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Keywords:

  • adolescence;
  • dopamine;
  • dopamine receptors;
  • glutamate;
  • nucleus accumbens;
  • repeated ethanol administration

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Adolescence is a developmental period which the risk of drug and alcohol abuse increases. Since mesolimbic dopaminergic system undergoes developmental changes during adolescence, and this system is involved in rewarding effects of drugs of abuse, we addressed the hypothesis that ethanol exposure during juvenile/adolescent period over-activates mesolimbic dopaminergic system inducing adaptations which can trigger long-term enduring behavioural effects of alcohol abuse. We treated juvenile/adolescent or adult rats with ethanol (3 g/kg) for two-consecutive days at 48-h intervals over 14-day period. Here we show that intermittent ethanol treatment during the juvenile/adolescence period alters subsequent ethanol intake. In vivo microdialysis demonstrates that ethanol elicits a similar prolonged dopamine response in the nucleus accumbens of both adolescent and adult animals pre-treated with multiple doses of ethanol, although the basal dopamine levels were higher in ethanol-treated adolescents than in adult-treated animals. Repeated ethanol administration also down-regulates the expression of DRD2 and NMDAR2B phosphorylation in prefrontal cortex of adolescent animals, but not of adult rats. Finally, ethanol treatment during adolescence changes the acetylation of histones H3 and H4 in frontal cortex, nucleus accumbens and striatum, suggesting chromatin remodelling changes. In summary, our findings demonstrate the sensitivity of adolescent brain to ethanol effects on dopaminergic and glutamatergic neurotransmission, and suggest that abnormal plasticity in reward-related processes and epigenetic mechanisms could contribute to the vulnerability of adolescents to alcohol addiction.

Abbreviations used
BECs

blood ethanol concentrations

DA

dopamine

DOPAC

dihydroxyphenylacetic acid

DRD1 and DRD2

DA receptors D1 and D2

NAc

nucleus accumbens

NR2B

NMDA receptor 2B

PND

postnatal day

Adolescence is an important stage of development in which the brain undergoes neuromaturation, characterized by changes in neurotransmission, plasticity and synaptic remodelling in several brain regions, including prefrontal cortex, hippocampus and the limbic system (Giedd 2008). In general, brain regions that underlie attention, reward evaluation, affective discrimination, response inhibition and goal-directed behaviour undergo structural and functional reorganization throughout late childhood and early adulthood. Accordingly, adolescence is associated with specific behavioural characteristics such as increased sensation-seeking, high levels of exploration, novelty, social interactions and risky behaviour (Yurgelun-Todd 2007).

Heavy binge drinking is becoming increasingly frequent among high school students and teenagers from the USA and European Countries (e.g. Oesterle et al. 2004, 2008; Caamaño-Isorna et al. 2008), and research with human adolescents clearly suggests that alcohol abuse during the teen years has deleterious effects since the prevalence of both alcohol-related problems and neurological deficits is more frequently observed among adolescents (Clark et al. 2008; Oesterle et al. 2008). Indeed, we have demonstrated that intermittent ethanol treatment during the adolescence stage in rats induces prefrontal cortex and hippocampal damage by inflammatory processes, and causes important short and long-lasting cognitive and behavioural deficits (Pascual et al. 2007).

Human epidemiological studies have also demonstrated that early-onset alcohol use is associated with an increased risk of subsequent alcohol abuse and the development of alcohol disorders, including dependence (Grant and Dawson 1997; Hawkins et al. 1997; DeWit et al. 2000). Other studies have also demonsrated that the age of first encounter with psychoactive drugs is critical, given the greater probability to shift from use to abuse and to develop addiction (e.g. Anthony and Petronis 1995; Breslau and Peterson 1996; Patton et al. 2004). Although these studies suggest that adolescence is a stage that is vulnerable to the consequences of alcohol and other psychoactive drugs abuse, the pathogenic process leading to drug addiction is still far from being completely understood.

The mesolimbic dopamine (DA) system, consisting of the ventral tegmental area and the nucleus accumbens (NAc), as well as associated limbic structures, is known to be involved in the reward and reinforced effects of drugs of abuse, including alcohol (Koob and Weiss 1992; Robbins and Everitt 2002). Ethanol has been shown to activate the mesolimbic DA pathway by increasing the firing of mesolimbic DA neurons and the extracellular levels of DA in the NAc (Imperato and Di Chiara 1986; Brodie et al. 1990). In addition, glutamate receptors through glutamatergic inputs to DA neurons in the NAc and striatum are also important for both DA modulation and synaptic plasticity associated with addiction (Nestler 2002; Kauer and Malenka 2007). Experimental studies demonstrated that the glutamatergic and mesolimbic DA system undergoes marked transitional changes during adolescence. For example, overproduction of NMDA (Guilarte 1998) and DA receptors, D1, D2 and D4 (Tarazi and Baldessarini 2000), increased in the DA synthesis and turnover in the NAc (Anderson et al. 1997; Spear 2000), and also in DA transporter densities (Tarazi et al. 1998). Moreover, elevated basal levels of extracellular DA have been noted in adolescent rodents in comparison with young adult rats (Badanich et al. 2006). In addition, frontal cortical regions are known to undergo developmental reorganization and functions, including an intense rewiring which underlies maturation and cognitive processing (Giedd 2004; Toga et al. 2006).

