Increased Hippocampal Excitability in the 3xTgAD Mouse Model for Alzheimer's Disease In Vivo

Abstract

Mouse Alzheimer's disease (AD) models develop age- and region-specific pathology throughout the hippocampal formation. One recently established pathological correlate is an increase in hippocampal excitability in vivo. Hippocampal pathology also produces episodic memory decline in human AD and we have shown a similar episodic deficit in 3xTg AD model mice aged 3-6 months. Here, we tested whether hippocampal synaptic dysfunction accompanies this cognitive deficit by probing dorsal CA1 and DG synaptic responses in anaesthetized, 4-6 month-old 3xTgAD mice. As our previous reports highlighted a decline in episodic performance in aged control mice, we included aged cohorts for comparison. CA1 and DG responses to low-frequency perforant path stimulation were comparable between 3xTgAD and controls at both age ranges. As expected, DG recordings in controls showed paired-pulse depression; however, paired-pulse facilitation was observed in DG and CA1 of young and old 3xTgAD mice. During stimulus trains both short-latency (presumably monosynaptic: 'direct') and long-latency (presumably polysynaptic: 're-entrant') responses were observed. Facilitation of direct responses was modest in 3xTgAD animals. However, re-entrant responses in DG and CA1 of young 3xTgAD mice developed earlier in the stimulus train and with larger amplitude when compared to controls. Old mice showed less DG paired-pulse depression and no evidence for re-entrance. In summary, DG and CA1 responses to low-frequency stimulation in all groups were comparable, suggesting no loss of synaptic connectivity in 3xTgAD mice. However, higher-frequency activation revealed complex change in synaptic excitability in DG and CA1 of 3xTgAD mice. In particular, short-term plasticity in DG and CA1 was facilitated in 3xTgAD mice, most evidently in younger animals. In addition, re-entrance was facilitated in young 3xTgAD mice. Overall, these data suggest that the episodic-like memory deficit in 3xTgAD mice could be due to the development of an abnormal hyper-excitable state in the hippocampal formation.

Full text

Increased Hippocampal Excitability in the 3xTgAD Mouse
Model for Alzheimer’s Disease
In Vivo
Katherine E. Davis, Sarah Fox, John Gigg*
Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
Abstract
Mouse Alzheimer’s disease (AD) models develop age- and region-specific pathology throughout the hippocampal
formation. One recently established pathological correlate is an increase in hippocampal excitability in vivo. Hippocampal
pathology also produces episodic memory decline in human AD and we have shown a similar episodic deficit in 3xTg AD
model mice aged 3–6 months. Here, we tested whether hippocampal synaptic dysfunction accompanies this cognitive
deficit by probing dorsal CA1 and DG synaptic responses in anaesthetized, 4–6 month-old 3xTgAD mice. As our previous
reports highlighted a decline in episodic performance in aged control mice, we included aged cohorts for comparison. CA1
and DG responses to low-frequency perforant path stimulation were comparable between 3xTgAD and controls at both age
ranges. As expected, DG recordings in controls showed paired-pulse depression; however, paired-pulse facilitation was
observed in DG and CA1 of young and old 3xTgAD mice. During stimulus trains both short-latency (presumably
monosynaptic: ‘direct’) and long-latency (presumably polysynaptic: ‘re-entrant’) responses were observed. Facilitation of
direct responses was modest in 3xTgAD animals. However, re-entrant responses in DG and CA1 of young 3xTgAD mice
developed earlier in the stimulus train and with larger amplitude when compared to controls. Old mice showed less DG
paired-pulse depression and no evidence for re-entrance. In summary, DG and CA1 responses to low-frequency stimulation
in all groups were comparable, suggesting no loss of synaptic connectivity in 3xTgAD mice. However, higher-frequency
activation revealed complex change in synaptic excitability in DG and CA1 of 3xTgAD mice. In particular, short-term
plasticity in DG and CA1 was facilitated in 3xTgAD mice, most evidently in younger animals. In addition, re-entrance was
facilitated in young 3xTgAD mice. Overall, these data suggest that the episodic-like memory deficit in 3xTgAD mice could be
due to the development of an abnormal hyper-excitable state in the hippocampal formation.
Citation: Davis KE, Fox S, Gigg J (2014) Increased Hippocampal Excitability in the 3xTgAD Mouse Model for Alzheimer’s Disease In Vivo. PLoS ONE 9(3): e91203.
doi:10.1371/journal.pone.0091203
Editor: Christian Holscher, University of Lancaster, United Kingdom
Received July 5, 2013; Accepted February 11, 2014; Published March 12, 2014
Copyright: ß2014 Davis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors are grateful for RCUK funding to KED (BBSRC PhD studentship), SF (BBSRC CASE PhD studentship) and JG (BBSRC grant BB/D011159/1;
Royal Society grant RSRG 24519). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have the following interests. JG is an in vivo electrophysiology consultant for GlaxoSmithKline Neurodegeneration, China.
This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.
* E-mail: j.gigg@manchester.ac.uk
Introduction
Alzheimer’s disease (AD) is characterised phenotypically by
profound declarative memory deficits. The hippocampus, vital for
both the formation and retrieval of declarative memory, is one of
the first areas affected by AD pathological hallmarks of amyloid-
beta (Ab), extracellular plaques and tau neurofibrillary tangles
[1,2]. Interestingly, however, overt plaque burden correlates
poorly with cognitive decline in AD patients [3]. A more recent
hypothesis is that intracellular oligomeric amyloid species, present
before the accumulation of extracellular plaque pathology, may
instead play a pivotal role in disease progression [4,5]. However,
despite extensive research examining the causes of AD pathology
and characterising behavioural phenotypes, there is still little
knowledge about the physiological basis for memory loss in
pathological states such as AD [6].
The hippocampal formation is comprised of the dentate gyrus
(DG), hippocampus proper (CA fields), subiculum (SUB), para-
subiculum, presubiculum and entorhinal cortex (EC; medial and
lateral divisions; MEC and LEC). CA1 and subiculum are the
principal output structures of the hippocampus [7] and EC
provides the interface between incoming and outgoing information
from surrounding cortex [8]. Hippocampal output can return to
surrounding neocortex via deep layers of EC and/or re-enter the
hippocampal formation via projections from deep to superficial
EC [9,10]. The latter reverberation (or ‘re-entrance’) is considered
to be pivotal in memory formation, possibly acting as a
comparator mechanism to allow processed input to be evaluated
alongside new information and/or as a memory consolidation
mechanism during sleep [7,11–13]. Selective lesions to all
hippocampal formation structures have profound effects on
memory performance [14–18]. Functional abnormalities have
also been detected in the hippocampus during memory encoding
in human AD patients [19]. It is of major interest, therefore, to
determine the pathophysiological profile of the hippocampal
formation in AD models and to elucidate, in particular, whether
reverberation (re-entrance) occurs in control and AD model mice.
The triple transgenic mouse model (3xTgAD) carries familial
AD transgenes for Amyloid Precursor Protein (APP
SWE
), Pre-
senilin-1(M146V) and an additional tauopathy mutation
(Tau
P301L
). This model develops Aband tau pathology targeted
to the hippocampus and other medial temporal lobe structures in a
manner temporally and spatially similar to human AD [20,21].
The model develops cognitive deficits at a young age [21,22] and,
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as we have demonstrated recently, a specific decline in episodic-
like memory from 3 months that becomes a complete deficit at 6
months of age [23,24]. 3xTgAD mice also show abnormalities in
basal synaptic transmission and deficits in LTP in CA1 hippo-
campus in vitro [21]. However, to date, there have been no
electrophysiological recordings in vivo in the 3xTgAD mouse and,
to our knowledge, there is only one in vivo account in another AD
model examining evoked responses [25]. Thus, the hippocampal
system in AD mouse models has largely been examined only in the
reduced brain slice preparation [21,26], that is, without the
substantial bi-directional cortical connectivity that also contributes
to memory processes [27]. In the 3xTgAD mouse, only CA3-CA1
connectivity has been examined directly in vitro [21], therefore,
there is a need to examine basic synaptic transmission more widely
in hippocampal formation and also hippocampal network function
through examining response reverberation.
