Ž .Brain Research 840 1999 23–35www.elsevier.comrlocaterbres

Research report

Alterations in synaptic transmission and long-term potentiation inhippocampal slices from young and aged PDAPP mice

John Larson a,d,) , Gary Lynch b, Dora Games c, Peter Seubert c

a Center for the Neurobiology of Learning and Memory, UniÕersity of California, IrÕine, CA 92697, USAb Department of Psychiatry and Human BehaÕior, UniÕersity of California, IrÕine, CA 92697, USA

c Elan Pharmaceuticals, 800 Gateway BouleÕard, South San Francisco, CA 94080, USAd The Psychiatric Institute, Department of Psychiatry, College of Medicine, UniÕersity of Illinois at Chicago, Chicago, IL 61612, USA

Accepted 1 June 1999

Abstract

Synaptic transmission and plasticity were studied in the CA1 field of hippocampal slices from young and aged transgenic miceŽ .over-expressing a mutant form of the human amyloid precursor protein PDAPP mice . The transgenic mice at 4–5 months of age, prior

Ž .to the formation of amyloid-b peptide deposits in these animals, differed from non-transgenic control mice in three respects: 1Ž . Ž . Ž .paired-pulse facilitation PPF was enhanced; 2 responses to high frequency stimulation bursts were distorted; 3 long-term potentiation

Ž .LTP decayed more rapidly. More striking was the profound reduction in the size of synaptic responses and frequent loss of fieldpotentials that were found in the transgenic mice at 27–29 months, an age at which they exhibit numerous amyloid plaques, neuriticdystrophy, and gliosis. Control mice at these ages did not show such dramatic effects. PPF was reduced in aged transgenic mice,compared to aged controls; however, LTP was still in evidence, although direct comparisons of its induction conditions in aged transgenicand control mice were compromised by the profound differences in field potentials between the two groups. These results point to two

Ž .conclusions: 1 altered synaptic communication appears in PDAPP mice in advance of amyloid plaque formation and probably involvesŽ .changes in presynaptic calcium kinetics; 2 the disturbances in synaptic transmission that appear when abundant plaques and

Alzheimer’s-like neuropathology are present in the transgenic mice are not necessarily accompanied by a disproportionate loss oflong-term synaptic plasticity. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Alzheimer’s disease; b-Amyloid; Amyloid precursor protein; Paired-pulse facilitation; Transgenic; Long-term potentiation; CA1

1. Introduction

A substantial body of evidence indicates that alteredŽ .cellular metabolism of the amyloid beta peptide Ab

contributes to the neuropathology and cognitive deficitsw xthat develop in Alzheimer’s disease 1 . The Ab peptide,

Ž .derived from the amyloid precursor protein APP , is themajor proteinaceous component of senile plaques that are

w xprominent in the brains of Alzheimer’s patients 1 . Plaqueformation is most pronounced in forebrain structures suchas hippocampus, entorhinal cortex and neocortex, areas

w xthat are critical for memory storage 2 . Genetic predisposi-tion to the disease co-segregates with mutations in the APP

) Ž .Corresponding author. The Psychiatric Institute MrC 912 , Depart-ment of Psychiatry, College of Medicine, University of Illinois at Chicago,1601 W. Taylor St., Chicago, IL 60612, USA. Fax: q1-312-413-4569;E-mail: jrlarson@psych.uic.edu

w xgene itself 3–5 that lead to increased production ofw xamyloidogenic forms of the Ab peptide 6–8 and also in

w xthe genes encoding the presenilins 9,10 , proteins that alsow xaffect the cellular production of Ab 11,12 . The mutations

in Presenilin-1 are though to be responsible for the major-ity of cases of early-onset, familial Alzheimer’s disease.

Several distinct lines of transgenic mice that incorporatevarious forms of the APP gene have been generated andfound to reproduce many aspects of Alzheimer’s disease

w xneuropathology 13–18 . The present study utilized thew xPDAPP line 109 described by Games et al. 14 . These

mice carry a transgene constructed from the human mutantŽ .V717F APP gene and show accumulation of Ab peptide

w x w xand amyloid plaques in an age- 14 and brain region- 19dependent manner. These Ab-containing plaques resemble

w xthose of Alzheimer’s disease both histochemically 14 andw xultrastructurally 20 . These animals also show age-depen-

dent astrocytosis, microgliosis, neuritic dystrophy, andsynaptic degeneration similar to Alzheimer’s disease.

