Neuroligins Mediate Excitatory and Inhibitory Synapse Formation

The balance between excitatory and inhibitory synapses is a tightly regulated process that requires differential recruitment of proteins that dictate the specificity of newly formed contacts. However, factors that control this process remain unidentified. Here we show that members of the neuroligin (NLG) family, including NLG1, NLG2, and NLG3, drive the formation of both excitatory and inhibitory presynaptic contacts. The enrichment of endogenous NLG1 at excitatory contacts and NLG2 at inhibitory synapses supports an important in vivo role for these proteins in the development of both types of contacts. Immunocytochemical and electrophysiological analysis showed that the effects on excitatory and inhibitory synapses can be blocked by treatment with a fusion protein containing the extracellular domain of neurexin-1β. We also found that overexpression of PSD-95, a postsynaptic binding partner of NLGs, resulted in a shift in the distribution of NLG2 from inhibitory to excitatory synapses. These findings reveal a critical role for NLGs and their synaptic partners in controlling the number of inhibitory and excitatory synapses. Furthermore, relative levels of PSD-95 alter the ratio of excitatory to inhibitory synaptic contacts by sequestering members of the NLG family to excitatory synapses. The balance between excitatory and inhibitory synapses is a tightly regulated process that requires differential recruitment of proteins that dictate the specificity of newly formed contacts. However, factors that control this process remain unidentified. Here we show that members of the neuroligin (NLG) family, including NLG1, NLG2, and NLG3, drive the formation of both excitatory and inhibitory presynaptic contacts. The enrichment of endogenous NLG1 at excitatory contacts and NLG2 at inhibitory synapses supports an important in vivo role for these proteins in the development of both types of contacts. Immunocytochemical and electrophysiological analysis showed that the effects on excitatory and inhibitory synapses can be blocked by treatment with a fusion protein containing the extracellular domain of neurexin-1β. We also found that overexpression of PSD-95, a postsynaptic binding partner of NLGs, resulted in a shift in the distribution of NLG2 from inhibitory to excitatory synapses. These findings reveal a critical role for NLGs and their synaptic partners in controlling the number of inhibitory and excitatory synapses. Furthermore, relative levels of PSD-95 alter the ratio of excitatory to inhibitory synaptic contacts by sequestering members of the NLG family to excitatory synapses. Synapse formation is a tightly regulated process that involves the recruitment of specific cell adhesion molecules and scaffolding proteins to newly formed contacts between an axon and a dendrite (1Kim E. Sheng M. Nat. Rev. Neurosci. 2004; 5: 771-781Crossref PubMed Scopus (1209) Google Scholar, 2Craig A.M. Boudin H. Nat. Neurosci. 2001; 4: 569-578Crossref PubMed Scopus (123) Google Scholar, 3Washbourne P. Dityatev A. Scheiffele P. Biederer T. Weiner J.A. Christopherson K.S. El-Husseini A. J. Neurosci. 2004; 24: 9244-9249Crossref PubMed Scopus (151) Google Scholar). In the brain, excitatory and inhibitory synaptic transmission is mainly mediated by two neurotransmitters: glutamate, which is released at excitatory glutamatergic synaptic contacts, and GABA, 1The abbreviations used are: GABA, γ-aminobutyric acid; GFP, green fluorescent protein; HA, hemagglutinin; NLG, neuroligin; VGAT, vesicular GABA transporter; VGLUT, vesicular glutamate transporter; DIV, days in vitro; E/I, excitatory/inhibitory; TBST, Tris-buffered saline with 0.1% Tween 20; mEPSC, miniature excitatory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current; AMPA, α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate. 1The abbreviations used are: GABA, γ-aminobutyric acid; GFP, green fluorescent protein; HA, hemagglutinin; NLG, neuroligin; VGAT, vesicular GABA transporter; VGLUT, vesicular glutamate transporter; DIV, days in vitro; E/I, excitatory/inhibitory; TBST, Tris-buffered saline with 0.1% Tween 20; mEPSC, miniature excitatory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current; AMPA, α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate. which is released at inhibitory GABAergic synapses. Initial transformation of a contact to either an excitatory or inhibitory synapse is thought to be controlled by spatial and temporal changes in protein content. This process is critical because an appropriate balance between excitatory and inhibitory synapses is required for proper neuronal excitability and function (2Craig A.M. Boudin H. Nat. Neurosci. 