Neuroligin3 splice isoforms shape inhibitory synaptic function in the mouse hippocampus

Abstract

Synapse formation is a dynamic process essential for the development and maturation of the neuronal circuitry in the brain. At the synaptic cleft, trans-synaptic protein–protein interactions are major biological determinants of proper synapse efficacy. The balance of excitatory and inhibitory synaptic transmission (E-I balance) stabilizes synaptic activity, and dysregulation of the E-I balance has been implicated in neurodevelopmental disorders, including autism spectrum disorders. However, the molecular mechanisms underlying the E-I balance remain to be elucidated. Here, using single-cell transcriptomics, immunohistochemistry, and electrophysiology approaches to murine CA1 pyramidal neurons obtained from organotypic hippocampal slice cultures, we investigate neuroligin (Nlgn) genes that encode a family of postsynaptic adhesion molecules known to shape excitatory and inhibitory synaptic function. We demonstrate that the NLGN3 protein differentially regulates inhibitory synaptic transmission in a splice isoform–dependent manner at hippocampal CA1 synapses. We also found that distinct subcellular localizations of the NLGN3 isoforms contribute to the functional differences observed among these isoforms. Finally, results from single-cell RNA-Seq analyses revealed that Nlgn1 and Nlgn3 are the major murine Nlgn genes and that the expression levels of the Nlgn splice isoforms are highly diverse in CA1 pyramidal neurons. Our results delineate isoform-specific effects of Nlgn genes on the E-I balance in the murine hippocampus. Synapse formation is a dynamic process essential for the development and maturation of the neuronal circuitry in the brain. At the synaptic cleft, trans-synaptic protein–protein interactions are major biological determinants of proper synapse efficacy. The balance of excitatory and inhibitory synaptic transmission (E-I balance) stabilizes synaptic activity, and dysregulation of the E-I balance has been implicated in neurodevelopmental disorders, including autism spectrum disorders. However, the molecular mechanisms underlying the E-I balance remain to be elucidated. Here, using single-cell transcriptomics, immunohistochemistry, and electrophysiology approaches to murine CA1 pyramidal neurons obtained from organotypic hippocampal slice cultures, we investigate neuroligin (Nlgn) genes that encode a family of postsynaptic adhesion molecules known to shape excitatory and inhibitory synaptic function. We demonstrate that the NLGN3 protein differentially regulates inhibitory synaptic transmission in a splice isoform–dependent manner at hippocampal CA1 synapses. We also found that distinct subcellular localizations of the NLGN3 isoforms contribute to the functional differences observed among these isoforms. Finally, results from single-cell RNA-Seq analyses revealed that Nlgn1 and Nlgn3 are the major murine Nlgn genes and that the expression levels of the Nlgn splice isoforms are highly diverse in CA1 pyramidal neurons. Our results delineate isoform-specific effects of Nlgn genes on the E-I balance in the murine hippocampus. Neuroligin proteins (NLGNs) were the first identified binding partners of α-latrotoxin receptors, neurexin proteins (NRXs), and localize at postsynaptic sites to regulate synapse maturation and function (1Sudhof T.C. Synaptic neurexin complexes: a molecular code for the logic of neural circuits.Cell. 2017; 171 (29100073): 745-76910.1016/j.cell.2017.10.024Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Four Nlgn genes (Nlgn1, Nlgn2, Nlgn3, and Nlgn4) encode trans-synaptic adhesion proteins (NLGN1, NLGN2, NLGN3, and NLGN4) that contain extracellular cholinesterase-like domains and transmembrane and PDZ-binding motif–containing intracellular domains. Whereas the intracellular domain is important for NLGN binding with postsynaptic scaffold