J Comp Physiol A DOI 10.1007/s00359-014-0952-9

REVIEW

Glia in Drosophila behavior L. Zwarts · F. Van Eijs · P. Callaerts 

Received: 28 March 2014 / Revised: 2 October 2014 / Accepted: 7 October 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Glial cells constitute about 10 % of the Drosophila nervous system. The development of genetic and molecular tools has helped greatly in defining different types of glia. Furthermore, considerable progress has been made in unraveling the mechanisms that control the development and differentiation of Drosophila glia. By contrast, the role of glia in adult Drosophila behavior is not well understood. We here summarize recent work describing the role of glia in normal behavior and in Drosophila models for neurological and behavioral disorders. Keywords  Drosophila · Glia · Behavior · Invertebrate · Brain

Introduction The Drosophila central and peripheral nervous systems (CNS, PNS) are composed of neurons and glia that make up 90 and 10 % of the total cell number, respectively (Goodman and Doe 1993). The existence of glial cells in insects has been known for a long time, but their high heterogeneity, and therefore difficult classification, has led to the fact that invertebrate glia have been less well studied than their vertebrate counterparts (Wigglesworth 1959; Strausfeld 1976; Saint Marie and Carlson 1983). However, the development of genetic and molecular tools in Drosophila more

L. Zwarts · F. Van Eijs · P. Callaerts (*)  Laboratory of Behavioral and Developmental Genetics VIB Center for the Biology of Disease, Center for Human Genetics, KULeuven, O&N IV Herestraat 49, Box 602, 3000 Louvain, Belgium e-mail: [email protected]‑kuleuven.be

than 20 years ago has facilitated the discovery of glialspecific markers and genes, and has helped to clarify the roles played by glial cells in the nervous system. While the mechanisms involved in glial differentiation and development have formed the subject of multiple reports, their functional role in the regulation of behavior has only gained attention more recently. In this review, we will first discuss the classification of the different glial cell types and then discuss their known roles in the regulation of adult behavior. In a number of cases where adult behavioral phenotypes were observed, it has not been unambiguously demonstrated that the underlying defect is not primarily developmental in origin. We have systematically identified this throughout the text. For more in-depth information on development and differentiation of Drosophila glia we refer to excellent recent reviews (Hartenstein 2011; Stork et al. 2012).

Classification of glial cells Ramón y Cajal already suggested that glial cells may be as heterogeneous as neurons based on the wide diversity of morphologies he observed in Golgi impregnated preparations (Cajal 1909). More than 80 years later, the use of Drosophila enhancer trap lines, such as gal4 lines, has made it possible to identify markers for all subsets of glial cells thereby enabling detailed studies of the morphology of glial cells (Klambt and Goodman 1991; Nelson and Laughon 1993; Ito et al. 1995; Awasaki et al. 2008; Awasaki and Lee 2011). Based on their morphological and topological features, glial cells are divided into five different types that fall into three classes according to their association with the basic regions of the CNS: the surface, the cortex and the neuropile (see Fig. 1).

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J Comp Physiol A

Fig. 1  Classification of glial cell in the adult brain. In the first panels, glia are visualized using glia subtype-specific gal4-lines driving expression of UAS-gfp-cd8 (green) (Awasaki and Lee 2011). In the second panels anti-bruchpilot (nc82) staining labels the different neuropils in the adult brain (magenta). The third panels show the overlay between both stainings. a Subperineurial glia (gli-gal4); b perineu-

rial glia (NP6293-gal4); c cortex glia (NP2222-gal4); d wrapping glia (Mz97-gal4); e ensheathing glia (NP6520-gal4); f astrocyte-like glia (Eaat1-gal4); g wrappig glia (Mz97-gal4): close up of the antennal lobe region H. Ensheathing glia (NP6520-gal4): close up of the antennal lobe region I. Ensheathing glia (NP6520-gal4): close up of the mushroom body lobe region. The scale bars represent 100 μm

• Surface glia extend sheath-like processes that wrap around the entire brain. This class of glia can be further subdivided in subperineurial and perineurial glia. Subperineurial glia have a flattened shape and form a thin monolayer, covering the surface of the late larval and adult CNS. Cells of the subperineurial layer are interconnected via septate junctions, spot adherens junctions, as well as gap junctions and function as the Drosophila CNS blood–brain barrier (Pereanu et al. 2005; Awasaki et al. 2008; Stork et al. 2008). The number of subperineurial glia in the Drosophila brain is remarkably low: one adult brain hemisphere is surrounded by fewer than 50 cells (Hartenstein 2011). Perineurial glia have an elongated or multilobulated shape, are smaller than the subperineurial glia and form the outermost cellular sheath surrounding the nervous system. The function of perineurial glia is unknown, but it has been suggested that they may contribute to the function of the blood–brain barrier (Hartenstein 2011). • Cortex glia (also known as cell body-associated glia while in the optic lobe they have been called satellite

glia (Tix et al. 1997; Edwards and Meinertzhagen 2009) have a small, rounded cell body and nucleus and are embedded within the cell cortex where they encapsulate neuronal somata and neuroblasts, thereby forming a scaffold (trophospongium) (Dumstrei et al. 2003; Pereanu et al. 2005). A single cortex glial cell can ensheath many neuronal cell bodies. Cortex-associated glia are also in close association with the blood–brain barrier and oxygen-supplying tracheal elements (Halter et al. 1995; Pereanu et al. 2005). • Neuropile glia form the most diverse class of glia. Cell bodies of this glia type have a flattened shape and are situated on the interface between cortex and neuropile. A high concentration of cell bodies is seen around the different compartments of the central complex. Neuropil-associated glia form sheaths around the synaptic neuropil (ensheathing glia) or they can infiltrate the neuropil (astrocyte-like or reticulate glia) (Awasaki et al. 2008; Stork et al. 2008; Hartenstein 2011). Another type of neuropil-associated glia are the wrapping glia that form sheaths around major tracts of neurites in the

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J Comp Physiol A

periphery. Ensheathing glia have been shown to successfully clear degenerating axons from the injured brain (Doherty et al. 2009), while astrocyte-like glia fulfill an important role in the clearance of neurotransmitters (Rival et al. 2004). Neuropil-associated glia also provide guidance cues for axons (Hidalgo et al. 2006; Spindler et al. 2009) and play a role in neuronal apoptosis (Sonnenfeld and Jacobs 1995; Kurant et al. 2008).

