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Acupuncture attenuates alcohol dependence through activation of endorphinergic input to the nucleus accumbens from the arcuate nucleus

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Science Advances  04 Sep 2019:
Vol. 5, no. 9, eaax1342
DOI: 10.1126/sciadv.aax1342
  • Fig. 1 Effects of acupuncture on ethanol withdrawal tremor.

    Rats on the chronic ethanol diet (ethanol group) received an ethanol-containing diet, and their pair-fed rats (control group) received an isocaloric diet with dextran substituted for ethanol. Rats received acupuncture at 2 hours after withdrawal from chronic ethanol consumption. Tremor was measured after acupuncture. (A and B) Representative force signals (filter, 10 to 22 Hz) at 0- and 2-hour time points in the control (n = 5) or ethanol (n = 5) group (A) and tremor activity expressed as a percentage of baseline tremor power at different time points (B). (C) Mechanical acupuncture instrument (MAI). (D) Effect of acupuncture on ethanol withdrawal tremor. Tremor was measured in rats given acupuncture at 2 hours after withdrawal from chronic consumption of an ethanol liquid diet. Another group of control diet rats (Con-control group) and ethanol diet rats (Con-ethanol group) did not receive any needle insertion. Con-control, n = 8; HT7-control, n = 7; Con-ethanol, n = 7; HT7-ethanol, n = 7; LI5-ethanol, n = 6. Graphs represent mean ± SEM. #P < 0.05 versus Con-control group, *P < 0.05 versus Con-ethanol group. Photo credit: Suchan Chang, College of Korean Medicine, Daegu Haany University.

  • Fig. 2 Involvement of accumbal β-endorphin in acupuncture inhibition of ethanol withdrawal tremor.

    (A and B) Effect of naloxone on HT7 inhibition of ethanol withdrawal tremor. Representative examples of force signals in the ethanol group (filter: 10 to 22 Hz) (A) and tremor activity, expressed as mean values of tremor power for 15 min in the control and ethanol groups (B). Tremor was measured in ethanol-dependent rats given acupuncture at 30 min after intraperitoneal injection of naloxone. #P < 0.05 versus saline-control group (n = 8), *P < 0.05 versus saline-ethanol group (n = 7), $P < 0.05 saline + HT7-ethanol group (n = 7); naloxone + HT7-ethanol group, n = 7; naloxone-ethanol, n = 9. (C and D) Effect of HT7 acupuncture on extracellular β-endorphin levels in the NAc. Basal β-endorphin levels of ethanol groups were significantly lower than those of all other groups (###P < 0.001 versus all other groups, n = 6). Accumbal β-endorphin levels were measured in rats given acupuncture at 2 hours after withdrawal from chronic consumption of an ethanol liquid diet. ***P < 0.001 versus all other groups, n = 6 per group. (E) Effect of intra-NAc infusion of β-endorphin on ethanol withdrawal tremor. Tremor was measured in rats given intra-NAc infusions of β-endorphin at 2 hours after withdrawal from chronic consumption of an ethanol liquid diet. ###P < 0.001 versus saline-control group, **P < 0.01 and ***P < 0.001 versus saline-ethanol group; saline-control group, n = 6; saline-ethanol group, n = 7; β-ED(0.25)-ethanol group, n = 6; β-ED(0.5)-ethanol group, n = 5. Graphs represent mean ± SEM.

  • Fig. 3 Effects of acupuncture on hypothalamic ARC neuron activities.

    (A and B) Immunohistochemical staining of c-Fos in ARC neurons of control rats (a) and rats given acupuncture at HT7 or LI5 (b and c). A significant increase in the number of c-Fos–positive cells in the ARC was shown in rats subjected to HT7 acupuncture compared to control rats or rats subjected to LI5 acupuncture [#P < 0.05 versus Con, n = 6 per group; (B)]. c-Fos–positive cells (d) in ARC neurons labeled with DiI (e) in HT7 acupuncture–treated rats (f). Scale bars, 50 μm (200×). (C) Typical waveforms of spontaneous and evoked activity of ARC neurons before and during HT7 acupuncture in control (n = 7) or ethanol (n = 7) diet rats. Left, single action potential; middle and right, the continuous single action potential of real-time (waveform) (bottom) and peri-stimulus time histogram (one bin width per second, top). (D) Basal firing rates in the control and ethanol diet rats before acupuncture stimulation. **P < 0.01 versus control group. (E) Effect of acupuncture on firing rate of control and ethanol diet rats. Single-cell activity was analyzed by graphic recording for 20 s at rest and 10 s during acupuncture treatment. *P < 0.05 versus control group. Graphs represent mean ± SEM (n = 7 per group).

  • Fig. 4 Effects of acupuncture or intra-NAc infusions of β-endorphin on anxiety-like behavior in the elevated plus maze in ethanol-dependent rats.

