Pharmacological Reports 66 (2014) 28–33
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Drinking of flavored solutions by high preferring (WHP) and low preferring (WLP) alcohol-drinking rats Wanda Dyr *, Edyta Wyszogrodzka, Paweł Mierzejewski, Przemysław Bien´kowski Department of Pharmacology and Physiology of the Nervous System, Institute Psychiatry and Neurology, Warszawa, Poland
A R T I C L E I N F O
Article history: Received 11 February 2013 Received in revised form 17 June 2013 Accepted 25 June 2013 Available online 31 January 2014 Keywords: Alcohol-preferring WHP rats Alcohol-nonpreferring WLP rats Tastes preference
A B S T R A C T
Background: Selective breeding alcohol-preferring (P) and alcohol-nonpreferring (NP) rats showed a strong preference for the sucrose solutions, whereas P rats intake greater amounts than NP rats. The aim of this study was the estimation of selectively bred ethanol-preferring (WHP – Warsaw High Preferring) and ethanol-nonpreferring (WLP – Warsaw Low Preferring) rats for their preference for various tastes. Methods: The oral drinking of the following substances was studied at a range of concentrations: sucrose (0.5–64.0 g/100 ml), NaCl (0.025–3.2 g/100 ml), citric acid (0.008–2.048 g/l), and sucrose octaacetate (0.002–0.512 g/l) solutions. Separate groups of 7–8 rats from each line were investigated of each of the four tastes. The investigated solutions were presented continuously keeping water and food always available. Concentrations of the various flavors were doubled every 48 h. Results: Rats from WHP and WLP lines clearly revealed the preference for the sucrose solution and the highest preference was at the 4.0 and 8.0 g/100 ml sucrose concentration. Similar to sucrose, both lines exposed strong preference for the NaCl solution and this preference enhanced together with the increase of the NaCl concentration. Nevertheless their preference for the NaCl solutions decreased when the concentration of NaCl reached 1.600 g/100 ml. Both lines of rats did not differ in citric acid or sucrose octaacetate intake at any of the concentrations studied. Conclusion: Selective breeding of rats (WHP) for high and rats (WLP) for low ethanol drinking is favorably correlated with the drinking of sweet and salty solutions and negatively correlated with the consumption of sour and bitter tastes. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction The oral consumption of alcohol is accompanied by chemosensory perception of flavor, which plays an important role in its acceptance and rejection in both human and laboratory animals. A positive association between alcohol intake and consumption of a sweet solution was observed in heterogeneous laboratory rodents [29] and also in humans [24]. Unselected Wistar rats with high intake of a 0.1 g/100 ml saccharin solution consumed more of 2% and 6% of an ethanol solution when compared to rats that had low saccharin intakes [17]. The connection between saccharin intake and ethanol preference was observed in rodent strains/lines genetically developed for alcohol preference or nonpreference. Larger amount of saccharin
* Corresponding author. E-mail address: [email protected] (W. Dyr).
consumption was observed in C57BL mice that intake much more of ethanol than DBA/2J mice [14,33]. Similar, selectively bred alcoholpreferring AA (Alko Alcohol) [12] and P (alcohol preferring) [16,20] rats drank more of the saccharin solutions when compared to their alcohol-avoiding counterparts ANA (Alco, nonalcohol) and NP (nonpreferring) rats, respectively [24,29,31]. Studies of high-(UChB) and low-(UChA) alcohol-drinking rats showed that long-term exposure to a 10% alcohol solution containing 0.2% saccharin induced a significant increase in the alcohol consumption of UChB rats after the saccharin was removed. However, under similar conditions, the alcohol consumption of UChA rats returned to a low level. These results are significant because they reveal an association between saccharin and alcohol preference. Furthermore, these results suggest that the different rodent genotypes might be implicated in alcohol aversion [34]. It is said that the same differential preferences for saccharin solution were found in each case support the statement that the connection between sweet and alcohol preference is not accidental.
