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Oral operant ethanol self-administration in 5-HT1b knockout mice
Behavioural Brain Research 102 (1999) 211 – 215
Short communication
Oral operant ethanol self-administration in 5-HT1b knockout mice Fred O. Risinger *, Angela M. Doan, A. Coryn Vickrey Department of Beha6ioral Neuroscience, L470, Portland Alcohol Research Center, Oregon Health Sciences Uni6ersity, 3181 SW Sam Jackson Park Road, Portland, OR 97201 -3098, USA Received 11 September 1998; received in revised form 15 December 1998; accepted 30 December 1998
Abstract The present experiment examined oral ethanol self-adminstration in 5-HT1b knockout (KO) mice and 5-HT1b wild-type (WT) control mice using a continuous access operant procedure. After lever press training, adult 5-HT1b KO and 5-HT1b WT mice were placed in operant chambers on a 23 h per day basis with access to food (FR1), 10% v/v ethanol (FR4), and water from a sipper tube. KO mice displayed higher rates of responding on the ethanol-associated lever compared to WT mice. KO mice also consumed greater amounts of water. Food responding was the same in both genotypes. Following 30 sessions, ethanol concentration was altered every 5 days. Response patterns were determined using 0, 5, and 20% v/v ethanol concentrations. Ethanol responding (0, 5, 10, and 20% v/v) was also examined after the addition of 0.15% saccharin. KO mice and WT mice showed similar response rates for all ethanol concentrations. Since KO mice showed greater levels of ethanol responding only for unsweetened 10% v/v ethanol, and showed modest ethanol self-adminstration overall, the present results are not consistent with the notion that 5-HT1b KO have a generally greater preference for ethanol than 5-HT1b WT mice. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Ethanol self-adminstration; Reinforcement; Continuous access; 5-HT1b knockout mice
Several recent studies have drawn attention to serotonin 5-HT1b receptor involvement in ethanol intake. Ethanol preferring P rats display lower 5-HT1b receptor binding in a number of brain regions compared to ethanol non-preferring NP rats [5], and 5-HT1b receptor binding is increased in rats chronically fed ethanol [6]. Quantitative trait loci (QTL) studies of ethanol drinking have indicated a provisional QTL on mouse chromosome 9, in a location near the 5-HT1b receptor gene [7]. Further indication of a specific role for 5HT1b receptors in ethanol drinking is encouraged by the finding that homozygous mutant mice lacking 5HT1b receptors drink substantially larger amounts of ethanol compared to their wild-type counterparts and show higher ethanol/water preference ratios in a twobottle home cage procedure [2]. These 5-HT1b KO mice were developed by homologous recombination, and are * Corresponding author. Tel.: +1-503-4942016; fax: + 1-5034946877. E-mail address: [email protected] (F.O. Risinger)
maintained on an inbred 129/Sv-ter background [12]. KO mice do not show 5-HT1b receptor specific binding in the brain, and do not show locomotor stimulation produced by the 5-HT1a/b receptor agonist RU 24969 [8]. These mice appear developmentally normal, show ambient locomotor activity levels the same as WT mice in an open field test, and do not differ from WT mice in the light/dark test of anxiety [8]. However, KO mice show heightened aggression in the resident intruder test [8,12]. In addition to drinking more ethanol, KO mice display reduced sensitivity to ethanol-induced ataxia [2]. KO mice do not differ from WT mice in ethanol withdrawal convulsions or in ethanol clearance [2]. These mice also do not show differential development of ethanol-induced conditioned taste aversion, but demonstrate reduced acquisition of ethanol-induced conditioned place preference [9]. In the present study, we examined 5-HT1b KO mice and 5-HT1b WT mice in a continuous access procedure utilizing 23-h/day sessions [10]. In this procedure, ethanol preferring
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F.O. Risinger et al. / Beha6ioural Brain Research 102 (1999) 211–215
