Dopamine D4 Receptor-Knock-Out Mice Exhibit Reduced Exploration of Novel Stimuli

RESULTS

Open field test

Mice were tested on 3 separate days in the open field, a forced exploration paradigm (Welker, 1957; Adams and Geyer, 1985; Belzung, 1992) in which escape from novelty is impossible. D4R+/+ and D4R−/− mice showed no differences in locomotor activity over the 3 d of testing (Fig. 2). Both genotypes exhibited locomotor habituation over time, showing reduced levels of activity on the second (F_(1,76) = 6.52; p < 0.01) and third (F_(1,76) = 6.04; p < 0.02) days relative to the first day (Table1). D4R−/− mice exhibited reduced behavioral responses to novelty relative to D4R+/+ mice, as indicated by fewer entries into the center (F_(1,111) = 4.4; p < 0.04), and a trend to spend less time in the center (F_(1,111) = 3.14; p = 0.08). The novelty of the center of the open field diminishes over time (Fowler, 1965), but its anxiogenic properties do not (Adams and Geyer, 1985). Therefore, entries into the center primarily reflect exploratory behavior on the first day of open field testing, but primarily reflect anxiety-related behavior after repeated testing. We assessed the effect size of the phenotypic difference in center entries for each day. The largest effect size was observed on the novel day of testing, and decreased each subsequent day: day one (ES = 0.7), day two (ES = 0.4), and day three (ES = 0.1). The phenotypic difference in center entries on day one primarily reflects a reduction in approach or exploratory behavior in D4R−/− mice (ES = 0.7), but on day three primarily reflects a reduction in anxiety in D4R−/− mice (ES = 0.1).

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Fig. 2.

Motor activity. Total locomotor activity in the emergence, novel object, and open field tests is shown for D4R+/+ and D4R−/− mice. Values are means ± SEM. *p < 0.05 versus D4R+/+ group with ANOVA.

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Table 1.

Open field measures

We applied an ANCOVA to center entries, with locomotor activity as a covariate, to assess whether phenotypic differences in behavioral responses to novelty and locomotor activity were independent. In the present experiment, ANCOVA revealed that the decreases in center entries exhibited by D4R−/− mice remained significant, even after accounting for the variance associated with the levels of locomotor activity. Thus, D4R−/− mice were less behaviorally responsive to novelty than D4R+/+ mice in 3 d of open field testing.

Emergence test

Mice were assessed in the emergence test, a free exploration paradigm (Welker, 1957; Adams and Geyer, 1985; Belzung and Le Pape, 1994) in which animals can explore the open field or retreat into a cylinder. D4R−/− mice were less behaviorally responsive to the novel environment than D4R+/+ mice. Relative to D4R+/+ mice, D4R−/− mice had significantly longer latencies to emerge from the cylinder (F_(1,132) = 4.62; p < 0.03), spent significantly more total time inside the cylinder (F_(1,132) = 29.81; p< 0.005), made fewer entries into the cylinder (F_(1,132) = 12.35; p< 0.002), and had significantly larger MTIC values (F_(1,132) = 6.12; p < 0.02) (Fig. 3). Additionally, a significant genotype × sex interaction with respect to entries into the cylinder was indicated. Post hoc tests showed that D4R+/+ female mice made more transitions than D4R+/+ male mice (F_(1,60) = 21.57; p < 0.0001) and D4R−/− female mice (F_(1,56) = 15.43; p < 0.0003), indicating that the reduction in entries into the cylinder exhibited by D4R−/− mice was caused by female mice (Fig.4). D4R−/− mice were also less active overall than D4R+/+ mice (F_(1,132) = 8.61; p < 0.01) (Fig. 2). Female mice were less active overall (F_(1,132) = 5.04;p < 0.03) (510.0 ± 27.4 vs 583.8 ± 23.5) and spent more time in the cylinder (F_(1,132) = 9.16; p < 0.004) than male mice, but these effects did not interact with genotype.

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Fig. 3.

