1 Department of Pharmacology and Physiology of the Nervous System, Institute of Psychiatry and Neurology, Al. Sobieskiego 1/9, PL-02957 Warsaw and
2 Department of Experimental and Clinical Pharmacology, Warsaw Medical University, ul. Krakowskie Przedmiescie 26/28, PL-00527, Warsaw, Poland
Received 2 September 1998; in revised form 9 December 1998; accepted 15 January 1999
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ABSTRACT |
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INTRODUCTION |
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We have recently shown that parameters of saccharin drinking behaviour were highly correlated with the initial acceptance of low ethanol concentrations in Wistar rats. However, this relationship disappeared during further weeks of ethanol presentation. In contrast, locomotor activity in an open field area did not predict any subsequent ethanol consumption (Koros et al., 1998). These findings suggest that certain behavioural features may predict only initial alcohol drinking behaviour. It could also be speculated that other behavioural parameters correlate with ethanol intake in later stages of alcohol self-administration (maintenance phase). Thus, one aim of the present study was to assess possible relationships between long-term ethanol self-administration and several behavioural parameters derived from the open field and the saccharin drinking tests.
The alcohol deprivation effect (ADE) was first described by Sinclair and Senter (1968) as a transient increase in ethanol consumption/preference after a period of forced abstinence. In rats, the ADE was reported to occur after 18 or 21, but not 1 or 7, days of access to alcohol (Sinclair and Senter, 1968; Sinclair, 1972
). More recently, Spanagel et al. (1996) have described a model of long-term free-choice alcohol self-administration with repeated deprivation episodes. In this latter study, rats showed the ADE after 2 months of access to water and three ethanol solutions (5, 10 and 20%, v/v). In contrast, we did not observe any changes in ethanol drinking after the deprivation episode, which followed a 50-day period of alcohol drinking (Koros et al., 1998
). Thus, another aim of the present study was to examine the pattern of ethanol consumption after repeated episodes of deprivation in the rats used by Koros et al. (1998).
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MATERIALS AND METHODS |
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Study design
Table 1 presents a general design of the study. One week after the completion of the initial saccharin drinking and the open field tests, the ethanol drinking tests began. For the first 22 days, the animals were exposed to increasing concentrations of ethanol (28%, v/v) and tap water in a two-bottle choice situation. Then, for the next 28 days, the animals were presented with two different ethanol solutions (8 and 16%, v/v) and tap water (a three-bottle choice test). The drinking tubes were rotated daily to prevent position preference. After 50 days of continuous access to alcohol and water the rats were deprived of alcohol for 5 days. During the deprivation period water was available in all three bottles. After the 5-day deprivation period, both alcohol solutions were presented again along with water for the next 28 days. [The correlations between the initial behavioural tests, ethanol self-administration during the first 50 days and the ADE after the first ethanol deprivation were described in detail by Koros et al. (1998).] After the completion of six three-bottle ethanol drinking/deprivation cycles, saccharin drinking and the open field tests were performed again (Table 1
).
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Saccharin drinking test
During the 48-h saccharin drinking test, the animals were given the choice between a saccharin solution (0.1% w/v; saccharin sodium salt, Aldrich, Gillingham, UK) and tap water. The position of bottles was changed after 24 h to prevent place preference. For each fluid, an intake measure was obtained, averaged across 2 days of choice and corrected for body weight of the subject (ml/kg/24 h).
It has been reported that the increase in total fluid intake (TFI) when saccharin is offered is a better predictor of subsequent ethanol intake than absolute saccharin consumption (Kampov-Polevoy et al., 1995). Thus, the increase in the TFI in the presence of saccharin was calculated as the percentage difference between the TFI when saccharin was available and a control TFI when only water was available (Kampov-Polevoy et al., 1995
). The mean water intake (ml/kg/24 h) during the 2 days preceding the saccharin drinking test was treated as the control TFI.
