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Alcohol consumption alters dopamine transporter sites in Wistar–Kyoto rat brain
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Alcohol consumption alters dopamine transporter sites in Wistar–Kyoto rat brain X. Jiao a , W.P. Paré b , S.M. Tejani-Butt a,⁎ a
University of the Sciences in Philadelphia, Department of Pharmacology and Toxicology (Box 118), 600 South 43rd Street, Philadelphia, PA 19104, USA b V.A. Medical Center, Perry Point, MD 21902, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
Even though animal and human studies show alterations in dopamine transporter (DAT)
Accepted 5 December 2005
sites after alcohol withdrawal, the role of DAT in influencing either alcoholic or depressive behavior has not been examined extensively. Given that the Wistar–Kyoto (WKY) rat is a putative animal model of depressive behavior, the present study examined the effects of
Keywords:
chronic alcohol consumption on DAT sites in WKY versus Wistar (WIS) rats. Brains from
Alcoholism
both strains were sectioned for autoradiographic analysis of [3H]-GBR12935 binding to DAT
DAT
sites after 24 days of alcohol exposure. The results indicated that WKY rats consumed a
Depression
greater amount of alcohol (P b 0.001) than WIS rats did throughout the experiment.
WKY rat
Autoradiographic analyses of discrete brain regions indicated that alcohol consumption increased DAT sites in a greater number of brain areas in WKY compared to WIS rats. In
Abbreviations:
WKY rats, the binding of [3H]-GBR12935 to DAT sites was increased in the basolateral, central
ANOVA, analysis of variance
and lateral nuclei of the amygdala, lateral nucleus of the hypothalamus, olfactory tubercle,
CPu, caudate–putamen
caudate–putamen, nucleus accumbens and substantia nigra (P b 0.05) and decreased in the
DA, dopamine
ventromedial nucleus of the hypothalamus and the CA1 region of the hippocampus. In WIS
DAT, dopamine transporter
rats, alcohol consumption increased DAT sites in the CA1 region of the hippocampus,
GABA, gamma-aminobutyric acid
basolateral nucleus of the amygdala, ventral tegmental area and substantia nigra, and
NAc, nucleus accumbens
decreased DAT sites in the lateral and ventromedial hypothalamus and dentate gyrus.
NMDA, N-methy-D-aspartic acid
These results indicate a strain dependent alteration in DAT sites which may be related to
QAR, quantitative autoradiography
altered dopamine neurotransmission in select brain regions following alcohol consumption.
S–D, Sprague–Dawley SN, substantia nigra SNCD, substantia nigra pars compacta SNR, substantia nigra pars reticulata VTA, ventral tegmental area WIS, Wistar WKY, Wistar–Kyoto
⁎ Corresponding author. Fax: +1 215 895 1161. E-mail address: [email protected] (S.M. Tejani-Butt). 0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.12.009
© 2005 Elsevier B.V. All rights reserved.
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1.
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Introduction
While clinical depression is commonly diagnosed in patients suffering from alcohol abuse, depressed patients are found to be more prone to alcohol misuse. Studies based on large community surveys indicate considerable similarities in symptoms between these two psychiatric disorders (Carpenter and Hasin, 1999; Regier et al., 1990). It has been noted that alcohol abuse may induce episodes of depressive symptoms during a withdrawal period in which relapse can be triggered, reflecting an attempt to selfmedicate with alcohol consumption (Carpenter and Hasin, 1999; Koob and Le Moal, 1997; Regier et al., 1990). It has been hypothesized that one abnormality increases the risk of the other, and therapy that treats one condition may cure the other (Laine et al., 1999; Lynskey, 1998). Given epidemiological and clinical reports which indicate a high rate of co-morbidity between depressive illness and alcohol dependence, it has been suggested that depression and alcohol addiction may be linked in terms of the neurobiological mechanisms that underlie these two psychiatric disorders (Markou et al., 1998; O'Malley and Krishnan-Sarin, 1998). The basis of clinical depression and alcoholism has been credited to the dysfunctioning of various neurotransmitter/neuromodulator systems such as dopamine (DA), norepinephrine, serotonin, gamma-aminobutyric acid (GABA), glutamate, neuropeptides and hormones (Markou et al., 1998; Ollat et al., 1988). The mesocorticolimbic DA system, long considered as one of the neurochemical systems related to reward, includes but is not limited to psychotropic drug reward, nondrug motivational attributes and inherent reward (Koob, 1992). Both in vivo and in vitro studies have reported that drugs of abuse such as cocaine, amphetamine and alcohol can stimulate DA neurotransmission, while withdrawal from these chemicals reduces DA release, alters pre- and post-synaptic receptors and decreases DA neurotransmission (Di Chiara and Imperato, 1988; Zhou et al., 1995). Although the role of the mesolimbic DA system in the psychopathology of mood disorders is far from understood, both clinical and preclinical studies suggest that DA may play an important role in depression and other neuropsychiatric disorders (Bowden et al., 1997; Brown and Gershon, 1993; Ebstein et al., 1997; Kapur and Mann, 1992; Lambert et al., 2000; Roth and Elsworth, 1995). The DA transporter (DAT) is located on DA neurons and plays an important role in maintaining DA homeostasis in the synapse via an active transport process (Bonhomme and Esposito, 1998; Gainetdinov et al., 1998; Nirenberg et al., 1997). While the DAT in terminal regions represents a key mechanism for regulating synaptic levels of DA, the DAT in the cell body area serves a critical role in modulating DA transmission (Nirenberg et al., 1997). Homozygote DAT knock-out mice exhibited behaviors that were profoundly altered by disruption of the DAT gene (e.g. spontaneous hyperlocomotion). Neither cocaine nor amphetamine treatment could enhance locomotor activity, or DA release in these animals (Giros et al., 1996; Jones et al., 1998). Even though several laboratories have attempted to find suitable animal models that demonstrate depressive behavior
as well as increased voluntary alcohol consumption, the findings thus far have been variable (Nurnberger et al., 2002; Overstreet et al., 1992; Perfumi et al., 1999; Rezvani et al., 2002). As a proposed animal model of depressive behavior, the Wistar–Kyoto (WKY) rat strain is hyper-responsive to stress and exhibits alterations in physiological, neurochemical and endocrine indices that are similar to those reported in clinical depression (Lahmame et al., 1997; Lopez-Rubalcava and Lucki, 2000; Paré, 1989a). Given that this strain shows “depressive” behaviors as measured by the Forced Swim Test (FST), Elevated Plus Maze Test (EPM), Open Field Test (OFT) and other behavioral measures (defensive burying, passive avoidance, etc.) (Paré, 1989b, 1993, 1994; Paré and Tejani-Butt, 1996; Paré et al., 1999; Tejani-Butt et al., 2003), it was suggested that this rat strain may represent a suitable model in which to study the neurochemical mechanisms that may underlie depressive behavior and increased alcohol consumption (Paré et al., 1999). In support of this hypothesis, when rats were offered a free choice of water and alcohol, the WKY strain preferred ethanol solution compared to the Sprague–Dawley (S–D) rat strain (Paré et al., 1999). We have previously reported that WKY rats exhibited altered DAT distribution pattern in select brain regions compared to S–D and Wistar (WIS) rats (Jiao et al., 2003). In addition, antidepressant drugs such as bupropion and nomifensine that target DAT sites, selectively increased locomotor activity in WKY rats, with upregulation of DAT sites in several mesocorticolimbic regions (Jiao et al., 2006; Tejani-Butt et al., 2003). Given the critical role of the DA system in reward and reinforcement, the aim of the present study was to examine the effects of chronic alcohol consumption on DAT sites in WKY versus WIS rats using the technique of quantitative autoradiography (QAR).
