Journal of Traditional Chinese Medicine ›› 2026, Vol. 46 ›› Issue (3): 552-560.DOI: 10.19852/j.cnki.jtcm.2026.03.003
• Original Articles • Previous Articles Next Articles
Role of gamma-aminobutyric acid-ergic neurons in the thalamic reticular nucleus in regulating sleep-wake cycles in mice treated with acupuncture for insomnia
LU Xiaoxiao1,2, XU Yipeng3, ZHOU Minjie3, ZHANG Chengshun3, CAI Dingjun3(
), ZHAO Zhengyu3()
- 1
Acupuncture and Tuina School ,Chengdu University of Traditional Chinese Medicine Chengdu 611137, China
2Department of acupuncture and moxibustion ,First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine Guiyang 550001, China
3Acupuncture and Tuina School ,Chengdu University of Traditional Chinese Medicine Chengdu 610075, China
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Received:2025-02-12Accepted:2025-07-22Online:2026-06-15Published:2026-06-08 -
Contact:ZHAO Zhengyu, CAI Dingjun, Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, China. zhaozhengyu@cdutcm.edu.cn;
CAI Dingjun, Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, China. djcai@cdutcm.edu.cn, Telephone: +86-28-61703327 -
About author:First author contact:LU Xiaoxiao and XU Yipeng are co-first authors and contributed equally to this work
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Supported by:National Natural Science Foundation of China: Mechanism Study on the Cholinergic-gamma-aminobutyric acid-ergic Neural Regulatory Effect of the Thalamic Reticular Nucleus in Acupuncture Regulating Sleep-Wake disturbance(81774434);Circadian-Controlled Microglia-Neuron Interactions Mechanism within the Thalamic Reticular Nucleus Underlying Acupuncture Amelioration of Insomnia-Induced Attention Deficits(82274659);Sichuan Province Science and Technology Program: Study on the Exosome-Mediated Mast Cell-Neural Modulation Mechanism Underlying Acupoint Sensitization(2019YJ0330);Circadian-Controlled Neuro-Immune Regulation Mechanism of Chrono-Acupuncture for Neuropathic Pain via SP-Mrgprb2R Pathway(2021YJ0177);Chengdu University of Traditional Chinese Medicine Foundation Program: Acupuncture Modulates Thalamic Reticular Nucleus Microglia-Neuronal Crosstalk in Sleep Disorder-Induced Attention Deficits(QJJJ2021005)
Cite this article
LU Xiaoxiao, XU Yipeng, ZHOU Minjie, ZHANG Chengshun, CAI Dingjun, ZHAO Zhengyu. Role of gamma-aminobutyric acid-ergic neurons in the thalamic reticular nucleus in regulating sleep-wake cycles in mice treated with acupuncture for insomnia[J]. Journal of Traditional Chinese Medicine, 2026, 46(3): 552-560.
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Figure 1 PSIS and voluntary wheel running test results after acupuncture treatment A: effect of acupuncture on pentobarbital-induced sleep latency; B: effect of acupuncture on pentobarbital-induced sleep duration; C: acrophase of circadian rhythms; D: representative double-plotted actograms from the Control group; the x-axis represents circadian time; Y‑axis: successive days; lights were on at 07:00 and off at 19:00; the figure shows locomotor activity rhythms for each group; E: representative double-plotted actograms from the PCPA group; F: representative double-plotted actograms from the MA group. The control group received daily i.p. normal saline (10 mL/kg); the PCPA group received daily i.p. PCPA (300 mg/kg) for 3 d; the MA group received identical PCPA treatment followed by daily MA (20 min/session, 3 d). Group differences were analyzed by ANOVA with Tukey HSD post-hoc test. PSIS: pentobarbital sodium-induced sleep test; PCPA: p-chlorophenylalanine; MA: manual acupuncture; ANOVA: analysis of variance; HSD: honestly significant difference; SEM: standard error of the mean. Data are mean ± SEM (n = 6). Compared with the Control group, aP < 0.01; compared with the PCPA group, bP < 0.01.
