- Email: [email protected]
Drinking of flavored solutions by high preferring (WHP) and low preferring (WLP) alcohol-drinking rats
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). *<0.05 WHP and WLP vs. water in according to concentration of sucrose.
A significant interaction of concentration with flavors [F(7, 220) = 54.7968, p < 0.001], indicating that the degree of preference for the NaCl solution depended on the NaCl concentration. Generally, when the concentration of NaCl was increased, the NaCl consumption also increased. However, consumption of the NaCl solution dropped rapidly at concentrations of 1.6 and 3.2 g/ 100 ml. The water consumption was initially small (no more than 220 200
*
whp rats Flavor wlp rats Flavor wlp rats water whp rats water
80
*
180
140
*
100 80
*
Liquid intake/48 h (ml)
Liquid intake/48 h (ml)
160
120
Fig. 3 shows the volumes of citric acid solution and water consumed by the WHP and WLP rats. An ANOVA analysis of the data showed a main effect of the flavor F(1, 252) = 135.733, p < 0.001, and there was a significant interaction between the concentration and the flavor F(8, 252) = 16.356, p < 0.001. When the concentration of citric acid was increased (up to 0.128 g/l), the consumption of the citric acid solution was between 20 and 40 ml/ 48 h for both of the rat lines and was corresponding to concurrently available water consumption. The water intake under the same conditions was not much higher. Increasing the citric acid solution concentration from 0.256 to 2.048 g/l caused a decrease in citric acid consumption. Consumption of the citric acid solution almost completely ceased at concentrations of 1.024 and 2.048 g/l. However, at concentrations of 0.512, 1.024 and 2.048 g/l, the citric acid consumption was significantly less than the water consumption in both WHP and WLP rats (t-test p < 0.001). 3-Way ANOVA did not reveal interactions between the line, concentration and flavor tested F(8, 252) = 1.278, p > 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). *<0.05 WHP and WLP vs. water in according to concentration of NaCl.
*
*
* 0,000 0,025 0,050 0,100 0,200 0,400 0,800 1,600 3,200
#
60
60
20
#
0
* *
0,000
0,008
0,016
0,032
0,064
0,128
*
0,256
0,512
*
*
*
1,024
2,048
Citric Acid concentration (g/L) Fig. 3. Mean (SEM) water and citric acid solutions 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). *<0.05 WHP and WLP vs. water in according to concentration of citric acid. #<0.05 WHP water vs. WLP water.
W. Dyr et al. / Pharmacological Reports 66 (2014) 28–33
90
whp rats Flavor wlp rats Flavor wlp rats Water whp rats water
80
*
*
Liquid itake/48 h (ml)
70
*
60
*
*
50
40
* 30
20
10
0
0,000 0,002 0,004 0,008 0,016 0,032 0,064 0,128 0,256 0,512
SOA concentration (g/L) Fig. 4. Mean (SEM) water and SOA solutions 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). *<0.05 WHP and WLP vs. water in according to concentration of SOA.
SOA consumption Fig. 4 shows the amount of SOA solution and water intake of the WHP and WLP rats. There was no significant interactions between the lines and concentrations tested F(8, 250) = 0.782, p > 0.1 indicating that the liquid consumption between the lines did not differ the tested of concentrations. A significant interaction of concentration with flavors [F(8, 250) = 14.966, p < 0.001] indicated that the degree of preference for the SOA solution depended on the SOA concentration. Additionally, there was no 3-way interaction between the line, concentration and flavor tested F(8, 250) = 1.4, p > 0.1. These data suggest that both lines of rats consumed similar amounts of SOA and water. Individual comparisons of the SOA intake of the WHP and WLP rats (using a t test) did not reveal significant differences in the SOA consumption between the rat lines at any concentration tested. A comparison of the consumption of the SOA solution and water indicated that at the 0.256 and 0.512 g/l of SOA, WLP and WHP rats consumed less of the bitter solution compared to water (t-test p < 0.001). Discussion The findings of the present experiment do not support the hypothesis of an association between the intake of EtOH and sweetened solutions. WHP and WLP rats showed a high preference for sucrose solution. Rats from P and NP lines showed a strong preference for the sucrose solution, but P rats consumed greater amounts of the solution when compared to NP rats and this study showed important discrepancies in the responses to EtOH and sweet solutions by selectively bred P and NP rats [32]. There were great differences in the largeness of intake of the two substances, where the sweet solutions were consumed in high amount compared to the EtOH solutions [32]. What is an important, the differences among the strains in EtOH consumption did not
31