2001; 4: 569-578Crossref PubMed Scopus (123) Google Scholar, 4Lee S.H. Sheng M. Curr. Opin. Neurobiol. 2000; 10: 125-131Crossref PubMed Scopus (88) Google Scholar, 5Ziv N.E. Smith S.J. Neuron. 1996; 17: 91-102Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 6Ziv N.E. Neuroscientist. 2001; 7: 365-370Crossref PubMed Scopus (10) Google Scholar). However, molecular events that control differentiation of a contact into either an excitatory or inhibitory synapse remain unknown.The postsynaptic density protein, PSD-95, is a molecule that is exclusively localized to glutamatergic synapses and regulates clustering of AMPA receptors through association with stargazin (7El-Husseini A.E. Schnell E. Chetkovich D.M. Nicoll R.A. Bredt D.S. Science. 2000; 290: 1364-1368Crossref PubMed Google Scholar, 8Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar). Through its third PDZ (PSD-95/Dlg/ZO-1) homology domain, PSD-95 also recruits neuroligin-1 (NLG1), a cell adhesion molecule involved in synapse formation (9Ichtchenko K. Hata Y. Nguyen T. Ullrich B. Missler M. Moomaw C. Sudhof T.C. Cell. 1995; 81: 435-443Abstract Full Text PDF PubMed Scopus (567) Google Scholar, 10Irie M. Hata Y. Takeuchi M. Ichtchenko K. Toyoda A. Hirao K. Takai Y. Rosahl T.W. Sudhof T.C. Science. 1997; 277: 1511-1515Crossref PubMed Scopus (601) Google Scholar, 11Brose N. Naturwissenschaften. 1999; 86: 516-524Crossref PubMed Scopus (53) Google Scholar). These findings indicate that association of PSD-95 with NLG1 is involved in excitatory synapse development. Recent work by Prange et al. (12Prange O. Wong T.P. Gerrow K. Wang Y.T. El-Husseini A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13915-13920Crossref PubMed Scopus (293) Google Scholar) showed that NLG1 can drive both excitatory and inhibitory presynaptic contact formation. These results suggested that NLGs are involved in inhibitory synapse formation. Our work also showed that the effects of NLG1 on postsynaptic differentiation were less dramatic. Overexpression of NLG1 modestly increased the total number of excitatory postsynaptic sites but did not enhance clustering of PSD-95 or AMPA receptors (12Prange O. Wong T.P. Gerrow K. Wang Y.T. El-Husseini A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13915-13920Crossref PubMed Scopus (293) Google Scholar). In contrast, coexpression of PSD-95 with NLG1 coordinated the maturation of pre- and postsynaptic elements and the recruitment of AMPA receptors (12Prange O. Wong T.P. Gerrow K. Wang Y.T. El-Husseini A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13915-13920Crossref PubMed Scopus (293) Google Scholar). Moreover, enhanced expression of PSD-95 induced changes in the number of excitatory versus inhibitory synapses and resulted in an overall increase in the ratio of excitatory/inhibitory (E/I) synaptic currents (12Prange O. Wong T.P. Gerrow K. Wang Y.T. El-Husseini A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13915-13920Crossref PubMed Scopus (293) Google Scholar). These findings provided the first evidence that assembly of specific postsynaptic elements can regulate a balance between excitatory and inhibitory synapses. Thus, abnormalities in the expression of and/or interactions between these molecules may result in aberrant synapse formation and a change in E/I ratio, which underlie complex psychiatric disorders (13Rubenstein J.L. Merzenich M.M. Genes Brain Behav. 2003; 2: 255-267Crossref PubMed Scopus (1670) Google Scholar). The detection of mutations in NLG3 and NLG4 in autistic patients suggests that synaptic imbalance may underlie autism (14Jamain S. Quach H. Betancur C. Rastam M. Colineaux C. Gillberg I.C. Soderstrom H. Giros B. Leboyer M. Gillberg C. Bourgeron T. Nyden A. Philippe A. Cohen D. Chabane N. Mouren-Simeoni M.C. Brice A. Sponheim E. Spurkland I. Skjeldal O.H. Coleman M. Pearl P.L. Cohen I.L. Tsiouris J. Zappella M. Menchetti G. Pompella A. Aschauer H. Van Maldergem L. Nat. Genet. 