BLA astrocytes are associated with withdrawal somatic signs during high-dose naloxone-precipitated morphine withdrawal revealed by 3D-AI behavioral analysis.

Relapse triggered by withdrawal memory is the important challenge in addiction therapy, with somatic withdrawal symptoms acting as its physiological substrate. Clarifying the relevant neural mechanisms is vital for relapse intervention, yet the link between basolateral amygdala (BLA) astrocytes and somatic withdrawal responses remains elusive. In this study, we combined 3D-AI behavioral analysis, in vivo calcium recording, and astrocyte specific inhibition using HPMCA2w/b (a calcium extrusion pump) in male C57BL/6J mice to study the role of BLA astrocytes in morphine withdrawal. We observed that BLA astrocytes were activated during morphine withdrawal conditioning. Inhibiting their intracellular calcium signaling reduced CPA scores. 3D-AI behavioral analysis showed a significantly locomotor suppression in withdrawal mice. Further, we identified two categories of behaviors: (1) jumping and standing, which were associated with the naloxone-induced withdrawal during the acquisition phase; (2) sniffing and self-grooming, which appeared during both naloxone-induced withdrawal during the conditioning phase and spontaneous withdrawal during memory retrieval phase. Astrocytic calcium signals were elevated during the occurrences of all four behaviors. Notably, astrocyte inhibition reversed the locomotor suppression during spontaneous withdrawal in post-conditioning and selectively suppressed jumping and standing during conditioning in withdrawal mice, but not affected sniffing or self-grooming. These findings indicate that BLA astrocytes are critical for association of context cues with aversive state of morphine withdrawal and they participate in some of somatic withdrawal signs during high-dose naloxone-precipitated morphine withdrawal. This study provides new insights into withdrawal for further research on opioid addiction.