Apomorphine hydrochloride

The alkaloid content of different extractive solutions (EW, DW, AW, NaDESs) was determined as described by Önal et al. [66 (link)]. The absorbance was recorded at 414 nm in a UV-Visible spectrophotometer. Apomorphine hydrochloride was used as standard (Merck, Darmstadt, Germany). Conventional and non-conventional solvent controls were performed in each determination to scan any possible interference. The experiment was performed in triplicate and the results were expressed as equivalent of Apomorphine hydrochloride per milliliter (µg ACE/mL). The extraction yield of each solvent was determined as the amount of chemical component per mL of extract.

Trans-astaxanthin, imipramine hydrochloride, p-chlorophenylalanine HCl (PCPA, an inhibitor of serotonin synthesis), apomorphine hydrochloride, kynuramine dihydrobromide, 4-hydroxyquinoline, clorgyline, deprenyl, 5-hydroxytryptamine, noradrenaline, dopamine, 5-hydroxyindoleacetic acid (5-HIAA) and 4-dihydroxyphenylacetic acid (DOPAC) were purchased from Sigma Chemical Co. (USA). Moclobemide hydrochloride and sodium carboxymethyl cellulose were provided by Beijing Institute of Pharmacology and Toxicology (China). For oral administration (via gavage, i.g.), Trans-astaxanthin was dissolved in 0.5% sodium carboxymethyl cellulose and moclobemide was dissolved in redistilled water. For intraperitoneal injection, imipramine and fluoxetine were dissolved in redistilled water. In acute experiments, the behavioral and neurochemical tests were conducted 1 h after Trans-astaxanthin (20, 40, 80 mg/kg, i.g.) treatment [37 (link)]. The effects of positive antidepressants such as moclobemide (20 mg/kg, i.g.), imipramine (10 mg/kg, i.p.) and fluoxetine (10 mg/kg, i.p.) were tested 1 h (meclobemide) and 30 min (imipramine and fluoxetine) respectively, after administration of the drugs as previously described [20 (link), 38 ].

This behavioral test was performed in a blinded fashion. The rats received a subcutaneous injection of 0.05 mg/kg apomorphine hydrochloride (Sigma) dissolved in 1% ascorbic acid and 0.9% NaCl. Immediately after apomorphine injection, the net number of rotations was recorded and assessed over 30 min. Rotational testing was performed in automatic rotameter bowls (Coulbourn Instruments Inc., PA, USA).

To induce parkinsonian mice, MPTP at a dose of 30 mg/kg/day was administrated intraperitoneally for 7 consecutive days. The control group received intraperitoneal treatment with 0.9% normal saline.
To induce 6-OHDA parkinsonian rats, chloral hydrate (400 mg/kg, i.p.) was intraperitoneally injected for anesthesia, and the rats were gently put in a stereotaxic frame (Narishige SN-3, Tokyo, Japan). And then, the skull was exposed and a burr hole was drilled in the skull with 4.3 mm posterior and 1.7 mm lateral to the bregma. A needle of a Hamition microsyringe was introduced into the medial forebrain bundle to a depth of 8.4 mm (Paxinos and Watson, 1998 ). A total dose of 14.5 μg 6-OHDA hydrochloride (H4381; Sigma, St. Louis, MO, United States) in 4 μl sterile saline containing 0.01% ascorbic acid was then microinjected into the right medial forebrain bundle at a rate of 1.0 μl/min. The needle was left in place for a further 10 min to prevent backflow of the drug. Rats were pretreated 30 min before the 6-OHDA infusion with 25 mg/kg desipramine to protect noradrenergic projections.
Three weeks after the surgery, the rats were subcutaneously received 0.2 mg/kg apomorphine hydrochloride (A4393, Sigma) dissolved in 0.1% ascorbate saline solution. The rats presenting more than 210 contralateral rotations in half an hour were considered successful parkinsonian rats.

ZD7288 was obtained from Tocris (Bristol, United Kingdom). 6-OHDA hydrochloride, MPTP and apomorphine hydrochloride were purchased from Sigma (St. Louis, MO, United States). Rabbit anti-HCN1 (AB5884), anti-HCN2 (AB5378) and anti-HCN4 (AB5808) polyclonal antibodies were obtained from Millipore (Massachusetts, United States). Rabbit anti-HCN3 antibodies (ab65705) were obtained from Abcam (Cambridge, United Kingdom). Histostain TM-Plus Kits used in DAB immunohistochemical staining was obtained from ZSGB-BIO (Beijing, China).
The data were expressed as mean ± SEM. Paired t-test was used to compare the difference of firing rate or CV before and after drug application. Student’s t-test and one-way ANOVA were used to analyze statistical comparisons between or among groups. Before t-tests or ANOVA, Levene’s test was performed to determine if the data is normally distributed. Chi-Square test was used to compare the fractions of different firing pattern types between normal and parkinsonian animals. The level of significance was presented by using a P-value of 0.05.