C57BL/6 mice show concentration-dependent responding for access to ethanol on an FR4 schedule of reinforcement. Ethanol non-preferring DBA mice do not respond for ethanol over the level of responding seen for plain water [10]. This study was intended to augment the results of Crabbe et al. [2], by demonstrating operant responding for access to oral ethanol in the KO mice. Adult male and female homozygous 5-HT1b KO ( − / − ) and 5-HT1b WT (+/ + ) mice were obtained from the Veterans Adminstration Medical Center, Portland, OR, where the animals were bred. Mice with the homozygous knockout mutation, which were developed by homologous recombination, lack the genes encoding for the 5-HT1b receptor [8,12]. KO mice and WT mice were trained over 20 sessions to produce four lever press responses (FR4) for access to 10% v/v ethanol in a procedure identical to one reported previously [10]. After training, subjects (n = 15 −/ − , 17 +/ +) were placed in mouse operant chambers (Med Associates ENV-003, enclosed in light/sound attenuating cubicles) for 23-h sessions. Each chamber was equipped with two levers on opposite walls, liquid dipper with a 0.02-ml cup, 20-mg pellet dispenser, drinking tube and house light. Ethanol was available from the dipper (FR4), 20 mg Noyes Formula A pellets from the pellet dispenser (FR1) and water from the drinking tube. During the first 30 consecutive sessions (designated as phase 1) 10% v/v ethanol was available. At the end of phase 1, the concentration of ethanol was changed every five sessions (designated as phase 2). The following v/v concentrations of ethanol were presented in the following order: 5, 20, 0. For phase 3, 0.2% w/v saccharin was used as the ethanol vehicle. Saccharin was used to mask the flavor of ethanol and promote higher rates of responding [10]. The concentration (% v/v) of ethanol was changed every five sessions in the following order: 0, 5, 10, 20. Statistical comparisons between genotypes focused on daily session response rates for ethanol and food responding, and water intake. Unweighted means analysis of variance with genotype as a factor was used for all comparisons. For analyses involving repeated measures, probability determinations were based on a Greenhouse-Geisser correction for inflated alpha. Ethanol responding in each genotype was also examined for the occurrence of an episodic pattern (i.e. bouts) of responding during each session. An ethanol bout consisted of four or more dipper presentations with 2 min or less between each dipper presentation [11]. Initial analyses indicated gender effects were minor and did not interact with either genotype or ethanol concentration, therefore this factor was removed from further analyses. During the course of the procedure, several subjects of each genotype showed drastic fluctuations in response rates which were unrelated to
changes in ethanol concentration. Therefore, for analysis purposes, data from subjects showing response changes greater than 2 S.D. from the mean of their genotype during any session were not used for analysis. Using this criterion, data from two − /− mice and three + /+ mice were excluded from the analyses reported below. Responses/session on the ethanol lever, responses/ session on the food lever, ml water intake/session, ethanol bouts/session, number of dippers per ethanol bout, and g/kg ethanol/session are given in Table 1. Data for each subject in phase 1 were averaged over five-session blocks. Data from phase 2 and phase 3 were averaged over the five sessions at each ethanol concentration. During phase 1, knockout mice responded more on the ethanol lever than wild-type mice. This response difference appeared specific for ethanol given that both strains showed similar response rates on the food lever. However, knockout mice also consumed more water, an observation suggesting a non-specific difference in fluid need. Genotype× Trial Block analysis of responding on the ethanol lever yielded a significant effect of Genotype (F(1,25)= 5.8, P B 0.02), but not Trial Block, or Genotype× Trial Block (Fs(5,125)5 1.8, PsB 0.1). KO mice also showed higher g/kg per session ethanol intakes than wild-type mice (F(1,25)= 4.6, P B 0.04). Differences in food lever responding were not noted between KO and WT mice (Genotype: F(1,25)= 1.5, PB 