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The Journal of Neuroscience, August 1, 2001, 21(15):5703-5714
Generation of Aggregated  -Amyloid in the Rat Hippocampus
Impairs Synaptic Transmission and Plasticity and Causes Memory
Deficits
Aline
Stéphan,
Serge
Laroche, and
Sabrina
Davis
Laboratoire de Neurobiologie de l'Apprentissage, de la
Mémoire et de la Communication, Centre National de la Recherche
Scientifique Unité Mixte de Recherche 8620, Université Paris Sud, 91405 Orsay, France
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ABSTRACT |
We injected a combination of the  -amyloids (As) A40 and
A43 to "seed" formation of amyloid deposits in the dorsal
dentate gyrus of rats in vivo, on the basis of a theory
of Jarrett and Landsbury (1993) . Rats were tested on several different
learning tasks, and synaptic transmission and plasticity were assessed in vivo. Between 7 and 16 weeks after injection, we
found aggregated amyloid material, reactive astrocytosis, microgliosis,
and cell loss around the sites of injection. Rats were impaired
specifically in working memory type tasks in accordance with the type
of memory deficit observed in the early stages of Alzheimer's disease.
Synaptic transmission and long-term potentiation, a candidate cellular mechanism for memory, were severely impaired in vivo.
Injections of the same dose of fragments individually did not induce
these effects. These findings suggest that aggregated amyloid material induces cognitive deficits similar to those observed in the early phases of Alzheimer's disease via an alteration in neuronal
transmission and plasticity.
Key words:
working memory; A40; A43; synaptic plasticity; Alzheimer's disease; dentate gyrus; senile plaque
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INTRODUCTION |
The neuropathology of Alzheimer's
disease (AD) consists of a complex milieu of senile plaques and
neurofibrillary tangles in the brain. Development of plaques and
tangles during the progression of the disease shows different
spatiotemporal patterns but leads eventually to lesions, loss of
neurons, and synaptic connections. Deposits of amyloid are an early and
critical event in the pathogenesis of AD (Selkoe, 1997), and they first
form in temporal cortical regions including the hippocampus (Hyman et
al., 1984; Ball et al., 1985; Duyckaerts et al., 1997), a region
implicated in the processing of information necessary for memory
formation (O'Keefe and Nadel, 1978; Hyman et al., 1986; Squire, 1986;
Wallenstein et al., 1998). Senile plaques consist of deposits of
extracellular -amyloid (A) filaments associated with dystrophic
dendrites and axons, activated microglia, and reactive astrocytes
(Itagaki et al., 1989; Selkoe; 1991). A is a 4 kDa peptide of 42/43
amino acids, harbored within the larger, transmembranal amyloid
precursor protein (APP) and located in the portion of the protein that
spans the transmembranal domain (Glenner and Wong, 1984; Kang et al., 1987). Under normal conditions, soluble A is cleaved between sites
16 and 17 within the protein (Esch et al., 1990; Sisodia, 1992) by an
unknown  -secretase, releasing it with a large (~90 kDa) N-terminal
portion of APP into the extracellular space. Under conditions in which
intact A peptide is released into the extracellular space, it is
proteolytically processed by cleavage at the N-terminal site by
-secretase (Seubert et al., 1993), identified as -site APP
cleaving enzyme (Sinha et al., 1999; Vassar et al., 1999), and
at the C-terminal site at either 40 or 43 by the  -secretase, identified as presenilin 1 (Annaert et al., 1999; Wolfe et al., 1999).
Senile plaques contain both A43, which aggregates more rapidly
(Suzuki et al., 1994), and the more abundant but soluble A40. Diffuse plaques, not associated with altered neurites and glia, appear
to be composed of the longer fragment, A43 (for review, see Dickson,
1997). Because the major -amyloid species observed in the CNS is
A40 and diffuse plaques consists of A43, the development of
hard-core senile plaques may occur by the conversion of diffuse plaques
into dense core plaques by amalgamation with circulating A40.