molecules, the extracellular domain confers the formation of excitatory and inhibitory synapses with NRX, its sole presynaptic binding partner. Therefore, precise combinations of NRX-NLGN interactions allow NLGN to diversify synapse identity. NLGN1 and NLGN2 are postsynaptic adhesion molecules localized to excitatory and inhibitory synapses, respectively. Overexpression, knockdown, and knockout approaches have revealed that NLGN1 is important for excitatory synaptic structure and transmission and synaptic plasticity, but not for inhibitory synaptic function (2Jiang M. Polepalli J. Chen L.Y. Zhang B. Südhof T.C. Malenka R.C. Conditional ablation of neuroligin-1 in CA1 pyramidal neurons blocks LTP by a cell-autonomous NMDA receptor-independent mechanism.Mol. Psychiatry. 2017; 22 (27217145): 375-38310.1038/mp.2016.80Crossref PubMed Scopus (46) Google Scholar, 3Chubykin A.A. Atasoy D. Etherton M.R. Brose N. Kavalali E.T. Gibson J.R. Südhof T.C. Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2.Neuron. 2007; 54 (17582332): 919-93110.1016/j.neuron.2007.05.029Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 4Shipman S.L. Nicoll R.A. A subtype-specific function for the extracellular domain of neuroligin 1 in hippocampal LTP.Neuron. 2012; 76 (23083734): 309-31610.1016/j.neuron.2012.07.024Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 5Futai K. Kim M.J. Hashikawa T. Scheiffele P. Sheng M. Hayashi Y. Retrograde modulation of presynaptic release probability through signaling mediated by PSD-95-neuroligin.Nat. Neurosci. 2007; 10 (17237775): 186-19510.1038/nn1837Crossref PubMed Scopus (209) Google Scholar, 6Futai K. Doty C.D. Baek B. Ryu J. Sheng M. Specific trans-synaptic interaction with inhibitory interneuronal neurexin underlies differential ability of neuroligins to induce functional inhibitory synapses.J. Neurosci. 2013; 33 (23426688): 3612-362310.1523/JNEUROSCI.1811-12.2013Crossref PubMed Scopus (34) Google Scholar). NLGN2 protein has specific functional roles in inhibitory synaptic transmission in the hippocampus (6Futai K. Doty C.D. Baek B. Ryu J. Sheng M. Specific trans-synaptic interaction with inhibitory interneuronal neurexin underlies differential ability of neuroligins to induce functional inhibitory synapses.J. Neurosci. 2013; 33 (23426688): 3612-362310.1523/JNEUROSCI.1811-12.2013Crossref PubMed Scopus (34) Google Scholar, 7Shipman S.L. Schnell E. Hirai T. Chen B.S. Roche K.W. Nicoll R.A. Functional dependence of neuroligin on a new non-PDZ intracellular domain.Nat. Neurosci. 2011; 14 (21532576): 718-72610.1038/nn.2825Crossref PubMed Scopus (75) Google Scholar, 8Nguyen Q.A. Horn M.E. Nicoll R.A. Distinct roles for extracellular and intracellular domains in neuroligin function at inhibitory synapses.eLife. 2016; 5 (27805570)10.7554/eLife.19236Crossref Scopus (27) Google Scholar). In contrast, it has been reported that NLGN3 protein localizes at both excitatory and inhibitory synaptic sites and regulates both synaptic functions (7Shipman S.L. Schnell E. Hirai T. Chen B.S. Roche K.W. Nicoll R.A. Functional dependence of neuroligin on a new non-PDZ intracellular domain.Nat. Neurosci. 2011; 14 (21532576): 718-72610.1038/nn.2825Crossref PubMed Scopus (75) Google Scholar, 8Nguyen Q.A. Horn M.E. Nicoll R.A. Distinct roles for extracellular and intracellular domains in neuroligin function at inhibitory synapses.eLife. 2016; 5 (27805570)10.7554/eLife.19236Crossref Scopus (27) Google Scholar, 9Etherton M. Foldy C. Sharma M. Tabuchi K. Liu X. Shamloo M. Malenka R.C. Sudhof T.C. Autism-linked neuroligin-3 R451C mutation differentially alters hippocampal and cortical synaptic function.Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (21808020): 13764-1376910.1073/pnas.1111093108Crossref PubMed Scopus (216) Google Scholar, 10Foldy C. Malenka R.C. Sudhof T.C. Autism-associated neuroligin-3 mutations commonly disrupt tonic endocannabinoid signaling.Neuron. 