Heller EA , Zhang W , Selimi F , Earnheart JC , Ślimak MA , Santos-Torres J , Ibañez-Tallon I , Aoki C , Chait BT , Heintz N .

5 R01 DA009618-09 NIDA NIH HHS , EY13079 NEI NIH HHS , MH091445-01 NIMH NIH HHS, RR00862 NCRR NIH HHS , RR022220 NCRR NIH HHS , Howard Hughes Medical Institute , U54 RR022220 NCRR NIH HHS , R01 DA009618 NIDA NIH HHS , P41 RR000862 NCRR NIH HHS , P30 EY013079 NEI NIH HHS , R21 MH091445 NIMH NIH HHS

	Figure 1. The inhibitory synapse affinity tag, Venus-GABAARα1, is functional in vitro.(A) Schematic of VGABAARα1, showing the N-terminal fusion of an affinity tag, Venus. (B) Representative GABA-evoked currents (left) and current amplitude quantification (right) in voltage clamped Xenopus oocytes after coinjection of the indicated GABR cRNA subunits. Values are expressed as mean ± SEM; n  = 5 oocytes per group (**p<0.01 t-test). (C) Schematic of the patch and stimulation electrodes used for paired-pulse recordings in cultured hippocampal neurons transduced with lentivirus encoding GABAARα1 (Lv-GARα1) or Venus-GABAARα1 (Lv-VGARα1) subunits. (D) Representative traces of GABAergic transmission in paired-pulse recordings in non-infected neurons and in neurons infected with the indicated lentivirus. Control traces in the presence of bicuculine are shown below each trace. (E) Quantification of the first eIPSC amplitudes and of the paired-pulse ratios obtained in the indicated neuronal cultures. Values are expressed as mean ± SEM; n  = 7−9 recorded cells per group.
	Figure 2. Transgenic expression of Venus-GABAARα1.(A) Strategy for Otx1 BAC modification with VGABAARα1. The red line shows the Southern blot probe used in (B). Scale: 2 kb. (B) Correct incorporation of Venus-GABRA1 cDNA into the Otx1 BAC is shown by southern blotting. The modified BAC (middle lane) contains an additional EcoR1 site. The right lane shows correct incorporation of the modified BAC into the mouse genome. The transgenic mouse genome contains a wild-type copy of the Otx1 regulatory region as well as the modified Otx1-Venus-GABRA1 BAC. (C) Cortical protein extract from wild type and Otx1-VGABAARα1 mice immunoblotted with anti-GABAARα1 antibody. Only the transgenic mouse expresses the fusion version of the GABAARα1 subunit (top band). (D) VGABAARα1 expression in cortical layers 5 and 6 pyramidal neurons of Otx1-VGABAARα1 mice is shown by GFP immunoreactivity. The fusion protein is localized to pyramidal cell soma in layers 5/6 and processes in layers 2/3. Scale: 500 µm. (E) Immunofluorescence shows the colocalization of VGABAARα1 (green) and NeuN (red), a neuronal marker, in layers 5 and 6 pyramidal neurons of Otx1- VGABAARα1 transgenic mice (left). VGABAARα1 is mainly localized to the perikarya of the cell soma as well as dendrites. A control Otx1 BAC transgenic mouse expresses soluble eGFP (right), which fills the cell soma. Scale: 100 µm. V: Venus. GAR: GABAA receptor.
	Figure 3. Venus-GABAARα1 localizes specifically to inhibitory synapses.(A) Light microscopy of fixed saggital sections from wild type and Otx1-VGABAARα1 transgenic mice treated with anti-GFP antibody and revealed with the DAB procedure. Transgenic, but not wild type mice express the fusion protein in layer 5/6 cortical pyramidal neurons. The fusion protein localizes to cell bodies (arrows) and processes (arrowheads) in cortex. Scale bars: 200 µm. (B) Immuno-electron microscopy shows VGABAARα1 expression (arrows) exclusively at inhibitory synapses by silver-intensified immunogold labeling (SIG). Inhibitory terminals immunoreactive for GAD65/67 are revealed with the DAB procedure (white asterisks). Asymmetric synapses (black asterisks) are immunonegative for both GAD and VGABAARα1. Scale: 500 nm. Cy: cytoplasm. Nu: nucleus. De: dendrite. V: Venus. GAR: GABAA receptor. (C) Within a total cortical area of 614.6 square microns 67 of the 134 inhibitory (symmetric) synapses were labeled by VGABAARα1, whereas none of the 200 excitatory (asymmetric) synapses were immunopositive for the fusion protein. (D) An average of 54% of inhibitory synapses were immunopositive for VGABAARα1, compared to 0% of the excitatory synapses. The data are presented as average ± SEM (t test).