1734-1140/$ – see front matter ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.pharep.2013.06.004
W. Dyr et al. / Pharmacological Reports 66 (2014) 28–33
29
P (alcohol preferring) rats drank more of the saccharin solutions when compared to their alcohol-avoiding counterparts NP (nonpreferring) rats, but intake of citric acid or sucrose octaacetate did not differ from each other at any of the concentrations tested and P rats showed negative associations with consumption of salty solutions [32]. In other investigation P and NP rats had similar daily intakes of salty and sour fluids [31]. WHP (Warsaw High Preferring) and WLP (Warsaw Low Preferring) rats were developed from Wistar rats at the Institute of Psychiatry and Neurology (Warsaw, Poland). WHP (highalcohol-drinking rats) voluntarily consume more than 5 g/kg/ 24 h. Conversely, WLP (low-alcohol-drinking rats) consume less than 2 g/kg/24 h [10,11]. Because WHP and WLP rats are selectively bred alcoholpreferring and alcohol-nonpreferring lines of rats, the question was whether WHP (Warsaw High Preferring) and WLP (Warsaw Low Preferring) rats would have similar consumption of sweet, salty, sour, and bitter solutions like P and NP lines of rats described in literature [32]. Then, the aim of the present study was an investigation on a range of concentrations of sucrose, NaCl, citric acid, and sucrose octaacetate (SOA) solutions offered in free choice with water during 48 h. This study was important because the obtained results would suggest any likeness or differences between WHP and WLP rats. The study was being approved by Local Ethic Committee of the Use Laboratory Animals in Warsaw, Poland (10/2010).
Labofeed rodent chow was continuously available. The animals were maintained on a reverse 12/12 h light/dark cycle throughout the experiment (lights on at 21:00). For the habituation period, both tubes were initially filled with distilled water for 7 days. The procedure to examine preference was started with the presentation of distilled water in both tubes for the first 48 h (baseline water consumption). Then, different concentrations of flavors were increased every 48 h in one of the tubes while the other tube remained filled with water. Every concentration was doubled at 48 h. To avoid a preference of sides, the positions of the tubes were changed every day and the volume of consumed fluid was refilled. The rats were weighed every 3rd day at the same time of day that the liquid measurements were assessed.
Materials and methods
Results
Subjects
Baseline water consumption
To establish whether the rats had a preference for sucrose, female WHP (n = 8) and WLP (n = 8) rats of the 50th generation selectively bred for ethanol preference were used. The initial body weight (mean SE) of the WHP females was 231.0 5.55 g. The initial body weight of the WLP females was 277.25 8.33 g. After finishing the experiments to determine whether the rats had a preference for sucrose, the same WHP and WLP rats were subjected to a washout for one week. After this washout, studies examining whether the rats had a preference for a NaCl solution were initiated. The initial body weight of the WHP rats (n = 8 females) was 256.5 6.88 g. The initial body weight of the WLP rats (n = 8 females) was 302.25 9.19 g. In a second group of female WHP and WLP rats (50th generation), preferences for citric acid and SOA solutions were assessed. The initial body weight (mean SE) of the WHP females (n = 8) was 209.75 5.5 g. The initial body weight of the WLP females (n = 8) was 235.5 14.25 g. One week after a washout of citric acid, experiments examining whether the rats had a preference for SOA solutions were initiated. The initial body weight of the WHP females was 249.5 9.06 g. The initial body weight of the WLP females was 257.25 9.81 g.
This study examined sucrose, NaCl, citric acid and SOA preferences. There were no significant differences between the measured water intakes of the WHP and WLP rats during the 48 h period before each flavored solution was tested (p > 0.05).
