You are on page 1of 6

Genes, Brain and Behavior (2011)

doi: 10.1111/j.1601-183X.2011.00692.x

Increased ethanol intake and preference in cyclin D2 knockout mice


P. Jaholkowski , P. Mierzejewski , P. Zatorski , A. Scinska , H. Sienkiewicz-Jarosz , L. Kaczmarek , J. Samochowiec , R. K. Filipkowski,1 and P. Bienkowski,,1
Laboratory of Molecular Neurobiology, Nencki Institute, Warsaw, Department of Pharmacology, Institute of Psychiatry and Neurology, Warsaw, Department of Otolaryngology,

Received 22 July 2010, revised 26 November 2010 and 14 February 2011, accepted for publication 19 March 2011

Faculty of Dentistry, Warsaw Medical University, Warsaw,


Department of Psychiatry, Pomeranian Medical Academy, Szczecin, and ** Department of Biological Psychology,

University of Finance and Management in Warsaw, Warsaw, Poland


1 Equal

senior authors.

*Corresponding author: Dr P. Bienkowski, Department of Pharmacology, Institute of Psychiatry and Neurology, Sobieskiego 9 Street, Warsaw PL-02957, Poland. E-mail: bienkow@ipin.edu.pl

Inhibitory effects of passive ethanol exposure on brain neurogenesis have been extensively documented in animal models. In contrast, a role of brain neurogenesis in ethanol self-administration has not been addressed, as yet. The aim of this study was to assess intake of, and preference for, ethanol solutions [216% (v/v)] in a mouse model of adult neurogenesis deciency based on permanent knockout (KO) of cyclin D2 (Ccnd2 ). Wild type (WT) and Ccnd2 KO mice did not differ in 2% and 4% ethanol intake. The KO group consumed signicantly more ethanol in g/kg when offered with 8% or 16% ethanol as compared with the WT controls. The WT and KO mice did not differ in 2% ethanol preference, but the KO group showed a signicantly higher preference for 416% ethanol. Animal and human studies have suggested that the low level of response to the sedative/hypnotic effects of alcohol is genetically associated with enhanced alcohol consumption. However, in this study, there were no between-genotype differences in ethanol-induced loss of righting reex. Previous reports have also suggested that high ethanol intake is genetically associated with the avidity for sweets and better acceptance of bitter solutions. However, the KO and WT mice consumed similar amounts of saccharin solutions and the KOs consumed less quinine (i.e. bitter) solutions as compared with the WTs. In conclusion, these results may indicate that Ccnd2 and, possibly, brain neurogenesis are involved in central regulation of ethanol intake in mice. Keywords: Ccnd2 , ethanol, mice, neurogenesis, quinine, saccharin, two-bottle choice test
2011 The Authors

Proliferation and differentiation of neuronal precursors (neurogenesis) in the adult brain occur in the subgranular zone of the hippocampal dentate gyrus and subventricular zone of the lateral ventricle. The two regions are responsible for the formation of new neurons in the hippocampus and olfactory bulb, respectively (Cameron et al . 1993; Eriksson et al . 1998; Lois & Alvarez-Buylla 1993). An increasing body of evidence indicates a role for brain neurogenesis in some forms of memory (Canales 2007; Shors et al . 2002; Zhao et al . 2008), stress-induced brain abnormalities and depressive-like behaviors in animal subjects (Jacobs et al . 2000; Sahay & Hen 2007). Inhibitory effects of passive ethanol administration on brain neurogenesis have been extensively documented in animal models (Crews et al . 2006; Nixon 2006) and it has been suggested that the level of brain neurogenesis can modulate ethanol intake and preference (see Canales 2007, for theoretical discussion). However, a possible role of brain neurogenesis in alcohol drinking behavior has not been addressed in experimental studies, as yet. For example, it is not known whether inhibition or elimination of adult brain neurogenesis could alter ethanol consumption. Recently, we have described a mouse model of adult neurogenesis deciency based on permanent knockout (KO) of cyclin D2 (Ccnd2 ) (Jaholkowski et al . 2009; Kowalczyk et al . 2004). In most mammalian cells, there is an expression of more than one cyclin D subtype. In those instances where only one cyclin D is expressed, its mutation produces signicant abnormalities in cell proliferation (Huard et al . 1999; Sherr 1995; Sicinski et al . 1996; Solvason et al . 2000). Permanent KO of the Ccnd2 gene results in virtually complete absence of proliferation of neuronal precursors in the adult mouse brain. The above decit is specic for brain neurons as non-neuronal precursors in the brain and neurogenesis in olfactory epithelium remain unaffected (Jaholkowski et al . 2009; Kowalczyk et al . 2004). A large number of genetic loci that inuence ethanol drinking have been mapped by using different genetic approaches (Bice et al . 2009; Mulligan et al . 2006). Although the Ccnd2 gene was not considered as one of the hot spots, the gene was signicant in a microarray meta-analysis aimed at identifying candidate genes for alcohol preference in mice (Mulligan et al . 2006). Given the inhibitory effects of ethanol on brain neurogenesis (Canales 2007; Crews et al . 2006) and the data linking the Ccnd2 gene with ethanol preference (Mulligan et al . 2006),

