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Neurobiology of Aging 33 (2012) 1481.e13–1481.e23

Age-dependent disruption in hippocampal theta oscillation in amyloid-�overproducing transgenic mice

Liam Scott, Jianlin Feng, Tamás Kiss, Elie Needle, Kevin Atchison, Thomas T. Kawabe,Anthony J. Milici, Éva Hajós-Korcsok, David Riddell, Mihály Hajós*

Pfizer Global Research and Development, Groton, CT, USA

Received 14 July 2011; received in revised form 7 November 2011; accepted 5 December 2011

Abstract

Transgenic mice are used to model increased brain amyloid-� (A�) and amyloid plaque formation reflecting Alzheimer’s diseaseathology. In our study hippocampal network oscillations, population spikes, and long-term potentiation (LTP) were recorded in APPswe/S1dE9 (APP/PS1) and presenilin1 (PS1) transgenic and wild type mice at 2, 4, and 8 months of age under urethane anesthesia.ippocampal theta oscillations elicited by brainstem stimulation were similar in wild type and PS1 mice at all age groups. In contrast,PP/PS1 mice showed an age-dependent decrease in hippocampal activity, characterized by a significant decline in elicited theta power and

requency at 4 and 8 months. Magnitudes of population spikes and long-term potentiation in the dentate gyrus were similar across groupst both 4 and 8 months. In APP/PS1 mice, soluble and insoluble A�, and hippocampal and cortical plaque load increased with age, and theisruption in hippocampal theta oscillation showed a significant correlation with plaque load. Our study shows that, using in vivolectrophysiological methods, early A�-related functional deficits can be robustly detected in the brainstem-hippocampus multisynapticetwork.

2012 Elsevier Inc. All rights reserved.

Keywords: LTP; Perforant path; Plaque histology; Electrophysiology; Nucleus pontis oralis; APP; PS1; EEG; Alzheimer’s disease; Biomarker

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1. Introduction

Alzheimer’s disease (AD) is a slowly progressing neu-rodegenerative illness characterized by cognitive decline,primarily affecting memory. The pathological hallmarks ofAD are extracellular plaques and vascular deposits consist-ing of amyloid-� (A�) protein, intracellular neurofibrillaryangles, neuronal loss, and the presence of chronic neuro-nflammation (Ittner and Gotz, 2011; Veerhuis, 2011). Cur-ently one of the critical efforts in AD research is to identifyiomarkers suitable for tracking disease progression anduiding the development of potential disease-modifyingherapies. Neurophysiological measurements are sensitive

* Corresponding author at: Translational Neuropharmacology, Section ofComparative Medicine, PO Box 208016, Yale University School of Med-icine, New Haven, CT 06520-8016, USA. Tel.: �1 860 501 1256; fax: �160 715 2349.

cE-mail address: mihaly.hajos@yale.edu (M. Hajós).

0197-4580/$ – see front matter © 2012 Elsevier Inc. All rights reserved.10.1016/j.neurobiolaging.2011.12.010

ools for detecting alterations in neuronal network activitiesn both experimental models and in psychiatric or neuro-ogical disorders (Javitt et al., 2008; Rossini et al., 2007). InD patients a number of aberrant neurophysiological sig-als have been detected, which could be related to increasesn A� levels, synaptic dysfunction, and/or morphologicalesions (Palop et al., 2006). For example, a correlation haseen revealed between the volume of the hippocampus, onef the brain regions showing early signs of neurodegenera-ion, and alterations of cortical electroencephalogram (EEG)ignals in mild cognitive impairment and AD (Babiloni etl., 2009).

The memory deficits associated with AD are thought toesult largely from synaptic failure leading to hippocampaletwork dysfunction (Selkoe, 2002). Neurophysiologicaletwork activities, including theta rhythm, have been aajor focus of studies of hippocampal function over de-

ades (Buzsáki, 2002). Theta oscillation, an approximately

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1481.e14 L. Scott et al. / Neurobiology of Aging 33 (2012) 1481.e13–1481.e23

sinusoidal (4–10 Hz) EEG activity that can be recorded inthe limbic circuitry, has been linked to various cognitiveprocesses and is considered to be a “natural tetanizer” con-tributing to long-term potentiation (LTP) (Buzsáki, 2002;Vertes, 2005). Furthermore, correlations between memoryformation and hippocampal theta have been demonstrated inboth experimental animals (Buzsáki, 2002; Lisman and Re-dish, 2009) and humans (intracranially recorded slow-thetaoscillation; Lega et al., 2011).

