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. Author manuscript; available in PMC: 2016 Sep 1.
Published in final edited form as: Alcohol Clin Exp Res. 2015 Aug 6;39(9):1680–1690. doi: 10.1111/acer.12819

CaMKIIα-GluA1 activity underlies vulnerability to adolescent binge alcohol drinking

Abigail E Agoglia 1,2, Sarah E Holstein 1, Grant Reid 1, Clyde W Hodge 1,2,3,4
PMCID: PMC4558330  NIHMSID: NIHMS702266  PMID: 26247621

Abstract

Background

Binge drinking during adolescence is associated with increased risk for developing alcohol use disorders (AUDs); however, the neural mechanisms underlying this liability are unclear. In this study, we sought to determine if binge-drinking alters expression or phosphorylation of two molecular mechanisms of neuroplasticity, calcium/calmodulin dependent kinase II alpha (CaMKIIα) and the GluA1 subunit of AMPA receptors (AMPAR) in addiction-associated brain regions. We also asked if activation of CaMKIIα-dependent AMPAR activity escalates binge-like drinking.

Methods

To address these questions, CaMKIIαT286 and GluA1S831 protein phosphorylation and expression were assessed in the amygdala and striatum of adolescent and adult male C57BL/6J mice immediately after voluntary binge-like alcohol drinking (blood alcohol > 80mg/dL). In separate mice, effects of the CaMKIIα-dependent pGluA1S831-enhancing drug tianeptine were tested on binge-like alcohol consumption in both age groups.

Results

Binge-like drinking decreased CaMKIIαT286 phosphorylation (pCaMKIIαT286) selectively in adolescent amygdala with no effect in adults. Alcohol also produced a trend for reduced pGluA1S831 expression in adolescent amygdala but differentially increased pGluA1S831 in adult amygdala. No effects were observed in the nucleus accumbens or dorsal striatum. Tianeptine increased binge-like alcohol consumption in adolescents but decreased alcohol consumption in adults. Sucrose consumption was similarly decreased by tianeptine pretreatment in both ages.

Conclusions

These data show that the adolescent and adult amygdalae are differentially sensitive to effects of binge-like alcohol drinking on plasticity-linked glutamate signaling molecules. Tianeptine-induced increases in binge-like drinking only in adolescents suggest that differential CaMKIIα-dependent AMPAR activation may underlie age-related escalation of binge drinking.

Keywords: Adolescence, alcohol, binge, CaMKII, GluA1

Introduction

Adolescence is a distinct developmental period that in humans occurs from the early teens to the early twenties. This developmental stage is characterized by increased exploration and risk-taking, and is the time at which experimentation with drugs of abuse, including alcohol, is usually initiated (Spear, 2000). Alarming data show that 25 – 37% of U.S. high school students engage in heavy episodic or binge drinking (Miller et al., 2007) at more than twice the rate of adults (Nelson et al., 2009), and a recent survey revealed that approximately 22% of 12th grade students had engaged in binge drinking in the last two weeks (Johnston LD, 2012). Binge drinking during adolescence is particularly troubling in light of evidence that individuals who initiate alcohol use during early adolescence are at substantially higher risk for the development of alcohol use disorders (AUDs) than those who initiate alcohol use as young adults (Hingson et al., 2006, Grant and Dawson, 1998). These data suggest that adolescent alcohol users are uniquely vulnerable to the development of AUDs, and indicate that a significant portion of the population is subject to this increased risk. However, the precise neurobiological mechanisms that mediate this effect have yet to be determined (Schramm-Sapyta et al., 2008).

In humans and in rodent models, adolescence is characterized by increased neuronal plasticity as the brain matures from childhood to adulthood (Selemon, 2013). This suggests that adolescents may be more vulnerable to the effects of alcohol on plasticity-linked cellular functions. Indeed, evidence indicates that adolescent rodents are more sensitive to alcohol-induced disruption of long-term potentiation (Pyapali et al., 1999), which is a cellular mechanism that underlies behavioral plasticity. Similarly, we have shown that adolescent mice are more sensitive than adults to acute alcohol-induced changes in ERK MAP kinase activation in the amygdala (Spanos et al., 2012) which is required for LTP in this region (Apergis-Schoute et al., 2005). Further, alcohol exposure blunts LTP in the amygdala (Stephens et al., 2005) where plasticity-linked proteins regulate alcohol-seeking behavior (Salling et al., 2014, Schroeder et al., 2008). Thus, increased understanding of alcohol-induced changes in signaling systems that regulate cellular plasticity may be of interest to identify mechanisms underlying adolescent vulnerability to development of AUDs.

Glutamate is the primary excitatory neurotransmitter in the mammalian brain and its receptors and cellular signaling pathways are required for adaptive plasticity. Calcium/calmodulin-dependent protein kinase II (CaMKII) is a 12-subunit protein expressed primarily in glutamatergic synapses, where it has a well-established role in synaptic plasticity, learning and memory (Lisman et al., 2002). CaMKII has been shown to be involved in alcohol consumption and reward. Mice expressing autophosphorylation-deficient CaMKIIα have been shown to drink less alcohol than wild type littermates (Easton et al., 2013a) and display altered conditioned place preference for alcohol (Easton et al., 2013b). We have shown that voluntary alcohol drinking increases expression of the active (phosphorylated) form of CaMKIIαT286 (pCaMKIIαT286) in the mouse amygdala, and that intra-amygdala inhibition of CaMKII activity reduces the positive reinforcing effects of alcohol (Salling et al., 2014). CaMKII is activated upon phosphorylation, allowing it to phosphorylate several downstream targets including the GluA1S831 subunit of the AMPA subtype of glutamate receptors (Derkach et al., 1999). Phosphorylation of GluA1S831 (pGluA1S831) is associated with increased stability of AMPARs in the synapse, which promotes synaptic plasticity and learning (Lee et al., 2010). Our work also shows that AMPAR activity in the amygdala is required for the reinforcing effects of alcohol (Salling et al., 2014). Together, these data suggest a critical role for CaMKIIα-GluA1 signaling in alcohol self-administration and reinforcement. However, age-dependent differences in sensitivity to alcohol-induced modifications of this system have not been explored.

To address this question, the present study was designed to test the hypothesis that adolescents are more sensitive to alcohol-induced changes in CaMKIIα and AMPAR GluA1protein phosphorylation and expression than adults. We chose to investigate protein changes in the amygdala and striatum based on our previous results as well as a wealth of literature indicating that these regions are functionally involved in alcohol self-administration and reinforcement (Hodge et al., 1992, Salling et al., 2014). To evaluate the functional involvement of this system in adolescent and adult alcohol drinking, we used the drug tianeptine, a systemic upregulator of CaMKIIa-dependent GluA1 activation (e.g., phosphorylation). A binge-like model of alcohol access was utilized in these experiments in order to most closely mimic the drinking patterns exhibited by adolescents in the United States.