One question that arises is the extent to which cognitive deficits
seen in early AD stem from abnormalities in the fibre pathways
projecting to the hippocampus and/or within hippocampal
subfields. Recent research has indicated the presence of early
myelination abnormalities in the 3xTgAD mouse Schaffer
collateral pathway, prior to the occurrence of Aband tau
pathology [28]. In human AD, tau pathology is restricted to the
stellate cells in EC layer II, and leads to degenerative changes in
the perforant path [29]. In addition, afferents from CA1 and SUB
to EC are major sites for Abpathology in human AD [16]; thus,
there is a strong possibility that information flow through the
hippocampal formation could become impaired in AD. However,
synaptic deficits first arise in AD models in vitro at an age prior to
overt extracellular plaque and tangle pathology, suggesting that
mechanisms such as intracellular Abaccumulation or abnormal-
ities in calcium homeostasis are major contributing factors to early
cognitive symptoms [21,26].
Here, we examined the functional state of the hippocampal
formation in the 3xTgAD mouse through the recording of
extracellular field potentials in vivo. Whereas most similar studies
focus on one region of hippocampus in isolation (e.g., CA1 or DG),
in this study we measured synaptic integrity and short-term
plasticity at three sites simultaneously, specifically, the granule cell
layer of DG, CA1 stratum radiatum (CA1sr) and CA1 stratum
lacunosum-moleculare (CA1slm), using a multi-site electrode
recording approach [9]. These sites were chosen as regions that
receive monosynaptic input from the perforant path and, for
CA1sr, polysynaptic feed forward input from DG via CA3. We
measured extracellular field excitatory post-synaptic potentials
(fEPSPs) in these target regions and performed current-source-
density (CSD) analyses on CA1-DG axis responses. Through this
we aimed to map synaptic current flow, indicative of synaptic-
activity throughout the laminar structure, to test the integrity of
synaptic connections within the EC-hippocampal tri-synaptic
circuit.
Electrophysiological recordings conducted in AD mouse lines in
vitro support a long-term decrease in synaptic function [21,30–32].
However, in vivo recordings support the presence of hyper-
excitability and epileptiform activity, at least in early pathological
stages, which are thought to contribute to cognitive decline
[33,34]. In addition, recent findings indicate the presence of
spontaneous seizure activity in the 3xTgAD model [35]. Thus, in
the current study, we hoped to further examine these contradic-
tory reports of decreased/increased synaptic function and test
whether our observed decline in episodic-like memory in the
3xTgAD model [23,24] correlates with excitability changes within
hippocampal circuits in vivo.
In contrast to in vitro reports, we found little evidence for
decreased functional connectivity in either young or old 3xTgAD
mice. Indeed, in measures of short-term plasticity we saw evidence
for increased DG and CA1sr excitability in 3xTgAD mice
compared to controls, particularly in young mice. This was
coupled, however, with relatively less facilitated fEPSP amplitudes
to a stimulus train alongside an enhanced re-entrant response into
the hippocampal circuit in 3xTgAD mice. Thus, our data support
increased hippocampal excitability at two loci in the 3xTgAD
mouse: firstly, EC layer II input to DG and CA1sr (the latter
presumably via propagation of DG output via CA3) in young and
aged mice; and secondly, facilitation of hippocampal re-entrance
in young 3xTgAD animals, presumably through potentiated deep-
to-superficial EC connectivity.
Materials and Methods
Animals
Triple-transgenic mice (3xTgAD) carrying APP
SWE
, PS1
M146V
and Tau
P301L
transgenes and matched controls were bred at the
University of Manchester from a colony donated by the La Ferla
group [21]. Mice were housed in same-sex and genotype groups of
5–6 individuals on a 12:12 light/dark cycle with access to food and
water ad libitum. The 3xTgAD colony was maintained through the
pairing of homozygous individuals and the presence of transgenes
was confirmed by genotyping a subset of 3xTgAD mice in each
cage. All procedures conformed to UK Home Office licensing
(project license PPL 40/3231) and were approved by the
University of Manchester Ethical Review Panel. Electrophysio-
logical data were collected from a ‘young’ sample of mice aged 4–6
months comprising 12 control (6 males: 6 female) and 11 3xTgAD
(5 males: 6 female) mice. The age of the young group was selected
to coincide with the early presence of hippocampal intracellular
Abpathology (plaques and tangles accumulate from approximate-
ly 9 months of age in 3xTgAD mice) and hippocampal-dependent
memory deficits [21–24]. A second ‘old’ sample of mice at 17–18
months of age comprised 5 control and 4 3xTgAD mice (all
female); at this age there is extracellular plaque and tau pathology
within hippocampus and subiculum. Thus, these groups allowed
us to explore the additive effect of both pathology and general
ageing on DG and CA1 responsiveness [20–22]. All female mice
had previously experienced a battery of behavioural testing
(spontaneous recognition tasks) which ceased at least a month
before electrophysiological recordings were conducted. Male mice
used for electrophysiology were experimentally naive. In the
present experiments data from young male and female mice were
combined as no sex differences were seen in either their basal
input/output response, or in their CSD profiles to a 5 Hz train
(data not shown).
Surgery and equipment
Mice were anaesthetised (urethane 30% w/v in dH
2
O, 1.6 g/
kg, i.p) and monitored until areflexia was achieved. Where reflexes
remained, a small additional dose of urethane (10% w/v in dH
2
O,
20–30 ml, i.p.) was given. The mouse was held in a stereotaxic
frame (Kopf, 1430, USA) with lamda and bregma in the same
horizontal plane to match the mouse atlas of Paxinos and Franklin
[36]. A homoeothermic heating blanket (Harvard Apparatus, UK)
and rectal probe maintained core body temperature at 37uC. A
midline scalp incision was made and the skull exposed. Craniot-
omies were drilled above CA1 (B–2 mm, ML 1.5 mm) for the
recording electrode and dorsal subiculum (B-3.85 mm, ML
1.5 mm) for the stimulating electrode. Recordings were made
using a linear multi-electrode recording array containing either 16
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contacts spaced100 mm, or 32 contacts spaced 50 mm apart (both
413 mm
2
contact area; NeuroNexus Tech, USA; there were no
clear differences in response profiles between the different contact
arrangements so data from different arrays were pooled.). The
array was lowered vertically into the brain until the tip was
approximately 2.5 mm below brain surface; at this depth
recording contacts spanned the CA1-DG axis fully ([9]; see
Figure 1A). A bipolar stimulating electrode (twisted 125 mm
diameter Teflon-insulated stainless-steel wires; Advent RM, UK)
was inserted 1.5 mm from brain surface at 30urelative to vertical
into the molecular layer of the mid antero-posterior part of the
subiculum (Figure 1A). At this position, stimulus pulses would
activate the hippocampus both directly (via activation of perforant
path (PP) fibres traversing subiculum) and indirectly (via re-
entrance through subicular output to deep layers of entorhinal
cortex which then activate superficial entorhinal PP output [9,10].
It was expected that upon stimulation, a characteristic pattern of
current sources and sinks would be identifiable across the CA1-
DG axis similar to those seen in the rat, reflecting the termination
pattern of PP inputs to DG and CA1 ([9]; see Figure 1B for typical
evoked laminar response and C for hippocampal circuit diagram).
Stimulation Protocols
Stimulus pulses were timed using a National Instruments card
(PCI-6071E, NI, UK) controlled using LabVIEW software (8.2,
NI, UK). These triggered a constant-current source (DS3,
Digitimer, UK) connected to the stimulating electrode. Stimulus
duration was set to 0.2 ms throughout the experiments. Stimulus
protocols consisted of single pulses (0.33 Hz), paired pulses
(PPulse) and trains of 20 pulses.
We first confirmed that the recording electrode spanned CA1sr,
CA1slm and the superior and inferior blades of DG. This was
achieved by monitoring the presence of stable evoked local field
potentials (LFPs) in these regions to single-pulse stimulation at
200 mA [9,10]. In all experiments, a characteristic DG molecular
layer positive-going LFP component was recorded with, in most
cases, a granule cell layer population spike (PS; latter visible with
high intensity PPulse or train stimulation; Figure 1B point C). The
CA1slm response (presumably from activation of direct EC layer
III input) was not seen in all experiments, likely due to variations
in the placement of recording/stimulating electrodes (Figure 1B
point B). CA1sr was characterised by a longer-latency negative-
going LFP (Figure 1B point A). Due to the fixed distance between
contacts in the recording array, upon identification of a typical
response in one layer, those in other layers could reliably be
confirmed due to the distance between contacts and LFP profile
(CA1sr 100 microns dorsal to CA1slm, CA1slm approximately
100 microns dorsal to the largest DG molecular layer response; see
Figure 1B for illustration of spacing).