Flavor solutions Solutions of sucrose (0.5–64.0 g/100 ml), NaCl (0.025–3.2 g/ 100 ml), SOA (0.002–0.512 g/l), and citric acid (0.08–2.048 g/l) were prepared in distilled water 1 or 2 days before use. To dissolve the two highest sucrose concentrations and all of the SOA concentrations, the solutions were heated and stirred. Procedure The rats were housed singly in cages with two graduated drinking tubes attached side-by-side on the front of the cage.
Data analysis The volumes (ml/48 h) of the flavored solutions and water consumed at each concentration were analyzed. A separate 3-way ANOVA was used for the studies examining sucrose, NaCl, citric acid and SOA preference. Statistical comparisons (t-test) were performed between the flavored solution at each concentration and the consumption of the concurrently available water (for both WHP and WLP rats). The baseline water consumption of the WHP and WLP rats in the absence of the flavored substance was compared using (t-test).
Sucrose consumption Fig. 1 shows the consumption of sucrose solutions and water by the WHP and WLP rats at each of the sucrose concentrations. There was no interaction between the lines and concentrations tested F(7, 219) = 1.559, p = 0.148, indicating that the liquid consumption between the lines did not differ between the tested concentrations. A significant interaction of concentration with flavors [F(7, 219) = 85.042, p < 0.001], indicating that the degree of preference for the sucrose solution depended on the sucrose concentration. Significant interaction of line with flavor was observed [F(1, 219) = 5.738, p < 0.05], indicating that both WHP and WLP rats had a similar sucrose intake. There were no 3-way interactions between the line, concentration, and flavor tested F(7, 219) = 1.69, p > 0.1. This result suggests that the WHP and WLP rats’ preference for the sucrose solution over water was similar. The comparison of sucrose solution and water intake at each concentration (using t-test) showed that WHP and WLP rats consumed more of the sucrose solution than water at all the sucrose concentrations tested (p < 0.001). NaCl consumption Fig. 2 shows the volume of NaCl solution and water consumption by WHP and WLP rats. An ANOVA analysis of the NaCl consumption showed that there was no interaction between the lines and concentrations tested F(7, 220) = 0.2724, p = 0.9641, indicating that the liquid consumption between the lines did not differ between the tested concentrations.
30
W. Dyr et al. / Pharmacological Reports 66 (2014) 28–33
350 whp rats Flavor wlp rats Flavor wlp rats Water whp rats water
Liquid intake/48 h (ml)
300
20 ml/48 h) and increased (60–80 ml/48 h) at NaCl concentrations of 1.6 and 32 g/100 ml. 3-Way ANOVA did not reveal interactions between the line, concentration and flavor tested F(7, 220) = 0.41, p > 0.1, suggesting that there were no differences in concentration-dependent NaCl consumption between lines. The comparison of the NaCl solution and water intake at each concentration (using t-test) showed that WHP and WLP rats have a preference for NaCl solution over water at several concentrations tested (0.025; 0.05; 0.1; 0.2; 0.4; 0.8; 3.2 g/100 ml NaCl) (p < 0.001). At a concentration of the 1.6 g/100 ml for the NaCl solution, the consumption of water and NaCl solution by WHP and WLP rats did not differ.
* *
250
*
200
* *
150
*
*
*
*
100
*
*
* 50
Citric acid consumption
* *
0 0,0
0,5
1,0
2,0
4,0
8,0
16,0
32,0
64,0
Sucrose concentration (g/100ml) Fig. 1. Mean (SEM) water and sucrose intakes (ml/48 h) by WHP and WLP rats (n = 7, 8). There are not significant differences between WHP and WLP rats in flavored solution intake at a given concentration (by t-test). * 0.2, suggesting that there were no differences in concentration-dependent citric acid solutions consumption between lines.