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 01698-4

( )J. Larson et al.rBrain Research 840 1999 23–3524

The present study represents the first electrophysiologi-cal characterization of this line. It employed hippocampal

Ž .slices prepared from young 4–5 months old and agedŽ .27–29 months old heterozygous PDAPP mice along withage-matched non-transgenic controls to investigate alter-ations in synaptic function associated with over-expressionof mutant human APP and Ab peptide secretionrdeposi-tion. Young transgenic mice express high levels of thehuman mutant APP and Ab but do not show deposition ofsignificant amounts of Ab peptide, whereas aged trans-genic mice continue to express equally high levels of APPand have large amounts of deposited Ab, forming plaques

w xin hippocampus and cortex 19 in addition to havingextensive neuritic dystrophy and synaptic degeneration.The studies addressed two related questions. First, doesover-expression of mutant APP and Ab affect synaptic

communication in the absence of amyloid deposits and, ifso, in what manner? Second, what types of transmissiondisturbances accompany plaque formation and is long-term

Ž .potentiation LTP particularly vulnerable? Given the hy-pothesized role of LTP in memory formation, these latterquestions are of evident importance in understanding thecauses of the memory and cognitive problems that charac-terize Alzheimer’s disease.

2. Materials and methods

2.1. Animals

w x Ž .PDAPP mice 14 aged 4–5 months young and 27–29Ž .months aged were obtained from a colony maintained at

Fig. 1. Input–output functions for CA1 dendritic field EPSPs evoked by Schaffer-commissural fiber stimulation in hippocampal slices from young controlŽ . Ž .and transgenic mice. A A series of responses each trace is an average of four consecutive records to stimulus durations ranging from 0.02 to 0.30 ms at

Ž . Žconstant current in a slice from a control mouse and a transgenic mouse. B Graph of field EPSP amplitude as a function of stimulus intensity current. Ž . Ž . Ž .times duration for the two experiments in panel A. C Comparison of average maximum field EPSP mean"S.E.M. in slices from control ns8 andŽ . Ž . Ž .transgenic mice ns8 . D Comparison of average mean"S.E.M. stimulus intensity required to evoke a field EPSP at least 1.0 mV in amplitude in

ŽU .control and transgenic mice. p-0.05 .

( )J. Larson et al.rBrain Research 840 1999 23–35 25

Ž .Charles River Laboratories Wilmington, MA . Non-trans-genic control mice were matched for age and geneticbackground.

2.2. Immunohistochemistry

The antibodies and immunohistochemical methods werew xas described previously 14,19 . Briefly, brains from trans-

genic and control mice, 27 months of age, were fixed in4% paraformaldehyde, sectioned at 40 mm on a vibratome,and stored in 30% glycerolr30% ethylene glycol in 40mM phosphate buffer at y208C before immunostaining.Amyloid plaques were visualized with an antibody specific

Ž .for Ab antibody 3D6 and neuritic plaques were detectedŽwith an antibody directed against human APP antibody

.8E5 . Coronal sections through the CA1 field of hip-pocampus were incubated with antibody at 48C, overnight.The sections were then reacted with the horseradish perox-

Ž .idase–avidin–biotin complex Vector Laboratories anddeveloped using 3,3X-diaminobenzidine as the chromagen.

2.3. Electrophysiology

Ž .Transverse hippocampal slices 400 mm thick wereprepared conventionally and maintained at 36"18C at theinterface between an atmosphere of humidified 95%

ŽO r5% CO and a perfusion medium consisting of in2 2.mM : NaCl, 124; KCl, 3; KH PO , 1.2; CaCl , 3.4,2 4 2

MgSO , 2.5; NaHCO , 26; D-glucose, 10; and Na-L-4 3Ž .ascorbate, 3 0.8 mlrmin . The animals were coded so that

the preparation of slices and electrophysiological experi-ments were performed blind with respect to genotype. A

Ž .glass micropipette filled with 2 M NaCl 5 MV wasplaced in stratum radiatum of field CA1b to record den-

Ž .dritic field excitatory postsynaptic potentials EPSPsŽevoked by bipolar stimulation electrodes 50 mm diameter.formvar-insulated twisted nichrome wires placed in stra-w xtum radiatum of fields CA1a and CA1c 21 . Electrodes

were positioned to evoke the maximum field EPSPs ineach slice. One or two slices exhibiting the largest fieldEPSPs were used from each animal. Field EPSP wave-

