All animal work was approved by the Committee for Animal Care of the Division of Comparative Medicine at the Massachusetts Institute of Technology and by the Institutional Animal Care and Use Committee at Georgia Institute of Technology. Mice were housed in groups no larger than five on a standard 12-h light/12-h dark cycle; all experiments were performed during the light cycle. Electrophysiology experiments were performed at Georgia Institute of Technology, male (1-3 month-old) WT mice (C57BL/6) were obtained from the Jackson laboratory. Mice were housed on a reverse 12 h light/12 h dark cycle and all experiments were performed during the dark cycle. Food and water were provided without restriction. For all experiments, mice from the same litter were divided into different conditions, respectfully. If additional groups were added, respective controls were always repeated concurrently.

Method Details

Surgical procedures Adult (2-3 month-old) mice were anesthetized with isoflurane and fixed in a stereotaxic frame. Ophthalmic ointment (Puralube Vet Ointment, Dechra) was applied to the eyes, and the scalp was shaved and sterilized with povidone-iodine (Dynarex) and 70% ethanol. A custom stainless steel headplate was fixed using dental cement (C&B Metabond, Parkell) and the target craniotomy site for LFP recordings was marked on the skull (in mm, from bregma: −2.0 anterior/ posterior, +/−1.8 medial/lateral for targeting CA1, −2.0 to −3.0 anterior/ posterior, +/−1.8 medial/lateral for targeting auditory cortex, and +1.3 to +1.4 anterior/posterior, +/− 1.0 medial/lateral for targeting prefrontal cortex). A craniotomy was later performed in 3-8 month-old mice. The day before or day of the first recording session, craniotomies (200-500 μm diameter) were made by thinning the skull with a dental drill and then making a hole with a 27-gauge needle. When not recording, the craniotomy was sealed with a sterile silicon elastomer (Kwik-Sil WPI).

Electrophysiology recordings During recordings, head-fixed animals ran on an air-floating 8-inch spherical treadmill. All animals had previously learned to maneuver on the treadmill until they were comfortable while occasionally receiving sweetened condensed milk (1:2 water dilution). Animals were on the ball for a maximum of 5 hs and had multiple periods of running and rest during this time. Single shank 32-channel probes (NeuroNexus) were advanced to the target location. Recording sites spanned 250 μm. For auditory cortex recordings, the probe was advanced at a 45° angle from vertical parallel to the coronal plane to a depth of 3-4.15mm. A series of 50 ms tones of 5, 10, 15, and 20 kHz were presented to detect auditory response in the mean LFP. For CA1 recordings, the probe was advanced vertically through the craniotomy to a depth of 1.14 – 2.05mm until hippocampal pyramidal layer electrophysiology characteristics were observed (large theta waves and sharp wave ripples, 150+ μV spikes on multiple channels). For prefrontal cortex recordings, the probe was advanced at a 20° angle from vertical, at a 49° angle from the coronal plane to a depth of 1.48-2.15mm. If data were collected at multiple depths during the same recording session; new depths were mapped in order to ensure the location of the recording sites remained in the target location (n = 9 recording depths from 9 sessions in 5 mice for AC and 12 recording depths from 10 sessions in 5 mice for CA1, n = 7 recording depths from 7 sessions in 4 mice for mPFC). Data were acquired with a sampling rate of 20 kHz using an Intan RHD2000 Evaluation System using a ground pellet as reference.

Auditory and visual stimuli for electrophysiology recordings Animals were presented with 10 s stimulation blocks interleaved with 10 s baseline periods. Stimulation blocks rotated between auditory-only or auditory and visual stimulation at 20 Hz, 40 Hz, 80 Hz, or with random stimulation (pulses were delivered with randomized inter-pulse intervals determined from a uniform distribution with an average interval of 25 ms). Stimuli blocks were interleaved to ensure the results observed were not due to changes over time in the neuronal response. 10 s long stimulus blocks were used to reduce the influence of onset effects, and to examine neural responses to prolonged rhythmic stimulation. All auditory pulses were 1 ms-long 10 kHz tones. All visual pulses were 50% duty cycle of the stimulation frequency (25 ms, 12.5 ms, or 6.25 ms in length). For combined stimulation, auditory and visual pulses were aligned to the onset of each pulse.

