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Keywords:

  • Alcohol Intake;
  • C57BL/6 Mice;
  • Dependence;
  • Alcohol Vapor;
  • Amygdala

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENT
  7. REFERENCES

Background: Studies in rodents have determined that intermittent exposure to alcohol vapor can increase subsequent ethanol self-administration, measured with operant and 2-bottle choice procedures. Two key procedural factors in demonstrating increased alcohol intake are the establishment of stable alcohol self-administration before alcohol vapor exposure and the number of bouts of intermittent vapor exposure. The present studies provide additional behavioral validation and initial pharmacological validation of this withdrawal-associated drinking procedure.

Methods: Studies at 2 different sites (Portland and Scripps) examined the effect of intermittent ethanol vapor exposure (3 cycles of 16 hours of ethanol vapor+8 hours air) on 2-hour limited access ethanol preference drinking in male C57BL/6 mice. Separate studies tested 10 or 15% (v/v) ethanol concentrations, and measured intake during the circadian dark. In one study, before measuring ethanol intake after the second bout of intermittent vapor exposure, mice were tested for handling-induced convulsions (HICs) indicative of physical dependence on ethanol. In a second study, the effect of bilateral infusions of the corticotropin-releasing factor (CRF) receptor antagonist d-Phe-CRF(12–41) (0.25 μg/0.5 μL) into the central nucleus of the amygdala (CeA) on ethanol intake was compared in vapor-exposed animals and air controls.

Results: Intermittent ethanol vapor exposure significantly increased ethanol intake by 30 to 40%, and the mice had higher blood ethanol concentrations than controls. Intra-amygdala infusions of d-Phe-CRF(12–41) significantly decreased the withdrawal-associated increase in ethanol intake without altering ethanol consumption in controls. Following the second bout of intermittent vapor exposure, mice exhibited an increase in HICs, when compared with their own baseline scores or the air controls.

Conclusions: Intermittent alcohol vapor exposure significantly increased alcohol intake and produced signs of physical dependence. Initial pharmacological studies suggest that manipulation of the CRF system in the CeA can block this increased alcohol intake.

MANY STUDIES EXAMINING self-administration of ethanol in rodents have employed the oral route, using operant and 2-bottle choice procedures, as a partial model of alcoholism in humans (Rhodes et al., 2005). When mice and rats have limited access to an ethanol solution with either 2-bottle choice (e.g., Finn et al., 2004; Ford et al., 2005; Grahame et al., 1999; Li et al, 1993; McBride and Li, 1998) or operant (e.g., Backstrom and Hyytiä 2005; Czachowski et al., 2002; Elmer et al., 1987; Funk et al., 2005; Middaugh et al., 2000; O'Dell et al., 2005; Risinger et al., 1998; Roberts et al., 2000; Tsiang and Janak, 2006) procedures, they can reliably self-administer doses of alcohol that are believed to be pharmacologically active.

Studies measuring alcohol intake in animals following a history of alcohol exposure have either reported increases or no change in preference for alcohol or alcohol self-administration in dependent animals (Begleiter, 1975; Deutsch and Koopmans, 1973; Deutsch and Walton, 1977; Ferko et al., 1979; Hardy and Deutsch, 1977; Marfaing-Jallat and Le Magnen, 1982; Myers et al., 1972; Roberts et al., 1996; Schulteis et al., 1996; Veale and Myers, 1969; Winger, 1988). Several factors may contribute to the variability among studies, including whether the animal learns to associate consumption of alcohol with relief from withdrawal symptoms, whether the animal perceives alcohol as a positive reinforcer before becoming physically dependent, and/or whether the withdrawal symptoms interfere with drinking behavior.

A more recent study demonstrated that if rats were allowed to perform a previously learned operant response for ethanol during withdrawal, significant increases in oral ethanol self-administration that were sufficient to block withdrawal symptoms were observed (Roberts et al., 1996). Subsequent studies determined that this increase in ethanol intake was more pronounced when the ethanol exposure (typically to ethanol vapor) to induce physical dependence was intermittent (O'Dell et al., 2004). Recent work in C57BL/6 (B6) mice also documented that intermittent ethanol vapor exposure significantly increased subsequent ethanol self-administration, measured during a limited access 2-bottle test (Becker and Lopez, 2004; Lopez and Becker, 2005) and operant (A. J. Roberts, unpublished) procedures. Notably, the increased ethanol intake persisted longer as the number of bouts of intermittent ethanol vapor exposure was increased (Lopez and Becker, 2005) and was selective for alcohol self-administration (i.e., mice trained to drink a sucrose solution did not increase their intake after intermittent ethanol vapor exposure, Becker and Lopez, 2004).

One system that appears to play a critical role in the enhanced ethanol self-administration subsequent to dependence involves the stress neuropeptide, corticotropin-releasing factor (CRF). Ethanol withdrawal is associated with disruptions in CRF functioning in humans and laboratory animals (Adinoff et al., 1996; Ehrenreich et al., 1997; Kreek and Koob, 1998; Olive et al., 2002; Pich et al., 1995; Valdez et al., 2003; Wilkins et al., 1992). In rats, the CRF receptor antagonist, d-Phe-CRF(12–41), attenuated increases in ethanol self-administration during withdrawal when administered into the cerebral ventricles (Valdez et al., 2002) as well as directly into the central nucleus of the amygdala (CeA; Funk et al., 2007). In neither case did d-Phe-CRF(12–41) affect ethanol self-administration in nondependent rats.

