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Selegiline alleviates the depressive-like behaviors of methamphetamine withdrawal syndrome through modulating mitochondrial function and energy hemostasis

Background: Methamphetamine (METH) is considered the second most commonly abused drug in the world. There is limited or no evidence concerning the effective treatment of METH withdrawal symptoms, such as depression and anxiety. Mode of action of selegiline (increase of the brain neurotransmitter activity) suggests that it might be useful in METH withdrawal syndrome treatment, being capable of diminishing the preference and depression involved in drug degeneration and addictive activities. Methods: Mice were randomly divided into 10 groups (n= 10): five METH-nondependent groups treated with normal saline intraperitoneal (i.p) for two weeks, to which, from the 15th day, selegiline (10, 20 and 40 mg/kg; i.p) or fluoxetine (5 mg/kg; i.p) was administrated for 10 consecutive days. Other groups injected METH (2 mg/kg, at 12-h intervals) for 14 days. From the 15th day, the 10-day period of METH abstinence started and the above-mentioned doses of selegiline or fluoxetine were injected. Then, the mice were evaluated for depression and biochemical assessments from the 25th day of the study. Results: Our data indicated that post-treatment with selegiline (10-40 mg/kg; i.p) for 10 days reversed METH-induced depressive-like behaviors in the forced swimming test (FST), tail suspension test (TST), and splash test with exerting no effects on the locomotor activity. Furthermore, none of the previously proposed treatments affected the behavioral abnormality in the control animals. Moreover, both selegiline and fluoxetine as standard antidepressant drug, substantially improved the levels of mitochondrial reduced glutathione (GSH), malondialdehyde (MDA), and adenosine triphosphate (ATP). Conclusion: Our findings demonstrated that selegiline produced antidepressant-like effects following METH withdrawal and prevented the mitochondrial dysfunction in the male mice.


Methamphetamine (METH)-induced withdrawal syndrome models:
In the present research, METH was initially dissolved in sterile normal saline (0.9%). IP administered of METH (2mg/kg) was done twice a day for 14 consecutive days as described by a previous study 35 .Subsequently, the treated animals were kept in cages for 10 days without any METH injections so that the withdrawal syndrome term would be induced in mice, which was confirmed through behavioral or molecular assessments. Saline injection in the control animals was done in order to exclude solvent effects as the sham group.
Experimental design: Mice were distributed into 10 groups (10 mice in each group): Group 1: control mice received normal saline; Group 2: animals which received METH (2 mg/kg) twice a day for 14 consecutive days so that the withdrawal syndrome could be induced; Group 3,4,5: in these groups, normal mice received 10, 20 and 40 mg/kg selegiline daily for 10 consecutive days after receiving of normal saline for two weeks ; Group 6: Normal mice received 5 mg/kg fluoxetine (FLX) on a daily basis for 10 consecutive days after receiving of normal saline for two weeks ; Group 7,8,9: METH-induced withdrawal mice received 10, 20 and 40 mg/kg selegiline on a daily basis for 10 consecutive days after induction of withdrawal syndrome in 14 days period time; Group 10: METH-induced withdrawal mice received 5 mg/kg FLX on a daily basis for 10 consecutive days after 14 days. At the end of the treatments, the animals were subjected to the behavioral test including OFT and TST, FST and Splash test between 10:00 AM and 14:00 PM. Ultimately, the animals were sacrificed under mild anesthesia according to our previous studies 36,37 and then, their hippocampi were dissected on an ice-cold surface and immediately immersed in liquid nitrogen, and then stored at -80 °C until mitochondrial function assay. The timeline of the procedure, treatment, behavioral and mitochondrial tests is represented in Figure.1. (Indexed in ISI and Scopus) https://ps.tbzmed.ac.ir

Forced swimming test (FST):
The immobility time was considered as a biomarker in depressive-like behavior in rodents 37 . Briefly, 26 days following the test, mice were put in cylinders containing water for 6 min. The immobility time for the period of the last 4 min was measured once the mice remained floating motionlessly in the water. It is worth noting that the reaction of each mouse was assessed by an observer who was not alert of the nature of the treatment.

Splash test:
Motivational and self-care difficulties were considered as anhedonia behaviors in the rodents 36 . Therefore, the grooming behavior of the mice was regarded as an indirect amount of spraying delicious solution intake (10% sucrose) on the dorsal of mice for 5 min by a blinded investigator 38 .Therefore, body grooming nose/face grooming, and head washing was measured in the grooming behaviors.

Tail suspension test (TST):
According to Can et al., study the decrease in the duration of immobility time was considered to assess the antidepressant-like efficacy of drug treatments in mice 39 .The animals were suspended by their tail for 6 min and the immobility posture period was considered as a depressive-like behavior when they were exposed to an inescapable situation 40 .

Open-field test (OFT):
The locomotor activity of the mice in response to withdrawal syndrome and different treatments was assessed using OFT based on our previous published study 41 ,in order to certify that the changes in the duration of immobility time were not due to the changes in the locomotor activity of mice. The animals were retained separately on the corner of the Plexiglas OFT box (50×50×40cm) that was dimly illuminated during the test and their activities Pharmaceutical Sciences (Indexed in ISI and Scopus) https://ps.tbzmed.ac.ir were documented with a camera for 5 min so that we could measure the distance they moved or their horizontal activity.