2003; 34: 27-29Crossref PubMed Scopus (1353) Google Scholar).NLG1 has additional homologs in rat including NLG2 and NLG3, which also contain a PDZ-binding site (10Irie M. Hata Y. Takeuchi M. Ichtchenko K. Toyoda A. Hirao K. Takai Y. Rosahl T.W. Sudhof T.C. Science. 1997; 277: 1511-1515Crossref PubMed Scopus (601) Google Scholar). Thus, PSD-95 may similarly control the action of NLG2 and NLG3 in neurons. Binding of NLGs to presynaptic neurexin-1β, a specific isoform of β-neurexin, is required for synaptic contact formation through trans-synaptic heterophilic protein interactions (9Ichtchenko K. Hata Y. Nguyen T. Ullrich B. Missler M. Moomaw C. Sudhof T.C. Cell. 1995; 81: 435-443Abstract Full Text PDF PubMed Scopus (567) Google Scholar, 11Brose N. Naturwissenschaften. 1999; 86: 516-524Crossref PubMed Scopus (53) Google Scholar, 15Missler M. Sudhof T.C. Trends Genet. 1998; 14: 20-26Abstract Full Text PDF PubMed Scopus (291) Google Scholar, 16Rao A. Harms K.J. Craig A.M. Nat. Neurosci. 2000; 3: 747-749Crossref PubMed Scopus (42) Google Scholar, 17Scheiffele P. Fan J. Choih J. Fetter R. Serafini T. Cell. 2000; 101: 657-669Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar). However, it remains unclear whether association of NLGs with β-neurexin is involved in the formation of both excitatory and inhibitory presynaptic contacts. Also, it remains unclear whether NLGs are localized to both excitatory and inhibitory synapses and whether PSD-95 modulates the E/I ratio through regulated distribution of these proteins.To elucidate the events that regulate synaptic specificity, we have analyzed the role of NLGs and PSD-95 in this process. We show that NLG1, NLG2, and NLG3 are capable of inducing both excitatory and inhibitory presynaptic contact formation. The effect on inhibitory synapses was blocked by a soluble form of neurexin-1β. Moreover, enhanced expression of PSD-95 induced clustering of NLG2 and NLG3 and shifted endogenous NLG2 from inhibitory to excitatory synapses. These results demonstrate that members of the NLG family are involved in establishing excitatory and inhibitory synapses; however, association with postsynaptic scaffolding proteins regulates the distribution of NLGs and controls the type of synapses formed.MATERIALS AND METHODScDNA Cloning and Mutagenesis—The hemagglutinin (HA)-tagged wild type NLG1 (1ab splice variant) amplified from mouse cerebellum was a gift from Dr. Peter Scheiffele (Columbia University). The generation of GW1 PSD-95 fused to GFP was previously described (18Craven S.E. El-Husseini A.E. Bredt D.S. Neuron. 1999; 22: 497-509Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 19El-Husseini A.E. Craven S.E. Chetkovich D.M. Firestein B.L. Schnell E. Aoki C. Bredt D.S. J. Cell Biol. 2000; 148: 159-172Crossref PubMed Scopus (237) Google Scholar). The generation of NLG2 and NLG3 constructs was carried out by PCR using oligonucleotides containing BglII and HindIII restriction sites (NLG2, GGGCCCAGATCTCGGGGAGGAGGGGGTCCC and GGGCCCAAGCTTCTATACCCGAGTGGTGGA; NLG3, GGGCCCAGATCTGCCAGTACCCAGGCCCCG and GGGCCCAAGCTTCTATACACGGGTAGTGGA) and subcloning the resulting fragments into pEGFP-C1 containing the NLG1 signal sequence followed by GFP (12Prange O. Wong T.P. Gerrow K. Wang Y.T. El-Husseini A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13915-13920Crossref PubMed Scopus (293) Google Scholar). The final HA-tagged versions were made by removing GFP (using AgeI and BglII) and inserting an HA tag.Neuronal Cell Culture and Transfections—Dissociated primary neuronal cultures were prepared from hippocampi of embryonic day 18 or 19 Wistar rats. The hippocampi were dissociated by enzymatic papain digestion followed by brief mechanical trituration. The cells were plated on poly-d-lysine (Sigma)-treated glass coverslips. The cultures were maintained in neurobasal medium (Invitrogen) supplemented with B27, penicillin, streptomycin, and l-glutamine as described by Brewer et