For transgenic mice studies, B6;C3-Tg (Prnp-MAPT*P301S) PS19Vle/J (RRID:IMSR_JAX:008169) and B6C3F1/J (RRID:IMSR_JAX:100010) were purchased in April 2016 from Jackson Laboratories (JAX) and bred at Yale to obtain littermates of wild-types (WT) and transgenics (PS19) . PS19 mice express a mutant human MAPT gene which results in a five-fold greater amount of human Tau proteins than the endogenous Tau produced naturally by mice. The PS19 mice were maintained in the hemizygous state, and a cohort of PS19 and WT littermates were randomly assigned to one of four experimental groups: WT, Vehicle; WT, AZD0530; PS19, Vehicle; PS19, AZD0530. Mouse genotyping was performed with a standard PCR assay as described on JAX website. DNA was extracted from ear tissue with REDExtract-N-Amp Tissue PCR kit (Sigma, XNAT) according to the manufacture’s protocol. There were two cohorts, each with the four groups, generated for these experiments (Supplementary Table S1). Average DOBs of the first and second cohorts are October 2016 and December 2018, respectively. Mice in the two cohorts were provided with chow formulated with either Vehicle or AZD0530 (depending on the experimental group) at 2 months of age and allowed to eat ad libitum until they were sacrificed at 9 months old and 11 months old, respectively. Similar results were obtained from the two cohorts in our behavioral tests.
For repetitive mild traumatic brain injury (rmTBI) plus stress studies, C57BL/6 J mice (RRID:IMSR_JAX: 000664) were purchased from JAX and bred for several generations at Yale. Only male mice were used for the combined rmTBI/stress model due to variation in chronic stress responses across the estrous cycle [26, 43], and to modulation of mouse TBI outcomes by sex [25, 57]. Since mice in the rmTBI/Stress study were dosed with AZD0530 by oral gavage, the mice were provided standard chow ad libitum. There were two cohorts with different treatment schedules for these experiments (Supplementary Table S1).
All protocols were approved by Yale Institutional Animal Care and Use Committee (IACUC). All animals were housed in groups with 2–4 animals per cage with access to food and water ad libitum. The housing light schedule had with a light period from 7 am to 7 pm and a dark period for the remaining 12 h.
Chronic Oral dose preparation of AZD0530
AZD0530 (saracatinib) was prepared as described . To generate chow containing AZD0530 for chronic dosing in PS19 experiments, the compound was incorporated into purified diet pellets by Research Diets, Inc. by dissolving the compound in a solution of 0.5% w/v Hydroxypropylmethylcellulose/ 0.1% w/v polysorbate 80 at 1.429 mg/ml. Vehicle pellets were purified diet pellets with control Vehicle solution (without drug). The dosage of the drug in the food was calculated to take into account the average amount of food eaten by a mouse in a single day per kg of weight  and adjusted to be equivalent to ingesting 5 mg/kg per day. Throughout the treatment period, the body weights of mice were monitored to ensure drug/food intake.
Brain tissue collection
Mice were euthanized with CO2 and perfused with ice-cold PBS for one and a half minutes. The brains were dissected and the hemispheres were divided. The hippocampus and cortex were dissected from the left hemisphere and were individually snap frozen in liquid nitrogen to be used for biochemical analysis. The right hemispheres were fixed in 4% paraformaldehyde in PBS for 24 h at 4 °C and then placed in PBS with 0.05% Azide to be used for immunohistochemistry.
Mouse brain protein extraction
Mouse brain protein extraction was performed as previously described  with modifications. The hippocampi were weighed and then homogenized with 20 strokes in ten-fold volume (w/v) of ice-cold 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, PhosSTOP, cOmplete-mini protease inhibitor cocktail (Roche), and 1 mM vanadate. After ultracentrifugation for 20 min at 100,000 x g at 4 °C, the supernatants were collected as TBS-soluble fractions, and the TBS-insoluble pellet was re-suspended in RIPA (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, PhosSTOP, cOmplete-mini (Roche), and 1 mM vanadate) at a volume equivalent to the amount used in the TBS extraction. The samples were incubated in RIPA for 30 min at 4 °C and then ultracentrifuged for 20 min at 100,000 x g at 4 °C. The supernatants were collected as RIPA-soluble fractions.
