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. 2007 Oct 5;318(5847):71-6.
doi: 10.1126/science.1146221. Epub 2007 Sep 6.

A neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice

Katsuhiko Tabuchi  1 , Jacqueline Blundell, Mark R Etherton, Robert E Hammer, Xinran Liu, Craig M Powell, Thomas C Südhof
Affiliations

A neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice

Katsuhiko Tabuchi et al. Science. .

Abstract

Autism spectrum disorders (ASDs) are characterized by impairments in social behaviors that are sometimes coupled to specialized cognitive abilities. A small percentage of ASD patients carry mutations in genes encoding neuroligins, which are postsynaptic cell-adhesion molecules. We introduced one of these mutations into mice: the Arg451-->Cys451 (R451C) substitution in neuroligin-3. R451C mutant mice showed impaired social interactions but enhanced spatial learning abilities. Unexpectedly, these behavioral changes were accompanied by an increase in inhibitory synaptic transmission with no apparent effect on excitatory synapses. Deletion of neuroligin-3, in contrast, did not cause such changes, indicating that the R451C substitution represents a gain-of-function mutation. These data suggest that increased inhibitory synaptic transmission may contribute to human ASDs and that the R451C knockin mice may be a useful model for studying autism-related behaviors.

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Figures

Figure 1
Figure 1
Generation and characterization of neuroligin-3 R451C KI and neuroligin-3 KO mice. (A and B) Representative immunoblots and summary graphs of protein levels in the brains of neuroligin-3 R451C KI mice (A) and neuroligin-3 KO mice (B). Selected synaptic proteins (NL1, neuroligin-1; NL3, neuroligin-3; Synaptop., synaptophysin; Syt1, synaptotagmin-1; Syb2, synaptobrevin-2) were analyzed by quantitative immunoblotting; two different neuroligin-3 antibodies were used (528B and 639B; arrows point to neuroligin-3 band; data shown are means ± SEMs; n=4 littermate pairs; * = p<0.05; ** = p<0.01; *** = p<0.005 by Student's t-test).
Figure 2
Figure 2
The R451C-substitution in neuroligin-3 increases the synaptic signal of the vesicular GABA transporter (VGAT) but not the number of inhibitory synapses. (A) Representative confocal immunofluorescence images of sections of the CA1 region of the hippocampus from littermate wild-type and R451C knockin mice double-labeled with antibodies to the vesicular glutamate-transporter VGlut1 (red; top panels) and the vesicular GABA-transporter VGAT (green; middle panels); the bottom panel depicts the merged image (for representative images from other brain areas or sections stained with other antibodies, see Figs. S6–S8). Scale bars in bottom panels that apply to all panels in a column. (B-E) Quantitation of the density of synaptic puncta that are above threshold by image analysis of confocal immunofluorescence sections from the CA1 and CA3 region of the hippocampus (B-D) and from the somatosensory cortex (E) from wild-type and R451C KI mice (top) or neuroligin-3 KO mice (bottom). Quantitations are from sections stained with antibodies to synaptophysin (B; labels all synapses) or to VGAT and VGlut (C-E; label only inhibitory or excitatory synapses, respectively). (F) Representative electron micrographs from layer 2/3 of the somatosensory cortex of wild-type (a) and R451C KI (b) mice (black arrowheads = asymmetric synapses; white arrowheads = symmetric synapses). (G-J) Quantitation of the density of asymmetric (excitatory) and symmetric (inhibitory) synapses (G), of the ratio of asymmetric to symmetric (H) synapses, and of the width of a synapse (J) and its synaptic vesicle numbers (K) in electron micrographs from 3 pairs of littermate wild-type and R451C KI mice (data shown in B-E and G-J are means ± SEMs; n=3 littermate pairs; * = p<0.05; ** = p<0.01; *** = p<0.005 by Student's t-test).
Figure 3
Figure 3
