Background: The analgesic effects of multiple electroacupuncture (EA) sessions and single EA sessions differ significantly in pain management. Area 24b (A24b) of the anterior cingulate cortex (ACC) is crucial in pain processing. EA relieves pain by targeting and modulating the neuronal activity within this subregion. However, whether the cumulative effect of EA antinociception is connected to A24b mechanisms has remained unclear.
Methods: In our study, we used the Complete Freund's Adjuvant (CFA) model to induce inflammatory pain and the Spared Nerve Injury (SNI) model to induce neuropathic pain, and adult male C57BL/6, FosTRAP, and FosTRAP:Ai9 mice were used as experimental subjects to investigate the cumulative effect of EA antinociception and whether multiple EA sessions and a single EA session regulate different neuronal populations in the A24b.
Results: We observed that EA effectively alleviated pain in mice, with three EA sessions yielding superior analgesic effects compared to a single session. Using chemical genetics combined with FosCreER technology to activate EA-TRAPed cells in the A24b, we found that pain relief was more pronounced with three EA sessions. Moreover, chemogenetic inhibition of EA-TRAPed cells in the A24b reversed the analgesic effects of a single EA session but not those of three EA sessions. Fluorescent in situ hybridization results indicated that three EA sessions significantly increased the number of GABAergic neurons in the A24b compared with a single session. Additionally, retrograde tracing revealed that the A24b circuit that monosynaptically innervates EA-TRAPed cells included projections from the central lateral nucleus (CL), lateral mediodorsal thalamic nucleus (MDL), lateral habenula (LHb), dorsal raphe nucleus (DR), caudal linear nucleus of the raphe (CLi), dorsal tuberomamillary nucleus (DTM), periventricular hypothalamic nucleus (Pe) and hippocampal fields CA1, CA2, and CA3. These findings suggest that multiple EA sessions and single EA sessions activated different neuronal populations in the A24b. The enhanced analgesic effect of multiple EA sessions may be attributed to an increase in the proportion of GABAergic neurons within the A24b.Conclusions: Multiple and single EA sessions recruit distinct neuronal populations in A24b, with the stronger analgesic effect of repeated EA linked to a higher proportion of GABAergic neurons in this region.
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2026 The Author(s). Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.
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