Materials and Methods

Animals

Male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were individually housed in standard Plexiglass cages with a small PVC pipe for environmental enrichment. Adolescent mice were postnatal day 21 (PND 21) and adults were (PND 63±2) upon arrival in our facility. Food and water were available ad libitum in home cages except where noted. The colony room was maintained on a 12-h light/dark cycle (lights on at 20:00 in the initial binge exposure experiment and lights on at 19:00 in the pharmacology experiments) at 21°C. All experimental manipulations and testing occurred during the dark cycle. All procedures were carried out in accordance with the NIH Guide to Care and Use of Laboratory Animals (NRC, 1996) and approved by the Internal Review Board of the University of North Carolina, Chapel Hill.

Experiment 1: Daily Binge Alcohol Exposure and Protein Changes in the Adolescent and Adult Mouse Brain

Binge Alcohol Exposure Procedure

Adolescent and adult mice (N=40) were allowed one week (PND 21–27) or (PND 63–69) to habituate to our colony and acclimate to the light/dark cycle (Fig 1A). During habituation, mice were handled and weighed daily. Mice were then brought to consume alcohol in a binge-like access procedure adapted from Rhodes et al. (2005). Beginning on PND 28 or PND 70, mice were weighed and home cage water bottles were removed at 11:00AM and replaced with a single drinking tube containing either 20% (v/v) alcohol (alcohol treated animals) or water (water control animals) for four hours, n=10/treatment/age. The drinking tubes consisted of a 10mL serological pipette fitted with a ball-bearing sipper tube and fastened to the wire cage lid with a medium binder clip. This limited access procedure was repeated daily for two weeks, ending on PND 42 for adolescents or PND 84 for adults.

Figure 1. Adolescent and adult mice achieve binge levels of alcohol consumption in a daily limited access procedure.

Figure 1

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(A) Timeline of binge-like drinking procedure. Adolescent and adult alcohol (B) and water (C) intake did not differ over two weeks of daily drinking sessions. (D) Alcohol drinking did not alter body weight in either adolescents or adults. (E) Blood ethanol concentration did not differ between alcohol-drinking adolescent and adult mice immediately following the drinking session on the last drinking day. Dashed line indicates the NIAAA criteria for a binge drinking session (≥ 80 mg/dL).

Tissue Collection

Immediately after the cessation of drinking, on the last day of testing, mice were rapidly decapitated and blood alcohol concentration (BAC) was assessed. Approximately 20µL of trunk blood was centrifuged to obtain 5µL of plasma for use in an AM1 Alcohol Analyzer (Analox Instruments, Lunenburg, MA). The brains were extracted from the skulls and flash-frozen in −30°C isopentane.

Western Blot Analysis

Brain regions of interest were dissected from coronal brain sections (1mm slices) using a 1mm sterile tissue punch (Stoelting, Wood Dale, IL) and homogenized in buffer (10ml: 1.1g sucrose, 50µL 1M HEPES buffer, 1:100 protease/phosphatase inhibitor cocktail, 1mL 10% SDS, 26.55mL ultra-pure water.) Protein concentration was measured using a calorimetric assay kit (Pierce Biotechnology, Rockford, IL). Protein (5µg) was diluted 4:1 with lithium dodecyl sulfate sample buffer (40–70% glycerol), 10:1 with sample reducing agent, vortexed, loaded onto a Nu-Page 4–15% Tris-glycine polyacrylamide gel (Invitrogen, Carlsbad, CA) for gel electrophoresis separation, and transferred to nitrocellulose membrane using an iBlot dry blotting system (Life Technologies, Grand Island, NY). Membranes were blocked with 3% albumin bovine serum (Sigma-Aldrich, St. Louis, MO) before being incubated with primary antibodies [rabbit anti-pCaMKIIT286 (1:2 500; Abcam, Cambridge, MA), -pGluR1S831 (1:1 000, EMD Millipore, Billerica, MA) -tGluR1 (1:1 000; Abcam) and mouse anti-tCaMKIIα (1:10 000; Millipore), in blocking solution at 4°C overnight and β-actin (mouse monoclonal, 1:5 000; Sigma) in blocking solution 1 h at room temperature] and washed before incubation with secondary antibodies (HRP-conjugated goat anti-rabbit and goat anti-mouse, 1:10 000; Jackson ImmunoResearch, West Grove, PA). Membranes were then visualized using enhanced chemiluminescence substrate (Pierce), and bands were quantified using optical density measurements (NIH/Scion Image).

Experiment 2: Pharmacological Manipulation of Intermittent Binge Drinking

Binge Alcohol or Sucrose Drinking Procedure

Mice (N=24/experiment) were allowed a one-week (PND 21–27) or (PND 63–69) habituation period (Fig 4A). During habituation, mice were handled and weighed daily and given saline injections to habituate to the injection procedure. Beginning on PND 28 (adolescent) or PND 70 (adult), mice were weighed and the home cage water bottles were removed at 10:00AM and replaced with a drinking tube containing 20% (v/v) alcohol (alcohol experiment) or 0.5% sucrose (sucrose experiment). Mice had access to alcohol or sucrose for four hours every other day. Beginning on PND 36 or PND 80, mice were treated with 0, 3, 10 or 17 mg/kg tianeptine (i.p.) 30 minutes prior to alcohol or sucrose tube access according to a Latin square dosing regimen. Drug pretreatment and drinking continued on PND 38, 40, and 42 (adolescent) or PND 82, 84, and 86 (adult). Tianeptine dose range and pretreatment time were determined from a study showing behavioral effects in alcohol-exposed rodents (Uzbay et al., 2006).

Locomotor Testing

On PND 46 or PND 90, mice were pretreated with either saline or the effective dose of tianeptine (10 mg/kg for adolescents, 17 mg/kg for adults) for 2 hours prior to a locomotor activity test (n=6/treatment/age). Open field activity was measured in Plexiglas activity monitor chambers (27.9 cm2; ENV-510, Med Associates, Georgia, VT). Two sets of 16 pulse-modulated infrared photobeams were located on opposite walls and recorded X–Y ambulatory movements. Distance traveled (in meters) throughout the session was quantified by assessing the mouse’s position in the open field every 100 miliseconds. Data from each chamber were collected by a computer.

Blood Alcohol Clearance

On PND 47–48 or PND 91–92, adolescent (n=6) and adult (n=6) mice were pretreated with the effective dose of tianeptine (10 mg/kg for adolescents, 17 mg/kg for adults) or saline. Thirty minutes later, mice were injected with 4.0 g/kg alcohol (20% w/v) i.p. Beginning at 10 minutes post-alcohol injection, mice were confined in clear Plexiglas restraint tubes (Braintree Scientific, Braintree, MA), tail blood was collected using a heparinized microcapillary tube. Additional samples were collected at 30 minutes, 1 hour, 2 hours and 4 hours post-alcohol injection. Blood samples were analyzed as in Experiment 1.

Drugs

Alcohol solutions (v/v) were prepared by diluting 95% ethanol (Pharmco Products Inc., Brookfield, CT) with tap water (for drinking) or 0.9% saline (for injection). The GluA1 modulator tianeptine (Tocris Bioscience; Ellisville, MO) was freshly dissolved in saline before each day of testing.

