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Cholinergic alterations following alcohol exposure in the frontal cortex of Aldh2-deficient mice models
BR A IN RE S E A RCH 1 2 95 ( 20 0 9 ) 3 7 – 4 3
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Research Report
Cholinergic alterations following alcohol exposure in the frontal cortex of Aldh2-deficient mice models Mostofa Jamal a,⁎, Kiyoshi Ameno a , Takanori Miki b , Weihuan Wang a , Mitsuru Kumihashi a , Toyohi Isse c , Toshihiro Kawamoto c , Kyoko Kitagawa d , Keiichi Nakayama e , Iwao Ijiri a , Hiroshi Kinoshita a a
Department of Forensic Medicine, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki, Kita, Kagawa 761-0793, Japan Department of Anatomy and Neurobiology, Faculty of Medicine, Kagawa University, Japan c Department of Environmental Health, University of Occupational and Environmental Health, Fukuoka, Japan d Department of Biochemistry, Hamamatsu University School of Medicine, Japan e Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Japan b
A R T I C LE I N FO
AB S T R A C T
Article history:
We investigated the effects of alcohol (EtOH) and acetaldehyde (ACe) on choline
Accepted 29 July 2009
acetyltransferase (ChAT) and acetylcholinesterase (AChE) in the frontal cortex of Aldh2-/-
Available online 5 August 2009
(KO) mice. KO mice were used as models of Aldh2-deficient humans to examine ACe effects. Brain samples were analyzed at 40 and 120 min after 2- and 4-g/kg intraperitoneal EtOH
Keywords:
administration by RT-PCR and Western blot. Wild-type (WT) mice exhibited a remarkable
Alcohol
decrease in ChAT and AChE mRNA expression at both time points only after 4-g/kg EtOH
Acetaldehyde
treatment compared with the naive control, whereas KO mice showed a considerable
Cholinergic marker
reduction in cholinergic markers after 2- and 4-g/kg EtOH treatment. The 4-g/kg EtOH-
Frontal cortex
induced decrease in ChAT and AChE RNA expression at both time points was significantly
In vivo microdialysis
greater than that in obtained with the administration of 2-g/kg at 40 min in WT mice. KO
Aldh2-/- mice
mice showed a significant difference in ChAT mRNA at 40 min between the EtOH groups. The findings regarding the ChAT mRNA levels are consistent with the results of Western blot in both types of mice, with some exceptions. EtOH-induced ChAT and AChE expression in KO mice was significantly lower than that in WT mice. This genotype effect occurred mostly at 40 min after EtOH dosing. Only ACe was quantified in the brains of KO mice, whereas EtOH was detected in both types of mice in vivo. These results suggest that EtOH and ACe combined or high EtOH alone alters cholinergic markers expression via changes in presynaptic and postsynaptic processes in the mice frontal cortex, thus indicating that central cholinergic neurons may be sensitive to EtOH and ACe. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Acetaldehyde (ACe), a highly toxic by-product of alcohol (EtOH) metabolism, is converted to acetic acid mainly by aldehyde
dehydrogenase 2 (Aldh2) in the metabolic pathway of EtOH. The biochemistry of people from some genetic backgrounds inhibits the processing of ACe into acetate. For example, approximately half of Asians have a deficiency of the low-Km
⁎ Corresponding author. Fax: +81 87 891 2141. E-mail address: [email protected] (M. Jamal). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.07.099
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Aldh2 isoenzyme, a deficiency that may directly contribute to excess ACe accumulation and thus may affect brain chemistry after EtOH intake (Wall et al., 1997). Inactive Aldh2 offers a possible protection against alcoholism, due to the buildup of ACe in the blood during EtOH metabolism (Wall et al., 1997). EtOH exerts a variety of adverse effects in the peripheral and central nervous system (CNS). However, the EtOH-induced toxic effect could be the direct effect of either EtOH or ACe. Thus, ACe is regarded by certain authors as playing a major role in producing several pharmacological, behavioral, and neurotoxic effects of EtOH (Quertemont et al., 2004; Zuddas et al., 1989). The cholinergic system in the cerebral cortex has been shown to play an essential role in sleep, attention, learning, and memory (Marukia et al., 2003). Reduced expression of the cholinergic function is associated with deterioration of cognitive functioning of some neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and autism (Dani and Bertrand, 2007). EtOH can alter behavioral parameters, such as learning and memory, with these changes being related to changes in cholinergic function (Beracochea et al., 1992). ACe, on the other hand, is responsible for some of the deleterious effects of EtOH due to a perturbation of cholinergic neurotransmission in the CNS (Kuriyama et al., 1987). The Aldh2 gene can influence the accumulation of ACe, which may also play a role in the pathogenesis of Alzheimer's disease (Wang et al., 2008). EtOH-induced changes in cholinergic transmission have been found to be variable in mice brains, having no effect in some cases (Kuriyama et al., 1987; Hsu etal., 1983) and resulting in the elevation of choline acetyltransferase (ChAT) in others (Durkin et al., 1982). However, Aldh2-/(KO) mice have proven to be an effective model for understanding the effects of ACe on cholinergic parameters in the brain because a greater amount of ACe is generated in the blood and tissues of such mice after drinking EtOH (Isse et al., 2005).
A more recent study in our laboratory has shown that the alteration of cholinergic markers is mediated by EtOH and ACe in the rat brain (Jamal et al., 2007a). To date, no study has been carried out to investigate possible changes in cholinergic function in high ACe-producing mice models. Accordingly, the purpose of our current approach was to examine the involvement of cholinergic neurotransmission in mediating the dose-dependent effects of EtOH in KO mice as a model of Aldh2-deficient humans. The Aldh2-deficient mouse has null mitochondrial aldehyde oxidation activity in the liver but maintains a normal level of cytosolic aldehyde oxidation activity (Kitagawa et al., 2000). To this end, we used RT-PCR and Western blot for assessing ChAT, which is the most specific marker for cholinergic neurons in the mice frontal cortex. In addition, acetylcholinesterase (AChE), a relatively specific cholinergic marker, was assayed by RT-PCR. The activation and inhibition of ChAT and AChE in cholinergic neurons play a major role in the maintenance of acetylcholine (ACh) levels. EtOH and ACe were analyzed simultaneously in vivo in the frontal cortex of freely moving C57B2/6J (WT) and KO mice by microdialysis, followed by head-space gas chromatography (GC).
2.
Results
Representative RT-PCR results for ChAT and AChE mRNAs and Western blot results for ChAT protein in the frontal cortex of KO mice are shown in Fig. 1. The results show a significant reduction in ChAT and AChE mRNA and ChAT protein levels at both time points, as compared with the naive control. Fig. 2 shows the ChAT mRNA and protein expression at 40 and 120 min after 2- and 4-g/kg EtOH treatment in WT and KO mice. For ChAT mRNA in WT mice, statistical analysis using one-way ANOVA showed a significant main effect of groups [F(4,20) = 8.507; P < 0.001]. Tukey–Kramer's post hoc
Fig. 1 – (A, C) RT-PCR analysis of ChAT and AChE and (B, D) Western blot analysis of ChAT. Lane 1, naive control; lane 2, EtOH 2 g/kg; lane 3, EtOH 4 g/kg. β-Actin was used as an internal control for mRNA expression.
