The main findings of these experiments clearly demonstrate that the potency of NTX to reduce self-administration of an ethanol solution is far greater when injected subcutaneously than when injected intraperitoneally. Via both routes of injection, NTX caused dose-related reductions of ethanol intake, and direct comparison of the dose—effect curves via comparison of ED50 values showed that subcutaneous NTX was approximately 30-fold more potent than intraperitoneal NTX. Cumulative records of the fluid deliveries obtained during the session and the bin analysis support other literature that show that the effective doses of NTX (subcutaneous or intraperitoneal) are characterized by early cessation of responding rather than delayed responding at the start of the operant session.
The route of opioid antagonist administration may have a substantial impact on preclinical experiments, although it is rarely emphasized in the literature regarding the effects of antagonists on self-administration of ethanol. Subcutaneous injections of 0.1—0.3 mg/kg naloxone (Hyytia and Kiianmaa, 2001
; Schwarz-Stevens et al., 1992
) or 0.1 mg/kg NTX (Holter and Spanagel, 1999
; June et al., 1998
) reduced ethanol-reinforced responding. Similar potency is observed in experiments where rats are allowed to consume ethanol during nonoperant conditions. For example, 0.1 mg/kg NTX reduced ethanol consumption when rats had limited access to ethanol from bottles in the home cage (Stromberg et al., 1998
). Intraperitoneal antagonist injections reduce ethanol-reinforced responding or ethanol consumption at doses that are at least 10-fold larger than the effective doses given via subcutaneous injection. In experiments wherein intraperitoneal antagonist dose—effect curves were identified, 1 mg/kg naloxone was required to reduce ethanol-reinforced responding (Hyytia and Sinclair, 1993
), and 1 mg/kg NTX was required to reduce ethanol drinking from bottles in the home cage (Hill and Kiefer, 1997
). It is important to note that intraperitoneal naloxone at 1 mg/kg does not have selective effects on ethanol consumption; this dose reduced bottle consumption of 15% sucrose, 0.6% saccharin, and water (Cichelli and Lewis, 2002
). In other studies, even larger intraperitoneal doses of NTX or naloxone (3 mg/kg or higher) had a marginal effect or no effect on responding for ethanol (Bienkowski et al., 1999
; Sharpe and Samson, 2001
; Williams, 2007
). Because the potency differences for NTX or naloxone across different routes of administration may differ by more than 10-fold, caution should be used when making comparisons of effective NTX doses across studies following systemic injections.
Our study is one of the only studies to directly compare the influence of route of opioid antagonist administration in rats. Only one other study has conducted a similar comparison (Mucha and Iverson, 1984
). In that study, subcutaneous injections of 0.5 mg/kg naloxone produced a large conditioned place aversion after only three pairings, and the aversion was greater than that observed after similar naloxone doses given via intraperitoneal injection. One explanation for this effect is that compounds injected via the intraperitoneal route are subject to first-pass metabolism by the liver due to absorption through portal circulation (Lukas et al., 1971
), and NTX undergoes substantial metabolism by the liver (Crabtree, 1984
). In our experiments, we show clear potency differences based on route of administration while controlling for NTX pretreatment time. In the literature, a broad range of pretreatment times exist. For example, naloxone has been given as an intraperitoneal injection immediately before an operant session in an appetitive-consumption model (Sharpe and Samson, 2001
), and NTX has been given as long as 70 min before a drinking session (Perfumi et al., 2003
). In our experiments, NTX was given 10 min before the operant session. Although we did not have the opportunity to measure blood levels of NTX, this pretreatment time should allow rapid entry of NTX into systemic circulation even via the intraperitoneal route. For instance, in one study, an intraperitoneal injection of 3 mg/kg NTX given 10 min before the session caused a dramatic rightward shift of the dose—effect curve for intravenous heroin self-administration in rats (Martin et al., 1996
). Our data are consistent with the above study; a dose of 3 mg/kg given intraperitoneally significantly reduced ethanol intake, and the pattern of fluid deliveries illustrated effects within the first 5 min of the operant session. Although the route of administration may influence the potency differences observed in the literature, other variables may play a role as well.
Variables related to motivational level may alter the potency of NTX. One motivational factor may be related to hunger or the feeding status of the subjects. In food-restricted rats (approximately 85% of free-feeding weight), an intraperitoneal injection of 30 mg/kg NTX had minimal effects on responding to ethanol (Williams, 2007
). In studies that directly compared the effects of opioid antagonists on consummatory behavior in food-restricted rats versus free-feeding rats, naloxone was at least 10-fold less potent in the food-restricted rats (Levine et al., 1995
; Rudski et al., 1994
). Rats that experienced repeated cycles of water restriction also showed resistance to the consumption-decreasing effects of NTX; 10–30 mg/kg reduced water consumption by no more than 40% (White and Holtzman, 2001
). Another motivational factor may be the taste or palatability of the solution. In studies of consummatory behavior, sensitivity to naloxone was increased when rats were feeding on sweet food or chow versus normal chow (Giruado et al., 1993
; Levine et al., 1995
). Although palatability influences the effects of opioid antagonist on consumption of food, the issue is not mentioned as frequently in studies regarding ethanol self-administration. However, palatability plays a role in these experiments due to the many different ethanol solutions used in the studies. For example, some researchers have explored the effects of opioid antagonists on sweetened ethanol solutions (e.g., Gardell et al., 1997
) or on lower concentrations of ethanol (e.g., Stromberg et al., 2002
). Stromberg et al (2002)
used a 6% vol/vol ethanol solution. This ethanol concentration equates to approximately 4.5% wt/vol ethanol, which may be more palatable than the 10% wt/vol solution often cited in the preclinical literature. At least one author calls for researchers to conform to a standardized way to calculate ethanol concentrations (Brick, 2006
). More recently, the addition of a sweetener to an ethanol solution has been proposed as part of a model for binge drinking (Ji et al., 2008
) and for adolescent drinking behavior (Maldonado et al., 2008
). Thus, palatability should be considered when examining the effects of opioid antagonists on ethanol consumption. Although palatability may play a role in the NTX potency differences observed across different studies in the literature, we controlled for palatability by administering subcutaneous and intraperitoneal NTX while rats were consuming the same sweetened ethanol solution.
In summary, opioid antagonists such as NTX are often used in preclinical work exploring the effects of ethanol. Inferences are often made about opioid receptor selectivity following systemic injections of NTX in rodents. The work presented here indicates that caution should be used when making these inferences and that the route of NTX administration should be considered an important variable when designing experiments using opioid antagonists.