Ž .Fig. 2. Paired-pulse and frequency facilitation in young control and PDAPP mice. A Field EPSPs recorded in response to paired-pulse stimulation at a 50Ž . Žms IPI in a slice from a control animal and a slice from a transgenic animal. Calibration bars: 1 mV and 10 ms. B Average facilitation mean"S.E.M.;

. Ž . Žns16 control, ns16 transgenic of the second response relative to the first of paired stimulation pulses at four IPIs. C Mean "S.E.M.; ns16 control,. Ž .ns15 transgenic responses to 10-pulse stimulation trains at 1, 5, and 10 Hz all responses normalized to the first pulse response in each train

ŽU UU .p-0.05; p-0.01 .

( )J. Larson et al.rBrain Research 840 1999 23–3526

Ž .forms were digitized by microcomputer 10 kHz , mea-sured using custom software, and stored on disk.

Input–output curves were generated by setting the stim-Ž .ulus intensity 3–40 mA to evoke a half-maximal field

EPSP at a stimulus duration of 0.10 ms and then varyingstimulus duration at 0.02 ms increments from 0.02 to 0.30ms. This yielded a stimulus charge-EPSP amplitude curvefor each pathway tested in each slice. The stimulus chargedelivery needed to evoke a 1.0 mV EPSP and the maximalEPSP amplitude were determined for each slice for com-parisons between groups.

Ž .Paired-pulse facilitation PPF curves were generated bytesting each pathway with twin pulses at inter-pulse inter-

Ž .vals IPI of 50, 100, 200, and 400 ms. Pulse pairs weregiven at 20 s intervals and four responses were averaged ateach IPI. The degree of facilitation was determined as thepercent increase in initial slope of the second responserelative to that of the first response in each pair. Frequencyfacilitation was assessed by delivering a train of 10 pulsesat 1.0, 5.0, and 10.0 Hz and expressing the facilitation ofresponse slope for pulses 2–10 as a percentage of theresponse to the first pulse of the train.

For LTP experiments, the standard testing cycle con-Ž .sisted of paired-pulse stimulation IPIs75 ms to one

electrode preceded 200 ms earlier by a single ‘‘priming’’pulse to the other electrode and a similar sequence with theelectrodes reversed 10 s later. The cycle was repeated at 20s intervals throughout the experiment. Priming stimulation

w xserves to reduce GABA receptor mediated IPSPs 22,23 .A

Paired-pulse responses were also collected without primingstimulation at selected time points during the experiment.Testing cycles were repeated for a baseline period of

Ž . w x10–15 min. Theta burst stimulation TBS 24 was used toinduce LTP and consisted of five or 10 bursts of fourpulses at 100 Hz, repeated at 5 bursts per second. One of

Ž .the pathways selected at random was then given a 10-burstTBS and testing cycles were resumed for 30–35 min. Thenthe other pathway was given a five-burst TBS and testingresumed for 60 min. During TBS, stimulus pulse durationwas increased from 0.1 to 0.2 ms and thereafter returned to0.1 ms. The magnitude of potentiation of EPSP slope wastabulated at 5 min intervals after TBS for the duration ofrecording and expressed as a percentage of the pre-TBSbaseline average value. Field potentials during TBS were

Ž .Fig. 3. Long-term potentiation in control and transgenic mice. A Results of individual experiments for a control and a transgenic mouse. Top panels showŽ . Žmeasurements of field EPSP slope normalized to the pre-TBS baseline average before and after a 10-burst TBS in one pathway of each slice each point

.is an average of four consecutive responses ; bottom panels show measurements before and after a five-burst TBS to the other pathways in the same slices.Ž . Ž . ŽB Comparison of average mean"S.E.M.; ns6 control, ns8 transgenic potentiation of response slope 60 min after TBS consisting of 10 bursts top

. Ž . ŽU .panel or five bursts bottom panel . p-0.05 .

( )J. Larson et al.rBrain Research 840 1999 23–35 27

recorded by computer and quantified by measuring thew xtotal area of negativity underlying the burst response 21 .

3. Results

3.1. Young mice

The over-expression of APP and Ab and lack of Ab

deposition in PDAPP mice aged 4–5 months have beenw xdescribed previously 14,19 .