BR A IN RE S E A RCH 1 2 95 ( 20 0 9 ) 3 7 – 4 3
39
Fig. 2 – ChAT mRNA and protein expression at 40 and 120 min after EtOH treatment. (A, C) WT mice and (B, D) KO mice. Data represent mean ± SE (n = 5). *P < 0.05 and **P < 0.01 indicate a significance of difference (one-way ANOVA Tukey–Kramer test).
analysis revealed that only 4-g/kg EtOH treatment reduced ChAT mRNA levels significantly at both 40 (P < 0.01) and 120 (P < 0.05) min, as compared with the naive control. The decrease in ChAT expression at 40 (P < 0.01) and 120 (P < 0.01) min in the 4-g/kg EtOH group was significantly greater than that at 40 min in the 2-g/kg EtOH group. These findings are consistent with the results of the Western blot analysis. An exception was that the reduction in ChAT protein levels was significantly greater in the 4-g/kg than in the 2-g/kg EtOH group at 120 min (P < 0.01). For ChAT mRNA in KO mice, there was a significant main effect of groups [F(4,20) = 47.791; P < 0.001]. The post hoc Tukey test showed that 2-g/kg EtOH treatment resulted in ChAT mRNA levels being significantly reduced at both 40 (P < 0.01) and 120 (P < 0.01) min. Similarly, 4-g/kg EtOH treatment significantly decreased ChAT mRNAs at both 40 (P < 0.01) and 120 (P < 0.01) min. The 4-g/kg EtOHinduced decrease in ChAT mRNA expression at 40 min was significantly greater than that at 40 (P < 0.01) and 120 (P < 0.01) min in the 2-g/kg EtOH group and at 120 min in the 4-g/kg EtOH group (P < 0.01). Our findings are also consistent with the results of the Western blot analysis. An exception was that no significant difference was found in ChAT protein levels between the EtOH groups at each time point. Fig. 3 shows the AChE mRNA expression at 40 and 120 min after 2- and 4-g/kg EtOH treatment in WT and KO mice. For AChE mRNA in WT mice, statistical analysis using one-way ANOVA showed a significant main effect of groups [F(4,20) = 13.576; P < 0.001]. Tukey–Kramer's test revealed that only the 4-g/kg EtOH treatment significantly reduced AChE mRNA levels at both 40 (P < 0.01) and 120 (P < 0.05) min when compared to the naive control. The 4-g/kg EtOH-induced decrease in AChE mRNA expression at 40 (P < 0.01) and 120 (P < 0.01) min was significantly greater than that at 40 min in the 2-g/kg EtOH
group. For AChE mRNA in KO mice, there was a significant main effect of groups [F(4,20) = 5.955; P < 0.003]. The 2-g/kg EtOH group showed a considerable decrease in AChE mRNA levels at
Fig. 3 – AChE mRNA expression at 40 and 120 min after EtOH treatment. (A) WT mice and (B) KO mice. Data represent mean ± SE (n = 5). *P < 0.05 and **P < 0.01 indicate a significance of difference (one-way ANOVA Tukey–Kramer test).
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40 (P < 0.01) and 120 (P < 0.05) min. Similarly, the 4-g/kg EtOH treatment significantly decreased AChE mRNA at 40 (P < 0.05) and 120 (P < 0.01) min. No significant difference was seen in AChE mRNA levels between the EtOH groups at either of these time points. Fig. 4 shows a comparison of ChAT and AChE mRNAs and ChAT protein expression between WT and KO mice. No genotype effect on ChAT and AChE mRNAs and ChAT protein levels was seen in the naive control. KO mice exhibited significantly lower ChAT mRNA expression than WT mice at 40 min after 2- (P < 0.01) and 4-g/kg (P < 0.01) EtOH treatment. KO mice also presented significantly lower ChAT protein expression than WT mice at 40 min in the 2-g/kg EtOH group (P < 0.01) and at 120 min in the 2- (P < 0.01) and 4-g/kg (P < 0.01) EtOH groups. AChE mRNA expression was also found to be remarkably lower in KO mice (P < 0.01) than in WT mice at 40 min following 2-g/kg EtOH exposure. Neither type of mice showed a statistical difference in AChE mRNA expression at either of these time points in the 4-g/kg EtOH group. As shown in Table 1, the brain EtOH concentrations were approximately 2-fold lower in the 2-g/kg than in the 4-g/kg EtOH group at 40 and 120 min in both mice. EtOH levels were significantly lower in WT mice than KO mice at 120 min in both the 2- and 4-g/kg EtOH groups (P < 0.01). No significant difference in EtOH levels was observed between WT and KO mice at 40 min for either dose. The brain ACe concentrations were approximately 1.5-fold lower in the 2-g/kg than in the 4-
Fig. 4 – Effect of genotype on cholinergic marker expression. Data represent mean ± SE (n = 5). *P < 0.05 and **P < 0.01, for the differences between WT and KO mice. C, naive control.
Table 1 – EtOH and ACe concentrations in the frontal cortex of WT and KO mice in vivo. EtOH (i.p.) 2g/kg
Time after Strain EtOH (mM) ACe (μM) EtOH injection 40 min 120 min
4g/kg
40 min 120 min
KO WT KO WT KO WT KO WT
24.7 22.6 17.9 10.2 51.3 47.3 38.3 22.7
± ± ± ± ± ± ± ±
3.5 3.2 2.3 1.9⁎⁎ 5.4 4.8 1.7 2.2⁎⁎
28.4 ND 24.6 ND 41.4 ND 36.5 ND
± 3.1 ± 3.4 ± 4.6 ± 3.5
Data represent mean ± SE (n = 5). ⁎⁎P < 0.01, for the differences between WT and KO mice. ND, not detected.
g/kg EtOH group at both time points in KO mice. No significant difference was observed for ACe concentrations at 120 min, as compared to that at 40 min following exposure to EtOH of either dose. Neither 2- nor 4-g/kg EtOH treatment produced a detectable level of ACe at either time point in the brains of WT mice.
3.
