Revisiting intragastric ethanol intubation as a dependence induction method for studies of ethanol reward and motivation in rats

Abstract

Background: The purpose of this study was to re-examine intragastric ethanol intubation as a dependence induction method that effectively induces physical dependence upon ethanol over a short time period, is devoid of intrinsic stress artifacts, inexpensive, and easy to implement.

Methods: Male Wistar rats were subjected to ethanol dependence induction via intragastric ethanol intubation. Ethanol solution (final concentration 20%, made up in a dietary liquid vehicle consisting of powdered milk, sucrose, and water) was intubated 4 times per day, at 4-hour intervals, for 6 consecutive days (for a total of 10 g/kg/day). The utility of this procedure was evaluated for inducing physical dependence, determined by daily and final withdrawal ratings. Anxiety-like behavior associated with ethanol dependence history was examined using the elevated plus-maze (EPM) test, conducted 5 days after ethanol withdrawal. To evaluate whether potential stress-like effects of intragastric intubation per se produce lasting effects on behavior, experimentally naive rats were compared with vehicle-intubated rats for anxiety-like behavior on the EPM.

Results: Blood alcohol levels reached stable levels between 200 and 250 mg%, measured 1 hour after the second and third ethanol intubation on days 2, 4, and 6. Ethanol-treated rats developed significant somatic withdrawal signs, recorded daily between 10 and 12 hours after the last ethanol administration. At 5 days postwithdrawal, ethanol-treated rats showed significant anxiety-like behavior, measured by decreased open arm time and open arm entries on the EPM, compared with vehicle controls. Additionally, ethanol postdependent rats showed decreased open arm time compared with experimentally naive rats. EPM performance did not differ between vehicle-intubated and naive rats. No withdrawal seizures were observed and mortality rate was near zero.

Conclusions: These findings suggest that intragastric ethanol administration produces a behavioral profile consistent with ethanol dependence (i.e., significant withdrawal signs after termination of ethanol exposure and elevated anxiety-like behavior persisting beyond completion of physical withdrawal), and that the intubation procedure itself does not produce lasting nonspecific anxiety-like effects. Thus, under the conditions employed here, this procedure provides an effective tool for inducing and evaluating the consequences of ethanol dependence in animal models of ethanol reward and motivation.

Figure 1

Diagram illustrating the procedure for inducing ethanol dependence. On day 1, rats were administered a total of 11.0 g/kg ethanol in four fractional doses of 3.0, 3.0, 2.5, and 2.5 g/kg ethanol, with doses administered at 4 h intervals. On days 2–6, 12 h after the last intubation on the preceding day, rats received a total of 10.0 g/kg ethanol in four fractional doses of 3.0, 2.5, 2.5, and 2.0 g/kg, again separated by 4 h intervals. Control rats received intragastric administration of vehicle at volumes and time intervals identical to those in ethanol-intubated rats. Tail bleeds were collected on days 2, 4, and 6 immediately before the first daily ethanol intubation and 1 h after the second and third daily intubation. Between 10–12 h after the last ethanol intubation, behavioral signs of withdrawal were rated daily.

Figure 2

Blood alcohol levels measured on days 2, 4, and 6 immediately before the first daily intubation (a) and 1 h after the second (b) and third (c) daily intubation. ^**p < 0.01, ^***p < 0.001, different from vehicle group; ^††p < 0.01, ^†††p < 0.001, different from the first tail bleed of the ethanol group.

Figure 3

Overall withdrawal severity (sum of somatic withdrawal scores across the four behavioral signs of ethanol withdrawal) measured between 10–12 h after the last daily ethanol intubation during the six-day ethanol dependence procedure. (Inset) Somatic withdrawal signs measured on day 6 of the ethanol dependence procedure. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, different from vehicle group; ^†p < 0.05, ^††p < 0.01, different from overall withdrawal score of the ethanol group rated on day 1. VLR, ventro-medial limb retraction.

Figure 4

Effect of intragastric ethanol intubation on body weight over the six-day ethanol dependence procedure in rats. ^*p < 0.05, different from vehicle group.

Figure 5

Effect of ethanol withdrawal measured five days after completion of the ethanol dependence procedure on behavioral anxiety in the elevated plus-maze, reflected in mean (± SEM) for (A) the percentage of total arm time spent in the open arms and (B) open arm entries. ^*p < 0.05, different from naive group; ^†p < 0.05, different from vehicle group.

Figure 5

Effect of ethanol withdrawal measured five days after completion of the ethanol dependence procedure on behavioral anxiety in the elevated plus-maze, reflected in mean (± SEM) for (A) the percentage of total arm time spent in the open arms and (B) open arm entries. ^*p < 0.05, different from naive group; ^†p < 0.05, different from vehicle group.