Once a characteristic laminar LFP profile was achieved, a
current-voltage (input-output) response curve was recorded for
each LFP component by applying pairs of pulses at 50 ms intervals
over a range of current intensities (50–600 mA). The first pulse of
each pair was analysed and used to plot the curve. Thereafter, for
PPulse and train protocols, the stimulus current was set to the
value required to evoke a half-maximal response (i.e., 50% of the
value required to elicit the maximum fEPSP in CA1sr); this was
typically in the range 100–200 mA.
To examine short-term plasticity, PPulses were delivered at
inter-pulse-intervals (PPI) of 25, 50, 100, 200, 500 and 1000 ms.
All pairs were separated by 3 seconds and repeated 20 times. To
examine the effects of repetitive stimulation and re-entrance into
the hippocampal circuit, a single train of 20 pulses was applied at a
frequency of 5 Hz. This frequency was chosen to provide a
comparison with PPulse intervals and we predicted that 5 Hz
trains would not produce any long-term plastic changes [9,10].
Upon completion of all stimulation protocols, lesions were
created to mark electrode placements displaying particular
response components [37]. Mice were perfused transcardially
with 0.2 M sodium phosphate buffer and 4% paraformaldehyde
and brains removed for fixation. Electrode placements were
confirmed from 30 mm thick, Nissl-stained sagittal brain sections.
Data Acquisition and Analysis
Signals were amplified at source through an AC-coupled
headstage (x20 gain) and further amplified for a total gain of
500x (Recorder64 system, Plexon, USA). Signals were filtered
(0.1 Hz–6 kHz) and sampled at 10 kHz per channel (12-bit
resolution).
Raw fEPSP response amplitudes to single-pulse stimulation
were plotted to describe the current-response relationship (input-
output; I/O) in control and 3xTgAD mice. In addition, PPulse
and train data were normalised to the first pulse and results shown
as a percentage change for both amplitude and latency to peak/
trough to demonstrate any facilitation or depression of responses.
Finally, train data were subjected to 1D CSD analysis [38] using
custom Matlab software (see below).
All numerical data were analysed using Prism (v5; Graphpad).
Mixed ANOVA with Bonferroni post hoc comparisons were
applied for identification of pair-wise genotype differences for each
of the three measures (I/O, PPulse and trains) for each response
(CA1sr, CA1slm and DG). Due to sex differences in the number of
animals present in each sample, young and old data sets were
analysed separately to control for the small female-only older
mouse group; however, young male and female data were then
pooled as no sex difference was seen for any measure (data not
shown). If recordings became unstable during an experiment,
stimulation protocol data were excluded for that animal. Final
sample sizes for each hippocampal layer in each stimulation
protocol are shown in Table 1.
Amplitude, Slope and Latency measurements
Amplitude and the latency of evoked responses were extracted
through custom Matlab software (Mathworks, version 7.0). In
brief, each response was first visually inspected to ensure it had the
expected shape with no noise or other artefacts. Five cursor points
were then marked on different components of a mean response
(see below) averaged over the 20 stimulus-response repeats.
Thereafter, the programme used this five-point template to
calculate the amplitude and latency of each original response.
For train data, the waveform of each pulse was measured ‘by
hand’ to account for the rapid changes in response profile
associated with repetitive stimulation. In both cases, five points on
each response were marked as follows: point 1 as the sample point
before the start of the stimulus artefact; points 2 and 3 surrounded
the response onset and points 5 and 6 surrounded the response
peak (or trough).
For response amplitude (mV), the mean value between points
2–3 was subtracted from the corresponding maximal (response
peak) or minimal (response trough) value between points 4 and 5.
For latency to response peak/trough (ms), the centre timestamp
between points 4 and 5 was subtracted from that for point 1. We
did not attempt to measure latency to response onset, as there was
seldom a defined level period in the recording between the
stimulus artefact and response. All data presented in this paper are
the average of 20 pulses. For DG population spikes (PS), the
probability of evoking a PS was calculated out of 20 sweeps for
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Figure 1. The hippocampal tri-synaptic circuit. A: Hippocampal formation with approximate electrode placements marked. Input and output
pathways with principal direction of synaptic flow (arrows) and termination of fibre pathways from Schaffer collaterals [A], PP layer III [B] and PP layer
II [C] are marked. Evoked activity at these points corresponds to CA1sr [A], CA1slm [B] and DG responses [C]. Stimulating electrode placement was in
the deep dendritic layer of subiculum (S). B: Typical laminar field response to single-pulse stimulation at half-maximal current intensity. A, B and C
correspond to responses recorded within regions A–F in Figure 3. C: Block diagram of the connectivity displayed in [A] for reader’s clarity. Dashed
lines represent the presence of sparse regional connectivity. Calibration applies to both profiles in B.
doi:10.1371/journal.pone.0091203.g001
Table 1. Summary of data included in statistical analyses.
Young (4–6 months)
Input/Output Paired Pulse Trains
Control 3xTgAD Control 3xTgAD Control 3xTgAD
CA1sr n = 12 n = 11 n = 12 n = 10 n = 10 n = 9
CA1slm n = 12 n = 11 n = 12 n = 10 n = 7 n = 8
DG n=12 n=11 n=12 n=10 n=11 n=9
Old (17–18 months)
Input/Output Paired Pulse Trains
Control 3xTgAD Control 3xTgAD Control 3xTgAD
CA1sr n=5 n=4 n=5 n=4 n=5 n=3
CA1slm n = 5 n = 4 n = 5 n = 4 n = 5 n = 3
DG n=5 n=4 n=5 n=4 n=5 n=3
doi:10.1371/journal.pone.0091203.t001
Hippocampal Hyperexcitability in 3xTgAD Mouse
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each PPI in animals that demonstrated PS at half-maximum
stimulation.
Current Source Density Analysis
Current Source Density (CSD) analysis when applied to
extracellular recordings taken from a laminar structure provides
a highly detailed, anatomically aligned, spatiotemporal map of
current sinks and sources produced through synaptic activity.
Single responses from train data were analysed by one-
dimensional CSD analysis in a custom built Matlab programme,
estimating the second-order spatial derivative using the formula of
Freeman and Nicholson [38]. For this the distance between
electrodes was either 100 or 50 mm and the degree of spatial
smoothing applied was 2 as we analysed neighbouring contacts. It
was assumed that (a) the major extracellular currents ran in
parallel to the recording electrode (along the CA1-DG axis) and (b)
tissue conductivity was spatially homogenous across the recording
array (that is, any residual currents would represent local synaptic
sinks and sources). CSD values at locations between those
calculated for each electrode position were estimated by linear
interpolation (20 steps) to produce a smooth and continuous
mapped depiction of synaptic currents. Mapped CSD values are
presented here in arbitrary units: current sources were mapped as
reds, oranges and yellows; current neutral regions in light green;
and regions showing current sinks as dark green to dark blue. The
colour map for each CSD plot was normalised to itself (max/min)
to represent the current gradient running from its maximum
positive current (hot colours), to the maximum negative current
(cool colours) with green equivalent to resting state or neutral.
Results
Current-response relationship
To examine the integrity of CA1 sr/slm-DG hippocampal
circuitry, amplitude and latency of responses to activation of PP
fibres were determined over a range of current intensities. There
were no significant differences in amplitude measurements
between control and 3xTgAD animals for CA1sr, CA1slm or
DG responses in either young or old mice (Figure 2A–F; genotype
(2) by PPI (5) mixed ANOVA). However, there was a significant
effect of current intensity on all response amplitudes in all animals
(F (4,28) = 5.74–23.06 P,0.0001), with higher currents eliciting
larger responses as would be expected by recruitment of PP fibres
with increasing current. In addition, DG population spikes
developed from 200 mA stimulus current onwards in 5/12 control
and 8/11 3xTgAD young mice (data not shown). In old mice, two
of the four 3xTgAD animals developed DG PS at high current
intensities; however, PS were not seen in old control mice (data not
shown). While these observations in DG PS frequency are merely
qualitative for the purpose responses to single stimuli, their
occurrence was strain-dependent in 3xTgADs and controls during
PPulse stimulation, as described below.
To investigate differences in response latency between 3xTgAd
and control mice we compared responses from each hippocampal
layer at a stimulus intensity of 200 mA (typical half-maximal
stimulation current; see Table 2). The order of latencies (to
response peak or trough) was consistent with our expectations for
the associated synaptic delays in vivo of CA1sr, CA1slm and DG in
response to PP stimulation as used here (i.e., fastest for DG and
CA1slm; refer to Figure 1C) and no genotype differences were
found (data not shown). Thus, from the similar I/O amplitude and
relative response latency patterns seen with this stimulus protocol,
we concluded that there were no genotype differences in
functional connectivity during low-frequency activation in the
PP fibre pathways from EC layers II and III traversing subiculum
to the hippocampal circuit in both young and old animals.