0.2; Genotype× Trial Block: F(5,125)= 1.7, P B 0.2). However, knockout mice drank more water (Genotype: F(1,25)= 21.7, PB 0.001). Both genotypes showed similar number of ethanol bouts per session (Genotype: F(1,25)=2.9, PB 0.1), and had similar numbers of dippers presented for each bout (Genotype: F(1,22)= 0.6, PB 0.4). During phase 2, each genotype responded similarly on the ethanol lever at each ethanol concentration (Genotype: F(1,25)= 0.01, PB 0.9; Genotype × Ethanol Concentration: F(2,50)= 1.4, P B 0.2). Analysis of ethanol lever responding for the sessions with ethanol available (5 and 20% v/v) also did not yield a significant effect of genotype (F(1,25)= 0.3, PB0.5). Ethanol intake (g/kg per session) was similar between genotype at each ethanol concentration (i.e. 5 and 20% v/v) (Genotype: F(1,25)= 0.3, P B 0.6). Ethanol intake was higher at the 20% v/v ethanol concentration for both genotypes (i.e. Ethanol Concentration: F(1,25)= 11.6, PB 0.001; Genotype× Ethanol Concentration: F(1,25)= 0.5, P B 0.5). Responding on the food lever was also similar between genotypes at each ethanol concentration (Genotype: F(1,25)= 1.4, PB 0.2, Genotype× Ethanol Concentration: F(2,50)= 1.4, PB0.2), and remained stable throughout this phase (Ethanol Concentration: F(2,50)= 1.5, P B 0.2). However, KO mice continued to drink greater amounts of water at each ethanol concentration (Genotype: F(1,25)=9.9,
F.O. Risinger et al. / Beha6ioural Brain Research 102 (1999) 211–215
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Table 1 Mean ( 9S.E.M.) ethanol responses, g/kg ethanol intake, food responses, ml water intake, and number of ethanol bouts per session during phases 1–3a Phase 1
Ethanol responses (FR4) KO WT
Session 1–5
6–10
11–15
130 (23) 72 (22)
106 (24) 60 (23)
102 (15) 37 (14)
Ethanol (g/kg) KO WT Food responses (FR1) KO WT
2.0 (0.4) 1.2 (0.3) 337 (43) 250 (41)
1.6 (0.4) 0.9 (0.4) 276 (29) 232 (28)
16–20
86 (14) 39 (14)
1.6 (0.2) 0.6 (0.2) 292 (29) 241 (28)
1.3 (0.2) 0.6 (0.2) 250 (20) 235 (19)
21–25
26–30
112 (18) 56 (18)
116 (29) 76 (28)
1.7 (0.3) 0.9 (0.3) 251 (15) 230 (14)
1.9 (0.5) 1.2 (0.5) 238 (13) 219 (13)
Water intake (ml) KO WT
6.3 (0.5) 4.1 (0.4)
5.8 (0.3) 4.0 (0.3)
6.2 (0.3) 3.9 (0.3)
6.0 (0.3) 4.1 (0.3)
5.9 (0.3) 4.2 (0.3)
5.6 (0.3) 4.2 (0.2)
Ethanol bouts KO WT
0.9 (0.6) 2.3 (0.6)
0.5 (0.4) 1.9 (0.4)
0.3 (0.3) 1.3 (0.3)
0.4 (0.4) 1.2 (0.4)
0.9 (0.4) 1.3 (0.4)
1.7 (1.2) 2.8 (1.2)
Ethanol bout size KO WT
6.5 (0.9) 6.6 (0.9)
5.7 (0.6) 6.9 (1.2)
5.6 (0.3) 5.4 (0.3)
6.2 (1.0) 5.7 (1.1)
5.7 (0.5) 7.5 (0.8)
5.9 (0.4) 7.0 (0.3)
Phase 2
Ethanol responses (FR4) KO WT
Ethanol (% v/v) 0
5
20
157 (94) 205 (91)
132 (48) 110 (46)
164 (53) 110 (51)
Ethanol (g/kg) KO WT Food responses (FR1) KO WT
– – 245 (17) 236 (17)
1.0 (0.4) 0.9 (0.4) 242 (14) 224 (13)
5.3 (1.9) 3.7 (1.8) 277 (22) 230 (21)
Water intake (ml) KO WT
5.6 (0.3) 4.7 (0.3)
5.6 (0.3) 4.2 (0.3)
5.4 (0.3) 4.2 (0.3)
Ethanol bouts KO WT
2.2 (1.4) 3.1 (1.3)
1.4 (1.5) 3.4 (1.5)
3.0 (1.2) 2.4 (1.2)
Ethanol bout size KO WT
5.9 (0.2) 7.4 (0.7)
5.7 (0.4) 7.1 (0.8)
5.9 (0.4) 7.1 (1.3)
Phase 3
Ethanol responses (FR4) KO WT Ethanol (g/kg) KO WT
EtOH (% v/v) 0
5
10
20
314 (121) 485 (116)
323 (141) 545 (136)
325 (100) 465 (97)
271 (65) 274 (63)
– –
2.6 (1.2) 4.4 (1.2)
5.2 (1.7) 7.4 (1.6)
8.5 (2.1) 8.7 (2.1)
F.O. Risinger et al. / Beha6ioural Brain Research 102 (1999) 211–215
214 Table 1 (Continued) Phase 3
Food responses (FR1) KO WT Water intake (ml) KO WT
EtOH (% v/v) 0
5
10
20
241 (17) 239 (16)
233 (20) 258 (19)
237 (17) 230 (16)
242 (15) 221 (15)
4.5 (0.5) 3.3 (0.4)
4.4 (0.5) 2.9 (0.5)
4.4 (0.4) 2.9 (0.4)
4.8 (0.3) 3.6 (0.3)
Ethanol bouts KO WT
7.9 (2.4) 10.7 (2.2)
7.7 (2.8) 12.6 (2.6)
8.5 (2.5) 12.3 (2.3)
6.6 (1.8) 6.8 (1.6)
Ethanol bout size KO WT
7.0 (1.0) 15.9 (6.0)
6.7 (0.8) 12.2 (3.9)
7.2 (0.8) 11.3 (2.3)
7.1 (0.8) 8.4 (0.8)
a During phase 1, 10% v/v ethanol in water was available for 30 consecutive sessions. During phase 2 (15 consecutive sessions), the concentration of ethanol was changed every 5 days with 5, 20 and 0% v/v presented in that order. During phase 3 (20 consecutive sessions) 0.2% w/v saccharin was presented along with ethanol given in the following concentrations: 0, 5, 10, 20% v/v.