Figure 1 PSIS and voluntary wheel running test results after acupuncture treatment A: effect of acupuncture on pentobarbital-induced sleep latency; B: effect of acupuncture on pentobarbital-induced sleep duration; C: acrophase of circadian rhythms; D: representative double-plotted actograms from the Control group; the x-axis represents circadian time; Y‑axis: successive days; lights were on at 07:00 and off at 19:00; the figure shows locomotor activity rhythms for each group; E: representative double-plotted actograms from the PCPA group; F: representative double-plotted actograms from the MA group. The control group received daily i.p. normal saline (10 mL/kg); the PCPA group received daily i.p. PCPA (300 mg/kg) for 3 d; the MA group received identical PCPA treatment followed by daily MA (20 min/session, 3 d). Group differences were analyzed by ANOVA with Tukey HSD post-hoc test. PSIS: pentobarbital sodium-induced sleep test; PCPA: p-chlorophenylalanine; MA: manual acupuncture; ANOVA: analysis of variance; HSD: honestly significant difference; SEM: standard error of the mean. Data are mean ± SEM (n = 6). Compared with the Control group, aP < 0.01; compared with the PCPA group, bP < 0.01.
Figure 2 Acupuncture-evoked activation of TRN GABA neurons A: virus injection for GCaMP6m expression in the TRN and post hoc brain slice validation; A1: schematic illustration of virus injection targeting the TRN in Vgat-Cre mice; A2: representative immunofluorescence image showing GCaMP6m expression (green) within the TRN, the scale bar represents 250 μm; B: representative heatmap displaying single-trial GCaMP6m fluorescence changes (Δf/f, %) across individual trials aligned to acupuncture stimulation onset; C: averaged calcium traces showing elevated GCaMP6m signals (red) following acupuncture compared to EGFP controls (blue). Fiber photometry recordings were performed during MA stimulation. TRN: thalamic reticular nucleus; GABA: gamma-aminobutyric acid-ergic; EGFP: expressing only green fluorescent protein; MA: manual acupuncture; SEM: standard error of the mean. Data are presented as mean ± SEM. Statistical analysis was performed using paired t-test comparing Δf/f values between baseline and MA stimulation in GCaMP6m expressing mice.
Figure 2 Acupuncture-evoked activation of TRN GABA neurons A: virus injection for GCaMP6m expression in the TRN and post hoc brain slice validation; A1: schematic illustration of virus injection targeting the TRN in Vgat-Cre mice; A2: representative immunofluorescence image showing GCaMP6m expression (green) within the TRN, the scale bar represents 250 μm; B: representative heatmap displaying single-trial GCaMP6m fluorescence changes (Δf/f, %) across individual trials aligned to acupuncture stimulation onset; C: averaged calcium traces showing elevated GCaMP6m signals (red) following acupuncture compared to EGFP controls (blue). Fiber photometry recordings were performed during MA stimulation. TRN: thalamic reticular nucleus; GABA: gamma-aminobutyric acid-ergic; EGFP: expressing only green fluorescent protein; MA: manual acupuncture; SEM: standard error of the mean. Data are presented as mean ± SEM. Statistical analysis was performed using paired t-test comparing Δf/f values between baseline and MA stimulation in GCaMP6m expressing mice.
Figure 3 Chemogenetic inactivation of TRN GABA neurons and sleep-wake monitoring A: schematic diagram of the chemogenetic inactivation of TRN GABA neurons in Vgat-Cre mice and subsequent EEG detection after acupuncture treatment. Combined EEG, EMG, and video recordings were used to determine the sleep-waking patterns; B: experimental strategy for assessing the effect of MA on the chemogenetic inactivation of TRN GABA neurons; Viral injections were administered at day −21 (3 weeks before intervention initiation), EEG were conducted after completion of acupuncture on day 5 (indicated by the arrow). Control (Cont) group: received normal saline (NS, blue dots) injections; PCPA group: Received p-chlorophenylalanine (PCPA, red dots) injections, followed by NS injections at days 3-5; MA group: received PCPA injections, then received acupuncture (triangles) combined with NS injections; MA + hM4Di group: received PCPA injections, then received acupuncture combined with clozapine N-oxide (CNO, yellow dots) injections (to trigger chemogenetic silencing of the TRN via hM4Di, 30 min before each MA session). Symbol legend: Red dots: PCPA; blue dots: NS; yellow dots: CNO; triangles: acupuncture; C: the effects of virus transfection are validated by post hoc sectioning. The scale bar represents 250 μm. TRN: thalamic reticular nucleus; GABA: gamma-aminobutyric acid-ergic; EEG: electroencephalography; EMG: electromyography; MA: manual acupuncture.