To date, however, despite growing evidence, it is not known exactly how
development of the senile plaque may specifically contribute to
development of the cognitive dysfunction that severely affects memory
processes associated with the disease. Because the pathology of AD is
not known to develop naturally in aged rodents, we have capitalized on
a theory postulated by Jarrett and Landsbury (1993) suggesting that
small quantities of A43 could accelerate "nucleation or seeding"
of senile plaques if in the presence of a metastable level of soluble,
circulating A40. In these experiments we have tested the hypothesis
that injections of a combination of both peptides into the dentate gyrus produces aggregated amyloid material and have examined whether this causes disruption of synaptic transmission and plasticity and
impairs learning.
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MATERIALS AND METHODS |
All experiments were performed in strict accordance with
recommendations of the European Union (86/609) and the French
National Committee (87/848). Male Wistar and Sprague Dawley rats
(n = 104) weighing between 300 and 400 gm were used in
all experiments. They were housed individually in a
temperature-controlled vivarium set on a 12 hr light/dark cycle (lights
on, 8:00 A.M.).
Drugs and groups
A40 and A43 (Bachem) were dissolved in
acetylnitrile (35%) and trifluoroacetate (0.1%). Drugs were made up
in aliquots of 10 µl and stored at  20°C.
In the first experiment, our aim was to test in vivo the
seeding hypothesis suggested by Jarrett and Landsbury (1993) and to
determine whether the formation of aggregated amyloid material had a
functional effect on synaptic plasticity and learning. To this end, we
made focal injections (two per hemisphere, bregma, 3.2 and
4.8 mm; midline, ±1.2 and 2.8 mm; 3.2 and 3.6 mm below the brain
surface) around the dorsal region of the dentate gyrus where
electrophysiological recordings were conducted. Five groups of rats
were injected with variations of combinations of peptides and differing
quantities. The rationale for this was based on A aggregation
kinetics of the relative ratio of A40 and A43, which shows that
A43 aggregates more rapidly than A40 and that the relative
concentration of A43 could be an important factor in A deposition
(Snyder et al., 1994). Therefore, to test the seeding hypothesis, one
group of rats was injected with a combination of A40 (10 µg in 1 µl) and A43 (5 µg in 1 µl; total quantity per site was 15 µg
in 2 µl per site). Two other groups of rats were injected with
individual fragments at the same dose (A40, 10 µg; A43, 5 µg)
in 1 µl per site to control for the effects of the individual
fragments, and two further groups with double the dose of A40 (20 µg) and A43 (10 µg) in 2 µl per site were injected. The
aggregation kinetics of the individual peptides described by Snyder et
al. (1994) suggests that A43 is capable of self-aggregating
but not A40. Our protocol was therefore designed to control for
whether the combined fragments need A43 or whether any effect is
caused by an increase in the concentration of peptides. Control rats
consisted of a group of noninjected rats and rats injected with 2 µl
per site of the vehicle solution to assess the potential effect of
toxicity induced by the vehicle solution alone.
Because of the results, we then focused more specifically on the
effects of the combined fragments on the neuropathology, electrophysiology, and learning in a second set of experiments in which
we used a more distributed injection protocol [modified from Jarrard
(1989)] of the combined A fragments to incorporate a more
widespread region of the dorsal dentate gyrus. In these experiments
eight bilateral sites were used, and the injecting syringe was lowered
into the dentate gyrus at the following coordinates (anteroposterior,
bregma, 2.4, 3.0, 3.0, 3.8, 4.0, 4.8, and 4.8; midline, ±1.0,
1.4, 3.0, 2.6, 2.6, 2.6, 3.9, and 2.5 mm). To maintain the same
total quantity of peptides injected, the concentration of A40 was
increased to 20 µg/µl, and A43 was increased to 10 µg/µl,
with a total reduced volume of 0.25 µl per site. In all injections,
whether focal or distributed, there was no evidence of flocculation of
the peptide during injection, indicating a soluble state of the
peptides during injection.
