SIK3 and Wnk converge on Fray to regulate glial K+ buffering and seizure susceptibility.

Glial cells play a critical role in maintaining homeostatic ion concentration gradients. Salt-inducible kinase 3 (SIK3) regulates a gene expression program that controls K+ buffering in glia, and upregulation of this pathway suppresses seizure behavior in the eag, Shaker hyperexcitability mutant. Here we show that boosting the glial SIK3 K+ buffering pathway suppresses seizures in three additional molecularly diverse hyperexcitable mutants, highlighting the therapeutic potential of upregulating glial K+ buffering. We then explore additional mechanisms regulating glial K+ buffering. Fray, a transcriptional target of the SIK3 K+ buffering program, is a kinase that promotes K+ uptake by activating the Na+/K+/Cl- co-transporter, Ncc69. We show that the Wnk kinase phosphorylates Fray in Drosophila glia and that this activity is required to promote K+ buffering. This identifies Fray as a convergence point between the SIK3-dependent transcriptional program and Wnk-dependent post-translational regulation. Bypassing both regulatory mechanisms via overexpression of a constitutively active Fray in glia is sufficient to robustly suppress seizure behavior in multiple Drosophila models of hyperexcitability. Finally, we identify cortex glia as a critical cell type for regulation of seizure susceptibility, as boosting K+ buffering via expression of activated Fray exclusively in these cells is sufficient to suppress seizure behavior. These findings highlight Fray as a key convergence point for distinct K+ buffering regulatory mechanisms and cortex glia as an important locus for control of neuronal excitability.

SCN VIP Neurons Are Essential for Normal Light-Mediated Resetting of the Circadian System.

The suprachiasmatic nucleus (SCN) synchronizes circadian rhythms in behavior and physiology to the external light cycle, but the mechanisms by which this occurs are unclear. As the neuropeptide vasoactive intestinal peptide (VIP) is important for circadian light responses, we tested the hypothesis that rhythmic VIP-producing SCN neurons mediate circadian light responses in male and female mice. Using in vivo fiber photometry over multiple days, we found daily rhythms in spontaneous calcium events of SCN VIP neurons that peaked during the subjective day and were disrupted by constant light. The light-evoked calcium responses peaked around subjective dusk and were greater during the subjective night. Using novel VIP sensor cells, we found that the activity patterns in SCN VIP neurons correlated tightly with spontaneous and NMDA-evoked VIP release. Finally, in vivo hyperpolarization of VIP neurons attenuated light-induced shifts of daily rhythms in locomotion. We conclude that SCN VIP neurons exhibit circadian rhythms in spontaneous and light-responsive activity and are essential for the normal resetting of daily rhythms by environmental light.SIGNIFICANCE STATEMENT Daily rhythms in behavior and physiology, including sleep/wake and hormone release, are synchronized to local time by the master circadian pacemaker, the suprachiasmatic nucleus (SCN). The advent of artificial lighting and, consequently, light exposure at night, is associated with an increased risk of disease due to disrupted circadian rhythms. However, the mechanisms by which the SCN encodes normal and pathological light information are unclear. Here, we find that vasoactive intestinal peptide (VIP)-producing SCN neurons exhibit daily rhythms in neuronal activity and VIP release, and that blocking the activity of these neurons attenuates light-induced phase shifts. We conclude that rhythmic VIP neurons are an essential component of the circadian light transduction pathway.

Bidirectional regulation of glial potassium buffering - glioprotection versus neuroprotection.

Glia modulate neuronal excitability and seizure sensitivity by maintaining potassium and water homeostasis. A salt inducible kinase 3 (SIK3)-regulated gene expression program controls the glial capacity to buffer K+ and water in Drosophila, however upstream regulatory mechanisms are unknown. Here, we identify an octopaminergic circuit linking neuronal activity to glial ion and water buffering. Under basal conditions, octopamine functions through the inhibitory octopaminergic G-protein-coupled receptor (GPCR) OctßR to upregulate glial buffering capacity, while under pathological K+ stress, octopamine signals through the stimulatory octopaminergic GPCR OAMB1 to downregulate the glial buffering program. Failure to downregulate this program leads to intracellular glia swelling and stress signaling, suggesting that turning down this pathway is glioprotective. In the eag shaker Drosophila seizure model, the SIK3-mediated buffering pathway is inactivated. Reactivation of the glial buffering program dramatically suppresses neuronal hyperactivity, seizures, and shortened life span in this mutant. These findings highlight the therapeutic potential of a glial-centric therapeutic strategy for diseases of hyperexcitability.