Considering that the mesocorticolimbic dopaminergic system undergoes developmental changes during the juvenile/adolescent period, and the impact of ethanol on this system, we postulate that binge alcohol drinking would affect the dopaminergic and glutamatergic responses differently in adolescents than in adults, and this different response in adolescents might induce long-lasting changes which could result in alcohol abuse and dependence. To address this hypothesis, we used the previous animal model of intermittent binge ethanol treatment in juvenile/adolescent rats (Pascual et al. 2007) and we analysed (i) the voluntary ethanol consumption in adult animals, (ii) the levels of DA, dihydroxyphenylacetic acid (DOPAC), and glutamate in the NAc after a challenged dose of ethanol, (iii) the levels of expression of DA receptors D1 and D2 and the phosphorylation of NR2B-containing the NMDA receptors in the NAc, prefrontal cortex, hippocampus and striatum. Finally, because dopaminergic and glutamatergic inputs have been shown to induce chromatin remodelling in striatal neurons (Li et al. 2004; Schroeder et al. 2008) and recent evidence suggests that histone acetylation and chromatin-remodelling events, are involved in drug-related behavioural sensitization and reward (Schroeder et al. 2008), we have also evaluated the possible changes in histone H3 and H4 acetylation, as a possible mechanism for long-term neurochemical alterations.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Subjects

Forty male Wistar rat pups on postnatal day (PND) 25 and forty adult male Wistar rats (Harlan, Barcelona, Spain), weighing 60–70 g and 200–225 g, respectively, were used as subjects in these experiments. The colony room was humidity controlled and maintained on a 12 h light/12 h dark cycle (lights on at 8 am). Pups remained housed with their respective dams until PND 21. On PND 21, pups were weaned and housed in groups of four rats per cage. Food and tap water were available ad libitum. All animal experiments were carried out in accordance with the guidelines approved by the European Community Council Directive (86/609/ECC) and by the Spanish R.D. 1201/2005.

Ethanol administration

All animals, 25-day old pups and adult rats, were given a single intraperitoneal (i.p.) injection of 25% (v/v) ethanol (3 g/kg) in isotonic saline, or saline, in a pattern where the injections (eight doses) were given on two consecutive days with gaps of two days without injections for two weeks. Specifically, adolescent animals (65 ± 9 g) were injected at 25, 26, 29, 30, 33, 34, 37 and 38 PND, as previously described (Pascual et al. 2007). Repeated ethanol treatment did not significantly affect the degree and the rate of body weight gain when compared with the saline control group. Thus, after eight injections of saline or ethanol administration, body weight was 140 ± 10 g for the saline group and 136 ± 8 g for the alcohol treated group.

In some experiments, animals received eight injections of saline followed by an i.p. injection of ethanol (3 g/kg) (acute ethanol) or saline (saline group in the microdialysis experiments). The total volume injected between the first and the last (8th) dose of 25% ethanol (3 g/kg) for the adolescent and adult animals was approximately 0.8–2 mL and 2.8–3.4 mL, respectively. In the acute ethanol or saline groups, the animals were habituated to i.p. injection to diminish the increase in DA in the NAc associated with stress from the injection.

Twenty-four hours after the last (8th) dose of saline or ethanol, animals were killed by decapitation, the brains from adolescent (PND 40) and adult rats were collected, and the cerebral cortex, hippocampus, striatum and NAc were dissected and stored at −80°C until use.

Alcohol self-administration

Voluntary ethanol consumption was performed in young adult animals (PND 60) which were treated with ethanol during juvenile/adolescence (PND 25 to PND 38), as described above. Specifically, the experiment started 3 weeks after the last (8th) saline or ethanol injection. The method for the acquisition of voluntary ethanol consumption followed the procedure described by Font et al. (2006). Briefly, alcohol presentation (24 h) commenced by increasing concentrations of ethanol every 2 days (2–10%, v/v), leaving an ethanol-free day between different concentrations (Stage1). Water was available ad libitum during all the phases of this experiment, and the alcohol drinking solutions were freshly prepared from 96% (v/v) ethanol diluted with tap water. The positions of the alcohol and water bottles were switched after every recording session to avoid any specific location preference. After 10 days of 24-h exposure to 10% (v/v) ethanol solution, with an ethanol-free day between 24-h ethanol sessions (Stage 2), animals were subjected to a forced ethanol-consumption paradigm for 1 week (Stage 3). During this week, the only liquid the animals had available was 10% (v/v) ethanol solution. Following this forced-access period, rats were returned to a 24-h two-bottle choice between ethanol (10%) and water for 1 week, leaving an ethanol-free day between 24-h ethanol sessions (Stage 4). During this week, alcohol preference scores (on days 1, 3, 5, 7) expressed as, 10% ethanol intake (mL)/total fluid intake (mL) × 100 were assessed. Afterwards, animals were switched to a limited-access procedure consisting of a 60-min ethanol (10%, v/v) exposure a day (at 7–8 pm), until intake reached stable levels (approximately 10 days) (Stage 5). Drinking bottles were removed at the end of the session, and the amount of fluid consumed was measured. The intake of total ethanol (g of pure ethanol per kg of body weight) was calculated as the daily average across the ten measuring days.