Rats were anesthetized with sodium pentobarbital (60 mg/kg) and
supplemented throughout the surgery as required. They were placed in a
stereotaxic frame, the skin overlaying the skull was retracted to drill
holes, and injections were made using a Hamilton syringe, lowered
slowly into place. The needle was left in place for 2 min before the
injection was started, and then the fragments were injected slowly over
a period of ~8 min. The needle was left for ~2-5 min before
retracting slowly to prevent backwash up the needle tract. When all
injections were made, the skull was swabbed, and the skin overlying the
skull was closed with surgical staples. Topical antiseptic
(exoseptoplix) was applied to the suture, and a 4 d course of
antibiotic treatment with teramycin was given to stem the development
of infection.
Behavior
Rats were tested on a battery of behavioral tasks on the radial
arm maze and the water maze. The radial arm maze stood 70 cm from the
floor and consisted of an octagonal platform from which eight arms (60 cm long; 12 cm wide) radiated. At the ends of each arm, a food cup was
located into which a single noyes pellet could be placed as
reinforcement. The water maze was 1.5 m in diameter and 0.6 m
in height. The pool stood 0.8 m above the ground, was painted
white, and was filled to a depth of 30 cm with water at 23 ± 2°C, made opaque with the addition of a little milk. A circular
escape platform (10 cm in diameter) was placed in a constant position,
submerged 1 cm below the water surface.
Spatial learning on the radial arm maze. Four weeks after
surgery, food was reduced progressively until rats reached a target weight of ~85% of their free-feeding weight. When the target weight had been reached, rats were habituated to the maze for 3 d. They were placed in the center of the platform of the maze and allowed to
explore all arms freely for 10 min. To aid exploration, noyes pellets
were scattered along all arms. To test spatial learning, four of the
eight arms were chosen in a pseudorandom manner for each rat and
baited, and these remained constant throughout the training period.
Rats were given eight consecutive trials a day for 7 d and were
tested until they had visited all the correct arms or for a maximum
time of 4 min/trial. Learning was assessed by the number of reference
memory errors (entry into nonbaited arms) or working memory errors
(entry into any previously visited arms during that trial).
Spatial working memory tested on the radial arm maze. This
was tested on the same radial arm maze, located in a different experimental room. By the use of a classical task in which all eight
arms were baited, rats had to enter each arm only once to recover a
food pellet. Rats were given one trial a day for 12 d, and errors
were scored as reentries into previously visited arms.
Delayed match-to-place. Rats were tested on the same maze,
located in another room and fitted with doors to block entry into arms.
Each session consisted of a sample trial and a test trial. During the
sample trial, all arms were blocked except for one. On the test trial,
all arms were made accessible, and the correct choice was to enter the
arm that was visited in the sample trial. The selection of the sample
arm was chosen randomly and was different for each session. The first
part of this experiment was to train the rats on the task, with no
delay between the sample trial and the test trial. When rats had
learned the task (making ~1 error/trial), different delays were
introduced (10, 20, 60, and 180 min) in addition to the zero delay. A
maximum of four sessions were given per day.
Spatial learning in the water maze. Rats were given two
trials a day for 7 d to learn the location of a hidden platform.
They were released into the pool from randomly selected positions
around the perimeter and given a maximum swim time of 90 sec to find the platform. If they did not find the platform in this time, they were
guided to it. They were left on the platform for 30 sec before being
removed. At the end of the training period, rats were tested on a probe
trial in which the platform was removed and they were allowed to swim
freely for 60 sec. Latency to escape the platform was measured during
the acquisition phase of learning and the time spent in each quadrant
of the pool during the probe trial.
ANOVA with repeated measures was used to assess group
differences and interaction between group and learning on errors and escape latencies, and one-way ANOVA was used on the target quadrant in
the probe trial and in the spatial match-to-place task.
Electrophysiology
Between 7 and 16 weeks after the injections, rats were
anesthetized with Urethane carbamate (1.5 mg/kg) and placed in a
stereotaxic frame to record dentate gyrus field EPSPs (fEPSPs) and to
induce long-term potentiation (LTP). Standard surgical and LTP-inducing procedures were followed using those described previously (Davis et
al., 2000). In brief, stimulating electrodes were placed in the angular
bundle of the perforant path (between 7.6 and 8.0 mm from bregma and
between 4.4 and 4.2 mm from the midline), and two recording electrodes,
staggered 200 µm tip to tip to maximize stable recording of the
evoked response, were placed in the hilus of the dentate gyrus (4.2 mm
from bregma; 2.5 mm from the midline), under electrophysiological guidance.