2.
Results
2.1.
Alcohol drinking behavior
When offered a free choice of water and alcohol solution, WKY consumed a larger amount of alcohol compared to WIS rats on a daily basis over the whole period of treatment (Fig. 1a). The average alcohol consumption was significantly higher (nearly 200%) in WKY rats throughout the 24-day experimental period, Student's t test, P b 0.001 (Fig. 1b).
2.2.
Effects of alcohol on DAT sites in terminal field areas
2.2.1.
Nucleus accumbens (NAc)
Alcohol consumption altered the specific binding of [3H]GBR12935 to DAT sites in the NAc in WKY rats. The binding to DAT sites was significantly increased in the alcohol group in the core region of the NAc, F(1,26) = 8.99, P b 0.01, Tukey HSD test, P b 0.01 as well as in the shell region of the NAc, F(1,27) = 11.31, P b 0.01, Tukey HSD test, P b 0.05 (Fig. 2) compared to the control nonalcohol group. In contrast, alcohol did not affect the binding to DAT sites in WIS rats in either sub-area of the NAc.
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Fig. 1 – Alcohol consumption in WKY and WIS rats over 24-days. (a) Data are expressed as the mean of measurement from 7–8 rats from each strain on a daily basis. (b) Data are expressed as the mean ± SEM of measurement over 24 days from 7–8 rats from each strain, Student's t test, P b 0.001.
2.2.2.
Olfactory tubercle
A significant increase in the specific binding of [3H]-GBR12935 to DAT sites, F(1,25) = 8.60, P b 0.01, Tukey HSD test, P b 0.05 (Fig. 3) was observed in the olfactory tubercle in WKY rats that received alcohol compared to the control group. In contrast, alcohol did not alter DAT binding sites in WIS rats.
2.2.3.
Caudate–putamen (CPu)
Alcohol consumption significantly increased the specific binding of [3H]-GBR12935 to DAT sites in the CPu in WKY, F
Fig. 2 – Effects of alcohol consumption on the binding of [3H]GBR12935 to DAT sites in the NAc in WKY and WIS rat strains. Data are expressed as the mean ± SEM of measurements from 7–8 rats from each strain, with determinations made in quadruplicate sections from each brain. Significant differences between treatment groups within strain, Tukey HSD test, *P b 0.01 in the core and *P b 0.05 in the shell.
(1,28) = 6.88, P b 0.05, Tukey HSD test, P b 0.01 (Fig. 4) but not in WIS rats.
2.2.4.
Hippocampus (CA1 region)
Alcohol consumption significantly altered the binding of [3H]GBR12935 to DAT sites, F(1,24) = 15.55, P b 0.001, in the CA1 region of hippocampus in both strains (Table 1). While alcohol consumption decreased DAT sites in WKY rats, Tukey HSD test, P b 0.05; it increased DAT sites in WIS rats, Tukey HSD
Fig. 3 – Effects of alcohol consumption on the binding of [3H]-GBR12935 to DAT sites in the olfactory tubercle in WKY and WIS rat strains. Data are expressed as the mean ± SEM of measurements from 7–8 rats from each strain, with determinations made in quadruplicate sections from each brain. Significant differences between treatment groups within strain, Tukey HSD test, *P b 0.05; #P b 0.05 between WKY and WIS alcohol consumption groups.
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2.3.
Effects of alcohol on DAT sites in cell body areas
2.3.1. Substantia nigra (compacta (SNCD) and reticulata (SNR)) Alcohol consumption increased the specific binding of [3H]GBR12935 to DAT sites in the SNCD F(1,26) = 45.23, P b 0.001, Tukey HSD test, P b 0.01 as well as in the SNR, F(1,27) = 18.00, P b 0.001, Tukey HSD test, P b 0.05 (Fig. 5) in both rat strains.
2.3.2.
Ventral tegmental area (VTA)
While alcohol consumption did not alter the binding of [3H]GBR12935 to DAT sites in WKY rats, it significantly increased the binding to DAT sites in WIS rats, F(1,27) = 10.40, P b 0.01, Tukey HSD test, P b 0.05 (Fig. 5).
Fig. 4 – Effects of alcohol consumption on the binding of [3H]-GBR12935 to DAT sites in the CPu in WKY and WIS rat strains. Data are expressed as the mean ± SEM of measurements from 7–8 rats from each strain, with determinations made in quadruplicate sections from each brain. Significant differences between treatment groups within strain, Tukey HSD test, *P b 0.01; #P b 0.01 between WKY and WIS control groups.
3.
Discussion
3.1.
Strain differences in alcohol consumption behavior
We have previously reported that the distribution of DAT sites in the mesolimbic regions was significantly different in WKY rats when compared to S–D and WIS rats (Jiao et al., 2003). We suggested that a genetic difference in the normal distribution
Table 1 – [3H]-GBR12935 binding to DAT sites (Plate 30) test, P b 0.05. Alcohol did not have any effect in the CA2,3 region of the hippocampus in either rat strain.
2.2.5.
Dentate gyrus
While the binding was not affected in WKY rats, alcohol consumption reduced the specific binding of [3H]-GBR12935 to DAT sites in WIS rats, F(1,25) = 10.13, P b 0.01, Tukey HSD test, P b 0.05 (Table 1).
2.2.6.
Hypothalamus
In the lateral hypothalamus, alcohol consumption significantly altered the specific binding of [3H]-GBR12935 to DAT sites in both rat strains, F(1,26) = 12.76, P b 0.01 (Table 1). While alcohol consumption increased the binding to DAT sites in WKY rats, Tukey HSD test, P b 0.05, it reduced the binding to DAT sites in WIS rats, Tukey HSD test, P b 0.05. In the ventromedial hypothalamus, alcohol consumption significantly reduced the binding to DAT sites in both strains, F(1,27) = 16.69, P b 0.001, Tukey HSD test, P b 0.05 (Table 1). However, there was no effect in the dorsomedial hypothalamus in either rat strain.