Based on the above, the purpose of the present studies was to use a slight modification of the procedures published by Drs. Becker and Lopez (Becker and Lopez, 2004; Lopez and Becker, 2005) to document the increased alcohol intake in 2 different laboratories affiliated with the Integrative Neuroscience Initiative on Alcoholism (INIA-West; Portland and Scripps). Additional studies confirmed that the intermittent ethanol vapor exposure produced signs of physical dependence and that the increased ethanol intake following intermittent ethanol vapor exposure produced a corresponding increase in blood ethanol concentration (BEC), when compared with mice exposed to air in the vapor chambers. Finally, we asked whether intra-amygdala infusion of the CRF antagonist d-Phe-CRF(12–41) would significantly decrease alcohol intake only in the animals exposed to intermittent alcohol vapor.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENT
  7. REFERENCES

Animals

Ethanol naïve male B6 mice, approximately 6 to 8 weeks old at the initiation of the studies, were used. B6 mice were purchased from Jackson Laboratories (Bar Harbor, ME or Davis, CA) and were housed in the Department of Comparative Medicine at Oregon Health & Science University (OHSU, Portland site) or obtained from The Scripps Research Institute breeding colony and housed in the Institute for Childhood and Neglected Diseases Building (Scripps site), both of which are AAALAC accredited. Upon arrival, mice were maintained in groups of 4 in standard polycarbonate or polysufone shoebox cages with EcoFresh (Portland) or Harlan Sani-Chips (Scripps) bedding and ad libitum access to Purina Test Diet 5001 (Portland) or Harlan Teklad LM-485 (Scripps) food and water under a 12:12 hours light/dark cycle (lights on at 06:00) at 21±1°C. All procedures were approved by the corresponding Institutional Animal Care and Use Committee and adhered to NIH guidelines.

General Methods

All studies examined the effect of intermittent ethanol vapor exposure on 2-hour limited access ethanol preference drinking in B6 mice. Mice were acclimated to a 12:12 reverse light/dark cycle (off at 09:00 hours) for 1 to 2 weeks before the initiation of baseline drinking. All animals were given free access to food and water throughout the experiment. Body weights were recorded at the beginning of each experiment, once per week during the periods of home cage drinking (which corresponded to cage change), and daily during the cycles of intermittent ethanol vapor exposure. Details for each experiment are provided after the “Data Analysis” section and are summarized in Table 1. Slight variations in the general procedure were conducted at each site to optimize the procedure and to avoid a complete duplication of effort.

Table 1.   Summary of Experimental Design
ExperimentSiteEthanol solution (%)Ethanol accessBouts of intermittent exposureAdditional experimental manipulation(s)
  1. BEC, blood ethanol concentration; CRF, corticotropin-releasing factor; HIC, handling-induced convulsion.

1Portland102h, 1 h after lights off2None
2Scripps152h, 3 h after lights off22-wk period of abstinence between Tests 1 and 2
3Portland152h, 3 h after lights off2HIC measured before Test 2; BEC measured during Test 2; 2-wk period of abstinence and resumption of ethanol intake (Test 3)
4Scripps152h, 3 h after lights off22-wk period of abstinence between Tests 1 and 2; bilateral infusion of CRF antagonist or vehicle during Test 2

Briefly, B6 mice were acclimated to 2 drinking tubes containing water for at least 2 days before initiating limited access ethanol consumption. Mice were then allowed unlimited access to 1 bottle containing a 10 or 15% v/v alcohol solution (depending on study) and to 1 containing tap water for 2 hours, beginning 3 hours after lights off. In Portland, drinking tubes were standard 25 mL graduated cylinders, with volumes read to the nearest 0.1 mL. At Scripps, drinking tubes were nalgene conical tubes fitted with sipper tubes, with drinking volumes inferred from tube weights (to the nearest 0.1 g). Average volume (or weight) depleted from tubes in control cages without mice were subtracted from individual values for each mouse for each day to control for spillage or evaporation. During the 2-hour period of ethanol access, one of the tubes containing tap water was replaced with a tube containing an ethanol solution. The position of the ethanol tube was switched every second day to control for side preferences. Consumption of water and ethanol was recorded (to the nearest 0.1 mL or 0.1 g) immediately before and after the period of ethanol access each day.

After baseline drinking stabilized (approximately 7 days), separate groups of mice were exposed in inhalation chambers to a series of 3 cycles of 16 hours of ethanol vapor (or air) exposure that were separated by 8-hour withdrawal periods. Ethanol vapor exposure was adjusted to yield target BECs of 1.5 to 2.0 mg/mL in the animals while they were exposed to ethanol vapor. Following the last cycle of ethanol exposure, both groups of mice were again allowed to consume either ethanol or water voluntarily for a minimum of 6 days (2-hour limited access, beginning 3 hours into the dark cycle). Then, mice were subjected to a second bout of intermittent ethanol vapor (identical to the first bout: 3 cycles of 16 hours of ethanol vapor+8 hours air) or air exposure inside inhalation chambers. After a single respite day during which mice drank only water, both groups of mice were again allowed to consume either ethanol or water for a minimum of 6 days (2-hour limited access sessions).

Ethanol Vapor Inhalation Exposure

Following the stabilization of voluntary baseline ethanol consumption, mice were divided into 2 cohorts of multiple withdrawal (MW) and air control (Control) that were matched on their baseline ethanol consumption. Immediately before placement into the inhalation chambers, the MW groups were weighed and injected daily with 1.5 g/kg of ethanol (20% v/v) in normal saline that included pyrazole HCl (68.1 g/kg; Sigma Chemical Company, St. Louis, MO). Pyrazole inhibits alcohol dehydrogenase and stabilizes BECs. The Control group was weighed and injected daily with the same dose of pyrazole in saline.