Characteristics of hippocampal theta oscillation havebeen studied using various methods, spanning from hip-pocampal brain slice preparations to multiunit recordingsfrom animals during various behavioral tasks. One of theexperimental models used to investigate hippocampal net-work activity in rodents involves electrical stimulation ofkey brain regions contributing to theta oscillation. Highfrequency stimulation of the brainstem nucleus pontis oralis(nPO) elicits robust hippocampal theta oscillation in anes-thetized rats, where both the power and frequency has beenshown to be stimulation current-dependent. The frequencyof theta oscillation in this preparation spans ranges associ-ated with arousal and attention as well as movement relatedtheta frequencies (for review see McNaughton et al., 2007).Notably, it has been shown that a variety of drugs thatdisrupt cognitive functions, regardless of their synapticmechanisms, diminish or eliminate elicited hippocampaltheta oscillation.

The aim of the present study was to identify and char-acterize neurophysiological markers in A� overproducing

ice (carrying double mutations for A� precursor proteinnd presenilin 1) by measuring elicited theta oscillation andTP in the hippocampal circuitry which could reflect A�-

related synaptic dysfunction. Such neurophysiological sig-nals could serve as surrogate markers of cognitive function-ing in animal models of disease, and eventually, aspredictors of pharmacological response to potential disease-modifying treatments for AD.

2. Methods

2.1. Transgenic animals

We compared electrophysiological markers in doubletransgenic mice (APP/PS1) carrying transgenes for amy-loid-� protein precursor (APPK670N,M671L-A�PPswe) andpresenilin 1 (PSEN1G384A) relative to littermate control

ice carrying only the presenilin 1 transgene (PS1) andheir wild type (WT) background strain C57B1/6 � SJL F1.riefly, this APP/PS1 line has A� deposition beginning at 4

months of age with progressive age-related accumulation asdemonstrated by both immunohistochemistry and biochem-ical enzyme-linked immunosorbent assay (ELISA) mea-surements. There is a qualitative increase in plaque loadfrom 4 to 12 months of age in cortical and hippocampalregions. The striatum, thalamus, hypothalamus, amygdala,

brain stem, and cerebellar regions showed little plaque de-

position until 12 months of age (Samaroo et al., 2011).These data are in agreement with those reported for asimilar APP/PS1 model (Holcomb et al., 1998).

2.2. Surgical procedures andelectrophysiological recordings

Mice were anesthetized with 1.5–1.7 g/kg urethane in-traperitoneally, under an approved animal use protocol andin compliance with the Animal Welfare Act Regulations (9CFR Parts 1, 2, and 3) and with the Guide for the Care andUse of Laboratory Animals, National Institutes of Healthguidelines. The animals were placed in a Kopf stereotaxicframe on a temperature-regulated heating pad (Harvard Ap-paratus, Holliston, MA, USA) set to maintain body temper-ature at 37 °C–38 °C.

2.2.1. Stimulation-evoked hippocampal theta oscillationLocal field potentials were recorded from the CA1 region

of the left hippocampus (Fig. 1A), 2.0 mm posterior and 1.5mm lateral from bregma and 1.5 mm ventral from corticalsurface (Paxinos and Franklin, 2001), using a bipolar, con-centric recording electrode (NE-100X, Rhodes Medical In-struments, Woodland Hills, CA, USA) and a Grass P55 ACdifferential preamplifier with filters set between 0.3 and 300Hz (Grass Technologies, West Warwick, RI, USA). Elec-trical stimulation of the nPO, 4.0 mm posterior and 1.2 mmlateral from bregma and 3.3 mm ventral from cortical sur-face, was performed using a bipolar concentric electrode(NE-100X, Rhodes Medical Instruments). During each ex-periment, spontaneous and stimulation-induced local fieldpotentials were continuously monitored, and data were dig-itized at 1 kHz using the Power 1401 AD converter andstored using the Spike2 software package (Cambridge Elec-tronic Design). In a further exploratory study conducted ina small number of animals a 16-site silicon recording elec-trode was used (A1x16-10 mm-100-177-T15 NeuroNexusTechnologies, Inc., Ann Arbor, MI, USA; see next para-graph for full methodology). Stimulation consisted of a trainof 0.3 msec square pulses delivered over 6 seconds at 250Hz, repeated every 100 seconds. Stimulus-response curveswere obtained using increasing stimulus intensities of 0.00to 0.22 mA in 0.02 mA increments. Curves were repeated 5times per animal, with the first 2 used as conditioning curvesand the last 3 for analysis. At the end of each recordinganimals were euthanized, and the brain rapidly removedwith hemibrains either frozen for biochemical analysis orstored in 10% paraformaldehyde for immunohistochemistry.