Strain
Hippocampus CA1
Hypothalamus Lateral (LH) Dorsomedial (DM) Ventromedial (VM) Amygdala Basolateral (BLA) Lateral (LA) Central (CE)
Control
Alcohol
4132.601 4268.382 4709.419 3433.618
± ± ± ±
258.925 232.955 446.629 278.33 b
5069.813 3515.146 3547.621 2817.097
± ± ± ±
247.387 a 109.879 b 176.095 a 203.558
WIS WKY WIS WKY WIS WKY
5118.995 3324.597 4328.597 3348.315 5169.013 5332.575
± ± ± ± ± ±
358.749 245.611 b 243.848 136.574 b 379.712 320.868
3902.374 4427.994 3718.94 3614.635 3505.722 3949.042
± ± ± ± ± ±
402.928 a 248.682 a 228.733 295.458 395.296 a 388.065 a
WIS WKY WIS WKY WIS WKY
5432.034 5073.301 5016.74 4669.003 4009.797 3383.873
± ± ± ± ± ±
170.584 228.269 163.81 340.837 370.949 194.088
6659.392 6565.153 4900.716 6075.42 3954.449 4787.15
± ± ± ± ± ±
180.086 a 400.9 a 302.654 317.587a,b 323.819 297.054 a
WIS WKY Dentate gyrus WIS (DG) WKY
Amygdala
In the basolateral nucleus, alcohol treatment led to higher binding of [3H]-GBR12935 to DAT sites in both strains, F (1,28) = 26.95, P b 0.001, Tukey HSD test, P b 0.01 in WKY and P b 0.001 in WIS strains, respectively (Table 1). Alcohol consumption also increased the specific binding of [3H]GBR12935 to DAT sites in the lateral nucleus of the amygdala, F(1,27) = 4.72, P b 0.05, Tukey HSD test, P b 0.01 as well as in the central nucleus of the amygdala in WKY rats F(1,25) = 4.84, P b 0.05, Tukey HSD test, P b 0.01 (Table 1) but not in WIS rats.
2.2.7.
Regions of interest
Effects of alcohol consumption on the binding of [3H]-GBR12935 to DAT sites in the hippocampus (CA1 and dentate gyrus), hypothalamus (lateral, dorsomedial and ventromedial) and amygdala (basolateral, lateral and central) in WKY and WIS strains. Data are expressed as the mean ± SEM of measurements from 7–8 rats from each strain, with determinations made in quadruplicate sections from each brain, specific comparison was conducted using Tukey HSD test. a P b 0.05, significant differences between treatment groups within strain. b P b 0.05, between strains with the same treatment.
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3.2. Alcohol consumption selectively modulates DAT sites in WKY rats in terminal field regions
Fig. 5 – Effects of alcohol consumption on the binding of [3H]-GBR12935 to DAT sites in the cell body areas in WKY and WIS rat strains. Data are expressed as the mean ± SEM of measurements from 7–8 rats from each strain, with determinations made in quadruplicate sections from each brain. Significant differences between treatment groups within strain, Tukey HSD test, *P b 0.01 in the substantia nigra pars compacta (SNCD), and *P b 0.05 in the substantia nigra pars reticulata (SNR) and ventral tegmetal area (VTA).
of DAT sites in terminal versus cell body regions may be responsible for the selective behaviors observed in WKY rats (Jiao et al., 2003; Paré, 2000). In a recent study, WKY rats were found to have lower basal DA levels in the prefrontal cortex and a higher DA turnover rate in the striatum and NAc compared to WIS rats (De La Garza and Mahoney, 2004). Thus, it is possible that differences in the DA pathway may play a role in the unique behavioral and emotional characteristics observed in this particular rat strain. Since a hypodopaminergic function is implicated in voluntary alcohol drinking behavior and since depression may function as a predisposing condition for alcohol abuse, the present study examined the effects of chronic alcohol consumption on DAT sites in WKY versus WIS rat strain. WKY rats consumed about 200% more alcohol suggesting an alcohol-preferring behavior compared to the WIS rat under basal condition. In a previous study, alcohol consumption significantly increased percent time spent in the open arms in the EPM, increased locomotion expressed as reduced response latency in the OFT and significantly reduced the severity of gastric stress-ulcers in WKY rats (Paré et al., 1999). Taken together, these results suggest that alcohol may be producing an anxiolytic effect in WKY rats. Antidepressant treatment not only improved their behavior as tested by the EPM, but also decreased their alcohol intake (Paré et al., 1999). The evidence from these studies supports the hypothesis that depressive states may contribute to increased voluntary alcohol consumption and the alcohol-preferring behavior observed in WKY rats may suggest a self-medication strategy similar to that observed in some populations of depressed subjects.
The results of this study indicated that while alcohol consumption increased the binding of [3H]-GBR12935 to DAT sites in both strains, the magnitude of the effect as well as the number of brain regions influenced by alcohol was much greater in WKY compared to WIS rats. At the present time, the reason for this selective pattern of distribution of DAT sites in the brains of WKY rats is not well understood. It is possible that different DA signaling pathways aimed at maintaining adequate DA transmission in these regions may produce a compensatory alteration in DAT sites. Given previous reports that the DAT can be regulated by alterations in DA transmission or by neuropharmacological interventions (Wilson et al., 1996; Xia et al., 1992), it is possible that alterations in DAT sites may reflect a corresponding alteration in DA concentration in functional brain regions, resulting in physiological and psychological changes associated with mood and affect. Thus, an increase in DAT sites may lead to a net deficit of DA in certain brain regions. Alternatively, an increase in DAT sites may be observed in response to abnormally high levels of DA in some brain regions. A decrease in DAT sites may indicate either a DA deficiency or lower DAT gene expression in select brain areas. Alternately, a decrease in DAT sites may be an adaptive response to abnormally low levels of DA in some brain areas. Since D2 auto-receptors negatively control DA release and synthesis (Mortensen and Amara, 2003; Rahman et al., 2003), the differences seen in the cell body areas may reflect a strain dependent alteration in DAT sites and D2 auto-receptors in WKY and WIS rats. Alcohol consumption increased DAT sites in WKY rats in several mesolimbic areas that are associated with reinforcement, emotion and locomotion. As the key reward structure, the NAc not only receives DA innervations from the VTA, but also receives glutamatergic inputs from the hippocampus, amygdala, cortex and thalamus (Tupala and Tiihonen, 2004). Previous in vivo microdialysis studies suggested that voluntary alcohol consumption enhances DA activity in the NAc(Di Chiaraand Imperato, 1988; Katner and Weiss, 2001). Thus, the increase in DAT sites in WKY ratsintheNAccouldberelatedtoincreasedDAlevelsstimulatedby voluntary alcohol consumption. Alternately, the alcohol-induced increase in DAT sites may result in a functional deficit in the availability of DA, providing a mechanism for this rat strain's selfmedicating behavior. The hippocampus is part of the limbic system that is associated with memory functions that drive drug and alcohol dependency (Miller, 1991). Since spontaneous DA release in the limbic reward centers is one of the proposed mechanisms of relapse (O'Malley and Krishnan-Sarin, 1998), a decrease in DAT sites in the CA1 may be related to higher DA transmission. Alternately, alcohol may be inducing an up-regulation of D2 auto-receptors that may inhibit DA synthesis and release in these areas.
