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Ethanol and acetaldehyde differentially alter extracellular dopamine and serotonin in Aldh2-knockout mouse dorsal striatum: A reverse microdialysis study
NeuroToxicology 52 (2016) 204–209
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Ethanol and acetaldehyde differentially alter extracellular dopamine and serotonin in Aldh2-knockout mouse dorsal striatum: A reverse microdialysis study Mostofa Jamala,* , Kiyoshi Amenoa , Takanori Mikib , Naoko Tanakaa , Asuka Itoa , Junichiro Onoc , Ayaka Takakuraa , Mitsuru Kumihashia , Hiroshi Kinoshitaa 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 Anesthesiology and Emergency Medicine, Faculty of Medicine, Kagawa University, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 September 2015 Received in revised form 15 December 2015 Accepted 15 December 2015 Available online 19 December 2015
Dopamine (DA) and serotonin (5-HT) seem to be involved in several of the effects of ethanol (EtOH). Acetaldehyde (AcH), especially in the brain, induces effects that mimic those of EtOH. The purpose of this study was to investigate the effects of local perfusion of EtOH and AcH on extracellular DA and 5-HT in the dorsal striatum of Aldh2-knockout (Aldh2-KO) and wild-type (WT) mice. Aldh2-KO mice were used as a model of aldehyde dehydrogenase 2 deficiency in humans to examine the effects of AcH. Mice were perfused with Ringer’s solution (control), EtOH (100, 200, or 500 mM) and AcH (100, 200, or 500 mM) into the dorsal striatum. Dialysate samples were collected every 5 min, and then analyzed with HPLC coupled to an ECD. We found that local perfusion with 500 mM EtOH increased extracellular levels of DA (p < 0.05) in both Aldh2-KO and WT mice, while 5-HT levels remain unchanged. EtOH at a dose of 200 mM also increased DA in WT mice, but this was limited to a 30–40-min time-point. In contrast, perfusion with 200 and 500 mM AcH decreased both DA and 5-HT (p < 0.05) in Aldh2-KO mice, but this decrease was not found in WT mice at any AcH dose, indicating an effect of AcH on DA and 5-HT levels. There were no genotype effects on the basal levels of DA and 5-HT. These results indicate that high EtOH can stimulate DA, whereas high AcH can depress both DA and 5-HT in the dorsal striatum of mice. ã 2015 Elsevier Inc. All rights reserved.
Keywords: Ethanol Acetaldehyde Dopamine Serotonin Reverse microdialysis Aldh2-KO mice
1. Introduction Ethanol (EtOH) can affect brain function by interacting with many neurotransmitter systems, including GABA (Aguayo, 1990), glutamate (Möykkynen and Korpi, 2012; Lovinger et al., 1989), and nicotinic cholinergic receptors (Yu et al., 1996). EtOH exerts a dual effect depending on its dosage: a large dose produces behavioral inhibition, while a low dose causes behavioral stimulation ska, 2005; Crabbe et al., 1982). (Tambour et al., 2006; Budzin AcH (acetaldehyde) may have the same effect: a high dose induces sedative as well as memory-impairing effects (Quertemont and Didone, 2006), whereas a low dose produces stimulation (Tambour et al., 2006). Several isozymes of aldehyde dehydrogenase (ALDH) have been identified. Mitochondrial ALDH2 is the major enzyme for AcH removal. ALDHs are highly expressed in the liver and stomach, but are also widely distributed in other tissues, including
* Corresponding author. Fax: +81 87 891 2141. E-mail addresses: [email protected], [email protected] (M. Jamal). http://dx.doi.org/10.1016/j.neuro.2015.12.011 0161-813X/ ã 2015 Elsevier Inc. All rights reserved.
the brain (Lassen et al., 2005; Quintanilla et al., 2005; Zimatkin et al., 1992). If ALDH functions normally, AcH does not accumulate in the tissues following EtOH intake, because ALDH oxidizes AcH to acetic acid very quickly. Therefore, we used Aldh2-knockout (Aldh2-KO) mice that lack the expression of human mitochondrial ALDH2, resulting in the accumulation of AcH, which is often considered a highly toxic compound that induces a range of unpleasant symptoms. Dopamine (DA) and serotonin (5-HT) play a number of roles in humans and other animals. In particular, it has a functional role in the pleasure/reward, learning and memory regions of the brain (Sharot et al., 2009; Bressan and Crippa, 2005; Buhot et al., 2000). DA release in the dorsal striatum has long been known to mediate the rewarding effect of stimulant drugs, including cocaine and EtOH (Veeneman et al., 2012; Haleem et al., 2005). 5-HT release may also contribute to mood regulation and reward functions in the striatum (Seymour et al., 2012), and has also been linked to the effect of EtOH on the brain (Lovinger, 1997). Acute EtOH exposure increases the release of 5-HT in the nucleus accumbens of rats (Yoshimoto et al., 1992), suggesting that 5-HT may contribute to
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the rewarding effect of EtOH. Several reports clearly show that high concentrations of EtOH induce mesolimbic DA neurons in rats (Tuomainen et al., 2003; Tang et al., 2003; Yim et al., 1997; Yoshimoto et al., 1992; Imperato and Di Chiara, 1986). AcH inhibits a variety of cellular processes, including protein synthesis and the secretion of glycoprotein in the livers and pancreases of rats (Majumdar et al., 1986; Tuma et al., 1980). It has also been shown that AcH is effective in inhibiting the induction of long-term potentiation (Abe et al., 1999); however, no study has thus far investigated the direct effect of AcH on DA and 5-HT levels in the dorsal striatum of mice. Here, we have chosen Aldh2-knockout (Aldh2-KO) mice to investigate the local effects of EtOH or AcH on extracellular DA and 5-HT levels in vivo. To this aim, we chose three doses of EtOH (100, 200, and 500 mM) and AcH (100, 200, and 500 mM) to perfuse directly into the dorsal striatum of Aldh2-KO and wild-type (WT) mice. Reverse microdialysis in combination with high-performance liquid chromatography and an electrochemical detector (HPLC-ECD) were employed in conscious, freely moving mice.
t-butanol as an internal standard and then EtOH and AcH concentrations were measured by a gas chromatography autosampler (Head-space GC, Shimadzu, Japan). The dialysate levels of EtOH and AcH were compared to the standard concentrations of EtOH and AcH.