P B0.001). As in phase 1, the genotypes did not differ in the number of ethanol bouts per session (Genotype: F(1,25)=0.2, PB0.7; Genotype × Ethanol Concentration: F(2,50)=1.6, PB 0.2) or in bout size (Genotype: F(1,21)=2.0, P B0.1). As expected, overall response rates per session on the ethanol lever were increased when saccharin was added [10]. For example, analysis of lever responses per session for plain water (0% concentration from phase 2) compared to lever responses per session for 0.2% w/v saccharin (0% concentration from phase 3) showed a significant overall increase in responding (F(1,25)= 37.7, PB0.001), which was similar in both genotypes (i.e. Genotype: F(1,25) = 0.6, P B0.5; Genotype×Saccharin Concentration: F(1,25) = 3.0, P B 0.1). Repeated measures analysis of phase 3 responding on the ethanol lever showed a significant effect of Ethanol Concentration (F(3,75)= 4.5, P B0.02) but not of Genotype (F(1,25)=0.4, P B 0.5) or Genotype× Ethanol Concentration (F(2,50)=1.2, P B 0.3). Analysis of ethanol lever responding in sessions when ethanol was available (5, 10 and 20% v/v) revealed a significant effect of Ethanol Concentration (F(2,50) =6.0, P B 0.02) but not Genotype (F(1,25) = 0.8, PB 0.3) or Genotype × Ethanol Concentration (F(2,50) =2.6, P B 0.1). Analysis of g/kg per session intakes also showed a significant effect of Ethanol Concentration (F(2,50) = 27.0, PB 0.001), but not of Genotype (F(1,25) = 0.4, PB 0.5) or Genotype ×Ethanol Concentration (F(2,50) =1.2, PB 0.3). Food responding was the same in both genotypes (Genotype: F(1,25)=0.0, P B0.1; Genotype× Ethanol Concentration: F(3,75) = 2.0, P B 0.1), and remained stable over changes in the concentration of ethanol (F(3,75)= 0.9, PB 0.4). Analysis of water intake yielded a significant effect of Genotype (F(1,25) = 5.1,
PB 0.03) and Ethanol Concentration (F(3,75)=4.4, PB 0.01) but no Genotype× Ethanol Concentration interaction (F(3,75)=0.7, P B 0.5). Analysis of ethanol bouts/session revealed a significant effect of Ethanol Concentration (F(3,69)= 3.6, PB 0.02) but not of Genotype (F(1,23)= 1.0, PB 0.3) or Genotype × Ethanol Concentration (F(3,69)= 1.5, PB 0.2). Likewise, ethanol bout size was similar for each genotype (Genotype: F(1,21)= 2.5, PB 0.1; Genotype× Ethanol Concentration: F(3,63)= 1.7, PB 0.2). Crabbe et. al. [2] reported that 5-HT1b knockout mice drink large amounts of ethanol compared to their wild-type counterparts. The level of ethanol self-adminstration in the aforementioned study, using a two-bottle home cage procedure, also indicated that 5-HT1b knockout mice drank ethanol at levels greater than that seen for the ethanol preferring C57BL strain. However, in the present study, KO mice showed modest rates of responding for 10% ethanol which were well below rates of responding seen in a similar procedure using C57BL mice [10]. KO mice did not display differential rates of responding on the ethanol lever when the concentration of ethanol was altered. Overall ethanol response rates in both KO mice and WT mice actually resembled ethanol non-preferring DBA mice [10]. Responses rates on the food lever were similar for both genotypes, however KO mice consumed more water during each phase suggesting higher fluid need in the KO genotype. Response rates for dipper presentation when only water was present in phase 2 were similar in both genotypes to rates seen for C57BL and DBA mice [10]. However, it is likely that carry-over effects from the training procedure might have influenced responding since subjects were trained to make lever press responses while water deprived. During phase 3, the
F.O. Risinger et al. / Beha6ioural Brain Research 102 (1999) 211–215
addition of saccharin to the ethanol solution promoted higher levels of responding on the ethanol lever. This finding is consistent with the behavior of C57BL mice and DBA mice in this procedure. However, genotype effects were not seen, and again indicate similar levels of ethanol preference in both KO mice and WT mice. Throughout the procedure KO mice or WT mice did not show substantial occurrence of episodic responding (bouts) of the kind seen in C57BL mice. Based on the modest rates of responding and the low levels of bouts, it is likely that neither KO mice or WT mice experienced appreciable blood ethanol levels during the procedure. Overall, this rather limited influence of genotype is not completely consistent with the finding that KO mice are ethanol preferring [2]. One limitation to this conclusion is the notion that neither strain consumed enough ethanol at any given time to experience significant reinforcing effects from ethanol. Additionally, operant procedures have in some cases yielded different conclusions from those reached using home cage drinking procedures regarding mouse strain/line preferences for ethanol. For example, BALB/cJ mice consume moderate amounts of ethanol in a two-bottle choice procedure [1], but do not show ethanol-reinforced behavior in an operant setting [3]. Another consideration for the present pattern of results is the amount of work needed to gain ethanol in the present procedure. Lower FR requirements are associated with larger ethanol intakes in rats [4]. It may be that the 129/SV-ter background strain is especially sensitivity to work requirement, which obscured detection of ethanol preference.
Acknowledgements This work was supported by a grant from the Alcoholic Beverage Medical Research Foundation and NIAAA grants AA10760, AA10520. Thanks are given to Drs John C Crabbe Jr, Tamara J Phillips and Rene´
.
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Hen for providing the mice. Portions of these data were presented at the 1997 annual meeting of the Research Society On Alcoholism (San Francisco, CA).