Prefrontal cortex histology During the final mPFC recording in each animal, the probe was coated with DiI (Thermo Fisher) and inserted to target depth. Mice were transcardially perfused with 4% paraformaldehyde in phosphate buffered saline (PBS) under anesthesia (isoflurane), and the brains were post-fixed overnight in 4% paraformaldehyde in 1xPBS. Brains were sectioned 100 μm thick with a Leica VT1000S vibratome (Leica). Sections were stained with 0.2% 1mMol DAPI (PanReac AppliChem) in 1xPBS and mounted onto microscopy slides with Vectashield mounting medium (VWR). Images were acquired on a Zeiss Axio Observer Z1 inverted epifluorescent microscope with the accompanying Zen Blue 2 software.

Spike sorting and single unit stability Chung et al., 2017 Chung J.E.

Magland J.F.

Barnett A.H.

Tolosa V.M.

Tooker A.C.

Lee K.Y.

Shah K.G.

Felix S.H.

Frank L.M.

Greengard L.F. A Fully Automated Approach to Spike Sorting. Spike detection and sorting was carried out using MountainSort automated spike sorting followed by manual curation based on visual inspection of waveforms and cross-correlograms (). Prior to manual curation, quality thresholds were applied to only include units with peak SNR greater than or equal to 1, less than 10% overlap with noise, and greater than 95% isolation against other units which resulted in well-isolated single units. To account for periods of instability in the recordings during which single units were lost, stability criteria were applied such that only stable periods (no sudden loss of a single unit’s firing rate) would be considered in analysis. Firing rate (FR) for each unit was computed over the course of the recording session. Firing rate was clustered into two distributions, low FR and high FR, using k-means clustering. For units with FR that dropped below 10% of the high FR mean, further analyses identified a stable recording period defined as the longest length of time that the FR was 2 standard deviations above the low FR mean.

LFP LFP was obtained by downsampling raw traces to 2kHz and bandpass filtering between 1-300Hz.

Power spectrum Power spectral density analysis was performed using multitaper methods from the Chronux toolbox (time-bandwidth product = 3, number of tapers = 5). LFP traces were divided into 10 s trials of each stimulation condition. The average power spectral density was computed for each animal (within the same recording day and recording depth) over these trials, referencing to a ground pellet in saline above the skull. Power spectral density analysis was initially computed for all recording sites in AC, CA1, and mPFC. From each recording depth, the traces with the largest 40 Hz peak in response to 40 Hz flicker stimuli were included in the analysis. The per-depth traces displayed in the presented data had the largest 40 Hz peak in response to auditory flicker stimuli.

Firing during flicker stimulation 1 / s t i m u l u s f r e q u e n c y sec), with 10 bins per cycle, to show spiking across trains of stimuli. Displaying spiking modulation over multiple cycles is typical for displaying modulation by oscillations ( Csicsvari et al., 1999 Csicsvari J.

Hirase H.

Czurkó A.

Mamiya A.

Buzsáki G. Oscillatory coupling of hippocampal pyramidal cells and interneurons in the behaving Rat. ( p e r i − s t i m u l u s s p i k e t i m e ) ∗ 2 π ∗ ( s t i m u l u s f r e q u e n c y ) and vector strengths and Rayleigh statistics were computed. Vector strength was computed using methods from the CircStat toolbox; the Rayleigh statistic was computed using the equation R S = 2 n V S 2 , where n is total spike count, and VS is vector strength ( Berens, 2009 Berens P. CircStat: A MATLAB Toolbox for Circular Statistics. Ma et al., 2013 Ma L.

Tai X.

Su L.

Shi L.

Wang E.