Emergence test. Exploratory measures of the emergence test are shown for D4R+/+ (n = 61) and D4R−/− mice (n = 75). The latency to emerge from the cylinder in seconds (a), the total time spent in the cylinder in seconds (b), the total number of entries into the cylinder (c), and the MTIC in seconds (d) are shown for both genotypes. Values are means ± SEM. *p < 0.05 versus D4R+/+ group with ANOVA; #p < 0.05 versus D4R+/+ group with ANCOVA.

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Fig. 4.

Cylinder entries: sex differences. The number of entries into the cylinder during the emergence test is shown for male D4R+/+ (n = 31), male D4R−/− (n = 43), female D4R+/+ (n = 28), and female D4R−/− mice (n = 29). Values are means ± SEM. *p < 0.05 versus female D4R+/+ group with ANOVA; +p < 0.05 versus male D4R+/+ group with ANOVA.

Appropriate ANCOVAs confirmed that the phenotypic differences for total time spent in (ES = 0.8) and number of entries into the cylinder (ES = 0.5) remained significant, independent of the level of locomotor activity. In contrast, the phenotypic differences for latency to emerge and for MTIC values were no longer significant, indicating that the differences in these measures were directly related to motor activity levels. Additionally, the main effect of sex on time spent in the cylinder was also dependent on the differences in locomotor activity. Thus, D4R−/− mice exhibited reduced behavioral responses to novelty compared to D4R+/+ mice, as indicated by the measures of time spent in the cylinder and number of entries into the cylinder.

Novel object test

Mice were tested in the novel object test, a free exploration paradigm (Belzung, 1992; Renner et al., 1992; Belzung and Le Pape, 1994) that provides animals the opportunity to explore a novel object in a non-threatening and familiar environment. Both genotypes spent a higher percentage of time and made a higher percentage of entries into the center after introduction of the cup, confirming that the novel cup elicited approach behavior. Both genotypes made comparable increases in the percentage of center entries after introduction of the cup. Nevertheless, D4R−/− mice were less behaviorally responsive to the novel cup than D4R+/+ mice (Fig. 5), because D4R−/− mice made a smaller increase in the percentage of time spent in the center after introduction of the cup than D4R+/+ mice (F_(1,127) = 19.24; p< 0.0001). Post hoc tests indicated that, although both genotypes spent significantly more time in the center after introduction of the cup, D4R−/− mice spent significantly less percent time in the center than D4R+/+ mice during this condition (F_(1,71) = 58.47; p < 0.0001). No interactions of sex and genotype were evident for these measures. Analyses using raw scores yielded the same conclusions as analyses using percentage scores for all measures. D4R−/− mice were also less active overall than D4R+/+ mice (F_(1,127) = 12.18; p< 0.0001) (Fig. 2). A sex difference in which female mice engaged in more locomotor activity than male mice was also observed (F_(1,127) = 6.19; p < 0.01) (1079.7 ± 53.6 vs 906.3 ± 42.0), but no interaction of sex and genotype was found.

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Fig. 5.

Novel object test. The percentage of time spent in the center region (a) and the percentage of center entries (b) are shown for D4R+/+ (n = 59) and D4R−/− mice (n = 72) for both environmental conditions of the novel object test. Values are means ± SEM. *p < 0.05 genotype × condition interaction with ANOVA; #p < 0.05 genotype × condition with ANCOVA.

Appropriate ANCOVAs testing for any relationship between locomotor activity and behavioral responses to novelty revealed that the increase in percentage of time spent in the center, after introduction of the cup, remained significantly decreased in D4R−/− mice (ES = 1.4). Thus, D4R−/− mice exhibited independent reductions in locomotor activity and behavioral responses to novelty relative to D4R+/+ mice in the novel object test.