Role of glia in normal adult behavior (summary in Table 1) Circadian rhythms Circadian clocks give organisms an innate sense of time so that they can cope with day/night changes in the environment. The molecular oscillators that constitute the circadian clock are strikingly similar between flies and mammals. They consist of transcriptional and translational feedback loops of clock proteins [Period (Per) and Timeless (Tim)] which regulate the activity of transcription factors [Clock (Clk) and Cycle (Cyc)] required for the expression of the clock genes (Jackson 2011; Ozkaya and Rosato 2012). Drosophila has ~150 clock neurons harboring a circadian oscillator (Nitabach and Taghert 2008). The ventral lateral neurons (LNvs) are of particular interest since they fulfill a crucial pacemaker role in Drosophila (Grima et al. 2004; Stoleru et al. 2004, 2005). The neuropeptide pigment dispersing factor (PDF), which is exclusively secreted by the LNvs, synchronizes the different clock neurons that make up the circadian neural circuit via its receptor PDFR (Renn et al. 1999; Lin et al. 2004; Lear et al. 2005; Nitabach et al. 2006; Shafer et al. 2008; Im and Taghert 2010). A number of papers indicate that glia constitute an intrinsic part of the network regulating circadian rhythmicity. The early discovery that both Drosophila neurons and glia show a cyclic expression of the clock gene per was the first indication that not only neurons, but also glia might be involved in the regulation of circadian rhythmicity (Siwicki et al. 1988; Zerr et al. 1990). Additional support for this hypothesis came from a study by Ewer and colleagues, who demonstrated that per expression in Drosophila glial cells was sufficient for the manifestation of a weak behavioral rhythm (Ewer et al. 1992). A recent study, however, showed that a glial knock down of the clock protein Per in adult flies (repo-gal4; UAS-period RNAi) did not affect rhythmicity (Ng et al. 2011). Given that the authors only tested the requirement of glial period expression for the execution of free-running activity rhythms (the maintenance of circadian locomotor activity in constant dark conditions after a period in light/dark conditions), glial period may still be required for the regulation of circadian light (or temperature) sensitivity or other types of circadian rhythm.

The observation of a rhythmic expression of the clock protein tim in glia (Peschel et al. 2006) and the apparent discrepancy concerning a possible role of per clearly indicates that a systematic analysis of the role of clock genes in glia is needed before any definitive conclusions can be drawn. Strong evidence for the role of glia in the regulation of normal behavioral circadian rhythmicity in Drosophila comes from work on the N-β-alanyl-biogenic amine synthetase Ebony. Previously it was reported that ebony shows rhythmic expression levels and that mutations in this gene disrupt circadian locomotor behavior (Newby and Jackson 1991; Claridge-Chang et al. 2001; Ueda et al. 2002). Suh and Jackson (2007) demonstrated that Ebony is rhythmic and under clock control, is exclusively in glia and is required therein to regulate circadian rhythmicity. The temporal patterns of per and tim expression and of immunostaining for PDF are normal in ebony mutants, thus indicating that ebony acts downstream of the clock to regulate circadian behavioral rhythms (Suh and Jackson 2007). The authors proposed that Ebony in glia might play a role in the regulation of circadian locomotor activity via the termination of biogenic amine transmitter action. There are three arguments that lend support to this hypothesis: (1) Ebony can conjugate β-alanine to several different biogenic amines, including dopamine, serotonin, histamine, tyramine, and octopamine (Richardt et al. 2002, 2003; Suh and Jackson 2007); (2) Ebony-expressing glia lie in close proximity to projections from dopamine- and serotoninexpressing neurons (Suh and Jackson 2007); (3) biogenic amines are known to regulate locomotor activity (Kume et al. 2005; Riemensperger et al. 2011). Whether this is indeed the case awaits further direct demonstration. The fact that glia play an important role in regulating circadian rhythmicity raises the key issue whether in this context circadian oscillations in glial cells are controlled by clock neurons or whether clock neurons are under control of a circadian input from glial cells. The work on ebony (Suh and Jackson 2007) is consistent with glia acting downstream of clock neurons. How this is regulated is not clear. It is not dependent on PDF (Suh and Jackson 2007). In studies of morphological plasticity displayed by glial cells in the visual system of Drosophila (Pyza and Górska-Andrzejak 2004; Damulewicz and Pyza 2011), it was proposed that ion transport peptide (ITP) secreted by the PDF-negative LNv clock neuron may be involved, but no experimental evidence to support this has as yet been given. In addition to glial circadian rhythmicity being controlled by the output of clock neurons, there is also convincing evidence for glia-to-neuron signaling in the circadian circuitry of Drosophila (Ng et al. 2011). In an attempt to unravel whether glial cells can physiologically regulate the neuronal circuitry driving circadian behavior, Ng and colleagues tested flies that harbored conditional genetic

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Gene/Drosophila model

13 Seugnet et al. (2011)

Glia surrounding the mushroom body neuropil Glia surrounding the mushroom body neuropil

Normal learning after sleep deprivation LOF: decreased homeostatic response to sleep deprivation

