Mice lacking the immediate early gene Egr3 respond to the anti-aggressive effects of clozapine yet are relatively resistant to its sedating effects

Abstract

Immediate early genes (IEGs) of the early growth response gene (Egr) family are activated in the brain in response to stress, social stimuli, and administration of psycho-active medications. However, little is known about the role of these genes in the biological or behavioral response to these stimuli. Here we show that mice lacking the IEG transcription factor Egr3 (Egr3-/- mice) display increased aggression, and a decreased latency to attack, in response to the stressful social stimulus of a foreign intruder. Together with our findings of persistent and intrusive olfactory-mediated social investigation of conspecifics, these results suggest increased impulsivity in Egr3-/- mice. We also show that the aggression of Egr3-/- mice is significantly inhibited with chronic administration of the antipsychotic medication clozapine. Despite their sensitivity to this therapeutic effect of clozapine, Egr3-/- mice display a marked resistance to the sedating effects of acute clozapine compared with WT littermate controls. This indicates that the therapeutic, anti-aggressive action of clozapine is separable from its sedating activity, and that the biological abnormality resulting from loss of Egr3 distinguishes these different mechanisms. Thus Egr3-/- mice may provide an important tool for elucidating the mechanism of action of clozapine, as well as for understanding the biology underlying aggressive behavior. Notably, schizophrenia patients display a similar decreased susceptibility to the side effects of antipsychotic medications compared to non-psychiatric controls, despite the medications producing a therapeutic response. This suggests the possibility that Egr3-/- mice may provide insight into the neurobiological abnormalities underlying schizophrenia.

Figure 1

Egr3–/– mice display heightened aggressive behavior. In the resident–intruder test, (a) Egr3–/– mice showed significantly increased aggressive behavior toward a foreign intruder compared to WT mice on two separate test sessions spaced several days apart. An ANOVA with repeated measures on total duration of aggression yielded a significant main effect of genotype (F(1,11)=29.40, p<0.001). Separate pairwise comparisons (t-tests) showed that Egr3–/– mice were significantly (*) more aggressive than controls on each test day (p=0.001 and p=0.010). (b) Egr3–/– mice also showed a significantly decreased latency to attack the intruder. An ANOVA with repeated measures showed a main effect of genotype (F(1,11)=20.33, p=0.001). Separate t-tests comparing Egr3–/– mice to WT controls on each test day were also significant (*) (p=0.001 and 0.002, respectively).

Figure 2

Acute low dose clozapine treatment reduces impulsivity in Egr3–/– mice but not overall aggression. (a) Analysis of total duration of aggressive behavior following acute administration of 1 mg/kg clozapine or vehicle to WT and Egr3–/– mice demonstrated a significant main effect of genotype (F(1,33)=11.04, p=0.002), but not of drug (F(1,33)=1.865, p=0.181). Individual comparison between Egr3–/– mice receiving vehicle and those receiving clozapine showed a trend toward decreased total duration of aggressive behavior that was not statistically significant (p=0.084). (b) However, 1 mg/kg clozapine significantly increased the latency to attack in Egr3–/– mice. (An ANOVA of latency to attack indicated a significant main effect of genotype (F(1,33)=84.78, p<0.001), and of drug (F(1,33)=9.632, p<0.004). Individual t-tests comparing clozapine vs vehicle-treated mice within each genotype showed that clozapine-treated Egr3–/– mice had significantly longer attack latencies relative to vehicle-treated Egr3–/– mice (F(1,33)=9.09, *p=0.005), while the drug had no significant effect on the attack latencies of the WT littermate controls.) (n=9 for each: Egr3–/– vehicle-treated, WT-vehicle treated, Egr3–/– clozapine-treated mice; mice). n=10 WT clozapine-treated

Figure 3

Acute higher-dose clozapine administration affects Egr3–/– mice in a manner different than WT mice. (a) Frames from a videotape of Egr3–/– and WT control mice following administration of either vehicle or 3.5 mg/kg clozapine show that this dose of clozapine produces immobility in WT mouse #1(circled). In contrast, the activity of an Egr3–/– mouse that has received 3.5 mg/kg of clozapine (#4), is not obviously distinguishable from WT or Egr3–/– mice that have received vehicle (#2 and #3, respectively). (See also Supplementary Figure S2 for video of response to 3.5 mg/kg, and Supplementary Figure S3 for response to 7 mg/kg). (b) Comparison of the effect of vehicle vs 3.5 mg/kg clozapine on the activity of Egr3–/– and WT littermate mice showed that clozapine affected Egr3–/– mice differently than WT mice (two-way ANOVA yielded a significant effect of genotype (F(1,29)=68.28, p<0.001), and of drug (F(1,29)=33.92, p<0.001), and a significant genotype by drug interaction (F(1,29)=6.96, p=0.013)). Individual t-tests indicated that vehicle-treated Egr3–/– mice are significantly more active than vehicle-treated WT controls. In addition, while acute administration of 3.5 mg/kg of clozapine profoundly repressed activity in WT mice (compare WT vehicle to WT clozapine 3.5 mg/kg), Egr3–/– mice remained as active as vehicle-treated WT mice following this dose of clozapine (*p<0.001; numbers in parentheses=n).

Figure 4

Acute haloperidol administration reduces the hyperactivity of Egr3–/– mice in a dose-dependent fashion. An overall ANOVA showed a significant main effect of genotype (F(1,39)=10.48, p=0.002) and a significant main effect of dose (F(3,39)=13.16, p<0.005). A one-way ANOVA across all Egr3–/– mice demonstrated a significant effect of haloperidol (p=0.003) and a pairwise comparison showed that a dose of 0.1 mg/kg was sufficient to reduced activity of Egr3–/– mice (p=0.028). A one way ANOVA across all WT mice demonstrated that haloperidol significantly reduced activity in controls (p=0.001), but a pairwise comparison demonstrated that 0.1 mg/kg did not affect activity in WT mice (p=0.897) (n.d.—not different. Single-digit numbers in parentheses=n).

Figure 5

Two weeks of chronic clozapine administration significantly reduced the aggressive behavior of Egr3–/– mice. An ANOVA showed significant effects of genotype (F(1,40)=7.603, p=0.009), and treatment (F(1,40)=4.120, p=0.039), and a significant genotype by treatment interaction (F(1,40)=4.120, p=0.049), suggesting that the effects of chronic clozapine treatment on aggression were different in Egr3–/– mice than in their WT littermates. A subsequent t-test confirmed that Egr3–/– mice receiving chronic clozapine showed significantly less aggression than vehicle-treated Egr3–/– mice (*p=0.005) (n=9 for each: Egr3–/– vehicle-treated, WT-vehicle treated, Egr3–/– clozapine-treated mice; n=10 WT clozapine-treated mice).