3.3. Alcohol consumption differentially modulates DAT sites in WKY rats in the VTA It was interesting to observe that alterations in DAT binding sites in the terminal fields versus the VTA were significantly
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different in the two strains after alcohol treatment. Since alcohol is reported to increase the firing rate of DA neurons in the VTA, it may explain the increased number of DAT sites in the VTA in WIS rats. However, alcohol consumption did not alter DAT sites in the VTA in the WKY rat strain. These results are in agreement with our previous report that while antidepressant drugs altered DAT sites in the cell body area for WIS rats, the treatment did not alter DAT sites in the VTA in the WKY rat strain (Jiao et al., 2006). The lack of an effect in the VTA in WKY rats may be due to a reduced functioning of the mesolimbic system and/or autoregulation. It is possible that alcohol induces an up-regulation of pre-synaptic D2 autoreceptors that may inhibit DA release via a negative feedback mechanism. Thus, the strain differences seen in the cell body areas may reflect a strain difference in the distribution of DAT sites and D2 auto-receptors in these brain areas. These possibilities are presently under investigation in our laboratory. The lack of an effect of alcohol on DAT sites in the VTA of WKY rats may also be attributed to other mechanisms as well. Alcohol-induced DAT alterations may be secondary to its effect on the neurochemical transmission of other pathways. For example, the interaction of glutamate and GABA with DA has been observed in the VTA, and is directly altered by alcohol administration to DA neurons (Goldstein, 1983; Saal et al., 2003). Given that high versus low doses of alcohol produce opposite effects on DAT site via GABA inter-neurons or DA neurons (Diana et al., 2003; Ollat et al., 1988), the observed differences in the VTA may be related to the amount of alcohol consumed by the two strains. Since alcohol consumption increased DAT sites in the SN (the cell body area of the nigrostriatal DA pathway) in both strains, it is possible that alcohol-induced responses of the nigrostriatal pathway may be similar in WKY and WIS rats. Based on our results, it would appear that WKY rats exhibit the normal accelerated motor behavior that is required to obtain a positive reinforcement, in a way that is similar to other strains (Paré, 1992a,b) and the increase in DAT sites in the SN may imply an increase in DA activity induced by alcohol. Given that an excited nigrostriatal DA pathway can stimulate locomotion in mice after alcohol exposure (Itzhak and Martin, 1999), our previous observation of increased locomotor activity in WKY rats after alcohol treatment (Paré et al., 1999) further supports the present data. In conclusion, the results of the present study indicate that WKY rats are more prone to alcohol consumption than WIS rats. Autoradiographic studies indicate that DAT sites in the WKY strain are more sensitive to modulation by chronic alcohol intake, particularly in the mesolimbic terminal field regions. Based on our previous and current results, we conclude that the WKY rat strain may be a suitable model in which to expand our studies on the role of the DA pathway in stress-induced alcohol consumption.
4.
Experimental procedures
4.1.
Animals
Male WKY and WIS rats were used in this study. WKY rats were raised in the Perry Point laboratory from the breeding stock originally obtained from Charles River Laboratories
(Kingston, NY). WIS rats were purchased from Harlan (Indianapolis, IN). Animals, 8 months of age at the time of experiment, were housed in single cages at 22 °C and placed on a 12-h light/dark cycle. Food and water were kept available during the whole day. 4.2.
Procedure
Rats from each strain were divided into the control (no-alcohol) group and the alcohol group (n = 7/8 per group). The alcohol preference method was based on the procedure established by Sandbak and Murison (1996) with minor modifications. The procedure utilized a progressive 3–7% (volume/volume) alcohol solution to elicit voluntary consumption of alcohol. 3% and 5% solution was presented during the first (days 1 to 7) and the second (days 8 to 14) 7-day periods, respectively, and 7% solution was presented during the rest of the time (days 15 to 24). Both alcohol solution and water were available during the experimental period until rats were sacrificed. Rats in the alcohol group were offered two sipper-type drinking tubes with tap water in one tube and with the alcohol solution in the other. The position (right/left) of the two tubes was switched daily to avoid any position-preference by rats. Since the alcohol solution was diluted ethanol without the addition of any flavor, and the position of the two bottles (water and alcohol) was switched daily, it eliminated the possibility of a flavor- and location-preference by the rats. Two sipper tubes containing water were presented to the control groups. The measurement of body weight and alcohol consumption was recorded at the same time on a daily basis. Alcohol consumption was recorded in terms of g alcohol per kilogram body weight. When body weights were recorded daily, there was no significant change in weights of WKY and WIS rats following alcohol consumption when compared to rats that received water over 24 days (data not shown). This experimental protocol was reviewed and approved by the Perry Point VAH Institutional Review Committee for the use of animal subjects. 4.3.
Brains section preparation
On Day 25, animals were sacrificed by decapitation between 0800 and 1000 and the brains were removed immediately and frozen at −80 °C until used. Brains were sectioned (16 μm) at −18 °C in a cryostat microtome and mounted on gelatin-coated microscope slides. Before storing at −80 °C, all slides were kept overnight at 4 °C under vacuum. Sections were sliced from plates 12, 30 and 42 according to the Brain Atlas of Paxinos and Watson (1998). Plates 12 and 30 represent terminal regions including the CPu, NAc, hippocampus, amygdala and hypothalamus and plate 42 represents the cell body areas including the ventral tegmental area (VTA), substantia nigra pars compacta (SNCD) and substantia nigra pars reticulata (SNR). 4.4.
[3H]-GBR12935 binding assay
To label DAT sites, quadruplicate sections were incubated for 23 h at 4 °C in buffer [50 mM NaH2PO4, 70 mM NaCl, 0.025% bovine serum albumin (BSA), 0.001% ascorbate and 1 μM cis-flupentixol, pH 7.5] containing 1.0 nM of [3H]-GBR12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine-3H, SA 43.5 Ci/ mmol, NEN Life Science Products, Boston, MA) for plate 12, and 2.0 nM of [3H]-GBR12935 for plates 30 and 42 (Jiao et al., 2003; Paxinos and Watson, 1998; Zavitsanou et al., 1996). Nonspecific binding was defined using 50 μM mazindol and represented less than 10% of total binding. The incubation was terminated by dipping the sections into ice-cold washing solution (2 h). The slides were dried in a stream of cold air and transferred into cassettes apposed to [3H] Hyper-film with calibrated [3H] standards. The exposure time was 3 weeks for plate 12 and 4 weeks for
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plates 30 and 42 (Jiao et al., 2006). Films were developed using Kodak GBX developer. 4.5.
Quantitation
The images were quantified using the Brain 3.0 computerized brain image analysis program for Macintosh (Gustafson, 1995). Nonspecific binding was subtracted from the total binding to provide the specific binding in the regions of interest. Data were expressed as mean ± SEM specific binding (fmol/mg brain protein). 4.6.
Statistics
Statistical analysis was performed with SigmaStat program for Windows. Difference between means for the quantitative autoradiographic data in individual regions was analyzed using two-way analysis of variance (ANOVA), with strain (two levels) and treatment (two levels) as independent variables. Where significant main effects (strain/treatment/strain-treatment) were reported (P b 0.05), a post hoc Tukey's test, with HSD P b 0.05, was conducted to locate significant differences between strain-treatment groups. All values of binding density were expressed as mean ± SEM. The alcohol consumption data were compared between WKY and WIS strains using Student's t test, P b 0.05.