Data Analysis

All analyses were performed using Prism v. 6.0 (GraphPad, La Jolla, CA). For drinking experiments, alcohol intake data were reported as grams of intake per kilogram of body weight and water intake data were reported as milliliters of fluid consumed per kilogram of body weight. Intake of both solutions was analyzed via two-way repeated measures ANOVA (Age × Day). BAC was analyzed via t-test to compare adolescent and adult values.

Western blot data were expressed as percent change from age-matched water drinking controls, and all protein levels were expressed as a ratio to β-Actin. Data were analyzed via independent t-tests comparing alcohol and water treated animals separately within each age group.

In the two tianeptine pretreatment experiments, alcohol and sucrose intake were analyzed via two-way repeated measures ANOVA (Age × Dose) respectively. Tukey’s LSD test was used for all post-hoc analyses. BEC was analyzed via two-way repeated measures ANOVA (Dose × Time) separately within each age. Open field locomotor data were collapsed into 20 minute time bins and activity was assessed via two-way repeated measures ANOVA (Dose × Time) separately within each age. Locomotor data were further analyzed for potential age or drug-induced differences in anxiety-like behavior in an open-field test. Thigmotaxis was evaluated by comparing distance (cm) traveled in the center zone (inner 25% of the area) to distance traveled in the periphery (outer 75% of the area) as previously described (Hodge et al., 2002). α was set at 0.05 for all comparisons.

Results

Experiment 1

Binge-like Alcohol Consumption

Adolescent and adult mice consumed equivalent amounts of alcohol and water over the two-week daily access period (adolescents averaged 5.01 ±.22 g/kg and adults averaged 5.09 ±.13 g/kg, Figures 1B and C). Alcohol drinking did not alter body weight in adolescents or adults (Figure 1D). Blood alcohol levels immediately after drinking on the last experimental day exceeded the NIAAA criteria for a binge drinking session (80 mg/dL), with no significant differences between the two age groups (Figure 1E) (NIAAA, 2004).

CaMKIIα Changes in the Amygdala

Western blot analysis of adolescent amygdala revealed that a two-week history of alcohol drinking significantly decreased the phosphorylation of CaMKIIαT286 by approximately 30%, t(17)= 1.916, p <0.05 (Figure 2A). No differences in total CaMKIIα expression emerged in adolescents (Figure 2B). In contrast, neither pCaMKIIαT286 nor tCaMKIIα were affected by alcohol drinking in the adult amygdala (Figure 2C and D).

Figure 2. CaMKII phosphorylation is altered by two weeks of daily binge-like alcohol drinking in the adolescent but not adult amygdala.

Figure 2

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(A) Phospho-CaMKIIαT286 (pCaMKIIαT286) was decreased in the amygdala of adolescent mice exposed to alcohol in the daily binge drinking procedure (*p <0.05). (B) Total CaMKIIα expression was not different between alcohol and water drinking adolescents. (C) pCaMKIIαT286 did not differ in the amygdala of adult mice exposed to alcohol or water in the daily binge drinking procedure. (D) tCaMKIIα expression was also not altered by alcohol treatment in adults.

CaMKIIα Changes in the Striatum

Analysis of the adolescent and adult nucleus accumbens via Western blot showed no effect of alcohol exposure on pCaMKIIαT286 in either age (Table 1). Similarly, pCaMKIIαT286 levels were unaltered by alcohol drinking in both ages in the dorsal striatum. Total expression of CaMKIIα was also unaffected by alcohol exposure in adolescents and adults in both regions.

Table 1.

Phospho-and total-CaMKIIα is unaffected by alcohol drinking in the adolescent and adult nucleus accumbens and dorsal striatum. Data are represented as the mean optical density/β-Actin for each age and treatment condition ±SEM.

pCaMKIIαT286 tCaMKIIα
Brain region Water Alcohol Water Alcohol
ADOLESCENT
Nucleus Accumbens 100±5 101±17 100±8 99±8
Dorsal Striatum 100±9 98±20 100±3 105±3
ADULT
Nucleus Accumbens 100±15 82±14 100±6 99±4
Dorsal Striatum 100±18 102±13 100±3 107±6
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GluA1 Changes in the Amygdala

To examine a downstream target of phosphorylated CaMKII activity, the GluA1 subunit of the AMPAR receptor was analyzed in the adolescent and adult amygdala. Western blot analysis showed that in the adolescent amygdala, a two-week history of alcohol drinking had a non-significant trend to decrease pGluA1Ser831, p= 0.11 (Figure 3A). In the adult amygdala, alcohol drinking significantly increased pGluA1Ser831 by approximately 65%, t (17)= 1.262, p =0.05 (Figure 3C). No effect of alcohol drinking on total GluA1 expression emerged in either age (Figure 3B and D).

Figure 3. Differential effects of a two-week history of daily binge alcohol exposure on GluA1 phosphorylation in the adolescent and adult amygdala.

Figure 3

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(A) Phospho-GluA1S831 (pGluA1S831) had a tendency to decrease in the amygdala of adolescent mice exposed to alcohol in the daily binge drinking procedure (#p <0.1). (B) Total GluA1 expression was not different between alcohol and water drinking adolescents. (C) pGluA1S831 was increased in the amygdala of adult mice exposed to alcohol in the daily binge drinking procedure (*p =0.05). (D) Total GluA1 expression was not different between alcohol and water drinking adults.

Experiment 2

Effect of Tianeptine Pretreatment on Binge-like Alcohol Consumption

Prior to drug treatment, baseline 4-hour every-other-day alcohol intake was significantly greater among adolescents (5.6 g/kg ±0.69) than adults (3.7 g/kg ±0.69) [Main effect of Age, F(1, 22) = 8.896, p <0.01]. Over the course of the entire experiment, adolescent body weight increased steadily from an average of 14g on PND 28 to an average of 21g on PND42. The rate of body weight gain did not change following tianeptine pretreatment and is consistent with the published literature for mouse body weight at these ages (Huang et al., 2012).

Following 30 minute pretreatment with tianeptine, a Dose × Age interaction emerged, F(3, 66) = 5.062, p <0.001. Tukey’s multiple comparisons test revealed that among adolescent mice, the 10mg/kg dose of tianeptine significantly increased alcohol consumption relative to vehicle (p <0.05, Figure 4B). Adolescent alcohol intake increased from 5.4 g/kg to 6.6 g/kg (22%). In contrast, the 17mg/kg dose of tianeptine significantly decreased alcohol consumption relative to vehicle in adults (p <0.05). Adult alcohol intake decreased from 4.2 g/kg to 3.2 g/kg (25%). Dose effects were only apparent during the last two hours and total four-hour intake period (data not shown.) A main effect of Age also emerged [F(1, 22) = 45.37, p <0.0001] with adolescents consuming significantly more alcohol than adults.

Figure 4. Tianeptine pretreatment alters alcohol, but not sucrose, intake in opposite directions in adolescent and adult male mice.

