One week before the implantation surgery, we injected 1.0 µl of virus solution into the hippocampal CA1 area of mice [antero-posterior: 1.90 mm, medio-lateral: 1.40 mm from the bregma, dorso-ventral: 1.25 mm (for Fig. 4 ), 1.60 mm (for Fig. 6 ) from skull surface] at a rate of 0.2 µl/min using a syringe (Hamilton, Model 80100, Knurled Hub NDL) with a blunt ended needle (25 gauges, 2.75″/69.9 mm, point style 3; Hamilton, Reno, NV, USA). We used viral vectors expressing GCaMP6f under the control of the regulatory element from the GFAP gene (AAV5/pZac2.1 gfaABC1D-cyto-GCaMP6f, prepared by Addgene, a gift from Dr. Baljit Khakh, Addgene plasmid # 52925) for Fig. 4 and GCAMP6s under the control of the regulatory element from the CaMKIIα gene [prepared as previously described (Kitanishi et al., 2015)] for Fig. 6 .
Aav5 pzac2.1 gfaabc1d cyto gcamp6f
Manufactured by Addgene
AAV5/pZac2.1 gfaABC1D-cyto-GCaMP6f is a recombinant adeno-associated virus (rAAV) construct. It contains the GCaMP6f calcium indicator gene under the control of the gfaABC1D promoter, packaged in an AAV5 viral capsid.
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3 protocols using aav5 pzac2.1 gfaabc1d cyto gcamp6f
1
Viral Vector Injections for Imaging
2
Organotypic Slices Viral Transduction and Imaging
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Muller organotypic slices were introduced to either AAV5 pZac2.1 gfaABC1D-cyto-GCaMP6f (Addgene #52925) or AAV1 pAAV.hSynapsin.SF-iGluSnFR.S72A (Addgene #106176) via microinjections using a glass pipette connected to Picospritzer (Parker Hannifin). Briefly, the virus particles were puffed from a pipette positioned in the CA1 area of the slice by brief pressure pulses (30 ms; 15 psi). The injection was performed 1 day after slice preparation and the expressing slices were imaged two weeks after that. The 2D imaging of either glutamate or calcium signals was performed at 100 Hz speed using a 488 nm excitation laser. To remove the bleed-through from the LISHI channel into the iGluSnFR/GCaMP channel, a band-pass filter into the detection path was added (Semrock FF03-525/50). Following that, a z-stack of Alexa Fluor 568 signal was acquired using a 560 nm excitation laser (step size 200 nm).
3
Viral Labeling of Astrocytes in Mouse Somatosensory Cortex
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Animals were anesthetized using a ketamine (10 mg/mL) xylazine (1 mg/mL) mixture and placed on a heating pad to maintain body temperature and faux tears were applied to the cornea. Animals (8 weeks of age) were placed in a stereotaxic apparatus and an incision was made down the midline of the scalp to expose the skull. A hole was drilled over the forelimb and hindlimb somatosensory cortex (S1: -0.4a-p, 1.9m-l), and a Hamilton syringe was lowered to (in mm from bregma: -0.7d-v) and viruses were injected bilaterally at 100 nL/min 48 . Mice were then sutured and left to heal for 2-3 weeks. AAV5-pZac2.1-gfaABC1d-cyto-GCaMP6f (Addgene), AAV8-GFAP-hM3D(Gq)-mCherry (UMN vector core), AAV8-GFAP-mCherry (UMN vector core) and AAV8-GFAP-mCherry-Cre (UMN vector core) viral constructs were used. For CNO experiments, C57BL/6J mice were injected with AAV8-GFAP-hM3D(Gq)-mCherry virus. In control conditions, a virus of AAV8-GFAP-mCherry was injected instead. For CB1R fl/fl mice experiments, AAV8-GFAP-mCherry-Cre was injected to delete CB1R from astrocytes (aCB1R -/-). AAV8-GFAP-mCherry was used as a control (aCB1R).
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