January 1, 2012;
The biochemical anatomy of cortical inhibitory synapses.
Classical electron microscopic studies of the mammalian brain
revealed two major classes of synapses, distinguished by the presence of a large postsynaptic density (PSD
) exclusively at type 1, excitatory synapses. Biochemical studies of the PSD
have established the paradigm of the synapse as a complex signal-processing machine that controls synaptic plasticity. We report here the results of a proteomic analysis of type 2, inhibitory synaptic complexes isolated by affinity purification from the cerebral cortex. We show that these synaptic complexes contain a variety of neurotransmitter receptors, neural cell-scaffolding and adhesion molecules, but that they are entirely lacking in cell signaling proteins. This fundamental distinction between the functions of type 1 and type 2 synapses in the nervous system has far reaching implications for models of synaptic plasticity, rapid adaptations in neural circuits, and homeostatic mechanisms controlling the balance of excitation and inhibition in the mature brain
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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.
Use of electron microscopy in the detection of adrenergic receptors.