Reduced fear and aggression and altered serotonin metabolism in Gtf2ird1-targeted mice

Abstract

The GTF2IRD1 general transcription factor is a candidate for involvement in the varied cognitive and neurobehavioral symptoms of the microdeletion disorder, Williams-Beuren syndrome (WBS). We show that mice with heterozygous or homozygous disruption of Gtf2ird1 exhibit decreased fear and aggression and increased social behaviors. These findings are reminiscent of the hypersociability and diminished fear of strangers that are hallmarks of WBS. Other core features of WBS, such as increased anxiety and problems with spatial learning were not present in the targeted mice. Investigation of a possible neurochemical basis for the altered behaviors in these mice using high-performance liquid chromatography analysis showed increased levels of serotonin metabolites in several brain regions, including the amygdala, frontal cortex and parietal cortex. Serotonin levels have previously been implicated in fear and aggression, through modulation of the neural pathway connecting the prefrontal cortex and amygdala. These results suggest that hemizygosity for GTF2IRD1 may play a role in the complex behavioral phenotype seen in patients with WBS, either individually, or in combination with other genes, and that the GTF2I transcription factors may influence fear and social behavior through the alteration of neurochemical pathways.

Figure 1. Targeted disruption of the Gtf2ird1 gene by homologous recombination

(a) Structural organization of the murine Gtf2ird1 gene (top), of the targeting vector (middle) and the targeted Gtf2ird1 locus (bottom). Grey boxes indicate coding exons. Restriction enzymes are ApaI (A), EcoRI (E), and KpnI (K). Black box represents position of probe used to screen embryonic stem (ES) cells (P). The loxP sites are represented by arrows; NEO, neomycin-resistance cassette; TK, thymidine kinase gene. Bent arrow represents the predicted translation start site. (b) Southern blot analysis of Gtf2ird1-targeted ES cells. Genomic DNA was digested with ApaI, fractionated on agarose gel and hybridized with ³²P-labeled probe. The 11-kb ApaI fragment corresponds to the WT locus and the 8.5-kb fragment to the targeted locus. (c) Real-time RT-PCR analysis of gene expression using brain cDNA from WT and targeted Gtf2ird1 mice. Gtf2ird1 and its two flanking genes (Gtf2i, Clip2) were assayed.

Figure 2. Gtf2ird1-targeted mice show mild growth retardation

Body weight analysis of adult Gtf2ird1 targeted mice. Adult male (+/+, n = 23; +/−, n = 23; +/+, n = 14) and female (+/+, n = 24; +/−, n = 28; +/+, n = 8) mice were analyzed for changes in body weight (mean age, 20.7 ± 2.6 weeks). Both male and female Gtf2ird1^−/− mice showed a significant decrease (P < 0.05) in body weight of approximately 15%. Although male and female Gtf2ird1^+/− mice also showed a decrease in body weight, the decrease was only statistically significant in the Gtf2ird1^−/− mice (*P < 0.05).

Figure 3. The resident-intruder test detects decreased aggression and increased social interaction in Gtf2ird1^+/− and Gtf2ird1^−/− mice

Both the Gtf2ird1^+/− and the Gtf2ird1^−/− mice showed a significant decrease in the number of aggressive interactions (a) and length of these aggressive interactions (b) as compared with WT mice. Both the Gtf2ird1^+/− and the Gtf2ird1^−/− mice also showed a significant increase in the number of social interactions including the time spent following the intruder (c) compared with WT mice. Gtf2ird1^−/− mice also displayed a significant increase in the number of followings (d) and an increased, but not significant time spent sniffing the intruder (e) compared with WT mice. A similar trend in noted for Gtf2ird1^+/− mice although this change was not statistically significant. Values are mean ± SEM (+/+, n = 17; +/−, n = 10; −/−, n = 18) (*P < 0.05, **P < 0.01).

Figure 4. Anxiety is decreased in Gtf2ird1^−/− mice

In the elevated plus maze, significant increases in the percentage of time spent in open arms (a) entries into the open arms (b) and head dips (c) were seen in Gtf2ird1^−/− mice compared with WT mice. In the novel object test, both Gtf2ird1^+/− and Gtf2ird1^−/− mice showed a significant increase in the time spent exploring an unfamiliar object (cube) (d). In the open field, both Gtf2ird1^+/− and Gtf2ird1^−/− mice showed a significant increase in the center to wall ratio (e) and Gtf2ird1^−/− mice showed a significant decrease in risk assessment (alarm scanning) (f). Values are mean ± SEM (+/+, n = 11; +/−, n = 12; −/−, n = 14) (*P < 0.05, **P < 0.01).

Figure 5. Gtf2ird1-targeted mice have deficits in cued but not contextual fear conditioning

In cued tests, there was no significant change in amount of freezing observed between genotypes in the altered context compared the baseline (pre-CS). Upon presentation of the auditory cue (post-CS), freezing in Gtf2ird1-targeted mice was decreased with both Gtf2ird1^+/− and Gtf2ird1^−/− mice displaying significantly less freezing than WT littermates. In contextual testing, Gtf2ird1-targeted mice did not differ significantly from WT littermates (data not shown). (*P < 0.01, **P < 0.05).

Figure 6. Gtf2ird1-targeted mice perform normally in the Morris water maze evaluation of visuospatial processing

Neither Gtf2ird1^+/− nor Gtf2ird1^−/− mice showed a significant difference in their performance compared to WT littermates (P > 0.05) during either the visible (V), hidden (acquisition) or the reversal phase (a). No significant differences were observed in either probe test (test administered at the end of the hidden platform trial is shown) (b). Values are mean ± SEM (+/+, n = 7; +/−, n = 11; −/−, n = 12).