Figure 3 Chemogenetic inactivation of TRN GABA neurons and sleep-wake monitoring A: schematic diagram of the chemogenetic inactivation of TRN GABA neurons in Vgat-Cre mice and subsequent EEG detection after acupuncture treatment. Combined EEG, EMG, and video recordings were used to determine the sleep-waking patterns; B: experimental strategy for assessing the effect of MA on the chemogenetic inactivation of TRN GABA neurons; Viral injections were administered at day −21 (3 weeks before intervention initiation), EEG were conducted after completion of acupuncture on day 5 (indicated by the arrow). Control (Cont) group: received normal saline (NS, blue dots) injections; PCPA group: Received p-chlorophenylalanine (PCPA, red dots) injections, followed by NS injections at days 3-5; MA group: received PCPA injections, then received acupuncture (triangles) combined with NS injections; MA + hM4Di group: received PCPA injections, then received acupuncture combined with clozapine N-oxide (CNO, yellow dots) injections (to trigger chemogenetic silencing of the TRN via hM4Di, 30 min before each MA session). Symbol legend: Red dots: PCPA; blue dots: NS; yellow dots: CNO; triangles: acupuncture; C: the effects of virus transfection are validated by post hoc sectioning. The scale bar represents 250 μm. TRN: thalamic reticular nucleus; GABA: gamma-aminobutyric acid-ergic; EEG: electroencephalography; EMG: electromyography; MA: manual acupuncture.
Figure 4 Power spectrum analysis of the EEG A: representative EEG and EMG traces in PCPA mice; Scale bar = 1 second; B: representative EEG and EMG traces following chemogenetic inactivation of TRN GABA neurons during MA; C: quantitative analysis of delta power band percentages; D: quantitative analysis of theta power band percentages; E: quantitative analysis of alpha power band percentages; F: quantitative analysis of beta power band percentages; G: quantitative analysis of gamma power band percentages. The hM4Di + MA group received PCPA and MA treatment plus stereotaxic injection of AAV2/9-EF1α-DIO-hM4Di into the TRN, with i.p. CNO (0.1 mL/10 g) administered 30 min before MA. EEG: electroencephalography; EMG: electromyography; PCPA: p-chlorophenylalanine; TRN: thalamic reticular nucleus; GABA: gamma-aminobutyric acid-ergic; MA: manual acupuncture; hM4Di: human M4 muscarinic receptor-based designer receptor exclusively activated by designer drugs; CNO: clozapine N-oxide; ANOVA: analysis of variance; HSD: honestly significant difference; SEM: standard error of the mean. Differences among groups were examined using one-way ANOVA followed by tukey's HSD test. Data are presented as mean ± SEM (n = 3). Compared with the Control group, aP < 0.01; compared with the PCPA group, bP < 0.01; compared with the Control group, cP < 0.05; compared with the PCPA group, dP < 0.05.
Figure 4 Power spectrum analysis of the EEG A: representative EEG and EMG traces in PCPA mice; Scale bar = 1 second; B: representative EEG and EMG traces following chemogenetic inactivation of TRN GABA neurons during MA; C: quantitative analysis of delta power band percentages; D: quantitative analysis of theta power band percentages; E: quantitative analysis of alpha power band percentages; F: quantitative analysis of beta power band percentages; G: quantitative analysis of gamma power band percentages. The hM4Di + MA group received PCPA and MA treatment plus stereotaxic injection of AAV2/9-EF1α-DIO-hM4Di into the TRN, with i.p. CNO (0.1 mL/10 g) administered 30 min before MA. EEG: electroencephalography; EMG: electromyography; PCPA: p-chlorophenylalanine; TRN: thalamic reticular nucleus; GABA: gamma-aminobutyric acid-ergic; MA: manual acupuncture; hM4Di: human M4 muscarinic receptor-based designer receptor exclusively activated by designer drugs; CNO: clozapine N-oxide; ANOVA: analysis of variance; HSD: honestly significant difference; SEM: standard error of the mean. Differences among groups were examined using one-way ANOVA followed by tukey's HSD test. Data are presented as mean ± SEM (n = 3). Compared with the Control group, aP < 0.01; compared with the PCPA group, bP < 0.01; compared with the Control group, cP < 0.05; compared with the PCPA group, dP < 0.05.
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