Catechol-O-methyltransferase-deficient mice exhibit sexually dimorphic changes in catecholamine levels and behavior

Abstract

Catechol-O-methyltransferase (COMT) is one of the major mammalian enzymes involved in the metabolic degradation of catecholamines and is considered a candidate for several psychiatric disorders and symptoms, including the psychopathology associated with the 22q11 microdeletion syndrome. By means of homologous recombination in embryonic stem cells, a strain of mice in which the gene encoding the COMT enzyme has been disrupted was produced. The basal concentrations of brain catecholamines were measured in the striatum, frontal cortex, and hypothalamus of adult male and female mutants. Locomotor activity, anxiety-like behaviors, sensorimotor gating, and aggressive behavior also were analyzed. Mutant mice demonstrated sexually dimorphic and region-specific changes of dopamine levels, notably in the frontal cortex. In addition, homozygous COMT-deficient female (but not male) mice displayed impairment in emotional reactivity in the dark/light exploratory model of anxiety. Furthermore, heterozygous COMT-deficient male mice exhibited increased aggressive behavior. Our results provide conclusive evidence for an important sex- and region-specific contribution of COMT in the maintenance of steady-state levels of catecholamines in the brain and suggest a role for COMT in some aspects of emotional and social behavior in mice.

Figure 1

Targeting of the COMT locus. (A) Sequence alignment of mouse and human COMT. Single and double asterisks indicate the membrane bound and soluble initiation methionines, respectively (by analogy to the rat and human clones). Arrowhead indicates codon 158 of the human gene where a met/val variation determines low or high activity of the enzyme (9, 31, 38). (B) Targeting of mouse COMT locus in embryonic stem cells and mice. A HindIII–SacI fragment was shown to encompass the entire set of coding exons of the COMT gene. For the construction of the targeting construct, part of this fragment, encompassing exons 2–4 (see below) was replaced by a cassette including the neo gene under the control of the phosphoglycerol kinase promoter. Cell culture, electroporation of A7ES cells, and generation of the chimeric mice were performed essentially as previously described (39). About 15% of the tested embryonic stem cell clones were positive for homologous recombination, and three clones were selected for karyotyping and injection into C57B6 blastocysts. Chimeric males were mated with C57B6 females, and DNA from tail biopsies of F₁ agouti coat pups was typed by Southern blotting and PCR at the comt genomic locus. F₁ heterozygous mice were mated, and F₂ mice of all three genotypes and of mixed 129/J/C57B6 background were obtained. Arrow lines indicate the diagnostic EcoRV restriction fragment. The probe used for Southern analysis is shown as a thick black line. (C) Genomic Southern blot analysis of tail biopsies. Genomic DNA was isolated from offspring obtained after breeding of heterozygous mice, digested with EcoRV, and probed with a ≈1-kb fragment adjacent to the right arm of the targeting construct or a 5′ coding portion of the mouse COMT cDNA, corresponding to exons 2–4. In the former case, wild-type and recombinant restriction fragments are 11.5 and 3.5 kb, respectively.

