A Circadian Clock in the Blood-Brain Barrier Regulates Xenobiotic Efflux.

Endogenous circadian rhythms are thought to modulate responses to external factors, but mechanisms that confer time-of-day differences in organismal responses to environmental insults/therapeutic treatments are poorly understood. Using a xenobiotic, we find that permeability of the Drosophila "blood"-brain barrier (BBB) is higher at night. The permeability rhythm is driven by circadian regulation of efflux and depends on a molecular clock in the perineurial glia of the BBB, although efflux transporters are restricted to subperineurial glia (SPG). We show that transmission of circadian signals across the layers requires cyclically expressed gap junctions. Specifically, during nighttime, gap junctions reduce intracellular magnesium ([Mg2+]i), a positive regulator of efflux, in SPG. Consistent with lower nighttime efflux, nighttime administration of the anti-epileptic phenytoin is more effective at treating a Drosophila seizure model. These findings identify a novel mechanism of circadian regulation and have therapeutic implications for drugs targeted to the central nervous system.

The Glial Perspective on Sleep and Circadian Rhythms.

While neurons and circuits are almost unequivocally considered to be the computational units and actuators of behavior, a complete understanding of the nervous system must incorporate glial cells. Far beyond a copious but passive substrate, glial influence is inextricable from neuronal physiology, whether during developmental guidance and synaptic shaping or through the trophic support, neurotransmitter and ion homeostasis, cytokine signaling and immune function, and debris engulfment contributions that this class provides throughout an organism's life. With such essential functions, among a growing literature of nuanced roles, it follows that glia are consequential to behavior in adult animals, with novel genetic tools allowing for the investigation of these phenomena in living organisms. We discuss here the relevance of glia for maintaining circadian rhythms and also for serving functions of sleep.

Modulation of sleep by trafficking of lipids through the Drosophila blood-brain barrier.

Endocytosis through Drosophila glia is a significant determinant of sleep amount and occurs preferentially during sleep in glia of the blood-brain barrier (BBB). To identify metabolites whose trafficking is mediated by sleep-dependent endocytosis, we conducted metabolomic analysis of flies that have increased sleep due to a block in glial endocytosis. We report that acylcarnitines, fatty acids conjugated to carnitine to promote their transport, accumulate in heads of these animals. In parallel, to identify transporters and receptors whose loss contributes to the sleep phenotype caused by blocked endocytosis, we screened genes enriched in barrier glia for effects on sleep. We find that knockdown of lipid transporters LRP1&2 or of carnitine transporters ORCT1&2 increases sleep. In support of the idea that the block in endocytosis affects trafficking through specific transporters, knockdown of LRP or ORCT transporters also increases acylcarnitines in heads. We propose that lipid species, such as acylcarnitines, are trafficked through the BBB via sleep-dependent endocytosis, and their accumulation reflects an increased need for sleep.

AANAT1 functions in astrocytes to regulate sleep homeostasis.

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.

Sleep is essential for our physical and mental health. A lack of sleep can affect our energy and concentration levels and is often linked to chronic illnesses and mood disorders. Sleep is controlled by an internal clock in our brain that operates on a 24-hour cycle, telling our bodies when we are tired and ready for bed, or fresh and alert to start a new day. In addition, the brain tracks the need for sleep and drives the recovery of sleep after periods of prolonged wakefulness ­ a process known as sleep-wake homeostasis. Chemical messengers in the brain such as dopamine and serotonin also play an important part in regulating our sleep drive. While dopamine keeps us awake, serotonin can both prevent us from and help us falling asleep, depending on the part of the brain in which it is released. Most research has focused on the role of different brain circuits on sleep, but it has been shown that a certain type of brain cell, known as astrocyte, may also be important for sleep regulation. So far, it has been unclear if astrocytes could be involved in regulating the need for recovery sleep after a sleep-deprived night ­ also known as rebound sleep. Now, Davla, Artiushin et al. used sleep-deprived fruit flies to investigate this further. The flies were kept awake over 12 hours (from 6pm to 6am), using intermittent physical agitation. The researchers found that astrocytes in the brains of fruit flies express a molecule called AANAT1, which peaked at the beginning of the night, declined as the night went on and recovered by morning. In sleep deprived flies, it inactivated the chemical messengers and so lowered the amount of dopamine and serotonin in the brain. However, in mutant flies that lacked AANAT1, both dopamine and serotonin levels increased in the brain after sleep deprivation. When AANAT1 was selectively removed from astrocytes only, sleep-deprived flies needed more rebound sleep during the day to make up for lost sleep at night. This shows that both astrocytes and AANAT1 play a crucial role in sleep homeostasis. Molecules belonging to the AANAT family exist in both flies and humans, and these results could have important implications for the science of sleep. The study of Davla, Artiushin et al. paves the way for understanding the mechanisms of sleep homeostasis that are similar in both organisms, and may in the future, help to identify sleep drugs that target astrocytes and the molecules they express.

Endocytosis at the Drosophila blood-brain barrier as a function for sleep.

Glia are important modulators of neural activity, yet few studies link glia to sleep regulation. We find that blocking activity of the endocytosis protein, dynamin, in adult Drosophila glia increases sleep and enhances sleep need, manifest as resistance to sleep deprivation. Surface glia comprising the fly equivalent of the blood-brain barrier (BBB) mediate the effect of dynamin on sleep. Blocking dynamin in the surface glia causes ultrastructural changes, albeit without compromising the integrity of the barrier. Supporting a role for endocytic trafficking in sleep, a screen of Rab GTPases identifies sleep-modulating effects of the recycling endosome Rab11 in surface glia. We also find that endocytosis is increased in BBB glia during sleep and reflects sleep need. We propose that endocytic trafficking through the BBB represents a function of sleep.

The Drosophila circuitry of sleep-wake regulation.

Sleep is a deeply conserved, yet fundamentally mysterious behavioral state. Knowledge of the circuitry of sleep-wake regulation is an essential step in understanding the physiology of the sleeping brain. Recent efforts in Drosophila have revealed new populations which impact sleep, as well as provided unprecedented mechanistic and electrophysiological detail of established sleep-regulating neurons. Multiple, distributed centers of sleep-wake circuitry exist in the fly, including the mushroom bodies, central complex and the circadian clock cells. Intriguingly, certain populations have been implicated in specific roles in homeostatic rebound sleep, occurring after sleep loss. In short, our knowledge of fly sleep circuitry advances towards a greater view of brain-wide connectivity and integration of the signals and correlates of the state of sleep.