Portland Site. Groups of 2 to 3 mice were placed into hanging wire mesh cages inside inhalation chambers (Flair Plastics, Portland, OR) that were housed inside a bank of climate control chambers (Weaver Technologies, Portland, OR) for 16 consecutive hours. The inhalation chambers can accommodate 3 tiers of mesh cages. Before placement of mice in the chambers, the chambers were brought to equilibrium concentrations of ethanol in air of 7 to 9 mg/L and 0.0 mg/L, for the MW and Control groups, respectively. Ethanol vapor was introduced into the chambers by atomization of 200 proof ethanol (Pharmco Products, Brookfield, CT) through a frit into a stream of laboratory air. The concentration was kept homogeneous by 3 internal fans. Chamber levels were monitored hourly via gas chromatography (Agilent 6890N GC, using a HP-PLOT Q column, Wilmington, DE) delivered through a gas-sampling valve.

Scripps Site. Ethanol vapor chambers consist of standard plastic mouse-sized shoebox cages, housing up to 4 mice per chamber (La Jolla Alcohol Research Inc., La Jolla, CA). Ethanol vapor was created by dripping 95% ethanol (Pharmco Products) into a 2,000 mL Erlenmeyer vacuum flask kept at 50°C on a warming tray. Air was blown over the bottom of the flask at a rate of 11 L/min. Concentrations of ethanol vapor were adjusted by varying the rate at which the ethanol was pumped into the flask, which in turn, was based on the BECs of the mice. Ethanol vapor was independently introduced into each sealed chamber through a stainless-steel manifold. Identical chambers into which air was pumped were used for control chambers.

At both the Portland and Scripps sites, water and food were available ad libitum throughout the period of ethanol vapor exposure. Following the 16 hours exposure, the MW groups were removed from the chambers and 20 μL tail blood samples were collected from all mice. Tails were nicked in the Control group, but no blood sample was taken. Animals in the MW group were then returned to their respective cage and moved into a separate control chamber (0.0 mg/L ethanol) for 8 hours to facilitate withdrawal. The cycle was repeated, for a total of 3 cycles, at which time the animals were rehoused in their initial cages and 2-hour limited access 2-bottle choice drinking was resumed following a single respite day when water was freely available.

BEC Analysis

Tail blood samples (20 μL) were analyzed immediately after collection.

Portland Site. The blood samples were diluted into 500 μL of a matrix of 4 mM n-propanol in deionized water. The 2.0 mL crimp top vial containing the blood sample in matrix was capped and vortexed thoroughly before analysis. Analysis was performed via ambient headspace sampling gas chromatography (Agilent 6890N GC, using a DB-ALC1 column) on a 30 μL aliquot. Six pairs of ethanol standards (0.1–3.0 mg/mL), which included n-propanol (internal standard), were run before the samples.

Scripps Site. Blood was collected in capillary tubes and emptied into Eppendorf tubes containing evaporated heparin and kept on ice. Samples were centrifuged and plasma was decanted into fresh Eppendorf tubes. The plasma was then injected into an oxygen-rate alcohol analyzer (Analox Instruments, Lunenburg, MA) for blood alcohol determination. Five pairs of ethanol standards (0.5–3.0 mg/mL) were run before the samples.

Handling-Induced Convulsion (HIC) Scoring

This procedure involves lifting the animal by the tail, gently turning it 180° if necessary, and observing the incidence and severity of a mild tonic or tonic/clonic convulsion. Handling-induced convulsion scores ranging from 1 to 3 required the gentle turn to elicit a tonic or clonic convulsion, whereas convulsions elicited by merely lifting the mouse by the tail were scored as 4 to 6 (see Finn and Crabbe, 1999). In one study, HIC scores were measured before the initiation of limited access ethanol intake (baseline) and following the second bout of intermittent ethanol vapor or air exposure during the respite day (i.e., before measuring postinhalation drinking). Additional details are presented in the methods to Experiment 3.

Data Analysis

Data are presented as the mean±SEM. Daily ethanol intake (g/kg) and preference ratio were calculated for each animal. Average baseline intake for each mouse was determined from the last 3 to 4 days of 2-hour ethanol intake before the first bout of exposure to intermittent ethanol vapor or air. Analysis of variance (ANOVA) examined the effect of treatment on ethanol intake, preference ratio (volume ethanol/total fluid volume), and BEC, with postinhalation drinking days as a repeated measures factor. Ethanol intake also was collapsed across postinhalation drinking periods (averaged across days in the drinking period) and analyzed for treatment effects with postinhalation drinking blocks (Tests 1–3) as a repeated measures factor. Peak ethanol intake for each test period was calculated as the average across the 3 days of highest intake (i.e., day on which intake was highest and the preceding and following days) and also was analyzed for treatment effects. Two-way ANOVA analyzed data for overall group (MW vs Control) differences, with time (day or Tests 1–3) as a repeated measures factor. Significant interactions were pursued with 1-way ANOVAs and Tukey's post-hoc tests. Based on our a priori hypothesis that the intermittent ethanol vapor procedure would differentially alter ethanol intake, the MW and Control groups were analyzed separately even in the absence of a significant interaction.

For the analysis of the HIC scores, area under the withdrawal curve (AUC) was calculated for each animal by summing the hourly HIC scores following removal from the inhalation chamber. Group differences (i.e., MW vs Control) in AUC were analyzed with ANOVA.

In the study for which BEC was determined following the 2-hour limited access ethanol session, correlational analyses were conducted between ethanol dose consumed and BEC. For all analyses, statistical significance was set at p≤0.05.

