Chronic Voluntary Alcohol Drinking Causes Anxiety-like Behavior, Thiamine Deficiency, and Brain Damage of Female Crossed High Alcohol Preferring Mice

Abstract

The central nervous system is vulnerable to chronic alcohol abuse, and alcohol dependence is a chronically relapsing disorder which causes a variety of physical and mental disorders. Appropriate animal models are important for investigating the underlying cellular and molecular mechanisms. The crossed High Alcohol Preferring mice prefer alcohol to water when given free access. In the present study, we used female cHAP mice as a model of chronic voluntary drinking to evaluate the effects of alcohol on neurobehavioral and neuropathological changes. The female cHAP mice had free-choice access to 10% ethanol and water, while control mice had access to water alone at the age of 60-day-old. The mice were exposed to alcohol for 7 months then subjected to neurobehavioral tests including open field (OF), elevated plus maze (EPM), and Morris water maze (MWM). Results from OF and EPM tests suggested that chronic voluntary drinking caused anxiety-like behaviors. After behavior tests, mice were sacrificed, and brain tissues were processed for biochemical analyses. Alcohol altered the levels of several neurotransmitters and neurotrophic factors in the brain including gamma-Aminobutyric acid (GABA), corticotropin-releasing factor, cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor. Alcohol increased the expression of neuroinflammation markers including interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and C-C chemokine receptor 2 (CCR2). Alcohol also induced cleaved caspase-3 and glial fibrillary acidic protein, indicative of neurodegeneration and gliosis. In addition, alcohol inhibited the expression of thiamine transporters in the brain and reduced thiamine levels in the blood. Alcohol also caused oxidative stress and endoplasmic reticulum (ER) stress, and stimulated neurogenesis.

Keywords: alcohol use disorder; endoplasmic reticulum stress; neurodegeneration; neuroinflammation; oxidative stress.

FIGURE 1

Effects of 7 months of voluntary alcohol drinking on anxiety-like behavior in female cHAP mice. After that, mice were subjected to Open Field (OF) test (A,B), Elevated Plus Maze (EPM) test (C,D), and Morris Water Maze (MWM) test (E). The total distance traveled (A) and the time spent in the center (B) in OF was measured and presented as the mean ± SEM (n = 7) in each group. The percentage of time spent in open arms (C) and percentage of entry numbers into open arms (D) in EPM were quantified and presented the mean ± SEM (n = 7). (E) The spatial learning and memory were evaluated by MWM. The total latency escape time was measured and presented as the mean ± SEM (n = 7). Unpaired student t-test was used to assess the difference between control and alcohol-exposed group. *p < 0.05 denotes a statistically significant difference from the control group.

FIGURE 2

Effects of chronic alcohol exposure on the levels of GABA and neurotrophic factors in the brain. (A) The concentration of GABA in the brain was determined by ELISA as described in the Materials and Methods. n = 7, *p < 0.05 denotes a statistically significant difference from the control. (B) The expression of CREB in the cerebrum was determined by immunohistochemistry (IHC). (C) The numbers of CREB-positive cells in the hippocampus or prefrontal cortex (PFC) were determined. The results were expressed as the mean ± SEM; n = 4, *p < 0.05 denotes a statistically significant difference from the control group. (D) The expression of CREB was determined by immunoblotting (IB). (E) The relative amounts of CREB were quantified and normalized to the expression of actin. (F) The expression of CRF, BDNF and MANF in the cerebrum was determined by IB. (G) The relative amounts of CRF, BDNF and MANF were quantified and normalized to the expression of actin. The results were expressed as the mean ± SEM, n = 4 for each group. *p < 0.05 denotes a statistically significant difference from the control group.

FIGURE 3

Effects of chronic alcohol exposure on oxidative stress and ER stress in the brain. (A) The expression of 4-HNE and DNP in the cerebrum was determined by IB. (B) The relative amounts of 4-HNE and DNP were quantified and normalized to the expression of actin. (C) The expression of ER stress markers, ATF-6, CHOP, Caspase-12, and XBP-1s in the cerebrum was determined by IB. The relative amounts of expression were quantified and normalized to the expression of actin. (D) The relative amounts of ER stress markers were quantified and normalized to the expression of actin. The results were expressed as the mean ± SEM; n = 4 for each group. *p < 0.05 denotes a statistically significant difference from the control group.