	Figure 4. Biochemical purification of a tagged inhibitory synaptic protein complex.(A) Immunoblotting of various proteins shows that detergent solubilized protein extract S3 was enriched in both inhibitory (VGABAARα1, GABAARα1, GABAARβ2/3, GABAARγ2) and excitatory (GluR2, PSD95) synaptic proteins, as well as mitochondria (COx). Gel filtration of fraction S3 enabled enrichment of synaptic protein complexes relative to intracellular proteins, as shown by the specific exclusion of the endoplasmic reticulum marker BIP, from the high molecular weight fractions (6–10). Protein concentration of each fraction was measured (top), and the void volume determined by the elution of Blue Dextran (2000 kDa). Identical results were obtained for endogenous proteins in fractions prepared from wildtype or Otx1-eGFP cortices (not shown). (B) Fractions 6–10 (red box in A) from Otx1-VGABAARα1 or Otx1-eGFP control were pooled and subject to co-immunopurification using an anti-eGFP antibody. Immunoblotting confirmed the specific presence of inhibitory synaptic proteins (VGABAARα1, GABAARα1, GABAARβ2/3, GABAARγ2) and the absence of excitatory synaptic (GluR2, PSD95) and mitochondrial (COx) proteins in the material immunopurified via VGABAARα1. Only soluble eGFP was detected in the control sample. IN: Input. FT: Flow-through. IP: Immunoprecipitate. V: Venus. GAR: GABAA receptor. Further biochemical experimental results are presented in Figure S1.
	Figure 5. Mass spectrometry identifies proteins present at tagged inhibitory synapses.(A) All peptides were evaluated individually, for their presence or absence in the sample isolated via VGABAARα1 or eGFP, using information from peptide fragmentation spectrum (MS/MS), peptide mass spectrum (MS), and peptide retention time in extracted ion chromatogram. An example is shown for peptide, GDDNAVTGTK, from GABAARβ2. V: Venus. GAR: GABAA receptor. (B) Schematic representation of the cortical inhibitory synaptic protein complex. These synapses contain a multitude of inhibitory receptors, as well as cell signaling and adhesion proteins, but are entirely lacking in cell signaling molecules. The localization of LHFPL4 and Neurobeachin is hypothetical. Complete information on each peptide is in Table 1 and Figure S2 and Table S1.
	Figure 6. Proteins identified by mass spectrometry are present at inhibitory synapses.(A) Immunoblotting of several proteins identified by mass spectrometry confirmed their presence in immunopurified inhibitory synapses. GABAARα2, neuroligin2 and neuroligin3 are present, while excitatory markers neuroligin1 and homer are absent from VGABAARα1-tagged inhibitory synapses. The abundant signaling molecule CaMKII is also absent. (B-F) Immunofluorescence studies confirm the colocalization of several proteins identified by mass spectrometry with VGABAARα1. Gephyrin is localized to inhibitory synapses on both the cell soma and axon initial segment (B), while PSD95 is markedly absent (C). GABAARβ2/3 (D), Nlgn2 (E) and Nlgn3 (F) also colocalize with VGABAARα1 in cortical pyramidal neurons. Scale: 10 µm. V: Venus. GAR: GABAA receptor.

