Journal: bioRxiv

Article Title: SYNPLA: A synapse-specific method for identifying learning-induced synaptic plasticity loci

doi: 10.1101/473314

Figure Lengend Snippet: a ) Diagram of SYNPLA targeting presynaptic myc-NRXN and. cLTP induces mobilization of GluA1 from an extrasynaptic pool and insertion into the post-synaptic density, decreasing the distance between the targeted proteins and permitting PLA. b ) Representative images of SYNPLA reactions performed on myc-NRXN expressing cultured rat hippocampal neurons at 14 days in vitro for the indicated conditions; PLA shown in gray. Scale bar: 10 μm. c ) Quantification of a single SYNPLA experiment (as in f ). The number of SYNPLA puncta detected in each field of view (round) and the average (square) ± SEM across all fields of view are shown; *** p<0.001, 1-way ANOVA followed by Tukey-Kramer post-hoc test. d ) Quantification of six independent experiments (indicated by different colors); the average number of SYNPLA puncta across 10-12 fields of view for each experiment (circles, normalized to CTRL) and the average PLA signal across experiments (square) ± SEM are shown; ** p<0.01, 1-way ANOVA (with Tukey-Kramer post-hoc test). e ) Diagram of SYNPLA in rat organotypic hippocampal slices. Sindbis virus expressing myc-NRXN is injected in the presynaptic CA3 region SYNPLA between myc-NRXN and endogenous GluA1 is measured in the postsynaptic CA1 region. f ) Representative images of SYNPLA under the indicated conditions in region CA1 of organotypic slice cultures. Scale bar: 10 μm. g ) Quantification of SYNPLA puncta in organotypic slices, from three independent experiments (indicated with different colors, 4 slices per condition (one circle per slice)); squares indicate average ± SEM across experiments; *** p<0.001, paired t-test.

Article Snippet: For Supplementary Fig. 2 different primary antibodies were used as negative controls: guinea pig anti-GluA1 (AGP-009, Alomone labs), rabbit anti-cFos (Cell signaling, #2250); all 1:100 dilution.

Techniques: Expressing, Cell Culture, In Vitro, Injection

Journal: bioRxiv

Article Title: SYNPLA: A synapse-specific method for identifying learning-induced synaptic plasticity loci

doi: 10.1101/473314

Figure Lengend Snippet: a ) Diagram of a general PLA reaction. Note that A and B must be sufficiently close to permit complementary binding and ligation of ABo1 and ABo2 and subsequent amplification to make a rolony. b ) Diagram of PLA targeting recombinant presynaptic myc-NRXN and postsynaptic HA-NLGN. c ) PLA rolonies (white dots, right) formed between postsynaptic cultured mouse hippocampal neuron expressing HA-NLGN + mCherry (red; middle) and co-cultured presynaptic neurons expressing myc-NRXN + GFP (green; left). Scale bar: 10 μm. d ) PLA puncta in each sample (circles) and average (squares) ± SEM for indicated expression and days in vitro (color indicates data acquired on the same day). *** p<0.001, 2-way ANOVA (see Methods); * p<0.05, 1-way ANOVA (with Tukey-Kramer post-hoc test). e ) PLA reaction between endogenous GluA1 and recombinant myc in cultured rat hippocampal neurons (exposed to cLTP, see ) expressing myc-NRXN and cytosolic fluorescent protein (green), subsequently immunostained for endogenous PSD-95 (red). Scale bars: 10 μm (left); 5 μm (right). f ) PLA puncta localize to synapses. In PSD-95 and myc-NRXN channels (from e , right), pixels were set to zero (masked) for values below a progressively increasing threshold (x axis). Puncta (PLA or randomly placed) display colocalization if non-zero pixels exist within 0.14 μm in both thresholded channels. For indicated threshold, ∼95% of PLA, while only ∼50% of randomly placed (mean ± SEM of 30 placements) puncta colocalized with pre- and post-synaptic markers. See Supplementary Fig. 3 and Methods for details.

Article Snippet: For Supplementary Fig. 2 different primary antibodies were used as negative controls: guinea pig anti-GluA1 (AGP-009, Alomone labs), rabbit anti-cFos (Cell signaling, #2250); all 1:100 dilution.

Techniques: Binding Assay, Ligation, Amplification, Recombinant, Cell Culture, Expressing, In Vitro