2. Material and methods
Each mouse was anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and placed in a stereotaxic apparatus (SR-5N, Narishige Scientific Instrument Lab, Tokyo, Japan) for the surgical implantation of a guide cannula (AG-4, Eicom, Japan). The guide cannula was positioned above the right dorsal striatum using the following coordinates: anterior, 0.2 mm to the bregma; lateral, 3 mm; depth, 3 mm according to the atlas of Paxinos and Franklin (Paxinos and Franklin, 2001). The guide cannula was secured to the skull with dental cement anchored by a stainless-steel screw, and then a dummy cannula was inserted into the guide cannula. After surgery, mice were kept in a separate cage where food and water were available ad libitum. The mice were kept warm until fully ambulatory using a heat lamp. The mice were allowed to recover for 24 h after stereotaxic surgery.
2.1. Animals All animal experiments were approved by the Kagawa University Animal Investigation Committee. Aldh2-KO mice were generated as previously reported (Kitagawa et al., 2000). These mice were maintained and backcrossed with the C57BL/6 J strain for more than 10 generations. They have the same genetic background, except for Aldh2. Two eight-week-old male/female pairs were obtained from the Department of Environmental Health at the University of Occupational and Environmental Health in Japan and were bred at the Kagawa University animal facility. Breeding pairs from this strain were used to generate the experimental groups. WT mice have the same genetic background as C57BL/6 J mice and were bred in our animal facility. All experiments were conducted with male mice. Each was 10– 12 weeks in age and weighed 24–28 g. All animals were housed in controlled temperature (21 3 C), humidity (50–70%) and light (12-h light-dark cycle) conditions. 2.2. Experimental groups Aldh2-KO and WT mice were each divided into seven experimental groups: (a) Ringer’s solution (control, n = 4), (b) EtOH (100 mM, n = 4), (c) EtOH (200 mM, n = 4) (d) EtOH (500 mM, n = 4), (e) AcH (100 mM, n = 5), (f) AcH (200 mM, n = 5) and (g) AcH (500 mM, n = 5). High doses of EtOH (1–5 g/kg) and AcH (1000 mM) can decrease DA levels in the rat brains (Budygin et al., 2001; Wang et al., 2007). Based on this observation, we chose perfusion with 100, 200 or 500 mM EtOH and 100, 200 or 500 mM AcH. AcH was purchased from Merck-Schuchardt, Hohenbrunn, Germany. All reagents used were of the highest quality available. A stock solution of 1 M EtOH and 1 mM AcH were prepared in Ringer’s solution. All prepared solutions were stored at 4 C until use.
2.4. Head-space GC A gas chromatograph equipped with a flame ionization detector (GC-2014, Shimadzu, Japan) combined with a head-space auto sampler (TurboMatrix 40, PerkinElmer) was used throughout the study. Chromatographic conditions, in short, were as follows: column, injector and detector temperatures 90, 110 and 200 C, respectively. The separation column was a SupelcowaxTM wide bore capillary column (60 m length, 0.53 nmm i.d., 2 mm film thickness, Supelco, Bellefonte, PA, USA). Nitrogen was used as the carrier gas at 50 kPa. 2.5. Surgical procedure
2.6. Microdialysis with direct EtOH and AcH perfusion In the morning on the day of the analysis, the dialysis experiments commenced with the insertion of a probe (A-I-4-2, Eicom) equipped with an active dialysis membrane (2 mm long; inner diameter, 0.20 mm; outer diameter, 0.22 mm; cutoff value, 50 kDa) constructed from hemicellulose dialysis tubing. Mice were caged individually in acrylic cages (width 30 cm, length 35 cm, depth 30 cm). The probe’s inlet was perfused continuously with Ringer’s solution (147 mM NaCl, 4 mM KCl, 2.25 mM CaCl2, pH 6.4) at a constant rate of 1 ml/min using a 1.0-ml gas-tight syringe (Hamilton, Reno, NV, USA). Samples were collected at 5 min intervals throughout the experiment. Immediately after obtaining a stable 4-sample baseline of DA and 5-HT, the striatum was perfused with control, EtOH (100, 200 or 500 mM) and AcH (100, 200 or 500 mM) in Ringer’s solution through the inlet of the probe for 60 min. The basal level (100%) was defined as the average output of four consecutive samples that did not differ by more than 4%. The in vitro recovery of DA and 5-HT from 2 mm membrane length probes was 15% (Shen et al., 2004) and the dialysate levels of DA and 5-HT were not corrected for the recovery.