Discussion
We carried out this study to investigate how genetic deficiency in Aldh2 affects the cholinergic neurons in the mouse frontal cortex following EtOH consumption. Brain ACe levels may be important in determining the effects of EtOH in the brain. We therefore chose genetically KO mice as models, since the deficiency may lead to a build-up of ACe levels in the brain after EtOH intake. One important and novel finding of this study is that KO mice demonstrated a decrease in both ChAT and AChE expression after 2- and 4-g/kg EtOH exposure. Another is that the 4-g/kg EtOH treatment in WT mice had the same profound effect in lowering the ChAT and AChE levels. These data, therefore, confirm our previous observations by demonstrating that EtOH and ACe exposure can decrease cholinergic marker expression in the rat brain (Jamal et al., 2007a). A number of studies have demonstrated EtOH and ACe concentrations in the mouse brain, but the majority of these works have employed brain homogenates (Isse et al., 2005; Zimatkin et al., 2006). While a few researchers have investigated cholinergic function after acute exposure to EtOH in the mouse brain, their results have not been consistent (Hsu et al., 1983; Durkin et al., 1982). This is the first report of simultaneous determination of both EtOH and ACe concentrations in vivo and cholinergic alterations in the frontal cortex of WT and KO mice following acute EtOH exposure. The in vivo microdialysis data revealed that frontal cortical EtOH concentrations in KO mice were approximately 2-fold lower in the 2-g/kg EtOH group compared to the 4-g/kg EtOH group (Table 1). EtOH concentrations in WT mice were found to be almost equal to those of KO mice at 40 min, although there was a tendency to decrease. These observations are correlated with those reported previously (Isse et al., 2005) and suggest that EtOH readily penetrates the mouse brain. Significantly lower levels of EtOH were seen at 120 min in
BR A IN RE S E A RCH 1 2 95 ( 20 0 9 ) 3 7 – 4 3
WT mice than in KO mice with both 2- and 4-g/kg EtOH treatments. One possible cause of this phenomenon is the faster rate of EtOH elimination in WT mice, in agreement with the previous report (Zimatkin and Buben, 2007). We next sought to determine the ACe concentrations in the frontal cortex of both mice in vivo. The brain ACe concentrations after 2-g/kg EtOH treatment in KO mice were approximately 1.5-fold lower than those in the 4-g/kg EtOH group. However, no significant difference was seen in ACe concentrations between 40 and 120 min for either dose of EtOH. In contrast, WT mice quickly eliminated ACe and showed concentrations below the detection limit at both time points in the 2-and 4-g/kg EtOH groups, which is in accordance with our previous work (Jamal et al., 2005). A previous study with KO mice found a large accumulation of ACe after EtOH intake by oral gavage (Isse et al., 2005). In fact, Aldh2 seems to play an important role in most of the ACe breakdown during EtOH metabolism, particularly in the liver (Bosron and Li, 1986). High peripheral ACe is able to cross the brain through the blood–brain barrier and to accumulate in the brain (Jamal et al., 2007b). Our results, however, indicate that Aldh2 deficiency leads to higher levels of brain ACe after acute exposure to EtOH. Furthermore, detection of EtOH and ACe was found in the present study to be dose-dependent in both WT and KO mice. Thus, the data do not rule out the possibility of the involvement other brain enzymes such as catalase and CYP2E1 (Zimatkin et al, 2006) in ACe formation. Further study is needed to confirm this conjecture. In order to verify whether the ChAT and AChE genes can be modulated when KO mice are exposed to EtOH, we performed RT-PCR and Western blot, as shown in Figs. 2 and 3. The study revealed that 2- and 4-g/kg EtOH treatment reduced ChAT and AChE mRNA levels significantly at 40 and 120 min in KO mice, while EtOH and ACe concentrations reached a maximum at 40 min followed by a decrease at 120 min. The findings regarding ChAT mRNA expression are consistent with the results of the Western blot analysis. Furthermore, decrease in ChAT mRNA, but not in protein levels, after EtOH treatment occurred in a time- and dose-dependent manner. No significant difference in the levels of AChE mRNA was noted between EtOH doses at any time point. These findings correlated with those in a previous report indicating that ACe induces a significant reduction in ChAT and AChE activity in the mouse brain (Kuriyama et al., 1987). Therefore, our data support the conclusion that combined exposure to both EtOH and ACe can alter cholinergic markers in the frontal cortex, through perturbation of both the presynaptic and postsynaptic cholinergic processes in the cortical cholinergic pathways. On the other hand, only 4-g/kg EtOH treatment in WT mice reduced the ChAT and AChE mRNA and ChAT protein levels at both 40 and 120 min, as shown in Figs. 2 and 3. The high EtOHinduced decrease in cholinergic marker expression was significantly greater than that observed at 2-g/kg EtOH. High EtOH concentrations were found after 4-g/kg EtOH exposure, while ACe was undetectable in the brain. A previous study has demonstrated a significant decrease in ChAT and AChE activity in the mouse cerebral cortex after acute exposure to 2-g/kg EtOH (Owasoyo and Iramain, 1981). Our present study revealed a significant decrease in both ChAT and AChE levels
41
only after high EtOH exposure. This discrepancy might be due to the mice strains and their genotype variations, which may have contributed to the difference in the sensitivity of the cholinergic function to EtOH (Hashemzadeh-Gargari and Mandel, 1989). The present data, however, demonstrate that high EtOH levels can also alter the central cholinergic parameters, via an alteration in both the presynaptic and postsynaptic process in the cortical cholinergic pathways. Indeed, perturbation of any one of these pathways is predicted to alter ACh release in cholinergic neurons. Our findings have demonstrated a profound effect of genotype on ChAT and AChE expression after EtOH treatment, as shown in Fig. 4. KO mice exhibited significantly lower ChAT, AChE mRNA, and ChAT protein levels than WT mice. Interestingly, that effect occurred mostly at 40 min after EtOH dosing, while both EtOH and ACe concentrations reached peak. There was no significant genotype effect on the levels of ChAT and AChE in the naive control. This result may indicate that KO mice are more sensitive than WT mice to EtOH-induced cholinergic alterations (Oyama et al., 2007). The enhanced sensitivity to EtOH in KO mice is consistent with previous findings (Fernandez et al., 2006). In addition, KO mice may be more susceptible than WT mice to EtOHinduced oxidative DNA damage (Kim et al., 2007). It is, however, conceivable that increased ACe levels in KO mice may contribute to a greater impairment of cholinergic markers expression. In summary, mice with Aldh2 deficiency exhibited a significant reduction in frontal cortex ChAT and AChE levels following 2- and 4-g/kg EtOH treatment, whereas WT mice showed a remarkable reduction in ChAT and AChE expression only after 4-g/kg EtOH. Moreover, there was a significant difference in cholinergic marker expression between genotypes after EtOH treatment. ACe was found only in the brains of KO mice, whereas EtOH was detected in both kinds of mice. These results clearly indicate that ACe may have a strong toxic effect on cortical cholinergic neurons in mice. Clearly, EtOH and ACe combined or high EtOH alone can alter cholinergic marker expression in the mouse frontal cortex.
4.
Experimental procedures
4.1.
Animals
KO mice were generated as reported previously (Kitagawa et al., 2000). They were obtained from the Department of Environmental Health, University of Occupational and Environmental Health, Japan, and then bred at the Kagawa University animal building. Male WT mice were purchased from CLEA Japan (Tokyo, Japan). All mice were housed in plastic cages with free access to standard laboratory food and water. Male mice were used at 10–12 weeks of age throughout this study, and weighed 24–28 g. The animal rooms were controlled for temperature (21 ± 3 °C), humidity (50%–70%), and light (12-h light–dark cycle). The Animal Investigation Committee of Kagawa University approved all experimental procedures and animal care involved in these studies.
42 4.2.
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Experimental groups
Animals (KO and WT mice) were each divided into three experimental groups: (a) naive control, (b) EtOH (2 g/kg), and (c) EtOH (4 g/kg). Mice (n = 5 per group) received an intraperitoneal (i.p.) injection of EtOH (2- and 4-g/kg body weight, 20% [v/v]) and the samples were collected at 40 and 120 min after the injection.
4.3.
Gene
Sequences 5′→3′
Size (bp)
ChAT
F-TACAGGCTTTACCAGAGACTGGTG R-AACTGGAGATGCAGAAGGTGATGG F-TACTCTGGACGAGGCG R-CGGTCGTATTATATCCCAGC F-TCATGAAGTGTGACGTTGACATCCGTAAAG R-CCTAGAAGCATTTGCGGTGCACGATGGAGG
433
AChE β-actin
Brain tissues
Mice were deeply anesthetized with diethyl ether inhalation and killed by decapitation. Frontal cortex (approximately 50 mg) were collected on ice and then stored at −70 °C until further assay.