Short-term synaptic plasticity
To assess short-term synaptic plasticity, we examined the effect
of varying PPI on the amplitude and latency of responses to half-
maximal stimulus current (range for all mice 100–200 mA).
Measurements from the second pulse were normalised to those
from the first and are presented as percentage change, such that
positive values represent paired-pulse facilitation (or a decreased
latency) and negative values represent paired-pulse depression (or
an increased latency). CA1sr, CA1slm and DG responses were
analysed in separate genotype (2) by PPI (6) mixed ANOVA with
Bonferroni post-hoc comparisons.
For young animals, there was a significant effect of PPI on the
amplitude of CA1sr and CA1slm responses (F(5,100) = 7.32, P,
0.0001 and F(5,100) = 4.93, P,0.0005, respectively) with maximal
facilitation seen at 25 and 50 ms intervals. While there were no
significant genotype differences for CA1slm responses (data not
shown), for CA1sr there was an interaction (F(5,100) = 2.54, P,
0.05) and a pair-wise genotype difference at 50 ms (t(20) = 3.27,
P,0.01; see Figure 3A). This was evident as 3xTgAD responses
facilitating significantly more than that for controls, in stark
contrast to the depression of CA1sr responses frequently seen in
AD models in vitro during LTP protocols [21,31,32]. For the DG
response, there was again a significant genotype difference (F
(1,100) = 11.80, P,0.005) and an interaction (F(5,100) = 4.88,
P,0.001). Furthermore, there were pair-wise genotype differences
at 25 ms (t(20) = 5.12, P,0.001) and 50 ms (t(20) = 3.54, P,0.01),
where response amplitudes facilitated in 3xTgAD mice and
depressed in controls (Figure 3B), suggestive of DG hyper-
excitability in this AD model. As expected, population spikes to
the second pulse (P2) were seen in the DG granule cell layer in 4/
10 3xTgAD and 6/12 controls during half-maximal amplitude PP
stimulation. However, 3xTgAD mice had a higher tendency to
exhibit PS at shorter PPI (25 ms–100 ms), whereas for control
animals this relationship was reversed (most PS in 200 ms–
1000 ms PP interval range; examples of paired-pulse responses at
50 ms and 200 ms PPI left panel Figure 3C, probability
distribution across PPI for PS to P2 displayed in right panel
Figure 3C).
To determine whether genotype differences in the velocity of
synaptic transmission could have contributed to observed short-
term amplitude differences, response latencies were examined for
each hippocampal layer in response to pairs of pulses (latency of
second pulse to peak/trough relative to the first). There was a
significant effect of PPI on latency for CA1sr (F(5,100) = 2.63, P,
0.05) with PPI 25 ms eliciting a slightly faster response for both
genotypes (3xTgAD 14.99%63.17 SEM faster and control
5.82%61.7 SEM faster than the first pulse). For DG, a significant
effect of PPI was also seen (F(5,100) = 7.41,P,0.001), with PPI
100 ms and 200 ms eliciting a slightly slower response in both
genotypes (data not shown); however, no effects of latency were
observed in CA1slm. Thus, despite a significant effect of PPI on
the time to peak/trough of the second pulse, there were no obvious
effects of AD pathology. To summarise the data from young
animals, we found an indication of increased short-term excitabil-
ity in 3xTgAD mice in the form of increased LFP amplitude in
CA1sr, and with DG facilitation to short interval delays; however,
there was no apparent change in overt hippocampal synaptic
connectivity as reflected in the absolute latencies of CA1sr and DG
molecular layer LFPs.
For old animals there was a significant effect of PPI on response
amplitude for CA1sr (F(5,35) = 2.54, P,0.05) and a significant
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interaction (F(5,35) = 5.94, P,0.0005). This manifested as a
significant pair-wise genotype difference at PPI 50 ms (t(7)
= 5.65,P,0.0001; Figure 4A) where 3xTgAD animals again
facilitated more than controls. There were no significant effects
for CA1slm; however, there was an interaction for DG (F(5,35)
= 3.46,P,0.05) and a pair-wise genotype difference at PPI 25 ms
(t(7) = 3.08), P,0.05; Figure 4B), where again 3xTgAD responses
facilitated in contrast to control responses which depressed. Of
note, 3xTgAD DG responses tended to depress more strongly than
controls at PPI of 200 ms or longer, however, these differences did
not reach significance. Also of interest, control DG appeared to
show little if any modulatory changes across the range of PPIs,
neither depressing nor facilitating (compare Figure 3B with
Figure 4B). We assume these were age-related changes in DG
function, as they present a different profile to that of aged 3xTgAD
animals. The implication of this reduction in paired-pulse
depression for DG with normal ageing is that it may be causative
in the known age-related impairment in pattern separation that
may, in turn, contribute to our observed age-related deficit in
episodic-like memory in older control mice ([23]; see Discussion).
For latency to peak/trough, there was a significant effect of PPI
for CA1sr (F(5,35) = 2.69, P,0.05), an interaction (F(5,35) = 4.23,
P,0.005) and a pair-wise genotype difference at PPI 50 ms
(t(7) = 3.99, P,0.05). In this case, 3xTgAD mice had a signifi-
cantly longer latency relative to P1, whereas control mice had a
slightly shorter latency (3xTgAD change 59.42% 627.83 SEM
slower, control 8.08%63.49 SEM faster). This difference can be
explained by the larger CA1sr amplitudes in 3xTgAD mice
compared to controls (3xTgAD responses have a longer period
from stimulus to response trough). For CA1slm, there was a
significant interaction (F(5,35) = 4.35, P,0.01) and a pair-wise
genotype difference at PPI 50 ms (t(7) = 3.01,P,0.05) with
3xTgAD mice showing a faster P2 response than control mice
(3xTgAD 10.79%64.28 SEM faster, control 1.47%65.26 SEM
slower). For DG, there was an effect of PPI only (F(5,35) = 3.02,
P,0.05). In these old animals, only one 3xTgAD mouse showed
evidence of a DG PS and, as per the young animals, this was more
prevalent at shorter PPI (PP25ms and PP50ms; data not shown).
Thus, as per the young data, it appeared that old 3xTgAD mice
demonstrate increased short-term plasticity and excitability in
CA1sr and DG, albeit to a smaller degree when compared to
younger mice.
To summarise the findings of the PPulse protocol, both young
and old animals showed response facilitation to PPulse stimulation
at short intervals in CA1sr; however, excitability in terms of
increases in DG PS to short PPI was most pronounced at a young
age. 3xTgAD mice had a significantly larger CA1sr response than
controls and this was seen in both young and old age groups,
suggesting a specific change at the CA1 level could alter
excitability in this region. In addition, 3xTgAD DG responses at
a short PPI were facilitated relative to controls (which depressed in
young controls), indicating changes in the DG synaptic network.
The DG paired-pulse depression in young controls appeared to be
absent in old controls, suggesting an ageing-related shift in DG
Figure 2. Input-output relationships in young and old 3xTgAD mice are similar to controls. A to F: Stimulus-response (I/O) curves in
young (4–6 month) and old (17–18 months) animals in relation to increasing input current intensity at CA1sr (A and D), CA1slm (B and E) and DG (C
and F). Error bars are (6SEM).
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function; this attenuation may have mechanistic implications for
age-related deficits in the pattern separation role carried out by
DG and episodic-like memory capacity in older mice.
Train stimulation
To examine neuronal reverberation within the hippocampal
formation, mice experienced low-frequency stimulation in the
form of a 20-pulse train, delivered at 5 Hz. Train data were
analysed in genotype (2) by pulse number (20) Mixed ANOVA as
follows: raw amplitudes and peak/trough latencies were examined
for each response (results labelled as ‘raw’) and data were also
converted to a percentage change from P1, as a measure of the
summated effect of train stimulation (labelled as ‘% change’). Data
from young and old mice were also subjected to CSD analyses to
identify the spatiotemporal relationships between current sources
and sinks along the CA1-DG axis and to shed light on the origins
and relative strength of the synaptic inputs to different laminae.
