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Proc Natl Acad Sci U S A. Aug 16, 2011; 108(33): 13764–13769.
Published online Aug 1, 2011. doi:  10.1073/pnas.1111093108
PMCID: PMC3158170
Neuroscience

Autism-linked neuroligin-3 R451C mutation differentially alters hippocampal and cortical synaptic function

Abstract

Multiple independent mutations in neuroligin genes were identified in patients with familial autism, including the R451C substitution in neuroligin-3 (NL3). Previous studies showed that NL3R451C knock-in mice exhibited modestly impaired social behaviors, enhanced water maze learning abilities, and increased synaptic inhibition in the somatosensory cortex, and they suggested that the behavioral changes in these mice may be caused by a general shift of synaptic transmission to inhibition. Here, we confirm that NL3R451C mutant mice behaviorally exhibit social interaction deficits and electrophysiologically display increased synaptic inhibition in the somatosensory cortex. Unexpectedly, however, we find that the NL3R451C mutation produced a strikingly different phenotype in the hippocampus. Specifically, in the hippocampal CA1 region, the NL3R451C mutation caused an ~1.5-fold increase in AMPA receptor-mediated excitatory synaptic transmission, dramatically altered the kinetics of NMDA receptor-mediated synaptic responses, induced an approximately twofold up-regulation of NMDA receptors containing NR2B subunits, and enhanced long-term potentiation almost twofold. NL3 KO mice did not exhibit any of these changes. Quantitative light microscopy and EM revealed that the NL3R451C mutation increased dendritic branching and altered the structure of synapses in the stratum radiatum of the hippocampus. Thus, in NL3R451C mutant mice, a single point mutation in a synaptic cell adhesion molecule causes context-dependent changes in synaptic transmission; these changes are consistent with the broad impact of this mutation on murine and human behaviors, suggesting that NL3 controls excitatory and inhibitory synapse properties in a region- and circuit-specific manner.

Keywords: synapse formation

Autism spectrum disorders (ASDs) constitute a heterogeneous group of neurodevelopmental diseases with a strong genetic component (13). The identification of multiple ASD candidate genes that encode synaptic proteins suggested that ASDs may involve impairments in synaptic transmission (412). In particular, numerous mutations in neuroligins, a family of postsynaptic cell adhesion molecules, have been identified in ASDs (47, 10, 12). Consistent with the hypothesis that synaptic dysfunction contributes to ASD pathogenesis (13, 14), mouse models of two ASD-associated neuroligin mutations exhibit functional changes in synaptic transmission (15, 16).

Neuroligins are not critical for the initial establishment of synapses but are required for normal synapse function (1720). Deletion of neuroligin-1 or -2 selectively impairs excitatory or inhibitory synaptic transmission, respectively (17, 18, 21, 22), whereas at least in the somatosensory cortex, deletion of neuroligin-3 (NL3) causes no major synaptic phenotype (15). Three ASD-relevant neuroligin mutations were characterized in mouse models: the R451C substitution in NL3 (15, 23), a loss of function mutation of NL4 (24), and a point mutation that was identified in the cytoplasmic tail of NL4 in an ASD patient but was analyzed in the context of NL3 (16). Characterization of NL3R451C knock-in mice uncovered a behavioral phenotype composed of social interaction deficits and increased spatial memory and an electrophysiological phenotype consisting of increased inhibitory synaptic transmission in the somatosensory cortex (15). However, an independently generated second line of NL3R451C mutant mice exhibited a different behavioral phenotype, although the physiological phenotype was not tested (23). Because behavioral properties of mice are background-dependent, differences in behavioral phenotypes between mouse lines are not surprising, but physiological properties of synapses are thought to be background-independent, raising the question of whether the two NL3R451C mutant mouse lines exhibit similar synaptic changes. In more general terms, ASD pathogenesis likely involves multiple brain areas, prompting the more important question of whether the NL3R451C mutation has similar or different effects on synaptic transmission in different brain regions and neuronal circuits. Thus, at present, two major questions emerge. First, does the NL3R451C mutation cause the same changes in synaptic transmission in different mouse lines, and second and more importantly, does the NL3R451C mutation change synaptic transmission similarly in all brain regions or act in a circuit-dependent, brain region-specific fashion?