Qin L. The neuronal responses to repetitive acoustic pulses in different fields of the auditory cortex of awake rats. 1 / S t i m u l u s f r e q u e n c y sec, α = 3), and z-scored. Neurons were aligned by their average stimulus phase preference in the analyzed four cycles. The single unit peri-stimulus time histograms (PSTH) for each stimulus frequency encompassed two stimulus cycles (where one cycle =sec), with 10 bins per cycle, to show spiking across trains of stimuli. Displaying spiking modulation over multiple cycles is typical for displaying modulation by oscillations (). PSTHs were computed for all single units by binning spikes for 1 stimulus cycles before and after the start of each light-on or audio-on pulse. No stimulation (baseline) histograms were calculated using randomly distributed pulse times, as in the random stimulation condition. Firing rate was computed in each bin by dividing the number of spikes per bin by the total time in that bin (the total number of pulses times the bin size). To quantify firing rate periodicity in relation to the stimulus frequency, the time interval between firing rate peaks was calculated for all single unit histograms. The peaks of each PSTH was the maximum firing rate within one stimulus interval. To quantify firing rate modulation by the stimulus and compute circular statistics, peri-stimulus spike times were converted into radians: (and vector strengths and Rayleigh statistics were computed. Vector strength was computed using methods from the CircStat toolbox; the Rayleigh statistic was computed using the equation, where n is total spike count, and VS is vector strength (). Differences in vector strength and Rayleigh statistic values were computed by taking the differences in these values between stimulus conditions for each unit. Heatmaps showing the firing rate response to flicker for all recorded single units were computed over four consecutive stimulus cycles. In order to show the response of all neurons, we show four consecutive stimulus cycles of each stimulation period. To do this, we aligned the 10 s presentation periods of each stimulus condition, and then excluded the first 100ms of each presentation period to prevent onset effects from obscuring entrainment. Then, we averaged spiking response over the next four stimulus cycles (200 ms for 20 Hz, 100 ms for 40 Hz, and 50 ms for 80 Hz) to obtain the firing rate response to flicker. Firing rate for each single unit was computed in 1ms bins, smoothed with a Gaussian windows proportional to each stimulus frequency (N =sec, α = 3), and z-scored. Neurons were aligned by their average stimulus phase preference in the analyzed four cycles.

Mean Firing Rate Mean firing rate was computed for each single unit for each stimulus condition. Only stable periods for each unit contributed to the mean FR calculation (see Spike sorting and single unit stability, above). Difference in mean firing rate between stimulus conditions was computed within each unit by taking the difference in mean FR in each condition for that unit.

40 Hz visual flicker stimulation protocol For biochemical and Immunohistochemical analysis, 5XFAD mice were placed in a dark chamber illuminated by a light-emitting diode (LED) bulb and were exposed to one of four stimulation conditions: dark, 8 Hz, 40 Hz (12.5 ms light on, 12.5 ms light off, 60 W), or random (light pulses were delivered with a random interval determined by a uniform distribution with a mean of 25 ms) stimulation for 1-h for seven days.

40 Hz auditory tone train stimulation protocol For biochemical, Immunohistochemical, or behavioral analysis, 5XFAD, APP/PS1, or P301S mice were placed in a dimly lit chamber in a quiet room insulated with sound-proof foam (McMaster-Carr, 5692T49). Speakers (AYL, AC-48073) were placed out-of-reach from the mouse above the chambers. Mice were exposed to one of five stimulation conditions: no tones, tones at 8 Hz, tones at 40 Hz, tones at 80 Hz, or tone delivered at random (auditory tones were delivered with a random interval determined by a uniform distribution with a mean of 25ms) stimulation. Tones for the stimulation conditions consisted of a 10 kHz tone that was 1 ms in duration and delivered at 60 dB. For electrophysiology recordings, after probe placement, the lights in the room were turned off and the animals were presented with alternating 10 s periods of audio-only and visual-audio stimulation interleaved with 10 s periods of no light or tones. For audio-only stimulation, a 10 kHz tone was played at 40 Hz with a 4% duty cycle. For visual-audio stimulation, the audio stimulation was accompanied with surrounding light flickered at 40 Hz for 10 s periods with a 50% duty cycle. Stimuli were presented in this manner for 20 min sessions, with 1-10 min pauses in between sessions to check on the animals’ behavior.

