6816 • The Journal of Neuroscience, July 19, 2017 • 37(29):6816–6836 Cellular/Molecular Unique versus Redundant Functions of Neuroligin Genes in Shaping Excitatory and Inhibitory Synapse Properties X Soham Chanda,1,2 XW. Dylan Hale,1 X Bo Zhang,1 Marius Wernig,2 and Thomas C. Su¨dhof1 1Department of Molecular and Cellular Physiology and Howard Hughes Medical Institute, and 2Institute for Stem Cell Biology and Regenerative Medicine and Department of Pathology, Stanford University School of Medicine, Stanford, California 94305 Neuroligins are evolutionarily conserved postsynaptic cell adhesion molecules that interact with presynaptic neurexins. Neurons express multiple neuroligin isoforms that are targeted to specific synapses, but their synaptic functions and mechanistic redundancy are not completely understood. Overexpression or RNAi-mediated knockdown of neuroligins, respectively, causes a dramatic increase or de- crease in synapse density, whereas genetic deletions of neuroligins impair synapse function with only minor effects on synapse numbers, raising fundamental questions about the overall physiological role of neuroligins. Here, we have systematically analyzed the effects of conditionalgeneticdeletionsofallmajorneuroliginisoforms(i.e.,NL1,NL2,andNL3),eitherindividuallyorincombinations,incultured mouse hippocampal and cortical neurons. We found that conditional genetic deletions of neuroligins caused no change or only a small change in synapses numbers, but strongly impaired synapse function. This impairment was isoform specific, suggesting that neuroligins arenotfunctionallyredundant.Sparseneuroligindeletionsproducedphenotypescomparabletothoseofglobaldeletions,indicatingthat neuroligins function in a cell-autonomous manner. Mechanistically, neuroligin deletions decreased the synaptic levels of neurotrans- mitter receptors and had no effect on presynaptic release probabilities. Overexpression of neuroligin-1 in control or neuroligin-deficient neurons increased synaptic transmission and synapse density but not spine numbers, suggesting that these effects reflect a gain-of- function mechanism; whereas overexpression of neuroligin-3, which, like neuroligin-1 is also targeted to excitatory synapses, had no comparable effect. Our data demonstrate that neuroligins are required for the physiological organization of neurotransmitter receptors in postsynaptic specializations and suggest that they do not play a major role in synapse formation. Key words: conditional knockout; neuroligin; primary neuronal culture; synapse development; synaptic transmission; synaptogenesis Significance Statement Human neuroligin genes have been associated with autism, but the cellular functions of different neuroligins and their molecular mechanisms remain incompletely understood. Here, we performed comparative analyses in cultured mouse neurons of all major neuroligin isoforms, either individually or in combinations, using conditional knockouts. We found that neuroligin deletions did not affect synapse numbers but differentially impaired excitatory or inhibitory synaptic functions in an isoform-specific manner. These impairments were due, at least in part, to a decrease in synaptic distribution of neurotransmitter receptors upon deletion of neuroligins. Conversely, the overexpression of neuroligin-1 increased synapse numbers but not spine numbers. Our results suggest that various neuroligin isoforms perform unique postsynaptic functions in organizing synapses but are not essential for synapse formation or maintenance. Introduction Nguyen and Su¨dhof, 1997). Mammals express four neuroligins Neuroligins (NLs) are postsynaptic cell adhesion molecules that (NL1 to NL4); of these, NL1, NL2, and NL3 are abundant and bind to presynaptic neurexins (Ichtchenko et al., 1995 and 1996; highly conserved in mice, whereas NL4 exhibits low abun- dance and poor conservation (Ichtchenko et al., 1996; Bolliger et al., 2008). Neuroligins form obligatory homodimers (Como- Received Jan. 15, 2017; revised May 10, 2017; accepted May 31, 2017. letti et al., 2003, 2006; Arac¸ et al., 2007; Fabrichny et al., 2007; Author contributions: S.C. and T.C.S. designed research; S.C. and W.D.H. performed research; S.C., W.D.H., B.Z., and M.W. contributed unpublished reagents/analytic tools; S.C. and W.D.H. analyzed data; S.C. and T.C.S. wrote the Chen et al., 2008) and may also assemble into NL1/NL3 or NL2/ paper. ThisworkwassupportedbygrantsfromtheNationalInstitutesofHealth(R37-MH-052804toT.C.S.;MH-092931 to M.W.), a postdoctoral grant award (Stanford, ChEM-H112878 to S.C.), and a National Science Foundation Grad- and Howard Hughes Medical Institute, and Institute for Stem Cell Biology and Regenerative Medicine and Depart- uate Research Fellowship (DGE-114747, to W.D.H.). ment