285
1 μL of 10 mM dNTP-mix, 0.5 μL of recombinant RNase inhibitor, and 1 μL of MMLV reverse transcriptase to a final volume of 20 μL. Total RNA was preincubated for 2 min at 70 °C prior to cDNA synthesis. The reverse transcription reaction was carried out for 60 min at 42 °C, followed by 5 min at 94 °C. The primer pairs used in the PCR were as shown in Table 2. PCR was performed in a final volume of 50 μL containing 2 μL of cDNA, 10 pmol of each primer, 0.5 μL of AmpliTaq DNA polymerase, and 14 μL of buffer containing 10 × PCR buffer, dNTP mix (10 mM), and MgCl2. Cycle parameters for ChAT included initial denaturation for 5 min at 94 °C, denaturation for 1 min at 94 °C, annealing for 1 min at 55 °C, and extension for 2 min at 72 °C, followed by a final extension for 3 min at 72 °C for 38 cycles. For AChE, initial denaturation was carried out for 2 min at 94 °C, denaturation for 1 min at 94 °C, annealing for 1 min at 55 °C, and extension for 1 min at 72 °C, followed by a final extension for 7 min at 72 °C for 35 cycles. For β-actin, initial denaturation was carried out for 5 min at 94 °C, denaturation for 1 min at 94 °C, annealing for 1 min at 54 °C, and extension for 2 min at 72 °C, followed by a final extension for 3 min at 72 °C for 24 cycles. PCR products were separated in a 2% agarose gel stained with ethidium bromide. Intensities in each band resulting from PCR amplification were analyzed by an LAS-1000plus luminoimaging analyzer (Fujix, Tokyo, Japan). The relative values were calculated by dividing the densitometric signals for ChAT and AChE by the signal obtained with the internal standard β-actin.
4.6. 4.5.
210
Microdialysis and head-space GC conditions
The microdialysis procedure and head-space GC (PerkinElmer, Norwalk, CT, USA) conditions were as previously described (Jamal et al., 2007b). In short, mice were implanted stereotaxically with a microdialysis probe (8 mm long, Eicom, Japan) with a 3-mm-long active dialysis membrane into the prefrontal cortex (coordinates relative from bregma: anterior, 2.0 mm; lateral, 1.0 mm; height, 3.0 mm) according to the atlas of Paxinos and Franklin (Paxinos and Franklin, 2001 [34]. One day after surgery, the experiments were begun. The probe was continuously perfused with Ringer's solution (147 mM NaCl, 4 mM KCl, 2.25 mM CaCl2) at a constant rate of 1 μL/min via syringe pump. Samples were collected at different time points for 10 min each after EtOH injection into vials containing 50 μL 0.002% t-butanol as an internal standard in 0.6 N perchloric acid and were then analyzed by head-space GC. The column, injector, and detector temperatures for the chromatography were 90, 110 and 200 °C, respectively. The separation column was a Supelcowax wide-bore capillary column (60 m length, 0.53 mm i.d., 2 μm film thickness, Supelco, Bellefonte, PA, USA). Nitrogen was used as the carrier gas at a flow rate of 20 mL/min. No artifactual ACe was detectable in the dialysate by this procedure. In vitro probe recovery of EtOH and ACe was 72.2% ± 3.6% and 52.6% ± 5.9%, respectively, and the values were corrected.
4.4.
Table 2 – Primer sequences used for the RT-PCR; F, forward; R, reverse; bp, base pair.
Western blot
RNA isolation and RT-PCR
Total RNA from the frontal cortex was extracted using 1.5 mL of ISOGEN (Wako Pure Chemical Industries, Japan), as described previously (Jamal et al., 2007a). The concentrations and purity of RNA samples were determined using a Pharmacia GeneQuant RNA/DNA spectrophotometer at a wavelength of 260 nm, and the integrity was confirmed by electrophoresis through 2% agarose gels stained with ethidium bromide. All samples were stored at −70 °C until analysis by RT-PCR. An 11-μg amount of each total RNA was used as a template for reverse transcription using 1 μL of oligo (dT)18 primer (Clontech, Takara Bio Com., Japan) in an appropriate amount of DEPC-treated water to a final volume of 13.5 μL. The synthesis reaction contained 4 μL of 5 × reaction buffer,
Frontal cortex was homogenized in 20 volumes (wt/vol) of 20 mM ice-cold Tris–HCl (pH 7.4) containing 0.32 mM sucrose as outlined previously (Jamal et al., 2007a). After centrifugation at 10,000 × g at 4 °C for 15 min, the supernatant was used for Western blot analyses. The protein content of the supernatant was determined by Bradford (Bio-Rad, Hercules, CA) using bovine serum albumin (Sigma Chemical, St. Louis, MO) as the standard. Samples were run on 10% SDS PAGE with molecular weight markers (Bio-Rad), then transferred electrophoretically to PVDF membranes. Goat anti-ChAT antiserum (AB144p, polyclonal; diluted 1:500, Chemicon International Inc., Temecula, CA, USA) and horseradish peroxidase-linked anti-goat IgG antibody were used for protein detection. The band intensities were evaluated by an LAS-1000plus lumino-imaging analyzer (Fujix).
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4.7.
Statistics
The data were analyzed by appropriate analysis of variance (ANOVA) procedures. Post hoc tests were carried out where appropriate using Tukey–Kramer's test. The Student's t-test was used to assess significant differences in cholinergic marker expression, and EtOH and ACe concentrations between genotypes. All statistics were carried out using SigmaStat (Systat, version 3.0) software on an IBM-compatible computer.
Acknowledgments This work was supported in part by the Grant-in-Aid for Scientific Research [Grand No. (c) 18590637, 20590681] from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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metabolism decreases acetylcholine release in medial frontal cortex of freely moving rats. Brain Res. 1039, 90–96. Jamal, M., Ameno, K., Ameno, S., Morishita, J., Wang, W., Kumihashi, M., Ikuo, U., Miki, T., Ijiri, I., 2007a. Changes in cholinergic function in the frontal cortex and hippocampus of rat exposed to ethanol and acetaldehyde. Neuroscience 144, 232–238. Jamal, M., Ameno, K., Uekita, I., Kumihashi, M., Wang, W., Ijiri, I., 2007b. Catalase mediates acetaldehyde formation in the striatum of free-moving rats. Neurotoxicology 28, 1245–1248. Kim, Y.D., Eom, S.Y., Ogawa, M., Oyama, T., Isse, T., Kang, J.W., Zhang, Y.W., Kawamoto, T., Kim, H., 2007. Ethanol-induced oxidative DNA damage and CYP2E1 expression in liver tissue of Aldh2 knockout mice. J. Occup. Health 49, 363–369. Kitagawa, K., Kawamoto, T., Kunugita, N., Tsukiyama, T., Okamoto, K., Yoshida, A., Nakayama, K., Nakayama, K., 2000. Aldehyde dehydrogenase (ALDH) 2 associates with oxidation of methoxyacetaldehyde; in vitro analysis with liver subcellular fraction derived from human and Aldh2 gene targeting mouse. FEBS Lett. 47, 6306–6311. Kuriyama, K., Ohkuma, S., Tomono, S., Hirouchi, M., 1987. Effects of alcohol and acetaldehyde on metabolism and function of neurotransmitter systems in cerebral cortical neurons in primary culture. Alcohol Alcohol 1 (Suppl.), S685–S689. Marukia, K., Izakic, Y., Akemac, T., Nomura, M., 2003. Effects of acetylcholine antagonist injection into the prefrontal cortex on the progress of lever-press extinction in rats. Neurosci. Lett. 351, 95–98. Owasoyo, J.O., Iramain, C.A., 1981. Effect of acute ethanol intoxication on the enzymes of the cholinergic system in mouse brain. Toxicol. Lett. 9, 267–270. Oyama, T., Isse, T., Ogawa, M., Muto, M., Uchiyama, I., Kawamoto, T., 2007. Susceptibility to inhalation toxicity of acetaldehyde in Aldh2 knockout mice. Front. Biosci. 12, 1927–1934. Paxinos, G., Franklin, K.B.J., 2001. The Mouse Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, San Diego, CA. Quertemont, E., Tambour, S., Bernaerts, P., Zimatkin, SM., Tirelli, E., 2004. Behavioral characterization of acetaldehyde in C57BL/ 6J mice: locomotor, hypnotic, anxiolytic and amnesic effects. Psychopharmacology 177, 84–92. Wall, T.L., Peterson, C.M., Peterson, K.P., Johnson, M.L., Thomasson, H.R., Cole, M., Ehlers, C.L., 1997. Alcohol metabolism in Asian-American men with genetic polymorphisms of aldehyde dehydrogenase. Anal. Int. Med. 127, 376–379. Wang, B., Wang, J., Zhou, S., Tan, S., He, X., Yang, Z., Xie, Y.C., Li, S., Zheng, C., Ma, X., 2008. The association of mitochondrial aldehyde dehydrogenase gene (ALDH2) polymorphism with susceptibility to late-onset Alzheimer's disease in Chinese. J. Neurol. Sci. 268, 172–175. Zimatkin, S.M., Pronko, S.P., Vasiliou, V., Gonzalez, F.J., Deitrich, R.A., 2006. Enzymatic mechanisms of ethanol oxidation in the brain. Alcohol Clin. Exp. Res. 30, 1500–1505. Zimatkin, S.M., Buben, A.L., 2007. Ethanol oxidation in the living brain. Alcohol Alcohol 42, 529–532. Zuddas, A., Corsini, G.U., Schinelli, S., Barker, J.L., Kopin, I.J., di Porzio, U., 1989. Acetaldehyde directly enhances MPP+ neurotoxicity and delays its elimination from the striatum. Brain Res. 501, 11–22.