2.2.2. Perforant path-evoked dentate gyrus LTPPerforant path was stimulated using a concentric bipo-

lar electrode and population spike in the cell-body layer ofthe dentate gyrus was recorded (Fig. 1B). A 16-site sili-con recording electrode (A1x16-10 mm-100-177-T15NeuroNexus Technologies, Inc.) was implanted to span thehippocampal formation, placed 2.0 mm posterior from and

1.5 mm lateral to bregma and the tip slowly lowered 1.9 mm

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from the cortical surface. The stimulating electrode wasplaced 0.5 mm anterior to and 2.5 mm lateral to lambda and1.4–1.9 mm from cortical surface. Initial responses wereobtained using 0.1 ms, 0.1-Hz stimuli at 0.05–0.7 mA. Alldata were digitized at 10 kHz using the Cerebus System andrecorded for offline analysis using NeuroExplorer (Black-Rock Microsystems, Salt Lake City, UT, USA). Recordingsites that resided in the cell-body layer of the dentate gyruswere identified for online analysis using the Power 1401 ADconverter and Spike2 software package (Cambridge Elec-tronic Design, Ltd.). Stimulation electrode position wasoptimized to give maximal population spike amplitude. Fol-lowing a 30-minute equilibration period stimulation-re-sponse relationship was determined using 3 cycles of in-creasing current, with 6 stimulations averaged for eachstimulation intensity. Stimulation strength was set to the

Fig. 1. Summary of protocols used in the present study. (A) Brainstem sbrainstem nucleus pontis oralis (nPO) was electrically stimulated andtransformation (FFT) of the LFP frequency of the highest power componefor each stimulation period (scale bars in the trace denote 1-second timpotentiation (LTP): the perforant path (PP) was electrically stimulated usinregistration a theta burst protocol was used to induce LTP. Responses tamplitude compared with baseline was measured (scale bars in the trace deDG granule cell layer) (Atlas images are modified from Paxinos and Fran

current required to produce 1-third of the maximal popula-

tion spike amplitude, the preparation was allowed to stabi-lize for 30 minutes prior to the recording of a 30-minutebaseline period and a 90-minute post-LTP period. LTP wasinduced using 2 sets of 6 trains with an intertrain interval of20 seconds, each train consisting of six 0.4 msec 400 Hzpulses, with 5 minutes between sets, using the lowest stim-ulation intensity required to produce maximal populationspike amplitude. At the end of each recording animals wereeuthanized, and the brain rapidly removed with hemibrainseither frozen for biochemical analysis or stored in 10%paraformaldehyde for immunohistochemistry.

2.3. Histological procedures

After conclusion of the electrophysiological experimentshemibrains stored previously in 4% paraformaldehyde were

ion-evoked hippocampal local field potential (LFP) theta oscillation: then the hippocampal CA1 region was recorded. Following fast Fourierk fr.), and total power in the � frequency band (3–12 Hz) were recorded0.2-mV amplitude). (B) Recording of population spikes and long-termtimuli and dentate gyrus (DG) response was recorded. Following baselinetimuli were recorded following LTP induction and increase of responsems time and 2-mV amplitude; black trace denotes recording closest to the01).

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processed and embedded in paraffin. Sections were cut at 5

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1481.e16 L. Scott et al. / Neurobiology of Aging 33 (2012) 1481.e13–1481.e23

�m. Slides with sections attached were deparaffinized andtreated with 88% formic acid for 4 minutes. The remainderof the protocol was done using a Dako Autostainer (Dako,North America, Inc., Carpinteria, CA, USA) to insure uni-form incubation times with each reagent. Briefly, the sec-tions were treated with an avidin and biotin solution toblock endogenous biotin, endogenous peroxidase wasblocked with a 3% hydrogen peroxide solution, and non-specific staining was blocked using a Dako serum-free pro-tein block (Dako, North America, Inc.). Biotinylated 6E10(Thermo Scientific, Emeryville, CA, USA) was reacted withthe tissue sections at 1:200 dilution for 30 minutes, rinsed,and then reacted with Vector Elite ABC (Burlingame, CA,USA). The immunoperoxidase reaction was visualized us-ing Diaminobenzidine (DAB) and counterstained with Lil-lie’s Modified hematoxylin. For evaluation of plaque load 3sections approximately 50 �m apart were analyzed. Slides

ere scanned into Aperio Scanscope and the resulting im-ges were processed using Aperio ImageScope Color De-onvolution method (Aperio Technologies, Inc., Vista, CA,SA) to determine the percentage of stained tissue (plaqueercent area). Location of the cortex as well as the hip-ocampus was determined visually.