parallel the pattern of intake of the sweet solutions, because preference for EtOH among the P and NP strains ranged from high to low and animals of both lines displayed high preferences for the sweetened solutions [32]. Goodwin’s and Amit’s studies [15] demonstrated the relationship between the consumption of EtOH and a sweetened solution in three non-selected strains of rats which differ in their ethanol preference: In the first phase of the experiment, all animals were presented with an ascending series of ethanol solution (2–10%) in free-choice with water, followed by a 10-day maintenance period 10% ethanol with water. In the second phase the same animals were presented with an ascending series saccharin–quinine (SQ) consumption. The results revealed an absence of a direct relationship between ethanol and SQ consumption. The ethanolnonpreferring Lewis rats showed a greater preference for the SQ solution than ethanol-preferring Wistar Kyoto rats that consistently intake significantly less SQ [16]. In the Sardinian alcohol-preferring (sP) and Sardinian nonpreferring (sNP) rats, a high degree of preference for a saccharin solution was observed, and there were only very small differences between the lines. These results may suggest that their intake of saccharin is controlled by mechanisms that are different from those that control their alcohol intake [1]. The authors concluded that saccharin drinking behavior in sNP rats deviates from the hypothesis that saccharin and ethanol intake may co-vary. Similarly, the orofacial response in ethanol-naive P and NP rats did not reveal differences in taste reactivity to a sucrose solution (0.3 M) [4]. But in comparison with the mice 129/J (129) strain, the C57BL6ByJ mice had higher preference for ethanol, sucrose, and citric acid. They had lower preferences for NaCl and similar preferences for capsaicin and quinine hydrochloride. The authors [2] think that these data are consistent with the hypothesis that the higher ethanol intake by B6 mice depends, in part, on higher hedonic attractiveness of its sweet taste component. There is direct evidence that EtOH may not be reliably compared with sweet tastes alone, because it appears to have both sweet and bitter taste properties. The data suggest that EtOH and sweet–bitter mixtures have similar gustatory properties [9]. In human, studies of ethanol and other tastants have been shown that 10% ethanol is perceived as bitter and 30% of the patients described it as sweet and or sour. Findings suggest that in human ethanol tastes both bitter and sweet and the relationship between sucrose and ethanol intakes previously found in animals and humans may result, at least partially, from similar taste responses elicited by sucrose and ethanol [30]. In our present study, WHP and WLP rats consumed higher amounts of NaCl. The intake of the NaCl solution enhanced when the concentration of NaCl was increased. The sP and sNP rats had been showing identical intakes of highly concentrated solutions of sodium chloride (sP-17.5 ml/kg; sNP-16.7 ml/kg at the 1.8% NaCl) under the two-bottle, free-choice regimen [7]. Intake of saline (NaCl) solutions and water by males of two inbred of mice C57BL/6J (C57) and 129/J was examined using 48-h, two-bottle preference tests. There was a large strain difference in saline intake: C57 mice that had no prior exposure to saline exhibited a greater intake of saline than C57 mice previously exposed to 0.30 M NaCl. Saline preferences of the 129/J mice were not affected by prior exposure to 0.30 M NaCl. The strain differences in mouse salt preference are due in part to postingestional consequences of saline consumption in C57 but not 129/J mice [3]. Our study found no differences in the consumption of citric acid and SOA solutions in WHP and WLP rats. This result is supported by studies of P and NP rats, which did not show differences in citric acid or sucrose octaacetate consumption [32]. The selectively bred mice (HDID-1, High Drinking in the Dark) reach very high blood
32
W. Dyr et al. / Pharmacological Reports 66 (2014) 28–33
ethanol concentrations (BECs) after drinking from a single bottle of 20% ethanol. These mice drink nearly 6 g/kg ethanol in 4 h and reach average BECs of more than 1.0 mg. HDID-1 mice were tested with the HS/Npt control stock to compare for preference for two concentrations each of quinine, sucrose, and saccharin. Results revealed that neither genotype showed either preference or avoidance for any tastant after high ethanol concentrations [8]. Our present study, using selectively bred alcohol-preferring WHP and alcohol-nonpreferring WLP rats may suggest that between them there are similar preferences for sweet, salt, sour and bitter solutions. In addressing the question of what commonality could exist between EtOH and sweet solutions, the answer does not appear to be simple. It is thought that enhanced initial chemosensory attraction to ethanol and sweet stimuli phenotypes is associated with genetic alcohol preference [5] and 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. Three independent sensory systems – taste, olfaction, and chemosensory irritation – are involved in the perception of flavor. Humans perceive alcohol as a combination of sweet and bitter tastes, odors, and oral irritation, all of which vary as a function of concentration [26]. Likewise, rodents detect the sweet (sweet-like) and bitter (quinine-like) taste [25] and odor volatiles. Rejection of bitter substances is common in many species and may function to protect an animal from ingestion of bittertasting toxins. Herbivorous guinea pigs (Cavia porcellus) showed weaker avoidance of quinine hydrochloride (the