An autism-associated point mutation in the neuroligin cytoplasmic tail selectively impairs AMPA receptor-mediated synaptic transmission in hippocampus
2011; Springer Nature; Volume: 30; Issue: 14 Linguagem: Inglês
10.1038/emboj.2011.182
ISSN1460-2075
AutoresMark R. Etherton, Katsuhiko Tabuchi, Manu Sharma, Jaewon Ko, Thomas C. Südhof,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle3 June 2011free access An autism-associated point mutation in the neuroligin cytoplasmic tail selectively impairs AMPA receptor-mediated synaptic transmission in hippocampus Mark R Etherton Mark R Etherton Department of Molecular and Cellular Physiology, Stanford University, Palo Alto, CA, USA Search for more papers by this author Katsuhiko Tabuchi Katsuhiko Tabuchi Department of Molecular and Cellular Physiology, Stanford University, Palo Alto, CA, USA Division of Cerebral Structure, Department of Cerebral Research, National Institute for Physiological Sciences, Okazaki, Japan Search for more papers by this author Manu Sharma Manu Sharma Department of Molecular and Cellular Physiology, Stanford University, Palo Alto, CA, USA Search for more papers by this author Jaewon Ko Jaewon Ko Department of Molecular and Cellular Physiology, Stanford University, Palo Alto, CA, USA Search for more papers by this author Thomas C Südhof Corresponding Author Thomas C Südhof Department of Molecular and Cellular Physiology, Stanford University, Palo Alto, CA, USA Howard Hughes Medical Institute, Stanford University, Palo Alto, CA, USA Search for more papers by this author Mark R Etherton Mark R Etherton Department of Molecular and Cellular Physiology, Stanford University, Palo Alto, CA, USA Search for more papers by this author Katsuhiko Tabuchi Katsuhiko Tabuchi Department of Molecular and Cellular Physiology, Stanford University, Palo Alto, CA, USA Division of Cerebral Structure, Department of Cerebral Research, National Institute for Physiological Sciences, Okazaki, Japan Search for more papers by this author Manu Sharma Manu Sharma Department of Molecular and Cellular Physiology, Stanford University, Palo Alto, CA, USA Search for more papers by this author Jaewon Ko Jaewon Ko Department of Molecular and Cellular Physiology, Stanford University, Palo Alto, CA, USA Search for more papers by this author Thomas C Südhof Corresponding Author Thomas C Südhof Department of Molecular and Cellular Physiology, Stanford University, Palo Alto, CA, USA Howard Hughes Medical Institute, Stanford University, Palo Alto, CA, USA Search for more papers by this author Author Information Mark R Etherton1, Katsuhiko Tabuchi1,2, Manu Sharma1, Jaewon Ko1 and Thomas C Südhof 1,3 1Department of Molecular and Cellular Physiology, Stanford University, Palo Alto, CA, USA 2Division of Cerebral Structure, Department of Cerebral Research, National Institute for Physiological Sciences, Okazaki, Japan 3Howard Hughes Medical Institute, Stanford University, Palo Alto, CA, USA *Corresponding author. Department of Molecular and Cellular Physiology, Neurology, and Psychiatry and Behavioral Sciences, Stanford School of Medicine Neuroscience Institute, 1050 Arastradero Road, B253, Palo Alto, CA 94304-5543, USA. Tel.: +1 650 721 1421; Fax: +1 650 498 4585; E-mail: [email protected] The EMBO Journal (2011)30:2908-2919https://doi.org/10.1038/emboj.2011.182 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Neuroligins are evolutionarily conserved postsynaptic cell-adhesion molecules that function, at least in part, by forming trans-synaptic complexes with presynaptic neurexins. Different neuroligin isoforms perform diverse functions and exhibit distinct intracellular localizations, but contain similar cytoplasmic sequences whose role remains largely unknown. Here, we analysed the effect of a single amino-acid substitution (R704C) that targets a conserved arginine residue in the cytoplasmic sequence of all neuroligins, and that was associated with autism in neuroligin-4. We introduced the R704C mutation into mouse neuroligin-3 by homologous recombination, and examined its effect on synapses in vitro and in vivo. Electrophysiological and morphological studies revealed that the neuroligin-3 R704C mutation did not significantly alter synapse