Figure 4

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(A) Timeline of tianeptine pretreatment binge drinking procedure. (B) Tianeptine dose-dependently increased alcohol intake in adolescent mice but decreased alcohol intake in adult mice (*p <0.05). (C) Tianeptine comparably decreased sucrose intake in adolescent and adult mice. The open-field locomotor behavior of adolescent (D) and adult (E) mice injected with the effective dose of tianeptine in each age did not differ from age-matched controls injected with vehicle. Adolescent (F) and adult (G) mice pretreated with tianeptine did not differ in blood alcohol concentration following an acute alcohol injection.

Selectivity of Tianeptine Effects

To assess behavioral (alcohol) specificity, we evaluated effects of tianeptine pretreatment on binge-like consumption of sucrose, a non-drug solution that has rewarding properties. Analysis of 4-hour sucrose consumption following 30-minute pretreatment with tianeptine revealed a main effect of Dose, F(3,63) =3.579, p <0.05, indicating that tianeptine decreased sucrose consumption similarly in adolescent and adult mice. No Age × Dose interaction emerged (p >0.05, Figure 4C).

To determine if tianeptine effects were associated with nonspecific locomotor effects, adolescent and adult mice were pretreated with either vehicle or the effective dose of tianeptine (10mg/kg for adolescents and 17mg/kg for adults) 2 hours prior to a 2 hour open-field locomotor activity test in order to correspond with the time interval during which tianeptine effects on alcohol drinking were observed. Analysis of cumulative locomotor activity in both adolescents and adults failed to detect any significant effect of tianeptine pretreatment (p >0.05, Figure 4D and E).

To evaluate anxiety-like behavior, locomotor data were analyzed for time spent in the center versus perimeter of the chamber. A main effect of zone emerged, such that mice in both age groups spent more time in the perimeter of the chamber than in the center zone, F(1, 10) = 504.0, p < 0.0001. Under vehicle treatment conditions, both adolescents and adults spent approximately 80% of their time in the perimeter zone. No significant effect of tianeptine pretreatment or age emerged in either zone (p > 0.05, data not shown.)

To determine if tianeptine affected alcohol clearance, adolescent and adult mice were pretreated with tianeptine 30 minutes prior to a 4g/kg intraperitoneal injection of alcohol. Analysis of blood alcohol samples collected 10, 30, 60, 120, and 240 minutes after the alcohol injection failed to reveal any significant effects of tianeptine pretreatment on BACs in either age (p >0.05, Figure 4F and G).

Discussion

Although human (Hingson et al., 2006, Grant and Dawson, 1998) and rodent studies (Pascual et al., 2009, Rodd-Henricks et al., 2002, Alaux-Cantin et al., 2013) indicate that adolescent alcohol exposure increases the risk for lifetime AUDs, the neurobiological mechanisms of this age-specific vulnerability remain to be determined. To address this gap in knowledge, we first evaluated effects of voluntary binge-like alcohol drinking on protein expression and activation (e.g., phosphorylation) of CaMKIIαT286 and the GluA1S831 subunit of AMPARs in adolescent and adult mice. To determine if CaMKIIα-GluA1 signaling mechanistically regulates age-dependent binge drinking, we next assessed the effects of systemic administration of the antidepressant Tianeptine, which potentiates AMPAR GluA1 activity via activation of CaMKIIα (Szegedi et al 2011), in adolescent and adult mice. Results indicate that voluntary binge-like alcohol drinking produces age-dependent differential effects on pCaMKIIαT286 and pGluA1S831 in the amygdala. Accordingly, positive modulation of CaMKII-dependent AMPA signaling via systemic Tianeptine administration increased binge-like alcohol drinking specifically in adolescent mice and reduced drinking in adults. Together, these data indicate that binge drinking produces age-dependent effects on CaMKIIα and GluA1 signaling that regulate the amount of alcohol consumed during binge drinking episodes.

Effects of Binge-Drinking on CaMKIIα and GluA1 Expression and Phosphorylation

Adolescent and adult male C57BL/6J mice voluntarily consumed similar doses of alcohol during the two-week exposure period, each achieving blood alcohol levels consistent with binge exposure (e.g., > 80 mg/dL). Binge drinking produced no effects on overall health as indexed by body weight comparisons to parallel water-only controls. The lack of age differences in alcohol intake in Experiment 1 was advantageous because it ruled out alcohol dose-dependent differences in protein expression, allowing us to more clearly interpret age-dependent differences (i.e., without the confound of differential alcohol dose).

Adolescent

Binge-like alcohol drinking produced an age-dependent decrease in pCaMKIIαT286 in adolescent amygdala with no change in total protein expression. Since pCaMKIIαT286 is a primary molecular mechanism of synaptic plasticity that is required for associative learning and memory processes mediated by the amygdala (Lisman et al., 2002, Rodrigues et al., 2004), binge alcohol-induced downregulation of CaMKIIα activity in the adolescent amygdala may underlie age-dependent inhibition of memory processes as previously observed following alcohol exposure (Broadwater and Spear, 2013, Spanos et al., 2012, Markwiese et al., 1998). This observation is highly significant from a translational perspective since estimates indicate that over 50% of college-age frequent binge drinkers have experienced significant associative memory loss, including complete blackouts (White and Hingson, 2013). Moreover, emerging adults (age18–24) who have experienced binge-induced blackouts have lower levels of glutamate in the anterior cingulate cortex as compared to light drinkers (Silveri et al., 2014). Thus, binge drinking may produce adolescent-specific cognitive deficits by disruption of basic molecular mechanisms of memory and plasticity in the amygdala.

At the molecular and cellular level, alcohol-induced inhibition of pCaMKIIαT286 expression in adolescents may disrupt numerous functions subserved by the kinase including regulation of membrane current, neurotransmitter synthesis and release, cytoskeletal organization, dendrite maturation, and gene expression (Colbran and Brown, 2004, Hanson and Schulman, 1992, Lisman et al., 2002, Metzger, 2010). Further, a crucial function of CaMKIIα is to phosphorylate AMPARs at the GluA1S831 site, which leads to potentiation of AMPA-mediated synaptic activity (Mammen et al., 1997, Barria et al., 1997), promotes AMPAR membrane insertion, and enhances their function (Colbran and Brown, 2004, Hanson and Schulman, 1992, Lisman et al., 2002). Here, we observed a trend for decreased pGluA1S831 following adolescent binge-like alcohol intake. Binge drinking may disrupt a variety of critical neural and behavioral functions in the adolescent brain via inhibition of CaMKIIα activity and downstream mechanisms. This suggests that binge-induced changes in CaMKIIα activity may be associated with subtle changes in AMPAR signaling.

Adult

In contrast to the adolescent, a two-week history of daily binge alcohol drinking specifically increased pGluA1S831 in adult amygdala with no change in pCaMKIIαT286 or total protein expression. These findings are consistent with previous evidence showing that pGluA1S831 is increased by voluntary 24-h home-cage drinking or low-dose operant alcohol self-administration in adult mice (Salling et al., 2014), and by chronic intermittent high-dose alcohol vapor in adult rats (Christian et al., 2012). Phosphorylation of the GluA1 subunit of the AMPAR results in increased single-channel conductance (Derkach, 2003, Derkach et al., 1999) and has been shown to be critical for the induction of LTP (Lee et al., 2010). Recent work in our lab has demonstrated that CaMKII and GluA1 activity in the amygdala functionally regulate alcohol self-administration (Salling et al., 2014). Thus, increased pGluA1S831 may be indicative of increased activity of AMPARs in the adult amygdala after alcohol exposure, and may contribute to long-term plastic adaptations to alcohol. Since CaMKIIα phosphorylation was not increased in adults, it is plausible binge-like alcohol drinking does not specifically alter CaMKII signaling in this age group. Thus, GluA1S831 phosphorylation may have been mediated by another alcohol-sensitive kinase, such as PKC (Besheer et al., 2006, Hodge et al., 1999, Ren et al., 2013, Wilkie et al., 2007). These results add weight to a growing body of evidence suggesting that glutamate signaling in the amygdala is a target of alcohol self-administration.