To address these questions, we have here reassessed the behavioral and physiological effects of the NL3R451C mutation in mice. In the physiological studies, we focused on the CA1 region of the hippocampus, which is arguably the best-studied brain area of the mammalian forebrain and among many other behaviors, has also been implicated in sociability (25). Strikingly, we find that the NL3R451C mutation, as assessed in both of the two independently generated mouse lines, caused a large increase in AMPA and NMDA receptor-mediated excitatory synaptic transmission and a dramatic change in NMDA receptor-mediated responses in the hippocampus, whereas the same mutation selectively increased inhibitory synaptic transmission in the somatosensory cortex. Thus, the NL3R451C mutation induces general context-specific alterations in synaptic function that result in region-specific changes in neural circuits with accompanying alterations in ASD-relevant mouse behaviors.

Results

NL3R451C Mutation Impairs Social Interactions.

Because deficits in social interaction represent one of the core criteria for diagnosis of ASDs, we reevaluated social behaviors in NL3R451C mutant mice. Previous analyses of social behaviors in NL3R451C mutant mice provided conflicting results (15, 23), prompting us to reassess the sociability of NL3R451C mutant mice in a different behavioral facility and institution using a three-chamber test (2628). In this test, the subject mouse is first habituated to the testing environment (habituation session), then exposed to a novel mouse in one of the target chambers and a novel object in the opposite chamber (sociability session), and finally, tested for preference for social novelty by replacing the no longer novel object in the opposite chamber with a new unfamiliar mouse.

In the initial habituation session, littermate NL3R451C mutant and WT mice did not exhibit a side preference for the left or right chamber. During the subsequent sociability session, both NL3R451C mutant mice and WT control mice displayed a significant preference for the cage containing the stranger mouse (Fig. 1A). During the final social novelty session, WT mice spent significantly more time sniffing at the novel stranger than at the now-familiar social object, whereas NL3R451C mutant mice did not (Fig. 1A).

Fig. 1.
NL3R451C knock-in mice exhibit social interaction impairments. (A) Three-chamber sociability test. Mice were first simultaneously exposed to an empty round wire container (pencil cup) as a novel object and a caged, unfamiliar mouse (Left). Afterward, ...

To further examine the sociability phenotype observed in the three-chamber test, NL3R451C mutant mice were tested in a caged adult social interaction test (15). In this task, NL3R451C mutant mice showed no change in the time of interaction with a novel inanimate object in the first phase of this test (Fig. 1 B and C). During the second phase of this test, however, mutant mice explored the unfamiliar caged mouse significantly less than WT controls during the entire time course of the second trial (Fig. 1B) (effect of genotype; F1, 306 = 4.47, P = 0.042). In addition, the mutant mice displayed a decreased time of interaction with the target mice during the total trial (Fig. 1C). Thus, two tests confirm a modest but significant social interaction deficit in NL3R451C mutant mice.

NL3R451C Mutation Increases AMPA Receptor-Mediated Synaptic Transmission in the CA1 Region of the Hippocampus.

To examine the effect of the NL3R451C mutation on synaptic transmission in other brain regions than the somatosensory cortex (15), we performed extracellular field recordings in the CA1 region of the hippocampus and additionally, analyzed NL3 KO mice in parallel. We plotted the slope of the field excitatory postsynaptic potential (fEPSP; mediated primarily by AMPA-type glutamate receptors) relative to the number of afferents stimulated, which was measured by the fiber volley amplitude. Input–output measurements using this approach showed that the NL3R451C mutation unexpectedly caused a large increase in excitatory synaptic transmission, whereas the NL3 KO had no effect (Fig. 2 A–C). To ensure that this effect was a direct effect of the NL3R451C mutation, we also analyzed the independently generated NL3R451C mutant mouse line (23) (referred to as R451C*) and observed the same phenotype (Fig. 2C). The observation that this synaptic change is reproducible in two independently generated lines of NL3R451C mutant mice but is absent from NL3 KO mice indicates that the enhancement of AMPA receptor-mediated synaptic transmission in the hippocampus represents a core effect of the NL3R451C mutation. Moreover, this effect is not caused by changes in presynaptic release probability, which was assessed by paired-pulse facilitation experiments (Fig. S1).

Fig. 2.
NL3R451C knock-in mutation increases excitatory synaptic transmission in the CA1 region of the hippocampus. (A) Representative traces and summary graph of input–output measurements performed by extracellular field recordings in acute hippocampal ...