2.3. Probe efficiency in vitro 2.7. HPLC conditions In vitro probe efficiency was measured to determine how much concentration of perfused EtOH and AcH diffused out through the probe. Three microdialysis probes were perfused with EtOH (100, 200 or 500 mM) or AcH (100, 200 or 500 mM) in Ringer’s solution individually at a flow rate of 1 ml/min. The probe was dipped into a tube containing Ringer’s solution in a 37 C water bath. Three consecutive dialysate samples were collected. Each sample was collected for a period of 15 min into a vial containing 15 ml 0.02%
In order to determine the concentrations of DA and 5-HT in the dialysate samples, we used an HPLC system equipped with an ECD300 (Eicom) and an autosampler (EAS-20, Eicom). The main operative conditions for HPLC were as follows: column (Eicom- PPODS II; 4.6 mm 30 mm), oven temperature of 25 C, detector, oxidation potential (+400 mV versus Ag/AgCl reference analytical electrode), mobile phase: 2% MeOH/100 mM phosphate buffer (pH
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5.4) containing 50 mg/l disodium EDTA and 500 mg/l sodium decane-1-sulfonate at a flow rate of 0.5 ml/min. The dialysis samples were collected in 5 min intervals by an autoinjector connected to the automated HPLC-ECD system. The chromatograms were recorded with a PowerChrom (AD Instruments, Sydney, Australia). 2.8. Verification of probe and cannula placement After the microdialysis experiments were completed, mice were given a lethal dose of sodium-pentobarbital (100 mg/kg) and decapitated. The brains were removed, and the location of the dialysis probe in each brain was verified by visual examination after sectioning the brain. Only data from mice with the correct probe placement were used. 2.9. Statistical analysis The data were normalized by taking the final four baseline microdialysis samples prior to perfusions, averaging them, and then presenting the data as a percentage of this baseline value. The results are expressed as the mean SE. Statistical analyses were performed by using a two-way analysis of variance (ANOVA) with repeated measures over time with treatments (Ringer’s solution, EtOH, AcH) as the independent factors. A post hoc Tukey–Kramer test was used for multiple comparisons. The level of significance of the post hoc tests was set at P < 0.05. All analyses were conducted using the statistical program StatView (J-4.5). 3. Results
increase in DA levels (main effect of treatment: df 3,108; F = 4.816; p < 0.05) compared to the control group and mice receiving perfusions of 100 and 200 mM EtOH. EtOH perfusions failed to change the levels of 5-HT in both WT (main effect of treatment: df 3,108; F = 1.345; p = 0.32) and Aldh2-KO mice (main effect of treatment: df 3,108; F = 0.879; p = 0.48) at any dose. The effects of 100, 200 or 500 mM AcH on DA and 5-HT levels were tested in WT and Aldh2-KO mice as shown in Fig. 2. For WT mice, treatment with 100, 200 or 500 mM AcH neither produced effect on DA (main effect of treatment: df 3,144; F = 0.510; p = 0.68) nor on 5-HT levels (main effect of treatment: df 3,144; F = 0.586; p = 0.64) compared to the control group. For Aldh2-KO mice, treatment with 200 and 500 mM AcH resulted in a significant decrease in DA levels (main effect of treatment: df 3,144; F = 9.203; p < 0.05) compared to the control group and mice receiving perfusions of 100 mM AcH. Similarly, perfusions with 200 and 500 mM AcH decreased extracellular levels of 5-HT (main effect of treatment: df 3,144; F = 8.946; p < 0.05) compared to the control group and mice receiving perfusions of 100 mM AcH. Fig. 3 shows a comparison of basal dialysate concentrations of DA and 5-HT between Aldh2-KO and WT mice. ANOVA showed no significant difference between genotypes in basal dialysate concentrations of DA (df 1,4; F = 0.100; p = 0.767) and 5-HT (df 1,4; F = 3.169; p = 0.111). These results revealed no differences between Aldh2-KO and WT mice in the basal levels of DA and 5-HT in the dialysates. Our data may not support a link between ALDH2 deficiency and DA or 5-HT concentrations in the striatum of mice. 4. Discussion
Table 1 shows the in vitro probe efficiency of EtOH at concentrations of 100, 200 or 500 mM (A) and AcH at concentrations of 100, 200 or 500 mM (B). The EtOH concentrations in the dialysates were in the range of 59–62% at doses of 100, 200 or 500 mM EtOH, indicating that 37–41% of the infused EtOH diffused out of the perfusion probe into the extracellular space. The AcH concentrations in the dialysates were in the range of 44–50% at doses of 100, 200 or 500 mM AcH, indicating that 50–56% of the infused AcH diffused out of the perfusion probe into the extracellular space. Fig. 1 shows the effects of 100, 200 or 500 mM EtOH perfusions on DA and 5-HT levels in WT and Aldh2-KO mice. For WT mice, perfusions with 500 mM EtOH resulted in a significant increase in DA levels (main effect of treatment: df 3,108; F = 19.742; p < 0.05) compared to the control group and mice receiving perfusions of 100 and 200 mM EtOH. Perfusions with 200 mM EtOH increased DA levels (p < 0.05) compared to the control group in WT mice, but this effect was limited to a 30–40 min time-point. For Aldh2-KO mice, perfusions with 500 mM EtOH resulted in a significant Table 1 Shows the in vitro probe efficiency of EtOH (A) and AcH (B) at three concentrations of EtOH and AcH. In vitro probe efficiency of EtOH EtOH (mM) in Ringer’s
Percent dialysate (x)
Percent diffusion (y)
100 200 500
59.28 3.00 61.36 6.19 62.16 5.85
y = 100 x y = 100 x y = 100 x
In vitro probe efficiency of AcH AcH (mM) in Ringer’s
Percent dialysate (x)
Percent diffusion (y)
100 200 500
44.43 11.28 50.22 14.94 50.12 13.97
y = 100 x y = 100 x y = 100 x
EtOH (100, 200, or 500 mM) and AcH (100, 200 or 500 mM) were perfused directly into the dorsal striatum of both Aldh2-KO and WT mice. The rationale for the use of high doses of EtOH and AcH in this experiment was to evoke the depressive effect of EtOH and AcH on DA and 5-HT neuronal activities. Interestingly, we found that local exposure of the striatum to 500 mM EtOH enhanced DA concentrations in both WT and Aldh2-KO mice. EtOH at a dose of 200 mM also increased DA in WT mice, but this was limited to a 30–40-min time-point. In contrast, local exposure to 200 and 500 mM AcH decreased DA and 5-HT levels in Aldh2-KO mice, but had no effect in WT mice. This result suggests that these positive (stimulatory) and negative (depressant) properties of EtOH and AcH, respectively, are mediated in part by the increment of DA or the decrement of both DA and 5-HT neuronal activities in the dorsal striatum. These results have already been partially published in an abstract (Jamal et al. Alc Alc 2014, 49, S1, i1-i69). Several researchers have looked for changes in the brain DA and 5-HT contents after systemic or local EtOH administration (Enrico et al., 2009; Jamal et al., 2003; Tuomainen et al., 2003; Tang et al., 2003; Budygin et al., 2001; Yim et al., 1997; Yoshimoto et al., 1992). Most of these studies show an increase in extracellular DA in the brain, though there are some inconsistencies. The present study found a similar increase in DA levels in both Aldh2-KO and WT mice (Fig. 1). However, it requires perfusion with a high concentration of EtOH (500 mM). This increase may occur by stimulating the release of DA through regulation of dopaminergic cell firing, synaptic release, or a combination of effects (Yim and Gonzales, 2000). This is in agreement with previous work that demonstrated that direct perfusion of 510 or 860 mM EtOH excites DA neurons in vivo in the rat striatum (Yim et al., 1997). This effect of EtOH was specific for DA concentration because no effect was observed on simultaneously monitored 5-HT levels, in accordance with our previous microdialysis finding in male Wistar rats (Jamal et al., 2003). Our results suggest that high EtOH acts directly on
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Fig. 1. Effect of local perfusion of EtOH through dialysis probe on DA and 5-HT levels in WT and Aldh2-KO mice (n = 4). *P < 0.05 versus Ringer’s solution, yP < 0.05 versus Et100 and zP < 0.05 versus Et 200. DA, dopamine; 5-HT, serotonin; Et100, ethanol 100 mM; Et200, ethanol 200 mM; Et500, ethanol 500 mM.