bitter plant secondary metabolite) than two nonherbivorous mouse species (Mus musculus and Peromyscus leucopus). Guinea pigs (and herbivores in general) have a generalized reduced bitter sensitivity [13]. Taste perception has several distinct components, including hedonics (affective value, or ‘‘pleasantness’’) and taste quality (i.e. sweet, sour, salty, bitter, umami and perhaps others). However, the hedonic and qualitative components of overall perception are not completely independent because some taste qualities are generally associated with positive hedonic value (e.g., sweet and umami), while other taste qualities are generally associated with negative hedonic value (e.g., bitter). In two-bottle tests, mice from the C57BL/6ByJ (B6) strain drink more umami taste compounds: monosodium L-glutamate (MSG) and inosine-50 -monophosphate (IMP) compared with mice from the 129P3/J (129) strain [18]. The question was whether this variation in consumption could be attributed to strain differences in the perception of taste quality of MSG and IMP and the results [27] of the studies have shown that the strain differences in MSG consumption are not due to variation in the perception of the taste quality of MSG. The differential intake of IMP likely reflects strain differences in the way the taste quality of IMP is perceived. MSG and IMP have been shown to have a complex taste consisting of taste components of different qualities [28,35,36]. Consequently, the hedonic value of MSG and IMP depends in part on the combined hedonic values of their qualitatively distinct taste components. The increased ingestive responses to the umami stimuli in B6 mice are accompanied by either unchanged or decreased neural responses to these stimuli [18]. Ethanol produces changes in taste nerve responses to salty, sour, sweet, and bitter stimuli by interacting with quality-specific taste receptors or with intracellular signaling effectors in taste receptors cells (TRCs) [21,22]. In rodents, CT taste nerve responses to NaCl are derived from at least two salt taste receptors/ion channels in fungiform TRCs [23]. The sweet taste receptor serves as a receptor for both sucrose and ethanol. Extremely important
studies by Lyall et al. [23] showed that phosphatidylinositol 4,5bisphosphate (PIP2) is a common intracellular effector for sweet, bitter, umami, and TRPV1t-dependent salt taste. PIP2 seems to directly regulate the taste receptor protein itself (the TRPV1 ion channel or its taste receptor variant, TRPV1t). Studies showed that P and NP rats differ in TRPV/TRPV1t expression, neural and behavioral responses to sweet and salty stimuli and chronic sucrose and ethanol exposure [6]. The findings revealed differential central neural representation of oral ethanol between genetically heterogeneous rats and P rats genetically selected to prefer alcohol [19]. In conclusion, our present results revealed an absence of a direct relationship between ethanol and sucrose consumption. WLP rats consumed higher amount of the sucrose solution at the 4 and 8 g/100 ml concentrations compared to WHP rats. However, both lines of rats showed a high preference for the sucrose solution at concentrations of 2–32 g/100 ml. The consumption of salt, acid and bitter solutions did not differ between the WHP and WLP rats. The intake of the salt solution increased at concentrations of 0.025 g/100 ml to 0.8 g/100 ml. Higher NaCl concentrations caused a rapid decrease in consumption. Citric acid and SOA solutions were not preferred over water by either WHP or WLP rats. These results may suggest that WHP and WLP rats share a preference for sucrose and NaCl solutions (at certain concentrations) but they do not prefer sour and bitter tastes. The findings of the present experiment do not support the hypothesis of an association between the intake of EtOH and sweet solutions. Both lines of rats, WHP and WLP showed a high preference for sucrose solutions. Conflict of interest No conflict of interest. Funding This paper was financially supported by Institute Psychiatry and Neurology, Warsaw Nr 501/004/12045. References [1] Agabio R, Carai MAM, Lobina C, Pani M, Reali R, Bourov I, et al. Dissociation of ethanol and saccharin preference in sP and sNP rats. Alcohol Clin Exp Res 2000;24:24–9. [2] Bachmanov AA, Tordoff MG, Beauchamp GK. Ethanol consumption and taste preferences in C57BL/6ByJ and 129/J mice. Alcohol Clin Exp Res 1996;20:201– 6. [3] Beauchamp GK, Fisher AS. Strain differences in consumption of saline solution by mice. Physiol Behav 1993;54:179–84. [4] Bice PJ, Kiefer SW. Taste reactivity in alcohol preferring and nonpreferring rats. Alcohol Clin Exp Res 1990;14:721–7. [5] Brasser SM, Silbaugh BC, Ketchum MJ, Olney JJ, Lemon CH. Chemosensory responsiveness to ethanol and its individual sensory components in alcoholpreferring, alcohol-nonpreferring and genetically heterogeneous rats. Addict Biol 2012;17:423–36. [6] Coleman J, Williams A, Phan TH, Mummalaneni S, Melone P, Ren Z, et al. Strain differences in the neural, behavioral, and molecular correlates of sweet and salty taste in naı¨ve, ethanol- and sucrose-exposed P and NP rats. J Neurophysiol 2011;106:2606–21. [7] Colombo G, Agabio R, Diaz G, Fa M, Lobina C, Reali