hAPPJ9 and hAPPJ20 mice express an hAPP minigene with the Swedish (K670N, M671L) and Indiana (V717F) mutations under the control of the PDGF β-chain promoter; hAPP and Aβ levels in hAPPJ20 mice are about double those in hAPPJ9 mice (Mucke et al., 2000
). hAPPJ20 mice were crossed with tau-deficient mice (Dawson et al., 2001
) as described (Roberson et al., 2007
) to produce offspring with two (Tau+/+
), one (Tau+/−
), or no (Tau−/−
) functional Tau alleles in the presence or absence of hAPP. hAPPJ9 mice were crossed with Tau−/−
mice for two generations to produce hAPPJ9/Tau−/−
mice do not have robust behavioral abnormalities (Chin et al., 2005
) unless challenged with additional AD-related insults, such as reduction of the Aβ-degrading enzyme neprilysin (Farris et al., 2007
) or overexpression of the tyrosine kinase Fyn (Chin et al., 2004
). Doubly transgenic hAPPJ9/Fyn mice exhibit prominent AD-related abnormalities (Chin et al., 2004
). To examine the role of tau in these mice, we separately crossed hAPPJ9 mice and mice overexpressing wild-type murine Fyn (line N8) (Kojima et al., 1997
) onto the Tau−/−
background and then bred hAPPJ9/Tau−/−
mice with Fyn/Tau−/−
TASD41 mice express hAPP with the Swedish and London (V717I) mutations from the Thy1 promoter (Rockenstein et al., 2001
). TASD41 mice were bred with the same Fyn transgenic line to produce TASD41/Fyn/Tau+/+
mice. TASD41 mice were also crossed with Tau−/−
mice to produce TASD41/Tau−/−
mice, which were then bred with Fyn/Tau−/−
mice to generate TASD41/Fyn/Tau−/−
mice from The Jackson Laboratory (stock number 004453) were crossed with Tau−/−
mice (Dawson et al., 2001
offspring were bred with Tau+/−
mice to produce SOD1G93A
, and SOD1G93A
mice were weighed regularly, and age at disease onset was defined as the point at which weight peaked before beginning to decline. Survival calculation was based on the age at which mice either died spontaneously or were killed for being unable to right themselves within 30 s after being placed on their sides.
A second tau-deficient line, in which the tau locus is replaced by green fluorescent protein (GFP) (Tucker et al., 2001
), was crossed with hAPPJ20 to produce hAPPJ20/Tau+/GFP
mice, heterozygous for tau. These mice were crossed with nontransgenic (NTG)/Tau+/GFP
mice to produce both hAPPJ20/Tau+/+
All lines had been backcrossed to a consistent strain background, C57BL/6J. Males and females were used for all experiments, and no sex-dependent effects were identified (data not shown). Animals were housed in a pathogen-free barrier facility with a 12 h light/dark cycle and ad libitum access to food and water. All experiments were approved by the Institutional Animal Care and Use Committees of the University of California, San Francisco or Baylor College of Medicine.
The water maze pool (122 cm diameter) contained opaque water with a 14 cm, square platform submerged 2 cm below the surface. For cued training sessions, a black-and-white striped mast was mounted above the platform. Mice were trained to locate the platform over six sessions (two per day, 4 h apart), each with two trials (15 min apart). The platform location was changed for each session. Hidden platform training began 3 d later, consisting of 10 sessions (two per day, 4 h apart), each with three trials (15 min apart). Platform location remained constant in hidden platform sessions, and entry points were changed semirandomly between trials. The day after hidden platform training was completed, a 60 s probe trial was performed. The platform was removed, and the entry point was in the quadrant opposite the original target quadrant. Performance was monitored with an EthoVision video-tracking system (Noldus Information Technology).
Elevated plus maze
The elevated plus maze consisted of two open arms and two closed arms equipped with rows of infrared photocells and a computer interface (Hamilton-Kinder). Mice were placed individually into the center of the maze and allowed to explore for 10 min. Beam breaks were quantified to calculate the amount of time spent and distance moved in each arm. The apparatus was cleaned with 0.25% bleach between the testing of each mouse to standardize odors.
