Somatosensorimotor and Odor Modification, Along with Serotonergic Processes Underlying the Social Deficits in BTBR T+ Itpr3tf/J and BALB/cJ Mouse Models of Autism

Abstract

Autism is a complex spectrum of disorders characterized by core behavioral deficits in social communicative behavior, which are also required for comprehensive analysis of preclinical mouse models. As animal models of the core behavioral deficits in autism, two inbred mouse strains, BTBR T+ Itpr3^tf/J (BTBR) and BALB/cJ (BALB), were compared with the standard social strain, C57BL/6J (B6), regarding a variety of behavioral factors underlying social communicative interactions, including olfactory and tactile sensory processes, social recognition abilities and behavioral expression strategies. Although both female BTBR and BALB mice can express social recognition and approach behavior depending on the stimuli they encounter, the available sensory modalities, along with modulation of the serotonergic system, differ between the two strains. BALB mice have deficits in using volatile olfactory cues and tactile information in a social context; they fail to exhibit a social approach to volatile cues and seek nonvolatile cues by exhibiting substantial sniff/contact behavior when allowed direct contact with social opponents. Systemic injection of the serotonin (5-HT1A) agonist buspirone has little effect on these social deficits, suggesting a congenitally degraded serotonergic system in BALB mice. In contrast, BTBR mice exhibit impaired body coordination and social motivation-modified olfactory signals, which are relevant to a reduced social approach. A systemic injection of the 5-HT1A agonist restored these social deficits in BTBR mice, indicating that a downregulated serotonergic system is involved in the social deficits exhibited by BTBR mice.

Keywords: autism model; odor signal; serotonin; social approach; tactile function.

Figure 1.

Representative images of somatosensory barrel maps in barrel cortex of B6, BALB, and BTBR mice. Representative maps of CO-stained tangential sections through cortical layer IV prepared from B6 (A), BALB (B), and BTBR (C) mice (scale bar= 1000 μm). The 20-μm-thick belt encircling the hollow was defined as the barrel wall. The area between barrel walls was defined as the septum. CO-staining revealed whisker-related pattern in B6 and BALB mice, while the distinctive barrel walls were fuzzy in BTBR mice. VGluT2 (D, E, F) and NeuN (G, H, I) double labeling of tangential sections from the cortex of these three strains were applied to simultaneously visualize the distribution of thalamocortical axons clusters and cortical neurons (scale bar= 100 μm). Unlike the discrete whisker-related VGlT2-positive patches in B6 mice, fuzzy VGluT2-staining patterns were found in BALB and BTBR mice. NeuN staining revealed ring-like organizations of cortical neurons that form a border around the barrel clusters in B6 mice (G), while the ring-like organizations formed by cortical neurons cramming in the septum without border spaces between barrel clusters in BALB mice (H). No clear segregation of cortical neurons was observed in BTBR mice (I). VGluT2 staining of coronal sections prepared from these three mouse strains depicted enriched VGluT2-positive cells in cortical layer IV of B6 (J) and BTBR mice (L), while buzzy VGluT2-staining patterns were found in BALB mice (K). NeuN staining revealed discrete, sectioned structures encircled each hollow in B6 mice (M), while enriched neuronal wall (but relatively chunky inside the barrel patches) at the septum of BALB mice (N). These neural patterning structures shown by NeuN labeling were absent in BTBR mice (O).

Figure 2.

Somatosensory performances of female B6 (blue), BALB (red), and BTBR (yellow) mice (each N=12). (A) The logarithm thresholds for withdrawal responses to filament stimulations on the hindpaws and (B) the withdrawal responsivity to 1g filament stimulation on the hindpaws of B6, BALB, and BTBR mice in the Von Frey touch sense test. (C) Time latencies that the subject mice licked and removed a piece of adhesive tape attached on the hind paws. (D) The gap distance crossed in B6, BALB, and BTBR mice in the gap-crossing test. (E) Latencies of time that subject mice spent to cross a narrow bridge in the dark (F) Motor coordination and balance of these mice during each trial and the average time on the rotarod, manifested as the time that mice could remain on the accelerating rotating rod. All data are presented as means ± S.E.M.; *p < 0.05 compared to B6.