Olfaction

Wallerian degeneration slow (Wlds)

Derailed 2 (Drl-2) Prevention of glia recruiting by damaged olfactory receptor neurons by expression of

Anachronism (ana)

No induction of synaptic plasticity upon deafferentation of projection neurons

Ensheathing glia

LOF: larval diminishment in olfactory response to chemoattract- – ants Glia surrounding glomeruli in the antennal lobe

Comas et al. (2004)

Kazama et al. (2011)

Sakurai et al. (2009)

Park et al. (1997)

Seugnet et al. (2011)

Grosjean et al. (2008)

LOF: males court and attempt to copulate with other males, – abnormal response to 7-tricosene cues Concentration-dependent effects on olfactory long-term memory Glia surrounding the mushroom body neuropil

Pereanu et al. (2007) Ghosh et al. (2011) Lehmann and Cierotzki (2010) Rival et al. (2004) Rival et al. (2006) Stacey et al. (2010) Nakano et al. (2001)

Bellen et al. (1992)

Genderblind (gb)

–

Glial cytoplasmic extensions that project into the neuropils

– –

–

LOF: rejection behavior of females towards courting males

LOF: aberrant flight behavior and abnormal photo- and negative geotactic Larval locomotor problems Decreased climbing capacity in a negative geotaxis assay LOF: stagger behavior, ability to run in straight line, higher motivation to start running LOF: flight problems, no touch-induced escape response, hypoactivity, hyperreactive to startle stimulus

Ng et al. (2011)

Kosmidis et al. (2011)

Suh and Jackson (2007)

Siwicki et al. (1988) Zerr et al. (1990) Ewer et al. (1992) Ng et al. (2011) Peschel et al. (2006)

References

Spinster (spin)

Excitatory amino acid transporter 1 (Eaat1)

Drop dead (drd)

Glial ablation Glial ablation in the adult brain

Couch potato (cpo)

Astrocyte-like glia

–

Ferritin 1 heavy chain (Fer1HCH) Maintenance of circadian rhythms Arrhythmic locomotor activity

–

Maintenance of circadian rhythms

Ferritin 2 light chain (Fer2LCH) Conditional manipulation of: Glial membrane ionic gradient Glial vesicle trafficking Glial calcium stores

–

Cyclic expressed in glia

Ebony (e)

Learning and Crammer (cer) memory Bunched (bun) Sleep bunched (bun)

Courtship

Locomotion

Type of glia

Cyclic expressed in glia – Glial expression sufficient for the manifestation of a weak behavioral rhythmicity (contradictory reports)

Behavioral function

Tim (timeless)

Circadian Per (period) rhythmicity

Behavior

Table 1  Role of glia in normal behavior

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manipulations affecting glial ionic gradient, glial vesicle trafficking (gliotransmission) or glial calcium stores. These manipulations all resulted in arrhythmic locomotor activity (Ng et al. 2011). This effect is accompanied by reduced PDF immunoreactivity in projections of the LNv neurons suggesting that it acts via transport or secretion of PDF. The effect of glial manipulation on PDF release by the clock neurons underscores the importance of glia-toneuron signaling in the regulation of circadian rhythmicity. In summary, current data support a model in which reciprocal interactions between glia and neurons are essential to regulate circadian rhythmicity. However, the signals that mediate glia-to-neuron communication remain to be identified. Lastly, not much is known about the molecular mechanisms within glial cells regulating the circadian patterns of gene transcription, protein synthesis and release. Some transcription factors, such as the cAMP response elementbinding protein (CREB) and nuclear factor kappa-B (NFκB) are known to show circadian oscillating patterns of activity in neurons and glial cells in which they may be responsible for the circadian expression of proteins like Ebony and Period in glia (Tanenhaus et al. 2012). While circadian CREB and NF-kB activity was shown to be under control of period expression in neurons, the molecule(s) responsible for the circadian activity of CREB and NF-κB in glia remain to be identified (Tanenhaus et al. 2012).

Locomotion: walk/flight Locomotor and flight behavior require a complex central integration of various inputs, such as visual cues and proprioceptive information, followed by the actual execution of the movement. Defects in either the central or peripheral nervous systems can lead to defective locomotion or flight. The involvement in the modulation of locomotion and flight is a better-studied role of glia in behavior. A direct demonstration of glia being required for adult locomotor behavior was made by targeted ablation of glia in the adult brain by expressing reaper under control of repo-gal4 and gal80ts. This leads to a dramatic decrease in climbing capacity in a negative geotaxis assay (Ghosh et al. 2011). One of the best-studied genes underlying the glial control of locomotor behavior encodes the glutamate transporter Eaat1. Glutamate is the main excitatory neurotransmitter in the vertebrate CNS, whereas in Drosophila it is assumed that acetylcholine is the primary neurotransmitter in the adult brain. However, a number of observations argue for a role for glutamate in the adult Drosophila brain as well. The glutamate transporter VGlut and ionotropic glutamate receptor subunits are expressed in the CNS and