Immunohistochemistry was performed as previously described  with slight modifications. Forty μm coronal sections of the right hemisphere were cut with a Leica VT1000S Vibratome. Antigen retrieval was performed on the forty μm free-floating sections by incubating three slices from each mouse in 1x Reveal decloaker buffer (Biocare Medical) in 24-well-plates for 10 min at 90 °C in an oven and then cooled down at room temperature for 10 min. The antigen retrieval step was done for PHF1, AT8, HT7, and GFAP stainings. Sections were permeabilized with 0.1% Triton X-100 at room temperature for 5 min for PHF1, HT7, and SV2A staining and for 30 min for CD68/Iba1, AT8, and GFAP. All sections were blocked with 10% donkey, horse, or goat serum in PBS for 1 h at room temperature. The sections were then incubated in primary antibody in 4% donkey, horse, or goat serum in PBS overnight at room temperature. For SV2A, HT7, and PHF1 stainings, primary antibodies were incubated at 4 °C rather than room temperature. The primary antibodies that were used include: PHF1 (gift from Dr. Peter Davies, Albert Einstein College of Medicine, Bronx, NY 1:250), SV2A (Abcam, Ab32942, 1:500), CD68 (Biorad, MCA1957, 1:900), Iba1 (Wako, 019–19,741, 1:500), AT8 (Invitrogen, MN1020, 1:500), GFAP (Abcam, Ab7260, 1:1000), pTyr18 (Medimabs, MM-0194-P, 1: 200), Tau (DAKO, A0024,1:5000), HT7 (Invitrogen, MN1000, 1:500), and NeuN (Millipore, ABN91, 1:500). The sections were then washed three times with PBS for 5 min each and then incubated for 1–2 h at room temperature in either donkey anti-rabbit or donkey anti-mouse fluorescent secondary antibodies in PBS (Invitrogen Alexa Fluor 1:500). After incubation, the sections were washed three times with PBS for 5 min. To quench autofluorescence for PHF1, AT8, and GFAP stainings, sections were dipped briefly in dH2O and then incubated in copper sulfate solution (10 mM copper sulfate, 50 mM ammonium acetate, pH 5) for 15 min before dipping back into dH2O and then placed in PBS . All sections were mounted onto glass slides (Superfrost, Fischer Scientific Company L.L.C.) and coverslipped with Vectashield (Vector) antifade mounting medium with DAPI.
Immunoblotting was performed as previously described  with modifications. In general, the RIPA-soluble fraction was mixed in 2x Laemmli Sample Buffer (Bio-Rad) with 0.5% β−mercaptoethanol. For Tau extracted from human brains, samples were diluted with 1x Laemmli Sample Buffer (containing no β−mercaptoethanol) to 10, 5% or 2.5% of their initial concentration to evaluate Tau concentration. The mixture was heated for 5 min at 95 °C and then loaded into precast 4–20% Tris-glycine gels (Bio-Rad) to be electrophoresed. The protein was then transferred with an iBlot 2 Transfer Device onto nitrocellulose membranes (Invitrogen IB23001) and then incubated in blocking buffer (Rockland) for 1 h at room temperature. Membranes were then incubated overnight at 4 °C in blocking buffer with primary antibodies: Fyn (Cell Signaling, 4023, 1:1000), pSRC (Tyr416) (Cell Signaling, 6943, 1:1000), β-actin (Cell Signaling, 3700, 1:2000), total Tau (HT7) (Invitrogen, MN1000, 1:1000) and p-Tau (Invitrogen, AT180, 1:1000). The next day, membranes were washed three times with TBST for 5 min and incubated in secondary antibodies (donkey anti-rabbit (800) and donkey anti-mouse (680), Li-Cor IR Dye) for 1 h at room temperature. Membranes were washed three times with TBST for 5 min, visualized with an Odyssey Infrared imaging system (Li-Cor), and then the immunoreactive bands were quantified with ImageJ software.