Blood ethanol measurements

Blood ethanol concentrations (BECs) were assessed in a separate group of adolescent and adult animals after a single dose of ethanol (3 g/kg i.p.). Periodically, tail blood samples were collected in heparinized tubes and centrifuged. Ethanol was determined using a spectrophotometric method (Sigma-Aldrich, Madrid, Spain). A single dose of ethanol to adolescent and adult animals resulted in a peak of BECs of 195 ± 12 mg/dL and 165 ± 35 mg/dL respectively, at 30 min post-injection.

Blood ethanol concentrations were also measured in adolescent and adult rats at Stage 4 (alcohol preference) and Stage 5 (limited-access) of the alcohol-self administration experiments. At Stage 4, BECs were determined between 8 and 9 am, while BECs were assessed immediately after the 60-min ethanol presentation at Stage 5 (limited-access procedure).

Intracranial microdialysis

At the end of ethanol treatment, and 24 h after the last ethanol injection, adolescent rats (PND 39) and adult rats were anesthetized using 2.5 g/L isophluorane inhalation and placed in a Kopft stereotaxis apparatus (David Kopf Instruments, Tujunga, CA, USA). A microdialysis guide (CMA/11) was placed unilaterally 1 mm above the shell of the NAc at the following coordinates for adolescent rats: AP +2.0, ML –0.6, DV –7.0 from duramater, and adult rats: AP +1.9, ML –1.0, DV –7.8 from duramater (Paxinos and Watson 1986; Fig. 2). The guide assembly was fixed to the skull with miniature stainless-steel screws and dental resin. After 48 h, microdialysis probes were lowered to the shell accumbens with 1 mm membrane tips and 0.24 mm o.d. The probe was perfused continuously with artificial cerebrospinal fluid (145 mM NaCl, 3.0 mM KCl, 2.26 mM CaCl2, 2 mM phosphate buffer, pH 7.4) at a constant rate of 2 μL/min. Six base-line samples were taken 2 h after the initial insertion; the final three samples were used to calculate the baseline neurotransmitter levels. Perfusate samples were collected in microcentrifuge tubes at 20-min intervals, which were placed on ice, and were either run immediately or stored at −80°C until analyzed to prevent degradation of neurotransmitters. Following the five baseline samples, animals received an injection of ethanol (3 g/kg, i.p.) or the equivalent volume of 0.9 g/L saline. After the drug challenge, sampling continued at 20 min intervals for an additional 140 min. After completing the dialysis experiments, rats were killed by decapitation. Brains were removed and frozen at −80°C. Brains were sliced on a cryostat into 25-μm sections on polysine-coated slides and inspected for probe placement in the NAc shell.

image

Figure 2.  The diagrams show the microdialysis probe location in the NAc shell for adolescent (a) and adult (b) rats according to Paxinos and Watson (1986). Only experiments with properly located microdialysis probes were included in the results. NAcC, NAc core; NAcS, NAc shell; CPU, caudate-putamen.

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Determination of DA and DOPAC

The content of DA and DOPAC in the microdialysis samples was analyzed by reverse-phase HPLC with electrochemical detection (Cauli et al. 2007). Samples were injected into a Rheodyne injector (20-μL loop), which was first run in a C18 pre-column (Waters Corp., Milford, MA, USA), and then in a 3.9 × 150-mm C18 pre-column with a particle size of 5-μm (Waters). The mobile phase consisted of 0.05 mM acetate/citrate buffer, 0.15 mM sodium EDTA, 0.4 mM sodium octyl sulfonate, 15 mL/L methanol, and 1.1 mM n-dibutylamine. Twenty microliter samples were manually injected with a Rheodyne 7125 injector (20-μL loop, Rheodyne Inc., Rohnert Park, CA, USA). The flow rate was kept at 1 mL/min. Theses conditions allowed monoamines to be detected within 12 min. Monoamines detection was performed by a colorimetric detector (Coulochem II Model 5200A; ESA). Chromatograms were processed using the Millennium 32 Waters software. The detection limit in 20 μL samples was 80 fmol/20 μL for DA.

Determination of glutamate

Glutamate contents in the microdialysis samples were analyzed using a Waters reverse-phase HPLC system with fluorescence detection and pre-column o-phtalaldehyde derivatization (Waters), as previously described (Canales et al. 2003). The HPLC system consisted of a solvent delivery system (Waters 515 pump) coupled to a fluorescence detector (Waters 474, excitation filter set at 340 nm and emission filter at 460 nm). Sample injections were performed using a Waters 717plus refrigerated Autosampler. Fifteen microliters of the collected dialysate samples was automatically derivatized by mixing with the working OPA solution and injecting into the HPLC system after 2 min. The column used was a reverse-phase C18, 5-μm particle size, 250 × 4.6 mm (ODS 2, Waters Spherisorb), and was maintained at a constant temperature. The chromatogram was performed with a gradient program of two mobile phases at a constant flow rate of 1 mL/min. Solution A was 95/5 (vol/vol) mixture of 50 mM sodium acetate buffer (pH 5.67) and methanol, to which 12.5 mL of isopropyl alcohol per liter were added; solution B was a 70/30 (vol/vol) methanol/water mixture. These conditions allowed amino acids to be detected within 24 min. Amino acids were quantified using the Millennium 32 software (Waters). Fresh standard solutions were prepared weekly and stored in the refrigerator at 4°C.