Genes, Brain and Behavior 2011 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society

Jaholkowski et al.

one could hypothesize that the permanent absence of brain neurogenesis in Ccnd2 KO mice can alter their preference for ethanol. In an attempt to test this hypothesis, we tested a free-choice ethanol (216%) drinking in wild type (WT) and Ccnd2 KO mice. In control experiments, possible mechanisms of between-genotype differences in ethanol drinking were addressed. The control experiments included, among others, testing of taste preferences and ethanol-induced loss of righting reex. Gustatory responses to sweet and bitter stimuli as well as sedative/hypnotic effects of ethanol are thought to modulate ethanol drinking in animal and human subjects (Duffy et al . 2004; Murphy et al . 2002; Schuckit 1994; Scinska et al . 2000; Youngentob & Glendinning 2009).

Table 1: Summary of two-bottle tests Fluids Baseline water 2% ethanol vs. water 4% ethanol vs. water 8% ethanol vs. water 16% ethanol vs. water Washout/water 0.0125 g/l saccharin vs. water 0.05 g/l saccharin vs. water 0.2 g/l saccharin vs. water 1.0 g/l saccharin vs. water Washout/water 18.75 M quinine vs. water 75 M quinine vs. water 300 M quinine vs. water 1200 M quinine vs. water
Tap In

Days drinking 8 4 4 4 4 8 2 2 2 2 8 2 2 2 2

Materials and methods


Subjects
The KO mice were obtained in the 129X1/SvJ background and crossed once with the C57BL/6J strain, then kept and bred continuously with each other as heterozygotes for >15 generations (Sicinski et al . 1996). In our laboratory, these mice were crossed once with BALB/cAnNCrl mice, then kept and bred continuously with each other as heterozygotes for >12 generations. Their homozygous progeny [/ (Ccnd2 KO) and +/+ (WT)], which was always littermates derived from several breeding pairs, was used in this study. Genotypes of all animals were veried after the completion of behavioral experiments (Jaholkowski et al . 2009; Kowalczyk et al . 2004). The KO and WT mice (34 months old) were kept in standard laboratory conditions (at 22 1 C, 60% relative humidity and a 12 h light/12 h dark cycle with lights on at 0700 h) with free access to lab chow (Labofeed H, WPiK, Kcynia, Poland) and tap water. All animals were singly housed in perforated stainless steel cages (15 15 15 cm, W L H, respectively) with two graduated drinking tubes mounted at both sides of each cage. The treatment of animals was in full accordance with the ethical standards laid down in respective European (directive no. 86/609/EEC) and Polish regulations. All experimental procedures were approved by a local ethics committee.

water was available in both drinking tubes. all the two-bottle tests, the drinking tubes were rotated daily to prevent position preference.

Groups of ethanol-naive KO and WT mice (n = 812 mice per group) were injected intraperitoneal (i.p.) with 3 or 4 g/kg ethanol [20% (v/v)]. After the ethanol injection, each animal was repeatedly placed on its back once every 30 second, up to a maximum of 600 second, until it was unable to right itself within 30 second (latency to loss of righting reex). Then, the mouse was left undisturbed until it regained the righting reex three times within a 30-second period (Christensen et al . 1996; Colombo et al . 1998).