the Drosophila NMDA receptor homologs are expressed in the mushroom bodies where they are required for olfactory memory formation (Schuster et al. 1993; Tomancak et al. 2002; Featherstone et al. 2005; Xia et al. 2005; Mahr and Aberle 2006; Grosjean et al. 2008). Glutamate has also been reported to play an inhibitory role in some neurons in the olfactory system, but their physiological effects have not been studied in detail (Liu and Wilson 2013). The Eaat1 gene encodes a high-affinity glutamate transporter expressed predominantly in glia throughout all developmental stages. The Eaat1 protein is absent from glial cell bodies, and is selectively located at the glial cytoplasmic extensions projecting into the neuropils (Rival et al. 2004). RNAi-mediated knock down of Eaat1 using Eaat1-gal4 resulted in flies that walked normally and had active neuromuscular junctions (NMJs), but flew poorly and showed no touch-induced escape response. Interestingly, Eaat1 knockdown flies are generally hypoactive but display strong startle-induced hyperreactivity. This phenotype is likely caused by glutamatergic excitotoxicity, as it was at least partially rescued by co-overexpression of the major human glutamate transporter hEEAT2 (Rival et al. 2004). The same authors investigated the effects of Eaat1 knock-down at the thoracic NMJ, a peripheral synapse. Although the NMJs are functional in the absence of Eaat1, the time courses of the recorded excitatory postsynaptic potentials were significantly increased, indicating a prolonged activation of muscle receptors by the neurotransmitter. This likely originates from the reduced glutamate buffering capacity of the dEAAT1-deficient NMJ (Rival et al. 2006). It is unclear if the observed effects of Eaat1 on locomotor behavior arise from developmental defects or from an adult-only requirement for Eaat1. However, further analyses of larval locomotor behavior indicate that the effects are due to alterations in glutamatergic signaling and not to developmental abnormalities or cell death from neurotoxicity (Stacey et al. 2010). Interestingly, Eaat1 expression is regulated by Fringe, a fucosyltransferase, that had previously been shown to promote Notch-Delta-mediated neuron–glia signaling during development (Thomas and van Meyel 2007; Stacey et al. 2010). Two genes with functions in glia were identified in independent screens for locomotor defects. Drosophila GIPC (dGIPC) (Kim et al. 2010) is the homolog of GAIP interacting protein C terminus (GIPC) that interacts with GAIP, a protein located on clathrin-coated vesicles that regulates G-protein signaling (De Vries et al. 1998a, b). dGIPC is expressed in the adult nervous system, mainly in glia but also in a subset of dopaminergic neurons. Knock down of dGIPC in both cell types resulted in a decrease in climbing ability, suggesting a role for glial endocytosis in locomotor behavior (Kim et al. 2010). Kinesin heavy chain (Khc) was identified in the “island assay”, an assay allowing the analysis of

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different locomotor parameters in flight response. Loss of Khc in glia resulted in a spastic phenotype in adult flies in which flies are defective in the jump response that precedes flight initiation. Khc was shown to be required mainly in the subperineural glia that form the blood–brain barrier to ensure normal neuronal function. Khc regulates Rab30-mediated transport of Neurexin IV, a well-known constituent of the blood–brain barrier (Schmidt et al. 2012). Finally, effects on adult locomotion have also been described for the couch potato and drop-dead genes. Couch potato was the first identified gene that suggested a regulatory role of glia in locomotor and flight behavior. cpo mutants show aberrant flight behavior and are abnormal in photo- and negative geotaxis (Bellen et al. 1992). However, since cpo is expressed in neurons and glia in the embryonic and adult peripheral and central nervous systems, the cell type responsible for the behavioral abnormalities remains unclear. Mutants for the drop-dead gene are initially behaviorally normal but within a week after eclosion, adult flies display a stagger behavior in which the flies appear to temporarily lose posture control (Buchanan and Benzer 1993). This also affects their ability to run in a straight line. Interestingly, these mutants show no difference in running performance, measured by different parameters such as maximum speed and total path length. However, they do show a higher motivation to start running than wild-type flies (Lehmann and Cierotzki 2010). Drop-dead mutants exhibit severe neurodegeneration. Prior to onset of neurodegeneration, adult cortex glia were found to have shortened processes that fail to completely wrap neurons. These observations suggest that glial dysfunction is at the basis of the observed behavioral abnormalities, although more recent studies indicate that the drop-dead gene may be required primarily during development and that neurodegeneration is secondary to tracheal system dysfunction (Lehmann and Cierotzki 2010; Sansone and Blumenthal 2013). Thus, the precise role of glia in the adult behavioral phenotypes of drop-dead mutants remains to be determined.

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dimorphic behavior (Ferveur 2010; Yamamoto and Koganezawa 2013). However, the involvement of glia has only more recently been the object of closer investigation. The genderblind (gb) gene encodes a glial amino acid transporter (Grosjean et al. 2008). gb mutants show no differences in heterosexual courtship behavior or copulation, but they also court and attempt to copulate with other males. Gb exerts its courtship modulating function in the adult brain rather than during development, since male– male courtship is induced within hours of RNAi induction targeting this gene. It was shown that male–male courtship in gb mutants is due to a changed response to chemosensory cues that are required to discriminate between sexes. Given that gb had previously been shown to regulate extracellular glutamate levels, and that extracellular glutamate leads to a suppression of glutamatergic synapse strength by the constitutive desensitization of glutamate receptors, it was proposed that an increase of glutamatergic synapse strength seems likely to be the cause of the homosexual behavior (Augustin et al. 2007). An elegant combination of genetic and pharmacological experiments corroborated this proposed mode of action of genderblind and revealed the role of glutamatergic neurotransmission in courtship. The Drosophila gene spinster (spin) (also called benchwarmer) was initially identified as a strong modulator of the rejection behavior of females towards courting males (Suzuki et al. 1997; Yamamoto et al. 1997; Dermaut et al. 2005). Spin mutant flies display CNS abnormalities including an extended abdominal ganglion and neurodegeneration. In the brain, spin is almost exclusively expressed in glial cells. These observations lead the authors to propose that spin acts in glial cells to mediate apoptosis of neurons in the CNS. The mechanism by which spin affects enhanced mate refusal is less clear, but a failure or loss of neuronal circuits or a general lack of brain homeostasis have been proposed (Nakano et al. 2001). Whether or not these phenotypes require spin in glia remains to be demonstrated.