.4. Biochemical procedures

.4.1. Homogenization of brain tissueThe frozen APP/PS1 hemibrains were sequentially ho-

ogenized in 5 volumes of Tris buffered saline (TBS,ediatech, Manassas, VA, USA) with protease inhibitors

Roche Diagnostics, Indianapolis, IN, USA), 5 volumes ofBS/1% Triton-X (TX)-100 with protease inhibitors and thenal pellet was homogenized in 10 volumes of 5 M guani-ine-HCl pH 8 (Sigma, St. Louis, MO, USA) and incubatedor 3 hours at room temperature. The TBS and TBS/TX-100omogenates were centrifuged at 175,000g for 30 minutes,

°C and the guanidine homogenate was centrifuged at16,000g for 1 hour, 4 °C. These supernatants constitutedhe soluble, detergent soluble, and insoluble A� fractions

respectively. TBS and TBS/TX-100 supernatants (extracts)were further mixed with equal volumes of 5 M guanidine-HCl pH 8 overnight at 4 °C. The guanidine treated super-natants and the guanidine extracted pellets were applied to60 mg HLB 96-well plates (Waters, Milford, MA, USA)and concentrated as described previously (Lanz andSchachter, 2006). The resulting lyophilized pellets werestored at �80 °C until needed for resuspension in blockingbuffer for analysis by the Delfia A� ELISA.

2.4.2. Delfia A� ELISAOptimal concentrations of APP/PS1 brain extracts were

determined by a serial dilution titration and sample dilutionswere incubated on 384 well plates prepared as follows:384-well plates (VWR, Bridgeport, NJ, USA) were coatedovernight at 4 °C with an anti-A�x-40 (4 �g/mL), ananti-A�x-42 (10 �g/mL) (Rinat, South San Francisco, CA,

USA), or 6E10 (4 �g/mL) (Signet Laboratories, Dedham, w

MA, USA) diluted in 0.05 M sodium carbonate/bicarbonate,pH 9.6, and then blocked for 2–4 hours at room temperaturewith 1% bovine serum albumin in phosphate-buffered salinewith 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO,USA). Standard curves were prepared from stock solutionsof species-specific A� peptides (Bachem Biosciences, Kingof Prussia, PA, USA) in blocking buffer. Standards andsamples were incubated on the coated and blocked plates for2 hours at room temperature. For detection biotinylated 4G8(0.2 �g/mL) or biotinylated antirodent A�1–40 (50 �g/mL)(Signet Laboratories) were incubated for 2 hours at roomtemperature. The signal was amplified by incubation witheuropium-conjugated streptavidin for 1 hour at room tem-perature followed by incubation with Delfia enhancementsolution at room temperature for 20 minutes in the dark.Plates were read on a Wallac, Victor plate reader (europium-Delfia reagents and equipment from PerkinElmer Life andAnalytical Sciences, Boston, MA, USA). Standards were fitto a fourth-order polynomial curve, and sample values wereextrapolated with GraphPad Prism 5.0 (GraphPad Software,Inc., La Jolla, CA, USA).

2.4.3. Analysis of electrophysiological dataFor nPO stimulation-evoked hippocampal theta oscilla-

tion Fast Fourier transformation was performed on each6-second stimulation period, using 1 to 5.096 seconds(0.25-Hz frequency resolution) following stimulation onset,to avoid inclusion of stimulation artifact in analysis. Peakfrequency and total power in the theta band (3–12 Hz) weredetermined. To establish correlation between evoked thetapower and plaque load, power values acquired for differentstimulation strengths were averaged across all stimulations.For perforant path stimulation evoked-dentate gyrus popu-lation spike experiments, all measures were taken using theaverage of the 2 channels in or closest to the cell body layerof the dentate gyrus, determined as those channels showingthe largest evoked population spike. For each animal, stim-ulation response curves were fitted to the data as sigmoidfunctions. The asymptotic value of the fitted function wastaken as maximal population spike amplitude and the stim-ulation strength required to produce a response that was50% of the asymptotic value (ES50) was also determined.

or characterization of LTP, data were averaged into 5-min-te bins and normalized to the average population spikemplitude of the baseline period.

.5. Statistical data analysis

For statistical evaluation of data by analysis of varianceANOVA), repeated measures ANOVA or t tests either theraphPad Prism 5.0 software (GraphPad Software, Inc.) or

he Matlab Statistics Toolbox (v7.1, R2009a, MathWorks,nc., Natick, MA, USA) was used. To assess statisticalignificance of regression analysis, p values of the F statistic

ill be given together with linear R statistic.

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1481.e17L. Scott et al. / Neurobiology of Aging 33 (2012) 1481.e13–1481.e23

3. Results

3.1. Elicited hippocampal theta oscillation in APP/PS1,PS1 and nontransgenic mice

High frequency electrical stimulation of the nPO elicitedhighly regular field potential oscillations in theta frequencyrange across the hippocampal formation, including CA1region and dentate gyrus in anesthetized APP/PS1 mice(n2 month � 6, n4 month � 9, n8 month � 6), PS1 (n2 month �, n4 month � 8, n8 month � 6), and WT (n2 month � 7, n4 month �

