BDSC #1104), repo-Gal4 (BDSC #7415), pdf-Gal4 (BDSC #6900), Eaat1-Gal4 (BDSC #8849), Lama-Gal4 (BDSC #35543), per0 (Smith and Konopka, 1981), yw;P{EPgy2}belEY08943 (BDSC #19945); P{PZ}belcap−1 ry506/TM3, ryRK Sb1 Ser1 (BDSC #11778), bel::GFP (Morin et al., 2001) (Kyoto Stock Centre #ZCL2696), UAS-CD8-GFP (BDSC #5137). Co-IP was performed with yw; tim-GAL4/+; UAS-HAcry/+ (Dissel et al., 2004). Flies were maintained on a standard cornmeal medium under LD 12:12 regime and at constant 23◦C.

# Co-immunoprecipitation, Mass Spectrometry Analysis, and Western Blot

Head extracts from overexpressing HACRY flies raised in 12:12 light:dark cycles and collected at Zeitgeber Time 24 (ZT24), before lights on, and after a 15-min light pulse were subjected to coimmunoprecipitation and Mass Spectrometry analysis as previously described (Mazzotta et al., 2013). Immunocomplexes were analyzed by Western blot using the following antibodies: rabbit polyclonal anti-BELLE (1:2000) (Johnstone et al., 2005) and mouse anti-HA (1:5000; Sigma Aldrich, St. Louis, MO, United States). Anti-rabbit IgG HRP (1:3000; BioRad Laboratories, Hercules, CA, United States) and anti-mouse IgG HRP (1:5000; Sigma Aldrich, St. Louis, MO, United States) were used as secondary antibodies.

# RNA Extraction and qRT-PCR

RNA was extracted using the reagent Trizol (Invitrogen) as indicated in manufacturer's instructions. For the analysis of belle expression, around 50 heads from flies reared in LD cycles for at least 3 days and collected at the indicated time points were used. For the analysis of transposons 30 mg of male or female gonadal tissues or heads were used. 1 µg of total RNA was used as a template for oligonucleotide dT-primed reverse transcription using SuperScriptIII RNaseH-reverse transcriptase (Invitrogen), according to manufacturer's instructions. qRT-PCR was performed in the 7500 Real-TimePCR System (Applied Biosystem) using SYBR green (Celbio) according to the manufacturer's instruction. For quantification of the transcripts, we used the 211Ct method as previously reported (Specchia et al., 2010). The primers used are listed in **Supplementary Table S1**.

#### Immunocytochemistry

fphys-10-00133 February 18, 2019 Time: 18:58 # 3

For the analysis of BELLE localization, heads from 7 days old males were fixed in 4% paraformaldehyde in phosphate buffer saline (PBS; pH 7.4) for 4 h, then they were washed in PBS twice and cryoprotected by incubation in 12.5% sucrose for 10 min and in 25% sucrose at 4◦C overnight. Material was embedded in Tissue-Tek, frozen in liquid nitrogen, and cryostat 20 µm sections were cut. The sections were washed in PBS for 30 min and five times in phosphate buffer (PB) with an addition of 0.2% Triton X-100 (PBT). After that, sections were incubated in 5% normal goat serum (NGS) with an addition of 0.5% bovine serum albumin (BSA) for 30 min first at room temperature, and then incubated with primary antibodies anti-REPO (mouse, 1:500, Hybridoma Bank), anti-Ebony (rabbit, 1:750, a gift from Dr. B. T. Hovemann, Ruhr Universität Bochum) and anti-GFP (rabbit, 1:1000, Novus Biologicals) for 24 h. Afterward, sections were washed six times in PBT/BSA, and blocked in 5% NGS for 45 min. After that, goat anti-mouse (conjugated with Cy3) and goat antirabbit (conjugated with Alexa 488) secondary antibodies (both 1:500, Jackson Immuno Research) were applied overnight in 4 ◦C. Finally, sections were washed twice in BSA, six times in PBT, and twice in PBS. Then, cryosections were mounted in Vectashield medium (Vector) and examined with a Zeiss Meta 510 Laser Scanning Microscope. About 30 brains or cryosections were checked.

For the analysis of PER in clock-neurons, flies were synchronized by LD cycles at least for 3 days and then collected under LD or DD conditions at the indicated time points. Whole flies were quickly fixed for 2 h in PFA 4%. About 10–12 brains were dissected in PBS and then treated as previously described (Vanin et al., 2012). Average fluorescence values are reported.

#### Male and Female Fertility Analysis

One young male was mated to three control virgin females, and three virgin females were mated with three control males. Ten individual males and 30 females were tested for each genotype. After 4 days, the crosses were transferred to a fresh vial. The parental flies were removed from the last vial after an additional 4 days. The number of the adult progeny from each vial was counted.

