Ethanol motivation and sensitivity were assessed in B6 × FVB and B6 × NZB F1 hybrids using a battery of tests including conditioned taste aversion, conditioned place preference, LORR, acute withdrawal severity and repeated alcohol injections followed by voluntary ethanol consumption (). B6 × FVB mice were less sensitive to the aversive and sedative, but not to the rewarding, effects of ethanol than B6 × NZB. In addition, the B6xFVB mice were more exploratory, less anxious and more active than B6 × NZB mice.
Differences in sensitivity to the aversive effects of ethanol could have a role in the development of the RAP and SAP behaviors seen in our models. Indeed, B6 × FVB mice developed a less severe ethanol-induced conditioned taste aversion than B6 × NZB mice. The relative strength of the conditioned stimulus, saccharin, is known to influence the development of conditioned taste aversion. Blednov et al. (2010)
report B6 × FVB and B6 × NZB both exhibit a high preference for saccharin (0.94 ± 0.01 and 0.88 ± 0.04, respectively); therefore, differences in saccharin preferences are not likely an explanation of the differences in ethanol-induced conditioned taste aversion. In addition to our findings, an inverse relationship between ethanol consumption and severity of ethanol-induced conditioned taste aversion has been described for many genotypes (Broadbent et al. 2002
; Chester et al. 2003
; Phillips et al. 2005
). However, it is unknown whether ethanol-induced conditioned taste aversion is produced by the rewarding or aversive effects of ethanol (Liu et al. 2009
). Additional conditioned taste aversion experiments would be helpful in determining if the conditioned taste aversion that develops in these hybrids is specific to ethanol. If it is specific to ethanol, one would expect the hybrids to develop a similar degree of conditioned taste aversion to lithium chloride. Another variant of conditioned taste aversion could be employed using two tastants where, on alternating days, one tastant is paired with ethanol injections and another tastant is paired with saline injections. Ethanol specificity would be supported if a taste aversion developed to the tastant paired with ethanol, but not to the tastant paired with saline.
B6 × FVB mice were less sensitive to the sedative effects of ethanol than B6 × NZB mice. Differences in ethanol-induced LORR can be due to differences in metabolism, sensitivity, or acute tolerance. We found no difference in rates of ethanol clearance after a single high dose, suggesting that the differences in LORR are not due to clearance. Blood alcohol levels at regaining of righting reflex were not different for B6 × FVB and B6 × NZB mice, indicating no difference between the hybrids in alcohol sensitivity at awakening. Therefore, it is likely that the difference in LORR is due to initial sensitivity to ethanol. B6 × FVB are less sensitive to the initial effects of ethanol than B6 × NZB. This implies that B6 × FVB develop greater acute tolerance than B6 × NZB.
B6 × FVB and B6 × NZB mice developed similar ethanol-induced conditioned place preference and both genotypes showed a motor stimulatory response to ethanol during the conditioning trials. These observations are consistent with both genotypes exhibiting similar sensitivity to the rewarding properties of ethanol. It is important to note that ethanol preference in general is likely influenced not only by pharmacological actions of ethanol, but also by its caloric value, taste, olfaction and palatability, whereas the final measure of reward in conditioned place preference (as well as conditioned taste aversion) paradigms takes place in the absence of ethanol (Bachmanov et al. 2003
; Belknap et al. 1993
; Kiefer et al. 1998
; McMillen and Williams 1998
). The issue of whether voluntary ethanol drinking is related to the reinforcing effects of ethanol has been addressed in a recent meta-analysis which revealed a positive relationship between ethanol drinking and ethanol-induced conditioned place preference (Green and Grahame 2008
Acute ethanol-induced withdrawal was higher for B6 × FVB mice than for B6 × NZB mice. However, it is pertinent to note that both genotypes showed a low HIC withdrawal severity. In the future, other tests of alcohol withdrawal severity such as withdrawal-induced anxiety could be measured. Since both hybrids show an initial high ethanol preference and a low severity of acute ethanol withdrawal, our results further support findings by Metten et al. (1998)
demonstrating a negative correlation between ethanol preference and acute ethanol withdrawal severity.
Although drug-induced locomotor sensitization is thought to reflect neural adaptations important in the development of addiction, ethanol-induced locomotor sensitization has been positively and negatively correlated with voluntary ethanol consumption in mice (Grahame et al. 2000
; Lessov and Phillips 1998
; Phillips et al. 1995
; Robinson and Berridge 2000
). Ethanol-induced locomotor sensitization is well known to be dependent upon genotype and ethanol dose (Lessov et al. 2001
; Phillips et al. 2005
). Under our conditions, neither genotype displayed ethanol-induced locomotor sensitization. Ethanol-experienced mice had suppressed locomotor activity as compared with saline-experienced mice.
In addition, injection of ethanol did not change the SAP and RAP behavioral phenotypes. However, ethanol preference and consumption were increased in B6 × NZB and B6 × FVB mice injected with ethanol as compared with saline injection. One interpretation of this finding is that mice are more sensitive to the stress associated with the previous experience of repeated saline injections. When comparing ethanol preference and consumption data from the first and second 9% ethanol presentations, B6 × FVB mice given repeated saline injections unexpectedly showed a small, but significant reduction. Other reports showed that pre-exposure to ethanol (via voluntary or forced ethanol consumption) can increase, decrease, or have no affect on subsequent ethanol consumption (Lessov et al. 2001
; Ufer et al. 1999
). Importantly, preliminary experience with equivalent ethanol doses did not change the SAP and RAP behavioral phenotypes. This suggests that initial differences in ethanol consumption play a minimal role in subsequent behavior. In contrast to the schedule carried out by Blednov et al. (2010)
, there were no periods of abstinence in this experimental schedule; therefore, they may not be necessary for the development of RAP seen in B6 × NZB mice. It will be important to test hybrid responses to different ethanol schedules (i.e., experience with high or low concentrations of ethanol, with or without periods of abstinence) to identify the conditions which produce RAP in B6 × NZB mice.
There were notable differences in behavior when handling the hybrids and subsequent behavioral tests (elevated plus maze, mirror chamber, and environmental novelty) validated that B6 × FVB mice were more exploratory, less anxious, and more active than B6 × NZB mice. Anxiety is often invoked as a predictor of ethanol consumption or more specifically, craving and relapse behavior. For example, the selected rat line Roman high-avoidance (low anxiety and high novelty seeking profile) shows a higher preference for ethanol than the Roman low-avoidance rats (high anxiety and low novelty seeking profile) (Fernández-Teruel et al. 2002
). Also, there are several reports indicating a major role for anxiety and stress in craving and relapse models (Heilig and Koob 2007
). To further understand ethanol-related behaviors in these hybrids, it will be important in the future to compare their behavioral responses before and after SAP/RAP, rather than under ethanol-naïve conditions.
These new genetic models of both stable, high consumption and experience-induced moderate drinking offer significant advantages to existing models. B6 × FVB mice drink high concentrations of ethanol with a high preference. Therefore, to test the validity of the SAP behavioral phenotype it is imperative to assess the development of tolerance and dependence after chronic voluntary consumption for this new mouse model. Factors important for experience-induced alcohol moderation, a negative alcohol deprivation effect, have not been fully explored. Neuronal circuitry underlies motivational aspects of ethanol consumption. Therefore it will crucial to identify neural networks important for the SAP and RAP behavioral phenotypes to better understand what neurobehavioral mechanisms could be responsible for SAP and RAP.