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1 advances.sciencemag.org/cgi/content/full/1/10/e /dc1 Supplementary Materials for Structural-functional connectivity deficits of neocortical circuits in the Fmr1 /y mouse model of autism Matthias G. Haberl, Valerio Zerbi, Andor Veltien, Melanie Ginger, Arend Heerschap, Andreas Frick This PDF file includes: Published 20 November 2015, Sci. Adv. 1, e (2015) DOI: /sciadv Fig. S1. High-field 11.7-T DT-MRI measurements of the white and gray matter of adult Fmr1 /y and wild-type littermate mice. Fig. S2. Tracing of the input to V1. Fig. S3. Schematic representation of the experimental strategy for the 3D anatomical registration of projection neurons into V1. Fig. S. Functional connectivity matrix of wild-type and Fmr1 /y mice. Legends for movies S1 and S2 Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/1/10/e /dc1) Movie S1 (.mp format). Slice view and 3D view, illustrating the 3D mouse brain model with the combined positions of all retrogradely (input; green) and anterogradely (local; red) labeled neurons in wild-type mice. Movie S2 (.mp format). Slice view and 3D view, illustrating the 3D mouse brain model with the combined positions of all retrogradely (input; green) and anterogradely (local; red) labeled neurons in Fmr1 /y mice.

2 Supplementary Materials A Eva WT Fmr1 -/y FMI Genu/Body EC Splenium/FMJ Eva Au CG HC M1 RS S1 S2 SN Thal Vi B Fmr1 KO AD AD FMI Genu/Body EC Splenium/FMJ Au CG HC M1 RS S1 S2 SN Thal Vi Fig. S1. High-field 11.7T DT-MRI measurements of the white and gray matter of adult Fmr1 -/y and wild-type littermate mice. Tensor images were collectively acquired in several horizontal planes from +2.0 to -.0 mm from the bregma, with an interplane distance of 0.5 mm (WT: n = 12; Fmr1 -/y : n = 7). Neither the first eigenvalue (A; Eva1, which represents the diffusion along the primary diffusion direction) nor the average diffusivity (B; AD, which represents the average of the diffusion along the three main directions and captures the size of the tensor) were significantly different for the corpus callosum, any cortical areas or the thalamus. Values were measured on individual planes and grouped into Splenium/Forceps major of the corpus callosum (FMJ), external capsule IC), Genu/Body of the corpus callosum, Forceps minor of the corpus callosum (FMI) for the corpus callosum, and per brain area for the cortex and thalamus. Data are presented as the mean ± SEM. Statistical significance was determined using multiple t-tests corrected for multiple comparisons using the Holm-Sidak method with alpha = Key: auditory cortex (Au), cingulate cortex (CG), hippocampus (HC), primary motor cortex (M1), retrosplenial cortex (RS), primary somatosensory cortex (S1), secondary somatosensory cortex (S2), substantia nigra (SN), visual cortex (Vi).

3 A/P: mm 2 Bregma A/P: mm A/P: mm A/P: mm V2ML 0 DLG A/P: mm A/P: mm A/P: mm A/P: mm LO A/P: mm A/P: mm A M2 Cg1 RSA LGP M2 IC LGP S1 BF S1 BF MCPO RSA Au1-8 V1 L1 2 L2/3 6 AuD Au1 100µm Au

4 B VO AI MCPO LGP IC A/P: mm A/P: mm A/P: mm A/P: mm LGP V2MM DLG 250µm Bregma A/P: mm A/P: mm V2ML - -6 V1-8 L1 0 2 L2/3 6 AuD A/P: mm 100µm SRad Contralateral Visual Cortex V1 Cortex Hippocampus RSA L1 L2 L6 2 A/P: mm A/P: mm Thalamus L6

5 Fig. S2 Tracing of the input to V1. Representative coronal sections of various brain areas with projecting cells sending input to V1, from one WT (A) and one Fmr1 -/y (B) mouse. Green cells: retrograde rabies virus RV G-eGFP (RV G) infected cells expressing egfp; Red cells: anterograde rabies virus RV G-mCherry(VSV G RtmC ) infected cells expressing mcherry; Blue cellular nuclei: DAPI staining. Key: L1-6: layers 1-6 of the neocortex; Au1: primary auditory cortex; AuD: secondary auditory cortex dorsal area; Cg1: cingulate cortex area 1; DLG: dorsal lateral geniculate nucleus of the thalamus; LGP: lateral globus pallidus; LO: lateral orbital cortex; M2: secondary motor cortex; MCPO: magnocellular preoptic nucleus; RSA: retrosplenial agranular cortex; S1 BF: primary somatosensory cortex barrel field; SRad: stratum radiatum of the hippocampus; : secondary visual cortex lateral area; V2ML: secondary visual cortex mediolateral area; V2MM: secondary visual cortex mediomedial area; VO: ventral orbital cortex. Scale bars: 1 mm (main images) and 25µm (insets), unless otherwise noted.