Concurrent 40 Hz auditory and visual stimulation protocol For biochemical, Immunohistochemical, or behavioral analysis, 5XFAD mice were placed in a dark chamber illuminated by an LED bulb and exposed to an auditory tone train, simultaneously. Mice were exposed to one of four stimulations: dark/quiet, 40 Hz light flicker, 40 Hz auditory tone train, concurrent 40 Hz light flicker and auditory tone, or random light flicker/tone stimulations.

Immunohistochemistry Mice were transcardially perfused with 4% paraformaldehyde in phosphate buffered saline (PBS) under anesthesia (2:1 of ketamine/xylazine), and the brains were post-fixed overnight in 4% paraformaldehyde in PBS. Brains were sectioned 40 μm thick with a Leica VT1000S vibratome (Leica). Sections were permeabilized and blocked in PBS with 0.3% Triton X-100 and 10% donkey serum at room temperature for 2-hs. Sections were incubated overnight at 4°C in primary antibody containing PBS with 0.3% Triton X-100 and 10% donkey serum. Primary antibodies were: anti-β -amyloid (Cell Signaling Technology; D54D2), anti-Iba1 (Wako Chemicals; 019-19741), anti-glial fibrillary acidic protein (GFAP)(Abcam; ab4674), anti-S100B (Abcam; ab868), anti-LRP1 (Abcam; 28320), DyLight 488 labeled Lycopersicon Esculentum (tomato) lectin (Vector laboratories; DL-1174), anti-amyloid oligomer (Millipore Sigma; AB9234), anti-phospho-tau (Ser396) (Cell Signaling Technology; 9632), anti-phospho-tau (Thr181) (Cell Signaling Technology, 12885), Hoechst 33342 (Thermo Fisher Scientific; H3570). The anti-Aβ antibody 12F4 was used because it does not react with APP, allowing us to determine whether our labeling was specific to Aβ, as well as allowing for co-labeling with Iba1. Anti-amyloid oligomer antibody AB9234 was used for co-labeling with LRP1. The following day, brain sections were incubated with fluorescently conjugated secondary antibodies (Jackson ImmunoResearch) for 2 hs at room temperature, and nuclei were stained with Hoechst 33342 (Invitrogen). Images were acquired using a confocal microscope (LSM 710; Zeiss) with a 40 × objective at identical settings for all conditions. Images were quantified using ImageJ 1.42q by an experimenter blind to treatment groups. For each experimental condition, two coronal sections from each animal were used for quantification. Scale bars are 50 μm unless otherwise noted in figure legends. ImageJ was used to measure the diameter of Iba1+ cell bodies and to trace the processes for length measurement. In addition, the Coloc2 plugin was used to measure co-localization of Iba1 and Aβ. Microglia processes arborization was quantified using Imarisx64 8.1.2 (Bitplane, Zurich, Switzerland). The ‘analyze particles’ function in ImageJ was used for counting plaque number and area, deposits of at least 10 μm were included and a set threshold was used for both control and experimental groups.

Vasculature- Aβ colocalization analysis ImarisColoc module was used to quantify colocalzation of signal between two separate source channels (i.e., Lectin and AB, Lectin and LRP1) in 3D. These source channels were thresholded to mask any intensity coming from noise or background signal. ImarisColoc then generates a new channel containing only voxels that colocalize within the thresholds set for the source channels, and presents the associated statistical analyses.

Microglia-Aβ clustering analysis IMARIS was used to analyze the microglial clustering pattern around amyloid plaques in 40uM slices. The surfaces module was utilized to detect and 3D render plaques (red) based on 12F4 signal. Iba1-positive microglia were then counted using the spots module, placing a sphere at the soma of each cell (green). Finally, the Spots Close To Surface XTension was run to find the subset of spots that are closer to the surface objects than the defined 25uM threshold, and exclusion of spots that fall outside this range. The algorithm measures the distance from the center of the spot to the nearest point of the surface object in 3D space, allowing for the quantification of microglial aggregation near plaques.

