Glutamate transporter 1 (GLT1) is the main astrocytic transporter that shapes glutamatergic transmission in the brain. However, whether this transporter modulates sleep/wake regulatory neurons is unknown. Using quantitative immunohistochemical analysis, we assessed perisomatic GLT1 apposition with sleep/wake neurons in the male rat following 6-h sleep deprivation (SD) or following 6-h undisturbed conditions when animals were mostly asleep (Rest). We found that SD decreased perisomatic GLT1 apposition with wake-promoting orexin (ORX) neurons in the lateral hypothalamus (LH) compared to Rest. Reduced GLT1 apposition was associated with tonic presynaptic inhibition of excitatory transmission to these neurons due to the activation of group III metabotropic glutamate receptors, an effect mimicked by a GLT1 inhibitor in the Rest condition. In contrast, SD resulted in increased GLT1 apposition with sleep-promoting melanin-concentrating hormone (MCH) neurons in the LH. Functionally, this decreased the postsynaptic response of MCH neurons to high frequency synaptic activation without changing presynaptic glutamate release. The changes in GLT1 apposition with ORX and MCH neurons were reversed after 3 h of sleep opportunity following 6-h SD. These SD effects were specific to orexin and MCH neurons, as no change in GLT1 apposition was seen in basal forebrain cholinergic or parvalbumin-positive GABA neurons. Thus, within a single hypothalamic area, GLT1 differentially regulates excitatory transmission to wake- and sleep-promoting neurons depending on sleep history. These processes may constitute novel astrocyte-mediated homeostatic mechanisms controlling sleep/wake behavior.Sleep/wake cycles are regulated by the alternate activation of sleep- and wake-promoting neurons. Whether and how astrocytes can regulate this reciprocal neuronal activity is unclear. Here we report that within the lateral hypothalamus, where functionally opposite wake-promoting orexin (ORX) neurons and sleep-promoting melanin-concentrating hormone (MCH) neurons co-distribute, the glutamate transporter GLT1, mainly present on astrocytes, distinctly modulates excitatory transmission in a cell-type specific manner and according to sleep history. Specifically, GLT1 is reduced around the somata of ORX neurons while increased around MCH neurons following sleep deprivation, resulting in different forms of synaptic plasticity. Thus, astrocytes can fine-tune the excitability of functionally discrete neurons via glutamate transport, which may represent novel regulatory mechanisms for sleep.

Dopamine (DA) and orexin neurons play important roles in reward and food intake. There are anatomical and functional connections between these two cell groups: orexin peptides stimulate DA neurons in the ventral tegmental area and DA inhibits orexin neurons in the hypothalamus. However, the cellular mechanisms underlying the action of DA on orexin neurons remain incompletely understood. Therefore, the effect of DA on inhibitory transmission to orexin neurons was investigated in rat brain slices using the whole-cell patch-clamp technique. We found that DA modulated the frequency of spontaneous and miniature IPSCs (mIPSCs) in a concentration-dependent bidirectional manner. Low (1 μM) and high (100 μM) concentrations of DA decreased and increased IPSC frequency, respectively. These effects did not accompany a change in mIPSC amplitude and persisted in the presence of G-protein signaling inhibitor GDPβS in the pipette, suggesting that DA acts presynaptically. The decrease in mIPSC frequency was mediated by D2 receptors whereas the increase required co-activation of D1 and D2 receptors and subsequent activation of phospholipase C. In summary, our results suggest that DA has complex effects on GABAergic transmission to orexin neurons, involving cooperation of multiple receptor subtypes. The direction of dopaminergic influence on orexin neurons is dependent on the level of DA in the hypothalamus. At low levels DA disinhibits orexin neurons whereas at high levels it facilitates GABA release, which may act as negative feedback to curb the excitatory orexinergic output to DA neurons. These mechanisms may have implications for consummatory and motivated behaviours.

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