*
*
whp rats Flavor wlp rats Flavor wlp rats Water whp rats water
#
40
* 20
40
0
NaCl concentration (g/100ml) Fig. 2. Mean (SEM) water and NaCl intakes (ml/48 h) by WHP and WLP rats (n = 7, 8). There are not significant differences between WHP and WLP rats in flavored solution intake at a given concentration (by t-test). *
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Drinking of flavored solutions by high preferring (WHP) and low preferring (WLP) alcohol-drinking rats Wanda Dyr *, Edyta Wyszogrodzka, Paweł Mierzejewski, Przemysław Bien´kowski Department of Pharmacology and Physiology of the Nervous System, Institute Psychiatry and Neurology, Warszawa, Poland
A R T I C L E I N F O
Article history: Received 11 February 2013 Received in revised form 17 June 2013 Accepted 25 June 2013 Available online 31 January 2014 Keywords: Alcohol-preferring WHP rats Alcohol-nonpreferring WLP rats Tastes preference
A B S T R A C T
Background: Selective breeding alcohol-preferring (P) and alcohol-nonpreferring (NP) rats showed a strong preference for the sucrose solutions, whereas P rats intake greater amounts than NP rats. The aim of this study was the estimation of selectively bred ethanol-preferring (WHP – Warsaw High Preferring) and ethanol-nonpreferring (WLP – Warsaw Low Preferring) rats for their preference for various tastes. Methods: The oral drinking of the following substances was studied at a range of concentrations: sucrose (0.5–64.0 g/100 ml), NaCl (0.025–3.2 g/100 ml), citric acid (0.008–2.048 g/l), and sucrose octaacetate (0.002–0.512 g/l) solutions. Separate groups of 7–8 rats from each line were investigated of each of the four tastes. The investigated solutions were presented continuously keeping water and food always available. Concentrations of the various flavors were doubled every 48 h. Results: Rats from WHP and WLP lines clearly revealed the preference for the sucrose solution and the highest preference was at the 4.0 and 8.0 g/100 ml sucrose concentration. Similar to sucrose, both lines exposed strong preference for the NaCl solution and this preference enhanced together with the increase of the NaCl concentration. Nevertheless their preference for the NaCl solutions decreased when the concentration of NaCl reached 1.600 g/100 ml. Both lines of rats did not differ in citric acid or sucrose octaacetate intake at any of the concentrations studied. Conclusion: Selective breeding of rats (WHP) for high and rats (WLP) for low ethanol drinking is favorably correlated with the drinking of sweet and salty solutions and negatively correlated with the consumption of sour and bitter tastes. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction The oral consumption of alcohol is accompanied by chemosensory perception of flavor, which plays an important role in its acceptance and rejection in both human and laboratory animals. A positive association between alcohol intake and consumption of a sweet solution was observed in heterogeneous laboratory rodents [29] and also in humans [24]. Unselected Wistar rats with high intake of a 0.1 g/100 ml saccharin solution consumed more of 2% and 6% of an ethanol solution when compared to rats that had low saccharin intakes [17]. The connection between saccharin intake and ethanol preference was observed in rodent strains/lines genetically developed for alcohol preference or nonpreference. Larger amount of saccharin
* Corresponding author. E-mail address: [email protected] (W. Dyr).
consumption was observed in C57BL mice that intake much more of ethanol than DBA/2J mice [14,33]. Similar, selectively bred alcoholpreferring AA (Alko Alcohol) [12] and P (alcohol preferring) [16,20] rats drank more of the saccharin solutions when compared to their alcohol-avoiding counterparts ANA (Alco, nonalcohol) and NP (nonpreferring) rats, respectively [24,29,31]. Studies of high-(UChB) and low-(UChA) alcohol-drinking rats showed that long-term exposure to a 10% alcohol solution containing 0.2% saccharin induced a significant increase in the alcohol consumption of UChB rats after the saccharin was removed. However, under similar conditions, the alcohol consumption of UChA rats returned to a low level. These results are significant because they reveal an association between saccharin and alcohol preference. Furthermore, these results suggest that the different rodent genotypes might be implicated in alcohol aversion [34]. It is said that the same differential preferences for saccharin solution were found in each case support the statement that the connection between sweet and alcohol preference is not accidental.