Ethanol-induced loss of righting reex

Open-eld test
The open-eld test was used to assess possible between-genotype differences in spontaneous locomotor activity. The open-eld test took place in a dimly lit experimental room with background white noise. An open-eld box (40 40 40 cm, W L H, respectively) had clear Plexiglass walls and a smooth stainless steel oor. The oor was divided into 16 identical squares. Four central squares were dened as a central sector (Swiergiel & Dunn 2007). A perspex coverage of the box allowed observation of mice with the aid of a video camera. Horizontal (crossings) and vertical (rearings) locomotor activity, central entries and time spent in the central sector were scored for 5 min (300 second) by a blinded observer. It is assumed that exploration of the central area may reect, at least partially, the level of neophobia (Swiergiel & Dunn 2007). Groups of ethanol-naive KO and WT mice (n = 6 mice per group) were used in the open-eld experiment. Each mouse was gently placed in the very center of the box. A crossing was scored when a mouse removed all four paws from one square and entered another. A rearing was scored when a mouse raised both front paws. A central entry was counted when a mouse entered one of the central squares from the peripheral one. Time spent in the central sector was dened as a cumulative time spent by an animal in the four central squares.

Two-bottle choice tests with ethanol, saccharin and quinine


Table 1 shows a summary of two-bottle choice tests. Basic aspects of the procedure were identical to those described in our previous report on Fosb KO mice (Korkosz et al . 2004). Ethanol drinking tests began after the assessment of baseline water drinking. Groups of ethanol-naive KO and WT mice (n = 9 mice per group) were exposed to increasing concentrations of ethanol [216% (v/v)] vs. tap water for 16 consecutive days. After completion of the ethanol tests and an 8-day washout period, saccharin drinking tests were initiated in the same animals. (Because of a limited number of Ccnd2 KO mice, the same subjects were used in the tests with ethanol, saccharin and quinine solutions.) The mice were given the choice between increasing concentrations of saccharin (0.01251.0 g/l) vs. tap water. At the end of this study, the subjects were exposed to increasing concentrations of quinine (18.751200 M; 1200 M = 0.038%) vs. tap water (Table 1). Fluid intake was measured daily at 1500 h and body weight was recorded twice a week. For each uid, an intake measure was obtained, averaged across a respective period of availability, and corrected for the body weight of a subject (g/kg for ethanol and ml/kg for other substances). Preference of each substance was calculated according to the following formula: [substance intake/(substance intake + water intake)] 100%. The drinking tubes were rotated daily to prevent position preference. All solutions were replaced completely every day.

Blood ethanol levels


In a preliminary experiment, we addressed possible betweengenotype differences in ethanol pharmacokinetics. Ethanol-naive WT and KO mice were injected i.p. with 1 g/kg ethanol. Subgroups of mice (n = 4) were killed by decapitation 5 or 15 min after the injection and trunk blood was collected in Eppendorf tubes. The samples were centrifuged for 5 min and serum (0.1 ml) was pipetted into 0.2-ml containers. Blood ethanol levels were determined with the aid of commercially available REA enzymatic assays and Abbott TDx autoanalyzer (Abbott Laboratories, Abbott Park, IL, USA) (Korkosz
Genes, Brain and Behavior (2011)

Cyclin D2 and ethanol intake


et al . 2005). Because of a limited number of Ccnd2 KO mice, blood ethanol levels in other time-points were not measured.

Drugs
The following substances were used: a stock ethanol solution [96% (v/v); Polmos, Zielona Gora, Poland], quinine hydrochloride (Sigma, Poznan, Poland) and saccharin sodium salt dihydrate (Aldrich Chemical, Gilingham, UK). Tap water was used to dilute the stock ethanol solution and to dissolve quinine and saccharin. The concentrations of saccharin and quinine refer to the free substance. All solutions were prepared daily and stored at room temperature.

A two-way analysis of variance (ANOVA; genotype time) was used to analyze changes in body weights in the course of the two-bottle tests. The two-way ANOVA (genotype concentration) with repeated measure on concentration was used to analyze ethanol, saccharin and quinine intake and preference. The two-way ANOVA was also used to compare total uid intakes (TFIs). The NewmanKeuls test was employed for individual post hoc comparisons of ethanol, saccharin and quinine intakes and preferences in the KO and WT mice. The NewmanKeuls test was selected as it is less conservative than the Tukeys test and more conservative than the LSD test. The Students t -test was used to analyze between-genotype differences in ethanolinduced loss of righting reex, open-eld behavior and blood ethanol levels. The Statistica 5.0 software package (StatSoft, Inc., Tulsa, OK, USA) was used in all analyses. P values less than 0.05 were considered signicant.

