Department of Human Anatomy and Physiology, University College Dublin, Dublin 2, Ireland
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ABSTRACT |
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Freir, Darragh B.,
Christian Holscher, and
Caroline E. Herron.
Blockade of Long-Term Potentiation by -Amyloid Peptides in
the CA1 Region of the Rat Hippocampus In Vivo.
J. Neurophysiol. 85: 708-713, 2001.
The effect of
intracerebroventricular (icv) injections of
-amyloid peptide
fragments A
[15-25], A
[25-35], and A
[35-25] were
examined on synaptic transmission and long-term potentiation (LTP) in
the hippocampal CA1 region in vivo. Rats were anesthetized using
urethan, and changes in synaptic efficacy were determined from the
slope of the excitatory postsynaptic potential (EPSP). Baseline
synaptic responses were monitored for 30 min prior to icv injection of
A
peptides or vehicle. High-frequency stimulation (HFS) to induce
LTP was applied to the Schaffer-collateral pathway 5 min or 1 h
following the icv injection. HFS comprised 3 episodes of 10 stimuli at
200 Hz, 10 times, applied at 30-s intervals. Normal LTP measured 30 min
following HFS, was produced following icv injection of vehicle
(191 ± 17%, mean ± SE, n = 6) or
A
[15-25; 100 nmol] (177 ± 6%, n = 6)
1 h prior to HFS. LTP was, however, markedly reduced by
A
[25-35; 10 nmol] (129 ± 9%, n = 6, P < 0.001) and blocked by A
[25-35; 100 nmol]
(99 ± 6%, n = 6, P < 0.001). Injection of the reverse peptide, A
[35-25], also impaired LTP at
concentrations of 10 nmol (136 ± 3%, n = 6, P < 0.01) and 100 nmol (144 ± 7, n = 8, P < 0.05). Using a different
protocol, HFS was delivered 5 min following A
injections, and LTP
was measured 1 h post HFS. Stable LTP was produced in the control
group (188 ± 15%, n = 7) and blocked by
A
[25-35, 100 nmol] (108 ± 15%, n = 6, P < 0.001). A lower dose of A
[25-35; 10 nmol]
did not significantly impair LTP (176 ± 30%, n = 4). The A
-peptides tested were also shown to have no significant
effect on paired pulse facilitation (interstimulus interval of 50 ms),
suggesting that neither presynaptic transmitter release or activity of
interneurons in vivo are affected. The effects of A
on LTP are
therefore likely to be mediated via a postsynaptic mechanism. This in
vivo model of LTP is extremely sensitive to A
-peptides that can
impair LTP in a time- ([25-35]) and concentration-dependent manner
([25-35] and [35-25]). These effects of A
-peptides may then
contribute to the cognitive deficits associated with Alzheimer's disease.
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INTRODUCTION |
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Alzheimer's disease (AD) is a
neurodegenerative disorder leading to loss of memory, progressive
decline in cognitive function, and premature death. One of the
hallmarks of AD is the presence of numerous senile plaques and
neurofibrillary tangles in the affected brain regions. The main
constituent of these plaques is -amyloid peptide (A
), a 39-42
amino acid peptide derived by proteolysis of amyloid precursor protein
(APP). There appears to be a correlation between amyloid load in the
cortex and disease progression on a cognitive level (Braak and
Braak 1997
). It is generally accepted that A
is involved in
neuronal degeneration; however, the mechanism of action and how it
causes dementia have yet to be determined.
It has been reported previously that the synthetic A-peptides
[22-35] and [25-35] produced neurotoxic effects in cell culture studies (Gray and Patel 1995
; Takadera et al.