Tau extraction from human brains
Pre-existing de-identified human autopsy brains were accessed for these studies under conditions considered exempt from Human Subjects regulation after review of the Institutional Review Board at Yale. Fresh frozen brain had been stored at − 80 C. The AD brain used in this study derived from a male, age 87, 23 h post-mortem interval, National Institute on Aging classification: A2, B3, C2 . The neurologically intact control brain had no signs or minimal signs of AD-associated histopathology, with Braak stage 0-II and CERAD neuritic plaque score of “none” or “sparse”. Tau was extracted based on a previously published protocol  with some modifications. Briefly, 11–12 g of cortical grey matter were dounce homogenized in 30 mL lysis buffer [10 mM Tris-HCl, 1 mM EDTA, 0.1% sarkosyl, 10% sucrose, freshly added 2 mM DTT, phosSTOP (Roche) and protease inhibitors (Roche)]. Throughout the extraction, lysates were kept on ice. Homogenates were centrifuged at 12,000 rpm at 4 °C for 12 min (Ti 45 rotor, Beckman Coulter). The supernatant was pooled, and the pellets were re-extracted and centrifuged twice more as above. The pooled supernatant was centrifuged again twice at 12,000 rpm at 4 °C for 12 min (Ti 45 rotor, Beckman) to remove debris. Then, the sarkosyl concentration was increased to 1% and samples were nutated for 1 h at room temperature (RT). The samples were centrifuged at 300,000 x g for 1 h at 4 °C (57,000 rpm, Ti 70 rotor, Beckman Coulter). The resulting pellet was washed with PBS supplemented with phosSTOP and protease inhibitors twice and then resuspended in PBS supplemented with phosSTOP and protease inhibitors. After sonication at 15% amplitude for 20 s with 0.5 s ON/0.5 s OFF pattern, the lysate was centrifuged at 100,000 x g for 30 min at 4 °C. The supernatant was discarded, and the pellet washed twice in PBS supplemented with phosSTOP and protease inhibitors. The pellet was once more resuspended in PBS supplemented with phosSTOP and protease inhibitors and sonicated at 30% amplitude for 60 s with 0.5 s ON/0.5 s OFF pattern. This was followed by a 100,000 x g spin for 30 min at 4 °C. The resulting supernatant contained the soluble Tau and was aliquoted and frozen at − 80 °C until further analysis or experimental use. The concentration of Tau in the extract was ~ 0.3 μg/μL by western blot analysis using recombinant human Tau.
In vitro tau seeding activity
Primary mouse neuronal culture was prepared as described . Pregnant mice were euthanized with CO2. Hippocampal and cortical tissues (1:1 ratio) were harvested from E17 embryos (both male and female) on ice cold Hibernate E media (BrainBits, HE) and digested in 0.05% Trypsin (Gibco), and 1 mg/mL DNase (Sigma DN25) in HBSS for 10 min at 37 °C. After incubation, neurons were triturated manually in Neurobasal-A media (Gibco) supplemented with B27, 1 mM sodium pyruvate, GlutaMAX, and 100 U/mL penicillin and 100 μL streptomycin (all from Gibco) at 37 °C. Dissociated neurons were spun at 250 x g at 4 °C for 6 min. Neurons were plated at 75,000 cells/well onto PDL-coated 96-well plates (Corning #354461) in the same Neurobasal-A media with supplements.
In vitro Tau seeding experiments were performed as previously described  with modifications. One week after primary neurons were plated onto PDL-coated 96-well plates (DIV7), Tau extracts (~ 150 ng of Tau /well) from human AD brains were seeded into wells. Neurons were also treated with 0.5 or 1 μM AZD0530 in high purity water. At DIV21, neurons were fixed with ice cold methanol for 30 min on ice and blocked with 10% normal donkey serum and 0.2% Triton X-100 in PBS for 30 min. Then, neurons were incubated with primary antibodies diluted in 1% normal donkey serum and 0.2% Triton X-100 in PBS overnight at 4 °C: Anti-MAP 2 (Cell Signaling, 4542, 1:150) and mouse Tau (T49) (Millipore sigma, MABN827, 1:500). The samples were washed three times with PBS and incubated in secondary antibodies (Invitrogen Alexa Fluor 1:500) diluted in 1% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h and DAPI.