Acknowledgments This study was supported by USPHS grant MH63002 (ST-B) and research funds from the Office of Research and Development, Medical Research Service, Department of Veteran Affairs (W. P.). The authors wish to thank Dr. J. Nissanov for his help in setting up the brain imaging system in our laboratory.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Alcohol consumption alters dopamine transporter sites in Wistar–Kyoto rat brain X. Jiao a , W.P. Paré b , S.M. Tejani-Butt a,⁎ a
University of the Sciences in Philadelphia, Department of Pharmacology and Toxicology (Box 118), 600 South 43rd Street, Philadelphia, PA 19104, USA b V.A. Medical Center, Perry Point, MD 21902, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
Even though animal and human studies show alterations in dopamine transporter (DAT)
Accepted 5 December 2005
sites after alcohol withdrawal, the role of DAT in influencing either alcoholic or depressive behavior has not been examined extensively. Given that the Wistar–Kyoto (WKY) rat is a putative animal model of depressive behavior, the present study examined the effects of
Keywords:
chronic alcohol consumption on DAT sites in WKY versus Wistar (WIS) rats. Brains from
Alcoholism
both strains were sectioned for autoradiographic analysis of [3H]-GBR12935 binding to DAT
DAT
sites after 24 days of alcohol exposure. The results indicated that WKY rats consumed a
Depression
greater amount of alcohol (P b 0.001) than WIS rats did throughout the experiment.
WKY rat
Autoradiographic analyses of discrete brain regions indicated that alcohol consumption increased DAT sites in a greater number of brain areas in WKY compared to WIS rats. In
Abbreviations:
WKY rats, the binding of [3H]-GBR12935 to DAT sites was increased in the basolateral, central
ANOVA, analysis of variance
and lateral nuclei of the amygdala, lateral nucleus of the hypothalamus, olfactory tubercle,
CPu, caudate–putamen
caudate–putamen, nucleus accumbens and substantia nigra (P b 0.05) and decreased in the
DA, dopamine
ventromedial nucleus of the hypothalamus and the CA1 region of the hippocampus. In WIS
DAT, dopamine transporter
rats, alcohol consumption increased DAT sites in the CA1 region of the hippocampus,
GABA, gamma-aminobutyric acid
basolateral nucleus of the amygdala, ventral tegmental area and substantia nigra, and
NAc, nucleus accumbens
decreased DAT sites in the lateral and ventromedial hypothalamus and dentate gyrus.
NMDA, N-methy-D-aspartic acid
These results indicate a strain dependent alteration in DAT sites which may be related to
QAR, quantitative autoradiography
altered dopamine neurotransmission in select brain regions following alcohol consumption.
S–D, Sprague–Dawley SN, substantia nigra SNCD, substantia nigra pars compacta SNR, substantia nigra pars reticulata VTA, ventral tegmental area WIS, Wistar WKY, Wistar–Kyoto
⁎ Corresponding author. Fax: +1 215 895 1161. E-mail address: [email protected] (S.M. Tejani-Butt). 0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.12.009
© 2005 Elsevier B.V. All rights reserved.
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1.
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Introduction
While clinical depression is commonly diagnosed in patients suffering from alcohol abuse, depressed patients are found to be more prone to alcohol misuse. Studies based on large community surveys indicate considerable similarities in symptoms between these two psychiatric disorders (Carpenter and Hasin, 1999; Regier et al., 1990). It has been noted that alcohol abuse may induce episodes of depressive symptoms during a withdrawal period in which relapse can be triggered, reflecting an attempt to selfmedicate with alcohol consumption (Carpenter and Hasin, 1999; Koob and Le Moal, 1997; Regier et al., 1990). It has been hypothesized that one abnormality increases the risk of the other, and therapy that treats one condition may cure the other (Laine et al., 1999; Lynskey, 1998). Given epidemiological and clinical reports which indicate a high rate of co-morbidity between depressive illness and alcohol dependence, it has been suggested that depression and alcohol addiction may be linked in terms of the neurobiological mechanisms that underlie these two psychiatric disorders (Markou et al., 1998; O'Malley and Krishnan-Sarin, 1998). The basis of clinical depression and alcoholism has been credited to the dysfunctioning of various neurotransmitter/neuromodulator systems such as dopamine (DA), norepinephrine, serotonin, gamma-aminobutyric acid (GABA), glutamate, neuropeptides and hormones (Markou et al., 1998; Ollat et al., 1988). The mesocorticolimbic DA system, long considered as one of the neurochemical systems related to reward, includes but is not limited to psychotropic drug reward, nondrug motivational attributes and inherent reward (Koob, 1992). Both in vivo and in vitro studies have reported that drugs of abuse such as cocaine, amphetamine and alcohol can stimulate DA neurotransmission, while withdrawal from these chemicals reduces DA release, alters pre- and post-synaptic receptors and decreases DA neurotransmission (Di Chiara and Imperato, 1988; Zhou et al., 1995). Although the role of the mesolimbic DA system in the psychopathology of mood disorders is far from understood, both clinical and preclinical studies suggest that DA may play an important role in depression and other neuropsychiatric disorders (Bowden et al., 1997; Brown and Gershon, 1993; Ebstein et al., 1997; Kapur and Mann, 1992; Lambert et al., 2000; Roth and Elsworth, 1995). The DA transporter (DAT) is located on DA neurons and plays an important role in maintaining DA homeostasis in the synapse via an active transport process (Bonhomme and Esposito, 1998; Gainetdinov et al., 1998; Nirenberg et al., 1997). While the DAT in terminal regions represents a key mechanism for regulating synaptic levels of DA, the DAT in the cell body area serves a critical role in modulating DA transmission (Nirenberg et al., 1997). Homozygote DAT knock-out mice exhibited behaviors that were profoundly altered by disruption of the DAT gene (e.g. spontaneous hyperlocomotion). Neither cocaine nor amphetamine treatment could enhance locomotor activity, or DA release in these animals (Giros et al., 1996; Jones et al., 1998). Even though several laboratories have attempted to find suitable animal models that demonstrate depressive behavior
as well as increased voluntary alcohol consumption, the findings thus far have been variable (Nurnberger et al., 2002; Overstreet et al., 1992; Perfumi et al., 1999; Rezvani et al., 2002). As a proposed animal model of depressive behavior, the Wistar–Kyoto (WKY) rat strain is hyper-responsive to stress and exhibits alterations in physiological, neurochemical and endocrine indices that are similar to those reported in clinical depression (Lahmame et al., 1997; Lopez-Rubalcava and Lucki, 2000; Paré, 1989a). Given that this strain shows “depressive” behaviors as measured by the Forced Swim Test (FST), Elevated Plus Maze Test (EPM), Open Field Test (OFT) and other behavioral measures (defensive burying, passive avoidance, etc.) (Paré, 1989b, 1993, 1994; Paré and Tejani-Butt, 1996; Paré et al., 1999; Tejani-Butt et al., 2003), it was suggested that this rat strain may represent a suitable model in which to study the neurochemical mechanisms that may underlie depressive behavior and increased alcohol consumption (Paré et al., 1999). In support of this hypothesis, when rats were offered a free choice of water and alcohol, the WKY strain preferred ethanol solution compared to the Sprague–Dawley (S–D) rat strain (Paré et al., 1999). We have previously reported that WKY rats exhibited altered DAT distribution pattern in select brain regions compared to S–D and Wistar (WIS) rats (Jiao et al., 2003). In addition, antidepressant drugs such as bupropion and nomifensine that target DAT sites, selectively increased locomotor activity in WKY rats, with upregulation of DAT sites in several mesocorticolimbic regions (Jiao et al., 2006; Tejani-Butt et al., 2003). Given the critical role of the DA system in reward and reinforcement, the aim of the present study was to examine the effects of chronic alcohol consumption on DAT sites in WKY versus WIS rats using the technique of quantitative autoradiography (QAR).