Immunoblot analysis

Brain tissue was homogenized in lysis buffer [10% Igepal CA-630 (Sigma-Aldrich, Madrid, Spain), 20 mM Tris-HCl pH8, 4 mM sodium chloride, 40 mM sodium fluoride and protease inhibitors]. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels, transferred to polyvinylidene fluoride membrane and incubated overnight at 4ºC with the following antibodies: anti-phospho-NMDAR2B (NR2B) (1 : 1000; Cayman Chem., Ann Arbor, MI, USA), anti-DRD1 (1 : 250; Santa Cruz Biotechnology, Madrid, Spain), anti-DRD2 (1 : 5000; Santa Cruz Biotechnology), anti-Lys9-acetyl-histone H3 (1 : 1000; Cell Signaling Technology, Hertfordshire, UK), anti-Lys12-acetyl-histone H4 (1 : 1000; Cell Signaling Technology), anti-GAPDH (1 : 5000; Chemicon, Hampshire, UK). Proteins were visualized either with alkaline phosphatase conjugate (Sigma-Aldrich) or an enhanced chemiluminescence system (ECL Plus, Amersham Pharmacia Biotech., Madrid, Spain). Band intensity of was quantified with the SigmaGel image analysis software version 1.0 (Jandel Scientific, Madrid, Spain).

Statistical analysis

The results are reported as mean ± SEM. The data obtained from alcohol shelf-administration were analyzed by two-way repeated anova measures. Unpaired Student’s t-test was used for comparison of the results obtained in control and ethanol-treated groups concerning body weight, western blot densitometric results, and basal levels of extracellular neurotransmitters. Two-way anova with Bonferroni’s post-hoc analysis were used for statistical analysis of BECs, extracellular neurotransmitter levels with time (time-course data), and between adolescent and adult animals in the levels of DRD1, DRD2, NR2B, or H3 and H4 acetylation.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Intermittent ethanol treatment during adolescence causes long-lasting increases on voluntary ethanol consumption

Our previous studies demonstrated that intermittent ethanol administration in juvenile/adolescent rats increases brain damage and causes long-term cognitive and behavioural consequences (Pascual et al. 2007). Therefore in order to extend our results, we evaluated whether intermitent ethanol administration during the juvenile/adolescent stage could predispose to alcohol consumption in later life. We analysed the alcohol consumption in young adult rats (PND 60) which had been treated with ethanol during juvenile/adolescence. For these experiments we measured alcohol consumption in a 5-stages paradigm. However, since no significant differences in either ethanol or water intake were found between saline- and ethanol-pretreated groups during Stages 1–3 (data not shown), the results on alcohol consumption and BECs at Stages 4 and 5 are shown only (Fig. 1), since these were the stages at which significant changes were observed.

image

Figure 1.  Intermittent ethanol treatment during adolescence induces voluntary ethanol consumption in adult animals. Rats received intermittent doses of ethanol (3 g/kg) or physiological saline over 2 weeks. Ethanol consumption was determined at the adult stage (PND 60), 3 weeks after the last ethanol administration. Adult animals were subjected to a procedure for the acquisition of voluntary ethanol consumption, which consisted of five stages (see Materials and Methods). Ethanol preference and ethanol intake were assessed at Stages 4 and 5, respectively. Stage 4: Ethanol preference and water intake were given in a 24-h two-bottle choice between ethanol (10%) and water for 1 week (days 1, 3, 5 and 7). On even days, only water was provided. Stage 5: 60-min restricted ethanol access. BECs of both stages are shown. Data given are means ± SEM of 8–10 animals.

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Figure 1 reveals a significant effect of sample with pre-treatment on ethanol preference (Stage 4) [F(1,48) = 19.03, p < 0.01], with no significant interactions being observed between pre-treatment and days [F(3,48) = 0.2184, p > 0.05]. Correspondingly, water intake on the days in which ethanol preference was being tested, was also significantly affected by pre-treatment [F(1,48) = 29.96, p < 0.01], again not showing any significant interactions between pre-treatment and days [F(3,48) = 0.6777, p > 0.05]. To further document alcohol intake, we determined BECs. The data revealed higher values for BECs in the ethanol-pretreated group than in saline-control group {significance for pre-treatment proven with anova [F(1,12) = 43.14, p < 0.01], which did not reveal significant interaction between pre-treatment and days of BEC determination [F(2,12) = 3.623, p > 0.05]}.

Figure 1 also represents the voluntary ethanol consumption for 60 min a day on 10 consecutive days (Stage 5). The two-way repeated anova measures (pretreatment group x day) revealed that the two main factors were statistically significant, pre-treatment group [F(1,12) = 7.2, p < 0.01] and day [F(9,108) = 14.24, p < 0.01], although there were no interactions between them. Assessment of BECs after 60-min ethanol presentation indicated significant main effect of pre-treatment group [F(1,20) = 61.31, p < 0.01], although there were no significant interactions between both variables [F(4,20) = 0.8747, p > 0.05].

The mean body weights from the first to the last week of voluntary ethanol consumption were similar in both groups of animals [PND 60: ethanol group: 143.50 ± 18.57 g vs. control group: 156.33 ± 12.66 g; t(1.615), p > 0.05; PND 105: ethanol group: 377.25 ± 29.99 g vs. control group: 362.67 ± 18.58 g; t(1.169), p > 0.05]. These results indicate that the ethanol-pre-treated group voluntarily consumed more ethanol than the saline pre-treated group, and that both groups changed their consumption over time in the same way.