1993
). The proposed mechanisms suggested include increases in
reactive oxygen species (Behl et al. 1994
) and increased
intracellular calcium levels (Mattson et al. 1992
). In
vivo injection of both A
[25-35] and A
[1-40] produces
neurodegeneration and neurite dystrophy similar to that seen in autopsy
samples of AD patients (Pike et al. 1991
, 1992
). APP is expressed and localized on glutamatergic
neurons (Ouimet et al. 1994
), while cellular damage in
the brains of patients with Alzheimer's disease is found predominantly
in areas that display glutamatergic synaptic plasticity in the adult
brain (Arendt et al. 1998
). Prolonged infusion of
synthetic A
into the brain can produce learning and memory deficits
in rats (Nitta et al. 1997
; Sweeney et al.
1997
), while overexpression of A
in transgenic mice is also
associated with cognitive decline (Chapman et al. 1999
;
Nalbantoglu et al. 1997
). A
neurotoxicity has also
been shown to be independent of plaque formation (Mucke et al.
2000
). Chronic infusion of low doses of A
[1-40] have also
been shown to reduce long-term potentiation (LTP) in vivo (Itoh
et al. 1999
).
Hippocampal LTP is an activity-dependent increase in the synaptic
response that is used as a cellular model of learning and memory. It is
dependent on Ca2+ influx and subsequent
activation of calcium-dependent second-messenger processes including
protein synthesis and phosphorylation. Experiments using transgenic
mouse models and gene targeting have shown a close association between
impaired hippocampal LTP and behavioral learning and memory deficits.
LTP may then represent a good model with which to examine the neuronal
mechanisms involved in diseases associated with cognitive decline. In
the CA1 region, LTP requires activation of
N-methyl-D-aspartate (NMDA)-type glutamate
receptors (Collingridge et al. 1983) and/or L-type
voltage-gated calcium channels (Grover and Teyler 1990
;
Morgan and Teyler 1999
). It has been used as a model of
activity-dependent synaptic plasticity that may underlie some forms of
learning and memory. Previous reports have indicated that
-amyloid
peptide can alter the activation kinetics of L-type calcium channels
(Ekinci et al. 1999
; Ueda et al. 1997
) in
addition to increasing NMDA receptor-mediated synaptic responses
(Wu et al. 1995b
). We decided therefore to examine the
effects of A
-peptides on hippocampal LTP and synaptic transmission.
In this report we document our investigation of the effects of
intracerebroventricular (icv) injections of several A
-peptides on
synaptic transmission and LTP in the CA1 region of the rat hippocampus
in vivo. The peptides examined included A
[15-25], which is part
of the extracellular hydrophilic domain and does not contain the active
sequence of A
(Pike et al. 1995
), as such
A
[15-25] was used as a control peptide. A
[25-35] is the
most neurotoxic fragment (Yankner et al. 1990
) that
comprises part of the membrane spanning region and includes a
hydrophopic domain. Finally, A
[35-25] was used to examine the
effects of reversing the sequence of the active region to determine
whether biological activity of the peptide was affected.
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METHODS |
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Male Wistar rats (150-200 g) were surgically prepared for acute
recordings. Rats were anesthetized with 1.5 g/kg urethan (ethyl carbamate) and placed in a stereotaxic device for surgery and recording. Heating pads (Braintree Scientific) were used to maintain the temperature of the animals at 37.0 ± 0.5°C. Small holes
were drilled in the skull at the sites of the reference, recording, and
stimulating electrodes. An additional hole was drilled to introduce a
cannula for icv injection of drugs/vehicle. The recording electrode was
positioned in the stratum radiatum area of CA1 (3 mm posterior to
bregma, 2 mm lateral to bregma). A bipolar stimulating electrode was
placed in the Schaffer collateral/commissural pathway, distal to the
recording electrode (4 mm posterior, 3 mm lateral). Physiological
(Leung 1980) and steriotaxic indicators were used to
lower electrodes slowly through the cortex into the hippocampus. Test
stimuli were delivered to the Schaffer-collateral commissural pathway
every 30 s. Electrodes were positioned to record a maximal evoked
field excitatory postsynaptic potential (EPSP). Baseline EPSPs were set
within the range 0.8-1 mV, which corresponded to 40% of the maximal
response. Rats were injected icv with A
-peptide fragments (100 or 10 nmol in 5 µL) or vehicle (distilled water; 5 µL). In each protocol
used, LTP was induced by applying high-frequency stimuli (HFS; 3 × 10 trains of 10 stimuli at 200 Hz) at a stimulus intensity that
evoked a field EPSP of approximately 1.5-2.0 mV, approximately 80% of
maximal EPSP. Stimulating (bi-polar stainless steel; 0.125 mm diam) and
recording (mono-polar stainless steel; 0.125 mm diam) electrodes were
obtained from Plastics One. Peptides were obtained from Bachem (Saffron
Walden, UK).