al. (37Brewer G.J. Torricelli J.R. Evege E.K. Price P.J. Neurosci. Res. 1993; 35: 567-576Crossref Scopus (1895) Google Scholar). Hippocampal cultures were transfected by lipid-mediated gene transfer using the Lipofectamine 2000 agent (Invitrogen) or by the calcium phosphate method (Clontech) at least 2 days prior to immunostaining.Immunocytochemistry—The coverslips were removed from culture wells and fixed in –20 °C methanol. The cells were washed three times with phosphate-buffered saline containing 0.3% Triton-X-100 before each antibody incubation. The following primary antibodies were used (immunoreactivity and dilution as indicated): HA (mouse, 1:1000; BABCO), GFP (guinea pig, 1:1000), NLG1 (mouse, 1:1000; gift from Dr. Nils Brose), NLG2 (goat, 1:50; Santa Cruz Biotechnology), VGLUT (rabbit, 1:1000; Synaptic Systems), VGAT (rabbit, 1:1000; Synaptic Systems), PSD-95 (1:500; Affinity BioReagents), and Shank (guinea pig, 1:500; gift from Dr. Morgan Sheng). Appropriate fluorescently conjugated secondary antibodies were used as previously described (12Prange O. Wong T.P. Gerrow K. Wang Y.T. El-Husseini A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13915-13920Crossref PubMed Scopus (293) Google Scholar). All of the antibody reactions were performed in blocking solution (2% normal goat or horse serum) for 1 h at room temperature or overnight at 4 °C. The coverslips were then mounted on slides (Frost Plus; Fisher) with Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL).Western Blotting and Application of Neurexin Fusion Protein—For immunoblotting, the protein samples were harvested in lysis buffer containing 25 mm Tris, 150 mm NaCl, 3 mm KCl, 1 mm EGTA, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride (Sigma), 0.8% Triton X-100, 0.2% SDS, and 1 protease inhibitor mixture tablet/10 ml (Roche Applied Science). The samples were boiled for 10 min in loading buffer (62.5 mm Tris-HCl, 2% SDS, 1% β-mercaptoethanol, 7.5% glycerol, 15 μm bromphenol blue), and the proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences). Nonspecific binding was blocked by incubating membranes with 3% bovine serum albumin in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h. After three washes in TBST, primary antibodies (HA: mouse, 1:1000; NLG2: goat, 1:250) were diluted in 3% bovine serum albumin in TBST, and the membranes were incubated for 1 h at room temperature. The membranes were washed three times in TBST, incubated with secondary antibody conjugated to horseradish peroxidase (anti-mouse, 1:2000, Amersham Biosciences; anti-goat, 1:5000, Santa Cruz Biotechnology). The blots were visualized by use of ECL (Pierce). Purification of soluble neurexin-1β fusion protein and the control FC-IgG protein was carried out as described by Ushkaryov et al. (38Ushkaryov Y.A. Hata Y. Ichtchenko K. Moomaw C. Afendis S. Slaughter C.A. Sudhof T.C. J. Biol. Chem. 1994; 269: 11987-11992Abstract Full Text PDF PubMed Google Scholar). For treatment using the purified proteins, the neurons were transfected with the appropriate construct. Transfection medium was then replaced with neurobasal medium containing either vehicle only (Hanks' balanced salt solution), neurexin-1β fusion protein, or FC-IgG (for each well of neurons, ∼8 μl of each was added to 500 μl of neurobasal medium).Imaging and Analysis—The images were acquired on a Zeiss Axiovert M200 motorized microscope by using a monochrome 14-bit Zeiss AxiocamHR charge-coupled device camera. In some experiments, the exposure times were individually adjusted to yield an optimum brightness of immunofluorescent clusters without saturation. In other experiments, the images were acquired with equal exposure and scaled to the same extent, without saturation. For analysis of cluster density (number), the images were analyzed in Northern Eclipse (Empix Imaging, Missasauga, Canada), by using custom written software routines as previously described (12Prange O. Wong T.P. Gerrow K. Wang Y.T. El-Husseini A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13915-13920Crossref PubMed Scopus (293) Google Scholar). Briefly, the images were processed at a constant threshold level (of 32,000 pixel values) to create a binary image, and