| Alcohol use disorders(AUDs)are common psychiatric diseases frequently seen in developed and developing countries that merit more attention from caregivers and have not been emphasized in treatments.According to the Diagnostic and Statistical Manual of Mental Disorders,5th Edition(DSM-5),around 36% of males and 22.7% of females reached the criteria of AUDs in the U.S.between 2012 and 2013.Recently,the gender difference among AUD patients is decreasing.Although most binge-drinking individuals do not meet the diagnostic criteria of AUDs,the incidence of AUDs is increasing along with the increased frequency of binge drinking.The most common ages seen in AUD patients are between 18 and 29 years old.Indeed,only 15% of AUD patients get treated appropriately in developed countries and most patients with AUDs do not receive proper treatments.Therefore,early interventions for AUD patients are always delayed and the average age of the first treatment for AUD patients is above the 30 s.Unfortunately,according to previous literature,AUDs are directly correlated to more than 60 diseases including trauma,alcoholic hepatitis,cardiovascular diseases,stroke,tumor,and gastrointestinal diseases.A few studies elucidate proper dosage of alcohol may reduce risks of cardiac events in males aged between40 and 60,however,the conclusion was suspected to be reliable in developed countries with a high incidence of cardiovascular disorders.Nonetheless,the risks induced by AUDs always overweigh the cardiovascular benefits even for this cohort.The prefrontal cortex is considered to control multiple behavior and function and is more vulnerable to alcohol than other cerebral cortex areas.According to the previous studies,the mouse model after chronic intermittent ethanol exposure developed diverse phenotypes associated with the deficits in the medial prefrontal cortex(mPFC)including the reduction of cognitive ability,working memory,impulsivity,and goal-directing behavior.Interestingly,according to the functional MRI and PET imaging for patients with AUDs,the white matter deficits,projection interruptions,and structural abnormalities were identified in the prefrontal cortex.Meanwhile,mitochondria are mainly located in the mPFC pyramidal neurons due to the high energy demanding status compared with other brain regions.Previous investigation has verified that the functional deficits in mitochondria of the prefrontal cortex could trigger working memory deficits.Therefore,we investigated the mitochondrial structure and function changes in the mPFC in a mouse model of AUD to elucidate potential pathogenesis underlying the behavioral deficits induced by AUDs.Purpose:We compared the function and morphology of mitochondria located in the medial prefrontal cortex of mice underlying chronic alcohol exposure treatment(n = 50)and air exposure(n = 50).We are aiming at displaying the molecular deficits induced by chronic intermittent alcohol exposure including oxidative phosphorylation dysfunction.We displayed the mitochondria morphology and function change in the anterior cingulate cortex(ACC)of the AUD mouse model.We further investigate the underlying biological mechanisms of the mitochondria functional and structural alterations under the AUD circumstance.Methods:We utilized the 6-week male C57BL/6J mice from the Jackson lab in our study.The AUD model was constructed by a chronic intermittent alcohol(CIA)vapor exposure over 4weeks combined with an intraperitoneal injection(1.5 g/kg/d alcohol solution)after 2-week habitualization.After a transient withdrawal for about 8 hours,we performed behavioral tests including open field,horizontal bar test,Y-maze spontaneous alternation,and novel object recognition test to evaluate the motility,cognitive ability,and working memory of the AUD mouse model.Mice that did not go through behavior tests were euthanized for molecular experiments.ACCs were sampled for serial block-face scanning electron microscopy study(300-350 slides per mitochondria).We also reconstruct mitochondria three-dimensional structure and analyze related parameters through R-construct.Meanwhile,we isolated mitochondria from the medial prefrontal cortex and utilized the Seahorse-XF96 analyzer to evaluate the respiratory capacity.We also performed western blotting(WB)and reverse transcription-quantitative polymerase chain reaction(RT-q PCR)to quantitate the fission and fusion proteins in the medial prefrontal cortex.We further validated the overexpression of Fis1 in ACCs via immunofluorescence.Results:1.Mitochondria morphology: At the end of the CIA exposure,the mean blood alcohol concentration(BAC)in the alcohol group was 219.72 mg/dl.The mitochondrial connectivity patterning in alcohol and air groups was observed and tracked in 300-350 continuing sections per mitochondria.