3.1.1. Input–output curÕesInput–output curves for field EPSPs were fairly similar

Ž . Žin young 4–5 months control and transgenic mice Fig.. Ž1A,B , although the maximum dendritic field EPSP Fig.

.1C was slightly smaller in slices from transgenic miceŽ .t s2.32, p-0.05 . The stimulus intensity level neces-30

Ž .sary to evoke a 1.0 mV response Fig. 1D was notŽ .different in the two groups t s0.73, p)0.40 .30

3.1.2. Paired-pulse and frequency facilitationExamples of PPF at a 50 ms IPI for typical control and

transgenic animals are shown in Fig. 2A. PPF curves wereŽ .generated from IPIs of 50, 100, 200, and 400 ms Fig. 2B .

Analysis of variance indicated significant main effects ofŽ . Žgenotype F s5.58, p-0.05 and IPI F s583.1,1,30 3,90. Žp-0.0001 and a significant interaction F s4.49, p3,90

.-0.01 . Newman–Keuls tests indicated that there wassignificantly greater facilitation in the transgenic mice atall IPIs.

Frequency facilitation was tested with trains of 10Ž .pulses at 1.0, 5.0, and 10.0 Hz Fig. 2C . Analysis of

variance indicated a significant main effect of genotype onŽfacilitation at all frequencies 1.0 Hz: F s9.34, p-1,29

0.005; 5.0 Hz: F s5.89, p-0.05; 10.0 Hz: F s1,29 1,29.6.98, p-0.05 . Frequency facilitation was significantly

greater for pulses 2–10 of each frequency train in theŽ .transgenic animals Newman–Keuls tests, p-0.01 .

Ž .Fig. 4. Responses to TBS in control and PDAPP mice. A Recordings of the first, second, and sixth burst in representative control and transgenic mice.Ž . Ž . ŽB Comparison of the total area of negativity evoked in response to a single burst. C Measurements of burst response area normalized to the first burst

. Ž . Ž . Ž .area for 10-burst TBS mean" S.E.M. . D Measurements of a positive afterpotential preceding each burst response after the first bursts 2–10 forŽU .10-burst TBS. p-0.05 .

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Ž . Ž .Fig. 5. Amyloid deposition and neuritic plaques in aged transgenic mice. Photomicrographs show tissue sections from aged transgenic panels A and B and non-transgenic panels C and D mice that wereŽ . Ž . Ž U .immunoreacted with antibodies to detect either b-amyloid panels A and C or neuritic plaques panels B and D . Abundant hippocampal amyloid plaques were deposited in CA1 panel A, , a region that

Ž . Ž . Ž .also contained numerous neuritic plaques panel B . Non-transgenic brains were devoid of both amyloid panel C and neuritic panel D plaques. Amyloid was visualized by reaction with the antibody 3D6,which recognizes the amino-terminus of the human Ab peptide. Neuritic plaques were detected by the anti-APP monoclonal antibody 8E5. Calibration bar: 100 mm.

( )J. Larson et al.rBrain Research 840 1999 23–35 29

3.1.3. LTPIn each slice tested for LTP, one pathway was first

given TBS consisting of 10 bursts and, 30 min later, thesecond pathway was given a five-burst TBS. Examples oftwo experiments are shown in Fig. 3A. Histograms in Fig.3B show the degree of potentiation 60 min post-TBS.Analysis of variance indicated a significant effect of geno-

Ž . Žtype F s9.78, p-0.01 and number of bursts F1,12 1,12.s6.10, p-0.05 , but no interaction between the two

Ž .factors F s 0.02, p ) 0.5 . Planned comparisons1,12

showed that the LTP remaining after 60 min was signifi-cantly smaller in the transgenic animals than in the con-

Žtrols for both five and 10 bursts protected t-tests, p-.0.05 .

