Disruption of Src Is Associated with Phenotypes Related to Williams-Beuren Syndrome and Altered Cellular Localization of TFII-I

Social approach behaviors are increased and social recognition is impaired in Src^thl/thl mice. A, Src^thl/thl mice spent more time sniffing a social cage versus a nonsocial cage (n = 9 WT males, n = 8 Src^thl/thl males). B, During the second phase of the test to measure social recognition, Src^thl/thl mice did not show a preference for a novel mouse versus a familiar mouse from the first phase (n = 9 WT males, n = 8 Src^thl/thl males). *p < 0.05, compared to the chamber with empty cage, between genotypes and within genotypes, respectively. C, In a direct social interaction test, Src^thl/thl mice showed an increased frequency of interactions with a stranger mouse. Data are presented as mean ± SEM (n = 9 WT males, n = 8 Src^thl/thl males). *p < 0.05, between genotypes. D, Src^thl/thl mice showed social dominance over their WT littermates in a tube test (n = 20 WT males, n = 20 Src^thl/thl males). *p < 0.05, between genotypes. E, Src^thl/thl mice were able to habituate to a smell over time and dishabituate toward a novel smell, demonstrating intact olfaction (n = 10 WT (5 males, 5 females), n = 9 Src^thl/thl (5 males, 4 females)). Data are presented as mean ± SEM.

Enhanced social reunion of female Src^thl/thl mutant mice. A, Time course of total number of USVs in WT and Src^thl/thl male mice when introduced to females for a 5 min trial. A gradual decline in total number of calls was observed across time but the total number of calls was still high, with no significant difference between WT and Src^thl/thl mice. Data are presented as mean ± SEM for each time point (n = 10 WT, n = 11 Src^thl/thl ). B, Time course of total number of USVs in WT and Src^thl/thl females when reunited with a litter mate for a 5 min trial. A gradual decline in total number of calls is observed across time with a significant difference between WT and Src^thl/thl females in total number of USVs produced in the first and second minutes (n = 11 WT, n = 10 Src^thl/thl ). C, There is a significant difference in the number of flat (Type 1) USVs produced by WT versus Src^thl/thl females at minute 1 (n = 11 WT, n = 10 Src^thl/thl ). D, There was no significant difference in the number of broken (Type 2) USVs produced by WT versus Src^thl/thl females across time (n = 11 WT, n = 10 Src^thl/thl ). E, There is a significant difference in the number of frequency modulated (Type 3) USVs produced by WT versus Src^thl/thl females at minutes 1 and 2 (n = 11 WT, n = 10 Src^thl/thl ). Data are presented as mean ± SEM for each time point. *p < 0.05 between genotypes.

Src^thl/thl mutant mice show hyperactivity in the open field. A, Src^thl/thl mutant mice showed an increase in total distance travelled in the open field compared with WT mice (n = 10 WT (5 males, 5 females), n = 16 Src^thl/thl (9 males, 7 females)). B, Percentage of time spent in the center of the arena versus the entire open field was decreased in Src^thl/thl (n = 10 WT (5 males, 5 females), n = 16 Src^thl/thl (9 males, 7 females)). *p < 0.05 between genotypes. C, Percentage time spent in the open arms versus the closed arms of the elevated plus maze showed no significant differences between genotypes (n = 13 WT (7 males, 6 females), n = 18 Src^thl/thl (9 males, 9 females)). Data are presented as mean ± SEM.

Balance and motor strength deficiency in Src^thl/thl mice and the inability to learn motor skills. Quantitative analysis of raised balance beam, grip strength, and rotarod task in WT and Src^thl/thl mice. A, Src^thl/thl mice needed significantly more time to cross the balance beam than did WT mice (n = 13 WT (7 males, 6 females), n = 12 Src^thl/thl (6 males, 6 females)). B, Src^thl/thl mice had significantly more hindfoot slips than did WT mice. C, Grip strength analysis revealed significantly reduced forelimb grip strength in Src^thl/thl mice compared with WT animals (n = 16 WT (9 males, 7 females), n = 12 Src^thl/thl (6 males, 6 females)). D, Grip strength analysis revealed significantly reduced combined forelimb and hindlimb grip strength in Src^thl/thl mice compared with WT animals (n = 16 WT (9 males, 7 females), n = 12 Src^thl/thl (6 males, 6 females)). E, Mice were tested on an accelerating rotarod with the same speed for 4 consecutive days. The latencies from rotation onset until the mice fell off the rod were measured. WT mice managed to stay significantly longer on the accelerating rotarod than Src^thl/thl mice on days 3 and 4. The falling latencies were similar in both groups on days 1 and 2. Falling latencies were compared within genotype between days 1 and 4 (n = 12 WT (6 males, 6 females), n = 12 Src^thl/thl (6 males, 6 females)). Data are presented as mean ± SEM. *p < 0.05.

Acoustic startle in response to high startling stimulus is increased in Src^thl/thl mice. Both the WT and Src^thl/thl mice exhibited similar startle amplitude responses to the low-intensity acoustic stimuli (70-105 dB). Src^thl/thl mice showed higher responses to the high-intensity stimuli (110-115 dB) (n = 8 WT (8 males), n = 8 Src^thl/thl (18 males)). *p < 0.05.

Impaired memory in Src^thl/thl mutant mice. A, Mice were assessed in the Morris water maze procedure (n = 12 WT (12 males), n = 12 Src^thl/thl (12 males)). Mean latency to reach a target platform in a visible platform session (days 1 and 2), and in a hidden-platform acquisition phase (days 3-8). B, Spatial memory retention was assessed in the probe trials administered 2 h after day 8 (n = 12 WT (12 males), n = 12 Src^thl/thl (12 males)). Mean number of platform crosses during probe trial 1 are shown. *p < 0.05. C, Freezing (as a percentage of total time) in a novel conditioning chamber (n = 24 WT (13 males, 11 females), n = 22 Src^thl/thl (12 males, 10 females)). After recording baseline freezing for 120 s, a tone was presented for 30 s (novel tone), after which mice were given a footshock and left inside the chamber for another 30 s (after shock). A subtle increase in freezing was observed during baseline and novel tone presentation in Src^thl/thl mice. *p < 0.05. D, Cued fear memory 24 h after pairing (n = 24 WT (13 males, 11 females), n = 22 Src^thl/thl (12 males, 10 females)). Pre-CS is freezing in the absence of the tone in a novel context, while CS is percentage freezing during tone presentation. Src^thl/thl mice showed impaired auditory fear conditioning, however, context was not affected. Data are presented as mean ± SEM, *p < 0.05.

Src^thl/thl mice have a deficit in short term object recognition. A, Mean time (s) spent exploring the displaced versus nondisplaced objects in the spatial change session (n = 12 WT (6 males, 6 females), n = 15 Src^thl/thl (8 males, 7 females)). B, Mean time spent exploring the novel item versus three familiar objects in the nonspatial change session (n = 12 WT (6 males, 6 females), n = 14 Src^thl/thl (7 males, 7 females). Data are expressed as mean ± SEM, *p < 0.05.

Altered cellular localization of TFII-I and TRPC3 in Src^{thl / thl} mouse brain tissue. Western blot analysis was carried out on fractionated cell lysates from WT and Src^thl/thl adult mouse whole brain. Representative blots are shown (n = 4 WT, n = 5 Src^thl/thl ). A, The nuclear fraction was probed with TFII-I antibody, with nucleolin as a loading control. B, The membrane fraction was probed with TRPC3 antibody, with n-cadherin as a control. C, Quantification of the Western blots. Data are expressed as mean ± SEM, *p < 0.05.