Experiment 1. Enhancement of Ethanol Intake (10% Ethanol Solution) With a Second Bout of Intermittent Alcohol Vapor Exposure

In Portland, mice were treated as described above with the following modification: ethanol preference drinking (2-hour limited access) was measured, beginning 1 hour after lights off. Two groups of mice, matched for baseline consumption, were exposed to 2 bouts of intermittent ethanol vapor exposure (n=20) or air (n=13). Intermittent alcohol vapor exposure was adjusted to produce BECs ranging from 1.0 to 1.5 mg/mL following each 16-hour exposure period.

Experiment 2. Enhancement of Ethanol Intake (15% Ethanol Solution) With a Second Bout of Intermittent Alcohol Vapor Exposure and Incorporation of a Period of Abstinence

At both the Portland and Scripps sites, the following modifications were made to facilitate the expression of increased drinking during withdrawal: All studies measured 2-hour limited access ethanol intake of a 15% solution in male B6 mice, beginning 3 hours after lights off. At Scripps, after the first bout of intermittent alcohol vapor (or air) exposure and 1 week of postinhalation limited access ethanol intake (Test 1), mice had a 2-week period of abstinence during which only water was available. Then, baseline ethanol intake was again determined for 1 week before the second bout of intermittent alcohol vapor (or air) exposure. Postinhalation drinking again was measured for 5 days. A total of 16 mice (8 per group) were tested.

Experiment 3. Withdrawal-Associated Drinking (15% Ethanol Solution) Produces Symptoms of Physical Dependence and Increased BEC Following Limited Access Ethanol Intake

In Portland, animals were treated as described in Experiment 2 with the exception that the period of abstinence was incorporated after the postinhalation drinking following the second bout of intermittent alcohol vapor exposure (Test 2). For this study, baseline HIC scores were measured before the initiation of baseline 2-hour ethanol intake and then were measured hourly for 12 hours following removal from the inhalation chambers after the second bout of intermittent ethanol vapor (or air) exposure (i.e., during the respite day before the subsequent Test 2 drinking sessions). During the second period of postinhalation drinking, orbital blood samples (20 μL) were taken immediately following one of the 2-hour drinking sessions in the MW and Control groups to assess BEC. After the period of postinhalation drinking (Test 2), a 2-week period of abstinence was incorporated, and then 2-hour limited access ethanol intake was resumed for another 2 weeks (Test 3). A total of 20 mice (MW=12, Control=8) were tested.

Experiment 4. Manipulation of Increased Drinking During Withdrawal (15% Ethanol Solution) With Bilateral Infusions of d-Phe-CRF(12–41) Into the CeA

At Scripps, mice were treated as described in Experiment 2 except that 1 week following the first bout of intermittent alcohol vapor (or air) exposure and postinhalation limited access ethanol intake, the mice were surgically prepared with chronic indwelling intracerebral cannulae (i.e., during the 2-week period of abstinence). Mice were allowed a 1-week period of recovery, another week of limited access ethanol intake and then a second bout of intermittent alcohol vapor (or air) exposure. After this final removal from the vapor chambers, mice were again exposed to the limited access 2-bottle choice procedure for 1 week before their first CeA injections and another week before their second CeA injections.

Mice (N=8 Control and 7 MW) anesthetized with an isoflurane vapor mixture were secured in a Kopf stereotaxic instrument fitted with a mouse adapter. A 27 gauge, 9.0 mm long stainless-steel cannulae was lowered within 1 mm of the CeA (AP: −1.22 mm, ML: ±2.3 mm, DV: −3.5 mm) and anchored to the skull with 1.6 mm stainless-steel screws and dental cement (Geristore Syringeable Paste, Den-Mat Corp., Santa Maria, CA). Stereotaxic coordinates were based on the atlas of Franklin and Paxinos (1997). Thirty gauge dummy cannulae were kept inside the guide cannulae when not in use.

For intracerebral injections, a 33-gauge stainless-steel injector made 1 mm longer than the guide cannulae was inserted. This was attached to tubing (0.01 ID, 0.03 OD in.) and a 0.5 μL solution was injected across a 60-second period through each cannula using Hamilton micropipettes. d-Phe-CRF(12–41) synthesized by Dr. Jean Rivier was dissolved in 2 × phosphate-buffered saline to a final concentration of 0.5 μg/μL (each mouse received 0.25 μg drug per hemisphere). The injector was left in place for an additional 60 seconds to allow efflux of the infusion material. The mouse was returned to its home cage for 15 minutes before 2-bottle choice testing. Half of the mice received d-Phe-CRF(12–41) and half received vehicle infusions 1 week following the second bout of intermittent ethanol vapor or air exposure, and then the groups were switched a week later. Therefore, each mouse received 2 infusions (1 of vehicle and 1 of CRF antagonist) in a counterbalanced order, separated by a 1-week interval. Upon conclusion of the experiment, brains were removed, placed in paraformaldehyde, and sliced. Cannulae tracks were visualized by microscopy and injection sites were determined by noting the location 1 mm below the tip of the cannulae.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENT
  7. REFERENCES

Experiment 1. Enhancement of Ethanol Intake (10% Ethanol Solution) With a Second Bout of Intermittent Alcohol Vapor Exposure