1734-1140/$ – see front matter ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.pharep.2013.06.004
W. Dyr et al. / Pharmacological Reports 66 (2014) 28–33
29
P (alcohol preferring) rats drank more of the saccharin solutions when compared to their alcohol-avoiding counterparts NP (nonpreferring) rats, but intake of citric acid or sucrose octaacetate did not differ from each other at any of the concentrations tested and P rats showed negative associations with consumption of salty solutions [32]. In other investigation P and NP rats had similar daily intakes of salty and sour fluids [31]. WHP (Warsaw High Preferring) and WLP (Warsaw Low Preferring) rats were developed from Wistar rats at the Institute of Psychiatry and Neurology (Warsaw, Poland). WHP (highalcohol-drinking rats) voluntarily consume more than 5 g/kg/ 24 h. Conversely, WLP (low-alcohol-drinking rats) consume less than 2 g/kg/24 h [10,11]. Because WHP and WLP rats are selectively bred alcoholpreferring and alcohol-nonpreferring lines of rats, the question was whether WHP (Warsaw High Preferring) and WLP (Warsaw Low Preferring) rats would have similar consumption of sweet, salty, sour, and bitter solutions like P and NP lines of rats described in literature [32]. Then, the aim of the present study was an investigation on a range of concentrations of sucrose, NaCl, citric acid, and sucrose octaacetate (SOA) solutions offered in free choice with water during 48 h. This study was important because the obtained results would suggest any likeness or differences between WHP and WLP rats. The study was being approved by Local Ethic Committee of the Use Laboratory Animals in Warsaw, Poland (10/2010).
Labofeed rodent chow was continuously available. The animals were maintained on a reverse 12/12 h light/dark cycle throughout the experiment (lights on at 21:00). For the habituation period, both tubes were initially filled with distilled water for 7 days. The procedure to examine preference was started with the presentation of distilled water in both tubes for the first 48 h (baseline water consumption). Then, different concentrations of flavors were increased every 48 h in one of the tubes while the other tube remained filled with water. Every concentration was doubled at 48 h. To avoid a preference of sides, the positions of the tubes were changed every day and the volume of consumed fluid was refilled. The rats were weighed every 3rd day at the same time of day that the liquid measurements were assessed.
Materials and methods
Results
Subjects
Baseline water consumption
To establish whether the rats had a preference for sucrose, female WHP (n = 8) and WLP (n = 8) rats of the 50th generation selectively bred for ethanol preference were used. The initial body weight (mean SE) of the WHP females was 231.0 5.55 g. The initial body weight of the WLP females was 277.25 8.33 g. After finishing the experiments to determine whether the rats had a preference for sucrose, the same WHP and WLP rats were subjected to a washout for one week. After this washout, studies examining whether the rats had a preference for a NaCl solution were initiated. The initial body weight of the WHP rats (n = 8 females) was 256.5 6.88 g. The initial body weight of the WLP rats (n = 8 females) was 302.25 9.19 g. In a second group of female WHP and WLP rats (50th generation), preferences for citric acid and SOA solutions were assessed. The initial body weight (mean SE) of the WHP females (n = 8) was 209.75 5.5 g. The initial body weight of the WLP females (n = 8) was 235.5 14.25 g. One week after a washout of citric acid, experiments examining whether the rats had a preference for SOA solutions were initiated. The initial body weight of the WHP females was 249.5 9.06 g. The initial body weight of the WLP females was 257.25 9.81 g.
This study examined sucrose, NaCl, citric acid and SOA preferences. There were no significant differences between the measured water intakes of the WHP and WLP rats during the 48 h period before each flavored solution was tested (p > 0.05).
Flavor solutions Solutions of sucrose (0.5–64.0 g/100 ml), NaCl (0.025–3.2 g/ 100 ml), SOA (0.002–0.512 g/l), and citric acid (0.08–2.048 g/l) were prepared in distilled water 1 or 2 days before use. To dissolve the two highest sucrose concentrations and all of the SOA concentrations, the solutions were heated and stirred. Procedure The rats were housed singly in cages with two graduated drinking tubes attached side-by-side on the front of the cage.