R, et al. g-Hydroxybutyric Acid Intake in Ethanol-preferring sP and -nonpreferring sNP rats. Physiol Behav 1998;64:197–202. [8] Crabbe JC, Spence SE, Brown LL, Metten P. Alcohol preference drinking in a mouse line selectively bred for high drinking in the dark. Alcohol 2011;45:427–40. [9] Di Lorenzo PM, Kiefer SW, Rice AG, Garcia J. Neural and behavioral responsivity to ethyl alcohol as a tastant. Alcohol 1986;3:55–61. [10] Dyr W, Kostowski W. Preliminary phenotypic characterization of the Warsaw high preferring (WHP) and Warsaw low preferring (WLP) lines of rats selectively bred for high and low ethanol consumption. Polish J Pharmacol 2004;56:359–65. [11] Dyr W, Kostowski W. Warsaw high-preferring (WHP) and Warsaw lowpreferring (WLP) lines of rats selectively bred for high and low voluntary
W. Dyr et al. / Pharmacological Reports 66 (2014) 28–33
[12] [13]
[14]
[15]
[16]
[17] [18]
[19]
[20] [21]
[22]
[23]
ethanol intake: preliminary phenotypic characterization. Alcohol 2008;42: 161–70. Eriksson K. Factors affecting voluntary alcohol consumption in the albino rat. Ann Zool Fennici 1969;6:227–65. Field KL, Beauchamp GK, Kimball BA, Mennella JA, Bachmanov AA. Bitter avoidance in guinea pigs (Cavia porcellus) and mice (Mus musculus and Peromyscus leucopus). J Comp Psychol 2010;124:455–9. Forgie ML, Beyerstein BL, Alexander BK. Combinations of taste factors and gender to opioid preference in C57BL and DBA mice. Psychopharmacology 1988;95:237–44. Goodwin FL, Amit Z. Do taste factors contribute to the mediation of ethanol intake? Ethanol and saccharin–quinine intake in three rat strains. Alcohol Clin Exp Res 1998;22:837–44. Goodwin FL, Bergeron N, Amit Z. Differences in the consumption of ethanol and flavored solutions in three strains of rats. Pharmacol Biochem Behav 2000;65:357–62. Gosnell BA, Krahn DD. The relationship between saccharin and alcohol intake in rats. Alcohol 1992;9:203–6. Inoue M, Beauchamp GK, Bachmanov AA. Gustatory neural responses to umami taste stimuli in C57BL/6ByJ and 129P3/mice. Chem Senses 2004;29: 789–95. Lemon CH, Wilson DM, Brasser SM. Differential neural representation of oral ethanol by central taste-sensitive neurons in ethanol-preferring and genetically heterogeneous rats. J Neurophysiol 2011;106:3145–56. Li TK, Lumeng L, McBride WJ, Murphy JM. Rodent lines selected for affecting alcohol consumption. Alcohol Alcohol 1987;1(Suppl.):91–6. Lyall V, Heck GL, Phan TH, Mummalaneni S, Malik SA, Vinnikova AK, et al. Ethanol modulates the VR-1 variant amiloride-insensitive salt taste receptor. I. Effect on TRC volume and Na+ flux. J Gen Physiol 2005;125:569–85. Lyall V, Heck GL, Phan TH, Mummalaneni S, Malik SA, Vinnikova AK, et al. Ethanol modulates the VR-1 variant amiloride-insensitive salt taste receptor. II. Effect on chorda tympani salt responses. J Gen Physiol 2005;125:587–600. Lyall V, Phan TH, Ren Z, Mummalaneni S, Melone P, Mahavadi S, et al. Regulation of the putative TRPV1t salt taste receptor by phosphatidylinositol 4,5-bisphosphate. J Neurophysiol 2010;103:1337–49.
33
[24] Kampov-Polevoy AB, Eick C, Boland G, Khalitov E, Crews FT. Sweet liking, novelty seeking, and gender predict alcoholic status. Alcohol Clin Exp Res 2004;28:1291–8. [25] Kiefer SW, Mahadevan RS. The taste of alcohol for rats as revealed by aversion generalization tests. Chem Senses 1993;18:509–22. [26] Mattes RD, DiMeglio D. Ethanol perception and ingestion. Physiol Behav 2001;72:217–29. [27] Murata Y, Beauchamp GK, Bachmanov AA. Taste perception of monosodium glutamate and inosine monophosphate by 129P3/J and C57BL/6ByJ mice. Physiol Behav 2009;98:481–8. [28] Ninomiya Y, Funakoshi M. Behavioural discrimination between glutamate and the four basic taste substances in mice. Comp Biochem Physiol 1989;92:365–70. [29] Overstreet DH, Kampov-Polevoy AB, Rezvani AH, Murrelle L, Halikas JA, Janowsky DS. Saccharin intake predicts ethanol intake in genetically heterogeneous rats as well as different rats strains. Alcohol Clin Exp Res 1993;17: 366–9. [30] Scinska A, Koros´ E, Habrat B, Kukwa A, Kostowski W, Bien´kowski P. Bitter and sweet components of ethanol taste in humans. Drug Alcohol Depend 2000;60: 199–206. [31] Sinclair JD, Kampov-Polevoy A, Stewart R, Li TK. Taste preferences in rat lines selected for low and high alcohol consumption. Alcohol 1992;9:155–60. [32] Stewart RB, Russell RN, Lumeng L, Li TK, Murphy JM. Consumption of sweet, salty, sour, and bitter solution by selectively bred alcohol-preferring and alcohol-nonpreferring lines of rats. Alcohol Clin Exp Res 1994;18:375–81. [33] Ramirez I, Sprott RL. Genetic mechanism of drinking and feeding. Neurosci Biobehav Rev 1978;2:15–26. [34] Tampier L, Quintanilla ME. Saccharin consumption and the effect of a longterm exposure to a sweetened alcoholic solution in high-(UChB) and low(UChA) alcohol-drinking rats. Alcohol 2005;37:47–52. [35] Yamamoto T, Yuyan N, Kato T, Kawamura Y. Gustatory responses of cortical neurons in rats. III. Neural and behavioral measures compared. J Neurophysiol 1985;53:1370–86. [36] Yamamoto T, Matsuo R, Kiyomitsu Y, Kitamura R. Taste effects of umami substances in hamsters as studied by electrophysiological and conditioned taste aversion techniques. Brain Res 1988;451:147–62.