5 Hz Train analysis: Young animals at 4–6 months of age
For young animals, there was a significant effect of pulse
number on the amplitude for all responses; however, for latency,
there was a significant effect of pulse number only for CA1slm and
DG responses and a genotype difference for CA1sr.
CA1sr (n = 10 control, n = 9 3xTgAD) showed a significant
effect of pulse number on amplitude (F(19, 323) = 5.94, P,0.0001
raw; F(19,323) = 6.18, P,0.0001%change; Figure 5A), that is,
subsequent pulses facilitated with respect to pulse 1. However,
despite the tendency for 3xTgAD mice to show less facilitation
throughout the train this genotype difference did not reach
significance. There was also no effect of pulse number on response
latency, however, there was a significant genotype difference
(F(1,323) = 11.01, P,0.005%change) and an interaction
(F(19,323) = 1.63, P,0.05), with Bonferroni pair-wise differences
at pulses 5–8 and 13 (t(17) = 3.071–3.725, P,0.05/P,0.01;
Figure 5B). In this case, 3xTgAD response latencies remained
the same or were slightly shorter compared to that of the first
pulse, whereas control latencies increased.
CA1slm response amplitudes (n = 7 control, n = 8 3xTgAD)
showed a significant effect of pulse number only (F(19, 247) = 5.96,
P,0.0001 raw; F(19, 247) = 6.07, P,0.0001, %change) as did
CA1slm latencies (F(19,247) = 3.37, P,0.0001 raw; F(19,
247) = 1.84, P,0.05%change) with progressive pulses evoking a
larger and slightly longer latency response (data not shown). There
were no genotype differences.
DG response amplitudes (n = 11 control, n = 9 3xTgAD)
showed a significant effect of pulse number (F(19,342) = 3.5, P,
0.0001 raw; F(19,343) = 10.36, P,0.0001%change) as did re-
sponse latencies (F(19, 342) = 3.69, P,0.0001 raw; F(19, 342) = 8,
P,0.0001), with subsequent responses depressing slightly and
latencies increasing compared to the first response. There were no
genotype differences for either response amplitude or latency (data
not shown).
In summary, there was a robust effect of pulse number on both
the amplitude and latencies of responses. We found no significant
evidence for genotype differences in the absolute amplitude of
CA1 and DG responses; however, CA1sr clearly demonstrated a
trend for poorer facilitation in 3xTgAD animals with an
accompanying genotype difference in CA1sr latency in 3xTgAD
mice.
Re-entrance into CA1 and DG: young mice
Half of the young mice from each genotype were classified as
‘excitable’ (5/11 control and 5/9 3xTgAD), that is, they showed
increasing levels of DG population spiking for subsequent pulses in
Table 2. Response latencies in hippocampal formation.
Control 4–6 mo 3xTgAD 4–6 mo Control 17–18 mo 3xTgAD 17–18 mo
Latency SEM n Latency SEM n Latency SEM n Latency SEM n
CA1sr 7.84 0.56 12 7.99 0.54 11 9.79 1.21 5 9.25 1.64 4
CA1slm 6.01 0.49 12 5.26 0.42 11 7.30 1.21 5 6.73 0.82 4
DG 8.03 0.65 12 7.46 0.36 11 6.69 2.24 5 7.81 1.26 4
Figures show peak response latencies (ms) in response to subiculum stimulation at 200 mA.
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the train and, in some cases, re-entrance into the hippocampal
circuit in both CA1 and DG (Figure 5 panels C and D; see arrows).
This is better illustrated using 1-dimensional CSD analyses, where
the evoked synaptic currents in each layer of the hippocampus can
be computed and visualised over time (see next section). All
animals that showed re-entrance did so by pulse 20: within the 5
excitable 3xTgAD animals, 3 showed clear re-entrance into CA1sr
(at pulses 10, 17 and 20; 15.7160.33 ms; mean latency 6SEM), 4
showed re-entrance into CA1slm (at pluses 15, 18 and twice by 20;
14.7160.12 ms) and 3 exhibited re-entrance into DG (at pulses 7,
15 and 20 pulses; 20.6961.98; Figure 5D). Re-entrance latencies
were similar in the 5 excitable control mice; CA1sr (n = 4;
17.561.5 ms) and CA1slm (n = 3; 15.4962.08 ms), whereas DG
had a shorter latency re-entrance wave (n = 2; 16.3262.22 ms).
However, in contrast to 3xTgAD mice, all re-entrance was seen at
pulse 20 of the train and not earlier in the sequence. It appears,
therefore, that young control and 3xTgAD mice (of 4–6 months of
age) can show self-sustained reverberation through the hippocam-
pal formation following repetitive low-frequency stimulation; in
addition, these responses tend to occur in 3xTgAD mice at an
earlier stage in the pulse sequence.
Current Source Density analysis: young mice
Stimulation of the PP with a 5 Hz frequency train elicited a
profile of current sources and sinks consistent with an electrode
traversing the CA1-DG axis, with positioning parallel to the
dendrites of pyramidal cells in CA1 and spanning DG [9]. Typical
CSD responses in and their layers of origin are outlined in
Figure 6A (control mouse response to the 5
th
pulse).
The CSD responses for control and 3xTgAD animals can be
viewed for pulses 1, 5, 10 and 20 in Figure 6 C and D. To
orientate the reader to the appearance of each synaptic
component, the CSD responses to pulse 1 are referred to using
the example in Figure 6A. In response to pulse 1, both 3xTgAD
and control animals show an early latency current sink in CA1slm
(Figure 6A: component 1) consistent with an excitatory synaptic
response to activation of perforant path (EC layer III tempero-
ammonic) terminals on the apical dendrites of CA1 pyramidal
cells. Simultaneous with this, a sink in the molecular layer of the
inferior blade of DG is seen (Figure 6A: component 2) and an
accompanying current source in the granule cell layer (Figure 6A:
component 3). For this control mouse, a further small current
source is seen in the CA1 pyramidal cell layer, perhaps
representing the passive source of a CA1slm sink-source pair
(Figure 6A: component 4). In both genotypes a weak sink is visible
in CA1 stratum oriens, consistent with synaptic delay from the DG
mossy fibres to CA3 and output of CA3 Schaffer collaterals onto
basal CA1 dendrites (Figure 6A: component 5). These CSD
patterns were typical of first pulse responses for all animals,
including those classified as ‘excitable’ (DG PS and/or re-
entrance) and in those that did not show any evidence of PS in
DG or CA1.
Figure 3. Increased short-term plasticity in young 3xTgAD mice. Panels A and B show fEPSP amplitude of pulse 2 normalised to pulse 1
(6SEM) for CA1sr [A], and DG [B]. No differences were observed for CA1slm (data not shown). Inserts show representative traces from single
experiments with pulse 1 (black line) and pulse 2 (grey line) taken from PP50ms in all examples. Pair-wise genotype differences are evident (**P,0.01;
***P,0.0001). Calibration 12.5 ms/2 mV. C: Left panel shows examples of population spikes (PS) evoked at 50 or 200 ms in control and 3xTgAD mice
(average of 20 sweeps each; calibration 10 ms/3 mV). Right panel shows the probability of evoking a PS to the second pulse of a stimulus pair versus
paired-pulse interval. Note that the PS probability is highest at short intervals for 3xTgAD and longer intervals for controls. Error bars are (6SEM).
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In response to pulse 5, a clear PS could be seen in the DG of the
example 3xTgAD animal and this DG population spike also
begins to emerge in the control animal (Figure 6, pulse 5 upward
arrow). Visible in the control mouse, there was also the
appearance of a slightly longer component extending the existing
CA1slm sink, perhaps within the region of CA1 where CA3
Schaffer collaterals synapse onto CA1sr (also visible in Figure 6C
pulses 10 and 20). It is likely that this response was also present in
the selected 3xTgAD mouse; however, the sink was likely obscured
by the large DG PS.
In response to pulse 10, long-latency re-entrance (presumably
transferring through EC) in the 3xTgAD animal is seen in the
form of a sink-source pair in CA1 cell layer at around 15 ms
latency (Fig. 6D: component 6). There is still a clear early PS in
DG, and there is a long-latency DG source, perhaps resulting from
the inferior blade cell layer.
By pulse 20, the example control animal is also showing a clear
long-latency CA1 current source; the CA1 response being
accompanied by a population spike in the 3xTgAD example
(Figure 6D upper black arrow). In the 3xTgAD profile the early
DG cell layer response is curtailed, by a presumed inhibitory after-
potential (Figure 6D: IAP); however, there is a clear re-entrant
population spike in the inferior blade of DG (Figure 6D lower
arrow). Figure 6B shows pulse 20 for the 3xTgAD animal in more
detail, where the re-entrance of the CA1 and DG response is
clearly reflected in the overlaid LFPs. Thus, the 3xTgAD animal
appears to demonstrate increased excitability in DG, as indicated
by a larger early response to activation of the PP.