Baseline ethanol intake stabilized at approximately 2.6 g/kg/2 h (Fig. 1) and did not differ between the MW and Control groups. Analysis of the daily intake data postinhalation (Tests 1 and 2) versus averaged baseline indicated that ethanol intake was higher in the MW versus Control group [F(1, 30)=8.27, p=0.007] and changed across time [F(15, 450)=9.50, p<0.001] (Fig. 1A). The trend for an interaction between time and group [F(15, 450)=1.60, p=0.07] and select post-hoc tests confirmed that postinhalation ethanol intake was higher in the MW versus Control group. An examination of daily postinhalation ethanol intake in the MW group indicated that peak ethanol intake increased to 3.7 g/kg/2 h (41% increase over baseline) following the first bout of intermittent alcohol vapor exposure, with a further increase to 4.4 g/kg/2 h (68% increase over baseline) following the second bout of intermittent alcohol vapor exposure. In contrast, there was a transient increase in ethanol intake in the Control group (40–44%) during Test 1 that did not increase further during Test 2.

image

Figure 1.  A second bout of intermittent ethanol vapor exposure yielded a greater increase in ethanol intake (10% ethanol solution), depicted as (A) daily intake and (B) averaged intake. In panel B, data are averaged over the last 3 d of baseline drinking or over the 7 to 8 d of drinking post each bout of intermittent ethanol vapor or air treatment. Values represent the mean±SEM for 20 (MW) or 12 to 13 (Control) mice per group. *p<0.05 versus Control group (panel A), *p<0.05, **p<0.01 versus respective baseline and Control group (Tests 1 or 2, panel B).

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Analyses conducted on the averaged baseline and postinhalation tests (Fig. 1B) documented that ethanol intake was significantly increased in the MW group [F(1, 30)=8.71, p=0.006]. The significant interaction between time and group [F(2, 60)=4.84, p=0.01] indicated that alcohol intake was differentially altered during the postinhalation tests in MW versus Control groups. Post-hoc tests confirmed that alcohol intake during Tests 1 and 2 was significantly greater in the MW group than respective values in Control mice as well as when compared with baseline (p≤0.05).

Analysis of peak ethanol intake during the postinhalation tests (not shown) was comparable with the averaged ethanol intake data. In the Control group, baseline ethanol intake (2.60 g/kg) increased to 3.34 g/kg during Test 2 (i.e., 28% increase). In the MW group, baseline ethanol intake (2.57 g/kg) increased by 66.5% during Test 2 (4.28 g/kg, p<0.01). Peak ethanol intake was higher in the MW than in the Control group [F(1, 30)=7.50, p=0.01] and was differentially altered during the postinhalation tests [F(2, 60)=4.88, p=0.01]. Peak ethanol intake was significantly higher during Test 2 (p=0.003) and tended to be higher during Test 1 (p=0.09) in the MW group versus the Control group.

Baseline preference ratio was high in both groups and did not differ significantly (not shown). The mean±SEM baseline preference ratio was 0.93±0.02 for the MW group and 0.91±0.03 for the Control group. Analysis of the collapsed baseline versus postinhalation preference ratio data indicated that there were no overall group differences. The lack of significant interaction between time and group indicated that postinhalation preference ratio was not differentially altered in the MW and Control groups.

Body weights in the MW and Control group are depicted in Table 2. Although baseline body weights were slightly lower in the MW (22.0±0.5 g) versus Control (23.5±0.5 g) group at the start of the study, there were no significant group differences at any time point. Ethanol vapor exposure produced BECs ranging from 0.92 to 1.32 mg/mL during the first bout of intermittent vapor exposure and 1.02 to 1.22 mg/mL during the second bout of intermittent vapor exposure.

Table 2.   Summary of Body Weight
ExperimentTime pointControl (g)MW (g)
  1. Depicted are the mean ± SEM body weights for each experiment, taken at baseline and at the beginning of each postinhalation drinking test (Tests 1, 2) or after a 2-week period of abstinence (Test 3). Within each experiment, there were no significant differences between groups in body weight at any of the time points.

  2. MW, multiple-withdrawal.

1Baseline23.5 ± 0.622.0 ± 0.5
Test 124.2 ± 0.422.2 ± 0.4
Test 224.7 ± 0.421.8 ± 0.3
2Baseline27.5 ± 0.627.2 ± 0.5
Test 129.1 ± 0.827.6 ± 0.8
Test 229.9 ± 0.828.6 ± 0.6
3Baseline23.2 ± 0.622.8 ± 0.3
Test 123.4 ± 0.721.8 ± 0.3
Test 223.4 ± 0.622.3 ± 0.3
Test 325.6 ± 0.525.2 ± 0.4
4Baseline27.6 ± 0.727.8 ± 0.9
Test 127.9 ± 0.927.0 ± 0.9
Test 228.1 ± 0.827.4 ± 1.0

Experiment 2. Enhancement of Ethanol Intake (15% Ethanol Solution) With a Second Bout of Intermittent Alcohol Vapor Exposure and Incorporation of a Period of Abstinence

The results of this experiment are shown in Fig. 2. Baseline ethanol intake stabilized at approximately 4.75 g/kg/2 h (Fig. 2A). Analysis of the daily intake data postinhalation versus averaged baseline indicated that ethanol intake was significantly higher in the MW versus the Control group during Test 2 [F(1, 14)=8.13, p=0.01]. Based on our a priori hypothesis, select post-hoc tests confirmed the increased ethanol intake in the MW group on days 2, 4, and 5 of Test 2. An examination of daily postinhalation ethanol intake during Test 2 indicated that peak ethanol intake increased to 6.4 g/kg/2 h (35% increase over baseline) in the MW group, whereas ethanol intake in the Control group remained at baseline values (range 3.5–4.6 g/kg/2 h).

image

Figure 2.  Enhancement of ethanol intake (15% ethanol solution) with a second bout of intermittent alcohol vapor exposure and incorporation of a period of abstinence. In panel B, data are averaged over the last 5 d of baseline drinking or over the 5 d of drinking post each bout of intermittent ethanol vapor or air treatment. A 2-wk period of abstinence occurred between Test 1 and Test 2. Depicted are the mean±SEM for 8 mice per group. *P<0.05 versus Control group (panel A), **P≤0.01 versus respective baseline and Control group (Test 2, panel B).

