Chronic Alcohol Exposure Induces Aberrant Mitochondrial Morphology and Inhibits Respiratory Capacity in the Medial Prefrontal Cortex of Mice

Abstract

Alcohol use disorder (AUD) is characterized as a chronic, relapsing disease with a pattern of excessive drinking despite negative consequences to an individual's life. Severe chronic alcohol use impairs the function of the medial prefrontal cortex (mPFC), which contributes to alcohol-induced cognitive and executive dysfunction. The mPFC contains more mitochondria compared to other cortical areas, which suggests mitochondrial damage may occur in AUD and trigger subsequent behavior change. Here, we identified morphological and functional changes in mitochondria in the mPFC in C57BL6/J mice after 8 h of withdrawal from chronic intermittent alcohol (CIA) exposure. Three-dimensional serial block-face scanning electron microscopy (SBFSEM) reconstruction revealed that CIA exposure elongated mPFC mitochondria and formed mitochondria-on-a-string (MOAS). Furthermore, alcohol significantly affected mitochondrial bioenergetics, including oxidative phosphorylation and electron transport, with inhibited aerobic respiration in mPFC mitochondria after CIA exposure. We also found decreased expression of fusion (mitofusin 2, Mfn2) and increased fission (mitochondrial fission 1 protein, Fis1) proteins in the mPFC of alcohol-treated mice. In sum, our study suggests that CIA exposure impairs mitochondrial dynamics and function in the mPFC.

Keywords: alcohol use disorder; fission; fusion; medial prefrontal cortex; mitochondria; morphology; respiratory capacity.

FIGURE 1

Chronic intermittent exposure to alcohol procedure and BAC. (A) Illustration of chronic intermittent alcohol exposure. (B) Process of tissue extraction for SBESEM study. (C) The red square displays ACC used in the SBFSEM study. (D) The red rectangle displays mPFC used in the Seahorse and WB studies.

FIGURE 2

Three-dimensional morphological changes of the ACC mitochondria. (A) Representative patterning of mitochondria reconstruction in three-dimensional SBFSEM study.*: Mayo Clinic Microscopy and Cell Analysis Core’s FEI Apreo VS SEM. (B) A series of 50-nm sections showing representative mitochondria in ACC from the alcohol and air groups. The 1st, 7th, and 14th sections were selected from the same sample in two groups. Yellow arrows indicate representative mitochondria in those sections. (C) Representative three-dimensional mitochondrial reconstruction in air and alcohol groups. (D) Quantification of mitochondria volume. (E) Quantification of disconnections (mitochondrial ends) per mitochondrial length. (F) Quantification of mitochondrial length. N = 8 mitochondria per mouse. Asterisks (*) represent significance exists between two groups (P < 0.05) after unpaired Student’s t-test.

FIGURE 3

Mitochondria respiratory capacity changes in the mPFC from chronic intermittent alcohol exposure. (A) The oxygen consumption rate of isolated mitochondria in mouse mPFC. States changed accompanied by four injections of ADP and mito-stress drugs. (B–E) Comparison of mitochondrial OCR values in different states between the alcohol and air groups. Asterisks (*) represent significance exists between the two groups (P < 0.05) after unpaired Student’s t-test.

FIGURE 4

Mitochondria fission and fusion protein level changes in the mPFC from chronic intermittent alcohol treatment. Western blotting bands of (A) fusion proteins (OPA1 and Mfn2) and (D) fission proteins (Fis1 and Drp1) in the alcohol and air groups. The relative intensity of (B,C) fusion proteins (OPA1 and Mfn2) and (E,F) fission proteins (Fis1 and Drp1). Asterisks (*) represent significance exists between the alcohol and air groups (P < 0.05) after unpaired Student’s t-test.