References [1] Belknap JK, Crabbe JC, Young ER. Voluntary consumption of ethanol in 15 inbred mouse strains. Psychopharmacology 1993;112:503 – 10. [2] Crabbe JC, Phillips TJ, Feller DJ, et al. Elevated alcohol consumption in null mutant mice lacking 5-HT1b receptors. Nat Genet 1996;14:98 – 101. [3] Elmer GI, Meisch RA, Goldberg SR, George FR. Fixed-ratio schedules of oral ethanol self-administration in inbred mouse strains. Psychopharmacology 1988;96:431 – 6. [4] Files FJ, Andrews CM, Lewis RS, Samson HH. Effects of ethanol concentration and fixed-ratio requirement on ethanol self-adminstration by P rats in a continuous access situation. Alcohol Clin Exp Res 1993;17:61 – 8. [5] McBride WJ, Chernet E, Russell RN, et al. Regional CNS densities of monoamine receptors in alcohol-naive alcohol-preferring P and -non-preferring NP rats. Alcohol 1997;14:141–8. [6] Nevo I, Langlois X, Laporte AM, et al. Chronic alcoholization alters the expression of 5-HT1A and 5-HT1B receptor subtypes in rat brain. Eur J Pharmacol 1995;281:229 – 39. [7] Phillips TJ, Crabbe JC, Metten P, Belknap JK. Localization of genes affecting alcohol drinking in mice. Alcohol Clin Exp Res 1994;18:931 – 41. [8] Ramboz S, Saudou F, Amara DA, et al. 5-HT1b receptor knockout: behavioral consequences. Behav Brain Res 1996;73:305 – 12. [9] Risinger FO, Bormann NM, Oakes RA. Reduced sensitivity to ethanol reward but not ethanol aversion, in mice lacking 5HT1B receptors. Alcohol Clin Exp Res 1996;20:1401 – 5. [10] Risinger FO, Brown MM, Doan AM, Oakes RA. Mouse strain differences in oral operant ethanol reinforcement under continuous access conditions. Alcohol Clin Exp Res 1998;22:677–84. [11] Samson HH, Tolliver GA, Pfeffer AD, Sadeghi K, Haraguchi M. Relation of ethanol self-administration to feeding and drinking in a non-restricted access situation in rats initiated to self-administer ethanol using the sucrose-fading technique. Alcohol 1988;5:375 – 85. [12] Saudou F, Amara DA, Dierich A, et al. Enhanced aggressive behavior in mice lacking 5-HT1b receptor. Science 1994;265:1875– 8.
Short communication
Oral operant ethanol self-administration in 5-HT1b knockout mice Fred O. Risinger *, Angela M. Doan, A. Coryn Vickrey Department of Beha6ioral Neuroscience, L470, Portland Alcohol Research Center, Oregon Health Sciences Uni6ersity, 3181 SW Sam Jackson Park Road, Portland, OR 97201 -3098, USA Received 11 September 1998; received in revised form 15 December 1998; accepted 30 December 1998
Abstract The present experiment examined oral ethanol self-adminstration in 5-HT1b knockout (KO) mice and 5-HT1b wild-type (WT) control mice using a continuous access operant procedure. After lever press training, adult 5-HT1b KO and 5-HT1b WT mice were placed in operant chambers on a 23 h per day basis with access to food (FR1), 10% v/v ethanol (FR4), and water from a sipper tube. KO mice displayed higher rates of responding on the ethanol-associated lever compared to WT mice. KO mice also consumed greater amounts of water. Food responding was the same in both genotypes. Following 30 sessions, ethanol concentration was altered every 5 days. Response patterns were determined using 0, 5, and 20% v/v ethanol concentrations. Ethanol responding (0, 5, 10, and 20% v/v) was also examined after the addition of 0.15% saccharin. KO mice and WT mice showed similar response rates for all ethanol concentrations. Since KO mice showed greater levels of ethanol responding only for unsweetened 10% v/v ethanol, and showed modest ethanol self-adminstration overall, the present results are not consistent with the notion that 5-HT1b KO have a generally greater preference for ethanol than 5-HT1b WT mice. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Ethanol self-adminstration; Reinforcement; Continuous access; 5-HT1b knockout mice
Several recent studies have drawn attention to serotonin 5-HT1b receptor involvement in ethanol intake. Ethanol preferring P rats display lower 5-HT1b receptor binding in a number of brain regions compared to ethanol non-preferring NP rats [5], and 5-HT1b receptor binding is increased in rats chronically fed ethanol [6]. Quantitative trait loci (QTL) studies of ethanol drinking have indicated a provisional QTL on mouse chromosome 9, in a location near the 5-HT1b receptor gene [7]. Further indication of a specific role for 5HT1b receptors in ethanol drinking is encouraged by the finding that homozygous mutant mice lacking 5HT1b receptors drink substantially larger amounts of ethanol compared to their wild-type counterparts and show higher ethanol/water preference ratios in a twobottle home cage procedure [2]. These 5-HT1b KO mice were developed by homologous recombination, and are * Corresponding author. Tel.: +1-503-4942016; fax: + 1-5034946877. E-mail address: [email protected] (F.O. Risinger)