Statistics

in g/kg and showed higher preference for the more concentrated ethanol solutions. The two-way ANOVA (genotype concentration) showed no effect of genotype on ethanol intake (F1,16 = 3.52, P > 0.05). An effect of concentration was highly signicant (F3,48 = 40.30, P < 0.001). There was also a signicant genotype concentration interaction (F3,48 = 4.10, P < 0.05) suggesting that ethanol concentration determined differences between the two groups. The NewmanKeuls test indicated that the WTs and KOs did not differ in 2% and 4% ethanol intake. However, the KOs consumed signicantly more ethanol when offered with 8% and 16% ethanol as compared with the WT group (Table 2). The ANOVA showed no effect of genotype (F1,16 = 3.75, P > 0.05) and no effect of concentration (F3,48 = 2.58, P > 0.05) on ethanol preference. However, a genotype concentration interaction was signicant (F3,48 = 3.90, P < 0.05). The NewmanKeuls test indicated that the WTs and KOs did not differ in 2% ethanol preference. However, the KOs showed signicantly higher preference for 4%, 8% and 16% ethanol as compared with the WT group (Table 2). The WTs and KOs did not differ in body weights and baseline water drinking (all P values > 0.05). Similarly, TFIs (ethanol + water) did not differ between the WT and KO animals (P values > 0.05; Table 2).

Saccharin intake and preference

Results
Ethanol intake and preference
Table 2 shows results of the two-bottle choice tests. In general, the KO mice consumed signicantly more ethanol

There was no difference between the two genotypes in saccharin intake (an effect of genotype: F1,16 = 0.40, P > 0.05). A genotype concentration interaction was not signicant (F3,48 = 1.02, P > 0.05). A highly signicant effect of concentration (F3,48 = 34.58, P < 0.001) reected a

Table 2: Consumption of water, ethanol, saccharin and quinine in WT and Ccnd2 KO mice WT mice Fluid Water Ethanol 2% 4% 8% 16% Saccharin 0.0125 g/l 0.05 g/l 0.2 g/l 1.0 g/l Quinine 18.75 M 75 M 300 M 1200 M Intake 188.0 10.5 ml/kg 1.1 0.3 g/kg 2.1 0.7 g/kg 4.3 1.7 g/kg 6.9 2.0 g/kg 119.0 17.7 ml/kg 124.8 18.8 ml/kg 163.8 18.5 ml/kg 236.3 25.9 ml/kg 96.0 12.9 ml/kg 101.1 17.9 ml/kg 115.4 13.7 ml/kg 23.6 4.2 ml/kg Preference (%) 38 8 33 10 34 12 30 9 64 7 68 7 79 7 93 2 52 5 55 8 66 6 13 2 TFI (ml/kg) 181.9 11.4 186.8 8.5 181.7 12.3 181.7 6.6 183.3 12.4 178.7 9.6 204.7 7.7 251.8 24.8 185.5 9.4 178.4 11.5 173.8 10.3 178.2 9.3 Intake 176.0 10.4 ml/kg 1.4 0.3 g/kg 3.1 0.6 g/kg 7.9 1.1 g/kg 12.4 1.5 g/kg 84.6 14.4 ml/kg 100.5 16.9 ml/kg 176.7 20.2 ml/kg 225.5 22.2 ml/kg 69.9 9.1 ml/kg 30.0 8.0 ml/kg 16.0 3.5 ml/kg 10.8 1.6 ml/kg

Ccnd2 KO mice
Preference (%) 48 11 52 11 73 10 60 9 50 9 61 2 80 5 93 2 39 6 16 5 9 2 51 TFI (ml/kg) 186.4 6.0 192.9 6.6 172.4 6.9 171.7 8.6 174.0 8.2 168.2 8.1 218.2 16.6 241.7 21.8 186.0 10.3 185.4 10.9 173.9 13.6 202.5 20.9

n = 9 mice per group. Preference = [substance intake/(substance intake + water intake)] 100%. Total water consumption from the two drinking tubes; all values expressed as means SE. P < 0.05; P < 0.01 vs. the WT group.
Genes, Brain and Behavior (2011)

Jaholkowski et al.

general tendency toward higher intake of more concentrated saccharin solutions in both WTs and KOs (Table 2). Similarly, there was no difference between the two genotypes in saccharin preference (an effect of genotype: F1,16 = 0.54, P > 0.05). A genotype concentration interaction was not signicant (F3,48 = 1.12, P > 0.05). A highly signicant effect of concentration (F3,48 = 23.05, P < 0.001) reected a general tendency toward higher preference for more concentrated sweet solutions (Table 2). Total uid intakes did not differ between the WT and KO animals (P values > 0.05).