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The average of the final 5 days of ethanol drinking before vapor exposure was used for the baseline measurement and the averages of the 5 days immediately following each of the 2 bouts of intermittent vapor exposure were used for Tests 1 and 2 (Fig. 2B). The significant interaction between time and group [F(2, 28)=8.9, p=0.001] suggested that postinhalation drinking was differentially altered in the MW versus Control groups. Further analyses revealed a significant effect of treatment only in the MW group [F(2, 14)=20.77, p<0.001] with a significant increase in Test 2 drinking in these mice relative to their baseline (p<0.01).

Baseline preference ratio was high in both groups and did not differ significantly (not shown). The mean±SEM baseline preference ratio was 0.84±0.01 for the MW group and 0.86±0.03 for the Control group. Analysis of the collapsed baseline versus postinhalation preference ratio data indicated that there were no overall group differences, nor was there a significant interaction between time and group. For example, preference ratios following the second bout of vapor exposure were 0.87±0.02 for the MW group and 0.89±0.01 for the Control group.

Body weights in the MW and Control group are depicted in Table 2. Baseline body weights were 27.5±0.6 g for the Control group and 27.2±0.5 g for the MW group at the start of the study. There were no significant group differences in body weight at any time point. Average BEC were 1.30 to 1.95 mg/mL during the first vapor exposure and 2.33 to 3.56 mg/mL during the second vapor exposure. These data are consistent with studies conducted in Portland in which intermittent vapor exposure producing BECs of either 1.25 mg/mL or 2.0 mg/mL both produced a significant increase in postinhalation consumption of a 15% ethanol solution (R. Dhaher and R. Hitzemann, unpublished).

Experiment 3. Withdrawal-Associated Drinking (15% Ethanol Solution) Produces Symptoms of Physical Dependence and Increased BEC Following Limited Access Ethanol Intake

Baseline ethanol intake stabilized at 3.3 g/kg/2 h and did not differ between Control and MW groups. Analysis of the daily intake data (Fig. 3A) confirmed that ethanol intake was significantly greater in the MW than in the Control group [F(1, 8)=12.78, p=0.007] and that intake increased significantly across time [F(35, 280)=1.69, p=0.01]. During Test 2, ethanol intake in the MW group was significantly greater on days 31 and 34 (p<0.01), when compared with values in the Control group. Following the 2-week period of abstinence, ethanol intake on days 51 and 54 was significantly greater in the MW versus Control group (p≤0.05).

image

Figure 3.  The effect of intermittent alcohol vapor exposure on limited access ethanol intake (15% ethanol solution), measured by (A) daily intake, (B) averaged intake, and (C) peak intake. In panel B, data are averaged over the last 3 d of baseline drinking, over the 7 d post each bout of intermittent ethanol vapor or air treatment (Tests 1 and 2), or over the 14 d postperiod of abstinence (Test 3). Values represent the mean±SEM for 7 to 8 (Control) or 11 to 12 (MW) mice per group. *At least p<0.05 versus Control group (panel A) *p<0.05, **p<0.01 versus respective baseline and Control group at respective Test (panels B and C) #p< 0.05 versus respective baseline (panel C).

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Analysis conducted on averaged baseline and postinhalation tests (Fig. 3B) revealed a trend for an overall group difference in ethanol intake [F(1, 16)=2.83, p=0.11]. Subsequent analysis of each group indicated that postinhalation ethanol intake tended to be higher than baseline values only in the MW group [F(3, 30)=2.47, p=0.08]. Post-hoc tests confirmed that intermittent alcohol vapor exposure significantly increased ethanol consumption over baseline and values in the Control group during the postinhalation Test 2, with a trend for increased ethanol intake in the MW group following a 2-week period of abstinence (Test 3).

Analysis of peak ethanol intake supported the increased alcohol intake in the MW group (Fig. 3C). Overall peak ethanol intake was significantly higher in the MW versus Control group [F(1, 16)=7.20, p<0.016] and increased significantly across time [F(3, 48)=10.075, p<0.001]. The trend for an interaction between group and time [F(3, 48)=2.24, p=0.096] suggested that peak ethanol intake was differentially altered in the MW versus Control groups. In the MW group, peak ethanol intake increased significantly across time [F(3, 30)=8.68, p<0.001]. Post-hoc tests confirmed that peak ethanol intake was increased significantly over baseline at each postinhalation time point (p<0.001) by 37.5% (Test 1) or 41% (Tests 2 and 3). Although peak ethanol intake was increased across time in the Control group [F(3, 18)=6.61, p<0.01], this effect was due to the increase over baseline in the postabstinence time point (Test 3, p<0.05) and may represent an alcohol deprivation effect (ADE).

Baseline preference ratio was high in both groups and did not differ significantly (not shown). The mean±SEM preference ratio was 0.86±0.03 for the MW group and 0.86±0.04 for the Control group. Analysis of the collapsed baseline versus postinhalation and abstinence preference ratio data indicated that there were no overall group differences, although there was a slight, but significant fluctuation in preference ratio across time [F(33, 495)=1.54, p<0.05]. The lack of significant interaction between group and time indicated that preference ratio was not differentially altered in the MW and Control groups.