Figure 2

(A) Northern blot analysis of mRNA isolated from the liver and the brain of homozygous and wild-type animals. Two mRNA species are observed in the liver, corresponding (by analogy to the rat and human gene) to two distinct sites of transcriptional initiation. The coding part of the COMT cDNA was used as probe. As a control the Northern blot was probed with a probe from rat β actin. (B) HVA/DOPAC ratio in the striatum, frontal cortex, amygdala, and hypothalamus of male and female homozygous and wild-type animals. Values are the average ± SEM for wild-type (shaded bar) and homozygous (solid bar) mice. Differences between wild-type and homozygous mice tested by Mann–Whitney u test were ∗∗∗, P < 0.0002, ∗∗, P < 0.002. The calculated HVA/DOPAC ratios for homozygous (Homo) versus wild-type (wt) animals are as follows: HVA/DOPAC_striatum = 0.05 ± 0.009 vs. 0.94 ± 0.71 (P = 0.0001); HVA/DOPAC_{frontal cortex/males} = 0.13 ± 0.04 vs. 2.77 ± 0.59 (P = 0.003); HVA/DOPAC_{frontal cortex/females} = 0.25 ± 0.09 vs. 3.05 ± 0.37 (P = 0.002); HVA/DOPAC_amygdala = 0.09 ± 0.009 vs. 1.56 ± 0.17 (P = 0.0002); HVA/DOPAC_{hypothalamus/males} = 0 vs. 0.41 ± 0.05 (P = 0.0006); and HVA/DOPAC_{hypothalamus/females} = 0 vs. 0.04 ± 0.008 (P = 0.002); It is notable that although the HVA/DOPAC ratio is decreased significantly in all of the brain areas tested, residual HVA is detectable, in several of these areas (not shown). One interpretation is that the residual methylation of dopamine and DOPAC is caused by the action of yet unidentified methyltransferases involved in the clearance of dopamine, whose activity is probably up-regulated in the absence of COMT. Further work is needed to verify this interpretation and possibly lead to better characterization of this pathway. Str, striatum; Fctx, frontal cortex; amygd, amygdala; Hyp, hypothalamus. The number of the animals tested (n =) is indicated.

Figure 3

Effect of COMT gene disruption on dopamine (DA), norepinephrine (NE), 5-hydroxytryptamine (5-HT), and 5-hydroxy indole acetic acid (5-HIAA). Shown are steady-state levels (in pg/μg protein) in the striatum, frontal cortex (Fctx), and hypothalamus (Hyp) of homozygous, heterozygous, and wild-type animals of both sexes (with the exception of striatum where heterozygous animals were not analyzed). Data were analyzed by two-way ANOVA. Dopamine levels for frontal cortex as shown by ANOVA were F(2,28) = 6.005, P < 0.01; norepinephrine levels for hypothalamus were P = 0.051 (a comparison of heterozygous to wild-type and homozygous males was short of significant). In the case of the striatum, data from males and females were pooled and differences between wild-type and homozygous animals were tested by Student’s t test. For all other areas, male and female data are presented even though no sex differences were present. The number of the animals tested (n = ) is indicated.

Figure 4

Effect of COMT gene disruption on emotionality and sensory gating. Homozygous females took longer to emerge into the light than did wild-type females (B), and they also spent less time ambulating in the light compartment (D). An ANOVA of genetype by latency to emerge was significant (2,29) = 6.056; P < 0.01 for females. Follow-up multiple comparison tests using the Bonferroni correction showed that female homozygous differed from both wild-type (P < 0.05) and heterozygous (P < 0.05) female mice, whereas the latter two groups did not differ from each other. A planned comparison of wild-type and homozygous females on total time spent in the lgiht was short of significant (not shown). However, planned comparison of wild-type and homozygous females on time spent ambulating in the light was significant [(20) = 2.12; P < 0.05], whereas the same comparison for ambulation in the dark was not (P = 0.56). Males did not differ on either measure of emotionality (A and C). Prepulse inhibition was examined for two prepulse dB levels; higher y-axos values represent greater percent inhibition. There were no effects of genetype for either males (E) or females (F) (∗, P < 0.05).

Figure 5

Effects of COMT gene disruption on aggressive behaviors. Data were analyzed by ANOVA for repeated measurements for the main effects of genotype and test day and their interaction. (A) Mean (+ SEM) latency to the first aggressive behavior act. There were overall genotype differences [F(2,9) = 5.057, P < 0.05], and heterozygotes showed significantly shorter latency to aggression (P < 0.05) compared with both wild-type and homozygotes, which were not different from each other. It also was found that latency decreased over 3 days [F(2,18)= 4.838, P < 0.05]. (B) Significant genotype differences were found in the total number of aggressive behavior bouts during 15-min tests [F(2,9) = 8.708, P < 0.01]. Heterozygous pairs were significantly more aggressive (P < 0.05) than both wild-type and homozygous pairs. The latter two groups were not different from each other, although further testing with an additional set of animals (that were not included in the present analysis because of differences in previous experiences) indicated a trend in which wild-type mice became more aggressive than homozygous mice after repeated behavioral tests for aggression (data not shown).