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Fig. 2. Effect of local perfusion of AcH through dialysis probe on DA and 5-HT levels in WT and Aldh2-KO mice (n = 5). *P < 0.05 versus Ringer’s solution, yP < 0.05 versus Et100 and zP < 0.05 versus Et 200. DA, dopamine; 5-HT, serotonin; AcH100, AcH 100 mM; AcH200, AcH 200 mM; AcH500, AcH 500 mM.
DAminergic nerve terminals, which may contribute to an increase in baseline DA in the dorsal striatum of mice. Low-dose EtOH (100 mM) had no effect on extracellular DA, whereas a moderate dose (200 mM) led to an inconsistent increase in DA, suggesting that the concentrations in this range (100– 200 mM) did not produce a consistent effect on the dialysate levels of DA in the dorsal striatum. Support for this argument can be
found in a previous report, which showed that 170 mM EtOH or more was required to increase the levels of extracellular DA in the rat striatum (Yim et al., 1997). The 500 mM EtOH-induced DA increment was lower in Aldh2-KO mice than in WT mice, and it could be argued that this lower effect on DA levels in Aldh2-KO mice could be ascribed to high AcH concentrations formed from catalase (Jamal et al., 2007; Hamby-Mason et al., 1997).
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Baseline DA and 5-HT (pg/µl)
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a profound increase in extracellular DA level in both Aldh2-KO and WT mice. EtOH (200 mM) also increased DA in WT mice, but this was limited to a 30–40-min time-point. Perfusion with 200 and 500 mM AcH decreased both DA and 5-HT levels in Aldh2-KO mice, but not in WT mice. This is the first report on Aldh2-KO mice to demonstrate that high EtOH and AcH directly increase baseline DA and decrease both DA and 5-HT, respectively, in the dorsal striatum.
Aldh2-KO
1
0.5 Conflict of interests All authors declare that there was no conflict of interests.
0 DA
5-HT
Fig. 3. Effect of genotype differences on the basal concentrations of DA and 5-HT.
High concentrations of EtOH perfused directly into the tissue may cause damage. We cannot exclude that perfusion with 200 or 500 mM EtOH, which elicited an increase in DA levels, causes damage to the DAminergic terminals surrounding the probe. However, a previous study measured striatal volume to examine the possible tissue damage by direct EtOH perfusion and found that EtOH at low to high concentrations (25–860 mM) did not increase the damage caused by the probe implantation (Yim et al., 1997). We show a concentration-related decrease in DA and 5-HT levels into the dorsal striatum of Aldh2-KO mice in the AcH groups (Fig. 2). Aldh2-KO mice lack active ALDH2 and therefore, a low AcH elimination rate may lead to a high AcH accumulation. The present study is in agreement with previous work in rats that demonstrated that systemic administration of AcH can reduce DA and 5-HT levels (Ward et al., 1997) and the amount of DA (Heap et al., 1995) in the brain. One might argue that this decrease in DA would indicate some kind of acute toxic effect on DAminergic neurons. AcH did not alter DA and 5-HT levels at any dose in WT mice, possibly due to the subsequent oxidation of AcH to acetate by active ALDH2. There have been various reports that brain 5-HT is involved in some of the actions of EtOH (Lovinger, 1997; Yoshimoto et al., 1992). A few studies exist regarding the effects of AcH on 5-HT neurons (Ward et al., 1997), and their results revealed a decrease in 5-HT in the nucleus accumbens in rats after systemic administration of AcH (20 or 100 mg/kg). In line with our observation of 5-HT levels, direct perfusion of AcH (200 or 500 mM) via reverse dialysis was shown to decrease 5-HT in the dorsal striatum of Aldh2-KO mice in a dose-dependent fashion. Findings concerning DA levels after AcH administration have been controversial. Some studies have shown that AcH microinjection at a high concentration (90 mM) decreased DA in the posterior ventral tegmental area, whereas at an intermediate dose (23 mM), DA levels increased (Deehan et al., 2013) or remained the same. For instance, perfusion of AcH (1–200 mM) in the rat nucleus accumbens via reverse microdialysis did not alter accumbal DA levels at any applied dose (Clarke et al., 2014). Another study showed that perfusion with AcH (75 mM) into the rat nucleus accumbens increased dialysate DA output (Diana et al., 2008). There are several factors that may account for the discrepancies between these findings and ours, including differences in AcH doses, placement of the dialysis probe, different strains of mice, and the administrative route used. Therefore, the present results lead us to suggest that striatal administration of high concentrations of AcH (200 or 500 mM) can decrease both DA and 5-HT in Aldh2-KO mice. In conclusion, we have shown that local reverse microdialysis administration of EtOH (500 mM) in the dorsal striatum produced
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NeuroToxicology
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Ethanol and acetaldehyde differentially alter extracellular dopamine and serotonin in Aldh2-knockout mouse dorsal striatum: A reverse microdialysis study Mostofa Jamala,* , Kiyoshi Amenoa , Takanori Mikib , Naoko Tanakaa , Asuka Itoa , Junichiro Onoc , Ayaka Takakuraa , Mitsuru Kumihashia , Hiroshi Kinoshitaa 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 Anesthesiology and Emergency Medicine, Faculty of Medicine, Kagawa University, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 September 2015 Received in revised form 15 December 2015 Accepted 15 December 2015 Available online 19 December 2015