Open field test
One week after the completion of the saccharin drinking test, the rats were used in the open field test. The open field apparatus consisted of four identical, computer-controlled cages (60 x 60 x 40 cm, L x W x H; COTM, Bialystok, Poland). Each cage was transected by two perpendicular, co-planar arrays of 16 infrared photocells which were intended to measure forward locomotion by determining the rat's position every 0.1 s (Bienkowski et al., 1997a; Koros et al., 1998
). The forward locomotion was defined as the distance (in in.) travelled by the rat during the 20-min test session. Another set of photocells located 15 cm above the cage floor measured the number of rearings. After the initial habituation of 20 min to the test room, each rat was introduced to the test cage for another 20 min. The cages were cleaned carefully between the recordings. The test sessions were conducted between 10:00 and 14:00 to avoid errors attributable to the variation in motor activity of the day activity cycle (File and Day, 1972
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Statistical analysis
Absolute changes (increases or decreases) in ethanol consumption ( g/kg) after repeated deprivation episodes were compared by a one-way ANOVA. In addition, the average ethanol drinking from pre- and post-deprivation days was analysed with a two-way ANOVA (ADE x episode). The NewmanKeuls test was used for post-hoc comparison. Multiple regression analysis was employed for testing the relationship between behavioural variables and ethanol intake. Due to the risk of a Type I error a probability below 0.01 was considered significant.
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RESULTS |
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The ADE calculated for changes in total ethanol intake
Figures 2A and B show the ADE expressed as the change in the total ethanol intake. One-way ANOVA indicated a significant difference between the subsequent ADEs [ADE24 h, F(5,23) = 3.52, P < 0.01; ADE72 h, F(5,23) = 3.37, P < 0.01; see Figs 2A and B
, respectively]. Visual inspection of the data and the post-hoc analysis revealed that both the ADE24 h and the ADE72 h values increased with repeated deprivation episodes. The above results were partially confirmed by two-way ANOVA, which revealed a trend towards significant ADE x episode interaction for the ADE72 h [F(5,230) = 2.78; 0.01 < P < 0.05].
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Correlational analysis
The mean (± SEM) weight of the rats after the completion of all ethanol self-administration procedures was 559 ± 16 g. The results of the saccharin drinking and the open field tests are summarized in Table 2. Parameters of saccharin drinking did not correlate with the open field behaviours (r values ranged from 0.18 to +0.10; P > 0.35).
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DISCUSSION |
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The ADE has been repeatedly shown after relatively short ethanol presentations in different rat strains (Sinclair and Senter, 1968; Sinclair, 1972
; Sinclair and Tiihonen, 1988
; Sinclair and Li, 1989
). The initial ADEs in the present study were relatively weak in comparison with the results cited above. Sinclair and Senter (1968), using Long-Evans rats, have shown that 1 and 7 days of ethanol (7%, v/v) access did not result in any significant ADE, whereas that for 21 days did. Similarly, Spanagel et al. (1996), using Wistar rats, have reported a significant ADE after a 3-day deprivation episode which followed 2 months of continuous access to water and three ethanol concentrations. In a more recent study from the same laboratory, Hölter et al. (1998), using the drinkometer system, did not find the ADE in Wistar rats pre-exposed to ethanol for 24 weeks. However, the baseline drinking in this latter study was relatively high (6.79 g/kg/24 h) and a ceiling effect might have occurred.
When the ADEs after repeated deprivation episodes were compared, a clear-cut difference between the initial and the last ADE was found. The last ADE24 h exceeded 1.5 g/kg and was fully comparable with the results reported previously by others (Sinclair, 1979; Spanagel et al., 1996
). Based on the above findings, one could hypothesize that the ADE in the present study increased with repeated deprivation episodes. However, at least two other possible explanations should be mentioned. First, the duration of ethanol exposure rather than the deprivation episodes might have been critical for the increase in the ADE magnitude. Second, the possibility exists that aged rats are more prone to increased ethanol drinking after deprivation. This latter hypothesis seems to be less likely, as Sinclair (1972) reported that young and old Wistar rats (aged 3 and > 6 months, respectively) showed a similar ADE after 18 days of access to water and ethanol solution (5%, v/v). Obviously, future studies with additional control groups are needed to clarify the above hypotheses.
