How the brain controls the need and acquisition of recovery sleep after prolonged wakefulness is an important issue in sleep research. The monoamines serotonin and dopamine are key regulators of sleep in mammals and in Drosophila. We found that the enzyme arylalkylamine N-acetyltransferase 1 (AANAT1) is expressed by Drosophila astrocytes and specific subsets of neurons in the adult brain. AANAT1 acetylates monoamines and inactivates them, and we found that AANAT1 limited the accumulation of serotonin and dopamine in the brain upon sleep deprivation (SD). Loss of AANAT1 from astrocytes, but not from neurons, caused flies to increase their daytime recovery sleep following overnight SD. Together, these findings demonstrate a crucial role for AANAT1 and astrocytes in the regulation of monoamine bioavailability and homeostatic sleep.

The dendritic trees of neurons result from specific patterns of growth and branching, and dendrite branches of the same neuron avoid one another to spread over a particular receptive field. Recognition molecules on the surfaces of dendrites influence these patterning and avoidance processes by promoting attractive, repulsive or adhesive responses to specific cues. The Drosophila transmembrane protein Turtle (Tutl) and its orthologs in other species are conserved members of the immunoglobulin superfamily, the in vivo functions of which are unknown. In Drosophila sensory neurons, we show that the tutl gene is required to restrain dendrite branch formation in neurons with simple arbors, and to promote dendrite self-avoidance in neurons with complex arbors. The cytoplasmic tail of Tutl is dispensable for control of dendrite branching, suggesting that Tutl acts as a ligand or co-receptor for an unidentified recognition molecule to influence the architecture of dendrites and their coverage of receptive territories.

Sleep is critical for many aspects of brain function and is accompanied by brain-wide changes in the physiology of neurons and synapses [1, 2]. Growing evidence suggests that glial cells contribute to diverse aspects of sleep regulation, including neuronal and metabolic homeostasis [3-5], although the molecular basis for this remains poorly understood. The fruit fly, Drosophila melanogaster, displays all the behavioral and physiological characteristics of sleep [1, 2], and genetic screening in flies has identified both conserved and novel regulators of sleep and wakefulness [2, 6, 7]. With this approach, we identified Excitatory amino acid transporter 2 (Eaat2) and found that its loss from glia, but not neurons, increases sleep. We show that Eaat2 is expressed in ensheathing glia, where Eaat2 functions during adulthood to regulate sleep. Increased sleep in Eaat2-deficient flies is accompanied by reduction of metabolic rate during sleep bouts, an indicator of deeper sleep intensity. Eaat2 is a member of the conserved EAAT family of membrane transport proteins [8], raising the possibility that it affects sleep by controlling the movement of ions and neuroactive chemical messengers to and from ensheathing glia. In vitro, Eaat2 is a transporter of taurine [9], which promotes sleep when fed to flies [10]. We find that the acute effect of taurine on sleep is abolished in Eaat2 mutant flies. Together, these findings reveal a wake-promoting role for Eaat2 in ensheathing glia through a taurine-dependent mechanism.

The architectures of dendritic trees are crucial for the wiring and function of neuronal circuits because they determine coverage of receptive territories, as well as the nature and strength of sensory or synaptic inputs. Here, we describe a cell-intrinsic pathway sculpting dendritic arborization (da) neurons in Drosophila that requires Longitudinals Lacking (Lola), a BTB/POZ transcription factor, and its control of the F-actin cytoskeleton through Spire (Spir), an actin nucleation protein. Loss of Lola from da neurons reduced the overall length of dendritic arbors, increased the expression of Spir, and produced inappropriate F-actin-rich dendrites at positions too near the cell soma. Selective removal of Lola from only class IV da neurons decreased the evasive responses of larvae to nociception. The increased Spir expression contributed to the abnormal F-actin-rich dendrites and the decreased nocifensive responses because both were suppressed by reduced dose of Spir. Thus, an important role of Lola is to limit expression of Spir to appropriate levels within da neurons. We found Spir to be expressed in dendritic arbors and to be important for their development. Removal of Spir from class IV da neurons reduced F-actin levels and total branch number, shifted the position of greatest branch density away from the cell soma, and compromised nocifensive behavior. We conclude that the Lola-Spir pathway is crucial for the spatial arrangement of branches within dendritic trees and for neural circuit function because it provides balanced control of the F-actin cytoskeleton.

The establishment of tissue architecture in the nervous system requires the proper migration and positioning of newly born neurons during embryonic development. Defects in nuclear translocation, a key process in neuronal positioning, are associated with brain diseases such as lissencephaly in humans. Accumulated evidence suggests that the molecular mechanisms controlling neuronal movement are conserved throughout evolution. While the initial events of neuronal migration have been extensively studied, less is known about the molecular details underlying the establishment of neuronal architecture after initial migration.

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory amino acid transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl-) channels, but the physiological role of Cl- conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl- channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. Here we investigated the effects of 5 additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behavior. Our results indicate that mutations resulting in decreased hEAAT1 Cl- channel activity but with functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl- homeostasis in glial cells for proper CNS function. We also identified what we believe is a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.

To identify novel regulators of nervous system development, we used the GAL4-UAS misexpression system in Drosophila to screen for genes that influence axon guidance in developing embryos. We mobilized the Gene Search (GS) P element and identified 42 lines with insertions in unique loci, including leak/roundabout2, which encodes an axon guidance receptor and confirms the utility of our screen. The genes we identified encode proteins of diverse classes, some acting near the cell surface and others in the cytoplasm or nucleus. We found that one GS line drove misexpression of the NF-kappaB transcription factor Dorsal, causing motor axons to bypass their correct termination sites. In the developing visual system, Dorsal misexpression also caused photoreceptor axons to reach incorrect positions within the optic lobe. This mistargeting occurred without observable changes of cell fate and correlated with localization of ectopic Dorsal in distal axons. We found that Dorsal and its inhibitor Cactus are expressed in photoreceptors, though neither was required for axon targeting. However, mutation analyses of genes known to act upstream of Dorsal revealed a requirement for the interleukin receptor-associated kinase family kinase Pelle for layer-specific targeting of photoreceptor axons, validating our screen as a means to identify new molecular determinants of nervous system development in vivo.

Developing neurons form dendritic trees with cell type-specific patterns of growth, branching and targeting. Dendrites of Drosophila peripheral sensory neurons have emerged as a premier genetic model, though the molecular mechanisms that underlie and regulate their morphogenesis remain incompletely understood. Still less is known about this process in central neurons and the extent to which central and peripheral dendrites share common organisational principles and molecular features. To address these issues, we have carried out two comparable gain-of-function screens for genes that influence dendrite morphologies in peripheral dendritic arborisation (da) neurons and central RP2 motor neurons.