This experiment examined the effects of acupuncture on ethanol withdrawal tremor in ethanol-dependent rats. Acupuncture was given for 20 s after withdrawals from chronic ethanol consumption. Ethanol withdrawal was precipitated by replacement of the ethanol-containing diet with the control liquid diet for 2 hours. To pharmacologically characterize the effects of acupuncture on ethanol withdrawal tremor, we gave rats an intraperitoneal injection of the nonselective opioid receptor antagonist naloxone (1.0 mg/kg) 30 min before acupuncture treatment. Rats also received intra-NAc infusions of β-endorphin (0.25 and 0.5 μg per side) to determine whether β-endorphin in the NAc can mimic acupuncture effect. As GABA neurons to the VTA projecting from the NAc were more abundant and more sensitive to μ-opioid receptor agonist in the shell than core (39), intra-infusions of β-endorphin were administered into the shell. Tremor was quantified in a real-time manner after acupuncture treatment by using a custom-made ATAMS (Fig. 2A), as previously described (40). In brief, the system was designed to monitor tremor activity through a force transducer (Grass Instruments, Braintree, MA, USA) mounted under a clear plastic cylinder (20 cm × 6 cm). The degree of tremor was measured for 15 min after placing each rat in the plastic cylinder. The second 5-min recording was performed for data. The signals from the force transducer were fed into bridged amplifiers (ETH-200, CB Sciences Inc., Dover, NH, USA), filtered between 10 and 22 Hz, and quantified using a LabChart and Scope program (ADInstruments). Power spectra for tremor were estimated by testing a tremogenic compound, harmaline. Harmaline, a derivative of β-carboline, is one of the most frequently used tremorgenic compound, and harmaline-induced tremor is regarded as a model for essential tremor in animals. It has been shown that subcutaneous or intraperitoneal injection of harmaline induces tremors within a few minutes in forelimb, hindlimb, head, or whole body, and the tremors last up to several hours (41). To find out specific oscillation frequencies to differentiate tremor-like activity from other movements, we assessed the motion powers during a 5-min pretreatment baseline and a 5-min posttreatment 10 min after subcutaneous injection of harmaline (10 mg/kg) in ethanol-naïve rats. On the basis of the results from validation of ATAMS using harmaline, ethanol withdrawal tremor was measured between 10 and 22 Hz. Tremor was expressed using the mean of motion power taken from a 5-min-long graphic recording.

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