HEK-293 Proximity Ligation Assay (PLA)
HEK-293 T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (100 U/mL). Cells were plated at 40,000 cells/well onto 8-well chamber slides (Thermo Scientific 154,941). Transient transfection with plasmids expressing human Tau (Origene, RC213312) and Fyn (Origene, RC224691) was performed using Lipofectamine 2000 transfection reagent (Invitrogen). Three hours later, 2 μM AZD0530 in DMSO or DMSO (vehicle) was added to the treatment or control wells. Twenty-four hours after treatment, cells were fixed in 4% paraformaldehyde in PBS at room temperature for 30 min and then washed 3X in PBS for 5 min and stored until PLA was performed.
Duolink In Situ Detection Reagents Green (Sigma DUO92014) were used for the PLA as described  with modifications. HEK-293 T cells were fixed on 8-well Chamber Slides and permeabilized/blocked with 10% normal donkey serum, 0.2% Triton X-100 in PBS for 30 min at room temperature. Wells were then incubated with primary antibodies Tau (DAKO, A0024, 1:4000) and Fyn15 (Santa Cruz, sc-434, 1:500) in 1% normal donkey serum in PBS overnight at 4 °C. The next day, after removing wells, the slides were washed 3x for 5 min in PBS and then incubated for 1 h at 37 °C in 8 μL Duolink In Situ PLA Prole Anti-Rabbit PLUS (Sigma DUO92002) and 8 μL Duolink In Situ PLA Probe Anti-Mouse MINUS (Sigma DUO92004) in 24 μL 1% normal donkey serum in PBS per sample. Slides were then washed 2X for 5 min with 1x Wash Buffer A (Sigma DUO82049) at room temperature. For the ligation step, slides were incubated for 1 h at 37 °C in 8 μL 5X Ligation buffer and 1 μL of Ligase in 32 μL high purity water. Then, slides were washed 2x for 5 min in 1x Wash Buffer A at room temperature. For the amplification step, the slides were incubated for 100 min at 37 °C in 8 μL 5x Amplification buffer and 0.5 μL polymerase in 31.5 μL high purity water per sample. The slides were then washed 2X for 10 min in 1x Wash Buffer B (Sigma DUO82049) and for 1 min in 0.01x Wash Buffer B and then 5 min in PBS at room temperature. For Tau and Fyn visualization, slides were incubated for 1 h at room temperature in the secondary antibodies donkey anti-rabbit conjugated with Alexa Fluor-568 and donkey anti-mouse conjugated with Alexa Fluor-647 (ThermoFisher 1:500) with 1% normal donkey serum in PBS. Then, slides were washed 4X for 5 min in PBS. Coverslips were mounted on the slides with Vectashield (Vector) antifade mounting medium with DAPI and stored at 4 °C until imaging.
Imaging and analysis
For imaging of sections from immunohistochemistry, Nikon Eclipse Ti Spinning Disc Confocal Microscope was used with a 40X 1.3 NA oil-immersion lens in Cargille immersion oil. A Zeiss AxioImager Z1 fluorescent microscope was used with a 5X objective. The dentate gyrus, CA1, and CA3 of mice were imaged and the percent positive area for each staining was analyzed with a macro in ImageJ or a pipeline in CellProfiler. For Iba1/CD68 images, z-stacks of the images were compressed into a maximum intensity projection with Volocity software before analyzing with CellProfiler. Iba1-positive area were identified with CellProfiler and masked over corresponding CD68 image. The percent of CD68-positive area within the Iba1 mask was calculated. Aperio ImageScope software was used for cresyl violet images.
For imaging of PLA, images were taken with Zeiss 800 confocal microscope was used with 20X 0.8 air-objective lens or Leica DMi8 with 20X 0.75 air-objective lens. Four pictures were taken per condition in each experiment. Z-stacks were compressed into a maximum intensity projection with ZEN software before analyzing with ImageJ. The area covered by Tau fluorescence was measured and masked over the corresponding PLA and Fyn image. The percent area of Fyn-Tau PLA-positive area within Tau-positive area and the percent of Fyn-positive area within Tau-positive area was calculated. The values were normalized to that of non-treated sample.
For the in vitro Tau seeding activity analysis, images were automatically taken using ImageXpress Micro XLS (Molecular Devices) (20X objective lens). Each experiment was performed in triplicate and four images were taken per well. With ImageJ, MAP 2-positive area was identified and masked over the corresponding T49 image. The percent of T49-positive area within the MAP 2 mask was calculated.