Table 4: Locomotor activity and central parameters in WT and Ccnd2 KO mice tested in the open-eld test WT mice Crossings Rearings Central entries Time spent in the central sector (seconds) 21.0 2.0 19.5 3.7 2.5 0.9 4.0 1.0

Ccnd2 KO mice
30.2 3.7 23.0 3.3 3.8 2.1 6.5 3.0

All values expressed as means SE. n = 6 mice per group.

Quinine intake and preference


There was a highly signicant difference between the two genotypes in quinine consumption (an effect of genotype: F1,16 = 29.94, P < 0.001). A genotype concentration interaction was also signicant (F3,48 = 10.44, P < 0.001). A highly signicant effect of concentration (F3,48 = 20.79, P < 0.001) reected a general tendency toward lower consumption of more concentrated bitter solutions in the WTs and KOs. The NewmanKeuls test indicated that the KO mice consumed signicantly less quinine (18.75300 M) as compared with the WT group (see Table 2 for details). There was a highly signicant difference between the two genotypes in quinine preference (an effect of genotype: F1,16 = 58.38, P < 0.001). A genotype concentration interaction was also signicant (F3,48 = 12.62, P < 0.001). A highly signicant effect of concentration (F3,48 = 23.53, P < 0.001) reected a general tendency toward avoidance of more concentrated quinine solutions in the WTs and KOs. The KO mice showed a signicantly lower preference for 75 and 300 M quinine than the WT controls (the NewmanKeuls test; see Table 2 for details). Total uid intakes did not differ between the WT and KO animals (P values > 0.05).

entries and time spent in the central sector; P values > 0.05; Table 4).

Blood ethanol levels


Blood ethanol levels measured 5 and 15 min after the administration of 1 g/kg ethanol did not differ between the KO (5 min, 113.5 10.4 mg% and 15 min, 95.5 3.7 mg%) and WT subjects (5 min, 102.0 9.3 mg% and 15 min, 88.5 8.0 mg%; P values > 0.05).

Discussion
Cyclin D2 gene was signicant in a microarray meta-analysis aimed at identifying candidate genes for alcohol preference in mice (Mulligan et al . 2006). To the best of our knowledge, this is the rst report linking Ccnd2 KO with free-choice ethanol drinking in mice. In this study, the Ccnd2 KO group consumed signicantly more ethanol than their WT counterparts. Notably, the between-genotype difference was most signicant for the two highest ethanol concentrations (8% and 16%). In line with the above, the KO mice preferred the highest ethanol concentrations over water, whereas the reverse was true for the WTs. In contrast, the KO mice consumed less quinine (18.75300 M) than the WT controls. The KOs avoided 75 and 300 M quinine (i.e. preferred water over quinine), whereas no avoidance of 75300 M quinine was observed in the WTs. In the control experiments, we further addressed possible mechanisms of the between-genotype difference in ethanol intake and preference. It has been postulated that oral ethanol self-administration is determined, at least partially, by bitter and sweet components of ethanol taste (Blizard 2007; Duffy et al . 2004; Koros et al . 1999; Scinska et al . 2000). Some reports have indicated a positive correlation between consumption of sweet solutions and ethanol preference in rodents (see Kampov-Polevoy et al . 2001 for review). However, saccharin intake and preference did not differ between the KO and WT group. Thus the betweengenotype difference in ethanol intake and preference may not be explained by different processing of sweet stimuli. In line with previous ndings (Agabio et al . 2000; BoguckaBonikowska et al . 2001; Korkosz et al . 2004; Koros et al . 1999; Kranzler et al . 2001), these results suggest that some basic mechanisms of ethanol and sweets preference can be dissociated in rodents.
Genes, Brain and Behavior (2011)

Ethanol-induced loss of righting reex


The onset and duration of ethanol-induced loss of righting reex did not differ between the KOs and WTs (P values > 0.05; Table 3).

Open-eld test
The WTs and KOs did not differ in horizontal (crossings) or vertical (rearings) locomotor activity. Similarly, the two genotypes did not differ in the central parameters (i.e. central
Table 3: Ethanol-induced sleep in WT and Ccnd2 KO mice WT mice 3 g/kg ethanol, i.p. Sleep latency (seconds) Sleep duration (seconds) 4 g/kg ethanol, i.p. Sleep latency (seconds) Sleep duration (seconds) 388.2 52.1 703.3 169.6 285.0 63.5 840.0 316.6

Ccnd2 KO mice
333.4 84.7 490.0 176.2 307.5 85.9 858.8 320.8

All values expressed as means SE. n = 812 mice per group.

