Syn-Cre-mCherry AAV (Serotype Retrograde)

Benzodiazepines, commonly used for anxiolysis, impair conditioned fear extinction, and the underlying circuit mechanisms are unclear. Using the ultra-short-acting benzodiazepine remimazolam, researchers here reveal its effects on the thalamic nucleus reuniens (RE) and interconnected hippocamposeptal circuits during fear extinction. Systemic or RE-specific administration of remimazolam impairs fear extinction by reducing RE activation of A type GABA receptors. Remimazolam enhances long-range GABAergic inhibition from the lateral septum (LS) to the RE, which is responsible for the impaired fear extinction. The RE projects to the ventral hippocampus (vHPC), which in turn sends projections characterized by feedforward inhibition to GABAergic neurons of the LS. This, combined with long-range GABAergic projections from the LS to the RE, together constitutes an overall positive feedback circuit construct that promotes fear extinction. RE-specific remimazolam counteracts the facilitation of fear extinction by disrupting this circuit. Thus, remimazolam in RE disrupts fear extinction mediated by the septum hippocampus, providing mechanistic insights into the dilemma of combining anxiolytics with extinction-based exposure therapy.

Here, the researchers introduced Cre-dependent exogenous Gabrg2 together with Gabrg2-shRNA (AAV-U6-Gabrg2-ShRNA-DIO-Gabrg2*-EGFP) into different subsets of RE projection neurons (Figure 1a). The AAV vector carrying Gabrg2-shRNA also has a double-floxed inverted orientation (DIO) coding sequence for shRNA-resistant Gabrg2-EGFP (read as Gabrg2*), which is only expressed in the presence of Cre recombinase (Figure 1b). Retrograde AAV expressing Cre (Retro-AAV-Syn-Cre-mCherry) was co-injected into vHPCs, where Cre recombinase is expressed in RE-vHPC projections, resulting in strong expression of Gabrg2-EGFP in a subset of RE neurons in the targeted vHPC region (Figure 1b). As a control, a retrograde AAV expressing only mCherry (Retro-AAV-Syn-mCherry) was co-injected into RE-vHPC projections, lacking Cre recombinase expression, and no Gabrg2-EGFP expression was shown in RE neurons. The shRNA in this AAV effectively eliminated the responsiveness to remimazolam in mice injected with the control Retro-AAV-Syn-mCherry. In contrast, in mice injected with Retro-AAV-Syn-Cre-mCherry, the virus restored the responsiveness to remimazolam. From an electrophysiological perspective, remimazolam had no effect on the excitability of RE neurons in the control Retro-AAV-Syn-mCherry group (Figure 1c-e), but significantly reduced the excitability of RE neurons in the Retro-AAV-Syn-Cre-mCherry group (Figure 1f-h). Behaviorally, systemic administration (ip) of remimazolam had no effect on fear extinction in the control Retro-AAV-Syn-mCherry group (Fig. 1i, j), but blocked extinction learning and disrupted subsequent retrieval sessions in the Retro-AAV-Syn-Cre-mCherry group (Fig. 1k, l). These results highlight the critical role of RE-vHPC projections in mediating the modulatory effects of remimazolam on fear extinction.

Figure 1. Reinstatement of Gabrg2 expression in RE-vHPC projectors restores remimazolam responsiveness in RE Gabrg2 knockdown mice. (Cheung H, et al., 2024)