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Research Report
Cholinergic alterations following alcohol exposure in the frontal cortex of Aldh2-deficient mice models Mostofa Jamal a,⁎, Kiyoshi Ameno a , Takanori Miki b , Weihuan Wang a , Mitsuru Kumihashi a , Toyohi Isse c , Toshihiro Kawamoto c , Kyoko Kitagawa d , Keiichi Nakayama e , Iwao Ijiri a , Hiroshi Kinoshita a a
Department of Forensic Medicine, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki, Kita, Kagawa 761-0793, Japan Department of Anatomy and Neurobiology, Faculty of Medicine, Kagawa University, Japan c Department of Environmental Health, University of Occupational and Environmental Health, Fukuoka, Japan d Department of Biochemistry, Hamamatsu University School of Medicine, Japan e Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Japan b
A R T I C LE I N FO
AB S T R A C T
Article history:
We investigated the effects of alcohol (EtOH) and acetaldehyde (ACe) on choline
Accepted 29 July 2009
acetyltransferase (ChAT) and acetylcholinesterase (AChE) in the frontal cortex of Aldh2-/-
Available online 5 August 2009
(KO) mice. KO mice were used as models of Aldh2-deficient humans to examine ACe effects. Brain samples were analyzed at 40 and 120 min after 2- and 4-g/kg intraperitoneal EtOH
Keywords:
administration by RT-PCR and Western blot. Wild-type (WT) mice exhibited a remarkable
Alcohol
decrease in ChAT and AChE mRNA expression at both time points only after 4-g/kg EtOH
Acetaldehyde
treatment compared with the naive control, whereas KO mice showed a considerable
Cholinergic marker
reduction in cholinergic markers after 2- and 4-g/kg EtOH treatment. The 4-g/kg EtOH-
Frontal cortex
induced decrease in ChAT and AChE RNA expression at both time points was significantly
In vivo microdialysis
greater than that in obtained with the administration of 2-g/kg at 40 min in WT mice. KO
Aldh2-/- mice
mice showed a significant difference in ChAT mRNA at 40 min between the EtOH groups. The findings regarding the ChAT mRNA levels are consistent with the results of Western blot in both types of mice, with some exceptions. EtOH-induced ChAT and AChE expression in KO mice was significantly lower than that in WT mice. This genotype effect occurred mostly at 40 min after EtOH dosing. Only ACe was quantified in the brains of KO mice, whereas EtOH was detected in both types of mice in vivo. These results suggest that EtOH and ACe combined or high EtOH alone alters cholinergic markers expression via changes in presynaptic and postsynaptic processes in the mice frontal cortex, thus indicating that central cholinergic neurons may be sensitive to EtOH and ACe. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Acetaldehyde (ACe), a highly toxic by-product of alcohol (EtOH) metabolism, is converted to acetic acid mainly by aldehyde
dehydrogenase 2 (Aldh2) in the metabolic pathway of EtOH. The biochemistry of people from some genetic backgrounds inhibits the processing of ACe into acetate. For example, approximately half of Asians have a deficiency of the low-Km
⁎ Corresponding author. Fax: +81 87 891 2141. E-mail address: [email protected] (M. Jamal). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.07.099
38
BR A IN RE S EA RCH 1 2 95 ( 20 0 9 ) 3 7 – 43
Aldh2 isoenzyme, a deficiency that may directly contribute to excess ACe accumulation and thus may affect brain chemistry after EtOH intake (Wall et al., 1997). Inactive Aldh2 offers a possible protection against alcoholism, due to the buildup of ACe in the blood during EtOH metabolism (Wall et al., 1997). EtOH exerts a variety of adverse effects in the peripheral and central nervous system (CNS). However, the EtOH-induced toxic effect could be the direct effect of either EtOH or ACe. Thus, ACe is regarded by certain authors as playing a major role in producing several pharmacological, behavioral, and neurotoxic effects of EtOH (Quertemont et al., 2004; Zuddas et al., 1989). The cholinergic system in the cerebral cortex has been shown to play an essential role in sleep, attention, learning, and memory (Marukia et al., 2003). Reduced expression of the cholinergic function is associated with deterioration of cognitive functioning of some neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and autism (Dani and Bertrand, 2007). EtOH can alter behavioral parameters, such as learning and memory, with these changes being related to changes in cholinergic function (Beracochea et al., 1992). ACe, on the other hand, is responsible for some of the deleterious effects of EtOH due to a perturbation of cholinergic neurotransmission in the CNS (Kuriyama et al., 1987). The Aldh2 gene can influence the accumulation of ACe, which may also play a role in the pathogenesis of Alzheimer's disease (Wang et al., 2008). EtOH-induced changes in cholinergic transmission have been found to be variable in mice brains, having no effect in some cases (Kuriyama et al., 1987; Hsu etal., 1983) and resulting in the elevation of choline acetyltransferase (ChAT) in others (Durkin et al., 1982). However, Aldh2-/(KO) mice have proven to be an effective model for understanding the effects of ACe on cholinergic parameters in the brain because a greater amount of ACe is generated in the blood and tissues of such mice after drinking EtOH (Isse et al., 2005).