Data analysis
Extracellular field potentials were amplified (×100), filtered at 5 kHz, digitized, and recorded using Mac Lab software acquisition system. The EPSP slope was used to measure synaptic efficacy. EPSPs are expressed as a percentage of the mean initial slope measured during the first 10 min of the baseline-recording period. Data were analyzed using ANOVA measured over 5 min prior to LTP induction and either at 25-30 or 55-60 min following the induction of LTP. The Tukey-Kramer multiple comparisons test was used to determine statistical significance.
Stimulation protocol
Field EPSPs were evoked in the CA1 region using low-frequency
stimulation (0.033 Hz) to obtain at least 30 min of stable baseline prior to icv injection of A-fragment or vehicle. At a time of either
5 min or 1 h following the icv injection, a series of
high-frequency stimuli were delivered to induce potentiation of the
synaptic response. Low-frequency stimulation was then used to evoke
EPSPs for a further period of 30 min to 1 h to monitor any change
in the synaptic response. Paired-pulse facilitation (PPF) with an inter-stimulus interval of 50 ms was also examined before icv injection
of A
-peptides and again 90 min following icv injection (30 min
following induction of LTP).
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RESULTS |
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In control experiments, icv administration of vehicle had no effect on baseline synaptic transmission, or PPF monitored for up to 1 h following injection. When HFS was applied 1 h following vehicle injection, LTP measured 191 ± 17% (n = 6, mean ± SE, P < 0.05, ANOVA, 30 min following HFS; Fig. 1). LTP of a similar magnitude was also produced following vehicle injection 5 min prior to HFS (LTP measured 1 h following HFS; 188 ± 15%; n = 7; Fig. 2).
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The effects of A[15-25], A
[25-35], and the reverse peptide
A
[35-25] were examined. Baseline synaptic transmission was
monitored for periods of up to 1 h following icv injection of
A
-peptides. There was found to be no significant effect on baseline
EPSPs evoked at low frequency (0.033 Hz, Figs. 1, 3, and 4) or on PPF (Table 1).
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Injection of A[15-25], 1 h before the application of HFS had
no significant effect on LTP (177 ± 6%, n = 6)
compared with the control group (191 ± 17%, n = 6; Fig. 1). A
[15-25] does not incorporate the
trans-membrane, neurotoxic sequence of A
and was a good
indication that injection of peptide was not causing a nonspecific
effect on synaptic transmission and plasticity.
Injection of the active neurotoxic portion of A, namely
A
[25-35] (Yankner et al. 1990
), however, was found
to significantly impair LTP in a time- and concentration-dependent
manner. When 10 nmol A
[25-35] was injected 5 min prior to HFS,
there was no effect on LTP (176 ± 30%, n = 4)
compared with control (188 ± 15%; n = 7), while
100 nmol was found to reduce significantly LTP (108 ± 15%,
n = 6, P < 0.001; Fig. 2). To examine
the effect of prolonging the application of A
[25-35], icv
injections were performed 1 h prior to the induction of LTP. Under
these conditions, the lower 10 nmol concentration of A
[25-35] was
now found to reduce significantly LTP (129 ± 9%,
n = 6, P < 0.01) when compared with
the control group (191 ± 17%; n = 6), while 100 nmol blocked LTP (99 ± 6%, n = 6, P < 0.001; Fig. 3). This
indicates that even short-term exposure to a dose of 100 nmol/5 µL of
A
[25-35] can impair LTP, although baseline synaptic transmission
remains unaffected.
