Two sets of experiments were designed to test our hypothesis (Figure 1). Experiment 1 was designed to identify the presence of MC4R on dopaminergic neurons in the SNpc by double staining of tyrosine hydroxylase (TH) and MC4R in brain sections of C57BL/6 mice (n = 6) (Figure 1A). Experiment 2 was designed to study whether the lateral ventricle administration of anti-nesfatin-1 antibody would induce nigrostriatal system degeneration in mice (n = 24, six mice per group) (Figure 1B). The following is the animal treatment for experiment 2.

Schematic illustration of the experimental design. (A) Double staining of TH and MC4R in the SNpc to demonstrate the presence of MC4R in the SNpc (n = 6). (B) Anti-nesfatin-1 antibody induces nigrostriatal system degeneration in mice (n = 24, six mice per group). The mice received the substances at the doses indicated by ICV injection for 14 days. The CSF was collected for the determination of the nesfatin-1 concentration. The SNpc and striatum were collected for the determination of TH, caspase-3, and p-ERK1/2 expression, BDNF concentration, mitochondrial morphology, and nuclear morphology. BDNF, brain-derived neurotrophic factor; CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; MAB 201, non-immune anti-mouse IgG antibody; MC4R, melanocortin 4 receptor; p-ERK1/2, phosphorylated ERK1/2; SHU 9119, MC4R receptor inhibitor; SNpc, substantia nigra pars compacta; Str, striatum; TH, tyrosine hydroxylase; ICV, intra-cerebroventricular.

To embed the guide cannula into the lateral ventricle, mice were fully anesthetized with chloral hydrate (10%, 10 mL/kg) (Keshi, Chengdu, China) by intraperitoneal (i.p.) injection and placed in a stereotaxic frame (RWD, Shenzhen, China). A longitudinal incision was made in the scalp to expose the surface of the skull. A cranial burr hole (1 mm) was drilled into the skull of the right hemisphere with the following coordinates: 0.3 mm posterior to bregma and 1.0 mm lateral to the midline (Paxinos and Franklin, 2001; Shen et al., 2017, 2020). Next, a stainless steel cannula (RWD, Shenzhen, China) was embedded at 3.2 mm vertical from the skull surface (Young et al., 2012). The cannula was fixed on the skull by a mixture of dental base acrylic resin powder and liquid (Pigeon Dental, Shanghai, China). On the experimental days, the dummy cannula was removed, and an injector (3.2 mm protrusion) was inserted into the guide cannula. ICV injection of drug solutions was performed manually at a rate of 0.5 μL/min (Dore et al., 2017) in freely moving animals through the cannula, which was connected to a 5 μL Hamilton microsyringe (Reno, NV, United States), and the injection cannula was kept in situ for an additional 3 min to avoid reflux of the solution along the injector track. The ICV injection technique is well established in this lab and was used in our previous studies (Shen et al., 2017, 2020).

After 1 week of recovery, 24 mice (8 weeks old) were randomly divided into four groups (six mice per group) and administered ICV injections once per day for 14 continuous days of the following: (1) control group: 2 μL saline; (2) non-immune anti-mouse IgG antibody group (MAB 201): 2 μL IgG1κ antibody (1.3 mg/ml) (Millipore, Darmstadt, Germany) (Evans et al., 2019); (3) MC4R receptor inhibitor group: 0.5 μL SHU 9119 (0.5 nmol) (Tocris, Bristol, United Kingdom) (Leckstrom et al., 2009); and (4) anti-nesfatin-1 group: 2 μL nesfatin-1 antibody (0.12 mg/mL) (Phoenix, Burlingame, CA, United States). Twenty-four hours after the last injection, the CSF sample was collected, and then the brain was removed from the skull. The right side of the SNpc was dissected to determine protein levels by Western blot or enzyme-linked immunosorbent assay (ELISA); the striatum was dissected for neurochemical analyses by high-performance liquid chromatography (HPLC). The collected samples were stored at −80°C for future analyses. The left side of the brain was fixed in 4% paraformaldehyde (PFA) for immunofluorescence staining.

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