DISCUSSION

Here we report that mice lacking the D4R exhibit reduced behavioral responses to novelty. In the open field test, D4R−/− mice made fewer entries into the center, but did not differ in overall levels of activity compared to D4R+/+ mice. In the emergence test, D4R−/− mice spent more time in the cylinder and made fewer entries into the cylinder than D4R+/+ mice. In the novel object test, D4R−/− mice made a smaller increase in percentage of time spent in the center after introduction of the cup relative to D4R+/+ mice. We have also replicated these basic findings in an independent group of mice (n = 61) (data not shown). These three tests were used to explore the extent to which changes in exploratory drive or anxiety accounted for the phenotypic differences in response to approach–avoidance conflicts. Differences in approach or exploratory behavior appear to account for much of the behavioral phenotype, rather than avoidance or anxiety-related behaviors, because the effect size was largest in the novel object test (ES = 1.4), intermediate in the emergence test (ES = 0.8, 0.5), and smallest in the open field test (ES = 0.1). The effect size for center entries on day three of the open field test was used for comparison because this measure primarily reflects avoidance or anxiety-related behavior, whereas center entries on day one are more indicative of approach or exploratory behavior. We also assessed effect sizes without including females to avoid any confound of sex, because only males were used in the repeated open field test. A similar pattern of effect sizes resulted for the novel object (ES = 1.5), emergence (ES = 0.8, 0.1), and open field tests (ES = 0.1). Thus, compared to D4R+/+ mice, D4R−/− mice exhibited the largest reductions in behavioral responses to novelty in the test that is maximally sensitive to approach behaviors (the novel object test), and the smallest reductions in the test that is most reflective of avoidance behavior (the open field test).

Our hypothesis that the D4R is involved in modulating behavioral responses to novelty in mice derived from observations of an association between polymorphisms of the D4R gene and personality inventory measures of the trait called novelty-seeking (Benjamin et al., 1996; Ebstein et al., 1996). Our finding that knock-out of the D4R results in diminished behavioral responses to novelty in mice is consistent with the hypothesis that a lack of D4R function may lead to decreased novelty-seeking in humans. A direct link, however, cannot be made between the present results and the findings of the human association studies (Benjamin et al., 1996; Ebstein et al., 1996), because mice were either wild-type or D4R-deficient, whereas the human subjects possessed different D4R alleles for which there is evidence for varying function (Asghari et al., 1995). A null allele for the D4R has been reported to occur in 2% of the general population (Nothen et al., 1994). Comparing the novelty-seeking trait in subjects with the null D4R allele to those with functional alleles could directly test the hypothesis that lack of function of the D4R is associated with diminished novelty-seeking in humans. The same D4R alleles associated with novelty-seeking have recently been associated with behavioral disorders suggested to be related to novelty-seeking, such as drug abuse (Kotler et al., 1997; Shields et al., 1998), pathological gambling (de Castro et al., 1997), and attention deficit hyperactivity disorder (LaHoste et al., 1996; Faraone et al., 1999). Further knowledge regarding the functional role of the D4R in behavioral responses to novelty could be an important step toward understanding the neurobiology of these disorders.

Mice lacking the D4R exhibited modest reductions in locomotor activity in the open field (nonsignificant), emergence, and novel object tests that were independent from the decreases in behavioral responses to novelty, as revealed by ANCOVA. This decrease in locomotor activity does not appear to result from a gross motor impairment, because D4R−/− mice have been reported to outperform D4R+/+ mice on the rotarod and are supersensitive to the locomotor-activating effects of ethanol, cocaine, and methamphetamine (Rubinstein et al., 1997). It has been widely reported that the vulnerability of rats to drugs of abuse can be predicted by their locomotor response to a novel environment (Hooks et al., 1991, 1994), with high responders to novelty acquiring amphetamine self-administration more readily (Piazza et al., 1989) and exhibiting greater behavioral activation in response to amphetamine (Hooks and Kalivas, 1994). One might predict that because D4R−/− mice exhibit less locomotor activity in a novel environment, they would show less behavioral activation to drugs of abuse than D4R+/+ mice. The supersensitivity of D4R−/− mice to the locomotor activation produced by ethanol, cocaine, and methamphetamine is thus surprising.

