Astrocytic Gi-GPCR activation enhances stimulus-evoked extracellular glutamate

Astrocytes perform critical functions in the nervous system, many of which are dependent on neurotransmitter-sensing through G protein-coupled receptors (GPCRs). However, whether specific astrocytic outputs follow specific GPCR activity remains unclear, and exploring this question is critical for understanding how astrocytes ultimately influence brain function and behavior. Here, we investigate the outputs of astrocytic Gi-GPCRs, a family of GPCRs which we previously showed is sufficient to increase slow-wave neural activity (SWA) during sleep when activated in cortical astrocytes1. We focus on two putative outputs by astrocytes in vivo, the regulation of extracellular glutamate and GABA, by combining fiber photometry recordings of the extracellular indicators iGluSnFR and iGABASnFR with astrocyte-specific chemogenetic Gi-GPCR activation. We find that Gi-GPCR activation does not change spontaneous dynamics of extracellular glutamate or GABA. However, Gi-GPCR activation does specifically increase visual stimulus-evoked extracellular glutamate. Together, these data point towards a complex relationship between astrocytic inputs and outputs in vivo that may depend on behavioral context. Further, they suggest an extracellular glutamate-specific mechanism underlying some astrocytic Gi-GPCR-dependent behaviors, including the regulation of sleep SWA.

Methods
All procedures were carried out in adult mice (C57Bl/6, P50–100, males and females). Mice underwent surgery to implant fiber optic cannulas as well as viral injections of relevant viruses including AAV-GFAP.SF-iGluSnFR.AI184S, AAV-GFAP-iGABASnFR2, AAV-GFAP-GCaMP6f, and AAV5-GFAP-hM4D(Gi)-mCherry in V1. Mice were first habituated to the recording setup. On the day of recording, mice were tethered to the recording setup via a patchcord and then injected I.P. with CNO (1 mg/kg) or saline (0.9%) immediately prior to recording. Four recordings were made in the following order: spontaneous (no LED, 10-min), LED flash stimuli (10-min), spontaneous (no LED, 30-min), LED flash stimuli (10-min) for a total of 1-hour. We used a Tucker-Davis Technologies RZ10X Processor with a Doric Lenses fluorescence mini-cube. A 473nm LED was used for the iGluSnFR and iGABASnFR2 excitation and a 405nm LED was used as an isobestic control. Both LEDs (Tucker-Davis Technologies, RZ10X Processor) went through a fluorescence mini-cube (Doric Lenses), and then through patchcords connected to a commutator to allow for free movement of the animal. After the commutator, a patchcord was connected to the fiber-optic cannula implanted in the animal. Fluorescence signals were reflected back through the mini-cube to a photoreceiver on the RZ10X Processor. The dataset provided contains the raw data that is outputted by the Synapse recording software.