Changes in DA and glutamate levels in nucleus accumbens shell of adolescent animals after ethanol administration: effects of multiple binge alcohol treatment

By using the microdialysis technique, we next evaluated the levels of DA, DOPAC and glutamate in the NAc shell after a challenge dose of ethanol (3 g/kg) to adolescent animals, which were pre-treated with either saline or ethanol during juvenile/adolescence stage. As Fig. 3 shows, acute ethanol administration significantly increased DA at 20 min in the adolescent saline group, while the levels of DA in the ethanol exposed group remained significantly elevated at 20–60 min post-injection (Fig. 3a). The overall analysis of the data revealed a significant effect of samples with time [F(7,72) = 16.42, p < 0.01], with treatment [F(2,72) =9.726, p < 0.01] and sample with treatment and time [F(14,72) = 5.021, p < 0.01]. Notably, we also observed that the baseline DA levels were significantly higher in animals treated with repeated ethanol injections than in the control saline animals [12.93 ± 2.04 nM vs. 6.92 ±1.25 nM; t(1.902), p < 0.05]. Furthermore, administration of ethanol increased the DOPAC levels in the NAc at 20 min post-injection in those animals pre-treated with ethanol, but no increase was observed in pre-treated saline group. The overall analysis of DOPAC revealed a non-significant time × treatment interactions [F(14,72) = 1.283, p > 0.05] in the extracellular levels of DOPAC in the NAc shell of adolescent animals (Fig. 3b). The basal DOPAC levels were not significantly different between animals pre-treated with saline or ethanol [81.86 ± 7.77 nM vs. 74.44 ± 10.18 nM; t(0.5691), p > 0.05].

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Figure 3.  Time-course effects of a challenged dose of saline or ethanol (3 g/kg, i.p.) on the extracellular levels of DA (a, d), DOPAC (b, e) and glutamate (c, f) in the NAc shell of adolescent (a–c) and adult (d–f) rats treated during the juvenile/adolescent period with saline (saline and acute ethanol group) or with intermittent ethanol (3 g/kg) (repeated ethanol) doses. All the results are shown as means ± SEM (n = 7).

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Intermittent ethanol treatment decreased glutamate levels in the NAc at 60–100 min post-injection (Fig. 3c). The overall analysis of glutamate revealed only a significant effect of sample with treatment [F(2,72) = 7.115, p < 0.01]. Analysis of the time-course data in the extracellular glutamate levels between the acute and the repeated ethanol treatment revealed a main effect of treatment [F(1,72) = 15.42, p < 0.01]. The analysis of baseline glutamate levels showed that these levels significantly increased in animals pre-treated with repeated ethanol injections when compared to saline animals [1530 ± 216.2 nM vs. 630.2 ±116.8 nM; t(3.273), p > 0.05].

Changes in DA and glutamate levels in nucleus accumbens shell of adult rats after ethanol challenged: effects of multiple alcohol treatment

To evaluate the potential differential dopaminergic response to ethanol in adolescent and adult animals, we assessed the levels of DA, DOPAC and glutamate in the NAc shell of adult animals after a challenge dose of ethanol (3 g/kg, i.p.). The results show that repeated administrations of ethanol (3 g/kg) in adult rats significantly increased the levels of DA and DOPAC in the NAc at 20–40 min and 20 min, respectively, while acute ethanol treatment increased the DA levels only at 20 min, and the DOPAC levels at 140 min (Fig. 3d and e). The overall analysis of DA revealed a significant effect of sample with time [F(7,72) = 5.962, p < 0.01] and sample with treatment with time [F(14,72) = 3.953, p < 0.01]. Furthermore, the overall analysis of DOPAC revealed a significant effect of sample with treatment [F(2,72) = 22.50, p < 0.01], with time [F(7,72) =3.043, p < 0.05] and sample with treatment and time [F(14,72) = 2.698, p < 0.01]. Analysis of the time-course data in the extracellular DA and DOPAC levels between the acute and the repeated treatments revealed no significant main effects (all p > 0.05). Analysis of the baseline DA and DOPAC levels in adult animals indicated no significant differences between animals treated with saline or repeated ethanol injections [DA: 6.05 ± 2.39 nM vs. 2.69 ± 1.17 nM; t(1.144), p > 0.05; and DOPAC: 44.56 ± 6.42 nM vs. 56.98 ± 11.11 nM; t(1.035), p > 0.05].

Acute ethanol treatment, either in saline or ethanol pre-treated animals, decreased glutamate levels at 40 min and 100 min post-injection, respectively (Fig. 3f). The overall analysis of glutamate revealed a significant effect of sample with time [F(7,72) = 2.376, p < 0.05] and sample with treatment with time [F(14,72) = 2.503, p < 0.01]. Again, the analysis of the time-course data in the extracellular glutamate levels between the acute and the repeated treatment revealed no significant main effects (all p > 0.05). Analysis of baseline glutamate levels indicated no significant differences between animals treated with either saline or repeated ethanol injections [784.8 ± 118.4 nM vs. 795.9 ± 126.4 nM; t(0.0638), p > 0.05].