A more recent study in our laboratory has shown that the alteration of cholinergic markers is mediated by EtOH and ACe in the rat brain (Jamal et al., 2007a). To date, no study has been carried out to investigate possible changes in cholinergic function in high ACe-producing mice models. Accordingly, the purpose of our current approach was to examine the involvement of cholinergic neurotransmission in mediating the dose-dependent effects of EtOH in KO mice as a model of Aldh2-deficient humans. The Aldh2-deficient mouse has null mitochondrial aldehyde oxidation activity in the liver but maintains a normal level of cytosolic aldehyde oxidation activity (Kitagawa et al., 2000). To this end, we used RT-PCR and Western blot for assessing ChAT, which is the most specific marker for cholinergic neurons in the mice frontal cortex. In addition, acetylcholinesterase (AChE), a relatively specific cholinergic marker, was assayed by RT-PCR. The activation and inhibition of ChAT and AChE in cholinergic neurons play a major role in the maintenance of acetylcholine (ACh) levels. EtOH and ACe were analyzed simultaneously in vivo in the frontal cortex of freely moving C57B2/6J (WT) and KO mice by microdialysis, followed by head-space gas chromatography (GC).
2.
Results
Representative RT-PCR results for ChAT and AChE mRNAs and Western blot results for ChAT protein in the frontal cortex of KO mice are shown in Fig. 1. The results show a significant reduction in ChAT and AChE mRNA and ChAT protein levels at both time points, as compared with the naive control. Fig. 2 shows the ChAT mRNA and protein expression at 40 and 120 min after 2- and 4-g/kg EtOH treatment in WT and KO mice. For ChAT mRNA in WT mice, statistical analysis using one-way ANOVA showed a significant main effect of groups [F(4,20) = 8.507; P < 0.001]. Tukey–Kramer's post hoc
Fig. 1 – (A, C) RT-PCR analysis of ChAT and AChE and (B, D) Western blot analysis of ChAT. Lane 1, naive control; lane 2, EtOH 2 g/kg; lane 3, EtOH 4 g/kg. β-Actin was used as an internal control for mRNA expression.
BR A IN RE S E A RCH 1 2 95 ( 20 0 9 ) 3 7 – 4 3
39
Fig. 2 – ChAT mRNA and protein expression at 40 and 120 min after EtOH treatment. (A, C) WT mice and (B, D) KO mice. Data represent mean ± SE (n = 5). *P < 0.05 and **P < 0.01 indicate a significance of difference (one-way ANOVA Tukey–Kramer test).
analysis revealed that only 4-g/kg EtOH treatment reduced ChAT mRNA levels significantly at both 40 (P < 0.01) and 120 (P < 0.05) min, as compared with the naive control. The decrease in ChAT expression at 40 (P < 0.01) and 120 (P < 0.01) min in the 4-g/kg EtOH group was significantly greater than that at 40 min in the 2-g/kg EtOH group. These findings are consistent with the results of the Western blot analysis. An exception was that the reduction in ChAT protein levels was significantly greater in the 4-g/kg than in the 2-g/kg EtOH group at 120 min (P < 0.01). For ChAT mRNA in KO mice, there was a significant main effect of groups [F(4,20) = 47.791; P < 0.001]. The post hoc Tukey test showed that 2-g/kg EtOH treatment resulted in ChAT mRNA levels being significantly reduced at both 40 (P < 0.01) and 120 (P < 0.01) min. Similarly, 4-g/kg EtOH treatment significantly decreased ChAT mRNAs at both 40 (P < 0.01) and 120 (P < 0.01) min. The 4-g/kg EtOHinduced decrease in ChAT mRNA expression at 40 min was significantly greater than that at 40 (P < 0.01) and 120 (P < 0.01) min in the 2-g/kg EtOH group and at 120 min in the 4-g/kg EtOH group (P < 0.01). Our findings are also consistent with the results of the Western blot analysis. An exception was that no significant difference was found in ChAT protein levels between the EtOH groups at each time point. Fig. 3 shows the AChE mRNA expression at 40 and 120 min after 2- and 4-g/kg EtOH treatment in WT and KO mice. For AChE mRNA in WT mice, statistical analysis using one-way ANOVA showed a significant main effect of groups [F(4,20) = 13.576; P < 0.001]. Tukey–Kramer's test revealed that only the 4-g/kg EtOH treatment significantly reduced AChE mRNA levels at both 40 (P < 0.01) and 120 (P < 0.05) min when compared to the naive control. The 4-g/kg EtOH-induced decrease in AChE mRNA expression at 40 (P < 0.01) and 120 (P < 0.01) min was significantly greater than that at 40 min in the 2-g/kg EtOH
group. For AChE mRNA in KO mice, there was a significant main effect of groups [F(4,20) = 5.955; P < 0.003]. The 2-g/kg EtOH group showed a considerable decrease in AChE mRNA levels at
Fig. 3 – AChE mRNA expression at 40 and 120 min after EtOH treatment. (A) WT mice and (B) KO mice. Data represent mean ± SE (n = 5). *P < 0.05 and **P < 0.01 indicate a significance of difference (one-way ANOVA Tukey–Kramer test).
40
BR A IN RE S EA RCH 1 2 95 ( 20 0 9 ) 3 7 – 43
40 (P < 0.01) and 120 (P < 0.05) min. Similarly, the 4-g/kg EtOH treatment significantly decreased AChE mRNA at 40 (P < 0.05) and 120 (P < 0.01) min. No significant difference was seen in AChE mRNA levels between the EtOH groups at either of these time points. Fig. 4 shows a comparison of ChAT and AChE mRNAs and ChAT protein expression between WT and KO mice. No genotype effect on ChAT and AChE mRNAs and ChAT protein levels was seen in the naive control. KO mice exhibited significantly lower ChAT mRNA expression than WT mice at 40 min after 2- (P < 0.01) and 4-g/kg (P < 0.01) EtOH treatment. KO mice also presented significantly lower ChAT protein expression than WT mice at 40 min in the 2-g/kg EtOH group (P < 0.01) and at 120 min in the 2- (P < 0.01) and 4-g/kg (P < 0.01) EtOH groups. AChE mRNA expression was also found to be remarkably lower in KO mice (P < 0.01) than in WT mice at 40 min following 2-g/kg EtOH exposure. Neither type of mice showed a statistical difference in AChE mRNA expression at either of these time points in the 4-g/kg EtOH group. As shown in Table 1, the brain EtOH concentrations were approximately 2-fold lower in the 2-g/kg than in the 4-g/kg EtOH group at 40 and 120 min in both mice. EtOH levels were significantly lower in WT mice than KO mice at 120 min in both the 2- and 4-g/kg EtOH groups (P < 0.01). No significant difference in EtOH levels was observed between WT and KO mice at 40 min for either dose. The brain ACe concentrations were approximately 1.5-fold lower in the 2-g/kg than in the 4-
Fig. 4 – Effect of genotype on cholinergic marker expression. Data represent mean ± SE (n = 5). *P < 0.05 and **P < 0.01, for the differences between WT and KO mice. C, naive control.
Table 1 – EtOH and ACe concentrations in the frontal cortex of WT and KO mice in vivo. EtOH (i.p.) 2g/kg
Time after Strain EtOH (mM) ACe (μM) EtOH injection 40 min 120 min
4g/kg
40 min 120 min
KO WT KO WT KO WT KO WT
24.7 22.6 17.9 10.2 51.3 47.3 38.3 22.7
± ± ± ± ± ± ± ±
3.5 3.2 2.3 1.9⁎⁎ 5.4 4.8 1.7 2.2⁎⁎
28.4 ND 24.6 ND 41.4 ND 36.5 ND
± 3.1 ± 3.4 ± 4.6 ± 3.5
Data represent mean ± SE (n = 5). ⁎⁎P < 0.01, for the differences between WT and KO mice. ND, not detected.
g/kg EtOH group at both time points in KO mice. No significant difference was observed for ACe concentrations at 120 min, as compared to that at 40 min following exposure to EtOH of either dose. Neither 2- nor 4-g/kg EtOH treatment produced a detectable level of ACe at either time point in the brains of WT mice.