Cyclin D2 and ethanol intake

More concentrated ethanol solutions taste bitter (Blizard 2007; Scinska et al . 2000) and reduced sensitivity to bitter tastants may promote ethanol intake (Duffy et al . 2004; Youngentob & Glendinning 2009). Given the above, we predicted that increased ethanol preference in the KO group could be associated with increased consumption (i.e. lower aversiveness) of quinine solutions. Contrary to our hypothesis, the KOs consumed less bitter solutions than their WT counterparts. Considering these ndings, one should remember that our data do not exclude the possibility that Ccnd2 KO is associated with a specic increase in sweetness and/or reduction in bitterness of ethanol solutions, which could not be detected in the two-bottle tests with saccharin or quinine. Animal and human studies have indicated a negative correlation between initial sensitivity to ethanol and levels of subsequent ethanol intake. Selectively bred alcohol preferring rats are less sensitive to the ataxic and sedative/hypnotic effects of ethanol than their alcohol non-preferring counterparts (Murphy et al . 2002). Sons of alcoholics show less alcohol-induced changes in subjective feelings, physiological measures and motor performance (Schuckit 1994). In general, the low level of response to alcohol is thought to be a genetically inuenced characteristic associated with an enhanced risk for future alcohol use disorders (Mayeld et al . 2008). In this study, there were no between-genotype differences in ethanol-induced loss of righting reex. Thus it seems that an increased propensity to consume ethanol solutions in Ccnd2 KO mice is not related to alterations in the sedative/hypnotic effects of ethanol. The open-eld experiment did not indicate any major difference between the KO and WT mice in spontaneous locomotor activity and the level of neophobia. It has been suggested that the anxiolytic-like effects of ethanol may contribute to the initiation of ethanol consumption and lead to self-medication of anxiety states in vulnerable individuals (Blanchard et al . 1993; Cosci et al . 2007; Kushner et al . 1990). Although it appears that the two genotypes do not differ in an open-eld neophobia, Ccnd2 KO mice may be more anxious in other models and/or be more sensitive to the anxiolytic-like effects of ethanol. Our data may indicate that increased ethanol intake in Ccnd2 KO mice is secondary to their altered sensitivity to the central rewarding effects of ethanol (although alternative explanations for these results cannot be ruled out). Functional links between brain dopamine reward systems and neurogenesis (see Borta & Hoglinger 2006 for review) suggest a good starting point for further studies addressing interactions among Ccnd2 , neurogenesis and ethanol reward in mice. Our study has some limitations. Female mice were not used and possible gender effects on ethanol intake were not addressed. Although blood ethanol levels did not differ between the two genotypes, ethanol levels were assessed only 5 and 15 min after the peripheral administration of 1 g/kg ethanol. Hence, between-genotype differences in metabolism and elimination of ethanol cannot be excluded. Another limitation is that the within-subject design was used for studying ethanol, saccharin and quinine intake. One may wish to verify the differences observed in this study in a
Genes, Brain and Behavior (2011)

between-subject experiment with separate groups of mice drinking different concentrations of ethanol, saccharin and quinine. Substantial individual differences were observed in ethanol-induced loss of righting reex. Although relatively large groups of animals were used (n = 812), it is possible that even larger groups are necessary to detect betweengenotype differences in loss of righting reex. One should remember that Ccnd2 KO may be associated with phenotypic abnormalities other than adult brain neurogenesis deciency (Sicinski et al . 1996). It is also possible that Ccnd2 KO changes expression of other genes, including those related to ethanol drinking. Last but not least, it can be expected that expression of many genes may affect the strength of any association between Ccnd2 and alcohol-related phenotypes.