2013; 78 (23583622): 498-50910.1016/j.neuron.2013.02.036Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 11Budreck E.C. Scheiffele P. Neuroligin-3 is a neuronal adhesion protein at GABAergic and glutamatergic synapses.Eur. J. Neurosci. 2007; 26 (17897391): 1738-174810.1111/j.1460-9568.2007.05842.xCrossref PubMed Scopus (191) Google Scholar, 12Shipman S.L. Nicoll R.A. Dimerization of postsynaptic neuroligin drives synaptic assembly via transsynaptic clustering of neurexin.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (23129658): 19432-1943710.1073/pnas.1217633109Crossref PubMed Scopus (38) Google Scholar). This unique ability alludes to a NLGN3 protein-specific molecular code that promotes its translocation to both excitatory and inhibitory sites. Splice insertion in Nlgn genes differentially regulates E-I balance and alters their binding affinity with presynaptic NRX proteins. In the extracellular cholinesterase-like domain, Nlgn genes have one or two splice insertion sites, Nlgn1 (A and B sites), Nlgn2 (A), and Nlgn3 (A1 and A2), leading to 2-4 theoretical splice isoforms. The splice insertion site B in NLGN1 determines its binding preference to NRXs (13Ichtchenko K. Hata Y. Nguyen T. Ullrich B. Missler M. Moomaw C. Südhof T.C. Neuroligin 1: a splice site-specific ligand for β-neurexins.Cell. 1995; 81 (7736595): 435-44310.1016/0092-8674(95)90396-8Abstract Full Text PDF PubMed Scopus (543) Google Scholar, 14Boucard A.A. Chubykin A.A. Comoletti D. Taylor P. Südhof T.C. A splice code for trans-synaptic cell adhesion mediated by binding of neuroligin 1 to α- and β-neurexins.Neuron. 2005; 48 (16242404): 229-23610.1016/j.neuron.2005.08.026Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar) and excitatory synaptic function (15Chih B. Gollan L. Scheiffele P. Alternative splicing controls selective trans-synaptic interactions of the neuroligin-neurexin complex.Neuron. 2006; 51 (16846852): 171-17810.1016/j.neuron.2006.06.005Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Similarly, NLGN2 contains a splice insertion at site A, which regulates inhibitory synaptic function (8Nguyen Q.A. Horn M.E. Nicoll R.A. Distinct roles for extracellular and intracellular domains in neuroligin function at inhibitory synapses.eLife. 2016; 5 (27805570)10.7554/eLife.19236Crossref Scopus (27) Google Scholar). However, to the best of our knowledge, the splice isoform–specific function of NLGN3 and the transcript levels of Nlgn splice isoforms at the single-cell level have not been addressed. In the present study, we assess the function of NLGN3 splice isoforms on excitatory and inhibitory synaptic transmission in CA1 pyramidal neurons in mouse organotypic slice cultures. Our results suggest that NLGN3 up- or down-regulates inhibitory synaptic transmission in a splice isoform–dependent manner. Furthermore, our single-cell RNA-Seq analysis reveals that Nlgn1 and Nlgn3 are the major Nlgn genes, and the expressions of Nlgn splice variants are highly distinct in hippocampal CA1 pyramidal neurons. The Nlgn3 gene contains two splice insertion sites (A1 and A2) that can yield four NLGN3 splice isoforms (Nlgn3Δ, A1, A2, and A1A2). NLGN3Δ lacks all splice insertions, whereas NLGN3A1, 3A2, and 3A1A2 receive insertion of A1, A2, or both A1A2 cassettes, respectively. To examine the potential roles of NLGN3 splice isoforms on excitatory and inhibitory synapse function, we biolistically transfected the Nlgn3 splice isoform genes in CA1 pyramidal cells of organotypic hippocampal slice cultures (Fig. 1). Transfection of NLGN3 splice variants did not alter gross cell morphology or levels of overexpressed NLGN3 splice variants identified by HA immunoreactivity (Fig. S1, A and B). Simultaneous electrophysiological recordings were made from transfected and neighboring untransfected neurons. CA1 pyramidal neurons overexpressing NLGN3Δ or 3A2 showed increased evoked inhibitory postsynaptic currents (IPSCs) compared with neighboring untransfected control neurons and a marked increase in excitatory postsynaptic currents (EPSCs), as reported previously (Fig. 1, A and C) (7Shipman S.L. Schnell E. Hirai T. Chen B.S. Roche K.W. Nicoll R.A. Functional dependence of neuroligin on a new non-PDZ intracellular domain.Nat. Neurosci. 