CLARITY immunostaining in brain slices Mice were perfused with ice-cold PBS (1X) followed by ice-cold 4% PFA, 1% glutaraldehyde in 1xPBS. Brains were dissected out and post-fixed in 4% PFA/1% glutaraldehyde solution for 72 hs at 4°C. Fixation was terminated by incubating brains in inactivation solution (4% acrylamide, 1 M glycine, 0.1% Triton X-100 in 1X PBS) for 48 hs at RT. After washing in 1xPBS, brains were sliced into 100uM coronal sections on a vibratome (Leica VT100S) in 1xPBS. Sections containing regions of interest (i.e., auditory cortex and hippocampus) were selected, with reference to the Allen Mouse Brain Atlas, and incubated in clearing buffer (pH 8.5-9.0, 200mM sodium dodecylsulfate, 20mM lithium hydroxide monohydrate, 4mM boric acid in ddH2O) for 2-4 hs, shaking at 55°C. Cleared sections were washed 3 x15mins in 1xPBST (0.1% Triton X-100/1XPBS) and put into blocking solution (2% bovine serum albumin/1xPBST) overnight at RT. Subsequently, three 1h washes in 1x PBST were performed, shaking at RT. Sections were incubated in weak binding buffer (pH 8.5-9.0, 37.75 mM Na2HPO4, 3.53 mM KH2PO4, 0.02% sodium azide in PBST) for 1 h at RT, then transferred to primary antibody, diluted to 1:100 in 1x weak binding buffer for 12 hs at 37°C. Reversal buffer (pH 7.4, 37.75 mM Na2HPO4, 3.53 mM KH2PO4 in 0.02% sodium azide in PBST) is then added in even aliquots over 6 hs, to equal the volume of primary antibody solution plus the volume of the tissue. Another set of 3x1 h washes in 1xPBST was conducted before sections were incubated for 12 hs at RT, in a mixture of Hoechst 33258 (1:250) (Sigma-Aldrich, 94403) and secondary antibody (1:100) in 1xPBS. Sections were then washed overnight in 1xPBS and incubated in RIMS (Refractive Index Matching Solution: 75 g Histodenz, 20mL 0.1M phosphate buffer, 60mL ddH2O) for 1 h at RT prior to mounting. Brain sections were mounted onto microscopy slides with coverslips (VWR VistaVision, VWR International, LLC, Radnor, PA) in RIMS. Images were acquired on a Zeiss LSM 880 microscope with the accompanying Zen Black 2.1 software (Carl Zeiss Microscopy, Jena, Germany). Z stack images were taken with a step size of 0.4-0.5 μm, pixel dwell 4.1 ms, averaging of 2, resolution 1024x1024 suitable for 3D reconstruction. Imarisx64 8.3.1 (Bitplane, Zurich, Switzerland) was used for 3-D rendering and analysis.

Whole mouse brain processing and clearing Park et al., 2018 Park Y.G.

Sohn C.H.

Chen R.

McCue M.

Yun D.H.

Drummond G.T.

Ku T.

Evans N.B.

Oak H.C.

Trieu W.

et al. Protection of tissue physicochemical properties using polyfunctional crosslinkers. Kim et al., 2015 Kim S.Y.

Cho J.H.

Murray E.

Bakh N.

Choi H.

Ohn K.

Ruelas L.

Hubbert A.

McCue M.

