3.2 Short-term DREADD specifically inhibit BLA astrocytes directly reverses mechanical pain and anxiety-like behaviors in DNP rats.
To investigate the regulation of BLA astrocytes on mechanical allodynia in the rats with DNP, we applied DREADD to manipulate BLA astrocytes. First, the rats were intraperitoneally injected with STZ to induce DNP. Then, we bilaterally injected the gfaABC1D-M4-EGFP virus into the BLA to inhibit BLA astrocytic activity. Three weeks after the gfaABC1D-M4-EGFP virus injection, the rats were exposed to a single dose of CNO, and pain was assessed over a 6-hour time course after CNO administration (Fig. 2A). We observed that mechanical allodynia was rapidly reversed within 1 hour, peaked at 3 hours and remained for 5 hours after CNO treatment in the DNP+gfaABC1D-M4-EGFP group. To test whether these effects were selectively mediated by CNO activation of M4-DREADD, we applied either saline to gfaABC1D-M4-expressing astrocytes or CNO in the rats where gfaABC1D-M4 expression was absent. In both cases, we observed no relief in mechanical allodynia (Fig. 2B). Compared with the MWT in the DNP+gfaABC1D-EGFP group, the MWT in the group of DNP rats expressing gfaABC1D-M4-EGFP was markedly increased by CNO (Fig. 2C).
Next, we used open field and elevated plus maze tests to observe the effect of inhibiting BLA astrocytes on anxiety-like behaviors in DNP rats. After intraperitoneal injection of CNO (1 mg/kg), there was no significant change in the total distance of the rats in the STZ+gfaABC1D-EGFP and STZ+gfaABC1D-M4-EGFP groups in the open field. However, compared with the STZ+gfaABC1D-EGFP group, the movement distance of the STZ+gfaABC1D-M4-EGFP group in the central area of the open field was significantly increased (Fig. 2D-2F). The elevated plus maze test showed that after CNO was administered, compared with the STZ+gfaABC1D-EGFP group, the STZ+gfaABC1D-M4-EGFP group significantly increased the activity time of the rats in the open arm, and the exploration frequency of entering the open arm was also significantly increased (Fig. 2G-2I).
Then, we detected the expression of GFAP in the BLA to determine the effects of CNO-induced gfaABC1D-M4-EGFP receptor activation on astrocyte activity. As expected, CNO treatment significantly attenuated GFAP fluorescence (Fig. 2J-2K) and the number of GFAP-positive cells (Fig. 2L) in the DNP rats expressing gfaABC1D-M4-EGFP compared with the DNP rats expressing gfaABC1D-EGFP.