Analysis of the responses to TBS are shown in Fig. 4.The total area of negativity underlying the first burst

Ž .response both five- and 10-burst TBS combined were not

Ž .different Fig. 4B; t s1.03, p)0.30 . Measurements of28

total area relative to the first burst for the 10-burst TBSpathways are shown in Fig. 4C. Analysis of variance

Žindicated an insignificant genotype main effect F s1,14. Ž2.28, p)0.10 , a significant effect of burst number F8,112

. Žs34.48, p-0.0001 , and a significant interaction F8,112.s4.30, p-0.001 . The area of responses to the laterŽ .bursts in the series bursts 6–10 were significantly larger

Žin slices from the control mice Newman–Keuls tests,.p-0.05 . Inspection of the burst response waveforms

suggested that a positive after-potential or ‘‘overshoot’’after each burst was larger in the slices from transgenic

Ž .mice Fig. 4A . This was quantified by measuring thevoltage present 2 ms prior to bursts 2–10 relative to the

Ž .DC potential present 2 ms prior to the first burst Fig. 4D .Analysis of variance confirmed that genotype had a signifi-

Ž .cant effect on the overshoot F s8.51, p-0.01 and1,14

Ž . ŽFig. 6. Input–output functions in slices from aged control and transgenic mice. A A series of responses each trace is an average of four consecutive. Ž .records to stimulus durations ranging from 0.02 to 0.30 ms at constant current in a slice from a control mouse and a transgenic mouse. B Graph of field

Ž . Ž .EPSP amplitude as a function of stimulus intensity current times duration for the two experiments in panel A. C Comparison of average maximum fieldŽ . Ž . Ž . Ž . Ž .EPSP mean"S.E.M. in slices from control ns9 and transgenic ns9 mice. D Comparison of average mean"S.E.M. stimulus intensity required

ŽU UUU .to evoke a field EPSP at least 1.0 mV in amplitude in control and transgenic mice. p-0.05; p-0.001 .

( )J. Larson et al.rBrain Research 840 1999 23–3530

that the overshoot was significantly higher in the trans-Žgenic mice for burst 2 and bursts 4–6 Newman–Keuls

.tests, p-0.05 .

3.2. Aged mice

3.2.1. Amyloid plaques in aged, transgenic micePrevious studies have documented the amyloid deposi-

tion and plaque formation in PDAPP mice aged 6–18w xmonths 14,19 . In order to extend these findings to the

ages studied in the present electrophysiological studies,tissue from transgenic and control mice, aged 27 months,were stained with antibodies labeling Ab and neuriticplaques. The results are shown in Fig. 5. Aged transgenic,but not control, mice showed extensive amyloid depositionand numerous neuritic plaques in the CA1 field of hip-

pocampus, as expected from the prior findings in some-what younger mice.

3.2.2. Input–output curÕesRepresentative input–output curves for slices from aged

Ž .27–29 months control and transgenic mice are shown inFig. 6A,B. The maximal EPSP was much smaller in the

Ž .transgenic animals Fig. 6C and higher stimulus intensi-Ž .ties were required to evoke a 1 mV EPSP Fig. 6D . Both

Žof these effects were statistically significant t s9.47,34.p-0.0001 and t s3.34, p-0.01; respectively . In ad-34

dition, responsive areas were more sharply localized inslices from transgenic mice; electrode placements resultingin no apparent field potentials were rare in the aged controltissue but common in the transgenic slices. The quantita-tive results shown in the figure are based on the bestresponses recorded from the animals tested and thus al-

Ž .Fig. 7. Paired-pulse and frequency facilitation in slices from aged control and transgenic mice. A Field EPSPs recorded in response to paired-pulseŽ .stimulation at a 50 ms IPI in a slice from a control and a slice from a transgenic. Calibration bars: 1 mV and 10 ms. B Average facilitation

Ž . Ž .mean"S.E.M.; ns18 control, ns18 transgenic of the second response relative to the first of paired stimulation pulses at four IPIs. C MeanŽ . Ž ."S.E.M. Responses to 10-pulse stimulation trains at 1, 5, and 10 Hz all responses normalized to the first pulse response in each train . At 5.0 Hz and

Ž .10.0 Hz, there were significant main effects of genotype on the degree of facilitation 5.0 Hz: F s5.08, p-0.05; 10.0 Hz: F s6.98, p-0.05 . All1,32 1,29Ž . ŽUU .responses showed greater facilitation in the controls than in the transgenics at 5.0 and 10.0 Hz Newman–Keuls tests, p-0.01 . p-0.01 .

( )J. Larson et al.rBrain Research 840 1999 23–35 31

Ž .Fig. 8. LTP in aged control and transgenic mice. A Results of individual experiments for a control and a transgenic mouse. Top panels showŽ . Žmeasurements of field EPSP slope normalized to the pre-TBS baseline average before and after a 10-burst TBS in one pathway of each slice each point

.is an average of four consecutive responses ; bottom panels show measurements before and after a five-burst TBS to the other pathways in the same slices.Ž . Ž . ŽB Comparison of average mean"S.E.M.; ns9 control, ns8 transgenic potentiation of response slope 60 min after TBS consisting of 10 bursts top

. Ž .panel or five bursts bottom panel .

most certainly represent the minimum difference betweengroups.