NL3R451C Mutation Enhances NMDA Receptor-Mediated Hippocampal Synaptic Transmission.

To further characterize the effects of the NL3R451C mutation on excitatory synaptic transmission, we analyzed the ratio of synaptic transmission mediated by NMDA- vs. AMPA-type glutamate receptors (the NMDA/AMPA ratio) in CA1 region pyramidal neurons (Fig. 3A). The NMDA/AMPA ratio was significantly increased in the hippocampus in NL3R451C mutant mice but was unchanged in NL3 KO mice (Fig. 3B). Conversely, the NMDA/AMPA ratio was not significantly altered by the NL3R451C mutation in layer 2/3 pyramidal neurons of the somatosensory cortex (Fig. 3 A and B). Analysis of the independently generated NL3R451C mutant mouse line (23) confirmed the increase in NMDA/AMPA ratio in the hippocampus (Fig. 3B). Because the input–output measurements (Fig. 2) showed that the NL3R451C mutation enhances AMPA receptor-mediated synaptic transmission, the enhanced NMDA/AMPA ratio implies that NMDA receptor-mediated transmission is increased in NL3R451C mutant mice to an even greater extent than AMPA receptor-mediated transmission.

Fig. 3.
NL3R451C knock-in mutation increases NMDA receptor-mediated synaptic responses and LTP in the hippocampus. (A) Sample traces of measurements of the ratio of NMDA vs. AMPA receptor-mediated synaptic currents. Analyses compared littermate WT and NL3R451C ...

The results obtained in the hippocampus differ dramatically from those results described for the somatosensory cortex, where an increase in spontaneous and evoked inhibitory synaptic transmission was detected (15). To reassess whether the NL3R451C mutation produces circuit-specific alterations of synaptic transmission, we directly compared spontaneous miniature synaptic events in acute slices from the hippocampus and somatosensory cortex of NL3R451C mutant and NL3 KO mice (Fig. S2). The NL3R451C mutation produced a significant increase in the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in the hippocampus without a change in miniature inhibitory postsynaptic currents (mIPSCs) but an increase in mIPSCs in the somatosensory cortex without a change in mEPSCs. These results confirm the circuit specificity of the NL3R451C mutant phenotype and agree with the above findings that the NL3R451C mutation enhances AMPA receptor-mediated synaptic transmission (Fig. S2). In addition, the mini recordings uncovered the only phenotype in NL3 KO mice that we detected in the current study, namely a decrease in mEPSC frequency in the hippocampus, which is opposite of the increase caused by the NL3R451C mutation, and an increase in the mIPSC frequency (Fig. S2).

Enhanced Long-Term Potentiation (LTP) in NL3R451C Mutant Mice.

The increased NMDA/AMPA ratio observed in NL3R451C mutant mice led us to hypothesize that NL3R451C mutant mice may have enhanced NMDA receptor-dependent LTP in the CA1 region of the hippocampus, a finding that could partly explain the increased spatial memory observed in these mice (15). To test this hypothesis, we assessed LTP in response to three stimulus trains (100 Hz for 1 s) using extracellular field recordings (Fig. 3C). Indeed, NL3R451C mutant mice exhibited significantly enhanced LTP (Fig. 3D). Quantitation of the degree of LTP at 55–60 min after induction showed an ~70% increase in NL3R451C mutant mice, whereas LTP in NL3 KO mice was unchanged (Fig. 3E).

NL3R451C Mutation Alters the Synaptic NMDA Receptor Subunit Composition in the Hippocampus.

The increased NMDA receptor-mediated synaptic transmission in the hippocampus from NL3R451C mutant mice could be because of either an increase in the number of NMDA receptors and/or a change in the NMDA receptor subunit composition, resulting in higher conductances (29, 30). We, thus, characterized the properties of NMDA receptors in NL3R451C mutant mice. The NMDA receptor current–voltage relationship was normal in NL3R451C mutant mice, implying that there is no change in the Mg2+ sensitivity of NMDA receptors (Fig. 4A). Interestingly, the decay time constant of NMDA receptor-mediated responses at +40 mV was significantly increased (Fig. 4B), suggesting a shift in NMDA receptor subunit composition (29, 30), with the most likely explanation being a switch from NR2A-type subunit to NR2B-type subunit containing NMDA receptors.