FIGURE 4

Effects of chronic alcohol exposure on the expression of activated caspase-3. (A) The expression of cleaved caspase-3 in the cerebrum was determined by IB. (B) The relative amounts of expression were quantified and normalized to the expression of actin. The result was expressed as the mean ± SEM; n = 4. *p < 0.05 denotes a statistically significant difference from the control group. (C) The expression of cleaved caspase-3 in the prefrontal cortex (PFC) and the dentate gyrus (DG) of the hippocampus was examined by IHC; bar = 50 μm. Arrows indicate cells that are positive for cleaved caspase-3. (D) The numbers of cleaved caspase 3-positive cells in the hippocampus and PFC were determined. The results were expressed as the mean ± SEM; n = 4.

FIGURE 5

Effects of chronic alcohol exposure on inflammatory cytokines/chemokines in the brain. (A) The expression of IL-6 and TNFα in the brain was determined with IB. (B) The relative expression of IL-6 and TNFα was quantified as described above. Each data point was the mean ± SEM; n = 4. *p < 0.05, statistically significant difference from the control group. (C) The expression of MCP-1 and CCR2 in the brain was determined with IB. (D) The relative expression of MCP-1 and CCR2 was quantified. Each data point was the mean ± SEM; n = 4. *p < 0.05, statistically significant difference from control group.

FIGURE 6

Effects of chronic ethanol exposure on astrocyte activation. (A) The expression of GFAP in the mouse brain was examined by IHC. The rectangle insets indicate an area of the hippocampus. (B) The rectangle insets in (A) are shown in higher magnifications. (C) The GFAP-positive cells in the hippocampus were quantified. The result was expressed as the mean ± SEM, n = 3 for each group. *p < 0.05 denotes a statistically significant difference from the control group. (D) The protein levels of GFAP were examined by IB. (E) The relative amounts of GFAP expression were quantified and normalized to the expression of actin. The result was expressed as the mean ± SEM, n = 4 for each group. *p < 0.05 denotes a statistically significant difference from the control group.

FIGURE 7

Effects of chronic alcohol exposure on the expression of thiamine transporters in the brain. (A) The expression of thiamine transporters (SLC19A2, SLC19A3, and OCT1) in the cerebrum was determined by IB. (B) The relative amounts of expression were quantified and normalized to the expression of actin. The results were expressed as the mean ± SEM, n = 4 for each group. *p < 0.05 denotes a statistically significant difference from the control group.

FIGURE 8

Effects of chronic alcohol exposure on the expression of G-protein-coupled estrogen receptor (GPR30) in the brain, and estradiol/progesterone levels in the blood. (A) The expression of GPR30 in the cerebrum was determined by IB. (B) The relative amounts of expression were quantified and normalized to the expression of actin. The results were expressed as the mean ± SEM, n = 4 for each group. *p < 0.05 denotes a statistically significant difference from the control group. The concentration of estradiol (C) and progesterone (D) in the blood was determined by ELISA as described in the Materials and Methods. n = 5 for estradiol test, and n = 8 for progesterone test.

FIGURE 9

Effects of chronic alcohol exposure on neurogenesis in the subventricular zone (SVZ) and dentate gyrus (DG) of the hippocampus. Both control and alcohol-exposed mice received BrdU injection. (A) BrdU-positive cells in the DG and SVZ were determined by IHC. Arrows indicate BrdU-positive cells; bar = 50 μm. The expression of doublecortin (DCX) in the DG of the hippocampus and Ki67 in the SVZ were examined by IHC. Arrows indicate DCX- and Ki67-positive cells; bar = 20 μm. (B) The number of DCX-, BrdU-, and Ki67-positive cells in the DG and SVZ was quantified as described in the “Materials and Methods” section. The results were expressed as mean ± SEM, n = 3 or 4 for each group. *p < 0.05 denotes a statistically significant difference from the control group.