Dopamine (DA) and serotonin (5-HT) seem to be involved in several of the effects of ethanol (EtOH). Acetaldehyde (AcH), especially in the brain, induces effects that mimic those of EtOH. The purpose of this study was to investigate the effects of local perfusion of EtOH and AcH on extracellular DA and 5-HT in the dorsal striatum of Aldh2-knockout (Aldh2-KO) and wild-type (WT) mice. Aldh2-KO mice were used as a model of aldehyde dehydrogenase 2 deficiency in humans to examine the effects of AcH. Mice were perfused with Ringer’s solution (control), EtOH (100, 200, or 500 mM) and AcH (100, 200, or 500 mM) into the dorsal striatum. Dialysate samples were collected every 5 min, and then analyzed with HPLC coupled to an ECD. We found that local perfusion with 500 mM EtOH increased extracellular levels of DA (p < 0.05) in both Aldh2-KO and WT mice, while 5-HT levels remain unchanged. EtOH at a dose of 200 mM also increased DA in WT mice, but this was limited to a 30–40-min time-point. In contrast, perfusion with 200 and 500 mM AcH decreased both DA and 5-HT (p < 0.05) in Aldh2-KO mice, but this decrease was not found in WT mice at any AcH dose, indicating an effect of AcH on DA and 5-HT levels. There were no genotype effects on the basal levels of DA and 5-HT. These results indicate that high EtOH can stimulate DA, whereas high AcH can depress both DA and 5-HT in the dorsal striatum of mice. ã 2015 Elsevier Inc. All rights reserved.
Keywords: Ethanol Acetaldehyde Dopamine Serotonin Reverse microdialysis Aldh2-KO mice
1. Introduction Ethanol (EtOH) can affect brain function by interacting with many neurotransmitter systems, including GABA (Aguayo, 1990), glutamate (Möykkynen and Korpi, 2012; Lovinger et al., 1989), and nicotinic cholinergic receptors (Yu et al., 1996). EtOH exerts a dual effect depending on its dosage: a large dose produces behavioral inhibition, while a low dose causes behavioral stimulation ska, 2005; Crabbe et al., 1982). (Tambour et al., 2006; Budzin AcH (acetaldehyde) may have the same effect: a high dose induces sedative as well as memory-impairing effects (Quertemont and Didone, 2006), whereas a low dose produces stimulation (Tambour et al., 2006). Several isozymes of aldehyde dehydrogenase (ALDH) have been identified. Mitochondrial ALDH2 is the major enzyme for AcH removal. ALDHs are highly expressed in the liver and stomach, but are also widely distributed in other tissues, including
* Corresponding author. Fax: +81 87 891 2141. E-mail addresses: [email protected], [email protected] (M. Jamal). http://dx.doi.org/10.1016/j.neuro.2015.12.011 0161-813X/ ã 2015 Elsevier Inc. All rights reserved.
the brain (Lassen et al., 2005; Quintanilla et al., 2005; Zimatkin et al., 1992). If ALDH functions normally, AcH does not accumulate in the tissues following EtOH intake, because ALDH oxidizes AcH to acetic acid very quickly. Therefore, we used Aldh2-knockout (Aldh2-KO) mice that lack the expression of human mitochondrial ALDH2, resulting in the accumulation of AcH, which is often considered a highly toxic compound that induces a range of unpleasant symptoms. Dopamine (DA) and serotonin (5-HT) play a number of roles in humans and other animals. In particular, it has a functional role in the pleasure/reward, learning and memory regions of the brain (Sharot et al., 2009; Bressan and Crippa, 2005; Buhot et al., 2000). DA release in the dorsal striatum has long been known to mediate the rewarding effect of stimulant drugs, including cocaine and EtOH (Veeneman et al., 2012; Haleem et al., 2005). 5-HT release may also contribute to mood regulation and reward functions in the striatum (Seymour et al., 2012), and has also been linked to the effect of EtOH on the brain (Lovinger, 1997). Acute EtOH exposure increases the release of 5-HT in the nucleus accumbens of rats (Yoshimoto et al., 1992), suggesting that 5-HT may contribute to
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the rewarding effect of EtOH. Several reports clearly show that high concentrations of EtOH induce mesolimbic DA neurons in rats (Tuomainen et al., 2003; Tang et al., 2003; Yim et al., 1997; Yoshimoto et al., 1992; Imperato and Di Chiara, 1986). AcH inhibits a variety of cellular processes, including protein synthesis and the secretion of glycoprotein in the livers and pancreases of rats (Majumdar et al., 1986; Tuma et al., 1980). It has also been shown that AcH is effective in inhibiting the induction of long-term potentiation (Abe et al., 1999); however, no study has thus far investigated the direct effect of AcH on DA and 5-HT levels in the dorsal striatum of mice. Here, we have chosen Aldh2-knockout (Aldh2-KO) mice to investigate the local effects of EtOH or AcH on extracellular DA and 5-HT levels in vivo. To this aim, we chose three doses of EtOH (100, 200, and 500 mM) and AcH (100, 200, and 500 mM) to perfuse directly into the dorsal striatum of Aldh2-KO and wild-type (WT) mice. Reverse microdialysis in combination with high-performance liquid chromatography and an electrochemical detector (HPLC-ECD) were employed in conscious, freely moving mice.
t-butanol as an internal standard and then EtOH and AcH concentrations were measured by a gas chromatography autosampler (Head-space GC, Shimadzu, Japan). The dialysate levels of EtOH and AcH were compared to the standard concentrations of EtOH and AcH.