To summarize our CSD findings in young animals, we saw a
pattern of sources and sinks revealed by CSD analyses that were
consistent across controls and 3xTgAD mice with the expected
connectivity of the layer II and III EC inputs to the hippocampal
formation. In addition, comparative excitability was increased in
DG and CA1 of 3xTgADs to both the direct effect of PP activation
and indirect, delayed re-entrant input, presumably also arriving
via PP afferents.
5 Hz train analysis: Old animals of 17–18 months of age
For old animals, there were no significant effects on the
amplitude or latency for any response (when analysed as
percentage change, i.e., normalized values); however, it is likely
that some effects could have been masked due to large variances in
the control sample (n = 5 control, n = 3 AD). Nevertheless,
analysing ‘raw’ response amplitudes did show genotype differenc-
es. In detail;
CA1sr response amplitudes showed a significant effect of
genotype for raw amplitude (F(1,114) = 6.96, P,0.05), with
3xTgAD animals showing little fEPSP facilitation following the
first stimulus pulse compared to controls (Figure 7A). There was a
trend for controls to show facilitation over the course of the train
but this effect did not reach significance. For pulse 20, the
3xTgAD fEPSP (early PP-evoked response) peaked at
9.8761.64 ms (n = 3) and for control animals at 8.6361.45 ms
(n =5); however, no long latency, re-entrant responses were seen in
either group.
CA1slm responses showed no significant genotype differences
for either fEPSP amplitude or latency (data not shown), however,
there was again a trend for CA1slm amplitude to remain steady
over the train in 3xTgAD mice (Figure 7B). Again, responses in
controls appeared to facilitate over the course of the train but this
group difference could have been masked by the large variance of
the control data set. The CA1slm response to pulse 20 peaked at
6.6962.75 ms (n = 4) in 3xTgAD and at 7.7161.26 ms (n = 5) in
control mice; however, no long-latency re-entrant responses were
seen.
DG responses showed a significant effect of pulse number on
response amplitude (F(19, 114) = 4.20, P,0.0001 raw; F(19, 114)
= 4.67, P,0.0001% change) as pulses depressed over each
subsequent trial; however, in contrast to young animals, there
was little evidence of DG PS activity (except for one 3xTgAD
animal: see Figure 7D). 3xTgAD early responses peaked faster
than those for control animals (5.7661.17 ms (n = 3) versus
7.4761.95 (n = 5) respectively); however, again there was a large
within group variance, perhaps due to an effect of age and sample
size. Also, as there were no long-latency DG responses this again
supports a lack of re-entrance in all old mice.
Laminar response profiles during pulse trains are shown in
Figure 7 panels C (control) and D (3xTgAD). In contrast to the
traces seen in young animals (Figure 5 panels C and D), very little
facilitation was seen in any cell layer. Thus, in summary, the
latencies to the initial response peak/trough in old animals were
comparable, if slightly longer (c.1ms) than those in young animals;
however, no long-latency response components were seen in CA1
and DG of old mice at delays that would be associated with
Figure 4. Increased short-term plasticity in old 3xTgAD mice.
Panels A and B as per Fig 3 for CA1sr [A] and DG [B]. Pairwise genotype
differences are *P,0.05; ***P,0.0001. Note lack of modulatory effect of
PPI in controls for DG.
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hippocampal re-entrance in younger animals. The lack of an effect
of pulse number in CA1 responses, and of PS activity in animals of
both genotypes, likely reflects age-related changes in excitability.
Current source density analysis: old animals
Mice aged 17–18 months demonstrated a pattern of synaptic
current sources and sinks consistent with activation of the EC PP
from layers II and III. This profile was similar to that seen in
young animals. However, whereas young 3xTgAD and control
mice demonstrated increasing excitability over a pulse train, the
CSD profiles of old animals remained largely unchanged (Figure 7
panels E and F). Both genotypes showed what appeared to be an
inhibitory after-potential in the DG granule cell layer following the
source seen in the main DG response (Figure 7 panels E and F).
Control mice showed a large sink in CA1 stratum oriens (smaller
in 3xTgAD) at a slightly longer latency than the CA1slm response,
consistent with a synaptic delay from the CA3 Schaffer collaterals.
In the example shown (Figure 7F), the 3xTgAD animal showed a
current source in CA1sr/stratum pyramidale.
To summarize the results from aged animals, CSD analyses
revealed that the expected synaptic components reflecting PP
inputs to hippocampus were present in animals of both genotypes;
however, progressive changes in excitability to 5 Hz stimulation
were not observed. There was a significant effect of genotype on
the absolute response amplitude evoked in CA1sr and a clear
trend for a similar lack of facilitation in CA1slm (although, not
significant). Thus, we found evidence for decreased short-term
potentiation in the form of train responses in old 3xTgAD mice,
above the general decline of amplitude seen in control mice in
normal aging.
Discussion
Alzheimer’s disease (AD) is characterised phenotypically by a
loss of episodic memory and pathologically by the stereotypical
accumulation of Aband tau protein. We have shown previously
that the 3xTg murine model for AD exhibits a selective deficit in
hippocampal-dependent, episodic-like memory at 6 months of age
with intact but declining performance at 3 months [23,24]. At six
months of age, 3xTgAD mice show pronounced intracellular
accumulation of Ab[20,21], strongly suggesting a causal role for
this AD pathology in our observed episodic deficits. In the present
study, we sought to examine whether this episodic-like memory
deficit is accompanied by changes in hippocampal synaptic
function through in vivo electrophysiological recordings in 3xTgAD
and control mice. A positive outcome would provide strong
evidence for a neural correlate of memory loss in early AD or
MCI. To this end, we examined synaptic responsiveness to single
pulses of increasing current intensity and short-term synaptic
plasticity through PPF and circuit reverberation (re-entrance) in
Figure 5. Changes in response latency and occurrence of hippocampal re-entrance during 5 Hz stimulus trains in young 3xTgAD
animals. A & B: fEPSP amplitude [A] and latency [B] for CA1sr response, expressed as a percentage change from the response to pulse 1. Pair-wise
genotype differences at *P,0.05 and **P,0.01. C & D: Example to show differential fEPSP responses to selected stimuli of a 5 Hz train in control [C]
and 3xTgAD [D] mice classified as ‘excitable’. Responses in ‘non-excitable’ animals during 5 Hz trains remained as per the response to pulse 1. Re-
entrance (longer latency responses, likely propagating through EC) are shown with upward pointing black arrows for pulse 20 in both genotypes and,
in this instance, by pulse 10 in [D]. Calibration bar [C,D] 2 mV by 10 ms.
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response to train stimulation in 3 regions of the hippocampal
circuit: CA1 stratum radiatum, CA1 stratum lacunosum-molecu-
lare and DG.
