Highlights d Ultrasound-induced neuromodulation is initiated by opening of TRPA1 in astrocytes d The Ca 2+ entry through TRPA1 causes a release of glutamate through Best1 channels d The released glutamate activates NMDA receptors in neighboring neurons

In the brain, a reduction in extracellular osmolality causes water-influx and swelling, which subsequently triggers Cl − - and osmolytes-efflux via volume-regulated anion channel (VRAC). Although LRRC8 family has been recently proposed as the pore-forming VRAC which is activated by low cytoplasmic ionic strength but not by swelling, the molecular identity of the pore-forming swelling-dependent VRAC (VRAC swell ) remains unclear. Here we identify and characterize Tweety-homologs (TTYH1, TTYH2, TTYH3) as the major VRAC swell in astrocytes. Gene-silencing of all Ttyh1/2/3 eliminated hypo-osmotic-solution-induced Cl − conductance (I Cl,swell ) in cultured and hippocampal astrocytes. When heterologously expressed in HEK293T or CHO-K1 cells, each TTYH isoform showed a significant I Cl,swell with similar aquaporin-4 dependency, pharmacological properties and glutamate permeability as I Cl,swell observed in native astrocytes. Mutagenesis-based structure-activity analysis revealed that positively charged arginine residue at 165 in TTYH1 and 164 in TTYH2 is critical for the formation of the channel-pore. Our results demonstrate that TTYH family confers the bona fide VRAC swell in the brain.

Tonic inhibition in the brain is mediated through an activation of extrasynaptic GABA receptors by the tonically released GABA, resulting in a persistent GABAergic inhibitory action. It is one of the key regulators for neuronal excitability, exerting a powerful action on excitation/inhibition balance. We have previously reported that astrocytic GABA, synthesized by monoamine oxidase B (MAOB), mediates tonic inhibition via GABA-permeable bestrophin 1 (Best1) channel in the cerebellum. However, the role of astrocytic GABA in regulating neuronal excitability, synaptic transmission, and cerebellar brain function has remained elusive. Here, we report that a reduction of tonic GABA release by genetic removal or pharmacological inhibition of Best1 or MAOB caused an enhanced neuronal excitability in cerebellar granule cells (GCs), synaptic transmission at the parallel fiber-Purkinje cell (PF-PC) synapses, and motor performance on the rotarod test, whereas an augmentation of tonic GABA release by astrocyte-specific overexpression of MAOB resulted in a reduced neuronal excitability, synaptic transmission, and motor performance. The bidirectional modulation of astrocytic GABA by genetic alteration of Best1 or MAOB was confirmed by immunostaining and in vivo microdialysis. These findings indicate that astrocytes are the key player in motor coordination through tonic GABA release by modulating neuronal excitability and could be a good therapeutic target for various movement and psychiatric disorders, which show a disturbed excitation/inhibition balance.