formation, but dramatically impaired synapse function. Specifically, the R704C mutation caused a major and selective decrease in AMPA receptor-mediated synaptic transmission in pyramidal neurons of the hippocampus, without similarly changing NMDA or GABA receptor-mediated synaptic transmission, and without detectably altering presynaptic neurotransmitter release. Our results suggest that the cytoplasmic tail of neuroligin-3 has a central role in synaptic transmission by modulating the recruitment of AMPA receptors to postsynaptic sites at excitatory synapses. Introduction Animal brains process information in vast parallel networks of neurons connected by synapses. Although synapses are primarily known for synaptic transmission, which consists of presynaptic neurotransmitter release and the postsynaptic reception of neurotransmitters, it should be noted that synapses fundamentally operate as intercellular junctions, wherein presynaptic and postsynaptic sides are connected by trans-synaptic cell-adhesion molecules. Neurexins and neuroligins are heterotypic cell-adhesion molecules that arguably constitute the best characterized trans-synaptic cell-adhesion pair (Ushkaryov et al, 1992; Ichtchenko et al, 1995). Presynaptic neurexins are differentially expressed in all neurons from three genes in two principal forms (α- and β-neurexins; Ullrich et al, 1995), whereas postsynaptic neuroligins are produced from four genes (NL1–NL4) that are largely co-expressed in most neurons (Ichtchenko et al, 1995). All neurexins and neuroligins bind to each other, although with distinct affinities dictated by isoforms and alternative splicing (Boucard et al, 2005; Chih et al, 2006; Comoletti et al, 2006; Graf et al, 2006; Arac et al, 2007). Neurexins and neuroligins are highly conserved evolutionarily except for NL4 which, at least in rodents, is poorly conserved and expressed at very low levels (Bolliger et al, 2008). Neurexins and neuroligins are essential for synapse function. Triple knockout (KO) of all α-neurexins (Missler et al, 2003) or triple KO of NL1, NL2 and NL3 (Varoqueaux et al, 2006) is lethal, and produces nearly complete inactivation of synaptic transmission. Even single KO's of α-neurexins (Missler et al, 2003) or of neuroligins (Chubykin et al, 2007) induce major phenotypes. The general importance of neurexins and neuroligins is confirmed by the observation of multiple mutations in the genes encoding neurexins and neuroligins in neurological disorders. In particular, heterozygous deletion of neurexin-1α severely predisposes to autism and schizophrenia (Feng et al, 2006; Sebat et al, 2007; Szatmari et al, 2007; Kirov et al, 2008; Marshall et al, 2008; Walsh et al, 2008; Yan et al, 2008; Zahir et al, 2008; Kim et al, 2008a; Bucan et al, 2009; Glessner et al, 2009; Rujescu et al, 2009), whereas a single missense mutation in NL3 (R451C) and a large number of frameshift and missense mutations in NL4 lead to autism and/or mental retardation with complete penetrance (Jamain et al, 2003; Laumonnier et al, 2004; Yan et al, 2005; Talebizadeh et al, 2006; Lawson-Yuen et al, 2008; reviewed in Südhof, 2008). Neuroligins are type I membrane proteins composed of a large extracellular domain homologous to cholinesterases, an extracellular O-glycosylated sequence, a single transmembrane region and a short cytoplasmic tail (Figure 1A). Neuroligin isoforms are differentially distributed among synapses in the same neuron (Song et al, 1999; Graf et al, 2004; Varoqueaux et al, 2004; Budreck and Scheiffele, 2007). The cytoplasmic tails of neuroligins are highly conserved, and include multiple sequence motifs that are present in all isoforms, raising the question of how the cytoplasmic sequences of neuroligins function. Among the conserved sequence motifs of cytoplasmic neuroligin tails, only the role of the C-terminal PDZ-binding sequence—which interacts with several PDZ domain proteins in vitro including PSD-95 (Irie et al, 1997)—is known, whereas the significance of the other sequence motifs remains unclear. Figure 1.R704C point mutation in the cytoplasmic tail of NL3 does not impair in vitro synaptogenic activity of NL3. (A) Diagram of the neuroligin domain structure (top; SP, signal peptide; EHD, esterase-homology