3.2.3. Paired-pulse and frequency facilitationFig. 7 shows waveforms evoked by paired pulses at a

Ž .50 ms IPI A and PPF curves for IPIs from 50 to 400 msŽ .B . In contrast to the results in young animals, PPF wassignificantly reduced in the aged, transgenic mice. Theanalysis of variance indicated significant main effects of

Ž . Žgenotype F s7.97, p-0.01 and IPI F s403.9,1,34 3,102. Žp-0.0001 and a significant interaction F s5.63,3,102

.p-0.01 . Planned comparisons indicated that PPF wassmaller in the transgenics at IPIs of 50, 100, and 200 ms.

Frequency facilitation was also significantly reduced inŽ .the aged transgenics at 5.0 and 10.0 Hz Fig. 7C .

3.2.4. LTPThe large difference in response size between control

and transgenic mice precluded a rigorous comparison ofLTP as this would require comparable stimulus conditionsand similar baseline responses. With this caveat, LTPexperiments were run in slices from both types of animal.Baseline response size was set to be about half-maximal as

Ždetermined from the IrO curves 1.5–2.0 mV for controls;.0.7–1.5 mV for transgenics . The results are shown in Fig.

Ž .8. LTP expressed as percent increase over baseline in-duced by TBS was comparable in both magnitude and timecourse under these conditions.

3.3. Effects of aging

A comparison of slices from young and aged non-trans-Ž .genic control animals was used to test for aging effects.

The maximal EPSP amplitude was greater in slices fromŽ .young animals t s5.97, p-0.0001 and the stimulus32

strength needed to evoke a 1 mV EPSP was significantlyŽ .lower t s2.84, p-0.01 . PPF was greater in the slices32

Žfrom aged animals at IPIs of 50, 100, and 200 ms New-.man–Keuls tests, p-0.01 . There were no differences in

ŽLTP at 60 min post-TBS at the two ages F s0.00,1,13.p)0.9 .

4. Discussion

Transgenic mouse models provide a unique opportunityto characterize the physiological consequences of the type

( )J. Larson et al.rBrain Research 840 1999 23–3532

of neuropathology that develops in Alzheimer’s disease.Ž .PDAPP mice express the human mutant V717F APP at

high and constant levels throughout their lifespans; deposi-tion of Ab peptide into amyloid plaques increases in anage- and brain region-dependent manner beginning at 8–12

w xmonths and continuing throughout aging 19 . The presentexperiments were designed to compare key electrophysio-logical properties in hippocampal slices of transgenic and

Ž . Žnon-transgenic mice at young 4–5 months and old 27–29.months ages, before and after the development of large

numbers of deposits of Ab peptide and subsequent degen-erative changes in hippocampus. The results show signifi-cant alterations in synaptic transmission in young mice anda dramatic loss of functional synapses in aged mice. Theseresults are substantially different than those obtained in thesmall number of other transgenic mouse models that have

w xbeen examined 25–27 .

4.1. Young mice

The transgenic animals denoted as young in the presentstudy over-express APP but have not yet deposited signifi-cant amounts of amyloid b-peptide. Input–output curvesfor field EPSPs evoked by Schaffer-commissural fiber

Ž .stimulation in field CA1 revealed a small 10% butsignificant decrease in the maximal response in the trans-genic mice. Larger differences were seen in the degree ofpaired-pulse and frequency facilitation of synaptic trans-mission, with the transgenic mice showing significantlygreater facilitation than the controls. It is noteworthy that