Mitochondrial and biochemical assessments:
Preparation of mitochondria: The mice were decapitated 24h after the completion of behavioral tests and their brains were fast divided out and rinsed utilizing isotonic PBS, and were soaked in the liquid nitrogen and kept at a -80 ºC freezer until the assays. The isolation of hippocampal mitochondria was performed as said by our prior investigation 37 .The final isolated mitochondria pellet obtained from a two-time centrifuge was re-suspended in Tris buffer. The uniformity of the experimental condition was done employing Coomassie blue method by adjusting to 0.5 mg mitochondrial protein per ml in each sample 42 .
GSH assay: GSH amount was measured using DTNB [5, 5'-dithiobis-(2-nitrobenzoic acid)] as the indicator 43 . 0.5 mL of supernatant was primarily added into 0.5 mL TCA (10%) and centrifuged in 8000 g for 5 min. Afterwards, 0.5 ml supernatant was added to 1.25 ml Tris buffer and 0.25 ml of DTNB (0.04%) in a total volume of 2.0 mL (pH 8.9). The developed color was measured at 412 nm with a spectrophotometer. GSH content was stated as µg mg -1 protein.

MDA assay:
The thiobarbituric acid reactive substances (TBARs) assay is widely utilized to measure lipid peroxidation or MDA level and tetra methoxy propane (TEP) as a standard of calibration curve 37 . In sum, 0.5 mL of supernatant was added into 2.5 mL TCA (20%) and centrifuged in 8000 g for 5 min and kept in temperature room for 10 min. 0.5 ml supernatant was then added to 2.5 ml sulphuric acid (0.05M) and 2 ml of TBA (0.2%). The developed yellow color was assessed at 532 nm with a spectrophotometer and MDA content was represented as µM.mg -1 protein.
Pharmaceutical Sciences (Indexed in ISI and Scopus) https://ps.tbzmed.ac.ir ATP assay: Briefly, 50 mg of hippocampus was homogenate with 0.5 mL TCA (6%) and centrifuged in 12000 g for 10 min in 4⸰C. Subsequently, potassium hydroxide (4M) was added to supernatant to reach pH=6.5 to neutralization and samples were immediately stored at -80° C.
Finally, the ATP levels were measured by ATP assay kit based on the phosphorylation of glycerol in order to generate a product that can be easily quantified by calorimetrically assay (OD 570 nm) based on the instructions of company.
Statistical Analysis: Results have been accessible as mean ± SD. All statistical analyses were done by SPSS 17 software. Statistical significance was performed by one-way ANOVA test, followed by the post-hoc Tukey test. P<0.05 was considered to be statistically significant.

Selegiline attenuated the depressive-like behaviours of METH withdrawal in mice
Our data in FST revealed no significant differences concerning the immobility time in the mice treated with selegiline (10,20&40mg/kg) according to our present protocol, compared to that in untreated animals (P>0.05; data not shown). Afterwards, the effect of selegiline on the behavioral assessment was assessed in our samples. Our results revealed that in METH withdrawal induced mice the immobility time significantly increased in the FST ( Fig.2a; p<0.05) and TST ( Fig.2b; p<0.05). In the splash test, in METH withdrawal induced mice the grooming activity time of the mice significantly decreased compared to the control counterparts ( Fig.2c; p<0.01). Moreover, no significant differences were identified concerning the locomotor activity in METH withdrawal induced mice compared to those in normal animals on the OFT (Fig.2d; p>0.05).

Depressant effects of METH withdrawal mice were reversed by selegiline treatment in mitochondrial function
Our data revealed that there was a significant decrease in mitochondrial reduced glutathione amounts in the hippocampus of METH withdrawal mice in comparison to the control group ( Fig.3a, ***p<0.001). Statistically analysis established significant differences among all treated groups in GSH level in the hippocampus following the administration of selegiline [F (9,20) =5.529, p<0.05; Fig.3a]. Also, a significant rise in mitochondrial GSH levels was found in the hippocampus of METH withdrawal mice compared to the control group based on one-way ANOVA analysis (Fig.3a, ***p<0.001). In addition, the administration of selegiline in normal rats did not induce significant difference in GSH amount in compared with the control animals ( Fig.3a, p>0.05).
Post-hoc analysis exhibited significant difference among experimental groups regarding mitochondrial MDA levels in the hippocampus after the treatment with selegiline [F(9,20)=12.093,***p<0.001; Fig.3b]. It revealed that a significant rise in mitochondrial MDA amounts in the hippocampus of animals following METH withdrawal mice compared to the control group (Fig.3b, ***p<0.001). According to our results, post-treatment with selegiline significantly decreased MDA levels compared to METH withdrawal mice (Fig.3b, ***p<0.001).
Statistical analysis showed a significant decline in mitochondrial ATP levels concerning the hippocampus of mice following METH treatment (Fig.3c, **p<0.01). The findings revealed that post-treatment with selegiline significantly increased ATP levels compared to METH withdrawal mice in the hippocampus (Fig.3c, #p<0.05). Furthermore, there are no significant difference between selegiline administration in normal mice and control groups in mitochondrial ATP amounts (Fig.3c, p>0.05). Similarly, we observed a significant rise in mitochondrial ATP levels in the hippocampus of METH withdrawal mice following selegiline treatment in comparison with METH withdrawal mice (Fig.3c, ***p<0.001).

Discussion:
For the first time, the results of the present study indicated that selegiline as a MAO inhibitor  Values are expressed as the mean ± SD and were analyzed using one-way ANOVA followed by Tukey's post hoc test (n=6-8). * P<0.05, ** P<0.01 and *** P<0.001 compared with control group. #P<0.05 and ##P<0.01 and ### P<0.001 compared with METH-withdrawal treated groups.