2011; 14 (21532576): 718-72610.1038/nn.2825Crossref PubMed Scopus (75) Google Scholar, 12Shipman S.L. Nicoll R.A. Dimerization of postsynaptic neuroligin drives synaptic assembly via transsynaptic clustering of neurexin.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (23129658): 19432-1943710.1073/pnas.1217633109Crossref PubMed Scopus (38) Google Scholar). In contrast, overexpression of NLGN3A1 or 3A1A2 resulted in reduced amplitude of IPSCs and increased amplitude of EPSCs compared with neighboring untransfected cells (Fig. 1, B and D). Paired stimulation of input fibers with a short interval (50 ms) induced paired-pulse facilitation (PPF) and depression (PPD) of EPSCs and IPSCs, respectively. NLGN3Δ or 3A2 transfection displayed both reduced AMPAR-PPF and GABAAR-PPD compared with untransfected neurons, consistent with previous work (5Futai K. Kim M.J. Hashikawa T. Scheiffele P. Sheng M. Hayashi Y. Retrograde modulation of presynaptic release probability through signaling mediated by PSD-95-neuroligin.Nat. Neurosci. 2007; 10 (17237775): 186-19510.1038/nn1837Crossref PubMed Scopus (209) Google Scholar, 7Shipman S.L. Schnell E. Hirai T. Chen B.S. Roche K.W. Nicoll R.A. Functional dependence of neuroligin on a new non-PDZ intracellular domain.Nat. Neurosci. 2011; 14 (21532576): 718-72610.1038/nn.2825Crossref PubMed Scopus (75) Google Scholar) (Fig. S1, C–E). As paired-pulse ratio (PPR) inversely correlates with presynaptic release probability, these results suggest that overexpression of NLGN3Δ and 3A2 can modulate presynaptic release probability. NLGN3 increased or decreased inhibitory synaptic transmission in a splice isoform–dependent manner, whereas all NLGN3 splice isoforms enhanced excitatory synaptic transmission. Next, we tested whether NLGN3 overexpression changes membrane excitability. The input-output relationship of CA1 pyramidal neurons transfected with any NLGN3 splice isoforms exhibited no significant difference compared with untransfected control neurons (Fig. S2). The findings above suggest that NLGN3 splice isoforms regulate synaptic but not membrane functions. Expression of NLGN3 at excitatory and inhibitory synapses has been observed in primary neurons, but in vivo NLGN3 expression has been studied only in the cerebellum and striatum (11Budreck E.C. Scheiffele P. Neuroligin-3 is a neuronal adhesion protein at GABAergic and glutamatergic synapses.Eur. J. Neurosci. 2007; 26 (17897391): 1738-174810.1111/j.1460-9568.2007.05842.xCrossref PubMed Scopus (191) Google Scholar, 16Uchigashima M. Ohtsuka T. Kobayashi K. Watanabe M. Dopamine synapse is a neuroligin-2-mediated contact between dopaminergic presynaptic and GABAergic postsynaptic structures.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (27035941): 4206-421110.1073/pnas.1514074113Crossref PubMed Scopus (43) Google Scholar, 17Baudouin S.J. Gaudias J. Gerharz S. Hatstatt L. Zhou K. Punnakkal P. Tanaka K.F. Spooren W. Hen R. De Zeeuw C.I. Vogt K. Scheiffele P. Shared synaptic pathophysiology in syndromic and nonsyndromic rodent models of autism.Science. 