Fig. 4.
NL3R451C mutation alters kinetics and composition of synaptic NMDA receptors. (A) Normalized sample traces (Left) and summary graph (Right) for NMDA receptor current voltage measurements. (B) Normalized sample traces (Left) and summary graph (Right) for ...

To probe this possibility, we measured the levels of selected synaptic proteins in the hippocampus by quantitative immunoblotting (Fig. 4C). Consistent with previous reports (15, 23), the NL3R451C mutation significantly reduced NL3 protein levels and had no effect on most synaptic proteins, including AMPA receptors. However, the NL3R451C mutation increased the concentrations of excitatory postsynaptic scaffolding proteins (PSD-95 and SAP-102) and doubled the concentration of the NMDA receptor subunit NR2B (Fig. 4C). No change in other synaptic proteins, particularly proteins specific for inhibitory synapses, was observed (Fig. S3C).

To further test whether the NL3R451C mutation alters postsynaptic glutamate receptor composition, we pharmacologically isolated the different components of NMDA receptor-mediated responses using ifenprodil, a selective antagonist of NR1/NR2B heterotetrameric NMDA receptors (31). In NL3R451C mutant mice, the ifenprodil sensitivity of NMDA receptor-mediated EPSCs was greater than in WT controls (Fig. S3), consistent with the hypothesis that the enhanced NMDA/AMPA ratio is, at least in part, secondary to a relative up-regulation of synaptic NR2B-containing NMDA receptors in CA1 pyramidal neurons. Thus, both the electrophysiological and biochemical results suggest that the NL3R451C mutation alters the synaptic NMDA receptor subunit composition, resulting in enhanced expression of NR2B-containing receptors.

NL3R451C Mutant Pyramidal Neurons Exhibit Changes in Synapse Structure and Dendritic Complexity.

Although previous studies in the somatosensory cortex failed to uncover major morphological changes in NL3R451C mutant neurons (15), subtle structural alterations may have been missed. To assess the structure of dendrites in NL3R451C mutant neurons, we performed Sholl analyses on biocytin-filled CA1 pyramidal neurons. Interestingly, we found that NL3R451C mutant pyramidal neurons had a significantly greater number of dendritic branch points in the stratum radiatum but not in the stratum oriens or stratum lacunosum moleculare (Fig. 5 A and B and Fig. S4). The density of spines on secondary dendrites, however, was unchanged (Fig. 5C), suggesting that, overall, the neurons contain more synapses in the stratum radiatum; this finding is in agreement with the observed increase in AMPA receptor-mediated synaptic transmission.

Fig. 5.
Changes in dendritic branching and synapse structure in NL3R451C mutant stratum radiatum of the CA1 region of the hippocampus. (A and B) Representative images (A) and summary graph of the number of dendritic branch points (B) of biocytin-filled pyramidal ...

To further assess the phenotype of the NL3R451C mutation, we examined by EM the structure of excitatory synapses in the stratum radiatum of NL3R451C mutant mice (Fig. 5D). We observed no major changes in the size of the postsynaptic density or number of docked vesicles in NL3R451C mutant mice (Fig. 5 E and F), but we detected significant reductions in spine area, bouton area, and total number of vesicles per bouton (Fig. 5 G–I). Thus, the NL3R451C mutation increases dendritic branching in the stratum radiatum of the CA1 region of the hippocampus without changing the density of synapses per branch length and renders the size, but not the junctional complex of synapses, significantly smaller.

Discussion

In the present study, we show that a single point mutation in NL3, the NL3R451C mutation that was associated with highly penetrant autism in a Swedish family (4), alters synaptic function in a circuit-dependent manner. Specifically, our findings are consistent with the following conclusions:

  • i) The NL3R451C mutant phenotype differs dramatically from the phenotype of the NL3 KO, suggesting that, despite the destabilization of NL3 protein by the R451C mutation that causes a loss of most NL3 protein (Fig. 4C), the remaining ~10% of NL3R451C mutant protein exerts a powerful gain of function effect on synaptic transmission.
  • ii) The NL3R451C mutation induces distinct phenotypes in the CA1 region of the hippocampus and layer 2/3 of the somatosensory cortex. In the former, it causes an enhancement of excitatory synaptic transmission and a change in the glutamate receptor composition of synapses; in the latter, it produces an enhancement of inhibitory synaptic transmission. Thus, the NL3R451C mutation does not act by the same mechanism in all synapses, but its effects are dependent on the synaptic context.
  • iii) In the CA1 region of the hippocampus, the NL3R451C mutation not only increased AMPA receptor-mediated synaptic transmission but also enhanced NMDA receptor-mediated transmission to an even larger degree, and it altered the subunit composition of postsynaptic NMDA receptors (Figs. 24). This finding suggests that the NL3R451C mutation not only increases synaptic transmission in a context-dependent fashion but also changes the properties of the affected synapses.
  • iv) Morphologically, the NL3R451C mutation enhanced the complexity of dendritic branching in the stratum radiatum of the hippocampal CA1 region (Fig. 5 A and B), thereby likely increasing synapse numbers and accounting for the increased excitatory synaptic transmission (Fig. 2 and Fig. S2). In NL3R451C mutant mice, the synapses in the stratum radiatum contain a relatively higher content of NR2B-type NMDA receptors (Fig. 4 B and C and Fig. S3), are morphologically smaller (Fig. 5 G–I), and exhibit greater LTP (Fig. 3 C–E). However, postsynaptic density (PSD) length and mEPSC amplitudes are normal. Thus, in the stratum radiatum, the NL3R451C mutation delays specific components of normal synaptic maturation while simultaneously increasing dendritic complexity.

A potential concern with a germline mutation introduced into mice by homologous recombination is that additional genetic changes are induced that may cause unrelated phenotypes. This concern was raised for the NL3R451C mutation, because different behavioral phenotypes were reported for two independently generated mouse lines (15, 23). However, we show here that, in two key electrophysiological tests, two independent mouse lines exhibit the same phenotype, suggesting that this phenotype truly reflects a functional change induced by the NL3R451C mutation.

The molecular basis of the NL3R451C mutant phenotype and its circuit specificity remains unclear. The circuit specificity of the phenotype suggests that the NL3R451C mutant protein does not produce the phenotype by interactions with canonical building blocks of all synapses—in which case, the phenotype should be similar in all neurons—but that its effect is dependent on the local protein environment unique to a particular synapse. Because in situ hybridizations indicate that NL3 is expressed in all brain regions and all neurons (17) (www.brain-map.org), it seems unlikely that the region specificity of the NL3R451C mutant phenotype is caused by region-specific NL3 expression patterns. Instead, it seems reasonable that the differential expression of NL3 ligands (known and unknown) is responsible for this phenotype. In support of this hypothesis, distinct region-specific expression patterns have been shown for neurexin isoforms and splice variants (32, 33). Moreover, other NL3 ligands likely contribute to NL3 function (20) and may also participate in the NL3R451C mutant phenotype; identification of such ligands will be required to clarify this issue.

The NL3R451C mutation was identified in a family with two affected sons with ASDs whose mutation was inherited from an asymptomatic heterozygous mother (4). The dramatic circuit-specific effects of the NL3R451C mutation on synaptic transmission could account for the diverse and variable symptoms observed in ASDs, which include not only impairments in social communication but also stereotypic behaviors, restricted interests, seizures, and changes in cognitive abilities ranging from common mental retardation to rare improvements in spatial or mathematical abilities. Genetic background effects and developmental influences likely differentially shape synaptic properties in different brain regions; in fact, the two autistic brothers carrying the NL3R451C mutation exhibited significant clinical heterogeneity: one brother had classical autism, whereas the other had Asperger's syndrome (4). The pervasive but distinct synaptic phenotype produced by the NL3R451C mutation in different brain regions suggests that the diverse clinical spectrum of ASD pathologies is secondary to region-specific susceptibilities that are modulated by genetic background effects and developmental or environmental influences.

Experimental Procedures

Mouse Husbandry and Genotyping.

Mice were genotyped as described (15). All animal protocols and husbandry practices were approved by the Institutional Animal Care and Use Committee at Stanford University. All experiments were performed on littermate WT and mutant male mice.

Behavioral assays were performed using an established three-chamber sociability and social novelty test (2628) and a caged adult social interaction text (15) as described in detail in SI Text.