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). *<0.05 WHP and WLP vs. water in according to concentration of sucrose.
A significant interaction of concentration with flavors [F(7, 220) = 54.7968, p < 0.001], indicating that the degree of preference for the NaCl solution depended on the NaCl concentration. Generally, when the concentration of NaCl was increased, the NaCl consumption also increased. However, consumption of the NaCl solution dropped rapidly at concentrations of 1.6 and 3.2 g/ 100 ml. The water consumption was initially small (no more than 220 200
*
whp rats Flavor wlp rats Flavor wlp rats water whp rats water
80
*
180
140
*
100 80
*
Liquid intake/48 h (ml)
Liquid intake/48 h (ml)
160
120
Fig. 3 shows the volumes of citric acid solution and water consumed by the WHP and WLP rats. An ANOVA analysis of the data showed a main effect of the flavor F(1, 252) = 135.733, p < 0.001, and there was a significant interaction between the concentration and the flavor F(8, 252) = 16.356, p < 0.001. When the concentration of citric acid was increased (up to 0.128 g/l), the consumption of the citric acid solution was between 20 and 40 ml/ 48 h for both of the rat lines and was corresponding to concurrently available water consumption. The water intake under the same conditions was not much higher. Increasing the citric acid solution concentration from 0.256 to 2.048 g/l caused a decrease in citric acid consumption. Consumption of the citric acid solution almost completely ceased at concentrations of 1.024 and 2.048 g/l. However, at concentrations of 0.512, 1.024 and 2.048 g/l, the citric acid consumption was significantly less than the water consumption in both WHP and WLP rats (t-test p < 0.001). 3-Way ANOVA did not reveal interactions between the line, concentration and flavor tested F(8, 252) = 1.278, p > 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). *<0.05 WHP and WLP vs. water in according to concentration of NaCl.
*
*
* 0,000 0,025 0,050 0,100 0,200 0,400 0,800 1,600 3,200
#
60
60
20
#
0
* *
0,000
0,008
0,016
0,032
0,064
0,128
*
0,256
0,512
*
*
*
1,024
2,048
Citric Acid concentration (g/L) Fig. 3. Mean (SEM) water and citric acid solutions 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). *<0.05 WHP and WLP vs. water in according to concentration of citric acid. #<0.05 WHP water vs. WLP water.
W. Dyr et al. / Pharmacological Reports 66 (2014) 28–33
90
whp rats Flavor wlp rats Flavor wlp rats Water whp rats water
80
*
*
Liquid itake/48 h (ml)
70
*
60
*
*
50
40
* 30
20
10
0
0,000 0,002 0,004 0,008 0,016 0,032 0,064 0,128 0,256 0,512
SOA concentration (g/L) Fig. 4. Mean (SEM) water and SOA solutions 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). *<0.05 WHP and WLP vs. water in according to concentration of SOA.
SOA consumption Fig. 4 shows the amount of SOA solution and water intake of the WHP and WLP rats. There was no significant interactions between the lines and concentrations tested F(8, 250) = 0.782, p > 0.1 indicating that the liquid consumption between the lines did not differ the tested of concentrations. A significant interaction of concentration with flavors [F(8, 250) = 14.966, p < 0.001] indicated that the degree of preference for the SOA solution depended on the SOA concentration. Additionally, there was no 3-way interaction between the line, concentration and flavor tested F(8, 250) = 1.4, p > 0.1. These data suggest that both lines of rats consumed similar amounts of SOA and water. Individual comparisons of the SOA intake of the WHP and WLP rats (using a t test) did not reveal significant differences in the SOA consumption between the rat lines at any concentration tested. A comparison of the consumption of the SOA solution and water indicated that at the 0.256 and 0.512 g/l of SOA, WLP and WHP rats consumed less of the bitter solution compared to water (t-test p < 0.001). Discussion The findings of the present experiment do not support the hypothesis of an association between the intake of EtOH and sweetened solutions. WHP and WLP rats showed a high preference for sucrose solution. Rats from P and NP lines showed a strong preference for the sucrose solution, but P rats consumed greater amounts of the solution when compared to NP rats and this study showed important discrepancies in the responses to EtOH and sweet solutions by selectively bred P and NP rats [32]. There were great differences in the largeness of intake of the two substances, where the sweet solutions were consumed in high amount compared to the EtOH solutions [32]. What is an important, the differences among the strains in EtOH consumption did not
31
