Higher sensitivity to ethanol's aversive properties in WLP (Warsaw Low Preferring) vs. WHP (Warsaw High Preferring) rats

Highlights

• There are no known reasons for high preference and low ethanol preference of the selected WHP and WLP rat lines.

• The purpose of the work was to study the sensitivity of WHP and WLP rats to aversive effects of ethanol.

• We showed difference between WHP and WLP rats in sensitivity to the aversive effects of alcohol at dose of 1.5 g/kg.

• A higher dose of ethanol, i.e. 2.0 g/kg, produced aversion in both groups of animals.

• Differences in sensitivity to aversive effects of ethanol are not associated with metabolism of alcohol.

Abstract

Ethanol can have both an aversive and rewarding effect, which may have a significant relationship to its individual preference. So far, the reasons for the high and low ethanol preference in the WHP (Warsaw High Preferring) and WLP (Warsaw Low Preferring) lines have not been found. WHP rats spontaneously drink over 5 g/kg/day of ethanol, while WLP rats drink under 2 g/kg/day. The purpose of the work was to study the sensitivity of WHP and WLP rats to the aversive effects of ethanol at doses of 1.5 g/kg and 2.0 g/kg in the conditioned taste aversion (CTA) procedure. Lower doses (0.5 and 1.0 g/kg, i.p. [intraperitoneally]) were tested earlier and only 1.0 g/kg produced a slight aversion in WLP rats. The secondary aim was to check the additional potential factors (blood ethanol concentration, pain sensitivity, anxiety-related behavior, learning, and memory) that may constitute an important differentiating feature of the WHP and WLP lines. For this purpose, the following tests were conducted: blood ethanol concentration, novel object recognition (NOR), flinch-jump, hot-plate, and elevated plus maze (EPM).

The 1.5 g/kg i.p. dose of ethanol caused the development of an aversion only in WLP rats and the aversion extinguished in the post-conditioning phase. The 2.0 g/kg i.p. dose of ethanol resulted in the development of an aversion in both the tested groups, with the aversion being maintained throughout the whole post-conditioning period only in the WLP rats. There were no differences between the lines in terms of the blood ethanol concentration and the EPM tests. WHP rats had a higher pain sensitivity compared to WLP rats in flinch-jump and hot-plate tests. WLP rats showed a shorter exploration time for both objects compared to WHP in the NOR test.

In conclusion, WHP and WLP rats differ in sensitivity to the aversive effects of ethanol. This difference may partially explain their opposite ethanol preference.

Introduction

Genetically selected lines of rats for a high or low alcohol preference are an important animal model for research on the mechanisms of alcohol dependence. The selection of WHP (Warsaw High Preferring) and WLP (Warsaw Low Preferring) rats with respect to a high and low ethanol preference started 27 years ago (Bisaga & Kostowski, 1993; Dyr & Kostowski, 2008). The initial non-inbred stock of 30 male and 50 female albino rats of Wistar origin was designated as generation G₀. The pairs with the highest ethanol consumption and the lowest ethanol intake were mated to start an ethanol high-preferring line (WHP) and an ethanol low-preferring line (WLP). Selective breeding was continued by the mating of males and females with the highest and lowest ethanol intake. Starting from the 10th generation, the general selection criteria were at least 5.0 g/kg/day for WHP rats and less than 2.0 g/kg/day for WLP rats. Animals that had no common progenitors up to three generations back were mated. At present, WHP rats drink more than 5 g of 10% ethanol per kg of body weight per day, while WLP rats drink less than 2 g/kg/24 h. Ethanol is also self-administered by WHP rats, and they show a higher motivation to obtain ethanol than WLP rats (Rok-Bujko, Dyr, & Kostowski, 2006). In free-choice conditions, blood ethanol level (BEL) reached 20–45 mg% in WHP rats, and 4–6 mg% in WLP rats (Dyr & Kostowski, 2008).

So far, it has not been possible to discover a mechanism explaining why WHP rats drink large amounts of ethanol while WLP rats avoid its consumption. Naïve WHP rats showed higher activity in a new environment than WLP rats (Acewicz et al., 2014). In addition, the WHP line as well as other lines selected for high ethanol consumption (P-Preferring and HAD-High Alcohol Drinking) showed reduced dopamine levels and its metabolites in the striatum, which – according to the theory of self-treatment – can partially explain the high consumption of ethanol (Dyr et al., 1998; Murphy, McBride, Lumeng, & Li, 1983).

The differences in ethanol preference may translate into a sensitivity to its aversive effects. The amount of ethanol consumed depends on the individual balance between its rewarding and aversive properties (Kutlu & Gould, 2016). Rats that are less sensitive to the aversive effects of ethanol in their youth are later more likely to become addicted to ethanol (Schramm-Sapyta et al., 2010).

The aversive effect of ethanol can be tested using the conditioned taste aversion (CTA) test. This test is based on pairing the ethanol effects with a naturally pleasant taste (e.g., saccharin solution). Animals that react negatively to ethanol develop an aversion to the taste associated with its effects. The CTA test is highly sensitive and has high reliability (Liu, Showalter, & Grigson, 2009). Many authors assume the existence of an inverse relationship between the strength of the CTA induced by ethanol and the volume of ethanol consumed in a free-choice test with water (Froehlich, Harts, Lumeng, & Li, 1988; Phillips et al., 2005; Roma, Flint, Higley, & Riley, 2006).

Our previous studies revealed that 1.0 g/kg of ethanol administered intraperitoneally (i.p.) produces a light and short-term aversion in WLP rats but not in WHP rats (Dyr et al., 2016). Higher doses of ethanol have not been tested as yet. One important factor that may influence the results of the CTA is taste. Our previous studies showed that WHP rats drank greater amounts of sugar and saccharin solutions in comparison to WLP rats (Dyr & Kostowski, 2000); however, our current experience and results do not support these findings. WHP and WHP rats seem to have a similar preference to a 0.1% saccharin solution.

The study is primarily intended as an extension to the higher doses of ethanol used in the previous study by Dyr et al. (2016). The aim of the work was to study sensitivity to the aversive effects of ethanol at doses of 1.5 g/kg and 2.0 g/kg i.p. in WHP and WLP rats. These higher doses of ethanol have never been tested before; thus, we still have no knowledge of the doses of ethanol that produce potentially aversive effects in WHP rats.

A secondary objective was to investigate the additional parameters that may differentiate between WHP and WLP rats, which have not been tested so far, and that may be relevant to the results of other tests, including the CTA. For this purpose, we decided to calculate the blood ethanol concentration after a single administration of ethanol. According to our hypothesis, WHP and WLP animals should not differ in blood ethanol concentration. To check whether there are any differences in working memory between WHP and WLP rats, we performed the novel object recognition test (NOR). Working memory is relatively simple to examine in animals. We assume that WHP and WPL animals will not differ in the NOR test, which will allow us to assume that WHP and WLP animals have similar cognitive abilities. To check pain sensitivity, we conducted the flinch-jump test and the hot-plate test. We also decided to investigate anxiety-related behavior using the EPM. Theoretically, different sensitivity to pain or increased anxiety-related behavior experienced during injection can affect the development of aversion in the CTA test. We assume that WHP and WLP animals will exhibit similar levels of pain sensitivity and similar levels of anxiety-related behavior.