Novel object recognition
Mice were transferred to the testing room and acclimated for at least 1 h before testing. The testing was performed in a 20 × 40 cm white plastic chamber under red light. On days 1 and 2, mice were habituated to the testing arena for 15 min. On day 3, each mouse was presented with two identical objects in the same chamber and allowed to explore freely for 10 min. Twenty-four hours after this training session, mice were placed back into the same arena for the test session, during which they were presented with an exact replica of one of the objects used during training along with a novel, unfamiliar object of different shape and texture. Object locations were kept constant during training and test sessions for any given mouse, but objects were changed semirandomly between mice. Arenas and objects were cleaned with 70% ethanol between each mouse. Behavior was recorded with a digital camcorder and time spent exploring each object was scored.
Pentylenetetrazole (PTZ) (Sigma) dissolved in PBS was used at a concentration of 5 mg/ml. A dose of 40 mg/kg was administered intraperitoneally. Each mouse was placed in a cage and observed for 20 min after administration, with video recording. An investigator analyzed the videotapes to quantify the time course and severity of seizures according to published scales (Racine, 1972
; Loscher et al., 1991
). Seizure severity scores were as follows: 0, normal behavior; 1, immobility; 2, generalized spasm, tremble, or twitch; 3, tail extension; 4, forelimb clonus; 5, generalized clonic activity; 6, bouncing or running seizures; 7, full tonic extension; 8, death.
For placement of electrodes, mice were anesthetized with Avertin, and four pairs of cranial burr holes were made; the most anterior were used for reference and ground, and others were positioned for bilateral recordings over the temporal, parietal, and occipital cortices. Teflon-coated silver wire electrodes (0.005 inch diameter) were implanted in the subdural space and connected to a microminiature connector (Omnetics Connector). All recordings were performed at least 7 d after surgery on mice freely moving in the test cage. Digital EEG activity with simultaneous video was recorded with Harmonie software, version 6.1c (Stellate Systems). EEG activity was monitored for a median of 8 h total per mouse in three 2–3 h sessions during a 1 week period. The number of abnormal epileptiform spikes (sharp positive or negative deflections with amplitudes exceeding twice the baseline and lasting 25–100 ms) in each hour of recording and the latency to detection and the number of seizures were scored. The severity of spontaneous seizures in each mouse was scored on a scale as follows: 0, no seizures; 1, only nonconvulsive seizures; 2, mixed convulsive and nonconvulsive seizures; and 3, all convulsive seizures.
Immunohistochemistry was performed on floating 30-μm-thick microtome sections (Palop et al., 2011). Primary antibodies were rabbit anti-calbindin D-28K (1:30,000; Swant) or rabbit anti-NPY (1:8000; Immunostar). Labeling was detected with a biotinylated secondary antibody and the Vectastain Elite avidin–biotin complex kit (Vector Laboratories). Images were acquired with a digital microscope (Axiocam; Carl Zeiss). Densitometric quantifications were performed with the Bioquant software package (Bioquant Image Analysis). Hippocampal remodeling was investigated in hAPPJ9/Fyn mice that had undergone PTZ challenge 20 min before being killed. Because PTZ does not induce changes in calbindin or NPY on this timescale (data not shown), the remodeling observed is attributable to previous spontaneous epileptiform activity, not to induced seizures.
Four- to 6-month-old mice were anesthetized with Avertin and decapitated. The brain was quickly removed and placed in ice-cold slicing solution containing the following (in mm): 2.5 KCl, 1.25 NaPO4, 10 MgSO4, 0.5 CaCl2, 26 NaHCO3, 11 glucose, and 234 sucrose, pH ≈ 7.4 (305 mOsm). Horizontal slices were prepared on a Vibratome-3000 at either 450 μm (for field recordings) or 350 μm (for whole-cell patch clamp) in the solution above. Slices were incubated for 30 min in standard artificial CSF (ACSF) (30°C) containing the following (in mm): 2.5 KCl, 126 NaCl, 10 glucose, 1.25 NaH2PO4, 1 MgSO4, 2 CaCl2, and 26 NaHCO3 (290 mOsmol; the pH was ~7.4 when gassed with a mixture of 95% O2/5% CO2) and then at room temperature for at least another 30 min before recording. No recordings were made from slices >5 h after dissection. For recording, individual slices were transferred to a submerged chamber in which they were maintained at 30°C and perfused with ACSF at a rate of 2 ml/min.