2. Material and methods
Each mouse was anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and placed in a stereotaxic apparatus (SR-5N, Narishige Scientific Instrument Lab, Tokyo, Japan) for the surgical implantation of a guide cannula (AG-4, Eicom, Japan). The guide cannula was positioned above the right dorsal striatum using the following coordinates: anterior, 0.2 mm to the bregma; lateral, 3 mm; depth, 3 mm according to the atlas of Paxinos and Franklin (Paxinos and Franklin, 2001). The guide cannula was secured to the skull with dental cement anchored by a stainless-steel screw, and then a dummy cannula was inserted into the guide cannula. After surgery, mice were kept in a separate cage where food and water were available ad libitum. The mice were kept warm until fully ambulatory using a heat lamp. The mice were allowed to recover for 24 h after stereotaxic surgery.
2.1. Animals All animal experiments were approved by the Kagawa University Animal Investigation Committee. Aldh2-KO mice were generated as previously reported (Kitagawa et al., 2000). These mice were maintained and backcrossed with the C57BL/6 J strain for more than 10 generations. They have the same genetic background, except for Aldh2. Two eight-week-old male/female pairs were obtained from the Department of Environmental Health at the University of Occupational and Environmental Health in Japan and were bred at the Kagawa University animal facility. Breeding pairs from this strain were used to generate the experimental groups. WT mice have the same genetic background as C57BL/6 J mice and were bred in our animal facility. All experiments were conducted with male mice. Each was 10– 12 weeks in age and weighed 24–28 g. All animals were housed in controlled temperature (21 3 C), humidity (50–70%) and light (12-h light-dark cycle) conditions. 2.2. Experimental groups Aldh2-KO and WT mice were each divided into seven experimental groups: (a) Ringer’s solution (control, n = 4), (b) EtOH (100 mM, n = 4), (c) EtOH (200 mM, n = 4) (d) EtOH (500 mM, n = 4), (e) AcH (100 mM, n = 5), (f) AcH (200 mM, n = 5) and (g) AcH (500 mM, n = 5). High doses of EtOH (1–5 g/kg) and AcH (1000 mM) can decrease DA levels in the rat brains (Budygin et al., 2001; Wang et al., 2007). Based on this observation, we chose perfusion with 100, 200 or 500 mM EtOH and 100, 200 or 500 mM AcH. AcH was purchased from Merck-Schuchardt, Hohenbrunn, Germany. All reagents used were of the highest quality available. A stock solution of 1 M EtOH and 1 mM AcH were prepared in Ringer’s solution. All prepared solutions were stored at 4 C until use.
2.4. Head-space GC A gas chromatograph equipped with a flame ionization detector (GC-2014, Shimadzu, Japan) combined with a head-space auto sampler (TurboMatrix 40, PerkinElmer) was used throughout the study. Chromatographic conditions, in short, were as follows: column, injector and detector temperatures 90, 110 and 200 C, respectively. The separation column was a SupelcowaxTM wide bore capillary column (60 m length, 0.53 nmm i.d., 2 mm film thickness, Supelco, Bellefonte, PA, USA). Nitrogen was used as the carrier gas at 50 kPa. 2.5. Surgical procedure
2.6. Microdialysis with direct EtOH and AcH perfusion In the morning on the day of the analysis, the dialysis experiments commenced with the insertion of a probe (A-I-4-2, Eicom) equipped with an active dialysis membrane (2 mm long; inner diameter, 0.20 mm; outer diameter, 0.22 mm; cutoff value, 50 kDa) constructed from hemicellulose dialysis tubing. Mice were caged individually in acrylic cages (width 30 cm, length 35 cm, depth 30 cm). The probe’s inlet was perfused continuously with Ringer’s solution (147 mM NaCl, 4 mM KCl, 2.25 mM CaCl2, pH 6.4) at a constant rate of 1 ml/min using a 1.0-ml gas-tight syringe (Hamilton, Reno, NV, USA). Samples were collected at 5 min intervals throughout the experiment. Immediately after obtaining a stable 4-sample baseline of DA and 5-HT, the striatum was perfused with control, EtOH (100, 200 or 500 mM) and AcH (100, 200 or 500 mM) in Ringer’s solution through the inlet of the probe for 60 min. The basal level (100%) was defined as the average output of four consecutive samples that did not differ by more than 4%. The in vitro recovery of DA and 5-HT from 2 mm membrane length probes was 15% (Shen et al., 2004) and the dialysate levels of DA and 5-HT were not corrected for the recovery.