parallel the pattern of intake of the sweet solutions, because preference for EtOH among the P and NP strains ranged from high to low and animals of both lines displayed high preferences for the sweetened solutions [32]. Goodwin’s and Amit’s studies [15] demonstrated the relationship between the consumption of EtOH and a sweetened solution in three non-selected strains of rats which differ in their ethanol preference: In the first phase of the experiment, all animals were presented with an ascending series of ethanol solution (2–10%) in free-choice with water, followed by a 10-day maintenance period 10% ethanol with water. In the second phase the same animals were presented with an ascending series saccharin–quinine (SQ) consumption. The results revealed an absence of a direct relationship between ethanol and SQ consumption. The ethanolnonpreferring Lewis rats showed a greater preference for the SQ solution than ethanol-preferring Wistar Kyoto rats that consistently intake significantly less SQ [16]. In the Sardinian alcohol-preferring (sP) and Sardinian nonpreferring (sNP) rats, a high degree of preference for a saccharin solution was observed, and there were only very small differences between the lines. These results may suggest that their intake of saccharin is controlled by mechanisms that are different from those that control their alcohol intake [1]. The authors concluded that saccharin drinking behavior in sNP rats deviates from the hypothesis that saccharin and ethanol intake may co-vary. Similarly, the orofacial response in ethanol-naive P and NP rats did not reveal differences in taste reactivity to a sucrose solution (0.3 M) [4]. But in comparison with the mice 129/J (129) strain, the C57BL6ByJ mice had higher preference for ethanol, sucrose, and citric acid. They had lower preferences for NaCl and similar preferences for capsaicin and quinine hydrochloride. The authors [2] think that these data are consistent with the hypothesis that the higher ethanol intake by B6 mice depends, in part, on higher hedonic attractiveness of its sweet taste component. There is direct evidence that EtOH may not be reliably compared with sweet tastes alone, because it appears to have both sweet and bitter taste properties. The data suggest that EtOH and sweet–bitter mixtures have similar gustatory properties [9]. In human, studies of ethanol and other tastants have been shown that 10% ethanol is perceived as bitter and 30% of the patients described it as sweet and or sour. Findings suggest that in human ethanol tastes both bitter and sweet and the relationship between sucrose and ethanol intakes previously found in animals and humans may result, at least partially, from similar taste responses elicited by sucrose and ethanol [30]. In our present study, WHP and WLP rats consumed higher amounts of NaCl. The intake of the NaCl solution enhanced when the concentration of NaCl was increased. The sP and sNP rats had been showing identical intakes of highly concentrated solutions of sodium chloride (sP-17.5 ml/kg; sNP-16.7 ml/kg at the 1.8% NaCl) under the two-bottle, free-choice regimen [7]. Intake of saline (NaCl) solutions and water by males of two inbred of mice C57BL/6J (C57) and 129/J was examined using 48-h, two-bottle preference tests. There was a large strain difference in saline intake: C57 mice that had no prior exposure to saline exhibited a greater intake of saline than C57 mice previously exposed to 0.30 M NaCl. Saline preferences of the 129/J mice were not affected by prior exposure to 0.30 M NaCl. The strain differences in mouse salt preference are due in part to postingestional consequences of saline consumption in C57 but not 129/J mice [3]. Our study found no differences in the consumption of citric acid and SOA solutions in WHP and WLP rats. This result is supported by studies of P and NP rats, which did not show differences in citric acid or sucrose octaacetate consumption [32]. The selectively bred mice (HDID-1, High Drinking in the Dark) reach very high blood
32
W. Dyr et al. / Pharmacological Reports 66 (2014) 28–33
ethanol concentrations (BECs) after drinking from a single bottle of 20% ethanol. These mice drink nearly 6 g/kg ethanol in 4 h and reach average BECs of more than 1.0 mg. HDID-1 mice were tested with the HS/Npt control stock to compare for preference for two concentrations each of quinine, sucrose, and saccharin. Results revealed that neither genotype showed either preference or avoidance for any tastant after high ethanol concentrations [8]. Our present study, using selectively bred alcohol-preferring WHP and alcohol-nonpreferring WLP rats may suggest that between them there are similar preferences for sweet, salt, sour and bitter solutions. In addressing the question of what commonality could exist between EtOH and sweet solutions, the answer does not appear to be simple. It is thought that enhanced initial chemosensory attraction to ethanol and sweet stimuli phenotypes is associated with genetic alcohol preference [5] and 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. Three independent sensory systems – taste, olfaction, and chemosensory irritation – are involved in the perception of flavor. Humans perceive alcohol as a combination of sweet and bitter tastes, odors, and oral irritation, all of which vary as a function of concentration [26]. Likewise, rodents detect the sweet (sweet-like) and bitter (quinine-like) taste [25] and odor volatiles. Rejection of bitter substances is common in many species and may function to protect an animal from ingestion of bittertasting toxins. Herbivorous guinea pigs (Cavia porcellus) showed weaker avoidance of quinine hydrochloride (the bitter plant secondary metabolite) than two nonherbivorous mouse