BEC was measured on day 34 (during Test 2), immediately after the 2-hour limited access to ethanol. Notably, the significant increase in ethanol intake corresponded to a significant increase in BEC (Fig. 4A). On the day that BEC was assessed, mean±SEM ethanol dose was 4.075±0.32 g/kg for the MW group versus 2.50±0.50 g/kg for the air controls [F(1, 16)=7.96, p=0.01], whereas BEC was 2.15±0.16 mg/mL for the MW group versus 1.07±0.20 mg/mL for the air controls [F(1, 16)=18.121, p=0.001]. Correlational analyses indicated that the ethanol dose consumed on day 34 was significantly positively correlated with BEC (r=0.76, n=18, p<0.001; Fig. 4B).

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Figure 4.  A second bout of intermittent alcohol vapor exposure significantly increases ethanol consumption and BEC. Panel A depicts mean±SEM BEC assessed on day 34 (during Test 2) in Control (n=7) and MW (n=11) mice, whereas data for individual animals are depicted in panel B. The ethanol dose consumed was significantly positively correlated with blood ethanol concentrations (BEC) (r=0.76, n=18, p<0.001). ***p=0.001 versus Control group.

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To determine whether 2 bouts of intermittent ethanol vapor exposure produced symptoms of physical dependence, HICs were measured at baseline (i.e., before initiation of baseline ethanol preference drinking) and following the second bout of intermittent ethanol vapor or air exposure (i.e., hourly for 12 hours following removal from the inhalation chambers and before any postinhalation drinking during Test 2). Hourly HIC scores were increased over baseline values in both groups (not shown). Area under the withdrawal curve, the index of ethanol withdrawal severity, was 20.94±1.87 (n=8) for the Control group and 31.86±2.19 (n=11) for the MW group. These values were significantly different [F(1, 17)=13.01, p=0.002], and represented an increase in AUC by 52% in the MW versus Control group.

At the beginning of the experiment, body weight did not differ between the animals subsequently divided into the Control (23.2±0.6 g) and MW (22.8±0.3 g) groups. There were no group differences in body weights at any time point (Table 2). Ethanol vapor exposure produced BECs ranging from 1.14 to 1.69 mg/mL during the first bout of intermittent vapor exposure and 1.46 to 1.50 mg/mL during the second bout of intermittent vapor exposure.

Experiment 4. Manipulation of Increased Drinking During Withdrawal (15% Ethanol Solution) With Bilateral Infusions of d-Phe-CRF(12–41) Into the CeA

The results of this experiment are shown in Fig. 5. The right panel shows the injection sites superimposed on brain slice drawings copied from the atlas of Franklin and Paxinos (1997) in the 14 mice (7 Control and 7 MW) for which injections were determined to be within the CeA. The average of the final 5 days of ethanol drinking before vapor exposure was used for the baseline measurement and the individual days of CeA infusions are shown for the vehicle and d-Phe-CRF(12–41) treatments (left panel). As daily ethanol intake for a comparable study conducted at the Scripps site are depicted in Fig. 2A and the microinjections were conducted during Test 2, the data for daily ethanol intake are not shown. Importantly, the significant increase in daily ethanol intake in the MW group that is depicted in Fig. 2A persisted in the present study for 2 weeks, allowing for microinjections to be conducted once per week.

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Figure 5.  Blockade of increased drinking during withdrawal with bilateral infusions of a CRF antagonist into the CeA. Left panel: mice received bilateral infusions of vehicle (0.5 μL/side) or d-Phe-CRF(12–41) (0.25 μg/0.5 μL/side) approximately 15 min before limited access ethanol preference drinking (15% solution) during Test 2 (Experiment 4). Mice received infusions of both treatments, separated by a 1-wk interval, with the order of infusions counterbalanced across weeks. Data are averaged over the last 5 d of baseline drinking and on the individual days of drug and vehicle infusions. Data for the noninfusion days are not shown, as they did not differ from those following vehicle infusions. Values represent the mean±SEM. Right panel: depicts the injection sites superimposed on brain slice drawings copied from the atlas of Franklin and Paxinos (1997) in the 14 mice (7 Control and 7 MW) for which injections were determined to be within the CeA. *p<0.05 versus respective baseline data.

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As there were no order effects for the drug infusions, analyses were conducted on the averaged baseline and averaged infusion data during Test 2 for mice in which cannulae placements were confirmed to be in the CeA. There was a significant interaction between treatment and group [F(2, 24)=11.7, p<0.001], suggesting that ethanol intake was differentially altered by the drug infusions in the MW versus Control groups. Further analyses revealed a significant effect of treatment only in the MW group [F(2, 12)=15.16, p<0.001], with a significant increase in ethanol intake following vehicle infusion (p<0.05) and a significant decrease in ethanol intake following d-Phe-CRF(12–41) infusion (p<0.05) when compared with baseline intake (Fig. 5). Ethanol intake on noninfusion days was comparable with that following vehicle infusion (not shown). The results of this experiment confirm the increase in ethanol drinking following 2 bouts of intermittent ethanol vapor exposure (i.e., the increase in vehicle-injected MW mice). Furthermore, they indicate that intra-CeA administration of a CRF antagonist reversed the increased drinking during withdrawal without altering ethanol intake in nondependent mice.

Baseline preference ratio was high in both groups and did not differ significantly (not shown). The mean±SEM baseline preference ratio was 0.91±0.02 for the MW group and 0.89±0.03 for the Control group. Analysis of the collapsed baseline versus postinhalation preference ratio data indicated that there were no overall group differences on nonmicroinjection days. Likewise, microinjection of d-Phe-CRF(12–41) did not alter preference ratio. On the test days, preference ratios of Control mice receiving vehicle were 0.90±0.05, Control mice receiving CRF antagonist were 0.92±0.03, MW mice receiving vehicle were 0.92±0.03, and MW mice receiving CRF antagonist were 0.93±0.03.