Learning and memory Courtship Mating behavior in Drosophila melanogaster involves a complex interplay that comprises distinct steps. First the male orients towards the female and chases her. Next, he taps the female abdomen with his foreleg and sings a courtship song. If the female is receptive, she will slow down upon which the male will lick her genitalia and attempt copulation. Finally, the female will either decide to allow copulation, by raising her wings and opening her vaginal plate, or to reject the male (Greenspan and Ferveur 2000). Multiple genes and neuronal circuits have been shown to play a role in different aspects of this complex and sexually

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Learning and memory is required to allow organisms to adapt to environmental changes, improve the ability to survive in novel situations, and avoid harmful stimuli. Memory processes are subdivided into protein synthesis-dependent and non-protein synthesis-dependent processes. Short-term memory, which lasts for less than an hour, as well as anesthesia-resistant memory, which lasts up to 24 h, are independent of protein synthesis. Middle-term memory on the other hand, lasting 1–4 h, relies on the translation of preexisting mRNA, while long-term memory requires de novo transcription and translation (Tully et al. 1994; DeZazzo and Tully 1995; Dubnau et al. 2003; Isabel et al. 2004).

J Comp Physiol A

The role of glia in learning and memory is poorly understood. The mushroom bodies are key neuropils in Drosophila for learning and memory. The Kenyon cells, intrinsic mushroom body neurons, form a close network with glia. Glial cells have been reported to reside both at the edges and within this neuropil, suggesting a tight interplay between both cell types (Ito et al. 1998). A study reporting the role of crammer (cer), a cathepsin inhibitor, in olfactory long-term memory provided the first proof that this interplay is functionally important (Comas et al. 2004). The Cer peptide is expressed in the mushroom body Kenyon cells and in surrounding glia. However, only overexpression of cer in the glial cells resulted in a decrease in longterm memory. Rescue experiments revealed that the role of Cer is dosage-sensitive. It was also shown that cer expression decreases in a narrow timeframe 3 h after long-term memory training. The authors speculate that this drop in cer leads to a transient activation of its cysteine protease targets (Comas et al. 2004). The observation that inhibition of different caspases in the hippocampus specifically blocks long-term memory formation in mice, while prolonged cysteine protease activation is associated with neurodegeneration in Alzheimer’s disease patients, suggest that protease inhibitors may constitute a conserved pathway in learning and memory (Callahan et al. 1998; Dash et al. 2000). A second study describes the involvement of mushroom body glia in learning. This study involves a sleep-sensitive form of memory formation and is discussed in the next paragraph (Seugnet et al. 2011).

Sleep Sleep is a universal and vital behavior and is defined by different behavioral changes including quiescence or immobility and an increase in arousal threshold. Sleep is under homeostatic regulation, meaning that periods of extended wakefulness will be followed by compensatory increases in sleep time or intensity (Shaw 2003). All these criteria have been investigated in Drosophila and display remarkable parallels to vertebrate sleep. Although convergence of different data sets suggests an interplay between glial control of drug addiction and circadian rhythmicity, a behavior closely linked to sleep, only one paper reports a role for glia in sleep (Haydon et al. 2009; Seugnet et al. 2011). Mutations in Bunched, a transcription factor regulating Notch activity, show a decreased homeostatic response to sleep deprivation (Seugnet et al. 2011). This effect on sleep homeostasis requires Bunched activity in the mushroom bodies. Given the link with Notch signaling, it was next shown that neuronal Delta in the mushroom bodies regulates sleep homeostasis and also plays a protective

role against sleep deprivation-induced learning impairments. The Notch receptor was shown to be expressed and required in glia. These results suggest that Notch mediates a neuron–glia signaling mechanism but the downstream pathway(s) remain unknown.

Olfaction Olfaction is crucial to the flies’ survival and indispensable for many behaviors. Proper odorant detection helps the fly find mates and food as well as suitable oviposition sites. The fly is equipped with a sophisticated olfactory system that allows it to identify hundreds of different odors (Vosshall 2000). In the adult fly, odor information is received by olfactory receptor neurons (ORNs) in the antennae and maxillary palps and sent to the glomeruli of the antennal lobe. These project further towards the mushroom bodies and the lateral horn via the projection neurons. Glia play an important role in development and function of the olfactory system. The genderblind gene encoding a glial amino acid transporter that regulates courtship behavior by controlling glutamatergic synaptic strength (see above) was also shown to affect olfactory behavior using an olfactory trap assay (Grosjean et al. 2008). It was concluded that, concomitant with defects in courtship, gb mutants have a more general defect in chemosensory processing. Glia have also been reported to play an important role in synaptic plasticity in the adult Drosophila brain, and more specifically in the antennal lobe (Kazama et al. 2011). Most ORNs from both the antennae and the maxillary palp project bilaterally to their target glomeruli. These antennal and maxillary glomeruli reside intermingled in the antennal lobe, where they are interconnected by local neurons. Plasticity following injury requires electrical activity of the ORNs. This plasticity is suppressed by overexpression of the neuroprotective Wallerian degeneration slow (Wlds) gene in these neurons. This results in failure of the Wlds-expressing damaged neurons to recruit glia. Furthermore, blocking endocytosis in glia by overexpression of Shibirets prevents ensheathing glia from engulfing the severed axons with a resultant loss of plasticity. Overall, these experiments demonstrate the important role of glia in adult brain plasticity.