domain; O-gly, O-glycosylation sequence; TMR, transmembrane region; PDZ, PDZ domain-binding motif) and alignment of neuroligin sequences surrounding the mutated residue R704C (bottom; arginine corresponding to R704 is shown in red typeface; intramembranous sequence, blue typeface; cytoplasmic sequence, black typeface; sequences show mouse NL1–NL4 and Drosophila neuroligin (NM_001170191.1)). (B) Co-immunoprecipitation experiment demonstrating that the R704C mutation does not block binding of NL3 to PSD-95. HA-tagged PSD-95 was transfected alone or together with Flag-tagged wild-type or R704C mutant NL3 into HEK293 cells, and co-immunoprecipitation of PSD-95 with NL3 was assayed. Data shown are representative immunoblot visualized by ECL. (C, D) Representative images (C) and quantitations (D) of artificial synapse formation on COS cells expressing mVenus only, or mVenus-fused wild-type NL3 (WT) or mutant NL3R704C (R704C). Synapses were quantified as the synapsin signal observed on the COS cells; transfection efficiency was measured as the mVenus signal. Scale bar=20 μm. (E, F) Representative images (E) and synapse quantitations (F) in neurons transfected with mVenus alone, or mVenus-fused wild-type (WT) or R704C-mutant NL3 (R704C). The synapse density per unit dendrite (E, left panels) or synapse size (E, right panels) was quantified based on the measurements of postsynaptic spines (as visualized with the mVenus signal) or presynaptic terminals (as measured by synapsin staining). Scale bar=5 μm. Data in (D, F) are mean values±s.e.m. (*P<0.05; ***P<0.001 by Student's t-test; (D) n=34 mVenus control-transfected, 27 WT NL3-transfected and 28 R704C NL3-transfected cells; (F) n=38 mVenus control-transfected, 41 WT NL3-transfected and 40 R704C NL3-transfected neurons; for both (D) and (F), three independent culture experiments were performed, with statistics based on the number of experiments and not the number of cells analysed). Download figure Download PowerPoint More than 20 mutations in NL4 were associated with autism (see references cited above). Most of these mutations predictably disrupt NL4 expression or folding, suggesting a loss-of-function mechanism during pathogenesis (Jamain et al, 2003; Laumonnier et al, 2004; Yan et al, 2005; Talebizadeh et al, 2006; Lawson-Yuen et al, 2008; reviewed in Südhof (2008) and Zhang et al (2009)). Some substitution mutations, however, do not indicate an obvious functional effect, raising the possibility that at least a subset of the described neuroligin mutations may be polymorphisms without functional consequence, or mediate gain-of-function effects. This is particularly true for the only mutation in a cytoplasmic residue of a neuroligin that has been described in autism, the R704C substitution (Yan et al, 2005). Although this substitution affects a highly conserved residue (Figure 1A), the arginine residue involved localizes close to the transmembrane region in a cluster of four positively charged residues, raising doubts about the significance of neutralizing a single charge in this cluster by the R704C mutation. In the present study, we have tested the significance of R704 and its substitution to cysteine using in vitro and in vivo approaches. We performed these experiments in NL3 because this isoform is well conserved evolutionarily and highly expressed, and because we previously showed that a different autism-associated point mutation in NL3, R451C, produces a gain-of-function phenotype with distinct effects on synaptic transmission that differ from the NL3 KO phenotype (Tabuchi et al, 2007). Thus, we could relate the R704C-mutant phenotype to that of the NL3 KO and of the R451C mutation. Our results reveal that although the R704C substitution does not detectably alter the activity of NL3 in synapse formation, it produces a dramatic deficit in AMPA receptor-mediated synaptic transmission. This synaptic transmission deficit implies that the conserved juxtamembranous sequence motif of neuroligins performs a critical function in regulating synaptic transmission by a postsynaptic mechanism, and by extension, that the corresponding mutation in human NL4 is pathophysiologically significant. Results The R704C substitution does not