Finally to determine differences in ethanol sensitivity between adolescents and adults, two-way anovas were conducted on the time-course and baseline data in both ethanol treatments (acute and repeated). The acute and repeated ethanol treatments increased DA levels at 20 min post-injection by 169% and 163%, respectively, in adolescent animals when compared to adults (Fig. 3a and d). The analysis of DA in the acute treatment revealed a significant effect of time [F(7,48) = 3.351, p < 0.01], although there were no significant interactions. The data of the repeated treatment showed a main effect of time [F(7,64) = 3.776, p < 0.01]. Analysis of the baseline DA levels revealed a significant effect of age and treatment [F(1,28) = 4.387, p < 0.05]. Furthermore, acute and repeated ethanol treatments decreased the glutamate levels at 20 min post-injection by 50.76% and 47.61%, respectively, when comparing values of adolescents with adults. However, the analysis of glutamate revealed no significant main effects (all p > 0.05) in the acute treatment and a main effect of age [F(1,64) = 10.15, p < 0.01] in the repeated ethanol administration (Fig. 3c and f). The analysis of the baseline glutamate levels also revealed a significant effect of age and treatment [F(1,20) = 55.08, p < 0.01].

Effect of intermittent ethanol treatment on DA and NR2B-NMDA receptors in different brain areas of adolescent and adult animals

Since changes in glutamatergic and dopaminergic neurotransmission and in the receptor density occur during adolescence (Anderson et al. 1997; Spear 2000; Tarazi and Baldessarini 2000), and both systems are important for drug addiction, we next investigated whether intermittent ethanol exposure during the juvenile/adolescence stages could affect the neurochemical remodelling contributing to the long-term behavioural consequences and to the vulnerability of adolescence to drug addiction. We therefore evaluated the levels of DA receptors D1 and D2 (DRD1, DRD2), as well as the phosphorylated NR2B subunit of the NMDA receptor in frontal cortex, hippocampus, striatum and NAc of both adolescent and adult rats treated intermittently with either saline or ethanol.

Western blotting analysis was used to assess the levels of these receptors. Figure 4 shows that the relative levels of DRD1, DRD2 and phosphorylated-NR2B, in most of the brain areas analysed, were higher in adolescents than in adults. Repeated doses of ethanol significantly decreased the protein expression of DRD1 from frontal cortex [t(4.120), p < 0.05] and the levels of DRD2 from frontal cortex [t(8.686), p < 0.01], striatum [t(6.687), p < 0.01] and NAc [t(5.210), p < 0.01] of adolescent rats, while no significant changes in the levels of these receptors were observed in adult animals with the same ethanol treatment (all p < 0.05) (Fig. 4a and b). However, the levels of DRD2 significantly decreased in hippocampus from both adolescent [t(6.660), p < 0.01] and adult [t(7.817), p < 0.01] animals treated with repeated doses of ethanol (Fig. 4a and b).

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Figure 4.  Western blotting and densitometric analysis of DRD1, DRD2 and phospho-NR2B (NR) in frontal cortex, hippocampus, striatum and NAc, obtained 24 h after the last ethanol or saline administration to the adolescent (a) and adult (b) animals treated intermittently with eight doses of physiological saline (S) or ethanol (3 g/kg) (RE) for 2 weeks. Blots were reproved with anti-GAPDH to show equal protein loading. A representative immunoblot is shown. Data are the mean ± SEM of four independent experiments. *p < 0.05, **p < 0.01, significant difference with respect to saline group.

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To compare if the effects of ethanol treatment on DRD1 and DRD2 levels in given brain areas were different in adolescent and adult animals, two-way anovas were conducted. The analysis of DRD1 demonstrated a significant effect of age and treatment in frontal cortex [F(1,8) = 16.98, p < 0.01], hippocampus [F(1,8) = 65.65, p < 0.01], striatum [F(1,8) = 19.63, p < 0.01] and NAc [F(1,8) = 7.128, p < 0.05], while the anova of the DRD2 data revealed a significant effect of age and treatment in frontal cortex [F(1,8) = 41.52, p < 0.01] and striatum [F(1,8) = 29.60, p < 0.01].

Because NMDA receptor is a major target of ethanol actions, and some of the effects of ethanol on this receptor are, in part, mediated by the regulation of the NMDA subunits composition and phosphorylation (Ron 2004), therefore we wondered whether the intermittent ethanol treatment in adolescent rats could modulate the phosphorylation state of the NR2B in different brain areas. As shown in Fig. 4(a), intermittent ethanol treatment during juvenile/adolescence stage significantly decreased the phosphorylation of NR2B in frontal cortex [t(5.220), p < 0.01], hippocampus [t(4.795), p < 0.01] and NAc [t(4.851), p < 0.01] when compared with untreated animals. No changes in the NR2B-phosphorylation were observed with repeated ethanol treatment in adult rats (all p > 0.05) (Fig. 4b). These results indicate that ethanol exposure during the adolescent period induces a brain region-specific inhibition of NR2B, and also suggest the sensitivity of the adolescence brain to the inhibitory effects of ethanol on the NR2B-NMDA receptors.

To determine differences between adolescent and adult animals in the levels of NR2B, two-way anovas were assessed in different brain areas of saline and ethanol-treated groups. The analysis revealed a significant effect of age and treatment in frontal cortex [F(1,8) = 11.07, p < 0.05], hippocampus [F(1,8) = 20.11, p < 0.01] and NAc [F(1,8) =8.044, p < 0.05].

