Disruption of visual circuit formation and refinement in a mouse model of autism

Abstract

Aberrant connectivity is believed to contribute to the pathophysiology of autism spectrum disorder (ASD). Recent neuroimaging studies have increasingly identified such impairments in patients with ASD, including alterations in sensory systems. However, the cellular substrates and molecular underpinnings of disrupted connectivity remain poorly understood. Utilizing eye-specific segregation in the dorsal lateral geniculate nucleus (dLGN) as a model system, we investigated the formation and refinement of precise patterning of synaptic connections in the BTBR T + tf/J (BTBR) mouse model of ASD. We found that at the neonatal stage, the shape of the dLGN occupied by retinal afferents was altered in the BTBR group compared to C57BL/6J (B6) animals. Notably, the degree of overlap between the ipsi- and contralateral afferents was significantly greater in the BTBR mice. Moreover, these abnormalities continued into mature stage in the BTBR animals, suggesting persistent deficits rather than delayed maturation of axonal refinement. Together, these results indicate disrupted connectivity at the synaptic patterning level in the BTBR mice, suggesting that in general, altered neural circuitry may contribute to autistic behaviours seen in this animal model. In addition, these data are consistent with the notion that lower-level, primary processing mechanisms contribute to altered visual perception in ASD. Autism Res 2017, 10: 212-223. © 2016 The Authors Autism Research published by Wiley Periodicals, Inc. on behalf of International Society for Autism Research.

Keywords: BTBR mouse; autism spectrum disorder; brain circuit; eye-specific segregation; lateral geniculate nucleus; synaptic patterning; visual system.

Figure 1

Retinal input to dLGN in the BTBR animals was smaller in cross‐sectional area and more rounded in shape, but with similar relative location, compared with that in the B6 animals. (A) Experimental design. CTB conjugated with green fluorophore was injected into one eye of the mouse, while CTB conjugated with red fluorophore injected into the other eye. CTB is taken up by retinal ganglion cells and then transported anterogradely by the optic nerve to the axon terminals in the dLGN. Domains with input from either contralateral or ipsilateral eye can be clearly recognised in dLGN, and given a certain species, they have characteristic shape, cross‐sectional area, and position. (B) Left panel: representative image of middle dLGN in the B6 animal. Green signal shows retina ganglion cell input from the contralateral eye, while red signal shows input from the ipsilateral eye. White signal shows DAPI nuclear staining. Right panel: compared with the B6 brain, retinal input to dLGN in the BTBR animals was smaller in cross‐sectional area and more rounded in shape. However, its location was relatively unchanged. Consistent with previous studies [Mercier, Kwon, & Douet, 2012], excessive separation of the two hippocampi was also observed in the BTBR brain. Scale bar: 400 μm.

Figure 2

BTBR animals showed deficits in eye‐specific segregation during neonatal development. (A) Representative images of dLGN receiving both contralateral (green signal) and ipsilateral (red signal) input from the two eyes, and the overlap between the two (yellow pixels), which became apparent after the image was thresholded and digitised. BTBR mice at P8 had increased intermingling between the retinal ganglion cell axons from left and right eyes compared with B6 control. Scale bar: 200 μm. (B) Quantification of the percentage of dLGN receiving overlapping inputs in the BTBR vs. B6 animals at P8. BTBR mice exhibit significantly more overlap than the B6 mice, regardless of the threshold used. Data are represented as mean ± SEM (n = 5 animals for the B6 group, and n = 7 animals for the BTBR group, P < 0.05 by Student's t‐test).

Figure 3

BTBR animals had less total cross‐sectional area of retinal field but greater percentage of ipsilateral input in the middle dLGN. (A) BTBR mice had significantly less total cross‐sectional area of retinal input compared with the B6 animals. (B) The percentage of ipsilateral domain to total dLGN area was significantly increased in the BTBR mice compared to the B6 controls. (C) There was no significant difference in the percentage of contralateral domain to total dLGN area between the two strains. Mean ± SEM (n = 5 animals for the B6 group, and n = 7 animals for the BTBR group, P < 0.05 by Student's t‐test).

Figure 4

Deficits in eye‐specific segregation continued into mature stage in the BTBR animals. (A) Retinogeniculate projection patterns visualised after injecting CTB into left and right eyes of the B6 and BTBR mice. BTBR mice at P30 had more intermingling (yellow pixels in thresholded images, overlap) between retinal ganglion cell axons from left and right eyes compared with the B6 control. Scale bar: 200 μm. (B) Quantification of the percentage of dLGN receiving overlapping inputs in the BTBR vs. B6 animals at P30. BTBR mice exhibit significantly more overlap than B6 mice, independent of the threshold used. Data are represented as mean ± SEM (n = 6 animals for each group, P < 0.05 by Student's t‐test).

Figure 5

Aberrant projections in the dLGN of the BTBR animals. dLGN from P30 B6 (A–C2) or BTBR (D–F2) mice. Boxed regions in lower magnification images (A and D) correspond to higher magnification images on the right with boxes of the same style (B–C2 and E–F2, respectively). Boxes with solid white line show examples of the contralateral domain, while boxes with dotted white line show the ipsilateral domain. Only the ipsilateral input (red signal) is shown in (B2) and (E2); similarly, only the contralateral input (green signal) is shown in (C2) and (F2). There were increased ipsilateral projections within the contralateral domain in the BTBR mice (E and E2) compared to the B6 mice (B and B2). Similarly, there were increased contralateral projections within the ipsilateral domain in the BTBR mice (F and F2) compared to the B6 mice (C and C2). Scale bar in (A) and (D): 100 μm, in (B–C2) and (E–F2): 50 μm.