3.
Discussion
We carried out this study to investigate how genetic deficiency in Aldh2 affects the cholinergic neurons in the mouse frontal cortex following EtOH consumption. Brain ACe levels may be important in determining the effects of EtOH in the brain. We therefore chose genetically KO mice as models, since the deficiency may lead to a build-up of ACe levels in the brain after EtOH intake. One important and novel finding of this study is that KO mice demonstrated a decrease in both ChAT and AChE expression after 2- and 4-g/kg EtOH exposure. Another is that the 4-g/kg EtOH treatment in WT mice had the same profound effect in lowering the ChAT and AChE levels. These data, therefore, confirm our previous observations by demonstrating that EtOH and ACe exposure can decrease cholinergic marker expression in the rat brain (Jamal et al., 2007a). A number of studies have demonstrated EtOH and ACe concentrations in the mouse brain, but the majority of these works have employed brain homogenates (Isse et al., 2005; Zimatkin et al., 2006). While a few researchers have investigated cholinergic function after acute exposure to EtOH in the mouse brain, their results have not been consistent (Hsu et al., 1983; Durkin et al., 1982). This is the first report of simultaneous determination of both EtOH and ACe concentrations in vivo and cholinergic alterations in the frontal cortex of WT and KO mice following acute EtOH exposure. The in vivo microdialysis data revealed that frontal cortical EtOH concentrations in KO mice were approximately 2-fold lower in the 2-g/kg EtOH group compared to the 4-g/kg EtOH group (Table 1). EtOH concentrations in WT mice were found to be almost equal to those of KO mice at 40 min, although there was a tendency to decrease. These observations are correlated with those reported previously (Isse et al., 2005) and suggest that EtOH readily penetrates the mouse brain. Significantly lower levels of EtOH were seen at 120 min in
BR A IN RE S E A RCH 1 2 95 ( 20 0 9 ) 3 7 – 4 3
WT mice than in KO mice with both 2- and 4-g/kg EtOH treatments. One possible cause of this phenomenon is the faster rate of EtOH elimination in WT mice, in agreement with the previous report (Zimatkin and Buben, 2007). We next sought to determine the ACe concentrations in the frontal cortex of both mice in vivo. The brain ACe concentrations after 2-g/kg EtOH treatment in KO mice were approximately 1.5-fold lower than those in the 4-g/kg EtOH group. However, no significant difference was seen in ACe concentrations between 40 and 120 min for either dose of EtOH. In contrast, WT mice quickly eliminated ACe and showed concentrations below the detection limit at both time points in the 2-and 4-g/kg EtOH groups, which is in accordance with our previous work (Jamal et al., 2005). A previous study with KO mice found a large accumulation of ACe after EtOH intake by oral gavage (Isse et al., 2005). In fact, Aldh2 seems to play an important role in most of the ACe breakdown during EtOH metabolism, particularly in the liver (Bosron and Li, 1986). High peripheral ACe is able to cross the brain through the blood–brain barrier and to accumulate in the brain (Jamal et al., 2007b). Our results, however, indicate that Aldh2 deficiency leads to higher levels of brain ACe after acute exposure to EtOH. Furthermore, detection of EtOH and ACe was found in the present study to be dose-dependent in both WT and KO mice. Thus, the data do not rule out the possibility of the involvement other brain enzymes such as catalase and CYP2E1 (Zimatkin et al, 2006) in ACe formation. Further study is needed to confirm this conjecture. In order to verify whether the ChAT and AChE genes can be modulated when KO mice are exposed to EtOH, we performed RT-PCR and Western blot, as shown in Figs. 2 and 3. The study revealed that 2- and 4-g/kg EtOH treatment reduced ChAT and AChE mRNA levels significantly at 40 and 120 min in KO mice, while EtOH and ACe concentrations reached a maximum at 40 min followed by a decrease at 120 min. The findings regarding ChAT mRNA expression are consistent with the results of the Western blot analysis. Furthermore, decrease in ChAT mRNA, but not in protein levels, after EtOH treatment occurred in a time- and dose-dependent manner. No significant difference in the levels of AChE mRNA was noted between EtOH doses at any time point. These findings correlated with those in a previous report indicating that ACe induces a significant reduction in ChAT and AChE activity in the mouse brain (Kuriyama et al., 1987). Therefore, our data support the conclusion that combined exposure to both EtOH and ACe can alter cholinergic markers in the frontal cortex, through perturbation of both the presynaptic and postsynaptic cholinergic processes in the cortical cholinergic pathways. On the other hand, only 4-g/kg EtOH treatment in WT mice reduced the ChAT and AChE mRNA and ChAT protein levels at both 40 and 120 min, as shown in Figs. 2 and 3. The high EtOHinduced decrease in cholinergic marker expression was significantly greater than that observed at 2-g/kg EtOH. High EtOH concentrations were found after 4-g/kg EtOH exposure, while ACe was undetectable in the brain. A previous study has demonstrated a significant decrease in ChAT and AChE activity in the mouse cerebral cortex after acute exposure to 2-g/kg EtOH (Owasoyo and Iramain, 1981). Our present study revealed a significant decrease in both ChAT and AChE levels
41
only after high EtOH exposure. This discrepancy might be due to the mice strains and their genotype variations, which may have contributed to the difference in the sensitivity of the cholinergic function to EtOH (Hashemzadeh-Gargari and Mandel, 1989). The present data, however, demonstrate that high EtOH levels can also alter the central cholinergic parameters, via an alteration in both the presynaptic and postsynaptic process in the cortical cholinergic pathways. Indeed, perturbation of any one of these pathways is predicted to alter ACh release in cholinergic neurons. Our findings have demonstrated a profound effect of genotype on ChAT and AChE expression after EtOH treatment, as shown in Fig. 4. KO mice exhibited significantly lower ChAT, AChE mRNA, and ChAT protein levels than WT mice. Interestingly, that effect occurred mostly at 40 min after EtOH dosing, while both EtOH and ACe concentrations reached peak. There was no significant genotype effect on the levels of ChAT and AChE in the naive control. This result may indicate that KO mice are more sensitive than WT mice to EtOH-induced cholinergic alterations (Oyama et al., 2007). The enhanced sensitivity to EtOH in KO mice is consistent with previous findings (Fernandez et al., 2006). In addition, KO mice may be more susceptible than WT mice to EtOHinduced oxidative DNA damage (Kim et al., 2007). It is, however, conceivable that increased ACe levels in KO mice may contribute to a greater impairment of cholinergic markers expression. In summary, mice with Aldh2 deficiency exhibited a significant reduction in frontal cortex ChAT and AChE levels following 2- and 4-g/kg EtOH treatment, whereas WT mice showed a remarkable reduction in ChAT and AChE expression only after 4-g/kg EtOH. Moreover, there was a significant difference in cholinergic marker expression between genotypes after EtOH treatment. ACe was found only in the brains of KO mice, whereas EtOH was detected in both kinds of mice. These results clearly indicate that ACe may have a strong toxic effect on cortical cholinergic neurons in mice. Clearly, EtOH and ACe combined or high EtOH alone can alter cholinergic marker expression in the mouse frontal cortex.
4.
Experimental procedures
4.1.