Overall, these data show differential age-dependent effects of binge drinking on CaMKIIαT286 and GluA1S831 phosphorylation between adolescent and adult mice. Although the specific mechanism(s) for these effects are unknown, it is plausible that well-documented age-dependent maturation of the target systems is partly accountable. For instance, CaMKIIα expression increases linearly in rat forebrain by 10-fold from PND 5 through 25 (Kelly and Vernon, 1985). Similarly, CaMKIIα mRNA increases 10-fold between PND 1 and 21 with an additional 5-fold increase by PND 90, which spans the adolescent and early adult periods examined in the present study. Additionally, the subcellular distribution of CaMKII shifts from primarily cytosolic to membrane localization during development (Sahyoun et al., 1985, Weinberger and Rostas, 1986). Developmentally regulated protein concentration and localization in neural tissue may influence the effects of alcohol on CaMKIIα phosphorylation.

Tissue for these experiments was collected immediately following the last drinking session, when mice had achieved a binge-level of alcohol consumption. Therefore, the results should be considered in the context of alcohol-induced brain protein changes in the presence of alcohol. The present findings were also selective for the amygdala, with no changes in pCaMKIIaT286 or pGluA1S831 observed in the nucleus accumbens or the dorsal striatum. Therefore, glutamatergic signaling in the striatum may be less relevant than in the amygdala in terms of the regulation of binge-like alcohol self-administration. The amygdala is a complex region comprised of several subnuclei, including the central, basolateral and lateral subregions, and these nuclei may play different roles in the regulation of alcohol drinking behavior (McCool et al., 2010, McBride, 2002). The current experiment is limited by the use of whole amygdala tissue, making it difficult to establish the relevance of different sub-nuclei in these findings. Future studies utilizing immunohistochemistry would clarify the contributions of different subregions of the amygdala to the effects observed in the present study.

Effects of the pGluA1S831 Upregulator Tianeptine on Binge-Drinking

In Experiment 2, adolescent animals consumed more alcohol than adult animals both at baseline and during drug treatment under vehicle conditions. This result is in contrast to Experiment 1, where adolescent and adult alcohol intake was equivalent. Although adolescents have been reported to consume more alcohol than adults in some studies (Holstein et al., 2011, Hargreaves et al., 2009), other reports have failed to find differences in adolescent and adult intake (Siegmund et al., 2005, Hefner and Holmes, 2007). Procedural differences between experiments are likely to account for discrepancies in age differences across studies. Interestingly, the studies above that failed to find age differences in alcohol self-administration utilized daily or continuous access procedures (as in Experiment 1) whereas the studies that demonstrated increased alcohol consumption during adolescence made use of intermittent drinking protocols (as in Experiment 2). These findings suggest that intermittent drinking procedures may be advantageous for experiments in which age differences in alcohol consumption are under investigation, whereas daily drinking procedures may be useful when dose differences between ages represent a confounding variable.

Binge alcohol consumption increased pGluA1S831 in the adult amygdala. Mimicking this effect via systemic pretreatment with the AMPAR positive modulator Tianeptine dose-dependently decreased binge-like alcohol consumption in adult mice. Since Tianeptine increases pGluA1S831 in a CaMKII-dependent manner (Szegedi et al., 2011), it may have substituted for a pharmacological effect of alcohol in adult mice, leading to decreased alcohol consumption. This finding is consistent with previous studies which have shown that tianeptine pretreatment decreases alcohol intake (Daoust et al., 1992) and attenuates alcohol withdrawal symptoms in adult rats (Uzbay et al., 2006). Moreover, alcohol self-administration increases pGluA1S831 expression in adult mouse amygdala where AMPAR activity is required for the positive reinforcing effects of alcohol (Salling et al., 2014) and increased non-NMDA glutamatergic signaling in the amygdala is associated with alcohol conditioned reward (Zhu et al., 2007). Importantly, tianeptine pretreatment also decreased sucrose consumption in adult mice, suggesting that tianeptine may globally reduce the consumption of palatable solutions. These findings add growing evidence that AMPAR activity is critical for the reinforcing effects of alcohol and other palatable solutions in adults.

In contrast to adults, binge-like alcohol drinking resulted in a trend for decreased pGluA1S831 in the adolescent amygdala. Opposing this alcohol-induced effect via systemic pretreatment with tianeptine increased binge-like alcohol consumption in this age group. Tianeptine may therefore have blocked a pharmacological effect of alcohol at AMPARs in adolescent mice, leading to a compensatory increase in alcohol drinking. The effects of tianeptine on adolescent binge drinking formed an inverted U-shaped dose-response curve, with an intermediate dose leading to increased alcohol consumption and a high dose returning to baseline. It is possible that testing a higher dose would reveal decreased alcohol consumption in adolescents, indicating a rightward shift of the dose-response curve in adolescents corresponding to the effect in adults. Significantly, tianeptine pretreatment produced comparable decreases in sucrose consumption in adolescent and adult mice, indicating that the increase in alcohol drinking seen after tianeptine pretreatment in adolescents is selective for alcohol and not generalized to other reinforcing solutions. The alterations in glutamate signaling reported here in the adolescent mouse may therefore be both selective for the adolescent brain and specific for alcohol. Together, these data provide evidence that CaMKIIα-GluA1 signaling differentially regulates binge-like drinking by adolescent and adult mice. Upregulation of AMPAR activity in the amygdala may promote increased drinking during adolescence but inhibit this behavioral pathology during adulthood. Future studies that target AMPAR activity specifically in the amygdala would clarify the anatomical basis of this age-dependent differential response.

Tianeptine pretreatment failed to significantly alter open-field activity in both adolescents and adults, and blood alcohol clearance was similarly unaffected by tianeptine pretreatment in both ages, indicating a lack of effect of tianeptine on gross locomotor activity and alcohol metabolism. It is possible that tianeptine and alcohol may interact to alter locomotor behavior in adolescent and adult mice. While no obvious locomotor effects were observed during drinking, this possibility limits the interpretation of the locomotor results. Tianeptine may alter the metabolism of lower doses of alcohol but not the higher doses tested here, limiting the interpretation of the blood alcohol clearance experiments. Additionally, the injections administered in these experiments may have represented a significant stressor in the experimental design, potentially contributing to the age differences observed in alcohol drinking. However, zone analysis of adolescent and adult locomotor behavior under vehicle conditions revealed no evidence for anxiety-like behavior in either age, and tianeptine pretreatment did not alter time spent in the center or perimeter of the open field. Therefore, stress-related explanations for these findings are unlikely.