Electrophysiology recordings were performed as described (15, 31) using acute slices from littermate WT or NL3R451C mutant mice (P28-40; details in SI Text). Extracellular field recordings were performed in 50 μM picrotoxin using patch pipettes (2–4 MΩ) filled with artificial cerebrospinal fluid and stimulation rates of 0.1 Hz (34). Whole-cell voltage-clamp recordings were performed with excitatory (117.5 mM CsMeSO4, 10 mM Hepes, 10 mM tetraethylammonium chloride (TEA-Cl), 15.5 mM CsCl, 1 mM MgCl2, 10 mM Na-phosphocreatine, 8 mM NaCl, 0.3 mM NaGTP, 4 mM MgATP, 5 mM EGTA, 1 mM QX-314) or inhibitory specific (120 mM CsCl, 10 mM Hepes, 5 mM NaCl, 1 mM MgCl2, 0.3 mM NaGTP, 3 mM MgATP, 10 mM EGTA, 5 mM QX-314) internal pipette solutions in artificial cerebrospinal fluid containing 50 μM picrotoxin (for recording excitatory events) or 10 μM 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (Tocris) and 50 μM AP5 (Tocris) (for recording inhibitory events). CA1 pyramidal neurons and layer 2/3 pyramidal neurons were patched based on their morphology and location. mEPSC/mIPSC recordings were performed in the presence of 0.5 mM tetrodotoxin (TTX) (31). NMDA/AMPA receptor ratios were analyzed in two steps for each neuron. First, stable synaptic responses were obtained at −70 mV (the amplitude of these responses was the AMPA-R–specific component); next, the holding potential was changed to +40 mV, and dual component EPSCs were collected. At 50 ms poststimulus, when the AMPA-R contribution was negligible, the amplitude of the dual component EPSC was interpreted as the NMDA-R–specific component. For NMDA/AMPA experiments in layer 2/3 pyramidal neurons, a stimulating electrode was placed in layer 1 of the somatosensory cortex. Extracellular LTP experiments were performed as described previously (35). NMDA receptor current/voltage relationships were examined in 50 μM picrotoxin and 10 μM NBQX with 5–10 traces at each holding potential. For NMDA receptor decay kinetics, at least 40 traces were averaged at +40 mV. The EPSC decay time constants were determined as the average-weighted mean of time constants in a double exponential fit. Ifenprodil experiments were performed in two stages. Stable NMDA-mediated responses were established at +40 mV for at least 3 min; then, 5 μM ifenprodil was bath-applied, and responses were recorded until ifenprodil block was complete.

Immunoblotting and Protein Quantitation.

Hippocampi from littermate mice (6 wk) were solubilized in PBS containing 1% Nonidet P-40, 0.1% SDS, and 1% deoxycholate, they were centrifuged at 20,000 × gav for 20 min, and the supernatants were analyzed by quantitative immunoblotting using 125I-labeled secondary antibodies (36).

Light Microscopy.

To quantify dendritic complexity by Sholl analysis, CA1 pyramidal cells were visualized after biocytin delivery during patch-clamp recording (37). Spine quantitations were done on patched neurons filled with dextran-conjugated Alexa-555 (Invitrogen).

EM.

Sixty-nanometer-thick sections were obtained from the stratum radiatum of the CA1 region of the hippocampus of two pairs of littermate WT and NL3R451C mutant mice. Sections were poststained with uranyl acetate and lead citrate, and they were examined with a FEI Tecnai transmission electron microscope at 120 kV accelerating voltage; digital images were captured with a 1 × 1 k SIS Morada CCD camera. Quantitative analyses were conducted on digital electron micrographs at magnifications of 18,500 to ~30,000, with average sample areas of 41 μm2 and 50 images/animal. The number of docked vesicles (defined as vesicles touching the presynaptic plasma membrane), number of vesicles per bouton, PSD length, and bouton and spine area were analyzed using MetaMorph software (Molecular Devices).

Statistical Analyses.

Pair-wise comparisons were analyzed by Student t test, whereas probability distributions were examined using the Kolmogorov–Smirnov test. All data represent mean ± SEM. For all experiments, the experimenter was blind to genotype throughout data collection and analysis.

Supplementary Material

Supporting Information:

Acknowledgments

This work was supported by National Institute of Mental Health Grant R37 MH052804 (to T.C.S.) and Simons Foundation Grant SF177850 (to T.C.S.).

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1111093108/-/DCSupplemental.

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