2.3. Probe efficiency in vitro 2.7. HPLC conditions In vitro probe efficiency was measured to determine how much concentration of perfused EtOH and AcH diffused out through the probe. Three microdialysis probes were perfused with EtOH (100, 200 or 500 mM) or AcH (100, 200 or 500 mM) in Ringer’s solution individually at a flow rate of 1 ml/min. The probe was dipped into a tube containing Ringer’s solution in a 37 C water bath. Three consecutive dialysate samples were collected. Each sample was collected for a period of 15 min into a vial containing 15 ml 0.02%
In order to determine the concentrations of DA and 5-HT in the dialysate samples, we used an HPLC system equipped with an ECD300 (Eicom) and an autosampler (EAS-20, Eicom). The main operative conditions for HPLC were as follows: column (Eicom- PPODS II; 4.6 mm 30 mm), oven temperature of 25 C, detector, oxidation potential (+400 mV versus Ag/AgCl reference analytical electrode), mobile phase: 2% MeOH/100 mM phosphate buffer (pH
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5.4) containing 50 mg/l disodium EDTA and 500 mg/l sodium decane-1-sulfonate at a flow rate of 0.5 ml/min. The dialysis samples were collected in 5 min intervals by an autoinjector connected to the automated HPLC-ECD system. The chromatograms were recorded with a PowerChrom (AD Instruments, Sydney, Australia). 2.8. Verification of probe and cannula placement After the microdialysis experiments were completed, mice were given a lethal dose of sodium-pentobarbital (100 mg/kg) and decapitated. The brains were removed, and the location of the dialysis probe in each brain was verified by visual examination after sectioning the brain. Only data from mice with the correct probe placement were used. 2.9. Statistical analysis The data were normalized by taking the final four baseline microdialysis samples prior to perfusions, averaging them, and then presenting the data as a percentage of this baseline value. The results are expressed as the mean SE. Statistical analyses were performed by using a two-way analysis of variance (ANOVA) with repeated measures over time with treatments (Ringer’s solution, EtOH, AcH) as the independent factors. A post hoc Tukey–Kramer test was used for multiple comparisons. The level of significance of the post hoc tests was set at P < 0.05. All analyses were conducted using the statistical program StatView (J-4.5). 3. Results
increase in DA levels (main effect of treatment: df 3,108; F = 4.816; p < 0.05) compared to the control group and mice receiving perfusions of 100 and 200 mM EtOH. EtOH perfusions failed to change the levels of 5-HT in both WT (main effect of treatment: df 3,108; F = 1.345; p = 0.32) and Aldh2-KO mice (main effect of treatment: df 3,108; F = 0.879; p = 0.48) at any dose. The effects of 100, 200 or 500 mM AcH on DA and 5-HT levels were tested in WT and Aldh2-KO mice as shown in Fig. 2. For WT mice, treatment with 100, 200 or 500 mM AcH neither produced effect on DA (main effect of treatment: df 3,144; F = 0.510; p = 0.68) nor on 5-HT levels (main effect of treatment: df 3,144; F = 0.586; p = 0.64) compared to the control group. For Aldh2-KO mice, treatment with 200 and 500 mM AcH resulted in a significant decrease in DA levels (main effect of treatment: df 3,144; F = 9.203; p < 0.05) compared to the control group and mice receiving perfusions of 100 mM AcH. Similarly, perfusions with 200 and 500 mM AcH decreased extracellular levels of 5-HT (main effect of treatment: df 3,144; F = 8.946; p < 0.05) compared to the control group and mice receiving perfusions of 100 mM AcH. Fig. 3 shows a comparison of basal dialysate concentrations of DA and 5-HT between Aldh2-KO and WT mice. ANOVA showed no significant difference between genotypes in basal dialysate concentrations of DA (df 1,4; F = 0.100; p = 0.767) and 5-HT (df 1,4; F = 3.169; p = 0.111). These results revealed no differences between Aldh2-KO and WT mice in the basal levels of DA and 5-HT in the dialysates. Our data may not support a link between ALDH2 deficiency and DA or 5-HT concentrations in the striatum of mice. 4. Discussion
Table 1 shows the in vitro probe efficiency of EtOH at concentrations of 100, 200 or 500 mM (A) and AcH at concentrations of 100, 200 or 500 mM (B). The EtOH concentrations in the dialysates were in the range of 59–62% at doses of 100, 200 or 500 mM EtOH, indicating that 37–41% of the infused EtOH diffused out of the perfusion probe into the extracellular space. The AcH concentrations in the dialysates were in the range of 44–50% at doses of 100, 200 or 500 mM AcH, indicating that 50–56% of the infused AcH diffused out of the perfusion probe into the extracellular space. Fig. 1 shows the effects of 100, 200 or 500 mM EtOH perfusions on DA and 5-HT levels in WT and Aldh2-KO mice. For WT mice, perfusions with 500 mM EtOH resulted in a significant increase in DA levels (main effect of treatment: df 3,108; F = 19.742; p < 0.05) compared to the control group and mice receiving perfusions of 100 and 200 mM EtOH. Perfusions with 200 mM EtOH increased DA levels (p < 0.05) compared to the control group in WT mice, but this effect was limited to a 30–40 min time-point. For Aldh2-KO mice, perfusions with 500 mM EtOH resulted in a significant Table 1 Shows the in vitro probe efficiency of EtOH (A) and AcH (B) at three concentrations of EtOH and AcH. In vitro probe efficiency of EtOH EtOH (mM) in Ringer’s
Percent dialysate (x)
Percent diffusion (y)
100 200 500
59.28 3.00 61.36 6.19 62.16 5.85
y = 100 x y = 100 x y = 100 x
In vitro probe efficiency of AcH AcH (mM) in Ringer’s
Percent dialysate (x)
Percent diffusion (y)
100 200 500
44.43 11.28 50.22 14.94 50.12 13.97
y = 100 x y = 100 x y = 100 x
EtOH (100, 200, or 500 mM) and AcH (100, 200 or 500 mM) were perfused directly into the dorsal striatum of both Aldh2-KO and WT mice. The rationale for the use of high doses of EtOH and AcH in this experiment was to evoke the depressive effect of EtOH and AcH on DA and 5-HT neuronal activities. Interestingly, we found that local exposure of the striatum to 500 mM EtOH enhanced DA concentrations in both WT and Aldh2-KO mice. EtOH at a dose of 200 mM also increased DA in WT mice, but this was limited to a 30–40-min time-point. In contrast, local exposure to 200 and 500 mM AcH decreased DA and 5-HT levels in Aldh2-KO mice, but had no effect in WT mice. This result suggests that these positive (stimulatory) and negative (depressant) properties of EtOH and AcH, respectively, are mediated in part by the increment of DA or the decrement of both DA and 5-HT neuronal activities in the dorsal striatum. These results have already been partially published in an abstract (Jamal et al. Alc Alc 2014, 49, S1, i1-i69). Several researchers have looked for changes in the brain DA and 5-HT contents after systemic or local EtOH administration (Enrico et al., 2009; Jamal et al., 2003; Tuomainen et al., 2003; Tang et al., 2003; Budygin et al., 2001; Yim et al., 1997; Yoshimoto et al., 1992). Most of these studies show an increase in extracellular DA in the brain, though there are some inconsistencies. The present study found a similar increase in DA levels in both Aldh2-KO and WT mice (Fig. 1). However, it requires perfusion with a high concentration of EtOH (500 mM). This increase may occur by stimulating the release of DA through regulation of dopaminergic cell firing, synaptic release, or a combination of effects (Yim and Gonzales, 2000). This is in agreement with previous work that demonstrated that direct perfusion of 510 or 860 mM EtOH excites DA neurons in vivo in the rat striatum (Yim et al., 1997). This effect of EtOH was specific for DA concentration because no effect was observed on simultaneously monitored 5-HT levels, in accordance with our previous microdialysis finding in male Wistar rats (Jamal et al., 2003). Our results suggest that high EtOH acts directly on
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Fig. 1. Effect of local perfusion of EtOH through dialysis probe on DA and 5-HT levels in WT and Aldh2-KO mice (n = 4). *P < 0.05 versus Ringer’s solution, yP < 0.05 versus Et100 and zP < 0.05 versus Et 200. DA, dopamine; 5-HT, serotonin; Et100, ethanol 100 mM; Et200, ethanol 200 mM; Et500, ethanol 500 mM.