2012; 338 (22983708): 128-13210.1126/science.1224159Crossref PubMed Scopus (194) Google Scholar). To ensure the expression of NLGN3 in the hippocampus, we performed immunohistochemistry against NLGN3 with the markers for excitatory and inhibitory synapses, vesicular glutamate transporter type 1 (VGluT1) and vesicular inhibitory amino acid transporter (VIAAT), respectively (Fig. 2). Our NLGN3 antibody, validated by Nlgn3 knockout tissue, detected punctate signals in the hippocampus (Fig. 2, A and B). The signals overlapped with VGluT1 and VIAAT puncta, indicating that NLGN3 proteins are targeted to both excitatory and inhibitory synapses, respectively (Fig. 2, C and D). To understand the mechanistic roles of NLGN3 splice isoforms in inhibitory synaptic transmission, we next performed immunocytochemistry to elucidate the subcellular localization of NLGN3Δ and 3A1A2, which displayed strong enhancement and suppression of IPSC, respectively. Excitatory synaptic sites were characterized by spine or VGluT1. Inhibitory synaptic sites were identified by the dendritic shaft proximal to VIAAT puncta. HA immunoreactivity illustrated that NLGN3A1A2 is highly concentrated in spines. In contrast, NLGN3Δ showed more diffuse expression in both spines and dendrites (Fig. 3A). The ratio of NLGNA1A2 signals between excitatory and inhibitory synapses was significantly higher than that of 3Δ (Fig. 3B). Next, we addressed whether these NLGN3 splice isoforms differentially promote excitatory and inhibitory synapses by comparing NLGN3/GFP and GFP (control) transfected neurons. Importantly, inhibitory synapse density was comparable among NLGN3Δ, 3A1A2, and control, whereas VIAAT signal intensity in 3Δ-expressing neurons was significantly higher than that of 3A1A2 and control (Fig. 3, C and D). The spine density or length was comparable among NLGN3Δ, 3A1A2, and control neurons (Fig. 3, E and F). The signal intensities of VGluT1 were markedly elevated in neurons overexpressing NLGN3Δ or 3A1A2 compared with control neurons (Fig. 3, G and H). These results suggest that differences in the subcellular localization of NLGN3Δ and 3A1A2 contribute to their distinct inhibitory synaptic functions. we tested whether NLGN3Δ or 3A1A2 overexpression changes expression of NLGN2 inhibitory NLGN protein A.A. Atasoy D. Etherton M.R. Brose N. Kavalali E.T. Gibson J.R. Südhof T.C. Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2.Neuron. 2007; 54 (17582332): 919-93110.1016/j.neuron.2007.05.029Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, S. Brose N. Neuroligin is localized to inhibitory J. PubMed Scopus Google Scholar). against and NLGN2 revealed that transfection of NLGN3 splice isoform has no on the level of NLGN2 at inhibitory synapses (Fig. This that the and we observed in 1 and 3Chubykin A.A. Atasoy D. Etherton M.R. Brose N. Kavalali E.T. Gibson J.R. Südhof T.C. Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2.Neuron. 2007; 54 (17582332): 919-93110.1016/j.neuron.2007.05.029Abstract Full Text Full Text PDF PubMed Scopus (401) Google are mediated by overexpressed NLGN3 and not to of of NLGN3 splice isoforms in CA1 pyramidal neurons. A, of dendritic for and VIAAT in CA1 pyramidal cells overexpressing A1A2 and control and spines and inhibitory synapses, respectively. the ratio of HA signals inhibitory synapse density VIAAT intensity spine density and spine length for dendritic of CA1 pyramidal cells overexpressing A1A2 and control VIAAT intensity is to the VIAAT intensity in neighboring inhibitory on the of dendritic for and VGluT1 in CA1 pyramidal cells overexpressing A1A2 and control transfected VGluT1 intensity for dendritic of CA1 pyramidal cells overexpressing A1A2 and control VGluT1 intensity is to the VGluT1 intensity in neighboring excitatory on the analysis of by or Finally, to understand the expression of Nlgn genes in CA1 pyramidal neurons, we from four neurons and performed single-cell The that the four cell were and to that of hippocampal pyramidal neurons from the (Fig. The expression of Nlgn genes was and with the single-cell RNA-Seq in the RNA-Seq by the for (Fig. The of Nlgn genes (Fig. that the expression of Nlgn1 and Nlgn3 are comparable but that of Nlgn2 is significantly than the two We also compared the expression of Nlgn splice isoforms in Nlgn Nlgn splice and that were not were (Fig. and their expression was and were the highly Nlgn1 splice isoforms (Fig. was the only isoform in the