Vassallo S.L.

et al. Stochastic electrotransport selectively enhances the transport of highly electromobile molecules. 5XFAD mouse brains were processed according to the SHIELD protocol (). Briefly, 5XFAD mice were transcardially perfused with ice-cold PBS followed by 20 mLs of SHIELD-OFF solution containing 4% PFA. Brains were dissected and post-fixed in the same solution for 24 hs at 4°C. Brains were then incubated overnight in SHIELD-OFF solution without PFA at 4°C. Brains were then incubated in the SHIELD-ON solution for 24 hs at 37°C. Following fixation, brains were incubated in an aqueous clearing solution containing 200mM sodium dodecyl sulfate (SDS), 20mM lithium hydroxide monohydrate, 40mM boric acid, pH 8.5-9.0. Brains were then cleared using SmartClear Pro (LifeCanvas Technologies, Cambridge, MA) based on stochastic electrotransport () for several days, until transparent.

Immunostaining of cleared whole hemispheres Kim et al., 2015 Kim S.Y.

Cho J.H.

Murray E.

Bakh N.

Choi H.

Ohn K.

Ruelas L.

Hubbert A.

McCue M.

Vassallo S.L.

et al. Stochastic electrotransport selectively enhances the transport of highly electromobile molecules. Murray et al., 2015 Murray E.

Cho J.H.

Goodwin D.

Ku T.

Swaney J.

Kim S.Y.

Choi H.

Park Y.G.

Park J.Y.

Hubbert A.

et al. Simple, scalable proteomic imaging for high-dimensional profiling of intact systems. Cleared hemispheres were stained with 15ul of beta-amyloid antibody conjugated with Alexa Fluor-488 (CST, #51374) over 2 days, using eFLASH, an unpublished technique that integrates stochastic electrotransport method () and SWITCH method ().

Light-sheet microscopy Immunostained samples were incubated with hPROTOS (3g diatrizoic acid, 5g N-methyl-d-gludamine, 125 g iohexol in 105ml DI-water) for optical clearing and then mounted to acrylic holder using 2% low-temperature melting agarose in hPROTOS. Custom-made light-sheet microscope equipped with 10 × CLARITY-optimized objective was used to image whole hemispheres using the 488 channel for beta-amyloid visualization and the 647 channel for autofluorescence.

Cleared whole brain image processing, plaque detection, and atlas alignment Acquired image data were illumination corrected using CIDRE, an open-source software package implemented in MATLAB, and the resulting processed images were stitched together using Terastitcher. Imaris (Bitplane, http://www.bitplane.com/imaris/imaris ) was used for 3D visualizations, and ImageJ (NIH, https://imagej.nih.gov/ij/ ) was used to create representative slice-by-slice 2D visualizations. Automated plaque detection was performed using a combination of the open-source ClearMap software, a custom cell classification neural network model, and Elastix. Candidate plaques were located as “spots” with ClearMap’s spot detection module. First, background subtraction was done slice-by-slice by using a gray-scale morphological top-hat transformation with a disk structure element with major and minor diameter pixel sizes of (21,21). Next, local maxima of the data are detected by applying a 3D maxima filter with disk structure element of size (7,7,4), and these local maxima are filtered with an intensity threshold of 100. The pixel volumes corresponding to each spot center location are also computed using a 3D watershed transform with spot centers as seed points. All candidate plaques with volume less than a sphere with 10-micron diameter were then filtered out. True plaques were identified from the candidate plaques using a convolutional neural network (CNN) model as a categorical plaque / non-plaque classifier implemented in Keras ( https://keras.io/ ) with a Theano backend ( https://github.com/Theano/Theano ). The CNN input is a 32-by-32 pixel bounding box centered at a candidate plaque center, and the output is a two element one-hot vector representing the plaque and non-plaque categories. The architecture consists of 12 total convolutional layers, each with a rectified linear unit (ReLU) activation and followed by batch normalization: 3 with 64 2x2 kernels, 3 with 128 2x2 kernels, followed by 3 with 192 2x2 kernels, 1 with 256 2x2 kernels, 1 with 256 1x1 kernels, and 1 with 2 1x1 kernels. 2x2 subsampling is done after the third, sixth, and ninth convolutional layer, and Dropout with a rate of 0.5 is applied after the last nine convolutional/batch normalization layers for regularization. After the final convolutional layer, global average pooling followed by softmax activation is applied to generate the final categorical vector. During training, a categorical cross entropy loss was used with the Adam optimizer with default parameters. The CNN was trained for 400 epochs with batch size of 64 on ∼10,000 manual plaque annotations augmented with random rotations, shears, and reflections using the Keras Image Data Generator. The resulting model was then used to classify plaques from detected spots for all samples. To perform atlas alignment, autofluorescence channel images were first downsampled to the atlas resolution, and then Elastix was used to calculate affine and B-spline transformation parameters to do 3D image registration, with the resampled autofluorescence image as the fixed image and the atlas as moving image. The resulting alignment parameters were applied on the plaque locations (output from the CNN model) to transform the plaques into the atlas space, after which a CSV file with plaque count and volume information for each brain region (segmentation according to the Allen Brain Atlas) is generated.