9, n8 month � 6) (Figs. 2 and 3A). Quantitative analysis oflicited theta oscillation in the hippocampus CA1 revealedhat stimulation intensity had a significant effect on therequency of oscillation in all 3 tested animal strains (WT:(11,228) � 30.01, p � 0.0001, � � 1.450; PS1: F(11,215) �3.16, p � 0.0001, � � 2.207; APP/PS1: F(11,228) �

26.60, p � 0.0001, � � 1.283). Analyzing peak frequenciescross different age groups, it has been found that age is aignificant factor in determining peak frequency in APP/S1 and WT but not PS1 animals (APP/PS1: F(2,228) �0.10, p � 0.0001, � � 0.176; WT: F(2,228) � 17.61, p �.0001, � � 0.154; PS1: F(2,215) � 1.442, p � 0.05; 2-way

ANOVA tested; Fig. 3B). Power of the evoked hippocampal

Fig. 2. Typical age-dependent deposition of amyloid plaques and change inin APP/PS1 mice. Representative micrographs containing hippocampus andwild type (WT) mouse, with overlaid signals recorded from 16 sites spanprogressive development of plaques is apparent with concomitant deteriorof active electrode sites, arrows on 4-month-old APP/PS1 point to 6E1

stimulation.

oscillation in the theta frequency band (3–12 Hz) showed arobust age-dependent decline in APP/PS1 mice (F(2,228) �60.92, p � 0.0001, �2 � 0.534), but not in PS1 or WT mice(Fig. 3B). Using multisite silicon recording electrodes it wasconfirmed that reduced field potential oscillations in re-sponse to nPO stimulation were not confined to a particularregion within the hippocampus; a qualitatively similar re-duction in theta power was present across the CA1 anddentate gyrus (DG) in APP/PS1 mice either at 4 or 8 monthage (n � 4; Fig. 2).

3.2. Perforant path stimulation-evoked population spikesand LTP in dentate gyrus

A different set of animals (WT: n4 month � 6, n8 month �6; PS1: n4 month � 6, n8 month � 6; APP/PS1: n4 month � 6,

8 month � 6) were tested using the LTP protocol to assessunctioning of perforant path (PP) synapses. Amplitude ofP stimulation-evoked population excitatory postsynapticotentials were recorded every 10 seconds in the dentateyrus. Stimulation-response relationship was first estab-ished and maximal evoked response amplitudes were iden-ified for all animals. A 2-way ANOVA showed no signif-cant effect of either age (F(1,30) � 0.25, p � 0.621, � �

m nucleus pontis oralis (nPO) stimulation-elicited hippocampal oscillationof the cortex of 2-, 4-, and 8-month-old APP/PS1 mice and an 8-month-oldross the cortex and hippocampus (stimulation strength was 0.06 mA). Aeta oscillation. Colored dots on 2-month-old APP/PS1 represent locationd amyloid plaques, red dotted lines denote beginning and end of nPO

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1481.e18 L. Scott et al. / Neurobiology of Aging 33 (2012) 1481.e13–1481.e23

0.008) or strain (F(2,30) � 0.71, p � 0.490, � � 0.044)(Fig. 4A). However, current required to produce a responseES50 was significantly affected by age (F(1,30) � 5.51, p �0.026, � � 0.128), but not by strain (F(2,30) � 2.02, p �.150, � � 0.094) as revealed by a 2-way ANOVA. A postoc Bonferroni test confirmed that in the APP/PS1 group-month-old animals required significantly less current tovoke 50% of the maximal response than 4-month-old an-mals (Bonferroni’s t test, t � 2.536, p � 0.05), whereasS50 in WT and PS1 groups was similar across age groups

(Fig. 4B). Comparing ES50 across strains in 8-month-oldanimals revealed a tendency of APP/PS1 mice to requireless current to drive the response (1-way ANOVA, F(2,15) �

Fig. 3. Age-dependent changes in nucleus pontis oralis (nPO) stimulation-theta oscillations in the hippocampal CA1 region as shown by representatiof nPO with 0.1-mA strength). (B) Power (lower panel) in the theta frequewild type (WT) groups. Peak frequency (upper panel) in APP/PS1 mice w

2.216, p � 0.144, � � 0.228). Potentiation of population p

pike amplitude was not found to be affected by strain atmonths (F(2,272) � 0.310, p � 0.740, � � 0.030; 2-ay repeated measures ANOVA) or at 8 months of age

F(2,289) � 1.390, p � 0.276, � � 0.121; 2-way repeatedeasures ANOVA) (Fig. 4D).