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The effect of reversing the sequence of the neurotoxic A fragment on
LTP and baseline synaptic transmission was also examined. When
A
[35-25] 10 and 100 nmol was injected 1 h prior to HFS, there was a significant reduction in LTP (136 ± 3%,
n = 6, P = 0.01) and (144 ± 7%,
n = 8, P < 0.05) compared with control
(191 ± 17%, n = 6; Fig.
4).
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PPF, induced by applying two stimuli at an inter-stimulus interval of
50 ms was recorded immediately prior to injection of A
fragments/vehicle and again 30 min following HFS. The PPF ratio was
measured as the slope of EPSP2/EPSP1 (Table 1). None of the A
peptides tested had a significant effect on PPF (paired
t-test), although there was a trend toward a decrease in facilitation.
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DISCUSSION |
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In this study we have shown for the first time in vivo that the
impairment of LTP by A[25-35] peptide is both time and
concentration dependent. This is demonstrated by the greater decrease
in LTP observed when tetanic stimulation was given 1 h and not 5 min post-injection. Also, 100 nmol A
[25-35] had a more powerful
blocking effect than 10 nmol A
[25-35]. LTP was impaired by the
most neurotoxic fragment, A
[25-35] (Yankner et al.
1990
), containing the lipophilic portion of A
(amino acids
28-35). Interestingly, A
[35-25], the "reverse" sequence
peptide, previously reported to have no effect on LTP in vitro
(Chen et al. 2000
), caused a marked reduction in
plasticity at both 10 and 100 nmol, while A
[15-25] had no effect
on LTP.
What are the potential mechanisms involved in the effects of
A-peptides on LTP? There are two types of channel known to be implicated in the induction of LTP that may be disrupted by active A
-peptide: namely the L-type voltage-gated calcium channel,
activated by the high stimulus frequencies of 200 Hz used in our study
to induce LTP (Grover and Teyler 1990
; Morgan and
Teyler 1999
) and/or the NMDA receptor/channel complex
(Collingridge et al. 1983
). Alteration of the activation
kinetics of either of these channels by A
could influence LTP induction.
A can cause the formation of calcium-permeable pores in cell
membranes (Engström et al. 1995
) and block the
fast inactivating potassium channel (Good and Murphy
1996
); both mechanisms would produce an increase in
intracellular calcium. In addition, the L-type calcium channel current
is increased by A
(Ueda et al. 1997
) acting via
mitogen-activated protein (MAP) kinase (Ekinci et al.
1999
). The disruption of intracellular calcium homeostasis is
likely to play a major role in LTP impairment. A
[1-40] has been
reported to cause a reduction in LTP in vivo (Cullen et al. 1997
), yet can also produce a potentiation of LTP in vitro,
possibly due to an increase in activation of the NMDA receptor
(Wu et al. 1995a
,b
). An increase in NMDA receptor
function has also been reported in hippocampal slices from transgenic
mice over expressing human APP (Hsiao et al. 1999
).
Depending on the degree of activation of the NMDA receptor, and
consequent calcium influx, it is possible to induce LTP or long-term
depression (Mulkey and Malenka 1992
). Prolonged
low level activation of the NMDA receptor/channel can also reduce the
ability to induce subsequent changes in synaptic plasticity
(Coan et al. 1989
). This may be due to a change in the
dynamics of activation of calcium calmodulin kinase (CaM KII) and
calcium/calcineurin (phosphatase 2B) by high and low levels of
intracellular calcium, respectively (Mulkey et al. 1993
,
1994
). Synaptic desensitization of the NMDA receptor and
down regulation of receptor function is known to depend on activation
of calcium/calcineurin (Tong et al. 1995
). A
has also
been reported to inhibit the late phase of LTP in vitro via a
calcium/calcineurin-dependent mechanism (Chen and Xie
1999
). Recently, the p38 MAP kinase pathway has been
implicated in the inhibitory actions of A
[25-35] on LTP in vitro
(Saleshando and O'Connor 2000
).