A potential limitation of these findings is the possibility for tianeptine activity at targets other than CaMKII/GluA1 signaling. Unlike most antidepressants, tianeptine has very low affinity for monoamine receptors or transporters (Pineyro et al., 1995) and the drug has not been found to interact directly with NMDA receptors (Svenningsson et al., 2007). However, activation of CaMKII also prompts the protein to bind with the NR2B subunit of the NMDA receptor (Lisman et al., 2012). Therefore, activation of CaMKII via tianeptine pretreatment could alter NMDA receptor activity as well as GluA1Ser831 phosphorylation. Additionally, a recent report suggests that tianeptine may have some affinity for the μ-opioid receptor (Gassaway et al., 2014), which could represent another mechanism for tianeptine’s effects on alcohol drinking. However, activation of μ-opioid receptors has been shown to be an upstream trigger of the signaling cascade that stimulates CaMKII activation (Garzon et al., 2008), and therefore may be part of the pGluASer831 potentiation seen after tianeptine treatment.

In conclusion, the findings from this study indicate that binge-like alcohol drinking impacts amygdala glutamate signaling systems of adolescent and adult animals in very different ways. Glutamate signaling in the amygdala, as indexed by CaMKIIα and GluA1 phosphorylation, appears to be downregulated by alcohol in adolescence and upregulated in adulthood. Results from this study suggest that CaMKIIα-GluA1 signaling in the adolescent amygdala is especially vulnerable to binge-induced insult. Understanding the age-dependent differences in the impact of binge alcohol drinking on glutamate signaling and how these systems, in turn, regulate intake is critical to understanding adolescent vulnerability to alcohol addiction.

Acknowledgments

This work supported by grants R37AA014983 and P60AA11605 from the National Institute on Alcohol Abuse and Alcoholism to CW Hodge.