.3. Amyloid plaque burden and A� levels inAPP/PS1 mice

Following completion of electrophysiological experi-ments, A� plaques were quantified in hemibrains of APP/

S1 mice. As it was expected, a drastic age-dependentncrease of plaque percent area was seen both in the hip-

hippocampal theta oscillation. (A) Stimulation of the nPO elicits regularl field potentials (LFPs) (horizontal bar under traces represent stimulationnd shows an age-dependent decrease in APP/PS1 mice, but not in PS1 or

shown to be affected by age.

elicitedve locancy ba

ocampus and cortex (Figs. 2 and 5). Amyloid plaques were

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1481.e19L. Scott et al. / Neurobiology of Aging 33 (2012) 1481.e13–1481.e23

apparent in each APP/PS1 mouse at 4 months age, althoughwith a different degree, and at 8 months a drastic increase ofplaque percent area was detected in all mice. Furthermore,hippocampal and cortical plaque load was found to correlate

Fig. 4. Analysis of perforant path (PP) stimulation-evoked dentate gyrus (Damplitude was comparable in wild type (circle), PS1 (triangle), and Arepresents an individual animal; mean and standard error of the meanpopulation spike amplitude (ES50) was not significantly different (1-way

S1, and WT animals at 4 months (gray). At 8 months (black) WT androups while APP/PS1 mice showed a decrease in ES50 (* p � 0.05). (C

(black) long-term potentiation (LTP) induction. (D) No statistically signat either 4 or 8 months of age (LTP was induced at time 0 minutes).

significantly (R � 0.9637, p � 0.001; Fig. 5B) across

PP/PS1 mice. Soluble and insoluble A� concentrationswere measured in 8-month-old APP/PS1 mice followingelectrophysiological recordings, resulting in 37 � 5 pmol/gn � 6) of soluble and 6332 � 750 pmol/g (n � 5) of

ulation spikes. (A) Maximal PP stimulation-evoked DG population spike(square) animals, and showed no age-dependent changes (each dot

en for each group). (B) Current required to produce 50% of maximalis of variance [ANOVA], followed by Bonferroni’s t test) in APP/PS1,

oups were not significantly different from their corresponding 4-monthesentative traces showing DG population spikes before (gray) and afterdifferences were found in PP LTP among WT, PS1, and APP/PS1 mice

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1481.e20 L. Scott et al. / Neurobiology of Aging 33 (2012) 1481.e13–1481.e23

an age-dependent increase in both soluble and insoluble A�levels. Soluble A� increased from 11 � 0.6 pmol/g (n �2) at 3 months old to 16 � 2 pmol/g (n � 12) at 6, and to68 � 15 pmol/g (n � 12) at 12 months of age, whilensoluble A� increased from 34 � 6 pmol/g (n � 12) at 3

onths old to 2964 � 39 pmol/g (n � 12) at 6 months, ando 79,719 � 5250 pmol/g (n � 12) at 12 months of age ashe plaque deposited. Therefore A� values of 8-month-oldnimals were in the range characteristic of these APP/PS1ice.In order to perform correlation analysis between neuro-

hysiological measures and plaque load, theta power and

Fig. 5. Cortical and hippocampal plaque load in APP/PS1 animals. Plaqueload in all APP/PS1 animals was determined in the cerebral cortex as wellas in the hippocampus. (A) Hippocampal plaque percent area was found toincrease drastically with age (each dot represents an animal colored ac-cording to its age group; medians, and 25th and 75th percentiles are shownfor all age groups); note the rapid onset of plaque load increase at around140 days of age. (B) Cortical and hippocampal plaque percent areas werefound to correlate well (R � 0.9637, note however, that at the 4-month ageroup all points are below the regression line). For each age group meannd standard deviation of surface ratio percentages were calculated andlotted (crosses: location gives mean in 2 dimensions, length of armsepresent standard deviation).

eak frequency values were averaged across stimulation

ntensities. This method is justified by the similar functionalorm of the input/output curves in this assay in all 3 mousetrains. Subsequent correlation analysis by fitting exponen-

ial functions in the form y � exp�x�a

b � revealed a trend

for negative correlation between peak frequency and hip-pocampal plaque load (R � 0.4268; a � 8.4108, b �

5.0929) and strong negative correlation between thetaand power and plaque load (R � 0.7815; a � 0.1232, b �0.0114) across all age groups based on an exponential fit

Fig. 6).

Fig. 6. Correlation between hippocamapal electrophysiological measuresand hippocampal plaque load in APP/PS1 mice. Collapsed electrophysio-logical measures, (A) peak frequency, and (B) theta power, are plotted asa function of hippocampal plaque load (note the logarithmic-linear scale;each dot represents an animal colored according to its age group, black:8-month; dark gray: 4-month; light gray: 2-month.). Exponential fits(dashed curves) showed significant correlation of theta power with plaqueload (R � 0.8) and a trend toward correlation of frequency with plaque load

(R � 0.4).