Role of glia in models for neurological and behavioral disorders (summary in Table 2) Neurodegeneration‑related locomotion defects Locomotor problems are an important feature of multiple neurodegenerative disorders, including Parkinson’s and

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Alzheimer’s disease and polyglutamine disorders, such as Huntington’s disease. In Drosophila models of these disorders multiple glial modulators of locomotor behavior have been identified. Polyglutamine disorders Expansion of CAG repeats is causal in different neurodegenerative disorders characterized by locomotor dysfunction including Huntington’s disease (HD), dentatorubral pallidoluysian atrophy (DRPLA) and spinocerebellar ataxias (SCAs). Several of the affected proteins are expressed in glia. For example, Huntingtin is found in glia of rodents and humans (Singhrao et al. 1998; Hebb et al. 1999). Furthermore, glia have a direct role in the pathogenesis of DRPLA (Hayashi et al. 1998; Yamada et al. 2002). Also in Drosophila, glia seem to play an important role in the toxicity of CAG repeat expansion (Kretzschmar et al. 2005). Glial-specific expression of truncated human ataxin-3 with or without polyglutamine expansions resulted in alterations in locomotor behavior that precede cell death. Importantly, neuronal overexpression of the same constructs gave similar behavioral defects but without the neurodegeneration, which strongly suggests that glial and neuronal compartments are both involved in the behavioral abnormalities but that neurodegeneration is primarily a glial-dependent process in this context. Overexpression of different forms of mutant Huntingtin (the first exon of human Htt with 93 CAG repeats and human Huntingtin 103Q) in a pan-glial manner using repogal4 leads to altered locomotor behavior in a negative geotaxis assay (Lievens et al. 2008; Tamura et al. 2009). Furthermore, glial expression of the first exon of human Htt with 93 CAG repeats also induces a bang-sensitive phenotype (Lievens et al. 2008). Overexpression of the first exon of human Htt with 93 CAG repeats in cortex glia subsets in the brain and peripheral glia at the neuromuscular junction using Eaat1-gal4 did not induce alterations in this assay. This suggests that the locomotor defects are caused by mHtt in distinct glia subsets that do not express dEaat1 (Lievens et al. 2008). It was previously shown that expression of the polyQcontaining domain of Htt or an extended polyQ peptide alone in Eaat1-positive glia resulted in decreased Eaat1 expression. This expression is normally regulated by the EGFR-Ras-extracellular signal-regulated kinase (ERK) signaling pathway, but the dEAAT1 upregulation by EGFR is abolished in the presence of polyQ peptides (Lievens et al. 2005). Furthermore, experiments in mice as well as patient data support that mHtt impairs glial glutamate uptake, thus leading to an increase in glutamate excitotoxicity (Arzberger et al. 1997; Behrens et al. 2002; Shin et al. 2005; Hassel et al. 2008). As Eaat1 has been shown to

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J Comp Physiol A

modulate locomotion, it might be involved in the impact of mHtt on this behavior. A second mechanism that has been implicated in glial mHtt pathology in Drosophila involves energy metabolism (Besson et al. 2010). In the brain, glia supply neurons with intermediate-energy substrates. Energetic defects and impairment of mitochondrial function are characteristic for Huntington’s disease. Mitochondrial uncoupling proteins (UCPs) are anion-carrier proteins located in the inner membrane of mitochondria that regulate mitochondrial metabolism and determine the way energy is supplied in cells. Overexpression of Drosophila UCP5 as well as human UCP2 has a positive effect on glial mHtt locomotion phenotypes (Besson et al. 2010). Finally, the Wingless pathway also seems to be involved in the modulation of locomotor behavior by glial mHtt (Dupont et al. 2012). Reduction of functional Armadillo/βCatenin signaling rescues the negative geotaxis and the bang-sensitive phenotypes caused by glial expression of the first exon of human Htt with 93 CAG repeats. Bangsensitive defects are correlated with an abnormal firing pattern of the giant fiber escape pathway after high-frequency electroconvulsive stimulation. This electrophysiological phenotype is observed in glial mHtt mutants and is rescued upon reduction of Armadillo/β-Catenin signaling (Dupont et al. 2012). Iron‑induced neurodegeneration Iron plays a role in the pathology of multiple neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. Its involvement relates to its requirement in the function of multiple iron-containing enzymes, but also in its labile form modulating oxidative stress and propagating lipid peroxidation (Zecca et al. 2004). Furthermore, mutations in several human iron metabolic genes form the basis of neurodegenerative disorders (Rouault and Tong 2008; Gregory et al. 2009), including neuroferritinopathy, a dominant adult-onset basal ganglia disease (Curtis et al. 2001; Levi et al. 2005). Ferritin Light Chain Homologue-2 (Fer2LCH) was shown to be upregulated in a Drosophila model of Parkinson’s disease and has been shown to have neuroprotective properties (Xun et al. 2008; Rival et al. 2009). In a study designed to evaluate a possible role of ferritin in glia, the two ferritin subunits were overexpressed in glia. Overall, this was well tolerated in young flies, but induces a significant decline in escape response at 5 weeks of age, and it affects circadian rhythmicity. These flies have glial iron deposits, but show no signs of neurodegeneration. The mechanism by which the iron deposits induce behavioral changes is unclear, but could be related to interference with iron bioavailability for cellular physiology (Kosmidis et al. 2011).