“Mitochondria on a string”(MOAS)was observed after reconstruction in alcohol group with teardrop-shaped mitochondria(0.5 μm in diameter)connected by a thin double membrane extending up to 5 μm long.The alcohol-treated mice displayed increased connectivity between mitochondrion compared to air mice(unpaired Student’s t-test,t(12)= 4.506,P<0.05).The average length of mitochondria in the alcohol group was 9.355 ± 1.67 μm compared with 3.938 ± 0.3161 μm in the air group.The mitochondrial average length was significantly higher in the alcohol group than the air group(unpaired Student’s t-test,t(12)= 3.677,P<0.05).However,mitochondria reconstruction of air and alcohol-treated mice(2 scans for each mouse)were similar in overall volume(unpaired Student’s t-test,t(12)= 0.1702,P>0.05).2.Functional tests of isolated mitochondria: After a comparison between the alcohol group and air group,we observed an overall decreased respiratory capacity in the CIA group.Significant differences were detected in basal level(unpaired Student’s t-test,t(9)=2.584,P<0.05),State Ⅲu(unpaired Student’s t-test,t(9)= 2.373,P<0.05),and State Ⅳo phases(unpaired Student’s t-test,t(9)= 2.796,P<0.05),indicating baseline respiration,phosphorylating respiration,maximum respiratory capacity were inhibited in the Alcohol group compared with control.Whereas,the OCR after ADP supplement(State Ⅲ)showed no significant change(unpaired Student’s t-test,t(9)= 1.958,P>0.05).3.Fission and fusion protein expression: According to WB results,Mfn2,one of the fusion proteins,significantly decreased in the alcohol group(unpaired Student’s t-test,t(6)= 3.331,P<0.05).Fission protein Fis1 was significantly increased in the alcohol group compared with the air control(unpaired Student’s t-test,t(6)= 4.333,P<0.01).However,OPA1 did not show significant changes after alcohol treatment(unpaired Student’s t-test,t(6)= 0.6933,P>0.05).Drp1 didn’t exhibit significance either(unpaired Student’s t-test,t(6)= 0.9853,P>0.05).According to the RT-q PCR results,Fis1 expression was significantly higher in the alcohol group compared with the air control(unpaired Student’s t-test,t(4)=4.655,P<0.01).Oppositely,the expression of fusion proteins including OPA1(unpaired Student’s t-test,t(4)= 5.560,P<0.01)and Mfn2(unpaired Student’s t-test,t(4)= 5.783,P<0.01)is lower than the air control.We further utilized confocal microscopy and verified the overexpression of Fis1 in mPFC from the alcohol-treated mouse.4.Evaluations of working memory,cognitive function,and motility: Working memory(unpaired Student’s t-test,t(28)= 3.364,P<0.01)and cognitive ability(unpaired Student’s t-test,t(20)= 2.977,P<0.01)in the alcohol-treated group is significantly lower than the air-treated group.However,the voluntary mobility parameters including total distance and movement duration recorded in the open-field test did not show the difference between those two groups(unpaired Student’s t-test,P>0.05).Meanwhile,motor coordination measured by the horizontal bar test did not show the difference between those two groups(unpaired Student’s t-test,P>0.05).Conclusions:1.CIA exposure may introduce MOAS change of mitochondria in the mPFC of mice.2.CIA exposure may trigger the interruptions of oxidative phosphorylation in the mPFC of the mouse brain.The deficits rely on the dysfunction of complex-I-associated respiration and the electron transport chain as well as the ATP biosynthesis.3.CIA exposure may induce fission and fusion protein changes in the mPFC of alcohol-treated mice.Fission proteins like Fis1 increased and fusion proteins like Mfn2 decreased in the alcohol group,which may correlate to the morphology change.4.CIA exposure may induce deficits of working memory and cognitive ability but does not influence the voluntary mobility function and motor coordination in alcohol-treated mice. |
