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. 2024 Feb 21;27(3):109289. doi: 10.1016/j.isci.2024.109289

Figure 1.

Figure 1

Experimental design and confirmation of selective inhibition of LMVN GABAergic neurons

(A) Schematic diagram of Cre-inducible AAV vector expressing hM4Di fused to mCherry under control of human synapsin I promoter. DIO, double-floxed inverted open reading frame; ITR, inverted terminal repeat; hSyn, human synapsin I promoter; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; pA, poly-A tail.

(B) Schematic of bilateral injection of AAV. The outline adapted from the brain atlas of Franklin and Paxinos22 represents a coronal section at the level of the medulla oblongata corresponding to 6.12 mm posterior to bregma. Red vertical lines and circles indicate the injection capillaries and targets, respectively.

(C) Representative images of the coronal brain sections of a GAD1/LMVN/M4 mouse. Confocal fluorescence images (red) and differential interference contrast (DIC) images (gray) are merged. (Left) Wide-field images of the coronal sections. Each value in the upper left corner indicates the anterior-posterior (AP) coordinate from bregma (mm). (Right) Magnified images corresponding to five frames with numbers in left wide-field images (C1-5). Fluorescence images are captured in z stack and projected with maximum intensity. Anatomical abbreviations are listed in Table 1. See also Figure S1.

(D) Representative projected z stack confocal images of fluorescence in situ hybridization (FISH) for Gad1 mRNA (Left, green) and immunohistochemistry (IHC) for hM4Di-mCherry (Middle, red) in the LMVN of a GAD1/LMVN/M4 mouse. Merged image of (Left) and (Middle) displays colocalization (Right).

(E) Fluorescence (Left, red) and Dodt gradient contrast (Right, gray) images of the LMVN of a GAD1/LMVN/M4 mouse in the same field of view. The patch pipette is seen on the right side.

(F) Representative recording from an hM4Di-expressing LMVN neuron of a GAD1/LMVN/M4 mouse in the current-clamp configuration. The solid bar indicates the duration of 5 μM CNO application (5 min). The dotted line denotes 0 mV. Spontaneous action potentials in LMVN neurons expressing hM4Di were inhibited by the application of CNO and recovered after washout.

(G) The mean firing rate of the LMVN neuron expressing hM4Di before (black circles, Baseline) and during (red circles, CNO) the application of CNO. Data obtained from the same cell are connected with a line. ∗p < 0.05, Wilcoxon signed-rank test.