Synaptic connectivity and the ‘tri-synaptic’ pathway
We found no significant genotype difference in the amplitude of
evoked responses to increasing input currents in 3xTgAD and age-
matched control mice at 4–6 or 17–18 months of age. This is in
contrast to previous in vitro reports in the same model and the
APP
SWE
mouse suggesting a depression in the input/output curve
upon the accumulation of intracellular Ab[21,31] but agrees with
other findings, showing no basal response change [30]. The
discrepancies between reports could be due to methodological
differences (in vitro slice recordings versus in vivo recordings in the
present study) and/or stimulation protocol. More specifically, in
the research cited above, single-pulse responses were examined
through direct stimulation of a monosynaptic pathway (Schaffer
collateral input to CA1 or PP input to DG [21,30,31]). In the
current study, responses were generated through stimulation
within the molecular layer of the subiculum, creating several
discrete responses: (1) an early mono-synaptic (‘direct’) response in
DG and CA1slm through activation of en passage PP fibres (the EC
layer II and III inputs to DG and CA1slm, respectively); (2) di-/tri-
synaptic responses in CA1sr and CA1so (presumably via
propagation through CA3); and (3) a longer-latency polysynaptic
(‘indirect’) response in DG and CA1. The latter represents ‘re-
entrance’ of synaptic activity, shown in the rat to depend on
activation of subicular/CA1 output that targets deep layers of EC,
from where recruitment of deep-to-superficial connectivity within
EC then allows ‘re-entrance’ of evoked activity into the DG and
hippocampus via the PP [9,10]. This re-entrant response is most
apparent during repetitive stimulation, suggesting that it requires
substantial temporal synaptic facilitation within EC. Thus, in our
in vivo 3xTgAD model, the combination of activating direct and
Figure 6. Laminar CSD profiles for responses to 5 Hz train stimulation in young mice. A: CSD from pulse 10 of control mouse with
approximate layer boundaries indicated (SO-stratum oriens, SP-stratum pyramidale, SR-stratum radiatum, SLM-stratum lacunosum-moleculare and
DG-Dentate gyrus and Alveus). Left side of all CSD panels represents point of stimulus onset. Current sources are yellow/red, current sinks light/dark
blues and neutral regions are in green. The overlaid numbers 1–6 seen in [A] and in [C–D] correspond to synaptic events as follows: slm sink (1), DG
molecular sink (2), DG granule cell source (3), CA1 source (4), SO sink (5) and CA1 long latency source (6). B: 3xTgAD CSD laminar profile with voltage
overlay of evoked response corresponding to pulse 20 in [D]. Long-latency re-entrance and PS can be seen in CA1. CSD figures from control [C] and
3xTgAD [D] mice correspond to the field response profiles for the pulses seen in Figure 5C and D. CSD analysis of pulses 1, 5, 10, and 20 of a 20-pulse
train shows progressive increases in excitability in CA1 and DG cell layers in control [C] and 3xTgAD [D] mouse. Black arrows point to short-early DG
PS (pulse 5), inferior blade DG PS (pulse 20 3xTgAD) and long-latency CA1 PS that appears to propagate away from CA1 through the alveus (pulse 20
3xTgAD). Presumed inhibitory-afterpotentials labelled as IAP. Scale bar is 20 ms. CSD figures are scaled to the same parameters for each pulse and
between genotypes.
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indirect pathways could, perhaps, have offset abnormalities
present in the monosynaptic CA3-CA1 pathway of the 3xTgAD
as found by Oddo et al. [21]. Due to a lack of any abnormality in
the basic synaptic connectivity of the 3xTgAD model shown here
in vivo, we propose that any differences found in the current study
for PPF or trains between the control and 3xTgAD mouse cannot
be attributed simply to a lower capacity or conductance of
damaged fibre pathways [28] as the current required to elicit a
half-maximum fEPSP was not different between genotypes. Thus,
in the following sections we discuss evidence for AD-specific
changes in short-term synaptic plasticity that are separate from the
basal synaptic abnormalities reported previously in the 3xTgAD
mouse [21].
Increased synaptic facilitation in the 3xTgAD model
supports the early development of hyper-excitability
To examine short-term synaptic plasticity, mice were subjected
to paired-pulse stimulation over a variety of intervals [39]. In DG,
the granule cell response to such short interval PPulse stimulation
is modulated through a combination of: (a) feed forward, GABAa-
dependent inhibition driven via PP input to local interneurons; (b)
pre-synaptic metabotropic glutamate receptor activity
(mGluR;[40,41]); and (c) feedback inhibition elicited from
recurrent interneuron collaterals in the vicinity of the activated
granule cell [41,42]. The net effect of these mechanisms is that DG
responses normally show paired-pulse depression to PP activation.
In the present study, control mice, as expected [42], exhibited
paired-pulse depression to short PPIs (25–50 ms). This depression
is presumably produced through polysynaptic GABAergic feed-
back, driven by the first pulse, causing hyperpolarisation of GCs
(mediated by GABA
A
and GABA
B
receptors[43,44]). This
inhibitory modulation not only results in depression of the field
response to PPulse stimulation, but also depresses the GC PS to
the second pulse of the pair; this effect wanes as the hyperpolar-
ising current decays during longer PPulse stimulus intervals (from
100 ms onwards; [45]). In agreement with the above mechanism,
we found control mice were most likely to display a PS to the
second pulse of a pair at PPIs of 100 ms or more, that is, at longer
intervals where presumably inhibition has subsided. In contrast,
we found the PPulse response profile of 3xTgAD mice in DG was
quite different from that of controls. Young and old 3xTgAD mice
showed pronounced synaptic facilitation at short PPIs (25 and
Figure 7. Train stimulation at 5 Hz in old animals. A. There is a significant genotype difference in raw fEPSP amplitude in old animals upon 5 Hz
frequency stimulation in CA1sr (P,0.05). B. CA1 responses did not facilitate following pulse 1 in old 3xTgAD animals (data shown for CA1slm). Control
[C] and 3xTgAD [D] representative responses for old animals. Note a lack of increase in response magnitude as the train progresses. 3xTgAD animal in
[D] was the only old animal to show DG PS. Calibration 2 mV/10 ms. CSD analysis of pulses 10 and 20 in representative old control [E] and 3xTgAD [F]
animals. CSD components are comparable to those seen in young mice at pulse 1 (Fig 6C & D; current sinks in blue, sources in red). A prolonged,
facilitating afterpotential is clearly visible from 20 ms in the DG of both control and 3xTgAD animals (marked IAP).
doi:10.1371/journal.pone.0091203.g007
Hippocampal Hyperexcitability in 3xTgAD Mouse
PLOS ONE | www.plosone.org 12 March 2014 | Volume 9 | Issue 3 | e91203

50 ms), often accompanied by a PS to the second pulse. In
particular, 3xTgAD mice were more likely to display a DG PS to
the second pulse at short inter-stimulus intervals with an apparent
increasing suppression of PS at longer paired-pulse intervals (that
in controls were more likely to elicit PS). We suggest that these
results demonstrate a long-term shift to hyper-excitability in the
layer II input to DG in these AD mice for high-frequency inputs.
In contrast, we saw a shift to increased depression for low-
frequency inputs at longer PPI and in the train stimuli. Overall,
these data suggest that DG in 3xTgADs may also show a more
pronounced ‘late’ inhibitory component compared to controls,
perhaps representing a homoeostatic compensatory mechanism to
ameliorate the impact of reduced ‘early’ inhibition. Such an
increase in late inhibition may also underlie the poor facilitation
shown by 3xTgADs to low-frequency train stimulation (see below).
When examining PPulse responses in CA1 sr and slm, we found
no overall genotype differences in PPF, and CA1 responses
facilitated relative to the first pulse at short intervals in accordance
with previous reports in young AD mice [30,46]. However, we
found 3xTgAD animals demonstrated significantly more facilita-
tion in CA1sr relative to controls at a PPI of 50 ms, in accordance
with the findings of Gengler et al at the same age [46]. In old
animals, the CA1sr response of both genotypes was shown to
facilitate at short intervals but, again, there was a significantly
increased CA1sr response in old 3xTgAD animals at PPI 50 ms.
Thus, it appears from examining the responses of DG (fEPSP and
PS data), that there is a subtle reduction in the short-term,
recurrent inhibitory feedback system in the 3xTgAD mouse at
short PPIs. It is also possible that an excess of excitability in DG
and subsequent polysynaptic propagation of ‘overly excitatory’
responses through CA3 to CA1sr could account for the facilitation
that we witnessed in CA1sr. Such a mechanism could contribute
to aberrant excitation and seizure-like activity, phenomena
witnessed in vivo in other AD mouse models [33–35].
Mechanisms for increased short-term excitability
Due to the unexpected differences between control and
3xTgAD mice (i.e., the facilitation seen in 3xTgAD PPulse
responses), it is worthwhile discussing mechanisms though which
the AD phenotype could exert a hyper-excitable effect. There is
little evidence for intra or extracellular Abpathology in the DG of
the 3xTgAD model and no tau pathology localised to the DG in
older animals [20,21]. This would suggest that any change in
inhibition is separate from these signature AD pathologies. Indeed,
in human AD, the DG is one of the hippocampal regions least
affected by pathology [1]. Previous research in other AD mouse
models (APP/PS1, APOE e4 and hAPP J20) has demonstrated a
loss of interneurons within CA1 and DG. Specifically, those
expressing calcium binding proteins parvalbumin, calbindin and
calretinin, resulting in alterations in GABAergic interneuron
populations and paralleling interneuron loss in human AD patients
[47–49]. Recently, degeneration of GABAergic interneurons in
DG expressing Neuropeptide Y was seen in a novel triple-
transgenic AD model (TauPS2APP mouse; [50]). Interestingly,
enhanced LTP to PP stimulation was seen in this triple mutation
mouse in DG (in vitro), therefore, it is possible that loss of
GABAergic tone in this and other models could contribute to
hyper-excitability originating in DG [33–35,50]. Although this
could account for the excessive facilitation seen in DG and CA1sr,
we know from immunohistological studies that the 3xTgAD
mouse model does not experience overt cell loss [20,21]. Thus,
there may be similar mechanisms present in the 3xTgAD mouse
detectable through the application of antibodies sensitive to GABA
interneurons expressing calcium-binding proteins. Alternative/
additional factors include the abnormal calcium homeostasis
linked to PS1mutations [26,51,52], a reduction in nicotinic a7
acetylcholine receptors (a7nAchRs [53]) and abnormalities in the
mGluR I-III families [54,55].