J Comp Physiol A

The Drosophila ApoD homologue, Glial Lazarillo The vertebrate Apolipoprotein D (ApoD) is a lipocalin associated with high-density lipoproteins in humans. Increases in ApoD are associated with a variety of neurological disorders, including Alzheimer’s disease, schizophrenia and stroke (Rassart et al. 2000). However, it was unclear whether ApoD played a toxic or protective role in these pathologies. Both ApoD and its Drosophila homologue Glial Lazarillo (GLaz) are predominantly expressed in glia throughout life (Sanchez et al. 2006). Glaz protects against oxidative stress and starvation, and regulates life span. Consequently, GLaz mutant males display problems in climbing behavior when aged for 21 days and proved more sensitive to the effects of the oxidative stress-inducing agent paraquat (Sanchez et al. 2006). GLaz mutant flies have increased concentrations of lipid peroxidation products, in line with the proposed protective role of lipocalins and GLaz against the effects of oxidative stress. Overexpression of GLaz was also shown to improve locomotor performance after periods of hyperoxia or hypoxia (Walker et al. 2006) and it has protective effects in a Drosophila model of Friedreich’s ataxia (Navarro et al. 2010). Friedreich’s ataxia Friedreich’s ataxia is the most common autosomal recessive ataxia (Harding 1981). This neurodegenerative disorder is caused by reduced expression of the mitochondrial protein frataxin and is accompanied by mitochondrial iron overload and dysfunction of the respiratory chain and Krebs cycle (Campuzano et al. 1996, 1997; Rotig et al. 1997; Delatycki et al. 1999). Targeted RNAi-induced knock down of frataxin in glia in a Drosophila model of Friedreich’s ataxia resulted in accumulation of fatty acids, reduced lifespan and increased sensitivity to oxidative stress. These flies also have a prominent defect in climbing ability accompanied by age-dependent neurodegeneration. It was also shown that Glial Lazarillo overexpression can significantly suppress these phenotypes and consistent with the protective role of GLaz against oxidative stress, an accumulation of lipid peroxidation products was measured in frataxin knock-down flies (Navarro et al. 2010). Addiction Addiction is a disorder characterized by excessive use of a drug to the point of compulsive drug seeking and consumption. This behavior has only relatively recently been the subject of studies in Drosophila. The vast majority of these studies focus on ethanol-related behaviors. Other drugs of abuse have not been extensively studied, although the effects of cocaine, nicotine and amphetamines were the

subjects of some reports. Only one study directly shows the involvement of glia in drug sensitivity (Bainton et al. 2005). Moody-A and moody-B, two GPCRs generated by alternative splicing, are co-expressed in the surface glia surrounding the adult and larval nervous system (Bainton et al. 2005). Moody is required for blood–brain barrier function in embryos (Schwabe et al. 2005). Either isoform alone is sufficient to maintain the integrity of the blood–brain barrier in the adult brain (Bainton et al. 2005). Furthermore, experiments conditionally expressing a moody-RNAi transgene show that the maintenance of this integrity is an active process in the adult brain rather than a developmental effect. Mutations in moody have effects on cocaine, nicotine and ethanol sensitivity, a behavior measured by drug-induced loss of negative geotaxis. Hypomorphic moodyEP1529 mutant flies show an increased sensitivity to ethanol and nicotine. In contrast, they are more resistant to the acute intoxicating effects of ethanol. Interestingly, while either Moody-A or Moody-B function is sufficient for survival and to maintain blood–brain barrier integrity, both Moody isoforms are required for normal cocaine sensitivity. Although no other reports examine the role of glia in addiction, combining the results of different studies has led to some hypotheses on other glial functions in addiction (Haydon et al. 2009). Studies in flies and mammals suggest connections between the circadian system and responses to drugs of abuse (Haydon et al. 2009). Mutations in per, Clk, cyc and doubletime (dbt), four Drosophila circadian genes, lead to elimination of cocaine sensitization as well as normal cocaine induction of tyrosine decarboxylase activity (Andretic et al. 1999). This study did not identify the relevant cell types. However, given that moody regulates cocaine response in glia and clock proteins have been shown to function in these cells (Suh and Jackson 2007), it was suggested that both findings might be interconnected (Haydon et al. 2009). Another interesting hypothesis involves the glial protein Ebony, an N-β-alanyl-biogenic amine synthetase involved in dopamine metabolism (Richardt et al. 2003). Since all Ebony-containing glia are Repo positive and Moody is localized within surface glia and other glial cells which contain the glial-specific transcription factor Repo, the expression patterns of both genes are expected to overlap in certain cells (Bainton et al. 2005; Suh and Jackson 2007; Haydon et al. 2009). Ebony is known to play a role in circadian control, while both glia and dopaminergic signaling regulate responses to drugs of abuse. Thus, the possibility exists that the Ebony-positive glial cells may help modulate neuronal responsiveness to such drugs (Haydon et al. 2009).

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13 Glial Lazarillo (GLaz)

Glial Lazarillo (ApoD homologue)

Seizures/bang-sensitive behavior

Addiction

Dupont et al. (2012)

Positive effect on glial mHtt phenotypes

Glial manipulation results in hyperex- Ensheathing glia at the larval NMJ Ueda et al. (2008) citability at the NMJ LOF rapid seizures at 38 °C and bang- Cortex glia sensitive behavior at room temperature

armadillo (arm) Focal adhesion kinase 56 (Fak56) Zydeco (zyd)

Melom and Littleton (2013)

Lievens et al. (2008)

Andretic et al. (1999)

Bainton et al. (2005)

Schmidt et al. (2012)

Kim et al. (2010)

Glial expression induces a bang-sensitive behavior

LOF: adult spastic phenotype, larval Subperineural locomotor abnormalities Surface glia LOF: alterations in cocaine, nicotine and ethanol sensitivity LOF: elimination of cocaine sensitiza- – tion and normal cocaine induction of tyrosine decarboxylase activity

Kosmidis et al. (2011b)

Expression of human Huntingtin: 93 CAG repeats

Doubletime (Dbt)

Cycle (cyc)

Clock (Clk)

Period (per)

Moody

Kinesin heavy chain (Khc)

GIPC and KHC (vesicle trafficking) GIPC (kermit)

Ferritin Light Chain Homologue-2 (Fer2LCH)

Dupont et al. (2012)

Locomotion: positive effect on glial mHtt phenotypes Locomotion: glial overexpression induces decline in escape response at 5 weeks of age LOF: decline in climbing behavior after 21 days or upon induction of oxidative stress GOF: improvement in locomotor performance after hyperoxia or hypoxia LOF: decrease in climbing ability

armadillo (arm)

Iron-induced neurodegeneration

Besson et al. (2010)

–

Lievens et al. (2008) Tamura et al. (2009)

Non eaat1 positive glia(?)