detectably alter in vitro activities of NL3 To evaluate the effect of the R704C substitution on NL3 function in vitro, we examined binding of wild-type and R704C-mutant NL3 to PSD-95 (Irie et al, 1997). Co-immunoprecipitation experiments showed that the R704C mutation did not disrupt PSD-95 binding (Figure 1B), as would be expected given the distance of the R704 residue to the PDZ domain-binding motif of NL3 (Figure 1A). Next, we tested the ability of overexpressed wild-type or R704C-mutant NL3 to increase synapse densities on transfected COS cells in the artificial synapse formation assay (Figure 1C and D; Scheiffele et al, 2000), and in transfected neurons (Figure 1E and F; Boucard et al, 2005). In both assays, wild-type and R704C-mutant NL3 potently enhanced the number of detectable synapses, probably by stabilizing transient synapses formed on the transfected cells (Chubykin et al, 2007). Although there was a trend for a lower activity by R704C-mutant NL3, its effectiveness was not statistically different from that of wild-type NL3. Thus, the NL3R704C mutation does not significantly perturb the in vitro activity of NL3. Generation of NL3 R704C (NL3R704C) KI mice Next, we introduced the R704C mutation by homologous recombination into the mouse NL3 gene (Figure 2A). NL3R704C-mutant mice exhibited no obvious survival phenotype (adult male offspring from matings of heterozygous females with wild-type males: wild type, n=24 mice; R704C, n=28 mice). Figure 2.Generation and characterization of NL3R704C knock-in mice. (A) Homologous recombination strategy. The structures of the wild-type NL3 gene (E6–E8=exons 6–8), the targeting vector (DT, diphtheria toxin cassette and NEO, neomycin-resistance cassette), the recombined allele containing NEO (third line) and the recombined allele after flp excision of NEO are shown from top to bottom. Asterisk in exon 8 indicates R704C point mutation. (B, C) Representative immunoblots (B) and summary graphs of protein levels (C) in wild-type and NL3R704C-mutant brains analysed by quantitative immunoblotting using 125I-labelled secondary antibodies (n=4 pairs). (D, E) Further immunoblotting analysis of the protein levels of glutamate receptor subtypes in wild-type and NL3R704C-mutant brains analysed by quantitative immunoblotting using 125I-labelled secondary antibodies (n=5 WT and 6 R704C mice). Abbreviations used in (C, E): Syt1, synaptotagmin-1; Syb2, synaptobrevin-2; NR1, NR2a and NR2b, NMDA receptor subunit 1, 2a and 2b, respectively. Data in (C, E) are mean values±s.e.m. (*P<0.05 by Student's t-test). Download figure Download PowerPoint Since previous data revealed that another autism-relevant neuroligin point mutation, the NL3R451C mutation, severely destabilized NL3 (Comoletti et al, 2004; Tabuchi et al, 2007; De Jaco et al, 2010), we quantified the levels of NL3 and of other synaptic proteins in NL3R704C-mutant mice. We detected only a modest decrease in NL3, but observed a small increase in the levels of the AMPA-type glutamate receptor subunit GluR1 (Figure 2B and C), suggesting a potential genetic interaction between GluR1 and NL3. To further explore the possibility that the R704C mutation alters glutamate receptor levels, we performed additional, more extensive quantitative immunoblotting analyses examining all major glutamate receptor subtypes (Figure 2D and E). These experiments confirmed the significant increase in GluR1 and additionally uncovered a significant increase in GluR3, suggesting that the R704C mutation increases AMPA-type glutamate receptor levels in the hippocampus. We then examined the effect of the NL3R704C mutation on brain development and synapse formation. Immunocytochemical analyses of brain sections failed to uncover major abnormalities (Figure 3). Specifically, we quantified synapse densities in three different brain regions (the CA1 and CA3 regions of the hippocampus or in the somatosensory cortex) using three independent markers (synaptophysin as a marker for all synapses, and the vesicular glutamate (vGlut1) and GABA transporters (vGAT) as markers for excitatory and inhibitory synapses, respectively). The results showed that both the overall synapse density and size and the density and size of excitatory