Assessment of autism-relevant behaviors in C57BKS/J leptin receptor deficient mice

Abstract

Gestational diabetes mellitus (GDM) was associated with greater autism risk in epidemiological studies. Disrupted leptin signaling may contribute to their coincidence, as it is found in both disorders. Given this we examined leptin receptor (Lepr) deficient (BKS.Cg-Dock7^m +/+ Lepr^db/J diabetic (db)) heterozygous (db/+) mice for autism-relevant behaviors. BKS db/+ females are lean with normal blood glucose, but they develop GDM while pregnant. We hypothesized BKS db/+ offspring might exhibit physiological and behavior traits consistent with autism. Adolescent body weight, fasting blood glucose, serum corticosterone, social preferences, self-grooming, marble burying, social dominance and cognitive flexibility of BKS db/+ mice was compared to C57BLKS/J (BKS) and C57BL/6J (BL6) mice. Male db/+ weighed more and had higher blood glucose and corticosterone relative to BL6, but not BKS mice. Also, male db/+ lacked social interaction preference, explored arenas less, and buried more marbles than BL6, but not BKS males. Male and female db/+ were more dominant and made more mistakes in water T-mazes locating a sunken platform after its position was reversed than BL6, but not BKS mice. Overall BKS db/+, particularly males, exhibited some autism-like social deficits and restrictive-repetitive behaviors relative to BL6, but BKS strain contributions to BKS db/+ behaviors were evident. Since BKS db/+ and BKS behavioral and physiological phenotypes are already so similar, it will be difficult to use these models in studies designed to detect contributions of fetal GDM exposures to offspring behaviors.

Keywords: Blood glucose; Cognitive flexibility; Corticosteroid; Gestational diabetes; Leptin receptor; Social dominance; Social preferences.

Fig. 1.. Timeline and mouse numbers used in the study.

Eight to 10 male and 8-9 female BL6, BKS and db/+ mice, 6-7 weeks old, were studied in four behavior tests with translational relevance to autism for 2 weeks before they were euthanized. Fasting blood glucose was measured and serum was collected for corticosterone measures at the time of euthanasia.

Fig 2.. Glucose tolerance tests at gestational day 15 in db/+, BKS and BL6 dams.

Pregnant female mice (2 each BKS or db/+, and 3 BL6) were fasted for 6 h starting at 0800h and then injected (i.p.) at 1400h with 1 g/kg glucose. a) The 15 min post-injection peak blood glucose was higher in db/+ and BKS than in BL6 dams (**p < 0.001), and db/+ dams also had higher blood glucose at 30 and 60 min (*p < 05) time points than BL6 or BKS. b) The area under curve (AUC) was greater for db/+ than BKS or BL6 (*p< 0.05). c) Analysis of only the 0, 30 and 60 min, consistent with prior reports (Yamashita et al., 2001; 2003) revealed db/+ blood glucose was higher at 30 and 60 min than either BL6 or BKS dams (*p< 0.05), and d) AUC was greater for db/+ than BKS or BL6 dams (*p <0.05).

Fig. 3.. Physiological parameters of db/+, BKS and BL6 mice with reference to representative db/db.

All data are shown as $\stackrel{‒}{\mathsf{X}}$ ± S.E.M., N = 8 BKS females and males and db/+ females, 9 BL6 females, 10 db/+ and BL6 males, 1 male and 1 female db/db unless otherwise noted. Mice were weighed before and after behavior tests. a) Initially and at final measure BKS females (circles, *p < 0.05 for each time) weighed less than BL6 or db/+ females, and all weighed less than a representative 8 wk old db/db female (dashed line at 38 g). b) By contrast male weights (squares) were not different at the study outset, but by day 15 db/+ males weighed more than BL6 or BKS males (*p < 0.005), and all male groups weighed less than a representative 8 wk old db/db male (dashed line at 36 g). c) Blood glucose was higher in male (squares) and female (circles) db/+ combined, than in male and female BL6 mice combined (*p <0.005), but not different from male or female BKS mice on day 15. All subjects had lower blood glucose than $\stackrel{‒}{\mathsf{X}}$ = 214 mg/dl (dashed line) found in the representative male (192 mg/dl) or female (235 mg/dl) db/db mice. c) Serum corticosterone in db/+ males (squares) was higher than in BL6 males (***p<0.001), and higher in female db/+ (*p <0.05), and BKS (*p<0.01) than BL6 females, in subsets of 5-6 randomly selected samples/group. All subjects had lower serum corticosterone than the representative female db/db mouse (dashed line) with 1852 ng/dl.