Phenotypes exhibited by knock-out mice of a mixed genetic background can result from the introduction of background genes linked to the mutated gene, rather than from the mutation itself. The present D4R−/− mice were generated using 129Sv/Ola stem cells crossed onto a C57BL/6 background (Rubinstein et al., 1997). The 129Sv/Ola strain performs normally relative to C57BL/6 mice in the open field test, the Morris water maze, the plus maze, and on coordination tests (Montkowski et al., 1997). In addition, 129Sv/Ola mice spend more time in the center of a novel open field than C57BL/6 mice (Montkowski et al., 1997), suggesting that the decrease in behavioral responses to novelty exhibited by D4R−/− mice is not caused by 129Sv/Ola background genes.A priori hypotheses reduce the likelihood of false positives when characterizing knock-out mice on a mixed background. The present experiments were undertaken to test the hypothesis the D4R is involved in modulating behavioral responses to novelty. Although it is conceivable that the reduction in behavioral responses to novelty observed in the D4R−/− mice results from 129Sv/Ola background genes, the observed phenotype is most likely caused by lack of the D4R. It will be important to confirm these findings in congenic strains of D4R+/+ and D4R−/− mice.

Differences in dopamine neurotransmission in D4R−/− mice are localized to brain areas that could mediate the observed reductions in behavioral responses to novelty. Although dopaminergic neurons of the substantia nigra provide most of the dopaminergic projections to the dorsal striatum, these neurons do not express D4Rs. Rather, D4Rs are densely localized on glutamatergic pyramidal cells of frontal cortex (Mrzljak et al., 1996; Ariano et al., 1997; Rubinstein et al., 1997) that project to the dorsal striatum and the substantia nigra (Carter, 1982). The stimulation of nigrostriatal nerve terminals (Westerink et al., 1992) in the dorsal striatum or of nigral neurons by glutamate induces dopamine release (Overton and Clark, 1992). Accordingly, dopamine synthesis and its conversion to DOPAC are increased in the dorsal striatum of D4R−/− mice (Rubinstein et al., 1997). Thus, frontal cortical D4Rs may alter the activity of nigrostriatal dopamine neurons by modulating the release of glutamate onto these neurons. Furthermore, D4Rs in the frontal cortex are likely to be under substantial influence of both noradrenergic inputs and dopaminergic inputs, because dopamine is only fivefold more potent at this receptor than epinephrine and norepinephrine, whereas dopamine is >100-fold more potent at D2 receptors than epinephrine and norepinephrine (Lanau et al., 1997).

Although effects of sex were neither predicted nor systematically studied in the present experiments, some sex differences in behavior were found. Female mice were more active overall than males in the novel object test, which is consistent with numerous reports that female mice are more active than male mice in many testing situations (Norton, 1977). However, in the emergence test, females were less active than males and spent more time in the cylinder than males, although an ANCOVA showed that these two effects were related. Only one interaction of genotype and sex was observed, in which female but not male D4R−/− mice made fewer entries into the cylinder than D4R+/+ mice in the emergence test. In both humans and rats, the modulation of the mesostriatal dopamine system and related behaviors by estrogen has been reported (Van Hartesveldt and Joyce, 1986), however, the human D4R association studies on novelty seeking have not reported gender effects (Benjamin et al., 1996; Ebstein et al., 1996; LaHoste et al., 1996;Kotler et al., 1997; de Castro et al., 1997; Shields et al., 1998;Faraone et al., 1999). Similarly, the important phenotypic differences observed in the present experiments did not interact significantly with the sex differences.

Our findings support the hypothesis positing a functional role for the D4R in behavioral responses to novelty. These results complement human genetic association studies implicating D4R polymorphisms in naturally occurring variations in the personality trait known as novelty seeking. Although it cannot be concluded that D4R-deficient mice are without abnormalities with regard to avoidance or anxiety-related behaviors, the present results are best explained by a reduction in approach or exploratory behavior. In conclusion, the D4R plays a role in modulating approach–avoidance responses in general and novelty-related exploration in particular.