2.2. Virus injection

AAV2/9 viruses were used and injected into C57 mice throughout the experiment, including rAAV2/9‐CaMKIIα‐hM4D(Gi)‐mCherry‐WPRE‐pA, rAAV2/9‐hSyn‐hM3D(Gq)‐EGFP‐WPRE‐pA, rAAV2/9‐CaMKIIα‐hChR2(E123T/T159C)‐mCherry‐WPRE‐hGH‐pA, rAAV2/9‐CaMKIIα‐EGFP‐WPRE‐pA, and AAV2/9‐CaMKIIα‐mCherry‐WPRE‐pA (BrainVTA, Wuhan). All viruses were injected into C57 mice. rAAV2/9‐CaMKIIα‐hChR2 (E123T/T159C)‐mCherry‐WPRE‐hGH‐pA were used and injected into M1 of GAD67‐GFP mice only in the neuron identification experiment in Figure 2. Mice were anesthetized with 1.25% tribromoethanol and fixed in a prone position in the adapter. The skull hair was cut off, and a line was cut along the midline of the scalp to expose the skull. Then, the tissue was removed. The target area was selected, and a hole was drilled. After opening the dura, a needle fixed to a stereotaxic instrument was filled with the target virus and slowly injected into the target area at a rate of 30 nL/min. After 10 min, the needle was slowly lifted, and mice were fed in the cage for 21 days before the experiment. In the optogenetic mice, an optical fiber was implanted above the virus injection site and fixed with dental cement.

Most neurons activated in the NTS induced by M1 optogenetic activation were excitatory neurons. (A) Representative images of c‐Fos‐positive and CaMKIIα neurons in the NTS after optogenetic activation of M1 with AAV2/9‐CaMKIIα‐ChR2 virus injected into M1 and AAV2/9‐CaMKIIα‐EGFP virus injected into NTS in C57 mice. Scale bar: 50 μm. (B) Representative images of c‐Fos‐positive and GABAergic neurons in the NTS after optogenetic activation of M1 with AAV2/9‐CaMKIIα‐ChR2 virus injected into M1 in GAD67‐GFP mice. Scale bar: 50 μm. (C) More excitatory neurons in the NTS were activated following optogenetic activation of M1 in the CaMKIIα group than in the GAD group (two‐tailed Student's unpaired t test, CaMKIIα, n = 5; GAD, n = 3, t = 5.907, **p < 0.01). Data are presented as the mean ± SD, and n indicates the number of biologically independent samples (mice) per group.