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1481.e21L. Scott et al. / Neurobiology of Aging 33 (2012) 1481.e13–1481.e23

4. Discussion

An age-dependent disruption of elicited hippocampaltheta oscillation in APP/PS1, but not PS1 or WT mice hasbeen shown, while LTP in the dentate gyrus was not af-fected by age in these mice; indicating that the brainstem-hippocampus multisynaptic pathway is more vulnerable tothe disruptive effects of amyloid pathology than the mono-synaptic perforant path innervation of the hippocampus.The presence of amyloid plaques at 4 and 8 months, but notat 2 months of age, the correlation between disrupted hip-pocampal theta oscillation and plaque load, as well as thepredicted high level of soluble A� at 8 months suggests thathe alterations in neurophysiological signals are related tohe disrupted A� processing in these transgenic mice.

It is well established that high frequency stimulation ofthe midbrain reticular formation, including the nPO, elicitshippocampal theta rhythm in both anesthetized and freelymoving rats (for review see McNaughton et al., 2007). Thepresent findings are the first demonstration of elicited thetaoscillation in anesthetized mice, providing a valuable invivo assay to assess hippocampal function in various trans-genic mice. Elicited theta oscillation is mediated, at least inpart, via glutamatergic, GABAergic, and cholinergic syn-apses, and both theta power and frequency are highly sen-sitive to manipulation of synaptic neurotransmission. It hasbeen shown that drugs known to disrupt hippocampus-dependent cognitive performance, such as the muscarinicreceptor antagonists atropine or scopolamine, N-methyl-D-aspartate receptor antagonist MK-801, nociceptin receptoragonist Ro 64-6198 or CB1 receptor agonist CP-55940reduce power, or at a high doses totally disrupt elicited thetaoscillation (Hajós et al., 2008; Kinney et al., 1999; Li et al.,2007; McNaughton et al., 2007; Siok et al., 2006; and Siokand Hajós, unpublished observations). In fact, it has beendemonstrated that the CB1 agonist induced decrease in thetapower and reduction in temporal coordination of simulta-neously recorded hippocampal neurons were correlated withmemory impairment in a hippocampus-dependent task, in-dicating that impaired oscillation/loss of neuronal syn-chrony was responsible for cannabinoid-induced memorydeficits (Robbe et al., 2006). Present findings demonstratean age-dependent decrease in power of elicited theta oscil-lation of the hippocampus in APP/PS1, but not in PS1 orWT mice. Because the recorded field theta power dependson the laminar location within the hippocampus (Buzsáki,2002; Kocsis et al., 1999), we also used multisite recordingelectrodes to avoid potential sampling errors due to possiblemorphological changes of the hippocampus, and confirmedthat reduction in the power of stimulated theta oscillations isapparent across all hippocampal layers. Therefore, we arguethat the drastically reduced hippocampal theta power indi-cates the compromised hippocampal-dependent cognitivefunction in APP/PS1 mice reported previously (Howlett et

al., 2004). These findings are also in line with observations t

by Villette et al. (2010), where microinjection of A� intothe hippocampus impaired behavioral performance and theassociated hippocampal theta oscillation in a rat visuospatialrecognition test. As cognitive deficits in APP/PS1 micedevelop at 6 months of age (Howlett et al., 2004), thepresently described disruption in hippocampal functioncould be an early surrogate signal of cognitive dysfunctionand be used for detecting and following pathologicalchanges in APP/PS1 mice.

The mechanisms underlying the significantly reducedpower of elicited hippocampal theta oscillation in APP/PS1mice are presently unknown. Stimulation-induced theta ac-tivity is highly dependent on cholinergic neurotransmission;enhanced cholinergic tone following administration of ace-tylcholine esterase inhibitors leads to enhanced theta powerin rats (Kinney et al., 1999), similar increases have beenseen following administration of muscarinic agonists,whereas blockade of muscarinic receptors abolishes or at-tenuates power of theta oscillation in rats (Hasselmo, 2006)and WT mice (Feng, Kiss, and Hajós, unpublished obser-vation). Although cholinergic neurons are highly vulnerablein AD patients and show neurodegeneration in the earlystages of the disease (Cuello et al., 2010), loss of cholinergicneurons have not been demonstrated in APP/PS1 mice(Wong et al., 1999). However, whether the reported altera-tions in cholinergic axon terminals in APP/PS1 mice (Hu etal., 2003) could contribute to the presently observedchanges in hippocampal physiology should be further eval-uated.