Figure 3.

Object and social recognition tests performed by female B6 (blue; N=14), BALB (red; N=12), and BTBR (yellow; N=12) mice using a habituation/dyshabituation paradigm throughout five trials. In the object recognition test (A), the subject mouse (B: B6, C: BALB, and D: BTBR) was confronted with an identical small object (Obj1) across the four trials (each 3 min) and then in the fifth trial, a novel object (novel) was placed. In the social recognition test (E), the subject mouse of either strain (F: B6, G: BALB, and H: BTBR) was confronted with a wire-mesh bin containing a stimulus mouse of either same strain with the subject (e.g., B6 subjects with B6 stimulus; circle) or different strain from the subject (e.g., B6 subjects with BALB stimulus; square). An identical stimulus mouse was exposed across the initial 4 trials and then in the fifth trial, a stimulus mouse was changed to a novel mouse (e.g., same: B6→B6, different: BALB→BTBR). Data are expressed as the mean investigation time ± S.E.M. Significant differences between trials compared with trial 1 (habituation), *p < 0.05, or trial 4 (dyshabituation), #p < 0.05, and between stimulus mouse (different vs. same), +p < 0.05.

Figure 4.

The percentages of social preference (time spent in social bin/total time spent in both empty and social bins; 50% = chance level) were calculated in B6 (B), BALB (C), or BTBR (D) mice that received a vehicle or buspirone injection (each N=12). Data are expressed as the mean percentages ± S.E.M. Significant differences between stimulus mouse (B6; blue, BALB; red, or BTBR; yellow) sessions compared to Empty session, +p < 0.05, and between vehicle (circle) vs. buspirone (square) injection, #p<0.05. n.s.; non-significant.

Figure 5.

Social interaction test performed by vehicle-injected subject mice of either strain, B6 (N=13), BALB (N=12), or BTBR (N=12) that were exposed to a stimulus mouse of either strain (B6; blue, BALB; red, or BTBR; yellow). Time spent sniff the stimulus mouse (A), time spent in physical contacts with each other (B), time spent sniff by the stimulus mouse (C), and the ratio that the subject mice exhibited evasion to the approaches of the stimulus mouse (D), in the social interaction test. Data are expressed as the mean values ± S.E.M. Significant differences between stimulus strains, *p < 0.05 and between subject strains, ^#p < 0.05. Significant main effects of stimulus (BALB) mice, ⁺p < 0.05.

Figure 6.

Social interaction test performed by mouse pairs of either subject mouse, B6 (vehicle N=13 and buspirone N=12)(A, D, G, and J), BALB (each N=12) (B, E, H, and K), or BTBR (each N=12) (C, F, I, and L) that were injected with either vehicle (circle) or buspirone (square), and stimulus mouse (B6; blue, BALB; red, or BTBR; yellow). Time spent in sniff the stimulus mouse (A, B, and C), time spent in physical contacts each other (D, E, and F), time spent in sniff by the stimulus mouse (G, H, and I), and the ratio of evasion to the approaches of the stimulus mouse (J, K, and L). Data are expressed as the mean values ± S.E.M. Significant differences between stimulus mice compared to B6 stimulus mouse, #p < 0.05; between vehicle vs. buspirone *p < 0.05; and between stimulus mice, +p < 0.05. n.s.; non-significant.

Figure 7.

The percentages of odor preference (time spent in social/total time spent in both empty and social) in B6 subject mice (each N=12) were calculated by B6 (blue; B), BALB (red; C), adult BTBR (yellow; D), or juvenile BTBR (green; E) stimulus mice. Data are expressed as the mean percentages ± S.E.M. Significant differences between stimulus mice compared to empty (grey), #p < 0.05, and between stimulus mice (vehicle (circle) vs. buspirone (square)), +p < 0.05.