Data analysis The volumes (ml/48 h) of the flavored solutions and water consumed at each concentration were analyzed. A separate 3-way ANOVA was used for the studies examining sucrose, NaCl, citric acid and SOA preference. Statistical comparisons (t-test) were performed between the flavored solution at each concentration and the consumption of the concurrently available water (for both WHP and WLP rats). The baseline water consumption of the WHP and WLP rats in the absence of the flavored substance was compared using (t-test).
Sucrose consumption Fig. 1 shows the consumption of sucrose solutions and water by the WHP and WLP rats at each of the sucrose concentrations. There was no interaction between the lines and concentrations tested F(7, 219) = 1.559, p = 0.148, indicating that the liquid consumption between the lines did not differ between the tested concentrations. A significant interaction of concentration with flavors [F(7, 219) = 85.042, p < 0.001], indicating that the degree of preference for the sucrose solution depended on the sucrose concentration. Significant interaction of line with flavor was observed [F(1, 219) = 5.738, p < 0.05], indicating that both WHP and WLP rats had a similar sucrose intake. There were no 3-way interactions between the line, concentration, and flavor tested F(7, 219) = 1.69, p > 0.1. This result suggests that the WHP and WLP rats’ preference for the sucrose solution over water was similar. The comparison of sucrose solution and water intake at each concentration (using t-test) showed that WHP and WLP rats consumed more of the sucrose solution than water at all the sucrose concentrations tested (p < 0.001). NaCl consumption Fig. 2 shows the volume of NaCl solution and water consumption by WHP and WLP rats. An ANOVA analysis of the NaCl consumption showed that there was no interaction between the lines and concentrations tested F(7, 220) = 0.2724, p = 0.9641, indicating that the liquid consumption between the lines did not differ between the tested concentrations.
30
W. Dyr et al. / Pharmacological Reports 66 (2014) 28–33
350 whp rats Flavor wlp rats Flavor wlp rats Water whp rats water
Liquid intake/48 h (ml)
300
20 ml/48 h) and increased (60–80 ml/48 h) at NaCl concentrations of 1.6 and 32 g/100 ml. 3-Way ANOVA did not reveal interactions between the line, concentration and flavor tested F(7, 220) = 0.41, p > 0.1, suggesting that there were no differences in concentration-dependent NaCl consumption between lines. The comparison of the NaCl solution and water intake at each concentration (using t-test) showed that WHP and WLP rats have a preference for NaCl solution over water at several concentrations tested (0.025; 0.05; 0.1; 0.2; 0.4; 0.8; 3.2 g/100 ml NaCl) (p < 0.001). At a concentration of the 1.6 g/100 ml for the NaCl solution, the consumption of water and NaCl solution by WHP and WLP rats did not differ.
* *
250
*
200
* *
150
*
*
*
*
100
*
*
* 50
Citric acid consumption
* *
0 0,0
0,5
1,0
2,0
4,0
8,0
16,0
32,0
64,0
Sucrose concentration (g/100ml) Fig. 1. Mean (SEM) water and sucrose intakes (ml/48 h) by WHP and WLP rats (n = 7, 8). There are not significant differences between WHP and WLP rats in flavored solution intake at a given concentration (by t-test). * 0.2, suggesting that there were no differences in concentration-dependent citric acid solutions consumption between lines.
*
*
whp rats Flavor wlp rats Flavor wlp rats Water whp rats water
#
40
* 20
40
0
NaCl concentration (g/100ml) Fig. 2. Mean (SEM) water and NaCl intakes (ml/48 h) by WHP and WLP rats (n = 7, 8). There are not significant differences between WHP and WLP rats in flavored solution intake at a given concentration (by t-test). *