Aoki, Use of electron microscopy in the detection of adrenergic receptors. 2000, Pubmed

Aoki, Use of electron microscopy in the detection of adrenergic receptors. 2000, Pubmed
Arancibia-Cárcamo, Ubiquitin-dependent lysosomal targeting of GABA(A) receptors regulates neuronal inhibition. 2009, Pubmed
Auer, Silencing neurotransmission with membrane-tethered toxins. 2010, Pubmed
Bayés, Neuroproteomics: understanding the molecular organization and complexity of the brain. 2009, Pubmed
Beavis, Using the global proteome machine for protein identification. 2006, Pubmed
Bedford, GABA(A) receptor cell surface number and subunit stability are regulated by the ubiquitin-like protein Plic-1. 2001, Pubmed
Blomberg, The structure of postsynaptic densities isolated from dog cerebral cortex. II. Characterization and arrangement of some of the major proteins within the structure. 1977, Pubmed
Budreck, Neuroligin-3 is a neuronal adhesion protein at GABAergic and glutamatergic synapses. 2007, Pubmed
Bürli, Single particle tracking of alpha7 nicotinic AChR in hippocampal neurons reveals regulated confinement at glutamatergic and GABAergic perisynaptic sites. 2010, Pubmed
Chen, Mass of the postsynaptic density and enumeration of three key molecules. 2005, Pubmed
Chen, GABAA receptor associated proteins: a key factor regulating GABAA receptor function. 2007, Pubmed
Chen, C-terminal modification is required for GABARAP-mediated GABA(A) receptor trafficking. 2007, Pubmed , Xenbase
Cherubini, GABA: an excitatory transmitter in early postnatal life. 1991, Pubmed
Cho, The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein. 1992, Pubmed
Collins, Molecular characterization and comparison of the components and multiprotein complexes in the postsynaptic proteome. 2006, Pubmed
Cox, Quantitative, high-resolution proteomics for data-driven systems biology. 2011, Pubmed
Delint-Ramirez, In vivo composition of NMDA receptor signaling complexes differs between membrane subdomains and is modulated by PSD-95 and PSD-93. 2010, Pubmed
Dunkley, A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: homogeneity and morphology of subcellular fractions. 1988, Pubmed
Earnheart, GABAergic control of adult hippocampal neurogenesis in relation to behavior indicative of trait anxiety and depression states. 2007, Pubmed
ECCLES, Excitatory and inhibitory synaptic action. 1959, Pubmed
ECCLES, The relationship between the mode of operation and the dimensions of the junctional regions at synapses and motor end-organs. 1958, Pubmed
Elias, Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. 2007, Pubmed
Fang, GODZ-mediated palmitoylation of GABA(A) receptors is required for normal assembly and function of GABAergic inhibitory synapses. 2006, Pubmed
Feng, Organization and dynamics of PDZ-domain-related supramodules in the postsynaptic density. 2009, Pubmed
Fritschy, GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. 1995, Pubmed
Fritschy, Epilepsy, E/I Balance and GABA(A) Receptor Plasticity. 2008, Pubmed
Fritschy, Gephyrin: where do we stand, where do we go? 2008, Pubmed
Gong, Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin of replication. 2002, Pubmed
GRAY, Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscope study. 1959, Pubmed
Hjelmeland, Solubilization of native membrane proteins. 1990, Pubmed
Huang, GABA and neuroligin signaling: linking synaptic activity and adhesion in inhibitory synapse development. 2008, Pubmed
Husi, Proteomic analysis of NMDA receptor-adhesion protein signaling complexes. 2000, Pubmed
Hutcheon, Organization of GABA receptor alpha-subunit clustering in the developing rat neocortex and hippocampus. 2004, Pubmed
Ibañez-Tallon, Dysfunction of axonemal dynein heavy chain Mdnah5 inhibits ependymal flow and reveals a novel mechanism for hydrocephalus formation. 2004, Pubmed
Ichtchenko, Structures, alternative splicing, and neurexin binding of multiple neuroligins. 1996, Pubmed
Keller, The gamma2 subunit of GABA(A) receptors is a substrate for palmitoylation by GODZ. 2004, Pubmed
Kelly, Synaptic proteins. Characterization of tubulin and actin and identification of a distinct postsynaptic density polypeptide. 1978, Pubmed