    (A) Effect of yohimbine on the time spent in the open arms. The percentage of time spent in the open arms was measured in ethanol-naïve rats 30 min after injection of yohimbine (5 mg/kg, intraperitoneally) (##P < 0.01 versus saline, n = 5 per group). (B) Effect of acupuncture on the time spent in the open arms. The percentage of time spent in the open arms was measured in rats given acupuncture at 2 hours after withdrawal from chronic consumption of an ethanol liquid diet. ###P < 0.001 versus Con-control group (n = 9) or HT7-control group (n = 9), *P < 0.05 versus Con-ethanol group (n = 9); HT7-ethanol group (n = 8). (C) Effect of intra-NAc infusions of β-endorphin on the time spent in the open arms. The percentage of time spent in the open arms was measured in rats given intra-NAc infusions of β-endorphin at 2 hours after withdrawal from chronic consumption of an ethanol liquid diet. #P < 0.05 Con-control group, *P < 0.05 versus Con-ethanol group, n = 6 per group. Graphs represent mean ± SEM of the percentage of time spent in the open arms.

  • Fig. 5 Effects of acupuncture or intra-NAc infusions of β-endorphin on ethanol self-administration in ethanol-dependent rats.

    (A) Experimental procedures. (B and C) Effect of acupuncture on ethanol self-administration in dependent rats. Ethanol self-administration was measured in rats given acupuncture at 2 hours after withdrawal from chronic consumption of an ethanol liquid diet. #P < 0.05 versus Con-control group; *P < 0.05 versus Con-ethanol group, n = 6 per group. (D and E) Effect of intra-NAc infusions of β-endorphin on ethanol self-administration in ethanol-dependent rats. Ethanol self-administration was measured in rats given intra-NAc infusions of β-endorphin at 2 hours after withdrawal from chronic consumption of an ethanol liquid diet. #P < 0.05 versus Con-control group; *P < 0.05 versus Con-ethanol group; Con-control group, n = 6; Con-ethanol group, n = 8; β-ED-ethanol group, n = 7. Graphs represent mean ± SEM of lever presses.

  • Fig. 6 Effects of intra-NAc infusions of β-endorphin on TH expression and phosphorylation in the VTA.

    (A and B) Immunohistochemical staining of TH in VTA neurons of the ethanol group given intra-NAc infusions of β-endorphin at 2 hours after ethanol withdrawal (c). Another group of control diet rats (a) (Con-control group, n = 7) and ethanol diet rats (b) (Con-ethanol group, n = 7) received artificial cerebrospinal fluid infusion in place of β-endorphin infusion. A significant increase in the number of TH-positive cells in the VTA was shown in rats (β-ED-ethanol group, n = 6) subjected to intra-NAc infusions of β-endorphin compared to control rats [#P < 0.05, Con-control versus Con-ethanol; *P < 0.05, Con-ethanol versus β-ED-ethanol; (B)]. Scale bar, 50 μm (200×). (C and D) Immunohistochemical staining of THser40, the phosphorylation form of TH, in VTA neurons of ethanol group given intra-NAc infusions of β-endorphin at 2 hours after ethanol withdrawal (c). Another group of control diet rats (a) (Con-control group, n = 7) and ethanol diet rats (b) (Con-ethanol group, n = 7) received artificial cerebrospinal fluid infusion in place of β-endorphin infusion. A significant increase in the number of THser40-positive cells was shown in the VTA of rats (β-ED-ethanol group, n = 6) subjected to intra-NAc infusions of β-endorphin compared to control rats [*P < 0.05, Con-ethanol versus β-ED-ethanol; (E)]. Scale bar, 50 μm (200×).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaax1342/DC1

    Fig. S1. Effects of chronic ethanol consumption on growth rate and BEC.

    Fig. S2. Measurement of ethanol withdrawal tremor.

    Fig. S3. Schematic localization of microdialysis probes and infusion sites.

    Fig. S4. Effects of intra-NAc infusions of β-endorphin on anxiety-like behavior in the elevated plus maze and acupuncture at HT7 on plasma corticosterone levels in yohimbine-treated rats.

    Fig. S5. Effects of HT7 acupuncture and intra-NAc infusions of β-endorphin on locomotor activity in the elevated plus maze.

    Fig. S6. Effects of intra-NAc infusions of β-endorphin on anxiety-like behavior in the elevated plus maze in control diet–fed rats.

    Fig. S7. Effects of HT7 acupuncture and intra-NAc infusions of β-endorphin on water self-administration.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Effects of chronic ethanol consumption on growth rate and BEC.
    • Fig. S2. Measurement of ethanol withdrawal tremor.
    • Fig. S3. Schematic localization of microdialysis probes and infusion sites.
    • Fig. S4. Effects of intra-NAc infusions of β-endorphin on anxiety-like behavior in the elevated plus maze and acupuncture at HT7 on plasma corticosterone levels in yohimbine-treated rats.
    • Fig. S5. Effects of HT7 acupuncture and intra-NAc infusions of β-endorphin on locomotor activity in the elevated plus maze.
    • Fig. S6. Effects of intra-NAc infusions of β-endorphin on anxiety-like behavior in the elevated plus maze in control diet–fed rats.
    • Fig. S7. Effects of HT7 acupuncture and intra-NAc infusions of β-endorphin on water self-administration.

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