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Science 338 (6103): 128-132

Copyright © 2012 by the American Association for the Advancement of Science

Shared Synaptic Pathophysiology in Syndromic and Nonsyndromic Rodent Models of Autism

Stéphane J. Baudouin1, Julien Gaudias1, Stefan Gerharz1,*, Laetitia Hatstatt1, Kuikui Zhou2, Pradeep Punnakkal1, Kenji F. Tanaka3,4, Will Spooren5, Rene Hen3, Chris I. De Zeeuw2,6, Kaspar Vogt1, and Peter Scheiffele1,{dagger}

1 Biozentrum of the University of Basel, Basel, Switzerland.
2 Department of Neuroscience, Erasmus MC, Rotterdam, Netherlands.
3 Department of Neuroscience, Columbia University, New York, NY, USA.
4 Department of Neuropsychiatry, School of Medicine, Keio University, Tokyo, Japan.
5 Hoffmann-La Roche, Basel, Switzerland.
6 Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Amsterdam, Netherlands.

Figure 1
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Fig. 1. Cell type–specific synaptic localization of NL3. (A) Nlgn3KO (Nlgn3STOP-tetO) mice lack NL3 protein expression, and Nlgn3PC mice (Nlgn3STOP-tetO::Pcp2tTA) show reexpression in cerebellar lysates. (B) Mossy fiber inputs (MF) in the inner granular layer (IGL) are relayed to PCs via parallel fibers (PF). Climbing fibers (CF) form synapses directly onto the proximal PC dendritic arborization. In Nlgn3PC mice, NL3 immunoreactivity is increased on PC dendrites (calbindin, CBP) and abolished in the IGL. (C) NL3 is apposed to vGluT1+ PF boutons and colocalizes with mGluR1α in CBP+ spines, similar to GluD2. (D) In glomeruli, NL3 is detected at PSD95+ and GABA-A receptor α2+ synapses and colocalizes with GFP+ terminals in GlyT2GFP transgenic mice.


Figure 2
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Fig. 2. Occlusion of mGluR-LTD in Nlgn3KO mice. (A) PF synaptic ultrastructure in Nlgn3KO mice by transmission electron microscopy (n ≥ 4 animals, 200 synapses per animal). (B) Cumulative distribution of mEPSCs (***P < 0.001, Kolmogorov-Smirnov test) and mean amplitude (n = 9 cells for wild-type and n = 21 cells for Nlgn3KO mice; ***P < 0.001, Mann-Whitney test). (C) mEPSC inter-event intervals, paired pulse ratios (n = 9 wild-type and n = 15 Nlgn3KO cells). (D) mGluR-LTD induced by 10 min of 50 μM DHPG (n ≥ 8 cells) and representative traces before (black) and after (red) DHPG stimulation. (E) Quantitative Western blot of basal and DHPG-induced phospho-GluA2 (normalized to GluA2/3 protein level, n ≥ 4 mice, *P < 0.03, t test). DHPG-induced phosphorylation is expressed as the ratio of treated to untreated samples (n ≥ 4 mice, **P < 0.01, t test).


Figure 3
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Fig. 3. Elevation of synaptic mGluR1α levels in Nlgn3KO mice. (A) Quantitative Western blot. Protein levels were normalized to tubulin and expressed relative to the wild type. (B) Quantitative Western blot of mGluR1α, GluD2, and GluA2/3 levels in the cerebellum (normalized to tubulin and expressed relative to the wild type, n ≥ 4, **P = 0.002, t test). (C) Quantitative line scan on cerebellar sections stained with antibodies to mGluR1α, Homer 1, and calbindin. (D) Line scan intensity ratios of mGluR1α/CBP and Homer1/CBP (n = 6 mice, 1200 synapses per genotype; mGluR1α, *P = 0.04; Homer, P = 0.6; t test). Reexpression of NL3 in PCs restores mGluR1α level (n = 4 mice, P = 0.4). There was no difference in Nlgn1KO mice (n = 6 mice, P = 0.9). (E) DHPG-induced phosphorylation of GluA2 expressed as the ratio of treated to untreated samples (normalized to GluA2/3 protein level, n ≥ 5 mice, * P < 0.05, t test).


Figure 4
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Fig. 4. Disrupted heterosynaptic competition and adult phenotypic rescue in Nlgn3KO mice. (A) vGluT2 puncta on distal PC dendritic tree (upper 20%, dashed line, anticarbonic anhydrase-related protein VIII, Car8) marked by green arrowheads (vGluT2+ clusters per 100 μm2, n = 6 mice per genotype, **P = 0.0023 for wild-type versus Nlgn3KO mice, t test). (B) Density of CF synapses along individual fibers (CRF+, n ≥ 3 mice, P = 0.6). (C) Evoked CF-PC transmission assessed by extracellular stimulation (n ≥ 7 cells from ≥ 4 mice, **P = 0.003, t test). (D) Counted runs, step times, and missed steps on the Erasmus ladder [n ≥ 10, *P < 0.05, **P < 0.01, analysis of variance (ANOVA), post hoc Tukey test]. The performance of Nlgn3PC mice over four training days is indistinguishable from that of wild-type mice (P = 0.2, ANOVA, post hoc Tukey test). (E) Adult reexpression of NL3. Doxycycline (100 μg/ml from embryonic day 0) was removed at P30, and mice were killed at P60. (F) (Left) Quantitative Western blot of mGluR1α in Nlgn3PCadult mice (at P60, n ≥ 5 mice, **P = 0.004). (Right) Quantification of vGluT2 ectopic puncta (at P60, n ≥3 mice, **P = 0.003, *P = 0.03).


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