2.
Results
2.1.
Alcohol drinking behavior
When offered a free choice of water and alcohol solution, WKY consumed a larger amount of alcohol compared to WIS rats on a daily basis over the whole period of treatment (Fig. 1a). The average alcohol consumption was significantly higher (nearly 200%) in WKY rats throughout the 24-day experimental period, Student's t test, P b 0.001 (Fig. 1b).
2.2.
Effects of alcohol on DAT sites in terminal field areas
2.2.1.
Nucleus accumbens (NAc)
Alcohol consumption altered the specific binding of [3H]GBR12935 to DAT sites in the NAc in WKY rats. The binding to DAT sites was significantly increased in the alcohol group in the core region of the NAc, F(1,26) = 8.99, P b 0.01, Tukey HSD test, P b 0.01 as well as in the shell region of the NAc, F(1,27) = 11.31, P b 0.01, Tukey HSD test, P b 0.05 (Fig. 2) compared to the control nonalcohol group. In contrast, alcohol did not affect the binding to DAT sites in WIS rats in either sub-area of the NAc.
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Fig. 1 – Alcohol consumption in WKY and WIS rats over 24-days. (a) Data are expressed as the mean of measurement from 7–8 rats from each strain on a daily basis. (b) Data are expressed as the mean ± SEM of measurement over 24 days from 7–8 rats from each strain, Student's t test, P b 0.001.
2.2.2.
Olfactory tubercle
A significant increase in the specific binding of [3H]-GBR12935 to DAT sites, F(1,25) = 8.60, P b 0.01, Tukey HSD test, P b 0.05 (Fig. 3) was observed in the olfactory tubercle in WKY rats that received alcohol compared to the control group. In contrast, alcohol did not alter DAT binding sites in WIS rats.
2.2.3.
Caudate–putamen (CPu)
Alcohol consumption significantly increased the specific binding of [3H]-GBR12935 to DAT sites in the CPu in WKY, F
Fig. 2 – Effects of alcohol consumption on the binding of [3H]GBR12935 to DAT sites in the NAc in WKY and WIS rat strains. Data are expressed as the mean ± SEM of measurements from 7–8 rats from each strain, with determinations made in quadruplicate sections from each brain. Significant differences between treatment groups within strain, Tukey HSD test, *P b 0.01 in the core and *P b 0.05 in the shell.
(1,28) = 6.88, P b 0.05, Tukey HSD test, P b 0.01 (Fig. 4) but not in WIS rats.
2.2.4.
Hippocampus (CA1 region)
Alcohol consumption significantly altered the binding of [3H]GBR12935 to DAT sites, F(1,24) = 15.55, P b 0.001, in the CA1 region of hippocampus in both strains (Table 1). While alcohol consumption decreased DAT sites in WKY rats, Tukey HSD test, P b 0.05; it increased DAT sites in WIS rats, Tukey HSD
Fig. 3 – Effects of alcohol consumption on the binding of [3H]-GBR12935 to DAT sites in the olfactory tubercle in WKY and WIS rat strains. Data are expressed as the mean ± SEM of measurements from 7–8 rats from each strain, with determinations made in quadruplicate sections from each brain. Significant differences between treatment groups within strain, Tukey HSD test, *P b 0.05; #P b 0.05 between WKY and WIS alcohol consumption groups.
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2.3.
Effects of alcohol on DAT sites in cell body areas
2.3.1. Substantia nigra (compacta (SNCD) and reticulata (SNR)) Alcohol consumption increased the specific binding of [3H]GBR12935 to DAT sites in the SNCD F(1,26) = 45.23, P b 0.001, Tukey HSD test, P b 0.01 as well as in the SNR, F(1,27) = 18.00, P b 0.001, Tukey HSD test, P b 0.05 (Fig. 5) in both rat strains.
2.3.2.
Ventral tegmental area (VTA)
While alcohol consumption did not alter the binding of [3H]GBR12935 to DAT sites in WKY rats, it significantly increased the binding to DAT sites in WIS rats, F(1,27) = 10.40, P b 0.01, Tukey HSD test, P b 0.05 (Fig. 5).
Fig. 4 – Effects of alcohol consumption on the binding of [3H]-GBR12935 to DAT sites in the CPu in WKY and WIS rat strains. Data are expressed as the mean ± SEM of measurements from 7–8 rats from each strain, with determinations made in quadruplicate sections from each brain. Significant differences between treatment groups within strain, Tukey HSD test, *P b 0.01; #P b 0.01 between WKY and WIS control groups.
3.
Discussion
3.1.
Strain differences in alcohol consumption behavior
We have previously reported that the distribution of DAT sites in the mesolimbic regions was significantly different in WKY rats when compared to S–D and WIS rats (Jiao et al., 2003). We suggested that a genetic difference in the normal distribution
Table 1 – [3H]-GBR12935 binding to DAT sites (Plate 30) test, P b 0.05. Alcohol did not have any effect in the CA2,3 region of the hippocampus in either rat strain.
2.2.5.
Dentate gyrus
While the binding was not affected in WKY rats, alcohol consumption reduced the specific binding of [3H]-GBR12935 to DAT sites in WIS rats, F(1,25) = 10.13, P b 0.01, Tukey HSD test, P b 0.05 (Table 1).
2.2.6.
Hypothalamus
In the lateral hypothalamus, alcohol consumption significantly altered the specific binding of [3H]-GBR12935 to DAT sites in both rat strains, F(1,26) = 12.76, P b 0.01 (Table 1). While alcohol consumption increased the binding to DAT sites in WKY rats, Tukey HSD test, P b 0.05, it reduced the binding to DAT sites in WIS rats, Tukey HSD test, P b 0.05. In the ventromedial hypothalamus, alcohol consumption significantly reduced the binding to DAT sites in both strains, F(1,27) = 16.69, P b 0.001, Tukey HSD test, P b 0.05 (Table 1). However, there was no effect in the dorsomedial hypothalamus in either rat strain.