dendrites of the cell of interest were outlined by using fluorescence signal. Only clusters with average pixel values two times greater than corresponding background pixel values were used for analysis. The number of stained clusters was measured as a function of dendritic length. For analysis of changes in NLG2 localization, puncta from the NLG2 channel were manually outlined, and the intensity of each punctum was subtracted from the dendritic background intensity value and multiplied by punctum area to obtain an integrated intensity. In the case of cells transfected with PSD-95 GFP, each punctum on the NLG2 channel was then scored for colocalization with either PSD-95 GFP or VGAT puncta. Mean intensity of NLG2 puncta colocalizing with PSD-95 GFP was then compared with that of NLG2 puncta colocalizing with VGAT, and a ratio was obtained. In the case of untransfected controls, the same process was conducted for VGLUT-positive or -negative NLG2 puncta. The ratios for PSD-95 GFP-transfected cells were then compared with those for untransfected controls. For statistical analyses, Mann-Whitney U or Wilcoxin signed ranks tests were used.Electrophysiology—Recording of miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) were performed at least 2 days post-transfection. Hippocampal neurons on coverslips were transferred to a recording chamber that was continuously perfused with extracellular solution (pH 7.4; 320–330 mOsm) containing 140 mm NaCl, 5.4 mm KCl, 1 mm MgCl2, 1.3 mm CaCl2, 25 mm HEPES, 33 mm glucose, and 0.0005 mm tetrodotoxin (Alomone Labs). Transfected cells were identified from their GFP signal under a fluorescence upright microscope. Patch pipettes were pulled from borosilicate glass capillaries (Sutter Instrument) and filled with an intracellular solution (pH 7.2; 300–310 mOsm), composed of 115 mm cesium gluconate, 17.5 mm CsCl, 10 mm HEPES, 2 mm MgCl2, 10 mm EGTA, 4 mm ATP, 0.1 mm GTP, and 0.1% Lucifer Yellow (Sigma). An Axopatch 200B amplifier (Axon Instruments) was used for the recording. The access resistance was monitored throughout each experiment. Recordings with a series resistance variation of more than 10% were rejected. No electronic compensation for series resistance was used. Whole cell patch clamp recordings were performed in voltage clamp mode, and the membrane potential was maintained at either the reversal potential for GABAA receptor-mediated mIPSCs (–60 mV) to isolate mEPSCs or at the reversal potential for ionotropic glutamate receptor-mediated mEPSCs (+10 mV) to isolate mIPSCs. The recorded spontaneous mIPSCs and mEPSCs were blocked completely by the GABAA receptor antagonist bicuculline (Sigma) and by the ionotropic glutamate receptor antagonist cyano-7-nitroquinoxaline-2,3-dione (Sigma), respectively (data not shown). Recordings were low pass filtered at 2 kHz, sampled at 10 kHz, and stored in a PC using Clampex 8.2 (Axon). The t test was used for statistical analysis.RESULTS AND DISCUSSIONNeuroligins Drive Excitatory and Inhibitory Presynaptic Contact Formation—Previous work showed that NLG1 drives excitatory synapse formation (17Scheiffele P. Fan J. Choih J. Fetter R. Serafini T. Cell. 2000; 101: 657-669Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar). However, recently we have revealed the surprising finding that NLG1 overexpression can also induce inhibitory presynaptic contact formation (12Prange O. Wong T.P. Gerrow K. Wang Y.T. El-Husseini A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13915-13920Crossref PubMed Scopus (293) Google Scholar). These results suggest that NLG1 may be involved in vivo in establishing both excitatory and inhibitory synapses. NLG2 and NLG3 are two other known members of the NLG family expressed in the brain (20Ichtchenko K. Nguyen T. Südhof T.C. J. Biol. Chem. 1996; 271: 2676-2682Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). To explore whether the effect on inhibitory synapses is unique to NLG1, we analyzed the effects of HA-tagged NLG2 (HA-NLG2) and NLG3 (HA-NLG3) on both excitatory and inhibitory synapse formation. For this analysis, days in vitro (DIV) 5 hippocampal neurons were transfected with HA-NLG2 and HA-NLG3, fixed at DIV 8, and stained for either