To identify whether ARC neurons projecting to the NAc were activated by HT7 acupuncture, we assessed the expression of c-Fos in ARC neurons. DiI (2%) (a fluorescent retrograde neuronal tracer; Life Technologies, MA, USA) in 0.1 μl per side over 5 min was infused bilaterally into the NAc (AP, 1.7; ML, ±0.75; and DV, 5.7 from bregma) using a 10-μl Hamilton syringe under intraperitoneal sodium pentobarbital anesthesia (50 mg/kg). The tracer was allowed to spread for at least 7 days. After acupuncture treatment, rats were sacrificed and brains were prepared for c-Fos immunohistochemistry. Preparation of the brain tissue began with transcardial perfusion of 4% paraformaldehyde in 0.1 M phosphate buffer under pentobarbital anesthesia (80 mg/kg) 1 hour after acupuncture stimulation. Subsequently, brains were removed, immersed in 4% buffered paraformaldehyde for 2 hours, allowed to stand in 30% sucrose overnight, and cryosectioned at 30 μm (between 3.3 and 3.6 mm posterior to bregma). The brain slices were then mounted on gelatin-coated glass slides. The ARC labeled by DiI was identified with an Olympus AX70 fluorescence microscope (Olympus, Tokyo, Japan). For c-Fos immunohistochemistry study, the brain slices were incubated in blocking solution containing 3% bovine serum albumin for 1 hour at room temperature and incubated with anti–c-Fos rabbit polyclonal antibodies (1:1000, Santa Cruz Biotechnology, Santa Cruz, USA) for 24 hours at 4°C, followed by incubation with a biotinylated donkey anti-rabbit Alexa Fluor 488 (1:500, Invitrogen, Grand Island, NY, USA). Last, the sections were mounted onto gelatin-coated slides. The numbers of c-Fos immunoreactive neurons were counted under 200× with a confocal laser scanning microscope (LSM700, Carl Zeiss, Oberkochen, Germany) and averaged from three to four brain slices per rat.

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