Strain
Hippocampus CA1
Hypothalamus Lateral (LH) Dorsomedial (DM) Ventromedial (VM) Amygdala Basolateral (BLA) Lateral (LA) Central (CE)
Control
Alcohol
4132.601 4268.382 4709.419 3433.618
± ± ± ±
258.925 232.955 446.629 278.33 b
5069.813 3515.146 3547.621 2817.097
± ± ± ±
247.387 a 109.879 b 176.095 a 203.558
WIS WKY WIS WKY WIS WKY
5118.995 3324.597 4328.597 3348.315 5169.013 5332.575
± ± ± ± ± ±
358.749 245.611 b 243.848 136.574 b 379.712 320.868
3902.374 4427.994 3718.94 3614.635 3505.722 3949.042
± ± ± ± ± ±
402.928 a 248.682 a 228.733 295.458 395.296 a 388.065 a
WIS WKY WIS WKY WIS WKY
5432.034 5073.301 5016.74 4669.003 4009.797 3383.873
± ± ± ± ± ±
170.584 228.269 163.81 340.837 370.949 194.088
6659.392 6565.153 4900.716 6075.42 3954.449 4787.15
± ± ± ± ± ±
180.086 a 400.9 a 302.654 317.587a,b 323.819 297.054 a
WIS WKY Dentate gyrus WIS (DG) WKY
Amygdala
In the basolateral nucleus, alcohol treatment led to higher binding of [3H]-GBR12935 to DAT sites in both strains, F (1,28) = 26.95, P b 0.001, Tukey HSD test, P b 0.01 in WKY and P b 0.001 in WIS strains, respectively (Table 1). Alcohol consumption also increased the specific binding of [3H]GBR12935 to DAT sites in the lateral nucleus of the amygdala, F(1,27) = 4.72, P b 0.05, Tukey HSD test, P b 0.01 as well as in the central nucleus of the amygdala in WKY rats F(1,25) = 4.84, P b 0.05, Tukey HSD test, P b 0.01 (Table 1) but not in WIS rats.
2.2.7.
Regions of interest
Effects of alcohol consumption on the binding of [3H]-GBR12935 to DAT sites in the hippocampus (CA1 and dentate gyrus), hypothalamus (lateral, dorsomedial and ventromedial) and amygdala (basolateral, lateral and central) in WKY and WIS strains. Data are expressed as the mean ± SEM of measurements from 7–8 rats from each strain, with determinations made in quadruplicate sections from each brain, specific comparison was conducted using Tukey HSD test. a P b 0.05, significant differences between treatment groups within strain. b P b 0.05, between strains with the same treatment.
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3.2. Alcohol consumption selectively modulates DAT sites in WKY rats in terminal field regions
Fig. 5 – Effects of alcohol consumption on the binding of [3H]-GBR12935 to DAT sites in the cell body areas in WKY and WIS rat strains. Data are expressed as the mean ± SEM of measurements from 7–8 rats from each strain, with determinations made in quadruplicate sections from each brain. Significant differences between treatment groups within strain, Tukey HSD test, *P b 0.01 in the substantia nigra pars compacta (SNCD), and *P b 0.05 in the substantia nigra pars reticulata (SNR) and ventral tegmetal area (VTA).
of DAT sites in terminal versus cell body regions may be responsible for the selective behaviors observed in WKY rats (Jiao et al., 2003; Paré, 2000). In a recent study, WKY rats were found to have lower basal DA levels in the prefrontal cortex and a higher DA turnover rate in the striatum and NAc compared to WIS rats (De La Garza and Mahoney, 2004). Thus, it is possible that differences in the DA pathway may play a role in the unique behavioral and emotional characteristics observed in this particular rat strain. Since a hypodopaminergic function is implicated in voluntary alcohol drinking behavior and since depression may function as a predisposing condition for alcohol abuse, the present study examined the effects of chronic alcohol consumption on DAT sites in WKY versus WIS rat strain. WKY rats consumed about 200% more alcohol suggesting an alcohol-preferring behavior compared to the WIS rat under basal condition. In a previous study, alcohol consumption significantly increased percent time spent in the open arms in the EPM, increased locomotion expressed as reduced response latency in the OFT and significantly reduced the severity of gastric stress-ulcers in WKY rats (Paré et al., 1999). Taken together, these results suggest that alcohol may be producing an anxiolytic effect in WKY rats. Antidepressant treatment not only improved their behavior as tested by the EPM, but also decreased their alcohol intake (Paré et al., 1999). The evidence from these studies supports the hypothesis that depressive states may contribute to increased voluntary alcohol consumption and the alcohol-preferring behavior observed in WKY rats may suggest a self-medication strategy similar to that observed in some populations of depressed subjects.
The results of this study indicated that while alcohol consumption increased the binding of [3H]-GBR12935 to DAT sites in both strains, the magnitude of the effect as well as the number of brain regions influenced by alcohol was much greater in WKY compared to WIS rats. At the present time, the reason for this selective pattern of distribution of DAT sites in the brains of WKY rats is not well understood. It is possible that different DA signaling pathways aimed at maintaining adequate DA transmission in these regions may produce a compensatory alteration in DAT sites. Given previous reports that the DAT can be regulated by alterations in DA transmission or by neuropharmacological interventions (Wilson et al., 1996; Xia et al., 1992), it is possible that alterations in DAT sites may reflect a corresponding alteration in DA concentration in functional brain regions, resulting in physiological and psychological changes associated with mood and affect. Thus, an increase in DAT sites may lead to a net deficit of DA in certain brain regions. Alternatively, an increase in DAT sites may be observed in response to abnormally high levels of DA in some brain regions. A decrease in DAT sites may indicate either a DA deficiency or lower DAT gene expression in select brain areas. Alternately, a decrease in DAT sites may be an adaptive response to abnormally low levels of DA in some brain areas. Since D2 auto-receptors negatively control DA release and synthesis (Mortensen and Amara, 2003; Rahman et al., 2003), the differences seen in the cell body areas may reflect a strain dependent alteration in DAT sites and D2 auto-receptors in WKY and WIS rats. Alcohol consumption increased DAT sites in WKY rats in several mesolimbic areas that are associated with reinforcement, emotion and locomotion. As the key reward structure, the NAc not only receives DA innervations from the VTA, but also receives glutamatergic inputs from the hippocampus, amygdala, cortex and thalamus (Tupala and Tiihonen, 2004). Previous in vivo microdialysis studies suggested that voluntary alcohol consumption enhances DA activity in the NAc(Di Chiaraand Imperato, 1988; Katner and Weiss, 2001). Thus, the increase in DAT sites in WKY ratsintheNAccouldberelatedtoincreasedDAlevelsstimulatedby voluntary alcohol consumption. Alternately, the alcohol-induced increase in DAT sites may result in a functional deficit in the availability of DA, providing a mechanism for this rat strain's selfmedicating behavior. The hippocampus is part of the limbic system that is associated with memory functions that drive drug and alcohol dependency (Miller, 1991). Since spontaneous DA release in the limbic reward centers is one of the proposed mechanisms of relapse (O'Malley and Krishnan-Sarin, 1998), a decrease in DAT sites in the CA1 may be related to higher DA transmission. Alternately, alcohol may be inducing an up-regulation of D2 auto-receptors that may inhibit DA synthesis and release in these areas.