maintained on an inbred 129/Sv-ter background [12]. KO mice do not show 5-HT1b receptor specific binding in the brain, and do not show locomotor stimulation produced by the 5-HT1a/b receptor agonist RU 24969 [8]. These mice appear developmentally normal, show ambient locomotor activity levels the same as WT mice in an open field test, and do not differ from WT mice in the light/dark test of anxiety [8]. However, KO mice show heightened aggression in the resident intruder test [8,12]. In addition to drinking more ethanol, KO mice display reduced sensitivity to ethanol-induced ataxia [2]. KO mice do not differ from WT mice in ethanol withdrawal convulsions or in ethanol clearance [2]. These mice also do not show differential development of ethanol-induced conditioned taste aversion, but demonstrate reduced acquisition of ethanol-induced conditioned place preference [9]. In the present study, we examined 5-HT1b KO mice and 5-HT1b WT mice in a continuous access procedure utilizing 23-h/day sessions [10]. In this procedure, ethanol preferring
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F.O. Risinger et al. / Beha6ioural Brain Research 102 (1999) 211–215
C57BL/6 mice show concentration-dependent responding for access to ethanol on an FR4 schedule of reinforcement. Ethanol non-preferring DBA mice do not respond for ethanol over the level of responding seen for plain water [10]. This study was intended to augment the results of Crabbe et al. [2], by demonstrating operant responding for access to oral ethanol in the KO mice. Adult male and female homozygous 5-HT1b KO ( − / − ) and 5-HT1b WT (+/ + ) mice were obtained from the Veterans Adminstration Medical Center, Portland, OR, where the animals were bred. Mice with the homozygous knockout mutation, which were developed by homologous recombination, lack the genes encoding for the 5-HT1b receptor [8,12]. KO mice and WT mice were trained over 20 sessions to produce four lever press responses (FR4) for access to 10% v/v ethanol in a procedure identical to one reported previously [10]. After training, subjects (n = 15 −/ − , 17 +/ +) were placed in mouse operant chambers (Med Associates ENV-003, enclosed in light/sound attenuating cubicles) for 23-h sessions. Each chamber was equipped with two levers on opposite walls, liquid dipper with a 0.02-ml cup, 20-mg pellet dispenser, drinking tube and house light. Ethanol was available from the dipper (FR4), 20 mg Noyes Formula A pellets from the pellet dispenser (FR1) and water from the drinking tube. During the first 30 consecutive sessions (designated as phase 1) 10% v/v ethanol was available. At the end of phase 1, the concentration of ethanol was changed every five sessions (designated as phase 2). The following v/v concentrations of ethanol were presented in the following order: 5, 20, 0. For phase 3, 0.2% w/v saccharin was used as the ethanol vehicle. Saccharin was used to mask the flavor of ethanol and promote higher rates of responding [10]. The concentration (% v/v) of ethanol was changed every five sessions in the following order: 0, 5, 10, 20. Statistical comparisons between genotypes focused on daily session response rates for ethanol and food responding, and water intake. Unweighted means analysis of variance with genotype as a factor was used for all comparisons. For analyses involving repeated measures, probability determinations were based on a Greenhouse-Geisser correction for inflated alpha. Ethanol responding in each genotype was also examined for the occurrence of an episodic pattern (i.e. bouts) of responding during each session. An ethanol bout consisted of four or more dipper presentations with 2 min or less between each dipper presentation [11]. Initial analyses indicated gender effects were minor and did not interact with either genotype or ethanol concentration, therefore this factor was removed from further analyses. During the course of the procedure, several subjects of each genotype showed drastic fluctuations in response rates which were unrelated to
changes in ethanol concentration. Therefore, for analysis purposes, data from subjects showing response changes greater than 2 S.D. from the mean of their genotype during any session were not used for analysis. Using this criterion, data from two − /− mice and three + /+ mice were excluded from the analyses reported below. Responses/session on the ethanol lever, responses/ session on the food lever, ml water intake/session, ethanol bouts/session, number of dippers per ethanol bout, and g/kg ethanol/session are given in Table 1. Data for each subject in phase 1 were averaged over five-session blocks. Data from phase 2 and phase 3 were averaged over the five sessions at each ethanol concentration. During phase 1, knockout mice responded more on the ethanol lever than wild-type mice. This response difference appeared