Locomotion: positive effect on glial mHtt phenotypes

Kretzschmar et al. (2005)

References

–

Type of glia

Drosophila UCP5 (Bmcp), human UCP2

Pan-glial expression of human ataxin-3: Locomotion: fast phototaxis assay, Buridan’s paradigm polyglutamine expanded and nonexpanded form Locomotion: negative geotaxis assay Expression of human Huntingtin: the first exon of human Htt with 93 CAG repeats and human Huntingtin 103Q

Polyglutamine disorders

Behavioral function

Gene/Drosophila model

Disorder

Table 2  Role of glia in models for neurological and behavioral disorders

J Comp Physiol A

J Comp Physiol A

Seizures/bang‑sensitive behavior Epileptic behavior is characterized by periods of hypersynchronous neuronal firing. In Drosophila, mutants have been described that display seizures following mechanical shock (bang-sensitive behavior) that are reminiscent of human epileptic seizures. A possible role of glia in this phenotype is supported by several studies. As mentioned earlier, glial expression of the first exon of human Htt with 93 CAG repeats can induce a bang-sensitive behavior (Lievens et al. 2008). Mutations in Focal Adhesion Kinase 56 (Fak56) display a bang-sensitive phenotype in addition to a decreased lifespan (Ueda et al. 2008). Endogenous Fak56 is abundantly present in ensheathing glia at the larval NMJ, and in Fak56 mutants, the structural integrity of the ensheathed neurons is affected. Furthermore, glial-specific manipulation of this gene results in hyperexcitability at the NMJ, while both neuronal and muscle manipulation had no effect. Therefore, these observations are consistent with a glia-specific requirement of Fak56 being causally linked to the bangsensitive phenotype. In mammals, changes in glial Ca2+ activity have been shown to precede epileptic events in mammals but the causal relationship is unclear. A Drosophila screen for genes involved in seizure susceptibility provided more proof for the involvement of glial Ca2+ homeostasis in bang-sensitive behavior. This screen identified zydeco, a gene encoding a glial-specific Na+/Ca2+, K+ exchanger (Guan et al. 2005). Mutations in zyd induce rapid seizures when flies are exposed to a temperature of 38 °C as well as when flies are briefly vortexed at room temperature (bangsensitive behavior) (Melom and Littleton 2013). These behavioral abnormalities can be rescued by expressing the Na+/Ca2+, K+ exchanger in cortex glia.

Conclusions and perspectives Until relatively recently, the mechanisms by which neurons modulate behavior were the main focus of studies in vertebrates and Drosophila. However, the importance of glia and of neuronal–glial interactions to preserve brain homeostasis and to regulate brain function is becoming increasingly clear. The overview provided here demonstrates that we are only at the beginning of understanding the role of glia in controlling adult behavior. The many tools, techniques and markers available make that Drosophila as a genetic model organism provides ample opportunities to study in detail the different glial cell types and their roles in the regulation of various processes, including behaviors, controlled by the brain. Furthermore, the conservation of many pathways and processes from Drosophila to human makes

it reasonable to assume that understanding the glial contribution to abnormal behaviors could guide us towards new treatment options for behavioral problems associated with psychiatric or neurodegenerative disorders. We highlight here several key questions for which we start to gain insight and where we can expect exciting new discoveries to follow in the near future. Several studies describe the importance of neuron–glia interactions in maintaining brain homeostasis to regulate normal behavior. Although some players have been identified or suggested such as gliotransmission and Notch-Delta signaling, the molecular mechanisms remain to be fully uncovered. Currently, five major types of glia in the adult Drosophila brain have been identified. However, data from other insects and careful analyses of the glial complexity in the optic lobe, suggest that the heterogeneity of glia in the Drosophila brain may be much more extensive than currently appreciated. The molecular characterization of glial cells at the single-cell level, the identification of molecular markers and genetic tools to mark additional types of glia will be important to fully describe the complexity of the glial compartment of the brain. In this context, it will also be exciting to discover whether or not Drosophila have the equivalent of mammalian microglia. Conceivably, this could either be a hitherto undescribed glial cell type or it could be that other glia are capable of an immune response. The study by Petersen et al. (2012) suggests that the latter could be the case. A few studies have shown that glia show differences depending on time of day, environmental conditions and ageing. Careful analyses of glia cell number and morphological plasticity during the day and over the lifetime of adult flies will be a first step towards discovering the molecular mechanisms and the environmental factors that contribute to these changes and how they are relevant for fly behavior under physiological and pathological conditions. In many studies to date, it is not clear whether the observed effects on adult behavior are due to the requirement of a glial expressed gene during development or specifically in the adult. Given the important role of glia in shaping and remodeling axonal projections and connectivity (Freeman 2006; Yamamoto et al. 2006; Awasaki et al. 2011; Hidalgo et al. 2011; Kuzina et al. 2011; Hakim et al. 2014), this will be an important issue to address in future studies. Several of the studies discussed in this review provide evidence that proper neurotransmitter clearance and metabolism are important roles of glia, similar to what is known in mammals. The power of Drosophila genetics should enable to systematically dissect which neurotransmitters are required for which behaviors and how glia maintain behavior within what can be defined as the normal range.

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Acknowledgments  The authors received financial support of VIB, IWT and FWO (grants G.0654.08 and G.0789.14). We are grateful to the anonymous reviewers for their critical input that helped improve this review.

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