BTBR T+tf/J mice: autism-relevant behaviors and reduced fractone-associated heparan sulfate

Abstract

BTBR T+tf/J (BTBR) mice have emerged as strong candidates to serve as models of a range of autism-relevant behaviors, showing deficiencies in social behaviors; reduced or unusual ultrasonic vocalizations in conspecific situations; and enhanced, repetitive self-grooming. Recent studies have described their behaviors in a seminatural visible burrow system (VBS); a Social Proximity Test in which avoidance of a conspecific is impossible; and in an object approach and investigation test evaluating attention to specific objects and potential stereotypies in the order of approaching/investigating objects. VBS results confirmed strong BTBR avoidance of conspecifics and in the Social Proximity Test, BTBR showed dramatic differences in several close-in behaviors, including specific avoidance of a nose-to-nose contact that may potentially be related to gaze-avoidance. Diazepam normalized social avoidance by BTBRs in a Three-Chamber Test, and some additional behaviors - but not nose to nose avoidance - in the Social Proximity Test. BTBR also showed higher levels of preference for particular objects, and higher levels of sequences investigating 3- or 4-objects in the same order. Heparan sulfate (HS) associated with fractal structures in the subventricular zone of the lateral ventricles was severely reduced in BTBR. HS may modulate the functions of a range of growth and guidance factors during development, and HS abnormalities are associated with relevant brain (callosal agenesis) and behavioral (reductions in sociality) changes; suggesting the value of examination of the dynamics of the HS system in the context of autism.

Figure 1

BTBR mice tested in the Visible Burrow System showed decreased approach front (a,b) and approach back (c,d) behaviors in the dark periods when compared to B6 (pictured in b and d). *p<0.05.

Figure 2

BTBR mice tested in the Visible Burrow System showed increased self grooming (a) and duration of time alone (b); and decreased allogrooming (c) and duration of time huddling (d) in the light periods, *p<0.05.

Figure 3

Same strain social proximity testing of male subjects (a) showed increased nose-to-anus (NA), crawl under (CU) and crawl over (CO) behavior; and decreased nose tip-to-nose tip (NN) and upright (U) behaviors in BTBR mice compared to B6 mice, *p<0.05. There were no significant strain differences in nose-to-head (NH) and jump escape (JE) behaviors. (b) B6 mice performing nose tip-to-nose tip behavior in the social proximity chamber.

Figure 4

Diazepam was administered to independent groups in the social proximity tests. Social approach testing (a) showed that in subjects administered the vehicle (VEH), B6 mice spend more time in the chamber containing a CD-1 stimulus mouse and BTBR mice spent more time in the chamber containing an empty cup, *p<0.05. Administration of diazepam (DZP) reversed the social approach behavior of the BTBR mice. In social proximity testing (b), subjects administered DZP displayed decreased crawl under (CU), upright (U) and jump escape (JE) behaviors, ^$p<0.05. Main effects for strain showed decreased nose tip-to-nose tip (NN), nose-to-head (NH) and U behaviors; and increased nose-to-anus (NA), crawl over (CO) and CU behaviors in BTBR mice compared to B6 mice, ^#p<0.05.

Figure 5

Laminin and n-sulfated heparan sulfate in the SVZ of B6 and BTBR mice. Laminin (red) labels meninges and vasculature in the SVZ and surrounding tissue of the intact and collapsed lateral ventricles, respectively of B6 (a) and BTBR (e) mice. Images (b) and (f) represent n-sulfated heparan sulfate, and (c) and (g) present merged laminin and heparan channels with magnified images of (d) and (h) illustrating the reductions in fractone number (arrows), size and heparan immunoreactivity of BTBR mice relative to B6 controls.