Nlgn2 gene (Fig. and were not detected in any of the four CA1 pyramidal neurons. Importantly, and 3A2, which exhibited increased inhibitory synaptic transmission, were the Nlgn3 splice isoforms in CA1 pyramidal neurons (Fig. The expressions of and 3A1A2 were significantly than that of The expression of these single-cell Nlgn splice isoforms was also by (Fig. protein–protein interactions are biological for synapse and function. are postsynaptic adhesion molecules that regulate excitatory and inhibitory synaptic transmission. we that NLGN3 regulates inhibitory synaptic transmission and excitatory and inhibitory synapse localization in a splice isoform–dependent manner. Our single-cell analysis revealed that and 3A2 are the Nlgn3 splice isoforms in hippocampal CA1 pyramidal neurons. The distinct subcellular localization of NLGN3Δ and 3A1A2 mechanisms splice isoforms synapse that the intracellular and transmembrane domains are between NLGN3 splice isoform their synapse through their unique extracellular domains. Similarly, the extracellular domain of NLGN2 changes in inhibitory synaptic function (6Futai K. Doty C.D. Baek B. Ryu J. Sheng M. Specific trans-synaptic interaction with inhibitory interneuronal neurexin underlies differential ability of neuroligins to induce functional inhibitory synapses.J. Neurosci. 2013; 33 (23426688): 3612-362310.1523/JNEUROSCI.1811-12.2013Crossref PubMed Scopus (34) Google Scholar). on our knowledge, NRXs are the only trans-synaptic family proteins that to interactions between NLGN3Δ, but not 3A1A2, and NRXs modulate inhibitory synaptic transmission in pyramidal neurons. We previously reported that postsynaptic NLGN2 can with presynaptic but not with to functional inhibitory synapses (6Futai K. Doty C.D. Baek B. Ryu J. Sheng M. Specific trans-synaptic interaction with inhibitory interneuronal neurexin underlies differential ability of neuroligins to induce functional inhibitory synapses.J. Neurosci. 2013; 33 (23426688): 3612-362310.1523/JNEUROSCI.1811-12.2013Crossref PubMed Scopus (34) Google that the specific binding of NLGN and NRX isoforms regulates functional synapse is that inhibitory not NRX isoforms that can to Therefore, NLGN3A1A2 localize to excitatory synapses. be performed to specific isoform interactions that inhibitory synaptic function. is protein interactions between NLGN3 and postsynaptic proteins. has been reported that protein between the extracellular domain of NLGN1 and are important for synaptic function E.C. S. Kim Y. Tabuchi K. R. Scheiffele P. Kim controls synaptic of glutamate through extracellular Natl. Acad. Sci. U.S.A. 2013; PubMed Scopus Google Scholar). NLGN3 splice variants have binding interactions with postsynaptic proteins that distinct functions on inhibitory synapses. has been that the levels of and their postsynaptic scaffold at excitatory and inhibitory synapses E-I balance excitatory and inhibitory neuroligin 2005; 48 Full Text Full Text PDF PubMed Scopus Google Scholar). are but it is to the that postsynaptic NLGN3A1 and A1A2 are strong and specific at excitatory synaptic sites and the protein interactions scaffold from inhibitory synapses to inhibitory synaptic transmission. The single-cell results demonstrate a gene expression of Nlgn splice isoforms in hippocampal CA1 pyramidal neurons obtained from Our findings are highly with the expression of the genes by the single-cell from neurons, indicating that the expression ratio of are NLGN2 has been at inhibitory synapses, its transcript levels were significantly than Nlgn1 and Nlgn3 (Fig. B and NLGN2 have unique and mechanisms compared with NLGN1 and The expression of and 3A1A2 were than of and 3A2, that these two splice isoforms are not the major functional Nlgn3 molecules work is to whether to neuronal as synaptic can alter the expression of Nlgn splice isoforms. A in E-I balance has been a of neurodevelopmental and reported in mouse models balance