versus inhibitory synapses were not detectably altered by the NL3R704C mutation (Figure 3A and B). Figure 3.NL3R704C-mutant mice exhibit no changes in hippocampal or cortical synapse size and density. Immunohistochemistry for presynaptic markers was performed in the CA1 and the CA3 regions of the hippocampus and in the somatosensory cortex (SSC). (A) Representative low- (top of each series) and high-resolution images (bottom of each series) of sections from the hippocampal CA1and CA3 regions and the SSC as indicated on the left of wild-type (NL3 WT) and NL3R704C-mutant mice, immunostained for synaptophysin (left panels) or for the vesicular glutamate and GABA transporters vGlut1 and vGAT (right panels). (B), Summary graphs of the synapse densities (top) and size (bottom) as measured by synaptophysin staining (left) or by vGlut1 and vGAT staining (right). Data in (B) are mean values±s.e.m. (n=3 pairs). No statistically significant difference between wild-type and R704C-mutant mice was detected in the analysed brain regions. Download figure Download PowerPoint Decreased excitatory synapse function in NL3R704C KI mice To test whether the NL3R704C mutation altered synaptic transmission, we performed whole-cell voltage-clamp recordings in hippocampal CA1 pyramidal neurons in acute brain slices. Strikingly, the NL3R704C mutation decreased the frequency but not the amplitude of spontaneous mEPSCs, whereas it had no significant effect on mIPSCs (Figure 4A–F). This selective deficit in mEPSC frequency suggests that the NL3R704C mutation may alter presynaptic release probability, excitatory synapse number or postsynaptic AMPA receptor responses. Figure 4.Decreased frequency of spontaneous mEPSCs in NL3R704C-mutant mice. (A, D) Sample traces of mEPSCs (A) and mIPSCs (D). (B, E) Cumulative probability plots of mEPSC (B) and mIPSC amplitudes (E). Insets display box plots of the amplitudes (numbers list number of cells recorded; mEPSC: WT=11 cells/4 mice; R704C=11 cells/4 mice; mIPSC: WT=13 cells/4 mice and R704C=11 cells/4 mice). (C, F) Cumulative probability plots of mEPSC (C) and mIPSC frequency, as measured by the inter-event intervals (F). Insets display box plots of the actual frequencies (n=same as for (B, E)). Data represent mean values±s.e.m. Statistical significance (*P<0.05) was evaluated with a KS-test (cumulative probability plots) and Student's t-test (box plots). Box plots represent median and inter-quartile range; vertical lines represent 10th and 90th percentiles. Download figure Download PowerPoint To further explore the possibility that the NL3R704C mutation causes a deficit in excitatory synaptic transmission, we measured excitatory synaptic strength by performing extracellular field recordings in the CA1 region of the hippocampus. In this assay, the slope of the postsynaptic fEPSP is measured as a function of the presynaptic fibre-volley amplitude (Figure 5A and B). Consistent with our mEPSC findings, the NL3R704C mutation produced a significant decrease in excitatory synaptic strength in the input–output experiments, as confirmed by measuring the slope of the linear fit for individual input–output experiments (Figure 5C). It is possible that the observed deficits are the result of changes in synaptic AMPA receptor subunit composition (the principal mediators of the synaptic response measured in Figure 5A–C). To exclude this possibility, we analysed the voltage dependence of synaptic AMPA receptor-mediated responses, but detected no change in the AMPA receptor rectification index (Figure 5D and E). Figure 5.NL3R704C mutation impairs AMPA receptor-mediated synaptic responses. (A–C) Sample traces (A), summary plots (B) and summary graph of the linear fit slopes (C) for input–output measurements obtained by extracellular field recordings in acute hippocampal slices from wild-type and NL3R704C-mutant mice (WT=13 slices/5 mice and R704C=14 slices/5 mice). (D) Current–voltage plot (I/V curve) for AMPA receptor-mediated synaptic responses. (E) AMPA receptor rectification index plotted as current at +40 mV relative to current at −40 mV (WT=6 cells/2 mice and R704C=4 cells/2 mice). Data are mean values±s.e.m. Statistical significance (*P<0.05) was evaluated by one-way ANOVA (B) or