Repeated alcohol treatment induces histone modification in adolescent rats, but not in adult rats

Because dopaminergic and glutamatergic inputs can induce chromatin remodelling in striatal neurons (Li et al. 2004; Schroeder et al. 2008) and recent evidence suggests that histone acetylation and chromatin-remodelling events, are involved in drug-related behavioural sensitization and reward (Schroeder et al. 2008), we next evaluated whether intermittent ethanol treatment during juvenile/adolescence could induce alterations to histone acetylation, causing permanent behavioural consequences, such as problems with alcohol abuse. For this propose, we measured the histone H3 (Lys9) and H4 (Lys12) acetylation in frontal cortex, hippocampus, striatum and NAc of adolescent and adult animals (Fig. 5).

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Figure 5.  Western blotting and densitometric analysis of the acetyl-histone H3 (Lys9) and the acetyl-histone H4 (Lys12) in frontal cortex, hippocampus, striatum and NAc, obtained 24 h after the last ethanol or saline administration to the adolescent (a) and adult (b) animals treated intermittently with eight doses of physiological saline (S) or ethanol (3 g/kg) (RE) for 2 weeks. Blots were reproved with anti-GAPDH to show equal protein loading. A representative immunoblot is shown. Data are the mean ± SEM of four independent experiments. *p < 0.05, **p < 0.01, significant difference with respect to saline group.

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Intermittent ethanol treatment significantly increased the acetylation of histones H3 and H4 in frontal cortex [H3: t(8.116), p < 0.01; H4: t(5.995), p < 0.01] and NAc [H3: t(6.857), p < 0.01; H4: t(3.549), p < 0.05] of adolescent animals without affecting the histone levels in hippocampus [H3: t(2.507), p > 0.05; H4: t(0.730), p > 0.05] (Fig. 5a). Strikingly, the same ethanol treatment significantly decreased the acetylations of both histones in striatum [H3: t(3.594), p < 0.05; H4: t(4.828), p < 0.01] (Fig. 5a). No changes in the acetylation of these histones were observed with repeated ethanol treatment in adult rats (all p > 0.05) (Fig. 5b). Two-way anova analysis was conducted to compare the effects of ethanol on H3 and H4 levels in adolescent and adult animals. The analysis of H3 revealed a significant effect of age and treatment in frontal cortex [F(1,8) = 17.42, p < 0.01], hippocampus [F(1,8) = 8.164, p < 0.05] and NAc [F(1,8) =11.61, p < 0.01], while the analysis of H4 showed a significant effect of age and treatment in striatum [F(1,8) = 13.40, p < 0.01].

These findings suggest that intermittent ethanol treatment during adolescence, but not in adults, induce changes in histone acetylation in some brain areas which might underlie the long-lasting neurobehavioral alterations observed in both previous results (Pascual et al. 2007) as well as in the present findings.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Using a binge-like ethanol administration paradigm designed to model the pattern of human adolescent alcohol use, the present study demonstrates that intermittent ethanol administration during the juvenile/adolescent period alters subsequent ethanol preference and intake. Our findings suggest that sensitization of the dopaminergic and glutamatergic systems could be involved in alcohol consumption, since although ethanol elicits a similar DA response in the NAc of both adolescent and adult animals pre-treated with multiple doses of ethanol, the basal extracellular DA levels are higher in adolescent than in adult animals pre-treated with ethanol. Repeated ethanol administration also down-regulates the expression of both DRD2 and NMDAR2B phosphorylation in some brain areas and alters histone acetylation in adolescent animals, but not in adult rats, effects that could participate in long-lasting neurobehavioral alterations and addictive-like behaviours.

A number of studies have demonstrated the vulnerability of the peri-adolescence/adolescence periods to ethanol-induced brain damage (Medina et al. 2007, 2008) and to the increased risk of alcohol-dependence and drug addiction (Anthony and Petronis 1995; Breslau and Peterson 1996; Patton et al. 2004). Our previous studies demonstrated that intermittent ethanol treatment during juvenile/adolescence results in hippocampal and cortical damage, and that these effects were associated with long-term cognitive and behavioral consequences (Pascual et al. 2007). The present results, although limited in scope since only one ethanol dose (3 g/kg) was studied, show that repeated ethanol administration during adolescence alters subsequent ethanol intake and preference, corroborating experimental and human epidemiological data (Grant and Dawson 1997; Hawkins et al. 1997; DeWit et al. 2000; Doremus et al. 2005; Vetter et al. 2007).

The neurobiological basis of adolescents’ vulnerability to develop alcohol abuse and addiction is not understood. However, if we consider that the brain undergoes prominent reorganization during adolescence (see review Spear 2002), it is possible that alcohol could sensitize brain regions and/or critical processes involved in the reinforcing effects of addictive drugs (e.g. mesocorticolimbic neural circuits) to increase the risk of alcohol-related problems later in life. The activation of the mesocorticolimbic DA transmission is involved in the rewards and the reinforcing actions of ethanol and other drug of abuse (Koob and Weiss 1992; Robbins and Everitt 2002). Ethanol dose-dependently increases DA release in the NAc shell, a region which has been attributed a fundamental role in the mechanism of drug addiction (Bassareo et al. 2003; Quintanilla et al. 2007). Here we show that although ethanol elicits a similar prolonged DA response in the NAc of both adolescent and adult animals pre-treated with multiple doses of ethanol, the levels of DA tend to be higher in adolescent (∼ 160%) than in adult animals, although there were no statistical differences between both groups. However, the accumbal DA baseline levels were significantly higher in adolescent than in adult animals treated with repeated ethanol injections. According with these results high extracellular basal levels of DA have been reported in adult animals which were chronically exposed to ethanol during adolescence (Badanich et al. 2007). A prolonged increase in the extracellular DA levels has been also shown in alcohol-preferring rats exposed to ethanol from PND 30 to PND 60, when compared with saline treated animals (Sahr et al. 2004). These results suggest that the high activity and sensitization of the dopaminergic system occurring during adolescence could mediate the increased likelihood of engaging in drug use initiation during adolescence. Supporting this fact, we found that the levels of both the basal extracellular DA and DA receptors, D1 and D2, are higher in adolescents than in adults, thus corroborating previous studies which demonstrated that the mesolimbic DA systems exhibit a pattern of overproduction throughout adolescence, and then declining in the adult stage (Spear 2000; Philpot and Kirstein 2004). Stressors of adolescence might also contribute to the high sensitization of the mesocorticolimbic DA system and the initiation of ethanol during adolescence (see review Spear 2002), since stressors can selectively activate the mesocorticolimbic DA projections (Wise 1996; Robbins and Everitt 2002). Nevertheless, a recent study demonstrates no association among early alcohol consumption and novelty seeking, anxiety and stress hormone levels in adolescent rat (Schramm-Sapyta et al. 2008), suggesting that not only stressors mediate the high alcohol consumption and the relapse-like behaviour in adolescent animals.