the vesicular γ-aminobutyric acid (GABA) transporter (VGAT), a marker for inhibitory synapses, or VGLUT, a marker for excitatory synapses. Remarkably, HA-NLG2 and HA-NLG3 overexpression significantly enhanced the average number of contacting presynaptic boutons, both excitatory (1.6 ± 0.1-fold for HA-NLG2 and 1.5 ± 0.2-fold for HA-NLG3) and inhibitory (3.0 ± 0.9-fold for HA-NLG2 and 2.7 ± 0.4-fold for HA-NLG3), when compared with GFP-transfected cells (Fig. 1). Other changes observed include enhanced numbers of dendritic filopodia (data not shown). These results demonstrate that various members of the NLG family exert similar effects on establishing new synaptic contacts, regardless of type.NLG-induced Inhibitory Synapse Formation Is Mediated by Neurexin-1β—The differential recruitment of proteins to their respective synaptic compartments is likely to be mediated by heterotypic trans-synaptic signaling. NLGs have been shown to associate with neurexin-1β, and this interaction leads to the recruitment of elements required for the structural reorganization of presynaptic compartments (10Irie M. Hata Y. Takeuchi M. Ichtchenko K. Toyoda A. Hirao K. Takai Y. Rosahl T.W. Sudhof T.C. Science. 1997; 277: 1511-1515Crossref PubMed Scopus (601) Google Scholar, 17Scheiffele P. Fan J. Choih J. Fetter R. Serafini T. Cell. 2000; 101: 657-669Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 20Ichtchenko K. Nguyen T. Südhof T.C. J. Biol. Chem. 1996; 271: 2676-2682Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 21Butz S. Okamoto M. Sudhof T.C. Cell. 1998; 94: 773-782Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). Work performed by Dean et al. (22Dean C. Scholl F.G. Choih J. DeMaria S. Berger J. Isacoff E. Scheiffele P. Nat. Neurosci. 2003; 6: 708-716Crossref PubMed Scopus (466) Google Scholar) demonstrated that synapse formation involves direct interaction between NLG1 and neurexin-1β and that the effects of NLG1 on excitatory synapses are blocked using an FC fusion protein containing the extracellular domain of neurexin-1β (17Scheiffele P. Fan J. Choih J. Fetter R. Serafini T. Cell. 2000; 101: 657-669Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar). However, it remains unclear how NLGs influence the maturation of GABA presynaptic terminals. Previous studies showed that β-neurexins are also expressed by inhibitory neurons (23Ullrich B. Ushkaryov Y.A. Sudhof T.C. Neuron. 1995; 14: 497-507Abstract Full Text PDF PubMed Scopus (356) Google Scholar). Moreover, recent work by Graf et al. (24Graf E.R. Zhang X. Jin S.X. Linhoff M.W. Craig A.M. Cell. 2004; 119: 1013-1026Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar) showed that expression of neurexin-1β in nonneuronal cells can drive clustering of NLGs and several other excitatory and inhibitory postsynaptic proteins in neurons. To examine whether NLG interaction with neurexin-1β is required for inhibitory synapse formation in vivo, DIV 6 neurons were transfected with either HA-NLG1 or HA-NLG2 and then incubated in a medium containing either vehicle solution, 30 μg/ml FC-IgG (control), or 30 μg/ml of a soluble form of neurexin-1β lacking splice site 4 fused to FC-IgG (NXN-FC; Supplemental Fig. 1). Two days post-transfection, the neurons were fixed and analyzed for induction of inhibitory synapses. Remarkably, inhibitory synapse formation mediated by either NLG1 or NLG2 was dramatically diminished upon incubation with NXN-FC (Fig. 2 and Supplemental Fig. 1). This was manifested by a decrease in the number of VGAT-positive puncta contacting dendrites of cells transfected with either construct as compared with vehicle-treated controls (Fig. 2, A and B; 34 ± 7% of control for NLG1 and 39 ± 9% of control for NLG2). Moreover, no significant effect of FC-IgG treatment was observed (Fig. 2B). These results show that a neurexin-1β-dependent interaction regulates NLG-induced inhibitory synapse formation. Significantly, treatment of GFP-transfected cells with NXN-FC resulted in a decrease in the number of inhibitory synapses (51 ± 9% of control) when compared with GFP cells treated with vehicle only (Fig. 2C and Supplemental Fig. 1C). This is further evidence that neurexin-1β is involved in the formation