Journal: eLife

Article Title: Alzheimer’s disease risk gene BIN1 induces Tau-dependent network hyperexcitability

doi: 10.7554/eLife.57354

Figure Lengend Snippet: ( A ) ICC experimental timeline: neurons were plated on day in vitro (DIV) 0, virally transduced on DIV 2, and immunostained at DIV 15, DIV 22, or DIV 27. ( B ) Representative images of primary hippocampal cultures: untransduced (left), AAV-mKate2 (center), or AAV-BIN1-mKate2 (right), showing Tau and BIN1 immunostaining at DIV 15 (top), DIV 22 (middle), or DIV 27 (bottom). Scale bar = 20 µm. ( C ) AAV-BIN1-mKate2 increased BIN1 levels ~ 8–9-fold in BIN1 group compared to BIN1 levels in untransduced or mKate2 groups ( n = 2–6 fields of view per coverslip, 60x magnification, two-way ANOVA, BIN-DIV interaction p=0.1123, main effect of AAV-BIN1-mKate2 ****p<0.0001, main effect of DIV p=0.6373, Tukey’s multiple comparisons test: DIV 15:UnTD vs. DIV 15:AAV-BIN1-mKate2, ****p<0.0001, DIV 22:UnTD vs. DIV 22:AAV-BIN1-mKate2, ****p<0.0001, DIV 27:UnTD vs. DIV 27:AAV-BIN1-mKate2, ****p<0.0001. ( D ) Representative images of primary hippocampal cultures at DIV 12: untransduced (left), AAV-mKate2 (center), or AAV-BIN1-mKate2 (right), showing NeuN immunostaining (top), mKate2 fluorescence (middle), or merge of both (bottom). Scale bar = 25 µm. ( E ) The total number of neurons per well did not change between untransduced, mKate2, or BIN1 groups ( n = 6–7 coverslips, 10 × 10 fields of view per coverslip, 20x magnification, from two different primary neuron harvests, one-way ANOVA, p=0.5157). ( F ) MEA experimental timeline: neurons were plated on day in vitro (DIV) 0, virally transduced on DIV 2, and recorded on DIV 12. ( G ) Primary neuronal hippocampal cultures grown on an MEA plate. Scale bar = 50 µm. ( H ) Representative local field potential (LFP) traces. ( I ) Representative raster plots of firing activity from five different neurons per group. ( J ) BIN1 increased action potential frequency ( n = 27–36 neurons per group from three different primary neuron harvests, normalized to the controls from each harvest, median frequency in controls = 388 mHz; unpaired Mann-Whitney U test; p=0.0233). ( K ) BIN1 increased burst firing frequency ( n = 27–36 neurons per group from three different primary neuron harvests, normalized to the controls from each harvest, median frequency in controls = 11.7 mHz; unpaired Mann-Whitney U test; p=0.0020). ( L ) The total number of active neurons per well did not differ between mKate2 and BIN1 expressing groups ( n = 9 MEA plates for each group from three different primary neuron harvests, unpaired Student’s t test; p=0.346). All data are expressed as mean ± SEM, *p<0.05, **p<0.01, and ****p<0.0001. All data are expressed as mean ± SEM.

Article Snippet: For 48-well plate multielectrode array recordings, neurons were plated at 30,000 per well in 48-well MEA plates (Axion Biosystems, M768-tMEA-48B-5).

Techniques: In Vitro, Immunostaining, Fluorescence, Activity Assay, MANN-WHITNEY, Expressing