Animals
KO mice were generated as reported previously (Kitagawa et al., 2000). They were obtained from the Department of Environmental Health, University of Occupational and Environmental Health, Japan, and then bred at the Kagawa University animal building. Male WT mice were purchased from CLEA Japan (Tokyo, Japan). All mice were housed in plastic cages with free access to standard laboratory food and water. Male mice were used at 10–12 weeks of age throughout this study, and weighed 24–28 g. The animal rooms were controlled for temperature (21 ± 3 °C), humidity (50%–70%), and light (12-h light–dark cycle). The Animal Investigation Committee of Kagawa University approved all experimental procedures and animal care involved in these studies.
42 4.2.
BR A IN RE S EA RCH 1 2 95 ( 20 0 9 ) 3 7 – 43
Experimental groups
Animals (KO and WT mice) were each divided into three experimental groups: (a) naive control, (b) EtOH (2 g/kg), and (c) EtOH (4 g/kg). Mice (n = 5 per group) received an intraperitoneal (i.p.) injection of EtOH (2- and 4-g/kg body weight, 20% [v/v]) and the samples were collected at 40 and 120 min after the injection.
4.3.
Gene
Sequences 5′→3′
Size (bp)
ChAT
F-TACAGGCTTTACCAGAGACTGGTG R-AACTGGAGATGCAGAAGGTGATGG F-TACTCTGGACGAGGCG R-CGGTCGTATTATATCCCAGC F-TCATGAAGTGTGACGTTGACATCCGTAAAG R-CCTAGAAGCATTTGCGGTGCACGATGGAGG
433
AChE β-actin
Brain tissues
Mice were deeply anesthetized with diethyl ether inhalation and killed by decapitation. Frontal cortex (approximately 50 mg) were collected on ice and then stored at −70 °C until further assay.
285
1 μL of 10 mM dNTP-mix, 0.5 μL of recombinant RNase inhibitor, and 1 μL of MMLV reverse transcriptase to a final volume of 20 μL. Total RNA was preincubated for 2 min at 70 °C prior to cDNA synthesis. The reverse transcription reaction was carried out for 60 min at 42 °C, followed by 5 min at 94 °C. The primer pairs used in the PCR were as shown in Table 2. PCR was performed in a final volume of 50 μL containing 2 μL of cDNA, 10 pmol of each primer, 0.5 μL of AmpliTaq DNA polymerase, and 14 μL of buffer containing 10 × PCR buffer, dNTP mix (10 mM), and MgCl2. Cycle parameters for ChAT included initial denaturation for 5 min at 94 °C, denaturation for 1 min at 94 °C, annealing for 1 min at 55 °C, and extension for 2 min at 72 °C, followed by a final extension for 3 min at 72 °C for 38 cycles. For AChE, initial denaturation was carried out for 2 min at 94 °C, denaturation for 1 min at 94 °C, annealing for 1 min at 55 °C, and extension for 1 min at 72 °C, followed by a final extension for 7 min at 72 °C for 35 cycles. For β-actin, initial denaturation was carried out for 5 min at 94 °C, denaturation for 1 min at 94 °C, annealing for 1 min at 54 °C, and extension for 2 min at 72 °C, followed by a final extension for 3 min at 72 °C for 24 cycles. PCR products were separated in a 2% agarose gel stained with ethidium bromide. Intensities in each band resulting from PCR amplification were analyzed by an LAS-1000plus luminoimaging analyzer (Fujix, Tokyo, Japan). The relative values were calculated by dividing the densitometric signals for ChAT and AChE by the signal obtained with the internal standard β-actin.
4.6. 4.5.
210
Microdialysis and head-space GC conditions
The microdialysis procedure and head-space GC (PerkinElmer, Norwalk, CT, USA) conditions were as previously described (Jamal et al., 2007b). In short, mice were implanted stereotaxically with a microdialysis probe (8 mm long, Eicom, Japan) with a 3-mm-long active dialysis membrane into the prefrontal cortex (coordinates relative from bregma: anterior, 2.0 mm; lateral, 1.0 mm; height, 3.0 mm) according to the atlas of Paxinos and Franklin (Paxinos and Franklin, 2001 [34]. One day after surgery, the experiments were begun. The probe was continuously perfused with Ringer's solution (147 mM NaCl, 4 mM KCl, 2.25 mM CaCl2) at a constant rate of 1 μL/min via syringe pump. Samples were collected at different time points for 10 min each after EtOH injection into vials containing 50 μL 0.002% t-butanol as an internal standard in 0.6 N perchloric acid and were then analyzed by head-space GC. The column, injector, and detector temperatures for the chromatography were 90, 110 and 200 °C, respectively. The separation column was a Supelcowax wide-bore capillary column (60 m length, 0.53 mm i.d., 2 μm film thickness, Supelco, Bellefonte, PA, USA). Nitrogen was used as the carrier gas at a flow rate of 20 mL/min. No artifactual ACe was detectable in the dialysate by this procedure. In vitro probe recovery of EtOH and ACe was 72.2% ± 3.6% and 52.6% ± 5.9%, respectively, and the values were corrected.
4.4.
Table 2 – Primer sequences used for the RT-PCR; F, forward; R, reverse; bp, base pair.
Western blot
RNA isolation and RT-PCR
Total RNA from the frontal cortex was extracted using 1.5 mL of ISOGEN (Wako Pure Chemical Industries, Japan), as described previously (Jamal et al., 2007a). The concentrations and purity of RNA samples were determined using a Pharmacia GeneQuant RNA/DNA spectrophotometer at a wavelength of 260 nm, and the integrity was confirmed by electrophoresis through 2% agarose gels stained with ethidium bromide. All samples were stored at −70 °C until analysis by RT-PCR. An 11-μg amount of each total RNA was used as a template for reverse transcription using 1 μL of oligo (dT)18 primer (Clontech, Takara Bio Com., Japan) in an appropriate amount of DEPC-treated water to a final volume of 13.5 μL. The synthesis reaction contained 4 μL of 5 × reaction buffer,
Frontal cortex was homogenized in 20 volumes (wt/vol) of 20 mM ice-cold Tris–HCl (pH 7.4) containing 0.32 mM sucrose as outlined previously (Jamal et al., 2007a). After centrifugation at 10,000 × g at 4 °C for 15 min, the supernatant was used for Western blot analyses. The protein content of the supernatant was determined by Bradford (Bio-Rad, Hercules, CA) using bovine serum albumin (Sigma Chemical, St. Louis, MO) as the standard. Samples were run on 10% SDS PAGE with molecular weight markers (Bio-Rad), then transferred electrophoretically to PVDF membranes. Goat anti-ChAT antiserum (AB144p, polyclonal; diluted 1:500, Chemicon International Inc., Temecula, CA, USA) and horseradish peroxidase-linked anti-goat IgG antibody were used for protein detection. The band intensities were evaluated by an LAS-1000plus lumino-imaging analyzer (Fujix).
BR A IN RE S E A RCH 1 2 95 ( 20 0 9 ) 3 7 – 4 3
4.7.
Statistics
The data were analyzed by appropriate analysis of variance (ANOVA) procedures. Post hoc tests were carried out where appropriate using Tukey–Kramer's test. The Student's t-test was used to assess significant differences in cholinergic marker expression, and EtOH and ACe concentrations between genotypes. All statistics were carried out using SigmaStat (Systat, version 3.0) software on an IBM-compatible computer.
Acknowledgments This work was supported in part by the Grant-in-Aid for Scientific Research [Grand No. (c) 18590637, 20590681] from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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