Student's t-test (C). Download figure Download PowerPoint One potential explanation for the observed decreases in mEPSCs and input–output relations in NL3R704C-mutant mice is that the presynaptic release probability is altered in NL3R704C-mutant mice. Although this explanation was previously suggested for NL1 (Futai et al, 2007), it would be difficult to reconcile with the postsynaptic localization of NL3. Two different tests of release probability, measurements of paired-pulse facilitation and of the use-dependent block of NMDA receptor responses by MK-801, failed to detect a change in NL3R704C-mutant mice (Figure 6). Thus, the deficit in excitatory synaptic transmission in NL3R704C-mutant mice is not a consequence of changes in presynaptic release probability. Figure 6.Normal presynaptic release probability in NL3R704C-mutant mice. (A, B) Sample traces (A) and summary graphs (B) of paired-pulse facilitation measurements obtained with 50 and 80 ms inter-stimulus (numbers list number of cells recorded; WT=13 cells/3 mice and R704C=11 cells/3 mice). (C, D) Sample traces (C) and summary graph (D) obtained during measurements of the presynaptic release probability using the progressive block of NMDA receptor-mediated responses by MK-801. NMDA receptor EPSCs were monitored at +40 mV before and after addition of MK-801 (25 μM); traces in (C) depict the 1st, 10th and 50th NMDA receptor EPSC following MK-801 application. Experiments were performed in three parts: stable NMDA receptor-mediated EPSCs induced by 0.1 Hz stimulation were established; stimuli were stopped as MK-801 was bath applied for 8 min; stimuli were resumed. The weighted τ of the NMDA receptor response decay was calculated using a double exponential function: A1exp(−t/τ1)+A2exp(−t/τ2) (WT=9 cells/4 mice and R704C=7 cells/4 mice). Data represent mean values±s.e.m. Statistical significance was excluded using Student's t-test. Download figure Download PowerPoint It is puzzling that the NL3R704C-mutant mice exhibit a decrease in excitatory mini frequency (Figure 4) and a decrease in evoked synaptic strength (Figure 5A–C), but no change in total excitatory synapse numbers as assessed by presynaptic staining for VGlut1 (Figure 3), because the first two observations would best be explained by a loss in synapse numbers, which is ruled out by the third observation. The limitation of staining for vGlut1 as a presynaptic marker for excitatory synapses, however, is that vGlut1 staining does not reveal whether excitatory synapses are functional. Thus, it is possible that the NL3R704C mutation decreases the number of mature, AMPA receptor containing excitatory synapses without dramatically affecting total excitatory synapse numbers. To address this question, we measured the relative ratio of NMDA versus AMPA receptor-mediated synaptic responses using whole-cell recordings in acute hippocampal slices (Figure 7A). Consistent with the hypothesis that the NL3R704C mutation causes a selective reduction in AMPA but not in NMDA receptor-mediated synaptic transmission, the NMDA/AMPA ratio was increased in NL3R704C-mutant mice (Figure 7B). In agreement with a lack of an effect on NMDA receptor-mediated synaptic transmission by the NL3R704C mutation, NMDA receptor-dependent long-term potentiation was unchanged in NL3R704C-mutant mice (Figure 8). Figure 7.Increased ratio of NMDA to AMPA receptor-mediated EPSC in NL3R704C-mutant mice. (A, B) Sample traces (A) and summary graph (B) of measurements of the ratio of NMDA versus AMPA receptor-mediated synaptic responses monitored in slices. The NMDA/AMPA ratio was determined by sequentially evaluating EPSC amplitudes at −70 mV (AMPA) and at +40 mV (NMDA) holding potential; NMDA receptor-mediated responses were measured at 50 ms post-stimulus (WT=12 cells/3 mice and R704C=13 cells/3 mice). Data represent mean values±s.e.m. (*P<0.05 by Student's t-test). Download figure Download PowerPoint Figure 8.Normal hippocampal LTP in NL3R704C-mutant mice. (A) Sample traces for extracellular field EPSP recordings performed before (1) and 60 min after (2) LTP induction with three 1 s stimulus trains at 100 Hz. (B) Summary graph for LTP experiments performed in wild-type and NL3R704C-mutant mice (WT=9 slices/5 