Fig. 5: Chemogenetic regulation of endogenous mGlu1 in brain tissue.
From: Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue
a Illustration of a sagittal section of the mouse brain and the enlarged image of the cerebellum (left), major components of the cerebellar cortical neuronal circuits (middle), and PF−Purkinje cell synapses (right). IP3R, inositol triphosphate receptor; TRPC, transient receptor potential channel canonical. b Illustration for preparing mGlu1(N264H) KI mice (mGlu1CBC/CBC) using CRISPR-Cas9 system. c Evaluation of the protein expression level of mGlu1, AMPAR and GluD2 in the cerebellum between mGlu1+/+ and mGlu1CBC/CBC by western blotting. Full scan images are shown in Source Data. d Quantification of the surface expression level of mGlu1 in acute cerebellar slices between mGlu1+/+ and mGlu1CBC/CBC using FITM-Cy3. Scale bars, 200 μm. (n = 5, P = 0.01835, 0.001975 for mGlu1+/+, mGlu1CBC/CBC, respectively). (Two-tailed Welch’s t-test **P < 0.01, *P < 0.05). See Supplementary Figure 10d for the merge images with Hoechst33342. e, f Conventional confocal (e) and super-resolution (f) microscopic images of immune-positive signals for mGlu1 (red) and calbindin (white) in the ML of mGlu1CBC/CBC cerebellar slices. Scale bars, 20 μm (e) and 2 μm (f). g Super-resolution microscopic images of immune-positive signals for mGlu1 (red), GluD2 (green), and Bassoon (blue). Scale bar, 1 μm. An enlarged view is shown in a right panel. Scale bar, 200 nm. h Representative CF-EPSC traces from mGlu1+/+ (top) and mGlu1CBC/CBC (bottom). In the left, an orientation of stimulus and recording electrodes is shown. i A histogram showing the percentage of the number of CFs innervating single Purkinje cells. [n = 32 cells from 5 mice (mGlu1+/+) or 30 cells from 5 mice (mGlu1CBC/CBC)]. j mGlu1-mediated slowEPSC data. By adjusting PF stimulus intensities, similar sizes of PF-evoked AMPA-EPSC (left traces) were obtained. Then, slowEPSC (right traces) were recorded by application of 2−10 times of PF stimuli at 100 Hz in the presence of AMPAR blockers. Amplitudes of slowEPSC were normalized by amplitudes of AMPA-EPSCs and plotted against the number of stimulations (right graph). [n = 9 cells from 3 mice (mGlu1+/+) or 13 cells from 4 mice (mGlu1CBC/CBC)]. Data are presented as mean ± s.e.m.
