Biochemical purification of a tagged inhibitory synaptic protein complex.

<p>(A) Immunoblotting of various proteins shows that detergent solubilized protein extract S3 was enriched in both inhibitory (VGABA_ARα1, GABA_ARα1, GABA_ARβ2/3, GABA_ARγ2) 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-VGABA_ARα1 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 (VGABA_ARα1, GABA_ARα1, GABA_ARβ2/3, GABA_ARγ2) and the absence of excitatory synaptic (GluR2, PSD95) and mitochondrial (COx) proteins in the material immunopurified via VGABA_ARα1. Only soluble eGFP was detected in the control sample. IN: Input. FT: Flow-through. IP: Immunoprecipitate. V: Venus. G_AR: GABA_A receptor. Further biochemical experimental results are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039572#pone.0039572.s001" target="_blank">Figure S1</a>.</p

The inhibitory synapse affinity tag, Venus-GABA_ARα1, is functional <i>in vitro</i>.

<p>(A) Schematic of VGABA_ARα1, showing the N-terminal fusion of an affinity tag, Venus. (B) Representative GABA-evoked currents (left) and current amplitude quantification (right) in voltage clamped <i>Xenopus</i> oocytes after coinjection of the indicated <i>GABR</i> 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 GABA_ARα1 (Lv-G_ARα1) or Venus-GABA_ARα1 (Lv-VG_ARα1) 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.</p

Mass spectrometry identifies proteins present at tagged inhibitory synapses.

<p>(A) All peptides were evaluated individually, for their presence or absence in the sample isolated via VGABA_ARα1 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 GABA_ARβ2. V: Venus. G_AR: GABA_A 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039572#pone-0039572-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039572#pone.0039572.s002" target="_blank">Figure S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039572#pone.0039572.s003" target="_blank">Table S1</a>.</p

Inhibitory synaptic proteins identified by mass spectrometry.

<p>Proteins were purified via VGABA_ARα1 or eGFP and identified by LC-MS/MS. Two replicate datasets were analyzed using the GPM Database and those proteins identified in the eGFP controls were excluded from the list. Proteins present in the dataset were confirmed to be present in the replicate by either MS or MS/MS, as indicated. GPM E value is the probability that an assignment occurs by chance. Total peptide number is the total number of peptides that match a given protein. When more than one homologue is reported, unique peptide no. corresponds to the number of peptide matches that are unique to the homologue.</p

Proteins identified by mass spectrometry are present at inhibitory synapses.

<p>(A) Immunoblotting of several proteins identified by mass spectrometry confirmed their presence in immunopurified inhibitory synapses. GABA_ARα2, neuroligin2 and neuroligin3 are present, while excitatory markers neuroligin1 and homer are absent from VGABA_ARα1-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 VGABA_ARα1. Gephyrin is localized to inhibitory synapses on both the cell soma and axon initial segment (B), while PSD95 is markedly absent (C). GABA_ARβ2/3 (D), Nlgn2 (E) and Nlgn3 (F) also colocalize with VGABA_ARα1 in cortical pyramidal neurons. Scale: 10 µm. V: Venus. G_AR: GABA_A receptor.</p

Venus-GABA_ARα1 localizes specifically to inhibitory synapses.

<p>(A) Light microscopy of fixed saggital sections from wild type and Otx1-VGABA_ARα1 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 VGABA_ARα1 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 VGABA_ARα1. Scale: 500 nm. Cy: cytoplasm. Nu: nucleus. De: dendrite. V: Venus. G_AR: GABA_A receptor. (C) Within a total cortical area of 614.6 square microns 67 of the 134 inhibitory (symmetric) synapses were labeled by VGABA_ARα1, 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 VGABA_ARα1, compared to 0% of the excitatory synapses. The data are presented as average ± SEM (t test).</p

Transgenic expression of Venus-GABA_ARα1.

<p>(A) Strategy for Otx1 BAC modification with VGABA_ARα1. The red line shows the Southern blot probe used in (B). Scale: 2 kb. (B) Correct incorporation of <i>Venus-GABRA1</i> 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-<i>Venus-GABRA1</i> BAC. (C) Cortical protein extract from wild type and Otx1-VGABA_ARα1 mice immunoblotted with anti-GABA_ARα1 antibody. Only the transgenic mouse expresses the fusion version of the GABA_ARα1 subunit (top band). (D) VGABA_ARα1 expression in cortical layers 5 and 6 pyramidal neurons of Otx1-VGABA_ARα1 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 VGABA_ARα1 (green) and NeuN (red), a neuronal marker, in layers 5 and 6 pyramidal neurons of Otx1- VGABA_ARα1 transgenic mice (left). VGABA_ARα1 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. G_AR: GABA_A receptor.</p