The second purpose of the present study was to evaluate correlations between long-term ethanol intake and parameters from the open field and saccharin drinking tests. Neither saccharin drinking nor the increase in the TFI in the presence of saccharin correlated with ethanol consumption or the ADE. This finding indicates that saccharin drinking is not a good predictor of ethanol intake in the maintenance phase of ethanol self-administration. Previously, Koros et al. (1998), using the same rats, have shown that the association between saccharin drinking and ethanol intake was limited to initial acceptance of low ethanol concentrations (26%, v/v). The correlation between saccharin and ethanol intake was also strongest for initial ethanol drinking in the study of Kampov-Polevoy et al. (1996). In our previous report (Koros et al., 1998), we used parameters of the saccharin drinking test performed before the start of the ethanol presentation (not shown here). In the present paper, we used these parameters again but, as expected, the results of the correlational analysis were negative. Thus, the parameters of the saccharin drinking test performed either before or after the period of ethanol presentation did not predict ethanol self-administration behaviour.
The obvious limitation of the present study is that only one strain of rats (Wistar) was used. It is possible that this rat strain does not possess the genetic diversity connecting long-term ethanol drinking and saccharin intake. As mentioned above, robust correlations between initial ethanol acceptance and saccharin intake have been found in the first part of the study (Koros et al., 1998). This finding was in agreement with the results of other short-term ethanol drinking experiments, including those done on genetically selected rats. Thus, it remains to be established if saccharin drinking might also predict long-term ethanol drinking behaviour in other rat strains.
In a recent clinical study, Kampov-Polevoy et al. (1997) have shown that a majority (80%) of their alcoholic patients were sweet likers, i.e. preferring high sucrose concentrations. In contrast, only 41% of the control group of non-alcoholic subjects preferred highly-concentrated sucrose solutions. Thus, it should be noted that our paradigm differs substantially from the clinical study mentioned above, as we have used only one concentration of saccharin. Interestingly, Sinclair et al. (1992) have reported that alcohol-preferring lines of rats accepted much higher sweet concentrations than their alcohol-non-preferring partners. The concentration of saccharin used by us (0.1%, w/v) is much preferred by naive rats (Kampov-Polevoy et al., 1996; Bienkowski et al., 1997b
). However, the study of Kampov-Polevoy et al. (1997) suggests that, in future preclinical research, even higher concentrations of saccharin should be used. Possibly, the positive association between saccharin and ethanol consumption exists only when drinking relatively high (possibly even aversive for some rats) concentrations of the sweetener is considered.
In line with many previous papers (Bisaga and Kostowski, 1993; Badishtov et al., 1995
; Fahlke et al., 1995
; Samson and Chappelle, 1995
; Nadal et al., 1996
), spontaneous locomotor activity during a single exposure to a novel environment failed to predict the ethanol intake. Interestingly, Gingras and Cools (1995) have even shown that their HR rats drank significantly less alcohol than LR rats. Moreover, no major differences in locomotor stimulant effects of dexamphetamine in the HR and the LR rats have been reported by the same authors (Gingras and Cools, 1997
). It is noteworthy that, in the study of Goeders and Guerin (1996), no association between the locomotor response to a novel environment and self-administration of a 0.125 mg dose of cocaine has been found.
In conclusion, the results of the present study indicate that neither saccharin consumption nor locomotor activity predicts long-term ethanol self-administration behaviour in Wistar rats. In addition, our findings suggest that the magnitude of the ADE may increase with repeated deprivation episodes.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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