Fig. 4.. Social interaction and social novelty tests of db/+, BKS and BL6 mice in 3 chamber arenas.

a) BL6 and BKS males (squares) spent more time in chambers with stranger males versus novel objects, while their female (circles) counterparts and db/+ mice did not (*p < 0.05). b) Female BL6 mice had lower social interaction time preference (stranger – novel object) than male BL6 (** p < 0.01), while among male mice, db/+ had lower preference for interaction than BL6 in an exploratory male only strain ANOVA (#p <0.05). c) BKS and BL6 males spent more time sniffing stranger mice than novel objects (*p <0.01), while females and db/+ mice did not. Further, BKS males spent more time sniffing strangers than BL6 or db/+ males (***p < 0.001), as well as sniffing novel objects than BL6 males or females (*p <0.05). BKS females also spent more time sniffing strangers than BL6 females (**p<0.01). d) However, there were no differences in social interaction sniffing preference (stranger-novel object) between BL6, BKS and db/+ mice. In social novelty tests e) Only db/+ males spent more time in chambers with new versus old strangers (*p <0.05). f) BKS female mice had lower social novelty preference (new stranger – old stranger) than BL6 or db/+ mice (*p <0.05). g) By measure of social sniffing, BKS males preferred old strangers, spending more time with them than any other group except BKS females (*p<0.05). h) Based on sniffing behavior, differences in social novelty preference were absent, however db/+ males trended toward spending more time sniffing new strangers than BKS males (p =0.0583). All data are shown as mean ± S.E.M., N = 8-9 females and 8-10 males. Preference ratio graphs are available in Fig S1.

Fig. 5.. Middle chamber time, chamber entry, self-grooming and marble burying differences among db/+, BKS and BL6 mice.

a) Time spent in middle chamber of test arena during social interaction (open symbols) and social novelty (closed symbols) test phases did not differ among mouse strains or sexes. b) Male BL6 made more chamber entries during both social interaction and social novelty preference tests than BKS mice (males and females) or db/+ males (*p <0.001). Chamber entries are useful for assessing exploratory activity and c) During social novelty tests, BKS mice (males and females) did more self-grooming than during the preceding social interaction tests. d) Female BL6 buried more flattened blue marbles than male BL6 (**p<0.0005), and male db/+ (*p<0.05) and BKS (***p < 0.0001) buried more than male BL6. Also female BKS buried more marbles than db/+ females (*p <0.05). Tests ran for a 30 min period under low red light (16 lx). Data are mean ± S.E.M., Sample sizes were BL6 females = 9, BL6 males = 10, BKS females = 8, BKS males = 8, db/+ females = 8, db/+ males = 10.

Fig 6.. Social dominance of db/+ males is greater than BL6 males in the tube test.

a) Male and female db/+ had higher average wins in 6 matches against 129S1/SvImJ same-sex competitors than BL6 mice (*p <0.05). Our tests also revealed that female mice do exhibit social dominance in this test. b) The duration of matches was longer for BKS females than BL6 females (*p <0.05). Data are mean ± S.E.M., N = 10 each BL6 and db/+ males, 8 BKS males and females, 9 BL6 and 5 db/+ females. Three db/+ females were not used in the social dominance test.

Fig 7.. Water T-maze reveals delays in BKS and db/+ initial spatial acquisition and reversal learning.

a) Female db/+ mice had fewer trials without errors than BL6 females on the first day of spatial acquisition learning (A1, *p <0.05). Male db/+ and BKS had fewer trials without errors than BL6 males (*p <0.05) on reversal learning day 1 (R1). b) On R1 db/+ and BKS females and males took longer to complete trials than BL6 females and males (*p <0.05, **p <0.001). c) On acquisition learning day 1 (A1) db/+ females made more mistakes than BL6 (*p < 0.001). More critically, BKS and db/+ females and males made more errors on R1 than BL6 males or females (*p <0.05, **p <0.001). d) On R1, BKS females made more perseverative errors than db/+ females (**p<0.005), or BL6 females (***p <0.0001), and BKS males made more than BL6 males (**p<0.05). e) Few regressive errors were made, and there were no strain differences. Data are mean ± SEM, N= 10 BL6 and db/+ males, 9 BL6 and 8 db/+ males and BKS males and females.