The present findings also show that repeated ethanol treatment decreases the levels of DRD2 in the prefrontal cortex, hippocampus, striatum and NAc of adolescent rats. Reductions in the levels of DRD2 have been reported in ethanol-preferring rats (Kanes et al. 1993; McBride et al. 1993), while the over-expression of DRD2 reduces both alcohol preference and intake (Thanos et al. 2001, 2004, 2005), suggesting that these receptors are involved in alcohol abuse. Indeed, DRD2 polymorphism has been associated with severe of alcohol dependence (Connor et al. 2002).

Glutamatergic transmission is vital for the control of the ventral tegmental area and may also be critical for the weighting of the novelty and importance of a stimulus, an essential output of this brain region. Considerable evidence demonstrates that glutamate-mediated excitatory neurotransmission and, particularly the NMDA receptor, play an important role in mediating the many behavioural actions of ethanol (see review, Krystal et al. 2003), and that the NMDA receptor subunits NR2A and NR2B, which play a key role in synaptic plasticity during brain development (Barria and Malinow 2002) are especially sensitive to ethanol inhibition (Masood et al. 1994). Our results demonstrate a differential sensitivity of ethanol-induced inhibition of NR2B-NMDA activity between adolescent and adult animals, since we found that while intermittent ethanol treatment decreases NR2B phosphorylation in prefrontal cortex, hippocampus and NAc of adolescent animals, no changes in the NR2B-phosphorylation were observed in adult animals. A recent study (Weitlauf and Woodward 2008) suggests that the ethanol-induced inhibition of the NMDA receptor activity selectively attenuates the NMDA-mediated transmission in the prefrontal cortex, effects which might be involved an abnormal function in the prefrontal cortex of alcoholics, and the impulsive behavior and lack of control over drinking that characterizes this disorder. Interestingly, prefrontal cortex reductions and abnormalities also occur in human adolescents with alcohol use disorders (Medina et al. 2008), and these abnormalities could be enhanced by the inhibition of the NR2B-NMDA activity, thus contributing to loss of control and alcohol addiction.

The results above suggest that alcohol exposure during adolescence can sensitize the mesocorticolimbic DA system to induce alterations in the dopaminergic and glutamatergic neurotransmissions which might affect the remodelling and functions of the adolescent brain. Strikingly, changes in dopaminergic and glutamatergic inputs have been shown to induce chromatin remodelling by histone modifications (Li et al. 2004; Schroeder et al. 2008) and these events have been associated with drug-related behavioural sensitization and reward (Li et al. 2004; Kumar et al. 2005; Fischer et al. 2007; McClung and Nestler 2008; Schroeder et al. 2008). Here we show that intermittent binge ethanol administration also induced changes in the acetylation of the histones H3 and H4 in frontal cortex, NAc and striatum. Therefore, on the bases of these results it is temping to speculate that ethanol-induced alterations in the dopaminergic and glutamatergic systems during adolescence can induce histone modifications, and these events participate in the long-term behavioural alterations induced by early alcohol consumption. Supporting this hypothesis, a recent study demonstrated that cocaine administration to adolescent rats reduced histone H3 methylation and causes long-term behavioural consequences (Black et al. 2006). Although little is known about the relation between binge ethanol and epigenetic mechanisms, it has been recently demonstrated that chromatin remodelling in the amygdala is also involved in chronic ethanol exposure and withdrawal effects (Pandey et al. 2008).

In summary, the evidence presented here suggests that intermittent ethanol administration during adolescence induces changes in dopaminergic and glutamatergic neurotransmissions. These changes could induce chromatin remodelling and abnormal plasticity in reward-related learning processes, events which might contribute to the long-lasting consequences of alcohol abuse and the vulnerability of adolescents to drug addiction.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We would like to thank M. March and E. Fernandez for their excellent technical assistance. We also thank M. Correa for her valuable help in the statistical analysis of the data on alcohol-self administration. This work was supported by General Direct. of Drugdependence (GV), The Spanish Ministry of Health (PNSD, G46923421), the Institute Carlos III, RTA Network (G03/005), Fundación de Investigación Médica Mutua Madrileña and the Spanish Ministry of Education and Science (SAF 2006-02178).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References