3.3. Alcohol consumption differentially modulates DAT sites in WKY rats in the VTA It was interesting to observe that alterations in DAT binding sites in the terminal fields versus the VTA were significantly
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different in the two strains after alcohol treatment. Since alcohol is reported to increase the firing rate of DA neurons in the VTA, it may explain the increased number of DAT sites in the VTA in WIS rats. However, alcohol consumption did not alter DAT sites in the VTA in the WKY rat strain. These results are in agreement with our previous report that while antidepressant drugs altered DAT sites in the cell body area for WIS rats, the treatment did not alter DAT sites in the VTA in the WKY rat strain (Jiao et al., 2006). The lack of an effect in the VTA in WKY rats may be due to a reduced functioning of the mesolimbic system and/or autoregulation. It is possible that alcohol induces an up-regulation of pre-synaptic D2 autoreceptors that may inhibit DA release via a negative feedback mechanism. Thus, the strain differences seen in the cell body areas may reflect a strain difference in the distribution of DAT sites and D2 auto-receptors in these brain areas. These possibilities are presently under investigation in our laboratory. The lack of an effect of alcohol on DAT sites in the VTA of WKY rats may also be attributed to other mechanisms as well. Alcohol-induced DAT alterations may be secondary to its effect on the neurochemical transmission of other pathways. For example, the interaction of glutamate and GABA with DA has been observed in the VTA, and is directly altered by alcohol administration to DA neurons (Goldstein, 1983; Saal et al., 2003). Given that high versus low doses of alcohol produce opposite effects on DAT site via GABA inter-neurons or DA neurons (Diana et al., 2003; Ollat et al., 1988), the observed differences in the VTA may be related to the amount of alcohol consumed by the two strains. Since alcohol consumption increased DAT sites in the SN (the cell body area of the nigrostriatal DA pathway) in both strains, it is possible that alcohol-induced responses of the nigrostriatal pathway may be similar in WKY and WIS rats. Based on our results, it would appear that WKY rats exhibit the normal accelerated motor behavior that is required to obtain a positive reinforcement, in a way that is similar to other strains (Paré, 1992a,b) and the increase in DAT sites in the SN may imply an increase in DA activity induced by alcohol. Given that an excited nigrostriatal DA pathway can stimulate locomotion in mice after alcohol exposure (Itzhak and Martin, 1999), our previous observation of increased locomotor activity in WKY rats after alcohol treatment (Paré et al., 1999) further supports the present data. In conclusion, the results of the present study indicate that WKY rats are more prone to alcohol consumption than WIS rats. Autoradiographic studies indicate that DAT sites in the WKY strain are more sensitive to modulation by chronic alcohol intake, particularly in the mesolimbic terminal field regions. Based on our previous and current results, we conclude that the WKY rat strain may be a suitable model in which to expand our studies on the role of the DA pathway in stress-induced alcohol consumption.
4.
Experimental procedures
4.1.
Animals
Male WKY and WIS rats were used in this study. WKY rats were raised in the Perry Point laboratory from the breeding stock originally obtained from Charles River Laboratories
(Kingston, NY). WIS rats were purchased from Harlan (Indianapolis, IN). Animals, 8 months of age at the time of experiment, were housed in single cages at 22 °C and placed on a 12-h light/dark cycle. Food and water were kept available during the whole day. 4.2.
Procedure
Rats from each strain were divided into the control (no-alcohol) group and the alcohol group (n = 7/8 per group). The alcohol preference method was based on the procedure established by Sandbak and Murison (1996) with minor modifications. The procedure utilized a progressive 3–7% (volume/volume) alcohol solution to elicit voluntary consumption of alcohol. 3% and 5% solution was presented during the first (days 1 to 7) and the second (days 8 to 14) 7-day periods, respectively, and 7% solution was presented during the rest of the time (days 15 to 24). Both alcohol solution and water were available during the experimental period until rats were sacrificed. Rats in the alcohol group were offered two sipper-type drinking tubes with tap water in one tube and with the alcohol solution in the other. The position (right/left) of the two tubes was switched daily to avoid any position-preference by rats. Since the alcohol solution was diluted ethanol without the addition of any flavor, and the position of the two bottles (water and alcohol) was switched daily, it eliminated the possibility of a flavor- and location-preference by the rats. Two sipper tubes containing water were presented to the control groups. The measurement of body weight and alcohol consumption was recorded at the same time on a daily basis. Alcohol consumption was recorded in terms of g alcohol per kilogram body weight. When body weights were recorded daily, there was no significant change in weights of WKY and WIS rats following alcohol consumption when compared to rats that received water over 24 days (data not shown). This experimental protocol was reviewed and approved by the Perry Point VAH Institutional Review Committee for the use of animal subjects. 4.3.
Brains section preparation
On Day 25, animals were sacrificed by decapitation between 0800 and 1000 and the brains were removed immediately and frozen at −80 °C until used. Brains were sectioned (16 μm) at −18 °C in a cryostat microtome and mounted on gelatin-coated microscope slides. Before storing at −80 °C, all slides were kept overnight at 4 °C under vacuum. Sections were sliced from plates 12, 30 and 42 according to the Brain Atlas of Paxinos and Watson (1998). Plates 12 and 30 represent terminal regions including the CPu, NAc, hippocampus, amygdala and hypothalamus and plate 42 represents the cell body areas including the ventral tegmental area (VTA), substantia nigra pars compacta (SNCD) and substantia nigra pars reticulata (SNR). 4.4.
[3H]-GBR12935 binding assay
To label DAT sites, quadruplicate sections were incubated for 23 h at 4 °C in buffer [50 mM NaH2PO4, 70 mM NaCl, 0.025% bovine serum albumin (BSA), 0.001% ascorbate and 1 μM cis-flupentixol, pH 7.5] containing 1.0 nM of [3H]-GBR12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine-3H, SA 43.5 Ci/ mmol, NEN Life Science Products, Boston, MA) for plate 12, and 2.0 nM of [3H]-GBR12935 for plates 30 and 42 (Jiao et al., 2003; Paxinos and Watson, 1998; Zavitsanou et al., 1996). Nonspecific binding was defined using 50 μM mazindol and represented less than 10% of total binding. The incubation was terminated by dipping the sections into ice-cold washing solution (2 h). The slides were dried in a stream of cold air and transferred into cassettes apposed to [3H] Hyper-film with calibrated [3H] standards. The exposure time was 3 weeks for plate 12 and 4 weeks for
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plates 30 and 42 (Jiao et al., 2006). Films were developed using Kodak GBX developer. 4.5.
Quantitation
The images were quantified using the Brain 3.0 computerized brain image analysis program for Macintosh (Gustafson, 1995). Nonspecific binding was subtracted from the total binding to provide the specific binding in the regions of interest. Data were expressed as mean ± SEM specific binding (fmol/mg brain protein). 4.6.
Statistics
Statistical analysis was performed with SigmaStat program for Windows. Difference between means for the quantitative autoradiographic data in individual regions was analyzed using two-way analysis of variance (ANOVA), with strain (two levels) and treatment (two levels) as independent variables. Where significant main effects (strain/treatment/strain-treatment) were reported (P b 0.05), a post hoc Tukey's test, with HSD P b 0.05, was conducted to locate significant differences between strain-treatment groups. All values of binding density were expressed as mean ± SEM. The alcohol consumption data were compared between WKY and WIS strains using Student's t test, P b 0.05.
Acknowledgments This study was supported by USPHS grant MH63002 (ST-B) and research funds from the Office of Research and Development, Medical Research Service, Department of Veteran Affairs (W. P.). The authors wish to thank Dr. J. Nissanov for his help in setting up the brain imaging system in our laboratory.
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