References

  1. Alaux-Cantin S, Warnault V, Legastelois R, Botia B, Pierrefiche O, Vilpoux C, Naassila M. Alcohol intoxications during adolescence increase motivation for alcohol in adult rats and induce neuroadaptations in the nucleus accumbens. Neuropharmacology. 2013;67:521–531. doi: 10.1016/j.neuropharm.2012.12.007. [DOI] [PubMed] [Google Scholar]
  2. Apergis-Schoute AM, Debiec J, Doyere V, Ledoux JE, Schafe GE. Auditory fear conditioning and long-term potentiation in the lateral amygdala require ERK/MAP kinase signaling in the auditory thalamus: a role for presynaptic plasticity in the fear system. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2005;25:5730–5739. doi: 10.1523/JNEUROSCI.0096-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barria A, Muller D, Derkach V, Griffith LC, Soderling TR. Regulatory phosphorylation of AMPA-type glutamate receptors by CaM-KII during long-term potentiation. Science. 1997;276:2042–2045. doi: 10.1126/science.276.5321.2042. [DOI] [PubMed] [Google Scholar]
  4. Besheer J, Lepoutre V, Mole B, Hodge CW. GABAA receptor regulation of voluntary ethanol drinking requires PKCepsilon. Synapse. 2006;60:411–419. doi: 10.1002/syn.20314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Broadwater M, Spear LP. Consequences of ethanol exposure on cued and contextual fear conditioning and extinction differ depending on timing of exposure during adolescence or adulthood. Behavioural brain research. 2013;256:10–19. doi: 10.1016/j.bbr.2013.08.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Christian DT, Alexander NJ, Diaz MR, Robinson S, Mccool BA. Chronic intermittent ethanol and withdrawal differentially modulate basolateral amygdala AMPA-type glutamate receptor function and trafficking. Neuropharmacology. 2012;62:2430–2439. doi: 10.1016/j.neuropharm.2012.02.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Colbran RJ, Brown AM. Calcium/calmodulin-dependent protein kinase II and synaptic plasticity. Curr Opin Neurobiol. 2004;14:318–327. doi: 10.1016/j.conb.2004.05.008. [DOI] [PubMed] [Google Scholar]
  8. Daoust M, Compagnon P, Legrand E, Mocaer E. Tianeptine, a specific serotonin uptake enhancer, decreases ethanol intake in rats. Alcohol and alcoholism. 1992;27:15–17. [PubMed] [Google Scholar]
  9. Derkach V, Barria A, Soderling TR. Ca2+/calmodulin-kinase II enhances channel conductance of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate type glutamate receptors. Proceedings of the National Academy of Sciences of the United States of America. 1999;96:3269–3274. doi: 10.1073/pnas.96.6.3269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Derkach VA. Silence analysis of AMPA receptor mutated at the CaM-kinase II phosphorylation site. Biophysical journal. 2003;84:1701–1708. doi: 10.1016/S0006-3495(03)74978-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Easton AC, Lucchesi W, Lourdusamy A, Lenz B, Solati J, Golub Y, Lewczuk P, Fernandes C, Desrivieres S, Dawirs RR, Moll GH, Kornhuber J, Frank J, Hoffmann P, Soyka M, Kiefer F, Schumann G, Peter Giese K, Muller CP, Treutlein J, Cichon S, Ridinger M, Mattheisen P, Herms S, Wodarz N, Zill P, Maier W, Mossner R, Gaebel W, Dahmen N, Scherbaum N, Schmal C, Steffens M, Lucae S, Ising M, Muller-Myhsok B, Nothen MM, Mann K, Rietschel M. alphaCaMKII autophosphorylation controls the establishment of alcohol drinking behavior. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 2013a;38:1636–1647. doi: 10.1038/npp.2013.60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Easton AC, Lucchesi W, Mizuno K, Fernandes C, Schumann G, Giese KP, Muller CP. alphaCaMKII autophosphorylation controls the establishment of alcohol-induced conditioned place preference in mice. Behavioural brain research. 2013b;252:72–76. doi: 10.1016/j.bbr.2013.05.045. [DOI] [PubMed] [Google Scholar]
  13. Garzon J, Rodriguez-Munoz M, Sanchez-Blazquez P. Do pharmacological approaches that prevent opioid tolerance target different elements in the same regulatory machinery? Current drug abuse reviews. 2008;1:222–238. doi: 10.2174/1874473710801020222. [DOI] [PubMed] [Google Scholar]
  14. Gassaway MM, Rives ML, Kruegel AC, Javitch JA, Sames D. The atypical antidepressant and neurorestorative agent tianeptine is a mu-opioid receptor agonist. Translational psychiatry. 2014;4:e411. doi: 10.1038/tp.2014.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grant BF, Dawson DA. Age of onset of drug use and its association with DSM-IV drug abuse and dependence: results from the National Longitudinal Alcohol Epidemiologic Survey. Journal of substance abuse. 1998;10:163–173. doi: 10.1016/s0899-3289(99)80131-x. [DOI] [PubMed] [Google Scholar]
  16. Hanson PI, Schulman H. Neuronal Ca2+/calmodulin-dependent protein kinases. Annual review of biochemistry. 1992;61:559–601. doi: 10.1146/annurev.bi.61.070192.003015. [DOI] [PubMed] [Google Scholar]
  17. Hargreaves GA, Monds L, Gunasekaran N, Dawson B, Mcgregor IS. Intermittent access to beer promotes binge-like drinking in adolescent but not adult Wistar rats. Alcohol. 2009;43:305–314. doi: 10.1016/j.alcohol.2009.02.005. [DOI] [PubMed] [Google Scholar]
  18. Hefner K, Holmes A. An investigation of the behavioral actions of ethanol across adolescence in mice. Psychopharmacology. 2007;191:311–322. doi: 10.1007/s00213-006-0646-2. [DOI] [PubMed] [Google Scholar]
  19. Hingson RW, Heeren T, Winter MR. Age at drinking onset and alcohol dependence: age at onset, duration, and severity. Archives of pediatrics & adolescent medicine. 2006;160:739–746. doi: 10.1001/archpedi.160.7.739. [DOI] [PubMed] [Google Scholar]
  20. Hodge CW, Mehmert KK, Kelley SP, Mcmahon T, Haywood A, Olive MF, Wang D, Sanchez-Perez AM, Messing RO. Supersensitivity to allosteric GABA(A) receptor modulators and alcohol in mice lacking PKCepsilon. Nature neuroscience. 1999;2:997–1002. doi: 10.1038/14795. [DOI] [PubMed] [Google Scholar]
  21. Hodge CW, Raber J, Mcmahon T, Walter H, Sanchez-Perez AM, Olive MF, Mehmert K, Morrow AL, Messing RO. Decreased anxiety-like behavior, reduced stress hormones, and neurosteroid supersensitivity in mice lacking protein kinase Cepsilon. The Journal of clinical investigation. 2002;110:1003–1010. doi: 10.1172/JCI15903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hodge CW, Samson HH, Haraguchi M. Microinjections of dopamine agonists in the nucleus accumbens increase ethanol-reinforced responding. Pharmacology, biochemistry, and behavior. 1992;43:249–254. doi: 10.1016/0091-3057(92)90665-3. [DOI] [PubMed] [Google Scholar]
  23. Holstein SE, Spanos M, Hodge CW. Adolescent C57BL/6J mice show elevated alcohol intake, but reduced taste aversion, as compared to adult mice: a potential behavioral mechanism for binge drinking. Alcoholism, clinical and experimental research. 2011;35:1842–1851. doi: 10.1111/j.1530-0277.2011.01528.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Huang C, Titus JA, Bell RL, Kapros T, Chen J, Huang R. A mouse model for adolescent alcohol abuse: stunted growth and effects in brain. Alcoholism, clinical and experimental research. 2012;36:1728–1737. doi: 10.1111/j.1530-0277.2012.01759.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Johnston LD OP, Bachman JG, Schulenberg JE. Monitoring the Future: national survey results on drug use, 1975–2011: volume I: secondary school students. 2012 [Google Scholar]
  26. Kelly PT, Vernon P. Changes in the subcellular distribution of calmodulin-kinase II during brain development. Brain research. 1985;350:211–224. doi: 10.1016/0165-3806(85)90265-2. [DOI] [PubMed] [Google Scholar]
  27. Lee HK, Takamiya K, He K, Song L, Huganir RL. Specific roles of AMPA receptor subunit GluR1 (GluA1) phosphorylation sites in regulating synaptic plasticity in the CA1 region of hippocampus. Journal of neurophysiology. 2010;103:479–489. doi: 10.1152/jn.00835.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lisman J, Schulman H, Cline H. The molecular basis of CaMKII function in synaptic and behavioural memory. Nat Rev Neurosci. 2002;3:175–190. doi: 10.1038/nrn753. [DOI] [PubMed] [Google Scholar]
  29. Lisman J, Yasuda R, Raghavachari S. Mechanisms of CaMKII action in long-term potentiation. Nature reviews. Neuroscience. 2012;13:169–182. doi: 10.1038/nrn3192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mammen AL, Kameyama K, Roche KW, Huganir RL. Phosphorylation of the alpha-amino-3-hydroxy-5-methylisoxazole4-propionic acid receptor GluR1 subunit by calcium/calmodulin-dependent kinase II. The Journal of biological chemistry. 1997;272:32528–32533. doi: 10.1074/jbc.272.51.32528. [DOI] [PubMed] [Google Scholar]
  31. Markwiese BJ, Acheson SK, Levin ED, Wilson WA, Swartzwelder HS. Differential effects of ethanol on memory in adolescent and adult rats. Alcoholism, clinical and experimental research. 1998;22:416–421. [PubMed] [Google Scholar]
