DOI: 10.5061/dryad.rbnzs7h8z
title: Alzheimer’s disease risk gene BIN1 induces Tau-dependent network hyperexcitability — MEA Axion Biosciences Maestro Recordings, Figure 6

description: Genome-wide association studies identified
 the BIN1 locus as a leading modulator of genetic risk in
 Alzheimer's disease (AD). One limitation in
 understanding BIN1's contribution to AD is its unknown
 function in the brain. AD-associated BIN1 variants are
 generally noncoding and likely change expression. Here, we determined the
 effects of increasing expression of the major neuronal isoform of human
 BIN1 in cultured rat hippocampal neurons. Higher BIN1 induced network
 hyperexcitability on multielectrode arrays, increased frequency of
 synaptic transmission, and elevated calcium transients, indicating that
 increasing BIN1 drives greater neuronal activity. In exploring the
 mechanism of these effects on neuronal physiology, we found that BIN1
 interacted with L-type voltage-gated calcium channels (LVGCCs) and that
 BIN1–LVGCC interactions were modulated by Tau in rat hippocampal neurons
 and mouse brain. Finally, Tau reduction prevented BIN1-induced network
 hyperexcitability. These data shed light on BIN1's neuronal function
 and suggest that it may contribute to Tau-dependent hyperexcitability in
 AD.
description: <strong>Multi electrode array
 cultures</strong> For 6-well multielectrode array
 recordings, neurons were plated at 100,000 per well in six-well MEA plates
 (ALA Scientific, ALAMEA-MEMMR5). 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). <strong>BIN1
 constructs and vectors</strong> A BIN1-mKate2 (GE
 Dharmacon, OHS5894-202501160) construct was developed to encode human BIN1
 isoform 1 (593 AA, the major neuronal isoform) tagged with mKate2
 (Evrogen, FP184, to allow for fluorescent visualization) at the C-terminus
 to allow for proper function of the N-terminal membrane-interacting BAR
 domain. A similar construct lacking the BAR domain (amino acids 32–273,
 BIN1-ΔBAR-mKate2) was produced as a BIN1 BAR domain deletion mutant. A
 construct encoding mKate2 only was used as a control. These constructs
 were then cloned into the CIGW vector (rAAV9-CBA-IRES-GFP-WPRE-rBG) (St
 Martin et al., 2007). Due to size limitations for efficient gene
 expression, the IRES-GFP was removed from the CIGW vector.
 Antisense oligonucleotide application
 Tau anti-sense oligonucleotide (ASO) sequences
 were adapted from DeVos
 et al., 2013 and produced by Integrated DNA Technology (Tau ASO:
 5-ATCACTGATTTTGAAGTCCC-3, Nontargeting control ASO:
 5-CCTTCCCTGAAGGTTCCTCC-3). ASOs were dissolved to 100 μM in 10 mM Tris
 with 0.1 mM EDTA and stored at −20°C until use. At DIV 6, one week before
 testing for both MEA experiments and PLA, neurons were treated with ASO to
 a final concentration of 1 μM. <strong>Axion
 Biosciences MEA</strong> Single neuron
 electrophysiological activity was recorded using an Axion Maestero
 recording system as in Savell
 et al., 2019a. Briefly, neurons were plated on the 48-well MEA
 (Axion Biosystems, M768-tMEA-48B-5) with 16 extracellular recording
 electrodes and a ground electrode per well at a density of 30,000 neurons
 per well in Neurobasal medium (5 μL) with 10% FBS (Atlanta Biologicals,
 S11550) and placed in a 37°C incubator with 5% CO<sub>2</sub>.
 After allowing neurons to attach to the plate for 2 hr, 300 μL serum-free
 Neurobasal (Life Technologies, 21103049) was added. The next day, AraC was
 added as with other experiments and a 50% medium change with BrainPhys
 (Stemcell Technologies Inc, 05790) supplemented with SM1 and L-glutamine
 was done at DIV 5. At DIV 6, neurons were treated with ASO to reduce Tau
 protein levels. At DIV 9, a 50% medium change was completed with
 supplemented BrainPhys, followed by a 50% medium change with supplemented
 Neurobasal at DIV 12. At DIV 13, neurons were recorded using Axion AxIS
 software for 15 min. Electrical activity was measured by an interface
 board at 12.5 kHz, digitized, and transmitted to an external computer for
 data acquisition and analysis in Axion AxIS Navigator software (Axion
 Biosystems). All data were filtered using dual 0.01 Hz (high pass) and
 5,000 Hz (low-pass) Butterworth filters. Action potential thresholds were
 set automatically using an adaptive threshold for each electrode
 (&gt;6 standard deviations from the electrode’s mean signal). Neuronal
 waveforms collected in Axion AxIS Navigator were exported to Offline
 Sorter (v. 4.0 Plexon). Offline Sorter automatically completes and plots
 PCA on waveforms for each electrode. Manual inspection of PCA, shape,
 inter-spike intervals, auto-correlograms, and cross-correlograms allowed
 us to distinguish between multiple units on a single electrode and to do
 per-neuron analyses. After waveforms were split into units, analysis of
 each unit’s action potential frequency and burst firing was completed in
 NeuroExplorer (v. 5.0, Plexon) using the built-in Burst Analysis function,
 with Poisson burst surprise = 5. Next, firing rates and bursting analysis
 were performed in NeuroExplorer (v. 5.0 Plexon). Researchers were blinded
 to experimental conditions performed in all MEA analyses.
 Statistics 
 Statistical distribution of data varied widely between data sets
 in this study, so we analyzed each data set for normality and analyzed
 using either parametric or non-parametric tests accordingly. The specific
 test used is indicated in the figure legend in each case. All statistical
 tests were performed with Prism 8 (GraphPad, v. 8.4.0).
description: An excel file with the plate layout is uploaded.