mice and R704C=10 cells/5 mice). (C) Summary graph for the magnitude of LTP at 55–60 min post-induction. Data represent mean values±s.e.m. Download figure Download PowerPoint Cultured neurons from NL3R704C KI mice The slice physiology results provide evidence for a selective change in excitatory synapse function in NL3R704C-mutant mice, and suggest a specific impairment in AMPA but not in NMDA receptor-mediated responses. To quantitatively assess this phenotype in a reduced system, we measured synaptic responses in cultured hippocampal neurons (Maximov et al, 2007). Strikingly, cultured hippocampal neurons from NL3R704C-mutant mice displayed a major, selective decrease in AMPA receptor-mediated responses, without a change in NMDA receptor-mediated glutamatergic responses or GABA receptor-mediated inhibitory responses (Figure 9A–C). Thus, the R704C mutation specifically reduced postsynaptic AMPA receptor function in cultured neurons, consistent with the observations obtained in acute slices. Figure 9.Decreased AMPA receptor-mediated synaptic responses in cultured neurons from NL3R704C-mutant mice. (A–C) Sample traces (left) and summary graphs (right) of AMPA (A), NMDA (B) and GABA receptor- (C) mediated synaptic responses recorded in cultured hippocampal neurons (AMPA: WT=19 cells/3 cultures, R704C=21 cells/3 cultures; NMDA: WT=17 cells/3 cultures, R704C=19 cells/3 cultures; GABA: WT=15 cells/3 cultures and R704C=18 cells/3 cultures). Data shown are absolute postsynaptic current amplitudes as recorded using the approach of Maximov et al (2007). Data represent mean values±s.e.m.; statistical significance (*P<0.05) was evaluated by Student's t-test. Download figure Download PowerPoint Discussion Our data show that a single amino-acid substitution in a conserved cytoplasmic residue of NL3, R704C, has no significant effect on its synaptogenic activity in vitro, but dramatically alters excitatory synapse function in vivo. Specifically, we show that KI mice carrying the NL3R704C mutation exhibit a discrete and selective impairment of AMPA receptor-mediated synaptic transmission in hippocampal pyramidal neurons. As measured by multiple independent approaches (intracellular recordings of spontaneous synaptic responses in acute slices, extracellular recordings of input/output curves in acute slices and intracellular recordings of evoked responses in cultured neurons; Figures 4, 5 and 9), the decrease in synaptic strength in NL3R704C-mutant synapses is selective for AMPA receptor-mediated excitatory transmission since we observed no change in NMDA receptor-mediated responses or in GABA receptor-mediated synaptic transmission. As a result, the NMDA/AMPA receptor EPSC ratio is increased (Figure 7). We observed no change in NMDA receptor protein levels (Figure 2), synapse numbers (Figure 3) or presynaptic release probability (Figure 6), but did observe a significant, possibly compensatory increase in the levels of GluR1 and GluR3 AMPA receptor subunits (Figure 2). The magnitude of the NL3R704C-mutant phenotype (40–50% decrease in AMPA receptor-mediated responses), and the fact that the R704C-mutant phenotype was consistently observed by multiple approaches and retained in cultured neurons, shows that it is robust. Thus, a single amino-acid substitution in the NL3 cytoplasmic tail caused a major effect on AMPA receptor-mediated synaptic transmission in the hippocampus. The changes we describe here are different from those previously observed in NL3R451C-mtuant and in NL3 KO mice (Tabuchi et al, 2007; Etherton et al, unpublished), suggesting a specific effect of different NL3 mutations on synaptic properties. The most plausible hypothesis to account for the phenotype of the NL3R704C mutation is that in NL3R704C-mutant mice, a significant subset of synapses contain no, or only few, functional postsynaptic AMPA receptors, but maintain normal postsynaptic NMDA receptors. Although this hypothesis would explain our observations, alternative hypotheses cannot be ruled out. For example, it is possible that the NL3R704C mutation leads to selective elimination of a small number of synapses that are particularly active, such that no synapse los
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