  32. Mcbride WJ. Central nucleus of the amygdala and the effects of alcohol and alcohol-drinking behavior in rodents. Pharmacology, biochemistry, and behavior. 2002;71:509–515. doi: 10.1016/s0091-3057(01)00680-3. [DOI] [PubMed] [Google Scholar]
  33. Mccool BA, Christian DT, Diaz MR, Lack AK. Glutamate plasticity in the drunken amygdala: the making of an anxious synapse. International review of neurobiology. 2010;91:205–233. doi: 10.1016/S0074-7742(10)91007-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Metzger F. Molecular and cellular control of dendrite maturation during brain development. Current molecular pharmacology. 2010;3:1–11. doi: 10.2174/1874467211003010001. [DOI] [PubMed] [Google Scholar]
  35. Miller JW, Naimi TS, Brewer RD, Jones SE. Binge drinking and associated health risk behaviors among high school students. Pediatrics. 2007;119:76–85. doi: 10.1542/peds.2006-1517. [DOI] [PubMed] [Google Scholar]
  36. Nelson DE, Naimi TS, Brewer RD, Nelson HA. State alcohol-use estimates among youth and adults, 1993–2005. American journal of preventive medicine. 2009;36:218–224. doi: 10.1016/j.amepre.2008.10.018. [DOI] [PubMed] [Google Scholar]
  37. NIAAA. NIAAA council approves definition of binge drinking. NIAAA Newsletter. 2004;3:3. [Google Scholar]
  38. NRC. Guide for the Care and Use of Laboratory Animals. Revised Edition. In: D. I. O. L. A. R. N. I. O. H, editor. National Academy of Sciences - National Research Council. Washington, D.C. 20402: Superintendent of Documents, U.S. Government Printing Office; 1996. [Google Scholar]
  39. Pascual M, Boix J, Felipo V, Guerri C. Repeated alcohol administration during adolescence causes changes in the mesolimbic dopaminergic and glutamatergic systems and promotes alcohol intake in the adult rat. Journal of neurochemistry. 2009;108:920–931. doi: 10.1111/j.1471-4159.2008.05835.x. [DOI] [PubMed] [Google Scholar]
  40. Pineyro G, Deveault L, Blier P, Dennis T, de Montigny C. Effect of acute and prolonged tianeptine administration on the 5-HT transporter: electrophysiological, biochemical and radioligand binding studies in the rat brain. Naunyn-Schmiedeberg's archives of pharmacology. 1995;351:111–118. doi: 10.1007/BF00169324. [DOI] [PubMed] [Google Scholar]
  41. Pyapali GK, Turner DA, Wilson WA, Swartzwelder HS. Age and dose-dependent effects of ethanol on the induction of hippocampal long-term potentiation. Alcohol. 1999;19:107–111. doi: 10.1016/s0741-8329(99)00021-x. [DOI] [PubMed] [Google Scholar]
  42. Ren SQ, Yan JZ, Zhang XY, Bu YF, Pan WW, Yao W, Tian T, Lu W. PKClambda is critical in AMPA receptor phosphorylation and synaptic incorporation during LTP. The EMBO journal. 2013;32:1365–1380. doi: 10.1038/emboj.2013.60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rhodes JS, Best K, Belknap JK, Finn DA, Crabbe JC. Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice. Physiology & behavior. 2005;84:53–63. doi: 10.1016/j.physbeh.2004.10.007. [DOI] [PubMed] [Google Scholar]
  44. Rodd-Henricks ZA, Bell RL, Kuc KA, Murphy JM, Mcbride WJ, Lumeng L, Li TK. Effects of ethanol exposure on subsequent acquisition and extinction of ethanol self-administration and expression of alcohol-seeking behavior in adult alcohol-preferring (P) rats: I. Periadolescent exposure. Alcoholism, clinical and experimental research. 2002;26:1632–1641. doi: 10.1097/01.ALC.0000036301.36192.BC. [DOI] [PubMed] [Google Scholar]
  45. Sahyoun N, Levine H, 3rd, Burgess SK, Blanchard S, Chang KJ, Cuatrecasas P. Early postnatal development of calmodulin-dependent protein kinase II in rat brain. Biochemical and biophysical research communications. 1985;132:878–884. doi: 10.1016/0006-291x(85)91889-3. [DOI] [PubMed] [Google Scholar]
  46. Salling MC, Faccidomo SP, Li C, Psilos K, Galunas C, Spanos M, Agoglia AE, Kash TL, Hodge CW. Moderate Alcohol Drinking and the Amygdala Proteome: Identification and Validation of Calcium/Calmodulin Dependent Kinase II and AMPA Receptor Activity as Novel Molecular Mechanisms of the Positive Reinforcing Effects of Alcohol. Biological psychiatry. 2014 doi: 10.1016/j.biopsych.2014.10.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schramm-Sapyta NL, Kingsley MA, Rezvani AH, Propst K, Swartzwelder HS, Kuhn CM. Early ethanol consumption predicts relapse-like behavior in adolescent male rats. Alcoholism, clinical and experimental research. 2008;32:754–762. doi: 10.1111/j.1530-0277.2008.00631.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schroeder JP, Spanos M, Stevenson JR, Besheer J, Salling M, Hodge CW. Cue-induced reinstatement of alcohol-seeking behavior is associated with increased ERK1/2 phosphorylation in specific limbic brain regions: blockade by the mGluR5 antagonist MPEP. Neuropharmacology. 2008;55:546–554. doi: 10.1016/j.neuropharm.2008.06.057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Selemon LD. A role for synaptic plasticity in the adolescent development of executive function. Translational psychiatry. 2013;3:e238. doi: 10.1038/tp.2013.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Siegmund S, Vengeliene V, Singer MV, Spanagel R. Influence of age at drinking onset on long-term ethanol self-administration with deprivation and stress phases. Alcoholism, clinical and experimental research. 2005;29:1139–1145. doi: 10.1097/01.alc.0000171928.40418.46. [DOI] [PubMed] [Google Scholar]
  51. Silveri MM, Cohen-Gilbert J, Crowley DJ, Rosso IM, Jensen JE, Sneider JT. Altered anterior cingulate neurochemistry in emerging adult binge drinkers with a history of alcohol-induced blackouts. Alcoholism, clinical and experimental research. 2014;38:969–979. doi: 10.1111/acer.12346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Spanos M, Besheer J, Hodge CW. Increased sensitivity to alcohol induced changes in ERK Map kinase phosphorylation and memory disruption in adolescent as compared to adult C57BL/6J mice. Behavioural brain research. 2012;230:158–166. doi: 10.1016/j.bbr.2012.02.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Spear LP. The adolescent brain and age-related behavioral manifestations. Neuroscience and biobehavioral reviews. 2000;24:417–463. doi: 10.1016/s0149-7634(00)00014-2. [DOI] [PubMed] [Google Scholar]
  54. Stephens DN, Ripley TL, Borlikova G, Schubert M, Albrecht D, Hogarth L, Duka T. Repeated ethanol exposure and withdrawal impairs human fear conditioning and depresses long-term potentiation in rat amygdala and hippocampus. Biological psychiatry. 2005;58:392–400. doi: 10.1016/j.biopsych.2005.04.025. [DOI] [PubMed] [Google Scholar]
  55. Svenningsson P, Bateup H, Qi H, Takamiya K, Huganir RL, Spedding M, Roth BL, Mcewen BS, Greengard P. Involvement of AMPA receptor phosphorylation in antidepressant actions with special reference to tianeptine. The European journal of neuroscience. 2007;26:3509–3517. doi: 10.1111/j.1460-9568.2007.05952.x. [DOI] [PubMed] [Google Scholar]
  56. Szegedi V, Juhasz G, Zhang X, Barkoczi B, Qi H, Madeira A, Kapus G, Svenningsson P, Spedding M, Penke B. Tianeptine potentiates AMPA receptors by activating CaMKII and PKA via the p38, p42/44 MAPK and JNK pathways. Neurochemistry international. 2011;59:1109–1122. doi: 10.1016/j.neuint.2011.10.008. [DOI] [PubMed] [Google Scholar]
  57. Uzbay T, Kayir H, Celik T, Yuksel N. Acute and chronic tianeptine treatments attenuate ethanol withdrawal syndrome in rats. Progress in neuro-psychopharmacology & biological psychiatry. 2006;30:478–485. doi: 10.1016/j.pnpbp.2005.11.013. [DOI] [PubMed] [Google Scholar]
  58. Weinberger RP, Rostas JA. Subcellular distribution of a calmodulin-dependent protein kinase activity in rat cerebral cortex during development. Brain research. 1986;394:37–50. doi: 10.1016/0165-3806(86)90080-5. [DOI] [PubMed] [Google Scholar]
  59. White A, Hingson R. The burden of alcohol use: excessive alcohol consumption and related consequences among college students. Alcohol research : current reviews. 2013;35:201–218. [PMC free article] [PubMed] [Google Scholar]
  60. Wilkie MB, Besheer J, Kelley SP, Kumar S, O'buckley TK, Morrow AL, Hodge CW. Acute ethanol administration rapidly increases phosphorylation of conventional protein kinase C in specific mammalian brain regions in vivo. Alcoholism, clinical and experimental research. 2007;31:1259–1267. doi: 10.1111/j.1530-0277.2007.00423.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zhu W, Bie B, Pan ZZ. Involvement of non-NMDA glutamate receptors in central amygdala in synaptic actions of ethanol and ethanol-induced reward behavior. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2007;27:289–298. doi: 10.1523/JNEUROSCI.3912-06.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]

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