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Fig. 2. Effect of local perfusion of AcH through dialysis probe on DA and 5-HT levels in WT and Aldh2-KO mice (n = 5). *P < 0.05 versus Ringer’s solution, yP < 0.05 versus Et100 and zP < 0.05 versus Et 200. DA, dopamine; 5-HT, serotonin; AcH100, AcH 100 mM; AcH200, AcH 200 mM; AcH500, AcH 500 mM.
DAminergic nerve terminals, which may contribute to an increase in baseline DA in the dorsal striatum of mice. Low-dose EtOH (100 mM) had no effect on extracellular DA, whereas a moderate dose (200 mM) led to an inconsistent increase in DA, suggesting that the concentrations in this range (100– 200 mM) did not produce a consistent effect on the dialysate levels of DA in the dorsal striatum. Support for this argument can be
found in a previous report, which showed that 170 mM EtOH or more was required to increase the levels of extracellular DA in the rat striatum (Yim et al., 1997). The 500 mM EtOH-induced DA increment was lower in Aldh2-KO mice than in WT mice, and it could be argued that this lower effect on DA levels in Aldh2-KO mice could be ascribed to high AcH concentrations formed from catalase (Jamal et al., 2007; Hamby-Mason et al., 1997).
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a profound increase in extracellular DA level in both Aldh2-KO and WT mice. EtOH (200 mM) also increased DA in WT mice, but this was limited to a 30–40-min time-point. Perfusion with 200 and 500 mM AcH decreased both DA and 5-HT levels in Aldh2-KO mice, but not in WT mice. This is the first report on Aldh2-KO mice to demonstrate that high EtOH and AcH directly increase baseline DA and decrease both DA and 5-HT, respectively, in the dorsal striatum.
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0.5 Conflict of interests All authors declare that there was no conflict of interests.
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High concentrations of EtOH perfused directly into the tissue may cause damage. We cannot exclude that perfusion with 200 or 500 mM EtOH, which elicited an increase in DA levels, causes damage to the DAminergic terminals surrounding the probe. However, a previous study measured striatal volume to examine the possible tissue damage by direct EtOH perfusion and found that EtOH at low to high concentrations (25–860 mM) did not increase the damage caused by the probe implantation (Yim et al., 1997). We show a concentration-related decrease in DA and 5-HT levels into the dorsal striatum of Aldh2-KO mice in the AcH groups (Fig. 2). Aldh2-KO mice lack active ALDH2 and therefore, a low AcH elimination rate may lead to a high AcH accumulation. The present study is in agreement with previous work in rats that demonstrated that systemic administration of AcH can reduce DA and 5-HT levels (Ward et al., 1997) and the amount of DA (Heap et al., 1995) in the brain. One might argue that this decrease in DA would indicate some kind of acute toxic effect on DAminergic neurons. AcH did not alter DA and 5-HT levels at any dose in WT mice, possibly due to the subsequent oxidation of AcH to acetate by active ALDH2. There have been various reports that brain 5-HT is involved in some of the actions of EtOH (Lovinger, 1997; Yoshimoto et al., 1992). A few studies exist regarding the effects of AcH on 5-HT neurons (Ward et al., 1997), and their results revealed a decrease in 5-HT in the nucleus accumbens in rats after systemic administration of AcH (20 or 100 mg/kg). In line with our observation of 5-HT levels, direct perfusion of AcH (200 or 500 mM) via reverse dialysis was shown to decrease 5-HT in the dorsal striatum of Aldh2-KO mice in a dose-dependent fashion. Findings concerning DA levels after AcH administration have been controversial. Some studies have shown that AcH microinjection at a high concentration (90 mM) decreased DA in the posterior ventral tegmental area, whereas at an intermediate dose (23 mM), DA levels increased (Deehan et al., 2013) or remained the same. For instance, perfusion of AcH (1–200 mM) in the rat nucleus accumbens via reverse microdialysis did not alter accumbal DA levels at any applied dose (Clarke et al., 2014). Another study showed that perfusion with AcH (75 mM) into the rat nucleus accumbens increased dialysate DA output (Diana et al., 2008). There are several factors that may account for the discrepancies between these findings and ours, including differences in AcH doses, placement of the dialysis probe, different strains of mice, and the administrative route used. Therefore, the present results lead us to suggest that striatal administration of high concentrations of AcH (200 or 500 mM) can decrease both DA and 5-HT in Aldh2-KO mice. In conclusion, we have shown that local reverse microdialysis administration of EtOH (500 mM) in the dorsal striatum produced
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