#### Behavioral Analysis

Locomotor activity of individual flies was recorded for 3 days in LD and 7 days in DD using the Drosophila Activity Monitoring System (TriKinetics, Inc., Waltham, MA, United States). Data were collected every 5 min and then analyzed in 30 min bins using spectral analysis and autocorrelation (Zordan et al., 2007). Morning anticipations was detected fly-by-fly examining the activity mean of 3 days under LD conditions (Vanin et al., 2012) and the Morning Index was calculated as in Seluzicki et al. (2014).

#### Statistical Analyses

For comparisons between two measurements a two-tailed Student's t-test was used to show the significance level of the replicated experiments. Expression profile's significance was evaluated by non-parametric ANOVA, Kruskal–Wallis test (p-value < 0.05) whereas putative period and phase of oscillation were identified by RAIN non-parametric test for independent samples (adjusted p-value < 0.05) (Thaben and Westermark, 2014).

# RESULTS

## dCRY Interacts With the RNA Helicase BELLE

In an experiment aimed at the identification of new genes involved in the circadian machinery in Drosophila, a coimmunoprecipitation assay followed by mass spectrometry analysis was performed on transgenic flies overexpressing a hemagglutinin (HA)-tagged form of dCRY (HACRY) raised in 12:12 light:dark cycles and collected at Zeitgeber Time 24 (ZT24), before lights on, and after a 15-min light pulse (Mazzotta et al., 2013).

A ∼85-kDa species was observed in the sample in the dark that was not present in the negative control (**Figure 1A**). This protein band was digested in-gel, and the peptide mixtures analyzed by liquid chromatography–mass spectrometry (LC-MS)/MS using an ESI-QTOF mass spectrometer (Wilm et al., 1996). Analysis of the MS/MS data using the MASCOT software yielded the identification of BELLE (**Supplementary Table S2**), a DEAD box RNA helicase involved in RNA metabolism at several levels (Linder and Jankowsky, 2011).

The presence of BELLE in the complex with HACRY was also confirmed by Western blot with an anti-BELLE antibody (Johnstone et al., 2005). By this procedure, BELLE was also detected after a 15 min of light pulse (**Figure 1B**). The same blot was hybridized also with an anti-HA antibody, in order to assess the specificity of the interaction (**Figure 1B**). The signal corresponding to BELLE was more intense in head collected in the dark; in fact after 15 min of light pulse the amount of BELLE was lower, in line with that of HACRY that decreased due to the well-known light-dependent degradation of CRY (Peschel et al., 2009).

## The RNA Helicase BELLE Is Expressed in the Fly Brain

It is known that BELLE is expressed in germ cells throughout all developmental stages and in larval neurons (Johnstone et al., 2005; Rolls et al., 2007), but little information was available regarding its expression in the adult fly head.

In order to characterize BELLE expression in the fly brain, we performed an immunocytochemistry experiment on flies carrying a GFP exon trap in belle (bel::GFP) (Morin et al., 2001). Flies were maintained in standard light–dark conditions, brains were dissected and an anti-GFP antibody was used to detect BEL::GFP expression. We observed BELLE expression in clock neurons and in glial cells in the optic lobe, with a predominantly cytoplasmic localization (**Figures 2**, **3**). In clock neurons, BELLE was expressed in either the small or the

FIGURE 2 | BELLE localizes in the main pacemaker cells in the brain of Drosophila melanogaster. Confocal images of whole mount of adult brain expressing BELLE fused with green fluorescent protein (bel::GFP). Scale bar: 10 µm. (A,D) Anti-GFP immunostaining (BEL::GFP). (B,E) Anti-PDF was used to label PDF-expressing ventral lateral neurons. Depends on optical section small LNv (s-LNv) or large LNv (l-LNv) are visualized. (C,F) BEL::GFP and PDF immune staining co-localize in the area of LNv cell bodies.

large ventral lateral neurons (s-LNvs and l-LNvs respectively) (**Figure 2**), PDF-expressing cells that act in promoting and governing the circadian locomotor behavior. In glia we observed BELLE expression in the epithelial glia and a weak labeling also in the lamina (**Figures 3D–F**): position and shape of these GFP- labeled cells suggest they may be R1-R6 photoreceptor terminals (**Figures 3D–F**); however, anti-REPO staining showed glia cells nuclei at the top of each structure (**Figure 3G**). Observed columnar shape is also similar to anti-EBONY staining (**Figure 3H**), which is a marker for epithelial glia (Richard et al., 2002), suggesting that BELLE may be located in epithelial glia in the lamina. Moreover, we observed immunostaining for BEL::GFP also in giant inner chiasm glia, which connects the medulla with the lobula complex in the optic lobe (Kremer et al., 2017). In these cells, both BELLE and PER are expressed (**Supplementary Figure S1**).

# Belle Expression Shows Circadian Features

In order to characterize the temporal expression of belle, mRNA and protein levels were analyzed during the 24 h. Wild-type flies white1118 were synchronized by 12:12 LD cycles and collected every 3 h, either in LD regime or at the third day after being transferred to constant darkness (DD).