References
Agabio, R., Carai, M.A., Lobina, C., Pani, M., Reali, R., Bourov, I., Gessa, G.L. & Colombo, G. (2000) Dissociation of ethanol and saccharin preference in sP and sNP rats. Alcohol Clin Exp Res 24, 2429. Bice, P., Valdar, W., Zhang, L., Liu, L., Lai, D., Grahame, N., Flint, J., Li, T.K., Lumeng, L. & Foroud, T. (2009) Genomewide SNP screen to detect quantitative trait loci for alcohol preference in the high alcohol preferring and low alcohol preferring mice. Alcohol Clin Exp Res 33, 531537. Blanchard, R.J., Magee, L., Veniegas, R. & Blanchard, D.C. (1993) Alcohol and anxiety: ethopharmacological approaches. Prog Neuropsychopharmacol Biol Psychiatry 17, 171182. Blizard, D.A. (2007) Sweet and bitter taste of ethanol in C57BL/6J and DBA2/J mouse strains. Behav Genet 37, 146159. Bogucka-Bonikowska, A., Scinska, A., Koros, E., Polanowska, E., Habrat, B., Woronowicz, B., Kukwa, A., Kostowski, W. & Bienkowski, P. (2001) Taste responses in alcohol-dependent men. Alcohol Alcohol 36, 516519. Borta, A. & Hoglinger, G.U. (2006) Dopamine and adult neurogenesis. J Neurochem 100, 587595. Cameron, H.A., Woolley, C.S., McEwen, B.S. & Gould, E. (1993) Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat. Neuroscience 56, 337344. Canales, J.J. (2007) Adult neurogenesis and the memories of drug addiction. Eur Arch Psychiatry Clin Neurosci 257, 261270. Christensen, S.C., Johnson, T.E., Markel, P.D., Clark, V.J., Fulker, D.W., Corley, R.P., Collins, A.C. & Wehner, J.M. (1996) Quantitative trait locus analyses of sleep-times induced by sedative-hypnotics in LSXSS recombinant inbred strains of mice. Alcohol Clin Exp Res 20, 543550. Colombo, G., Agabio, R., Lobina, C., Loche, A., Reali, R. & Gessa, G.L. (1998) High sensitivity to gamma-hydroxybutyric acid in ethanol-preferring SP rats. Alcohol Alcohol 33, 121125. Cosci, F., Schruers, K.R., Abrams, K. & Griez, E.J. (2007) Alcohol use disorders and panic disorder: a review of the evidence of a direct relationship. J Clin Psychiatry 68, 874880. Crews, F.T., Mdzinarishvili, A., Kim, D., He, J. & Nixon, K. (2006) Neurogenesis in adolescent brain is potently inhibited by ethanol. Neuroscience 137, 437445. Duffy, V.B., Davidson, A.C., Kidd, J.R., Kidd, K.K., Speed, W.C., Pakstis, A.J., Reed, D.R., Snyder, D.J. & Bartoshuk, L.M. (2004) Bitter receptor gene (TAS2R38), 6-n-propylthiouracil (PROP) bitterness and alcohol intake. Alcohol Clin Exp Res 28, 16291637. Eriksson, P.S., Perlieva, E., Bjork-Eriksson, T., Alborn, A.M., Nordborg, C., Peterson, D.A. & Gage, F.H. (1998) Neurogenesis in the adult human hippocampus. Nat Med 4, 13131317.

