2.1. Effect of mechanical acupuncture treatment on alcohol self-administration
We first tested whether mechanical acupuncture stimulation of HT7 and the nonacupuncture point had a suppressive effect on alcohol intake when using the self-administration operant chamber in alcohol self-administration rats (Fig. 1a). The administration of mechanical acupuncture at HT7significantly decreased the number of alcohol-induced lever presses in the alcohol-treated group (F (3, 26) = 7.256, p = 0.005, n = 7–8 per group) (Fig. 1b).
2.2. Stimulation at HT7 reduced the increased microglial activity and Sigma receptor level of the Hb in the alcohol self-administration rats
Next, we measured microglial activity and sigma receptor activity in the rat Hb (Fig. 2a). We investigated whether acupuncture stimulation could change microglial and sigma receptor activity markers. Alcohol self-administration significantly increased the Arginase-1 level in the Hb. The stimulation of HT7 by mechanical acupuncture significantly decreased Arginase-1 levels in the Hb in the alcohol self-administration group (F (3, 10) = 16.26, p = 0.0004, n = 3–4 per group) (Fig. 2b). The Iba-1 level also increased in the alcohol self-administration group. However, the stimulation of HT7, but not the nonacupuncture point, significantly decreased the Iba-1 level in the Hb in the alcohol self-administration group (F (3, 10) = 6.380, p = 0.0109, n = 3–4 per group) (Fig. 2c). The sigma receptor-1 level increased in the Hb in the alcohol self-administration group. However, the stimulation of HT7, but not the nonacupuncture point, significantly decreased the sigma1 receptor level in the Hb in the alcohol self-administration group (F (3, 9) = 6.93, p = 0.0103, n = 3–4 per group) (Fig. 2d).
2.3. Stimulation at HT7 increased the BDNF level in the mPFC and decreased TH in the VTA in the alcohol self-administration rats
In the next experiment, the expression levels of BDNF in the mPFC and TH in the VTA were measured when HT7 was stimulated in alcohol self-administration rats (Fig. 3a). Alcohol self-administration significantly decreased the BDNF level in the mPFC. The stimulation of HT7, but not the nonacupuncture point, by mechanical acupuncture significantly increased BDNF levels in the mPFC in the alcohol self-administration group (F (3, 10) = 37.64, p = 0.0001, n = 4 per group) (Fig. 3b and d). In addition, the expression of TH in the VTA was measured when HT7 was stimulated in alcohol self-administration rats. Alcohol self-administration significantly increased TH expression in the VTA. The stimulation of HT7, but not the nonacupuncture point, by mechanical acupuncture significantly decreased dopamine neuron expression in the VTA in the alcohol self-administration group (F (3, 10) = 29.11, p = 0.0001, n = 3–4 per group) (Fig. 3c and d).
2.4. Inhibition of pTrkB in the mPFC reduced the expression of microglia in the Hb, decreased TH expression in the VTA and reduced alcohol intake
We used ANA-12, an antagonist of TrkB, to determine how the BDNF increase in the mPFC affected sigma receptor and microglial activity in the Hb. As shown in Fig. 4, the administration of a low dose (50 nmol/µl) of ANA-12 did not significantly decrease arginase-1 in the Hb (Fig. 4a). However, the administration of 100 and 200 nmol/µl ANA-12 significantly decreased Arginase-1 in the Hb (F (3, 11) = 11.64, p = 0.0010, n = 3–4 per group) (Fig. 4b), and the administration of 200 nmol/µl ANA-12 dose significantly decreased TH levels in the VTA (F (3, 11) = 9.768, p = 0.0020, n = 3–4 per group) (Fig. 4c). Next, we tested whether the microinjection of 100 nmol/µl ANA-12 had a suppressive effect on alcohol intake in alcohol-addicted rats in the self-administration operant chamber. The microinjection of 100 nmol/µl ANA-12 into the mPFC significantly decreased the number of alcohol activation lever presses compared with the injection of vehicle (t = 2.586, p = 0.0271, n = 6 per group) (Fig. 4d).
2.5. Blocking the sigma receptor and inhibiting microglia in the Hb reduced the expression of TH in the VTA and reduced alcohol intake
We used a microglial inhibitor, minocycline (MINO), and a sigma receptor antagonist, BD1047 (BD), to determine how the activation of the sigma receptor and microglia in the Hb affected TH in the VTA region (Fig. 5a). The administration of a low dose (50 nmol/µl) of BD did not significantly decrease TH in the VTA. However, the administration of 100 and 200 nmol/µl BD significantly decreased TH in the VTA (F (3, 10) = 16.44, p = 0.0003, n = 3–4 per group) (Fig. 5b). Similarly, the administration of all doses (50, 100 and 200 nmol/µl) of MINO significantly decreased TH in the VTA (F (3, 10) = 32.05, p = 0.0001, n = 3–4 per group) (Fig. 5c). Next, we tested whether the microinjection of 50 nmol/µl MINO and 100 nmol/ml BD had a suppressive effect on alcohol intake in alcohol-addicted rats in the self-administration operant chamber. The microinjection of 50 nmol/µl MINO and 100 nmol/µl BD into the Hb significantly decreased the number of alcohol activation lever presses compared with the microinjection of vehicle (F (2, 16) = 3.089, p = 0.0734, n = 7 per group) (Fig. 5d).