Western blot Hippocampus and auditory cortex from 6-month-old tau P301S mice were dissected and homogenized in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitors. Lysates were incubated on ice for 15 min and spun at 12,000 RPM for 15 minutes. Then, supernatants were transferred to fresh tubes and analyzed for protein concentration (Bio-Rad Protein Assay). Equal amounts of protein (20 ug/lane) was resolved on a SDS-polyacrylamide gel and blotted onto a PVDF membrane. This membrane was incubated in blocking buffer containting 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% (v/v) Tween 20 (TBS-T) plus 5% dry milk (m/v) for 1 h at room temperature followed by incubation overnight at 4°C in primary antibodies and then secondary antibodies at room temperature for 1 h. Primary antibodies were anti-phospho-tau (Ser396) and anti-phospho-tau (Thr181). Secondary antibodies were LI-COR IRDye secondary antibodies. Signal intensities were quantified using ImageJ 1.46a and normalized to values of total tau Tau5 (Thermo Fisher Scientific; AHB0042).

Tau seeding activity assay Holmes et al., 2014 Holmes B.B.

Furman J.L.

Mahan T.E.

Yamasaki T.R.

Mirbaha H.

Eades W.C.

Belaygorod L.

Cairns N.J.

Holtzman D.M.

Diamond M.I. Proteopathic tau seeding predicts tauopathy in vivo. 2-month old tau P301S brain sections were homogenized in 1 × TBS supplemented with protease inhibitors (Roche complete mini tablets) using a probe sonicator (30% power; 15 pulses). After sonication, the lysates were centrifuged at 14,000 × g for 15 min to eliminate large, insoluble material. The supernatant was stored at −80°C and used for all future experiments. Protein concentration was determined using a Bio-Rad Protein Assay Dye. Fluorescence resonance energy transfer (FRET) biosensor cell lines described previously () were provided by Marc I. Diamond. Cells were grown in DMEM (Invitrogen) augmented with 10% FBS and 1 × penicillin/streptomycin and maintained at 37°C and 5% CO2 in a humidified incubator. For the assay, cells were plated in a 96-well plate at a density of 40,000 cells/well. Sixteen hs later, at 50% confluence, brain homogenate samples were transduced into cells using 1.2 μL Lipofectamine/well. After a 24 h incubation at 37°C, cells were harvested with 0.25% trypsin, fixed in 2% PFA (Electron Microscopy Services) for 15 min, and then resuspended in PBS. An LSR II HST-2 flow cytometer was used to measure the FRET signal within each cell. FRET quantification was accomplished using FlowJo version 10 software (TreeStar). Integrated FRET density was derived by multiplying the percentage of FRET-positive cells in each sample by the median FRET intensity of those cells.

ELISA Primary auditory cortices, medial prefrontal cortices, and hippocampi were isolated from 6-month-old 5XFAD males and subjected to Aβ measurement using Aβ 42 or Aβ 40 ELISA kits (Invitrogen) according to the manufacturer’s instructions. Insoluble Aβ was treated with 5M guanidine/50 mM Tris HCL (pH 8.0) buffer before ELISA measurement.