of inhibitory synapses in vivo.Fig. 2NLG-induced inhibitory synapse formation involves neurexin-1β. DIV 6 hippocampal neurons were transfected with either HA-NLG1 or HA-NLG2 and incubated with medium containing 30 μg/ml FC-IgG, 30 μg/ml FC-neurexin-1β peptide (NXN-FC), or Hanks' balanced salt solution (vehicle) only. The cells were fixed 2 days following treatment and then immunostained with HA and VGAT antibodies. A and B, addition of NXN-FC results in a decrease in the number of VGAT-positive terminals contacting dendrites of HA-NLG1 (vehicle, n = 10; FC-IgG, n = 8; NXN-FC, n = 11) or HA-NLG2 (vehicle, n = 10; FC-IgG, n = 13; NXN-FC, n = 10) transfected cells. B, quantification of changes in number of VGAT-positive terminals contacting transfected cells presented as a percentage of control (vehicle; dotted line). C, quantification of changes in number of VGAT-positive terminals contacting cells transfected with GFP. Each group has been normalized to vehicle only treatment (vehicle, n = 10; NXN-FC, n = 10). **, p < 0.005; *, p < 0.05. Scale bars, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Treatment with NXN-FC also induced aggregation of HA-NLG1 and HA-NLG2 (Supplemental Fig. 2). This phenomenon is consistent with that observed in the case of the integrin transmembrane cell-adhesion receptors, where ligand binding induces aggregation of the receptor (25Miyamoto S. Akiyama S.K. Yamada K.M. Science. 1995; 267: 883-885Crossref PubMed Scopus (788) Google Scholar). Interestingly, NLG clusters were present at sites positive for PSD-95 but lacking synaptophysin staining, indicating nonsynaptic localization (Supplemental Fig. 2, B and C). Similar coclustering of NLGs and PSD-95 by neurexin-1β was recently reported (24Graf E.R. Zhang X. Jin S.X. Linhoff M.W. Craig A.M. Cell. 2004; 119: 1013-1026Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar). The aggregation of NLGs and PSD-95 upon treatment with NXN-FC is intriguing because NLG expression alone was not sufficient to enhance PSD-95 clustering (Supplemental Fig. 2A). Further work is required to clarify these differences.Functional Effects of Neurexin-1β on Excitatory and Inhibitory Synapses—To examine functional correlates of the immunocytochemical changes induced by application of NXN-FC fusion protein, an electrophysiological approach was taken (Fig. 3). DIV 8–9 hippocampal neurons were transfected with either GFP alone or HA-NLG1 and incubated with either NXN-FC or FC-IgG for 2–3 days. Changes in mEPSCs and mIPSCs were compared by using whole cell voltage clamp recordings (Fig. 3A). Ectopic expression of HA-NLG1 in cells treated with control peptide (FC-IgG) significantly increases the frequency of mEPSCs and mIPSCs compared with GFP transfected cells (Fig. 3, B and C, upper panels). Strikingly, the effects on both mEPSCs and mIPSCs were blocked by treatment with NXN-FC. In addition, NXN-FC treatment reduced basal frequency of mEPSCs and mIPSCs in cells transfected with GFP alone. These results parallel our immunocytochemical data and support a novel role for β-neurexins in the induction of inhibitory synapses. Importantly, treatment of HA-NLG1 transfected cells with NXN-FC increases the ratio of E/I synaptic currents (∼4-fold), suggesting that β-neurexins play a more critical role in inhibitory synapse formation.Fig. 3Blocking neurexin-1β function diminishes NLG1-mediated excitatory and inhibitory synaptic function. Shown are electrophysiological recordings from cultured rat hippocampal neurons coexpressing HA-NLG1 and GFP, or GFP alone, and incubated with medium containing either 30 μg/ml FC-IgG or 30 μg/ml neurexin-1β-FC peptide (NXN-FC). Spontaneous mEPSCs and mIPSCs were recorded in voltage clamp mode at holding potentials of –60 mV and +10 mV, respectively. A, representative traces of mEPSCs (left) and mIPSCs (right) recorded from these neurons. B and C, both the mEPSC and mIPSC frequencies were enhanced in neurons expressing HA-NLG1 when compared with neurons transfected with GFP alone. In contrast, treatment of HA-NLG1 expressing cells with NXN-FC resulted in a significant reduction in both mEPSC and mIPSC frequency when compared with FC-IgG treated controls. Moreover, NXN-FC