Jaholkowski et al.
Huard, J.M., Forster, C.C., Carter, M.L., Sicinski, P. & Ross, M.E. (1999) Cerebellar histogenesis is disturbed in mice lacking cyclin D2. Development 126, 19271935. Jacobs, B.L., Praag, H. & Gage F.H. (2000) Adult brain neurogenesis and psychiatry: a novel theory of depression. Mol Psychiatry 5, 262269. Jaholkowski, P., Kiryk, A., Jedynak, P., Ben Abdallah, N.M., Knapska, E., Kowalczyk, A., Piechal, A., Blecharz-Klin, K., Figiel, I., Lioudyno, V., Widy-Tyszkiewicz, E., Wilczynski, G.M., Lipp, H.P., Kaczmarek, L. & Filipkowski, R.K. (2009) New hippocampal neurons are not obligatory for memory formation; cyclin D2 knockout mice with no adult brain neurogenesis show learning. Learn Mem 16, 439451. Kampov-Polevoy, A.B., Tsoi, M.V., Zvartau, E.E., Neznanov, N.G. & Khalitov, E. (2001) Sweet liking and family history of alcoholism in hospitalized alcoholic and non-alcoholic patients. Alcohol Alcohol 36, 165170. Korkosz, A., Kolomanska, P., Kowalska, K., Rogowski, A., Radwanska, K., Kaczmarek, L., Mierzejewski, P., Scinska, A., Kostowski, W. & Bienkowski, P. (2004) Dissociation of ethanol and saccharin preference in fosB knockout mice. Physiol Behav 82, 391395. Korkosz, A., Taracha, E., Plaznik, A., Wrobel, E., Kostowski, W. & Bienkowski, P. (2005) Extended blockade of the discriminative stimulus effects of nicotine with low doses of ethanol. Eur J Pharmacol 512, 165172. Koros, E., Piasecki, J., Kostowski, W. & Bienkowski, P. (1999) Development of alcohol deprivation effect in rats: lack of correlation with saccharin drinking and locomotor activity. Alcohol Alcohol 34, 542550. Kowalczyk, A., Filipkowski, R.K., Rylski, M., Wilczynski, G.M., Konopacki, F.A., Jaworski, J., Ciemerych, M.A., Sicinski, P. & Kaczmarek, L. (2004) The critical role of cyclin D2 in adult neurogenesis. J Cell Biol 167, 209213. Kranzler, H.R., Sandstrom, K.A. & Van Kirk, J. (2001) Sweet taste preference as a risk factor for alcohol dependence. Am J Psychiatry 158, 813815. Kushner, M.G., Sher, K.J. & Beitman, B.D. (1990) The relation between alcohol problems and the anxiety disorders. Am J Psychiatry 147, 685695. Lois, C. & Alvarez-Buylla, A. (1993) Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc Natl Acad Sci U S A 90, 20742077. Mayeld, R.D., Harris, R.A. & Schuckit, M.A. (2008) Genetic factors inuencing alcohol dependence. Br J Pharmacol 154, 275287. Mulligan, M.K., Ponomarev, I., Hitzemann, R.J., Belknap, J.K., Tabakoff, B., Harris, R.A., Crabbe, J.C., Blednov, Y.A., Grahame, N.J., Phillips, T.J., Finn, D.A., Hoffman, P.L., Iyer, V.R., Koob, G.F. & Bergeson, S.E. (2006) Toward understanding the genetics of alcohol drinking through transcriptome meta-analysis. Proc Natl Acad Sci U S A 103, 63686373. Murphy, J.M., Stewart, R.B., Bell, R.L., Badia-Elder, N.E., Carr, L.G., McBride, W.J., Lumeng, L. & Li, T.K. (2002) Phenotypic and genotypic characterization of the Indiana University rat lines selectively bred for high and low alcohol preference. Behav Genet 32, 363388. Nixon, K. (2006) Alcohol and adult neurogenesis: roles in neurodegeneration and recovery in chronic alcoholism. Hippocampus 16, 287295. Sahay, A. & Hen, R. (2007) Adult hippocampal neurogenesis in depression. Nat Neurosci 10, 11101115. Schuckit, M.A. (1994) Alcohol sensitivity and dependence. EXS 71, 341348. Scinska, A., Koros, E., Habrat, B., Kukwa, A., Kostowski, W. & Bienkowski, P. (2000) Bitter and sweet components of ethanol taste in humans. Drug Alcohol Depend 60, 199206. Sherr, C.J. (1995) D-type cyclins. Trends Biochem Sci 20, 187190. Shors, T.J., Townsend, D.A., Zhao, M., Kozorovitskiy, Y. & Gould, E. (2002) Neurogenesis may relate to some but not all types of hippocampal-dependent learning. Hippocampus 12, 578584. Sicinski, P., Donaher, J.L., Geng, Y., Parker, S.B., Gardner, H., Park, M.Y., Robker, R.L., Richards, J.S., McGinnis, L.K., Biggers, J.D., Eppig, J.J., Bronson, R.T., Elledge, S.J. & Weinberg, R.A. (1996) Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis. Nature 384, 470474. Solvason, N., Wu, W.W., Parry, D., Mahony, D., Lam, E.W., Glassford, J., Klaus, G.G., Sicinski, P., Weinberg, R., Liu, Y.J., Howard, M. & Lees, E. (2000) Cyclin D2 is essential for BCRmediated proliferation and CD5 B cell development. Int Immunol 12, 631638. Swiergiel, A.H. & Dunn, A.J. (2007) Effects of interleukin-1beta and lipopolysaccharide on behavior of mice in the elevated plus-maze and open eld tests. Pharmacol Biochem Behav 86, 651659. Youngentob, S.L. & Glendinning, J.I. (2009) Fetal ethanol exposure increases ethanol intake by making it smell and taste better. Proc Natl Acad Sci U S A 106, 53595364. Zhao, C., Deng, W. & Gage, F.H. (2008) Mechanisms and functional implications of adult neurogenesis. Cell 132, 645660.

Acknowledgments
The study was supported by the Institute of Psychiatry and Neurology and by the Ministry of Science and Graduate Education, Warsaw, Poland (Scientic Network No. 26/E-40/SN0023/2007 and Grant No. N30305131/1624).

Genes, Brain and Behavior (2011)