Fig. 7: Cell-type-specific activation of mGlu1 in the cerebellum using dA-CBC and AAVs.
From: Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue
a Schematic illustration of the two-component AAV system for Purkinje cells- or MLIs-specific expression of mGlu1. b Confocal microscopic images of immune-positive signals for GFP, Car8 and HA-tag in the cerebellar slice infected with AAV-L7-mGlu1CBC. The white squarer ROI is expanded on the right. Scale bars, 100 µm (in a large left panel) and 50 µm (in small right panels). c Evaluation of expression level of HA-tagged mGlu1, AMPAR and GluD2 in the AAV-infected or -uninfected cerebellum by western blotting. Full scan images are shown in Source Data. d Averaged data of Pd(sulfo-bpy)-induced chemLTD recordings from AAV-L7-mGlu1WT (black circles) and AAV-L7-mGlu1CBC (red circles). Insets, representative PF-EPSC traces just before (blue; t = −1 min) and 25 min after (black or red) drug washout in each mouse group. A right graph shows a cumulative probability of the degree of LTD at t = 35 min. [n = 7 cells from 5 mice (AAV-L7-mGlu1WT) or 7 cells from 5 mice (AAV-L7-mGlu1CBC)]. e Schematic illustration of major components of the cerebellar cortical neuronal circuits including MLI. An orientation of recording electrode is shown. f Confocal microscopic images of immune-positive signals for GFP, Car8, PV and HA-tag in the cerebellar slice infected with AAV-hSyn-mGlu1CBC. The white squarer ROI is expanded on the right. To obtain the MLI image, immunofluorescent signals in the Car8 image were subtracted from those in the PV image. Scale bars, 100 µm (in a large left panel) and 50 µm (in small right panels). g Spontaneous action potential (sAP)-mediated currents from MLIs infected with AAV-hSyn-mGlu1WT, AAV-hSyn-mGlu1CBC, or AAV-L7-mGlu1CBC with bath application of 10 µM Pd(sulfo-bpy). Upper: sAP-mediated currents before Pd(sulfo-bpy) treatment. Lower: sAP-mediated currents after Pd(sulfo-bpy) treatment. [n = 16 cells from 4 mice (AAV-hSyn-mGlu1WT, P = 0.4534), n = 22 cells from 4 mice (AAV-hSyn-mGlu1CBC, P = 4.613 × 10–5) or 15 cells from 4 mice (AAV-L7-mGlu1CBC, P = 0.2934)]. (two-sided Wilcoxon signed-rank test, ***P < 0.001, n.s. not significant). Data are presented as mean ± s.e.m.