3xTgAD field responses to low-frequency pulse trains are
depressed; however laminar synaptic connectivity
profiles appear normal and response re-entrance into the
hippocampal circuit is enhanced
Alongside responses to single and PPulse stimulation, we
examined the effects of low-frequency trains on presumed
monosynaptic and polysynaptic/re-entrant CA1 and DG respons-
es [7,11–13]. Young mice displayed synaptic excitability (increased
DG population cell spiking) and reverberation within the
hippocampal formation, in a subset of both 3xTgAD and control
animals. This confirms for the first time the presence of
hippocampal output and re-entrant pathways in the mouse, as
seen previously in rat ([9,10]. As an effect of genotype, small
changes in fEPSPs could be seen in CA1sr (and to a lesser extent in
CA1slm) in young 3xTgAD mice. Over a train of 20 pulses, the
fEPSP amplitude of 3xTgAD mice tended not to facilitate as much
as seen in controls (although there were no significant genotype
differences), further, they displayed relatively constant response
latencies over the train where response latencies in control mice
generally slowed. In the 3xTgAD mouse, this effect was
independent of response amplitude, thus, represented a faster rise
time to fEPSP peak.
CSD profiles in both 3xTgAD and control mice to train
stimulation showed a pattern of current sinks and sources
consistent with excitatory synapses onto DG molecular layer
(GC dendrites) and apical dendrites in CA1 [9,56]. If the
connectivity between subiculum, EC and hippocampus was intact
in the early stages of AD we expected to see: 1) a current sink in
DG, reflecting layer II EC PP inputs to the dendrites of granule
cells within the molecular layer and a positive-going fEPSP
(presumably a reversal of the population cell fEPSP); 2) a
concomitant current sink (reflected by a fast, negative-going local
field potential; LFP) within CA1slm, marking the excitatory input
from layer III PP (stimulated directly by the traversing of fibres
through subiculum); 3) a delayed CA1sr negative going LFP, of a
latency reflecting synaptic delay through the tri-synaptic connec-
tivity to EC, DG, CA3 and ending with the Schaffer collateral
input to CA1sr [9,10].
The presence of a clear CA1slm sink was variable in both
genotypes, likely due to the relative success of electrode placement
within the narrow CA1slm layer, rather than an abnormality in
the PP input. In all animals, a large DG current source could be
seen upon population spiking, thus, synaptic transmission through
PP layer II appeared intact in the 3xTgAD mouse. CSD sinks also
indicated that the (presumed) Schaffer collateral input from CA3
terminated in both stratum oriens and stratum radiatum of CA1,
suggesting that the connectivity of hippocampal fibre pathways are
functionally intact in the 3xTgAD mouse (but see [28]). Note that
we cannot discount a contribution from CA2 input to CA1 so/sr
[57]. However, the CA1sr latency would appear rather too long
for a di-synaptic response (EC layer III input to CA2 which then
projects to CA1 so/sr). Thus, the CA1sr response is much more
likely to reflect feed-forward excitation from DG via CA3 [9,10].
Animals that demonstrated response re-entrance also showed
progressive DG (and CA1 in 3xTgAD) population cell spiking and,
within a train of 20 pulses, there was evidence for hippocampal
excitability within each layer of the CA1-DG axis. For 3xTgAD in
particular, the re-entrant input to CA1 elicited PS, which could be
Hippocampal Hyperexcitability in 3xTgAD Mouse
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seen within as few as ten pulses of a train. However, 3xTgAD
excitatory responses were also frequently coupled with what were
presumably prolonged inhibitory after potentials (also seen in non-
excitable 3xTgAD mice), perhaps indicating abnormalities in the
balance of inhibitory feedback within hippocampus [33]. The
presence of putative inhibitory currents during a 5 Hz train is
supported by the lack of a DG second PS during seen PPulse
stimulation, where a 100 ms or more ISI suppressed DG PS
activity in 3xTgADs.
The highly specific topographic connectivity of the hippocam-
pal-entorhinal system [8–10,58–60] may also have meant that
subtle differences in recording and stimulating placements
produced a range of response profiles within each genotype;
however, placement of electrodes was consistent across all animals
at the targeted coordinates. Thus, the present response profiles
strongly suggest that there are subtle differences in the laminar
profile of hippocampal responses in 3xTgAD and control mice, in
response to low-frequency stimuli.
By 17–18 months of age, we found significant differences in the
size of CA1sr fEPSP amplitude generated in the 3xTgAD mouse;
further, their CA1 responses did not facilitate relative to the first
pulse of the train. This effect appeared to be independent of the
normal ageing process, as control mice retained some degree of
facilitation; therefore, we conclude that this change in the
3xTgAD mouse is due to AD-like pathological progression. The
latter likely includes the appearance of extracellular plaques and
hyperphosphorylated tau [20,21] in addition to a range of other
pathological markers, including increase in de-myelination [28],
inflammatory processes [20] and/or loss of synaptic density [32].
There was, however, a general decrease in excitability with age in
both genotypes, which could be due to an age-related increase in
inhibitory after-potentials, such as those after-hyperpolarisation
currents seen when recording intracellularly from hippocampal
slices derived from old animals [61]. The latter conclusion is
supported by the CSD profiles of aged animals which appear to
show: (a) a sustained sink current in DG; (b) the lack of
hippocampal re-entrance; and (c) a decrease in PS probability
during both PPulse and train stimulation. Due to the small
numbers of animals in the old group, conclusions must be drawn
with caution; nevertheless, it appears in the aged 3xTgAD mouse
there is augmentation of the usual decline in synaptic facilitation
associated with ageing. Of note, there is also an age-related
decrease in DG paired-pulse depression in control mice, a change
that may underlie the known ageing-related deficit in pattern
separation (see [62] for review) and, perhaps in turn, the age-
related deficit in episodic-like memory in older mice [23].
Conclusions
What do the present data contribute to our understanding of
early hippocampal dysfunction in AD? We found previously that
the 3xTgAD model exhibits an early deficit in episodic-like
memory that develops fully by 6 months of age [23,24]. We show
here that this behavioural deficit coincides with subtle abnormal-
ities in neuronal excitability and paucity of fEPSP facilitation seen
in the DG and CA1 regions, suggesting an imbalance of inhibitory
feedback in the model. However, our single-pulse responses show
no obvious evidence of degradation in hippocampal fibre
pathways [28]. Therefore, we suggest that cellular/synaptic
abnormalities (be it intracellular Abaccumulation, abnormal
calcium homeostasis or dysfunction of recurrent inhibition) are
mediating the effects witnessed here, at least in young mice. These
findings do not necessarily contradict deficits seen in LTP (which
are likely interlinked with intracellular mechanisms) seen in this
and other AD mouse models [21,25,32,46]. However, a major
conclusion from the present experiments is that hippocampal
synaptic integrity in the 3xTgAD model of familial human AD is
largely intact (as measured through single and paired-pulse low
frequency stimulation in vivo) but that responses to brief high-
frequency activation display hyper-excitability. If the 3xTgAD
model does indeed mirror the pathological progression of familial
human AD (bearing in mind that no familial patient would
experience multiple mutations in APP or PS1 together as in this
AD model), these results provide some hope that in the early
stages, the hippocampus is functionally complete. Thus, treat-
ments that can alleviate intracellular abnormalities that underlie
AD-related synaptic changes (such as chelation of excess calcium;
[26] or clearance of intracellular Ab[22]) may improve cognitive
function and quality of life in individuals at the onset of AD by
correcting the hippocampal hyper-excitability shown here by us
and others in AD models.
Author Contributions
Conceived and designed the experiments: KED JG. Performed the
experiments: KED. Analyzed the data: KED SF JG. Contributed
reagents/materials/analysis tools: SF. Wrote the paper: KED SF JG.
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