All images analyzed on ImageJ were uniformly thresholded for area analysis. All the imaging and analyses were conducted by a researcher who was blinded to the genotype and treatment type.
Morris water maze
The Morris water maze (MWM) paradigm was performed as previously described . When conducting all behavioral tests, the investigator was blinded to the mouse’s genotype and pharmacological treatment. Prior to behavioral tests, each mouse was handled for 5 min for 4 days to reduce anxiety. Mice were placed in a pool with a hidden, clear platform filled with water to 1 cm above the submerged platform. The hidden platform was placed in one of the four quadrants of the pool with the 4 drop zones directly across from the platform. At each of the four cardinal directions, a symbol, such as a plus or a cross, was placed as possible recognition flags. At the beginning of each day, mice were habituated in the behavior room for an hour before MWM began. For three consecutive days, two times each day, mice were dropped off facing the wall at four different drop zones (four trials between 9 am – 2 pm and four between 3 pm – 8 pm). Each trial was performed by alternating two mice (A1, A2, A1, A2, A1, A2 … etc). Latency was measured as the time that it took for the mouse to find and spend 1 s on the hidden platform. If there was a failure to reach the platform in 60 s, the mouse was guided to the platform and allowed to rest on it for 15 s. On the fourth day, a probe trial was performed, in which the platform was removed and mice was allowed to swim in the pool for 60 s.
In the subsequent trials (reverse learning and probe trials), the order in which the mice were placed in the pool was reversed, and the swim procedure was repeated with the hidden platform relocated diagonally from the initial platform location, and the drop zones were also altered to be directly diagonal from the forward swim drop zones.
After reverse learning and probe trials, a flag was placed atop of the hidden platform and mice were repeatedly placed in the pool. Time taken to reach the visible platform was recorded. When a consistent time for a mouse was reached, the last three times were averaged and the overall average of latency to hidden platform was used to exclude mice that were outliers from analysis due to visual impairments. Latencies and distance traveled for all trials were measured with the Panlab SMART Video Tracking Software.
Passive avoidance test
A Passive Avoidance Controller CAT 7551 was used to conduct the passive avoidance test as previously described  with slight modifications. The door delay was set to 90 s, and the shock intensity was set to 0.5 mA with a shock duration of 2 s. A mouse was placed into the white box with a light source overhead and given 5 min to cross through the door and into the opaque, black box after 90 s of acquisition in the white box. In trial 1, the mouse received a foot shock once it passed through to the black box. Trial 1 began at 10 am, after an hour of habituation in the behavior room. For trial 2, the mouse was placed back in the white box approximately 5 min after trial 1 and was shocked if it passed through to the black box. Twenty-four hours after trial 1, trial 3 was conducted with the shock intensity lowered to 0.0 mA. Mice were excluded if they failed to cross into the black box after 5 min during trial 1. Experimenter was blinded to the genotype of the mouse.
Rotarod performance test
The Rotarod test was performed as previously described . Mice were habituated in the behavior room for an hour before Rotarod test began at approximately 1 pm. Mice were placed atop a Rotarod (Economex Columbus Instruments) that was set to accelerate at 0.3 rpm/s until 4 rpm. Five trials were performed on each mouse with two-minute rests in between each trial. The time that each mouse stayed on the rod was recorded. Experimenter was blinded to the genotype of the mouse.
Repetitive Mild Traumatic Brain Injury (rmTBI) plus stress studies
The injury combined Closed Head Injury (CHI) and Chronic Variable Stress (CVS). Control mice received Sham-CHI and Sham-CVS treatments. There is a wide range of rodent head injury models [36, 47, 69]. These include lateral fluid percussion , controlled cortical impact , weight drop  and blast injury . The rmTBI model used here is CHI . This model offers multiple advantages, which include the capacity to titrate the intensity of impact, depth of injury and duration of impact. Most critically, it does not involve craniotomy, thereby facilitating multiple mild injuries and reducing infectious complications .
Multiple preclinical studies have explored the interaction of TBI with psychiatric disease [9, 41, 44, 45, 61]. Combined stress and TBI increased neuroinflammation, axonal injury and behavioral deficits . CVS induces Tau phosphorylation at Ser396 and Ser404 . Unpublished work by one of us (A.F.Z.) showed that stress preceding injury generated more severe behavioral deficits and greater neuroinflammation. The injury/stress model used in this study is based on these studies, and employed 14 days of CVS proceeding mild CHI on each day, with the side of the CHI alternating between days.
