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[Preprint]. 2023 Oct 10:2023.10.06.561261. [Version 1] doi: 10.1101/2023.10.06.561261

Figure 7: Selective chemogenetic inhibition of previously activated APP vs. AV cholinergic neurons in the VP abolishes approach to the appetitive odor, reversing behavior to strong avoidance.

Figure 7:

A. Workflow and timeline of behavior experiments using ADCD to target inhibitory DREADD’s for specific inhibition of previously activated subpopulations of VP cholinergic neurons. Chat-Cre × Fos-tTA/GFP mice were injected with ADCD and AAV-Syn-eGFP mice in the VP. Mice in the sham group were only injected with AAV-Syn-GFP but otherwise went through identical procedures as ADCD injected mice. Following recovery from surgery, mice were habituated (2 × 10 min) in the Y-Maze. Following arena habituation, mice were removed from a DOX diet, thus allowing for ADCD expression. Approximately 24-hours later, mice were exposed to either the appetitive odor (APP) or aversive odor (AV) in one arm of the Y-Maze, thus labeling activated VP cholinergic neurons with ADCD. Following odor exposure, mice were put back on a DOX diet, preventing further ADCD expression. The ADCD construct labels activated neurons with an inhibitory DREADD (ADCD-hM4Di). Approximately 24-hours later, all mice were injected IP with 0.1 mg clozapine to inhibit VP cholinergic neurons that were previously activated by the APP or AV odor . Approximately 15-minutes later, mice underwent an odor preference test for the odor to which they had been previously exposed.

B. ADCD-hM4Di mediated inhibition of VP cholinergic neurons previously activated in response to the appetitive odor (APP) not only blocks approach behavior, but reverses the approach behavioral response to active avoidance. Left: representative heatmap showing approach behavior to the APP odor in a sham mouse (injected with AAV-Syn-eGFP). Right: Representative heatmap in a mouse following inhibition of VP cholinergic neurons previously activated in response to the APP odor, now displaying active avoidance behavior in response to the APP odor (i.e., more time spent in the N arm).

C. Mice in the sham group (n = 6) exhibit approach to the APP odor. Inhibiting previously activated VP cholinergic neurons in response to APP with ADCD-hM4Di + 0.1 mg/kg clozapine (n = 7), leads to significantly more time in the saline paired arm, consistent with both a decrease in approach behavior and reversal of approach behavior to active avoidance. * p < 0.05.

D. ADCD-hM4Di mediate inhibition of VP cholinergic neurons previously activated in response to the aversive odor (AV). Left: Representative heatmap depicting avoidance of the AV odor in a control mouse. Right: ADCD inhibition of VP cholinergic neurons previously activated in response to the AV odor has no effect on innate avoidance of the AV odor and mice continue to exhibit avoidance behavior.

E. Mice in the sham group (n = 5) exhibit innate avoidance of the AV odor. Mice with ADCD-hM4Di + 0.1 mg/kg clozapine (n = 6) also exhibit avoidance of the AV odor. (* p < 0.05).