Kennedy, The postsynaptic density at glutamatergic synapses. 1997, Pubmed
Kennedy, Signal-processing machines at the postsynaptic density. 2000, Pubmed
Kim, SynGAP: a synaptic RasGAP that associates with the PSD-95/SAP90 protein family. 1998, Pubmed
Kittler, Modulation of GABAA receptor activity by phosphorylation and receptor trafficking: implications for the efficacy of synaptic inhibition. 2003, Pubmed
Levinson, Postsynaptic scaffolding molecules modulate the localization of neuroligins. 2010, Pubmed
Li, Two pools of Triton X-100-insoluble GABA(A) receptors are present in the brain, one associated to lipid rafts and another one to the post-synaptic GABAergic complex. 2007, Pubmed
Li, Septin 11 is present in GABAergic synapses and plays a functional role in the cytoarchitecture of neurons and GABAergic synaptic connectivity. 2009, Pubmed
Lisman, A mechanism for the Hebb and the anti-Hebb processes underlying learning and memory. 1989, Pubmed
Liu, Direct protein-protein coupling enables cross-talk between dopamine D5 and gamma-aminobutyric acid A receptors. 2000, Pubmed
Luscher, GABAA receptor trafficking-mediated plasticity of inhibitory synapses. 2011, Pubmed
McBain, Presynaptic plasticity: targeted control of inhibitory networks. 2009, Pubmed
McKernan, GABAA receptor subtypes immunopurified from rat brain with alpha subunit-specific antibodies have unique pharmacological properties. 1991, Pubmed
Medrihan, Neurobeachin, a protein implicated in membrane protein traffic and autism, is required for the formation and functioning of central synapses. 2009, Pubmed
Merchán-Pérez, Counting Synapses Using FIB/SEM Microscopy: A True Revolution for Ultrastructural Volume Reconstruction. 2009, Pubmed
Muir, NMDA receptors regulate GABAA receptor lateral mobility and clustering at inhibitory synapses through serine 327 on the γ2 subunit. 2010, Pubmed
Nusser, Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells. 1998, Pubmed
Olsen, GABA A receptors: subtypes provide diversity of function and pharmacology. 2009, Pubmed
Papadopoulos, Collybistin is required for both the formation and maintenance of GABAergic postsynapses in the hippocampus. 2008, Pubmed
Pirker, GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. 2000, Pubmed
Prior, Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein. 1992, Pubmed
Saiepour, Complex role of collybistin and gephyrin in GABAA receptor clustering. 2010, Pubmed
Schneider Gasser, Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons. 2006, Pubmed
Selimi, Proteomic studies of a single CNS synapse type: the parallel fiber/purkinje cell synapse. 2009, Pubmed
Shen, A critical role for alpha4betadelta GABAA receptors in shaping learning deficits at puberty in mice. 2010, Pubmed
Sheng, The postsynaptic architecture of excitatory synapses: a more quantitative view. 2007, Pubmed
Siekevitz, The postsynaptic density: a possible role in long-lasting effects in the central nervous system. 1985, Pubmed
Stürzebecher, An in vivo tethered toxin approach for the cell-autonomous inactivation of voltage-gated sodium channel currents in nociceptors. 2010, Pubmed , Xenbase
Südhof, Neuroligins and neurexins link synaptic function to cognitive disease. 2008, Pubmed
Szabadits, NMDA receptors in hippocampal GABAergic synapses and their role in nitric oxide signaling. 2011, Pubmed
Thomson, Mechanisms underlying synapse-specific clustering of GABA(A) receptors. 2010, Pubmed
Tretter, The clustering of GABA(A) receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor alpha 2 subunits to gephyrin. 2008, Pubmed
Tyagarajan, GABA(A) receptors, gephyrin and homeostatic synaptic plasticity. 2010, Pubmed
Walikonis, Identification of proteins in the postsynaptic density fraction by mass spectrometry. 2000, Pubmed
Wang, Neurobeachin: A protein kinase A-anchoring, beige/Chediak-higashi protein homolog implicated in neuronal membrane traffic. 2000, Pubmed
Wehner, Solubilization of benzodiazepine receptors from long- and short-sleep mice. 1993, Pubmed
Wiśniewski, Universal sample preparation method for proteome analysis. 2009, Pubmed