Žthe entire paired-pulse curve percent facilitation as a.function of the interval between pulses was shifted up-

ward and that facilitation was enhanced across the fulllength of the 10 pulse frequency facilitation series. Thispattern is also produced in Schaffer-commissural synapsesby lowering extracellular calcium concentrations or in-

w xcreasing the magnesiumrcalcium ratio 28,29 . In theseand related instances, the increase in facilitation is usuallyinterpreted as being due to individual pulses causing asmaller influx of calcium and thus a lesser depletion of thetransmitter pool. Smaller influxes could also result inproportionately larger residual calcium pools available topromote release to the next pulse. However, reductions incalcium influx reduce the size of single responses and thesmall differences in input–output curves suggest that thisdid not occur in the transgenic mice. A more likelyexplanation is that APP, or a released fragment of it,influences the processing of calcium after it has enteredthe terminal and triggered release. Recent work has shownthat APP modifies calcium fluxes and homeostasis in

w xdissociated cells 30,31 and the protein is known to bew xtransported within axons 32,33 to presynaptic terminals

w x34 . Possibly then, APP normally contributes to restora-tion of resting calcium levels inside terminals and thus tothe degree of facilitation elicited by repetitive action poten-

tials; over-expression or the presence of mutant APP would,according to this argument, subtly impair this function,resulting in larger residual calcium levels after a releaseevent.

The young transgenic mice also showed alterations inthe LTP induced by TBS. Although the potentiation ex-pressed immediately after TBS was comparable in trans-genic and control mice, the potentiation decayed morerapidly in slices from the transgenic animals. There aretwo general types of explanation for this: changes in thetriggering conditions for LTP or alterations in the mecha-nisms responsible for its stabilization. The latter involvesmolecular machinery presumably localized to individualsynapses whereas the former requires both local and cir-cuit-wide mechanisms. Clues to the role of the circuitrycan be obtained from the analysis of responses to the high

w xfrequency stimulation bursts used to induce LTP 21,22 .In the analysis of TBS responses, there were two signifi-

Ž .cant differences between the transgenics and controls: i alarger positive overshoot that was present after the first

Ž .and all succeeding bursts, and ii a diminution of laterŽ .responses 3–4 in later bursts in the train. Since the latter

difference was not evident in the five-burst TBS, it doesnot seem important for the effect on LTP, which was atleast as prominent in these pathways. The overshoot waspresent in these pathways, however. The overshoot is

Žlikely to be due to an inhibitory conductance mediated by.either GABA or GABA receptors that shunts the exci-A B

tatory currents evoked by the bursts and would reduce thedendritic depolarization thereby produced.

The abnormal overshoot does not necessarily indicate adirect effect of APP over-expression or mutation onGABAergic physiology; a simpler explanation is that theeffect is secondary to the above-discussed changes in thefrequency characteristics of the glutamatergic terminals.Feedforward and feedback GABAergic interneurons infield CA1 receive their primary innervation from theSchaffer-commissural projections or the recurrent collater-als of local pyramidal cells. Enhanced facilitation duringtheta bursts would presumably result in greater depolariza-tion of interneurons and thus a more intense inhibitoryresponse.

The argument that changes in frequency facilitationsecondarily increase inhibitory responses during bursts,and suppress LTP as a tertiary effect, emphasizes the pointthat such changes will modify a wide range of physio-logical activities.

4.2. Aged mice

The aged animals tested were 27–29 months old. Atthese ages the transgenic mice have deposited largeamounts of Ab peptide in plaques having dense coresresembling the neuritic plaques that are characteristic ofAlzheimer’s disease. In addition, these animals show pro-

( )J. Larson et al.rBrain Research 840 1999 23–35 33

found neuritic dystrophy and extensive activation of bothastrocytes and microglial cells. The most striking differ-ence observed electrophysiologically was that maximal

Ž .synaptic field potentials were much smaller 55% in thetransgenic than in the control aged mice. Qualitativelysimilar results were obtained from two aged transgenicmice when slices were prepared in medium containing the

Ž .glutamate receptor antagonist, kynurenic acid 1 mM ,suggesting that the differences found between transgenicand control mice were not due to excitotoxic damage

w xresulting from slice preparation 25 . The most obviousinterpretation of this effect is that large numbers of synapseshave degenerated or otherwise become non-functional insitu in the transgenic animals. Immunohistochemical stud-ies support this interpretation, showing a reduction ofsynaptophysin and microtubule-associated protein-2 in

w xtransgenic mice with age 14 . In fact, the magnitude of theeffect is most likely underestimated in the present datasince we analyzed only those slices and electrode positionsthat generated maximal responses; there was more variabil-ity among these in the slices from transgenic than fromcontrol mice. Indeed, field potential responses were muchmore localized in the transgenic slices and the occurrenceof ‘‘dead spots’’ near areas in which field potentials couldbe recorded suggest that such non-responsive areas may bethe result of amyloid plaque-associated neuropathology asthis hippocampal field is severely laden with amyloid inthe transgenic animals. Alternatively, neurons in the trans-genic mice may be chronically depolarized. Intracellularrecording studies will be needed to test this idea in thePDAPP mice.

