AAV1-TRE-Cre

The dorsal medial prefrontal cortex (dmPFC) plays a dual role in regulating drug-seeking and fear-related behaviors. Here, researchers explored the stability of a dmPFC cocaine-seeking assembly over 2 weeks and its effects on persistent cocaine-seeking and fear memory retrieval. In the first series of experiments, researchers trained TetTag c-fos-driven EGFP mice for cocaine self-administration and labeled neurons that were robustly activated with EGFP during an initial cocaine-seeking session on day 7. A follow-up seeking test was then performed 2 weeks later to examine assembly reactivation between seeking sessions by c-Fos immunostaining. In the second series of experiments, researchers co-injected viruses expressing TRE-cre and a cre-dependent inhibitory PSAM-GlyR into the dmPFC of male and female c-fos-tTA mice in order to "tag" cocaine-seeking or cued-fear assembly neurons with inhibitory chemogenetic receptors. The contribution of ensembles to subsequent cocaine seeking and fear recall during inhibition of labeled ensembles was then investigated by administering uPSEM792s, a selective ligand for PSAM-GlyR. In both sexes, there was a positive correlation between the persistence of cocaine seeking and the proportion of reactivated EGFP+ neurons within the dmPFC. The results suggest that cocaine and fear recall ensembles in the dmPFC are stable but largely mutually exclusive.

To test whether reactivation of the dmPFC drug-seeking ensemble is necessary for subsequent cocaine seeking, researchers used a chemogenetic ensemble tagging strategy in which c-fos-tTA mice were co-injected with AAV1-TRE-cre and cre-dependent inhibitory PSAM-GlyR (AAV-FLEX-PSAM-GlyR-EGFP) into the dmPFC (Figure 1A). A large number of neurons expressing EGFP were observed in the dmPFC of the labeled group, while negligible EGFP expression was detected in the dmPFC of the unlabeled group maintained with dox during the day 7 cocaine seeking session (Figure 1B). In addition, researchers determined the in vitro function of inhibitory PSAM-GlyR on dmPFC pyramidal neurons using whole-cell patch clamp electrophysiology (Figure 1C). Pressure injection of the PSAM-GlyR ligand uPSEM792s (50 nM) blocked action potential firing in EGFP+ neurons expressing PSAM-GlyR, but had no effect on EGFP- neurons, confirming that uPSEM792s selectively inhibits neuronal activity in cells expressing PSAM-GlyR.

Figure 1. Validation of expression and function of cre-dependent PSAM-GlyR. (Liu S, et al., 2024)