species (Mus musculus and Peromyscus leucopus). Guinea pigs (and herbivores in general) have a generalized reduced bitter sensitivity [13]. Taste perception has several distinct components, including hedonics (affective value, or ‘‘pleasantness’’) and taste quality (i.e. sweet, sour, salty, bitter, umami and perhaps others). However, the hedonic and qualitative components of overall perception are not completely independent because some taste qualities are generally associated with positive hedonic value (e.g., sweet and umami), while other taste qualities are generally associated with negative hedonic value (e.g., bitter). In two-bottle tests, mice from the C57BL/6ByJ (B6) strain drink more umami taste compounds: monosodium L-glutamate (MSG) and inosine-50 -monophosphate (IMP) compared with mice from the 129P3/J (129) strain [18]. The question was whether this variation in consumption could be attributed to strain differences in the perception of taste quality of MSG and IMP and the results [27] of the studies have shown that the strain differences in MSG consumption are not due to variation in the perception of the taste quality of MSG. The differential intake of IMP likely reflects strain differences in the way the taste quality of IMP is perceived. MSG and IMP have been shown to have a complex taste consisting of taste components of different qualities [28,35,36]. Consequently, the hedonic value of MSG and IMP depends in part on the combined hedonic values of their qualitatively distinct taste components. The increased ingestive responses to the umami stimuli in B6 mice are accompanied by either unchanged or decreased neural responses to these stimuli [18]. Ethanol produces changes in taste nerve responses to salty, sour, sweet, and bitter stimuli by interacting with quality-specific taste receptors or with intracellular signaling effectors in taste receptors cells (TRCs) [21,22]. In rodents, CT taste nerve responses to NaCl are derived from at least two salt taste receptors/ion channels in fungiform TRCs [23]. The sweet taste receptor serves as a receptor for both sucrose and ethanol. Extremely important
studies by Lyall et al. [23] showed that phosphatidylinositol 4,5bisphosphate (PIP2) is a common intracellular effector for sweet, bitter, umami, and TRPV1t-dependent salt taste. PIP2 seems to directly regulate the taste receptor protein itself (the TRPV1 ion channel or its taste receptor variant, TRPV1t). Studies showed that P and NP rats differ in TRPV/TRPV1t expression, neural and behavioral responses to sweet and salty stimuli and chronic sucrose and ethanol exposure [6]. The findings revealed differential central neural representation of oral ethanol between genetically heterogeneous rats and P rats genetically selected to prefer alcohol [19]. In conclusion, our present results revealed an absence of a direct relationship between ethanol and sucrose consumption. WLP rats consumed higher amount of the sucrose solution at the 4 and 8 g/100 ml concentrations compared to WHP rats. However, both lines of rats showed a high preference for the sucrose solution at concentrations of 2–32 g/100 ml. The consumption of salt, acid and bitter solutions did not differ between the WHP and WLP rats. The intake of the salt solution increased at concentrations of 0.025 g/100 ml to 0.8 g/100 ml. Higher NaCl concentrations caused a rapid decrease in consumption. Citric acid and SOA solutions were not preferred over water by either WHP or WLP rats. These results may suggest that WHP and WLP rats share a preference for sucrose and NaCl solutions (at certain concentrations) but they do not prefer sour and bitter tastes. The findings of the present experiment do not support the hypothesis of an association between the intake of EtOH and sweet solutions. Both lines of rats, WHP and WLP showed a high preference for sucrose solutions. Conflict of interest No conflict of interest. Funding This paper was financially supported by Institute Psychiatry and Neurology, Warsaw Nr 501/004/12045. References [1] Agabio R, Carai MAM, Lobina C, Pani M, Reali R, Bourov I, et al. Dissociation of ethanol and saccharin preference in sP and sNP rats. Alcohol Clin Exp Res 2000;24:24–9. [2] Bachmanov AA, Tordoff MG, Beauchamp GK. Ethanol consumption and taste preferences in C57BL/6ByJ and 129/J mice. Alcohol Clin Exp Res 1996;20:201– 6. [3] Beauchamp GK, Fisher AS. Strain differences in consumption of saline solution by mice. Physiol Behav 1993;54:179–84. [4] Bice PJ, Kiefer SW. Taste reactivity in alcohol preferring and nonpreferring rats. Alcohol Clin Exp Res 1990;14:721–7. [5] Brasser SM, Silbaugh BC, Ketchum MJ, Olney JJ, Lemon CH. Chemosensory responsiveness to ethanol and its individual sensory components in alcoholpreferring, alcohol-nonpreferring and genetically heterogeneous rats. Addict Biol 2012;17:423–36. [6] Coleman J, Williams A, Phan TH, Mummalaneni S, Melone P, Ren Z, et al. Strain differences in the neural, behavioral, and molecular correlates of sweet and salty taste in naı¨ve, ethanol- and sucrose-exposed P and NP rats. J Neurophysiol 2011;106:2606–21. [7] Colombo G, Agabio R, Diaz G, Fa M, Lobina C, Reali R, et al. g-Hydroxybutyric Acid Intake in Ethanol-preferring sP and -nonpreferring sNP rats. Physiol Behav 1998;64:197–202. [8] Crabbe JC, Spence SE, Brown LL, Metten P. Alcohol preference drinking in a mouse line selectively bred for high drinking in the dark. Alcohol 2011;45:427–40. [9] Di Lorenzo PM, Kiefer SW, Rice AG, Garcia J. Neural and behavioral responsivity to ethyl alcohol as a tastant. Alcohol 1986;3:55–61. [10] Dyr W, Kostowski W. Preliminary phenotypic characterization of the Warsaw high preferring (WHP) and Warsaw low preferring (WLP) lines of rats selectively bred for high and low ethanol consumption. Polish J Pharmacol 2004;56:359–65. [11] Dyr W, Kostowski W. Warsaw high-preferring (WHP) and Warsaw lowpreferring (WLP) lines of rats selectively bred for high and low voluntary