Chronic Variable Stress (CVS)
Stress was induced as described . The CVS was comprised of exposure to 5 different aversive stimuli over a 14 day period. The stimuli included: 3-min cold water swim at 16–18 °C, overnight food deprivation with access to water ad libitum, 3 h in a cage with 300 ml of water added, 3 h exposure to a cage tilted at 45 degrees, and 15 min immobilization in a flat-bottomed restraint chamber (Braintree Scientific Instruments). The mice were exposed to a set of three pre-randomized aversive stimuli on each given day during the 14 day period, in order to simulate the unpredictable nature of psychological trauma, while limiting habituation. Sham-CVS were transferred to single-housed cages for the same time period but not otherwise stressed. The CVS was conducted consistently between 8 am and 1 pm.
Closed Head Injury (CHI)
Within 1–3 h of CVS exposure on each of 14 consecutive days, isoflurane-based anesthesia was induced for 3 min with isoflurane (3.5% in oxygen (1.0 L/min) and maintained (3% in oxygen (1.0 L/min) until immediately after the impact. The head of the mouse was shaved and CHI was induced using a 5.0 mm diameter tip operated by an electromagnetic impactor (Leica Microsystems, Buffalo Grove, IL). The 5 mm diameter impactor tip was placed 5 mm lateral from the sagittal line, 5 mm caudal from the eye, at an angle of 20° from the vertical with an impact velocity 5 m/sec, impact depth of 1 mm and 100 msec dwell time. Sham-CHI mice were shaved and anesthetized in the same manner, but did not undergo impaction. The total anesthesia exposure during each procedure did not exceed 6 min for any mice, and no hypothermia was detected in this short period. The mice core body temperature was closely controlled and monitored using a heat pad (36.5–37.5 C), and a rectal probe. Mice were placed on a heating pad to maintain body temperature while receiving the impact, as well as during the post-injury recovery period. The mice underwent a total of 14 consecutive days of CHI injury, once per day. The site of injury alternated between right and left hemispheres on consecutive days to produce diffuse injury. By alternating sides on different days, the surgical procedure was substantially simplified and anesthesia time was kept to a minimum. Injury severity was assessed using time interval between injury and recovery of the righting reflex . Mice were returned to standard vivarium after restoration of their righting reflex, typically less than 5 min after CHI. The CHI was conducted between 3 pm and 7 pm.
Schedule for rmTBI/stress treatment with AZD0530
In the first treatment experiment, mice began treatment on Day 15 (24 h following the last injury on Day 14) with AZD0530 at 5 mg/kg/day in two equally divided doses by oral gavage for a 10 week period as described . The Vehicle for the drug was 0.5% wt/vol hydroxypropyl-methylcellulose (HPMC)/0.1% wt/vol polysorbate 80, and each dosing volume was approximately 250 μl .
In the second treatment experiment, the mice underwent a 10 week treatment starting on Day 121, or 107 days after the last day of injury.
Upon completion of the treatment period, both groups underwent a weeklong period of behavioral assays including Morris Water Maze (MWM), and novel object recognition, as described . Each mouse was handled for 5 min for the 5 days preceding the behavioral testing. The mice received the continued oral gavage treatment during the testing period.
Cresyl violet staining of rmTBI/stress brain
To assess tissue damage, coronal sections were stained with cresyl violet (Sigma Aldrich, C5042) for 10 min, washed in water for 3 min, de-stained in 95% ethanol for 10 min, and then dehydrated with 100% ethanol for 5 min twice and xylene for 5 min twice. Sections were then mounted with CytoSeal60 (ThermoFischer, 8310–4).
Quantification and statistical analysis
One-way ANOVA with Dunnett’s multiple comparisons test, two-way ANOVA with Sidak’s multiple comparisons test, t-test, or Wilcoxon match-pairs signed tests were performed as specified in the figure legends using GraphPad Prism 8. All n-values represent individual mice. For IHC, each data point represents the average of three brain sections from one animal. For behavioral tests, the number of trials that each data point represents can be found in the figure legends. Values are represented as mean ± SEM. Statistical significance is determined if p < 0.05.