6 0 A Tracer injections Retrograde tracing Sectioning B Slide scanning slices, 50μm thickness: >7.5mm tissue DLG V1 AuD Au1 C Overlay and Transfer to brain model Brain model D Connectivity calculations Distance Center of injection site #x y z comment Distance from injection site in m Mo M2 Ctx 363, Mo M2 Ctx 3291, Mo M2 Ctx 3635, Mo M2 Ctx 3595, Mo M2 Ctx 336, Mo M2 Ctx 3251, Mo M2 Ctx 333,11 Area Weight

7 Fig. S3. Schematic representation of the experimental strategy for the 3D anatomical registration of projection neurons into V1. (A) Stereotaxic injection permits introduction of the viral tracers necessary for retrograde labeling (green). The inclusion of an anterograde tracer (red) marks the site of injection. Following sufficient time for viral expression, the animal is sacrificed, the brain fixed and then subsequently sectioned to permit microscopy. (B) A slide scanning approach permits the rapid acquisition of sections from the entire fore-brain. (C) Labeled neurons are detected manually from individual sections overlaid with a brain atlas and assigned a numerical coordinate, used for the generation of a 3D model (D). The 3D model is then used to calculate connectivity features (distance of projection neurons (retrogradely labeled, green) from injection site calculated from centre of mass of red-labeled neurons).

8 A LGP L LGP R PF L PF R VPN L VPN R DH L DH R VH L VH R Au L Au R Mo L Mo R S1 L S1 R V1 L V1 R RS Pi L Pi R Am L Am R PtA L PtA R CPu L CPu R DLG L DLG R WT DH L DH R VH L VH R Au L Au R Mo L Mo R S1 L S1 R V1 L V1 R RS Pi L Pi R Am L Am R Fmr1 -/y PtA L PtA R CPu L CPu R DLG L DLG R LGP L LGP R PF L PF R VPN L VPN R 20 z-score 10 0 B Difference Fmr1 - WT Statistical differences PtA L PtA R CPu L CPu R DLG L DLG R LGP L LGP R PF L PF R VPN L VPN R -/y DH L DH R VH L VH R Au L Au R Mo L Mo R S1 L S1 R V1 L V1 R RS Pi L Pi R Am L Am R DH L DH R VH L VH R Au L Au R Mo L Mo R S1 L S1 R V1 L V1 R RS Pi L Pi R Am L Am R PtA L PtA R CPu L CPu R DLG L DLG R LGP L LGP R PF L PF R VPN L VPN R Fmr1 -/y > WT Fmr1 -/y > WT p < 0.05 p < 0.01 p < Fig S. Functional connectivity matrix of wild-type and Fmr1 -/y mice. (A) fmri measurements under light anaesthesia were performed in Fmr1 -/y (n=7) and WT (n=10) Mice. (B) The intracortical functional connectivity is reduced in Fmr1 -/y mice, in particular between the sensory cortical areas

9 (Au, Mo, S1, and V1). None of the analyzed areas showed a significant increase in functional connectivity. Key: Dorsal hippocampus (DH), ventral hippocampus (VH), auditory cortex (Au), motor cortex (Mo), primary somatosensory cortex (S1), primary visual cortex (V1), retrosplenial cortex (RS), Piriform Cortex (Pi), Amygdala (Amy), Pretectal Area (PtA), Caudate Putamen (CPu), lateral geniculate nucleus (DLG), globus pallidus (LGP), parafascicular nucleus (PF), ventral posterolateral nucleus and ventral posteromedial nucleus (VPN). Right (R) and left hemisphere (L). Supplementary Movie S1. Slice view and 3D view, illustrating the 3D mouse brain model with the combined positions of all retrogradely- (input; green) and anterogradely (local; red) labeled neurons in wild-type mice. Supplementary Movie S2. Slice view and 3D view, illustrating the 3D mouse brain model with the combined positions of all retrogradely- (input; green) and anterogradely (local; red) labeled neurons in Fmr1 -/y mice.