Studies on A� overproducing transgenic mice, includingPP/PS1 mice, produced rather conflicting findings with

espect to LTP (Randall et al., 2010). In APP/PS1 mice initro, an increased rate of decay in dentate gyrus LTP andormal CA1 LTP at 17–18 months of age were reportedGruart et al., 2008; Gureviciene et al., 2004). Although aeduced LTP has been reported in APP/PS1 mice at 8onths of age in vivo (Gengler et al., 2010), in vitro studies

id not find deficits in hippocampal LTP up to even 14onths of age (Fitzjohn et al., 2010; Volianskis et al.,

010). The present findings demonstrate the same magni-ude of LTP in the dentate gyrus of APP/PS1, PS1, and WTice, at both 4 and 8 months of age. Furthermore, eachouse group showed similar input-output relationships to

erforant path stimulation-induced population spikes. Inter-stingly, APP/PS1 mice required significantly less current at

months than at 4 months of age to elicit 50% of theopulation spike maximal response, whereas no changesere observed in PS1 or WT mice. This finding indicates an

ge-dependent enhancement of excitability of the perforantath-dentate gyrus pathway in APP/PS1 mice, likely to bessociated with an A� overproduction-related synaptic dys-unction (Palop et al., 2007).

Electrophysiological recordings in the current study werearried out in urethane anesthetized mice and it is possible

hat this anesthesia is a contributing factor to the observed

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1481.e22 L. Scott et al. / Neurobiology of Aging 33 (2012) 1481.e13–1481.e23

dysfunction of hippocampal oscillation. Therefore, hip-pocampal activity recorded over the sleep-wake cyclewould be particularly informative. Previous studies of stim-ulation-induced theta indicate that drugs reducing frequencyof elicited hippocampal theta oscillation reduce rapid eyemovement (REM) sleep (McNaughton et al., 2007; Siok etal., 2009). It has recently been reported that APP/PS1 miceshow a significant reduction in REM sleep (Jyoti et al.,2010), resembling some sleep abnormalities of AD patientssuch as a reduced REM sleep and a slowing of EEG duringREM (Montplaisir et al., 1998; Rossini et al., 2007). Be-cause it is presumed that activity of the nPO contributes toREM sleep regulation (Vertes et al., 2004; Xi et al., 2004),reduction in elicited hippocampal theta power and de-creased amount of REM sleep can both reflect the impact ofA� on the same or partly overlapping neuronal network. Itwould be also highly informative to evaluate hippocampalactivity in APP/PS1 mice during various behavioral or cog-nitive tasks. As we have shown, various drugs can modulatehippocampal theta oscillation in a stage- or behavior-depen-dent manner (Hajós et al., 2008; Kocsis et al., 2007), there-fore it would be critical to connect potential abnormalities inhippocampal theta activity to impaired cognitive function.

APP/PS1 mice showed age-dependent pathology in theform of amyloid plaque loads, soluble, and insoluble A�levels. Although the present study was not primarily de-signed to establish correlations between neurophysiologicaldysfunction and various pathological or biochemical mark-ers of A� we have seen a strong, nonlinear correlationbetween power of elicited hippocampal theta oscillation andhippocampal plaque load. Because our findings indicate thatat 4 months of age APP/PS1 mice show the largest variationin elicited theta power, these mice would be ideally suited toestablish relationships between A� pathology and disruptedhippocampal function.

It is important to note that the presently described ab-normality of theta oscillation in APP/PS1 mice occurs whenamyloid plaque formation can be first detected. Indeed,these changes precede those previously reported in A�overproducing mice in both behavioral assays and otherelectrophysiological measures (Gengler et al., 2010; Harriset al., 2010; Jyoti et al., 2010). Reduction in hippocampaltheta power is an easily quantifiable signal, which could beused to address underlying mechanisms of A� pathology.This electrophysiological measure is related to multisynap-tic pathway contributing to hippocampal oscillatory activityand seems to be more vulnerable to the disruptive effects ofamyloid pathology than LTP of the monosynaptic perforantpath-dentate gyrus pathway. Evaluating the role of varioussoluble A� peptides, including A� dimers, in the observedlectrophysiological deficits may provide further insightsnto the neural dysfunction underlying AD. Although theltimate clinical efficacy of A� lowering therapies in ADrogression will be seen in patients, preclinical in vivo

fficacy assays could address numerous questions about A�

lowering strategies. For example, the safety range of A�reduction should be evaluated because current findings in-dicate neuronal activity-dependent release of A�, whichmight have both physiological roles and contribution toAD-specific pathology (Bero et al., 2011). Furthermore,elicited hippocampal theta in APP/PS1 mice could be de-veloped to measure outcomes of potential A�-loweringherapeutics such as gamma secretase inhibitors and mod-lators or beta-secretase inhibitor compounds.

isclosure statement

All authors were full-time employees of Pfizer, Inc.The animal use protocol was approved and in compli-

nce with the Animal Welfare Act Regulations (9 CFR Parts, 2, and 3) and with the Guide for the Care and Use ofaboratory Animals, National Institutes of Health guide-

ines.

cknowledgements

During preparation of this work, Tamás Kiss was onabbatical leave from the KFKI Research Institute for Par-icle and Nuclear Physics of the Hungarian Academy ofciences. This research was supported by Pfizer Globalesearch and Development, Groton, CT, USA.

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