W. Dyr et al. / Pharmacological Reports 66 (2014) 28–33
[12] [13]
[14]
[15]
[16]
[17] [18]
[19]
[20] [21]
[22]
[23]
ethanol intake: preliminary phenotypic characterization. Alcohol 2008;42: 161–70. Eriksson K. Factors affecting voluntary alcohol consumption in the albino rat. Ann Zool Fennici 1969;6:227–65. Field KL, Beauchamp GK, Kimball BA, Mennella JA, Bachmanov AA. Bitter avoidance in guinea pigs (Cavia porcellus) and mice (Mus musculus and Peromyscus leucopus). J Comp Psychol 2010;124:455–9. Forgie ML, Beyerstein BL, Alexander BK. Combinations of taste factors and gender to opioid preference in C57BL and DBA mice. Psychopharmacology 1988;95:237–44. Goodwin FL, Amit Z. Do taste factors contribute to the mediation of ethanol intake? Ethanol and saccharin–quinine intake in three rat strains. Alcohol Clin Exp Res 1998;22:837–44. Goodwin FL, Bergeron N, Amit Z. Differences in the consumption of ethanol and flavored solutions in three strains of rats. Pharmacol Biochem Behav 2000;65:357–62. Gosnell BA, Krahn DD. The relationship between saccharin and alcohol intake in rats. Alcohol 1992;9:203–6. Inoue M, Beauchamp GK, Bachmanov AA. Gustatory neural responses to umami taste stimuli in C57BL/6ByJ and 129P3/mice. Chem Senses 2004;29: 789–95. Lemon CH, Wilson DM, Brasser SM. Differential neural representation of oral ethanol by central taste-sensitive neurons in ethanol-preferring and genetically heterogeneous rats. J Neurophysiol 2011;106:3145–56. Li TK, Lumeng L, McBride WJ, Murphy JM. Rodent lines selected for affecting alcohol consumption. Alcohol Alcohol 1987;1(Suppl.):91–6. Lyall V, Heck GL, Phan TH, Mummalaneni S, Malik SA, Vinnikova AK, et al. Ethanol modulates the VR-1 variant amiloride-insensitive salt taste receptor. I. Effect on TRC volume and Na+ flux. J Gen Physiol 2005;125:569–85. Lyall V, Heck GL, Phan TH, Mummalaneni S, Malik SA, Vinnikova AK, et al. Ethanol modulates the VR-1 variant amiloride-insensitive salt taste receptor. II. Effect on chorda tympani salt responses. J Gen Physiol 2005;125:587–600. Lyall V, Phan TH, Ren Z, Mummalaneni S, Melone P, Mahavadi S, et al. Regulation of the putative TRPV1t salt taste receptor by phosphatidylinositol 4,5-bisphosphate. J Neurophysiol 2010;103:1337–49.
33
[24] Kampov-Polevoy AB, Eick C, Boland G, Khalitov E, Crews FT. Sweet liking, novelty seeking, and gender predict alcoholic status. Alcohol Clin Exp Res 2004;28:1291–8. [25] Kiefer SW, Mahadevan RS. The taste of alcohol for rats as revealed by aversion generalization tests. Chem Senses 1993;18:509–22. [26] Mattes RD, DiMeglio D. Ethanol perception and ingestion. Physiol Behav 2001;72:217–29. [27] Murata Y, Beauchamp GK, Bachmanov AA. Taste perception of monosodium glutamate and inosine monophosphate by 129P3/J and C57BL/6ByJ mice. Physiol Behav 2009;98:481–8. [28] Ninomiya Y, Funakoshi M. Behavioural discrimination between glutamate and the four basic taste substances in mice. Comp Biochem Physiol 1989;92:365–70. [29] Overstreet DH, Kampov-Polevoy AB, Rezvani AH, Murrelle L, Halikas JA, Janowsky DS. Saccharin intake predicts ethanol intake in genetically heterogeneous rats as well as different rats strains. Alcohol Clin Exp Res 1993;17: 366–9. [30] Scinska A, Koros´ E, Habrat B, Kukwa A, Kostowski W, Bien´kowski P. Bitter and sweet components of ethanol taste in humans. Drug Alcohol Depend 2000;60: 199–206. [31] Sinclair JD, Kampov-Polevoy A, Stewart R, Li TK. Taste preferences in rat lines selected for low and high alcohol consumption. Alcohol 1992;9:155–60. [32] Stewart RB, Russell RN, Lumeng L, Li TK, Murphy JM. Consumption of sweet, salty, sour, and bitter solution by selectively bred alcohol-preferring and alcohol-nonpreferring lines of rats. Alcohol Clin Exp Res 1994;18:375–81. [33] Ramirez I, Sprott RL. Genetic mechanism of drinking and feeding. Neurosci Biobehav Rev 1978;2:15–26. [34] Tampier L, Quintanilla ME. Saccharin consumption and the effect of a longterm exposure to a sweetened alcoholic solution in high-(UChB) and low(UChA) alcohol-drinking rats. Alcohol 2005;37:47–52. [35] Yamamoto T, Yuyan N, Kato T, Kawamura Y. Gustatory responses of cortical neurons in rats. III. Neural and behavioral measures compared. J Neurophysiol 1985;53:1370–86. [36] Yamamoto T, Matsuo R, Kiyomitsu Y, Kitamura R. Taste effects of umami substances in hamsters as studied by electrophysiological and conditioned taste aversion techniques. Brain Res 1988;451:147–62.