Generation of Astrocyte-specific BEST1 Conditional Knockout Mouse with Reduced Tonic GABA Inhibition in the Brain.

Bestrophin-1 (BEST1) is a Ca2+-activated anion channel known for its role in astrocytes. Best1 is permeable to gliotransmitters, including GABA, to contribute to tonic GABA inhibition and modulate synaptic transmission in neighboring neurons. Despite the crucial functions of astrocytic BEST1, there is an absence of genetically engineered cell-type specific conditional mouse models addressing these roles. In this study, we developed an astrocyte-specific BEST1 conditional knock-out (BEST1 aKO) mouse line. Using the embryonic stem cell (ES cell) targeting method, we developed Best1 floxed mice (C57BL/6JCya-Best1em1flox/Cya), which have exon 3, 4, 5, and 6 of Best1 flanked by two loxP sites. By crossing with hGFAP-CreERT2 mice, we generated Best1 floxed/hGFAP-CreERT2 mice, which allowed for the tamoxifen-inducible deletion of Best1 under the human GFAP promoter. We characterized its features across various brain regions, including the striatum, hippocampal dentate gyrus (HpDG), and Parafascicular thalamic nucleus (Pf). Compared to the Cre-negative control, we observed significantly reduced BEST1 protein expression in immunohistochemistry (IHC) and tonic GABA inhibition in patch clamp recordings. The reduction in tonic GABA inhibition was 66.7% in the striatum, 46.4% in the HpDG, and 49.6% in the Pf. Our findings demonstrate that the BEST1 channel in astrocytes significantly contributes to tonic inhibition in the local brain areas. These mice will be valuable for future studies not only on tonic GABA release but also on tonic release of gliotransmitters mediated by astrocytic BEST1.

Astrocytes Render Memory Flexible by Releasing D-Serine and Regulating NMDA Receptor Tone in the Hippocampus.

BACKGROUND: NMDA receptor (NMDAR) hypofunction has been implicated in several psychiatric disorders with impairment of cognitive flexibility. However, the molecular mechanism of how NMDAR hypofunction with decreased NMDAR tone causes the impairment of cognitive flexibility has been minimally understood. Furthermore, it has been unclear whether hippocampal astrocytes regulate NMDAR tone and cognitive flexibility. METHODS: We employed cell type-specific genetic manipulations, ex vivo electrophysiological recordings, sniffer patch recordings, cutting-edge biosensor for norepinephrine, and behavioral assays to investigate whether astrocytes can regulate NMDAR tone by releasing D-serine and glutamate. Subsequently, we further investigated the role of NMDAR tone in heterosynaptic long-term depression, metaplasticity, and cognitive flexibility. RESULTS: We found that hippocampal astrocytes regulate NMDAR tone via BEST1-mediated corelease of D-serine and glutamate. Best1 knockout mice exhibited reduced NMDAR tone and impairments of homosynaptic and α1 adrenergic receptor-dependent heterosynaptic long-term depression, which leads to defects in metaplasticity and cognitive flexibility. These impairments in Best1 knockout mice can be rescued by hippocampal astrocyte-specific BEST1 expression or enhanced NMDAR tone through D-serine supplement. D-serine injection in Best1 knockout mice during initial learning rescues subsequent reversal learning. CONCLUSIONS: These findings indicate that NMDAR tone during initial learning is important for subsequent learning, and hippocampal NMDAR tone regulated by astrocytic BEST1 is critical for heterosynaptic long-term depression, metaplasticity, and cognitive flexibility.