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.