specific for ethanol given that both strains showed similar response rates on the food lever. However, knockout mice also consumed more water, an observation suggesting a non-specific difference in fluid need. Genotype× Trial Block analysis of responding on the ethanol lever yielded a significant effect of Genotype (F(1,25)= 5.8, P B 0.02), but not Trial Block, or Genotype× Trial Block (Fs(5,125)5 1.8, PsB 0.1). KO mice also showed higher g/kg per session ethanol intakes than wild-type mice (F(1,25)= 4.6, P B 0.04). Differences in food lever responding were not noted between KO and WT mice (Genotype: F(1,25)= 1.5, PB 0.2; Genotype× Trial Block: F(5,125)= 1.7, P B 0.2). However, knockout mice drank more water (Genotype: F(1,25)= 21.7, PB 0.001). Both genotypes showed similar number of ethanol bouts per session (Genotype: F(1,25)=2.9, PB 0.1), and had similar numbers of dippers presented for each bout (Genotype: F(1,22)= 0.6, PB 0.4). During phase 2, each genotype responded similarly on the ethanol lever at each ethanol concentration (Genotype: F(1,25)= 0.01, PB 0.9; Genotype × Ethanol Concentration: F(2,50)= 1.4, P B 0.2). Analysis of ethanol lever responding for the sessions with ethanol available (5 and 20% v/v) also did not yield a significant effect of genotype (F(1,25)= 0.3, PB0.5). Ethanol intake (g/kg per session) was similar between genotype at each ethanol concentration (i.e. 5 and 20% v/v) (Genotype: F(1,25)= 0.3, P B 0.6). Ethanol intake was higher at the 20% v/v ethanol concentration for both genotypes (i.e. Ethanol Concentration: F(1,25)= 11.6, PB 0.001; Genotype× Ethanol Concentration: F(1,25)= 0.5, P B 0.5). Responding on the food lever was also similar between genotypes at each ethanol concentration (Genotype: F(1,25)= 1.4, PB 0.2, Genotype× Ethanol Concentration: F(2,50)= 1.4, PB0.2), and remained stable throughout this phase (Ethanol Concentration: F(2,50)= 1.5, P B 0.2). However, KO mice continued to drink greater amounts of water at each ethanol concentration (Genotype: F(1,25)=9.9,
F.O. Risinger et al. / Beha6ioural Brain Research 102 (1999) 211–215
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Table 1 Mean ( 9S.E.M.) ethanol responses, g/kg ethanol intake, food responses, ml water intake, and number of ethanol bouts per session during phases 1–3a Phase 1
Ethanol responses (FR4) KO WT
Session 1–5
6–10
11–15
130 (23) 72 (22)
106 (24) 60 (23)
102 (15) 37 (14)
Ethanol (g/kg) KO WT Food responses (FR1) KO WT
2.0 (0.4) 1.2 (0.3) 337 (43) 250 (41)
1.6 (0.4) 0.9 (0.4) 276 (29) 232 (28)
16–20
86 (14) 39 (14)
1.6 (0.2) 0.6 (0.2) 292 (29) 241 (28)
1.3 (0.2) 0.6 (0.2) 250 (20) 235 (19)
21–25
26–30
112 (18) 56 (18)
116 (29) 76 (28)
1.7 (0.3) 0.9 (0.3) 251 (15) 230 (14)
1.9 (0.5) 1.2 (0.5) 238 (13) 219 (13)
Water intake (ml) KO WT
6.3 (0.5) 4.1 (0.4)
5.8 (0.3) 4.0 (0.3)
6.2 (0.3) 3.9 (0.3)
6.0 (0.3) 4.1 (0.3)
5.9 (0.3) 4.2 (0.3)
5.6 (0.3) 4.2 (0.2)
Ethanol bouts KO WT
0.9 (0.6) 2.3 (0.6)
0.5 (0.4) 1.9 (0.4)
0.3 (0.3) 1.3 (0.3)
0.4 (0.4) 1.2 (0.4)
0.9 (0.4) 1.3 (0.4)
1.7 (1.2) 2.8 (1.2)
Ethanol bout size KO WT
6.5 (0.9) 6.6 (0.9)
5.7 (0.6) 6.9 (1.2)
5.6 (0.3) 5.4 (0.3)
6.2 (1.0) 5.7 (1.1)
5.7 (0.5) 7.5 (0.8)
5.9 (0.4) 7.0 (0.3)
Phase 2
Ethanol responses (FR4) KO WT
Ethanol (% v/v) 0
5
20
157 (94) 205 (91)
132 (48) 110 (46)
164 (53) 110 (51)
Ethanol (g/kg) KO WT Food responses (FR1) KO WT
– – 245 (17) 236 (17)
1.0 (0.4) 0.9 (0.4) 242 (14) 224 (13)
5.3 (1.9) 3.7 (1.8) 277 (22) 230 (21)
Water intake (ml) KO WT
5.6 (0.3) 4.7 (0.3)
5.6 (0.3) 4.2 (0.3)
5.4 (0.3) 4.2 (0.3)
Ethanol bouts KO WT
2.2 (1.4) 3.1 (1.3)
1.4 (1.5) 3.4 (1.5)
3.0 (1.2) 2.4 (1.2)
Ethanol bout size KO WT
5.9 (0.2) 7.4 (0.7)
5.7 (0.4) 7.1 (0.8)
5.9 (0.4) 7.1 (1.3)
Phase 3
Ethanol responses (FR4) KO WT Ethanol (g/kg) KO WT
EtOH (% v/v) 0
5
10
20
314 (121) 485 (116)
323 (141) 545 (136)
325 (100) 465 (97)
271 (65) 274 (63)
– –
2.6 (1.2) 4.4 (1.2)
5.2 (1.7) 7.4 (1.6)
8.5 (2.1) 8.7 (2.1)
F.O. Risinger et al. / Beha6ioural Brain Research 102 (1999) 211–215
214 Table 1 (Continued) Phase 3
Food responses (FR1) KO WT Water intake (ml) KO WT
EtOH (% v/v) 0
5
10
20
241 (17) 239 (16)
233 (20) 258 (19)
237 (17) 230 (16)
242 (15) 221 (15)
4.5 (0.5) 3.3 (0.4)
4.4 (0.5) 2.9 (0.5)
4.4 (0.4) 2.9 (0.4)
4.8 (0.3) 3.6 (0.3)
Ethanol bouts KO WT
7.9 (2.4) 10.7 (2.2)
7.7 (2.8) 12.6 (2.6)
8.5 (2.5) 12.3 (2.3)
6.6 (1.8) 6.8 (1.6)
Ethanol bout size KO WT
7.0 (1.0) 15.9 (6.0)
6.7 (0.8) 12.2 (3.9)
7.2 (0.8) 11.3 (2.3)
7.1 (0.8) 8.4 (0.8)
a During phase 1, 10% v/v ethanol in water was available for 30 consecutive sessions. During phase 2 (15 consecutive sessions), the concentration of ethanol was changed every 5 days with 5, 20 and 0% v/v presented in that order. During phase 3 (20 consecutive sessions) 0.2% w/v saccharin was presented along with ethanol given in the following concentrations: 0, 5, 10, 20% v/v.