Neuroligin-3 R451C-knockin but not neuroligin-3 KO mice exhibit increased spontaneous inhibitory synaptic transmission. Recordings were performed in whole-cell patch-clamp mode in pyramidal neurons in layer 2/3 of the somatosensory cortex in acute slices. (A-F) Representative traces (A, C and E) and summary graphs of the amplitudes and frequency (B, D and F) of spontaneous miniature excitatory postsynaptic currents (mEPSCs; A and B) and inhibitory postsynaptic currents (mIPSCs; C and D) from R451C KI (A-D) or KO mice (E and F). (G and H) Representative traces (G) and summary graph for response to a locally applied GABA puff (50 µM injected at 5 psi for 1 s) in layer 2/3 of the somatosensory cortex. In G, responses are also shown in the presence of 50 µM picrotoxin to document their inhibitory nature (data shown are means ± SEMs; n=3 littermate pairs; total number of cells recorded are indicated within bars; * = p<0.05; ** = p<0.01; *** = p<0.005 by Student's t-test; all electrophysiological parameters are listed in Table S1).
Figure 4
Figure 4
Selective increase in inhibitory synaptic strength in neuroligin-3 R451C KI but not in neuroligin-3 KO mice. Representative traces (A and D) and summary graphs (C, D, E and F) of synaptic responses induced with increasing stimulus intensities applied with a local microelectrode in acute slices of the somatosensory cortex from littermate pairs of R451C KI mice (A, B, D, and E) or KO mice (C and F). Recordings were obtained in the whole-cell mode in layer 2/3. EPSCs (A-C) and IPSCs (D-F) were analyzed separately after pharmacological isolation. In A and D, arrows and vertical dashed lines indicate peaks measured for determining evoked response amplitude. Dotted horizontal lines represent baselines. All data were recorded in acute slices from littermate R451C-mutant and wild-type mice (data shown are means ± SEMs; n=4 or 3 littermate pairs for EPSCs (KI or KO), and 5 or 3 littermate pairs for IPSCs (KI or KO); * = p
Figure 5
Figure 5
Impaired social interaction behaviors in neuroligin-3 R451C KI mice. (A) Interacting time of individual wild-type and R451C KI mice exposed to a novel inanimate object in an unfamiliar cage (5 min). (B) Interacting time of mice that are exposed to an unfamiliar immobilized target mouse in a now familiar cage (5 min; procedure immediately follows A). (C) Interacting time of mice that are exposed simultaneously to a novel inanimate object and a novel, caged target mouse. (D) Social learning measured by monitoring the time of direct interactions of wild-type and R451C KI mice with the same freely moving juvenile target mouse on day 1 (1st Interaction) and day 4 (2nd Interaction for social learning). All data shown are means ± SEMs; n=19 male littermate pairs; only statistically significant differences between wild-type and R451C KI mice are specifically identified in the figure (* = p<0.05; ** = p<0.01; *** = p<0.001 by t-test or two-way ANOVA); a detailed statistical analysis for all parameters is provided in Table S2 (30).
Figure 6
Figure 6
Neuroligin-3 R451C KI mice exhibit enhanced spatial learning. (A) Morris water maze analysis of spatial learning in R451C KI and littermate wild-type control mice during the initial 7 days of training as measured by the distance traveled to reach a submerged platform. (B) Number of days of initial training required to reach the submerged platform in an average of 10 sec or less. (C) Number of crossings over the previous location of the target platform and over corresponding locations in the other 3 quadrants measured on day 8 after removal of the platform (probe trial). (D) Reversal learning experiment in which on day 9 after the probe trial the platform was moved to the opposite quadrant, and the learning of the new location of the platform by the mice was monitored. Learning is measured as distance traveled prior to mounting the newly localized target platform as a function of days of training. (E) Number of days of reversal training required to reach the submerged platform in an average of 10 sec or less. (F) Probe trial after reversal learning uncovers a large increase in learning abilities of the R451C KI mice. Only statistically significant differences between wild-type and R451C KI mice are identified in the panels (* = p<0.05; ** = p<0.01; *** = p<0.001; in A and D, Genotype = main effect of genotype, Day = main effect of day of training; Interaction = interaction between genotype and day). All data shown are means ± SEMs; n=19 male littermate pairs; see Fig. S14 Table S2 for all statistical comparisons.
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