Field EPSPs (fEPSPs) were evoked every 20 s with a parallel bipolar tungsten electrode (FHC) and recorded with glass electrodes (~3 MΩ tip resistance) filled with 1 m NaCl and 25 mm HEPES, pH 7.3. Recordings were filtered at 2 kHz (−3 dB, eight-pole Bessel), digitally sampled at 20 kHz with a Multiclamp 700A amplifier (Molecular Devices), and acquired with a Digidata-1322A digitizer and pClamp 9.2 software. Data were analyzed offline with pClamp9 software and OriginPro 8.0 (OriginLab). For recordings in area CA1, the stimulating electrode was placed in the stratum radiatum at the border of CA3 and CA1, and the recording electrode was placed ~150 μm away in CA1 stratum radiatum. Synaptic transmission strength in CA1 was assessed by generating input–output curves for the relationship between peak amplitude of the fiber volley and the initial slope of the resulting fEPSP. For each slice, the fiber volley amplitude and initial slope of the fEPSP responses were measured at stimulus strengths of 25–800 μA. For dentate gyrus recordings, the stimulating electrode was moved to the medial perforant path (MPP) in the dorsal blade; the recording electrode was also placed in the MPP ~150 μm closer to CA3 than the recording electrode. To overcome inhibitory feedforward and feedback circuits that are activated during induction of long-term potentiation (LTP) and that normally prevent robust LTP induction in the dentate, inhibition by GABAA receptors was blocked by addition of picrotoxin (50 μm; Tocris Bioscience) for field recordings from the dentate. Input–output curves were generated as in CA1. Stimulus strength was then adjusted to ~30% of the maximal fEPSP response for recordings that followed. Paired-pulse ratios were determined by evoking two fEPSPs 50 ms apart and dividing the initial slope of the second fEPSP by the initial slope of the first (fEPSP2/fEPSP1). After a 15 min stable baseline was established, LTP was induced in the dentate by theta burst stimulation (10 theta bursts were applied at 15 s intervals; each theta burst consisted of 10 bursts, at 200 ms intervals, of four 100 Hz pulses).
To evaluate epileptiform bursting, we adjusted the stimulus strength to evoke fEPSPs ~30% of maximal size (~150 μA), stimulated every 20 s in the stratum radiatum
near the CA1/CA3 border, and recorded field responses ~200 μm away in stratum pyramidale
of area CA1. Epileptiform activity was induced by superfusing bicuculline into the bath (10 μm). We quantified the strength of discharges by calculating the coastline burst index (CBI) (Korn et al., 1987
), which is a measure of the length of the outline of the burst waveform. The CBI is the summated distance between successive data points in a time window containing the entire burst using the formula, Σ√((Vi − Vi−1
), where Vi is field voltage at time point i
), and Vi-1
is field voltage at the previous time point (ti-1
). To correct for underlying baseline “noise,” the CBI for an equal duration of recording in the absence of bursts was subtracted from the burst CBI. This sensitive measure of burst intensity is useful for determining the effects of anti-epileptic interventions (Korn et al., 1987
; Tallent and Siggins, 1999
Whole-cell recordings were made from individual granule cells located within the dentate gyrus, identified under infrared differential interference contrast video microscopy with a Carl Zeiss Axioskop 2 FS plus microscope. Patch pipettes were pulled from borosilicate glass (WPI) and filled with the following (in mm): 120 Cs-gluconate, 10 HEPES, 0.1 EGTA, 15 CsCl2, 4MgCl2, 4 Mg-ATP, and 0.3 Na2-GTP, pH 7.25 (adjusted with 1 m CsOH; 285–290 mOsm; patch electrode resistance, 3–6 MΩ). Electrophysiological data were obtained from granule cells that had a holding current between −100 and 50 pA at −60 mV and a membrane input resistance (Rm) >100 MΩ. Series resistance was monitored throughout recordings; if it increased by >15 MΩ or varied by >15%, recordings were discarded. Voltage-clamp data were acquired with a Digidata-1322A digitizer and pClamp 9.2 software (Molecular Devices), low-pass filtered at 6 kHz (−3 dB, eight-pole Bessel), and digitally sampled at 10 kHz with a Multiclamp 700A amplifier (Molecular Devices).