Body weights in the MW and Control group are depicted in Table 2. Baseline body weights were 27.6±0.7 g for the Control group and 27.8±0.9 g for the MW group at the start of the study. There were no significant group differences in body weight at any time point. Average BEC were 1.33 to 1.75 mg/mL during the first vapor exposure and 1.38 to 1.72 mg/mL during the second vapor exposure.

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENT
  7. REFERENCES

Modification of the procedure recently published by Becker and Lopez (2004; Lopez and Becker, 2005) produced reliably increased drinking during withdrawal at the Portland and Scripps sites. Following the first bout of intermittent alcohol vapor exposure, 2-hour limited access (10% solution) intake was increased by approximately 30%. However, the increase was most pronounced in animals consuming the 15% ethanol solution and when 2-hour limited access ethanol consumption was measured beginning 3 hours after lights off. This time frame corresponds with recent data indicating that 2-hour ethanol intake was maximal when consumption was initiated at 3 hours after lights off (Rhodes et al., 2005). Incorporating a second bout of intermittent alcohol vapor exposure as well as a period of abstinence (before the second bout of vapor exposure) produced a more pronounced and persistent increase in alcohol intake. Averaged ethanol intake increased by 35% whereas peak ethanol intake increased by 40% over baseline values in the MW group. These data are consistent with recent findings (Lopez and Becker, 2005), in which withdrawal-associated drinking was more prominent following 2 or 4 bouts of intermittent alcohol vapor exposure and persisted longer following 4 bouts of intermittent vapor exposure. A persistent increase in ethanol intake would facilitate studies examining the neurobiological mechanisms underlying drinking during withdrawal.

The significant increase in ethanol intake in the MW group was consistent with the significant increase in BEC assessed immediately following the 2-hour period of ethanol access in Experiment 3. Notably, the 63% increase in ethanol dose consumed corresponded to a 101% increase in BEC, and values in individual animals were significantly positively correlated (r=0.76, p<0.001, n=18). The majority of the mice in the MW group achieved BECs≥2.0 mg/mL following the 2-hour period of ethanol access, which is an ethanol concentration that would be expected to produce visible signs of intoxication (e.g., Cronise et al., 2005; Rhodes et al., 2007).

Hourly HIC scores and AUC indicated that there was a significant increase in withdrawal severity in the MW versus Control group following the second bout of intermittent alcohol or air exposure. While we cannot rule out the possible contribution of the daily ethanol intake before our assessment of withdrawal (i.e., baseline and Test 1), AUC in the MW group was significantly higher that that in the Control group. As withdrawal has been used as an index of physical dependence in rodent models, the increased AUC in the MW group suggests that these animals were exhibiting a greater degree of physical dependence. As withdrawal was assessed on the respite day before the resumption of limited access ethanol intake during Test 2, we think that it is unlikely that the HIC scoring altered ethanol intake on subsequent days.

In some studies there was a transient increase in ethanol intake in the Control group following the bouts of air exposure. This transient increase might be due to a mild ADE, i.e., a transient increase in ethanol consumption and preference after a period of imposed ethanol abstinence (e.g., Heyser et al., 1997; McKinzie et al., 1998; Rodd-Henricks et al., 2000; Spanagel and Holter, 1999). Quality control assessments assured that no ethanol vapor had leaked into the air chamber (i.e., the transient increase in the air controls was not due to ethanol vapor contamination in the air chamber). Additionally, there was no further increase in ethanol intake in the air controls following the second bout of intermittent air exposure. Finally, the significant increase in peak ethanol intake in the Control group following the 2-week period of alcohol deprivation (i.e., Test 3; Fig. 3C) could reflect an ADE.

Pharmacological manipulation of drinking during withdrawal can be conducted to identify important neurochemical targets that contribute to the acquisition (i.e., drug administered during intermittent ethanol vapor or air exposure) or the expression (i.e., drug given during the postinhalation drinking period before each period of ethanol access) of this phenotype. The present results implicate neuroadaptations in the CRF system of the amygdala as one critical component underlying the expression of withdrawal-associated drinking. The amygdala has been implicated as an important brain site in the neuroadaptations associated with drug and ethanol dependence (Bruijnzeel and Gold, 2005). For example, CRF-like immunoreactivity content was elevated in the amygdala of rats 6 weeks after ethanol withdrawal (Zorrilla et al., 2001). Funk et al. (2007) have recently shown that administration of d-Phe-CRF(12–41) in the CeA reverses the increases in operant ethanol self-administration in rats following ethanol withdrawal. We have extended these results to a mouse model that measured limited access home cage ethanol intake, both highlighting the utility of this model for mechanistic studies and providing further evidence for a role of the CeA CRF system in the high ethanol consumption associated with dependence.

In conclusion, intermittent alcohol vapor exposure significantly increased alcohol intake and produced signs of physical dependence, suggesting that information gained with this procedure may help in furthering our understanding of the mechanisms underlying increased alcohol intake in dependent animals. The finding that pharmacological manipulation of the CRF system in the CeA altered the expression of increased drinking during withdrawal points to an important neurochemical target to pursue in future studies that could aid in the development of new strategies for the treatment of alcoholism.

ACKNOWLEDGMENT

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENT
  7. REFERENCES

The authors wish to thank Dr. Jean Rivier of the Salk Institute for providing d-Phe-CRF(12–41).

REFERENCES

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  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENT
  7. REFERENCES
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