and in autism spectrum Full Text Full Text PDF PubMed Scopus Google Scholar). and of Nlgn3 are with autism spectrum S. C. M. C. B. M. C. T. of the genes neuroligins NLGN3 and are with PubMed Scopus Google and that the autism Nlgn3 mutation E-I and synaptic (1Sudhof T.C. Synaptic neurexin complexes: a molecular code for the logic of neural circuits.Cell. 2017; 171 (29100073): 745-76910.1016/j.cell.2017.10.024Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). A of specific NLGN3 splice isoform functions elucidate their in molecular that were and by the and at the of and the The Nlgn3 mouse was a from Tanaka K.F. E.C. Scheiffele P. S. N. K. Hen R. a and new gene Psychiatry. Full Text Full Text PDF PubMed Scopus Google Scholar). hippocampal slice cultures were from of both as previously L. D. A for organotypic cultures of Neurosci. PubMed Scopus Google Scholar). The of four CA1 neurons were using the previously (6Futai K. Doty C.D. Baek B. Ryu J. Sheng M. Specific trans-synaptic interaction with inhibitory interneuronal neurexin underlies differential ability of neuroligins to induce functional inhibitory synapses.J. Neurosci. 2013; 33 (23426688): 3612-362310.1523/JNEUROSCI.1811-12.2013Crossref PubMed Scopus (34) Google Scholar). and analysis were on our The single-cell RNA-Seq is in in the were with were and with in were using a to were with for slice cultures transfected with NLGN3 splice isoforms were with and in were with by with A of primary against enhanced GFP NLGN2 M. Ohtsuka T. Kobayashi K. Watanabe M. Dopamine synapse is a neuroligin-2-mediated contact between dopaminergic presynaptic and GABAergic postsynaptic structures.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (27035941): 4206-421110.1073/pnas.1514074113Crossref PubMed Scopus (43) Google NLGN3 M. Ohtsuka T. Kobayashi K. Watanabe M. Dopamine synapse is a neuroligin-2-mediated contact between dopaminergic presynaptic and GABAergic postsynaptic structures.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (27035941): 4206-421110.1073/pnas.1514074113Crossref PubMed Scopus (43) Google VGluT1 and Y. M. T. T. E. Watanabe M. T. M. The is the major at excitatory presynaptic sites in the hippocampus and Neurosci. 2006; 26 PubMed Scopus Google and VIAAT and T. M. Watanabe M. of vesicular glutamate at cell synapses in mouse J. Neurosci. PubMed Scopus Google Scholar) and a of with and were for were with a with a and with hippocampal slice cultures were from of at in were transfected using a gene and were transfection in as previously (5Futai K. Kim M.J. Hashikawa T. Scheiffele P. Sheng M. Hayashi Y. Retrograde modulation of presynaptic release probability through signaling mediated by PSD-95-neuroligin.Nat. Neurosci. 2007; 10 (17237775): 186-19510.1038/nn1837Crossref PubMed Scopus (209) Google Scholar, 6Futai K. Doty C.D. Baek B. Ryu J. Sheng M. Specific trans-synaptic interaction with inhibitory interneuronal neurexin underlies differential ability of neuroligins to induce functional inhibitory synapses.J. Neurosci. 2013; 33 (23426688): 3612-362310.1523/JNEUROSCI.1811-12.2013Crossref PubMed Scopus (34) Google Scholar, Y. W. S. J. K. effects on in mouse hippocampal pyramidal 2017; PubMed Scopus Google Scholar, W. M. Y. Watanabe M. S. K. of synaptic through the Full Text Full Text PDF PubMed Scopus Google Scholar). recordings were made from a of CA1 pyramidal neurons, one transfected by and one untransfected IPSCs were evoked with a in the and at EPSCs were evoked at in the of electrophysiological are in the are reported as and were for was at for and The for the RNA-Seq and reported in is are in the The Watanabe for We D. for on the with neuroligin neurexin inhibitory postsynaptic excitatory postsynaptic paired-pulse facilitation paired-pulse depression enhanced GFP vesicular inhibitory amino acid transporter vesicular glutamate transporter type 1

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