P B0.001). As in phase 1, the genotypes did not differ in the number of ethanol bouts per session (Genotype: F(1,25)=0.2, PB0.7; Genotype × Ethanol Concentration: F(2,50)=1.6, PB 0.2) or in bout size (Genotype: F(1,21)=2.0, P B0.1). As expected, overall response rates per session on the ethanol lever were increased when saccharin was added [10]. For example, analysis of lever responses per session for plain water (0% concentration from phase 2) compared to lever responses per session for 0.2% w/v saccharin (0% concentration from phase 3) showed a significant overall increase in responding (F(1,25)= 37.7, PB0.001), which was similar in both genotypes (i.e. Genotype: F(1,25) = 0.6, P B0.5; Genotype×Saccharin Concentration: F(1,25) = 3.0, P B 0.1). Repeated measures analysis of phase 3 responding on the ethanol lever showed a significant effect of Ethanol Concentration (F(3,75)= 4.5, P B0.02) but not of Genotype (F(1,25)=0.4, P B 0.5) or Genotype× Ethanol Concentration (F(2,50)=1.2, P B 0.3). Analysis of ethanol lever responding in sessions when ethanol was available (5, 10 and 20% v/v) revealed a significant effect of Ethanol Concentration (F(2,50) =6.0, P B 0.02) but not Genotype (F(1,25) = 0.8, PB 0.3) or Genotype × Ethanol Concentration (F(2,50) =2.6, P B 0.1). Analysis of g/kg per session intakes also showed a significant effect of Ethanol Concentration (F(2,50) = 27.0, PB 0.001), but not of Genotype (F(1,25) = 0.4, PB 0.5) or Genotype ×Ethanol Concentration (F(2,50) =1.2, PB 0.3). Food responding was the same in both genotypes (Genotype: F(1,25)=0.0, P B0.1; Genotype× Ethanol Concentration: F(3,75) = 2.0, P B 0.1), and remained stable over changes in the concentration of ethanol (F(3,75)= 0.9, PB 0.4). Analysis of water intake yielded a significant effect of Genotype (F(1,25) = 5.1,
PB 0.03) and Ethanol Concentration (F(3,75)=4.4, PB 0.01) but no Genotype× Ethanol Concentration interaction (F(3,75)=0.7, P B 0.5). Analysis of ethanol bouts/session revealed a significant effect of Ethanol Concentration (F(3,69)= 3.6, PB 0.02) but not of Genotype (F(1,23)= 1.0, PB 0.3) or Genotype × Ethanol Concentration (F(3,69)= 1.5, PB 0.2). Likewise, ethanol bout size was similar for each genotype (Genotype: F(1,21)= 2.5, PB 0.1; Genotype× Ethanol Concentration: F(3,63)= 1.7, PB 0.2). Crabbe et. al. [2] reported that 5-HT1b knockout mice drink large amounts of ethanol compared to their wild-type counterparts. The level of ethanol self-adminstration in the aforementioned study, using a two-bottle home cage procedure, also indicated that 5-HT1b knockout mice drank ethanol at levels greater than that seen for the ethanol preferring C57BL strain. However, in the present study, KO mice showed modest rates of responding for 10% ethanol which were well below rates of responding seen in a similar procedure using C57BL mice [10]. KO mice did not display differential rates of responding on the ethanol lever when the concentration of ethanol was altered. Overall ethanol response rates in both KO mice and WT mice actually resembled ethanol non-preferring DBA mice [10]. Responses rates on the food lever were similar for both genotypes, however KO mice consumed more water during each phase suggesting higher fluid need in the KO genotype. Response rates for dipper presentation when only water was present in phase 2 were similar in both genotypes to rates seen for C57BL and DBA mice [10]. However, it is likely that carry-over effects from the training procedure might have influenced responding since subjects were trained to make lever press responses while water deprived. During phase 3, the
F.O. Risinger et al. / Beha6ioural Brain Research 102 (1999) 211–215
addition of saccharin to the ethanol solution promoted higher levels of responding on the ethanol lever. This finding is consistent with the behavior of C57BL mice and DBA mice in this procedure. However, genotype effects were not seen, and again indicate similar levels of ethanol preference in both KO mice and WT mice. Throughout the procedure KO mice or WT mice did not show substantial occurrence of episodic responding (bouts) of the kind seen in C57BL mice. Based on the modest rates of responding and the low levels of bouts, it is likely that neither KO mice or WT mice experienced appreciable blood ethanol levels during the procedure. Overall, this rather limited influence of genotype is not completely consistent with the finding that KO mice are ethanol preferring [2]. One limitation to this conclusion is the notion that neither strain consumed enough ethanol at any given time to experience significant reinforcing effects from ethanol. Additionally, operant procedures have in some cases yielded different conclusions from those reached using home cage drinking procedures regarding mouse strain/line preferences for ethanol. For example, BALB/cJ mice consume moderate amounts of ethanol in a two-bottle choice procedure [1], but do not show ethanol-reinforced behavior in an operant setting [3]. Another consideration for the present pattern of results is the amount of work needed to gain ethanol in the present procedure. Lower FR requirements are associated with larger ethanol intakes in rats [4]. It may be that the 129/SV-ter background strain is especially sensitivity to work requirement, which obscured detection of ethanol preference.
Acknowledgements This work was supported by a grant from the Alcoholic Beverage Medical Research Foundation and NIAAA grants AA10760, AA10520. Thanks are given to Drs John C Crabbe Jr, Tamara J Phillips and Rene´
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Hen for providing the mice. Portions of these data were presented at the 1997 annual meeting of the Research Society On Alcoholism (San Francisco, CA).
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