Icv injection of the active fragment of A may then cause disruption
of calcium homeostasis (Mattson et al. 1993
), including increased activation of the calcium-permeable NMDA channel and/or increased calcium influx directly or via the L-type calcium channel. The subsequent induction of LTP would then be precluded due to changes
in the level of channel phosphorylation. A
has also been shown to
increase calcium influx via the N-type calcium channel in cerebellar
granule cells (Price et al. 1998
).
It is unlikely that the deficit in LTP observed following our acute
application of low concentrations of A-peptides is due to cytotoxic
processes (Games et al. 1992
). The full sequence A
[25-35] has been shown to be necessary for peptide aggregation, with an emphasis on the importance of methionine residue
(M35) for the promotion and/or stabilization of
A
aggregation. This property also correlates with the neurotoxic
effects of this peptide on cultured cells (Pike et al.
1995
). Although we have shown that A
[35-25] causes a
deficit in LTP in vivo, it has been shown to have no effect on LTP in
vitro (Chen et al. 2000
). The reversal of the amino acid
sequence in A
[35-25] alters the aggregation properties of the
peptide from
-sheet to random coil formation, thereby reducing the
neurotoxic effects on cultured cells (Buchet et al.
1996
). It has, however, been reported previously that other peptides that are non-aggregating and non-toxic in cell culture can
produce a deficit in LTP in vitro. This suggests that it may not be
necessary for a peptide to aggregate to disrupt synaptic plasticity
(Chen et al. 2000
). In our study, the lower 10-nmol dose
of A
[35-25] had a greater inhibitory effect than the 100-nmol dose; however, when LTP was measured 30 min following induction, there
was little difference in the degree of potentiation observed with
either dose. The results obtained using A
[35-25] merit further investigation with attention to how this peptide might effect either
NMDA receptor-mediated responses or activity of the L-type Ca2+ channel. Our results suggest that the
lipophilic portion of the peptide (in the forward or reverse sequence)
can impair LTP. In contrast, A
[15-25] did not alter LTP when
compared with controls. This peptide, unlike A
[25-35] and
A
[35-25], does not contain the lipophilic fragment of
A
[1-40/42] that is found in senile plaques of AD patients. This
was a good indication that icv injections of peptide did not have an
unspecific blocking action on LTP.
The A-peptides tested had no effect on low-frequency synaptic
transmission recorded during a period of up to 1 h post icv injection. In addition, there was no significant change in PPF. Changes
in PPF can be associated with alterations in presynaptic release;
increases in PPF indicating a decrease in basal neurotransmitter release. In addition, any change in interneuron activity in vivo could also cause an alteration in PPF. Our results suggest that the
A
-peptides tested had a negligible effect on interneuron activity or
mechanisms of neurotransmitter release in the CA1 region in vivo. There
was, however, a trend toward a decrease in PPF that may suggest that
A
-peptides have subtle effects on these processes.
Our results suggest that A has an effect on the processes involved
in LTP in a short period of time, since injection of 100 nmol
A
[25-35] only 5 min before the application of tetanic stimulation to induce LTP causes a change of LTP to short-term
potentiation. This model of in vivo synaptic plasticity is
therefore extremely sensitive to
-amyloid. It is possible that this
system could provide a working model with which to investigate the
mechanism of action of A
-peptides and to develop possible
therapeutic interventions for the treatment of A
-induced
neurotoxicity, found in Alzheimer's disease. These results support the
theory that
-amyloid may be responsible at least in part, for the
neurodegeneration and decline in cognition associated with Alzheimer's disease.
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ACKNOWLEDGMENTS |
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This work was supported by Enterprise Ireland and the Department of Physiology, University College Dublin.
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FOOTNOTES |
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Present address and address for reprint requests: C. E. Herron, Dept. of Physiology, National University of Ireland, Earlsfort Terrace, Dublin 2, Ireland (E-mail: Caroline.Herron{at}ucd.ie).
Received 18 July 2000; accepted in final form 27 October 2000.
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REFERENCES |
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