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. 2020 Apr 1;9:e56054. doi: 10.7554/eLife.56054

Figure 5. The α1-AR-EPSC is eliminated by targeting of GluD1R-channels via CRISPR/Cas9.

(A) Membrane resistance (Rm, ΔV −65 to −120 mV) decreased after stimulation in transduced neurons from mice injected with AAV-Cas9 and AAV-empty (ctrl, p=0.0002, n = 13), but not in transduced neurons from mice injected with AAV-Cas9 and AAV-Grid1 (Grid1, p=0.562, n = 16). (B) Representative traces (left) and grouped data (right, p<0.0001, n = 15 and 16 and 7) demonstrating the presence of an α1-AR-EPSC in transduced neurons from control mice, but not from Grid1 mice. Neighboring non-transduced neurons from mice injected with AAV-Cas9 and AAV-Grid1 (non) had an α1-AR-EPSC that was indistinguishable from transduced neurons from control mice (p>0.999). (C) Current-voltage relationship of the α1-AR-EPSC from control (n = 13) and Grid1 (n = 16) grouped data. Shaded area represents mean ± SEM. (D) Targeting GluD1R reduced the inward current to noradrenaline (NA, INA,30 μM) as compared to transduced neurons from control mice and neighboring non-transduced neurons (p=0.004, n = 16 and 16 and 4). Inward INA in non-transduced neurons from mice injected with AAV-Cas9 and AAV-Grid1 was similar to transduced neurons from control mice (p=0.631). (E) Targeting GluD1R reduced the tonic inward current revealed by bath application of NASPM (100 μM, INSP) as compared to transduced neurons from control mice (p=0.009, n = 5 and 11). Line and error bars represent mean ± SEM, * denotes statistical significance, ns denotes not significant.

Figure 5—source data 1. Numerical data that were used to generate graphs in Figure 5.

Figure 5.

Figure 5—figure supplement 1. Design and testing of guide RNA targeting mouse Grid1.

Figure 5—figure supplement 1.

(A) A graphic representation of AAV vectors used in conjunction with AAV-Cas9 to encode eGFP reporter only (top, AAV-empty) and mouse Grid1 guide RNA with eGFP reporter (bottom, AAV-Grid1). (B) A graphic representation of the third exon of mouse Grid1, marked with the location of the gRNA (390F) and the overlapping Bgl I restriction site. Filled arrows represent the location of primers used for PCR amplification while the hollow arrow indicates the primer used for sequencing. (C) A nucleotide representation of gRNA target site with the translated peptide sequence underneath. The Bgl I recognition site (GCCNNNN^NGGC) is contained by the grey box and overlaps with the predicted Cas9 cleavage site (|). (D) Fragment analysis on Bgl I-digested Grid1 PCR products amplified from the dorsal raphe of mice injected with (AAV-Cas9 and AAV-empty) or (AAV-Cas9 and AAV-Grid1). ‘% PCR undigested’ refers to the amount of the undigested PCR product relative to the total PCR product as determined by AATI fragment analysis.