of Pathology, Stanford University School of Medicine, Stanford, CA 94305. E-mail: [email protected]. The authors declare no competing financial interests. DOI:10.1523/JNEUROSCI.0125-17.2017 Correspondence should be addressed to Soham Chanda, Department of Molecular and Cellular Physiology Copyright © 2017 the authors 0270-6474/17/376816-21$15.00/0 Chanda et al. • Neuroligins in Synapse Function J. Neurosci., July 19, 2017 • 37(29):6816–6836 • 6817 NL3 heterodimers (Budreck and Scheiffele, 2007; Poulopoulos et al., deconstruct the contributions of endogenous neuroligins in syn- 2012). Despite their homology and coexpression in the same neu- aptogenesis and synapse function and to demonstrate that neu- rons, neuroligins are differentially localized. NL1 is targeted to glu- roligins perform an essential role in organizing synapses but not tamatergic synapses, NL2 to GABAergic and cholinergic synapses, in the initial formation of synapses. and NL3 to both glutamatergic and GABAergic synapses (Song et al., 1999; Varoqueaux et al., 2004; Budreck and Scheiffele, 2007; Fo¨ldy et Materials and Methods al., 2013; Taka´cs et al., 2013). Constitutive NL123 triple-knock-out (KO) mice displayed ma- General experimental design. All animal experiments were performed with male and female newborn mice according to institutional guidelines jor impairments in synapse function but exhibited normal synapse and approved by the Administrative Panel on Laboratory Animal Care of morphology and density (Varoqueaux et al., 2006). Moreover, Stanford University School of Medicine. All experiments except for those conditional KOs (cKOs) of neuroligins in vivo in the striatum, in Figures 1, 3F–J, 4D, and 11A and B, were performed in a “blinded” cerebellum, or hippocampus did not greatly alter synapse num- fashion (i.e., the experimenter was unaware of whether a sample repre- bers (Rothwell et al., 2014; Zhang et al., 2015; Jiang et al., 2017); sented a test or control sample). only cKO of NL2 in adult prefrontal cortex caused a modest Mouse husbandry. A detailed description of generating NL cKOs (NL1 inhibitory synapse loss that developed after 6 weeks (Liang et al., single cKO, NL2 single cKO, NL3 single cKO, NL13 double cKO, NL23 2015). KOs of NL1 produce a major decrease in NMDA receptor double cKO, and NL123 triple cKO) mice is described in the studies by (NMDAR)-mediated and, to a lesser extent, in AMPA receptor Liang et al. (2015), Rothwell et al. (2014), and Zhang et al. (2015). (AMPAR)-mediated synaptic responses, causing a shift in the Neuronal culture. Hippocampal and cortical neurons were cultured from newborn mice as described previously (Maximov et al., 2007). Dis- NMDAR/AMPAR-ratio (Chubykin et al., 2007; Kim et al., 2008; sected hippocampi (Figs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) or cortices (Figs. 11, 12, Blundell et al., 2010; Soler-Llavina et al., 2011; Jedlicka et al., 13, 14) were digested for 30 min with 10 U/ml papain in HBSS buffer 2ϩ 2015; Jiang et al., 2017). KOs of NL2, conversely, impair synaptic supplemented with 2 mM Ca and 0.5 mM EGTA in an incubator, transmission in subsets of GABAergic synapses (Chubykin et al., washed with HBSS buffer, dissociated in plating media (MEM supple- 2007; Gibson et al., 2009; Poulopoulos et al., 2009; Rothwell et al., mented with 0.5% glucose, 0.02% NaHCO3, 0.1 mg/ml transferrin, 10% 2014; Liang et al., 2015; Zhang et al., 2015). Overall, these data FBS, 2 mML-glutamine, and 0.025 mg/ml insulin), and seeded on Matri- suggest that neuroligins are required for synapse maturation and gel (BD Biosciences) precoated coverslips placed inside 24-well dishes. function, but not for the initial formation of synaptic contacts. The day of plating was considered as 0 d in vitro (DIV0). After 24 h In some experiments using microRNA (miRNA)/short hairpin (DIV1), 75% of the plating media was replaced with neuronal growth RNA (shRNA)-mediated knockdown (KD) of neuroligins, how- media (MEM supplemented with 0.5% glucose, 0.02% NaHCO3, 0.1 mg/ml transferrin, 5% FBS, 2% B27 supplement, and 0.5 mML-glutamine). At ever, reduction in neuroligin expression dramatically decreased DIV2 (for hippocampal cultures) or DIV3 (for cortical cultures), 50% of synapse and spine numbers (Chih et al., 2005; Kwon et al., 2012; the medium was exchanged with fresh growth medium additionally sup- Shipman et al., 2011; but for different results, see Ko et al., 2011; plemented with 4 ␮M Ara-C (Sigma-Aldrich). The time course experi- Soler-Llavina et al., 2011). Moreover, the overexpression of ments for hippocampal and cortical synaptogenesis were performed neuroligins in cultured neurons uniformly increased synapse every other day starting from DIV4 until DIV16 (Figs. 1, 11A,B). Most of numbers (Dean et al., 2003;