Synaptic responses in aged, transgenic mice showedsignificantly reduced paired-pulse and frequency facilita-tion, compared to age-matched controls. This is in contrastto the enhanced facilitation observed in the young, trans-genic mice.

Although the large difference in baseline response am-plitude precludes a direct comparison between LTP inaged transgenic mice and controls, the finding that an LTPeffect of apparently normal magnitude and stability couldbe elicited in the transgenics indicates that the synapticmechanism for LTP is intact in these animals. Since thedistribution of Ab peptide deposits is not uniform and isconcentrated in amyloid plaques in this tissue, it wouldappear that there exist local populations of relatively nor-mal neurons adjacent to areas of amyloid accumulation.Although these neurons presumably express APP, this doesnot prohibit induction and stabilization of LTP. Clearly,hippocampal function in general would be compromisedby massive loss of normal synaptic function. It remains tobe determined whether or not amyloid production andsecretionrdeposition is limited to a population of patho-logical cells or is widespread in all neurons. If the latterwere correct, it would appear that the pathology does notcompletely prevent LTP mechanisms from becoming en-gaged.

The present results are in contrast to those of severalrecent investigations of similar phenomena in other trans-

w xgenic models. Hsia et al. 26 studied a separate lineŽ .expressing the same mutant transgene APP V717F and

found much greater losses in synaptic field potentials atŽ . Ž .young 1–4 months, 40% and older 8–10 months, 80%

ages. Histological analyses indicated significant reductionsof neuronal and synaptic densities in the transgenic mice,although not of sufficient magnitude or age-dependence to

w xaccount wholly for the electrophysiological results 26 .Nonetheless, stable LTP could be elicited even in the older

w xanimals. Chapman et al. 25 did not observe any differ-ences in basal synaptic transmission or PPF in mice over-

Žexpressing the ‘‘Swedish’’ double mutation APP K670N,.M671L at either 2–8 or 15–17 months of age. However,

Žthey found that LTP was selectively reduced decayed.more quickly in 15–17-month-old transgenic animals.

Changes that may take place as these animals age furtherhave yet to be investigated.

Another study reported that mice over-expressing theŽ .carboxy terminal 104 amino acid fragment of APP C104

w xhad impaired LTP but normal PPF 27 . Electrophysiologi-cal differences between that model and the present one arenot unexpected, since the pathologies present in the twomodels are very different. The expression of C104 doesnot result in the production or deposition of detectableamounts of the 4 kDa band characteristic of Ab in thebrain, and the C104 fragment appears to produce a cyto-toxicity distinct from the animal models in which holo-APPis expressed. The effects of intracellular over-production ofC104 lacking the normal amino terminal protein traffick-ing sequences present in APP, and the relevance of this toAlzheimer’s disease, are areas of ongoing debate andinvestigation. A further complication is that the studies of

w xNalbantoglu et al. 27 were performed at a single age; thusit is impossible to determine how much of the effect onLTP is developmentally related and how much of it islinked to age-dependent pathological changes.

In summary, young PDAPP mice show a small reduc-tion in the maximal size of field EPSPs, an enhancementof PPF, and decremental LTP. The effect on LTP may bedue to a change in the operation of the circuit mechanismsengaged to trigger potentiation rather than a deficit in thesubsynaptic mechanisms that express and stabilize theenhancement of transmission. These subsynaptic mecha-nisms are apparently intact in aged PDAPP mice despite alarge decrease in the number of functional synapses andthe presence of large amounts of amyloid deposits.

The results of these studies thus indicate that thehistopathological characteristics found in the PDAPPmouse have major functional consequences for the neu-ronal circuitry in the hippocampus. Demonstration that thefunctional changes are related to amyloidosis will requirethe emergence of therapeutic strategies to prevent or re-verse amyloid deposition. Successful demonstration ofpreservation or restoration of neuronal and synaptic func-

( )J. Larson et al.rBrain Research 840 1999 23–3534

tion by such an approach in these animals would providestrong impetus to application of this strategy in Alzheimer’sdisease.

Acknowledgements

ŽThis work was supported by grants from NIMH MH-. Ž .51151 and NIA AG-16508 .

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