EPSCs were recorded at a holding voltage of −60 mV, at which there are no currents through GABAA receptors. IPSCs were isolated by recording at a holding potential of +15 mV, the reversal potential of AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated currents. To block GIRK (G-protein-coupled inwardly rectifying K+ channel) channels activated by GABAB receptor activation, Cs+ was included in the recording pipette. Evoked PSCs were generated by a bipolar glass stimulating electrode filled with ACSF and placed in the molecular layer of the dentate gyrus within 100 μm of the patched granule cell. Stimulus pulses were delivered through a stimulus isolation unit (IsoFLEX; A.M.P.I.). To generate input–output relationships, the minimal stimulus intensity that evoked a synaptic current was first determined for a 40 μs stimulus, which was typically 50–100 μA. Stimulus duration was then increased in 20 μs intervals to vary input strength. Evoked (e) EPSCs that were ~80% of the maximal response were used for subsequent analysis. Spontaneous and miniature currents were analyzed with event detection software (wDetecta; J. R. Huguenard, Stanford University, Stanford, CA). The frequencies of spontaneous (s) and miniature (m) EPSCs were determined from the average frequency of 200 randomly selected sequential events for each cell. For amplitude, individual currents without other currents contaminating the decay phase were isolated. In each cell, eEPSCs and sEPSCs were first recorded at −60 mV, then eIPSCs and sIPSCs were recorded at +15 mV. Then, mEPSCs and mIPSCs were recorded after addition of tetrodotoxin. Calculations involving comparisons between various PSC types (e.g., sIPSC – mIPSC frequency) were made for individual cells in which both datasets were obtained.
To determine the NMDAR/AMPAR ratio, EPSCs were evoked in standard ACSF in the presence of 50 μm picrotoxin (picrotoxin was not used for other whole-cell recordings). Stimulus amplitude was adjusted to produce single-peaked responses with a short constant latency (2–3 ms, to ensure monosynaptic responses) and average amplitude of ~60 pA. Evoked EPSCs were recorded at −80 and +35 mV, and in some cells, the complete current–voltage (I–V) relationship was determined from −80 to +35 mV in 5 mV steps. Because steps to +35 mV can activate voltage-dependent currents that decay very slowly (t1/2 > 5 s), EPSCs were evoked at low frequency (once every 40 s) to minimize their accumulation and the accumulation of slowly decaying NMDAR-mediated currents. Three to five traces were used to generate a single average trace at each holding potential for final analysis. Occasional traces were contaminated by stimulus-evoked epileptiform events (resulting from disinhibition by picrotoxin), preventing the determination of peak monosynaptically evoked currents; these traces were not included in the final average trace used for analysis. The peak current at −80 mV, considered to be fully mediated by AMPAR attributable to magnesium block of NMDAR, was used to establish AMPAR-mediated responses. The time for AMPAR currents to decay fully to baseline was determined from the recordings at −80 mV, and a time window after that point was selected for measurement of the NMDAR current in recordings at +35 mV. This current was designated as the NMDAR measurement, and INMDA at +35mV/IAMPA at −80 mV was taken as the NMDAR/AMPAR ratio.
Investigators acquiring and scoring data were blinded to the genotype and treatment of mice. Data were analyzed with GraphPad Prism and SPSS 16.0. Unless specified otherwise in figure legends, statistical comparisons were made with the log-rank test for survival data, the exact test for categorical data, Kolmogorov–Smirnoff test for comparison of cumulative probability plots, and ANOVA with Bonferroni’s post hoc tests for other data. A p value <0.05 was considered significant. Bar graphs show mean ± SEM.