After a stable response was established, input-output (I-O) curves
were generated. After the I-O curves, a 30-60 min baseline was
recorded under low-frequency stimulation (0.033 Hz; 100 µsec pulses
delivered via a photically isolated constant-current unit). The test
stimulus intensity was chosen to evoke a population spike amplitude
between 1 and 3 mV. After the baseline, a tetanus, consisting of six
trains of pulses (400 Hz; 20 msec) with a 10 sec interval, repeated six
times at 2 min intervals, was delivered to induce LTP. After the
tetanus, low-frequency stimulation was resumed for a further 3 hr. All
responses were amplified and filtered between 0.1 Hz and 3 kHz. They
were displayed on a storage oscilloscope and fed via an interface to a
computer for storage and off-line analysis.
Electrophysiological responses were stored as averages of four
responses. Analysis of the response was performed by measuring the
maximal slope of the early rise phase of the fEPSP and the amplitude of
the population spike, measured from the negative peak to a tangent
drawn between the two positive peaks. All responses, including the
baseline responses, were normalized to the mean value of the baseline
and assessed with ANOVA and Student's t test. Analysis of
I-O curves was conducted using ANOVA with repeated measures, the mean
of all baseline responses, and a mean of 30 min of recording of the
EPSP and spike 30 min after tetanus; and the last 30 min of recording
was analyzed with a single-factor ANOVA.
Histology
Histology and immunohistochemistry were performed to assess the
presence of aggregated material and examine anatomical and neurochemical abnormalities induced by the different fragments. Nissl
and NeuN (Chemicon, Temecula, CA; 1:500 dilution) staining was used to
assess the extent of cell loss, and Thioflavin S (Sigma) was used for
the detection of aggregated amyloid material, using standard
procedures. Specific antibodies were used to assess gliosis (rabbit
polyclonal GFAP; Dako, Carpinteria, CA; 1:2000 dilution) and microglial
activation (OX-42; Serotec, Oxford, UK; 1:50 dilution), synaptic
terminals (anti-synaptophysin; Boehringer Mannheim, Mannheim, Germany;
1:200 dilution), and the presence of the peptide in tissue (anti--amyloid; Dako; 1:50 dilution). Immediately after the
electrophysiological recording, rats were perfused transcardially with
paraformaldehyde (4%) in PBS (0.1 M). The brains were
post-fixed for 12 hr, cryoprotected with 30% sucrose for between 24 and 72 hr, frozen, and stored at 20°C. Brains were cryosectioned at
40 µm on a freezing microtome.
For immunohistochemistry, immunostaining was performed on free-floating
sections. Sections were rinsed in PBS (0.1 M) three times
(10 min/wash), endogenous peroxidase activity was blocked by incubation
with H2O2 (3%) for 30 min,
and sections were then washed in PBS three times (10 min/wash).
Nonspecific epitopes were then blocked by incubation in 10% normal
goat serum and 0.1% Triton X-100 in PBS for 1 hr. Sections were
incubated in primary antibodies overnight at room temperature and then
washed three times in PBS for 10 min/wash. Secondary antibodies (goat
anti-rabbit biotin for GFAP and goat anti-mouse for all other
antibodies; 1:200 dilution) were applied for 90 min. Immunostaining was
visualized using an ABC elite system (Vector Laboratories, Burlingame,
CA; 1:100 dilution) and DAB oxidation. All sections were mounted on gelatin-coated slides and coverslipped. Visualization was made by light
microscopy using a Leica DMRB microscope. For Thioflavin S,
fluorescence visualization was made with a filter light (emission wavelength set to 490 nm).
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RESULTS |
Focal injections of amyloid peptides in the dorsal
dentate gyrus
The first experiment was conducted to test the theory of Jarrett
and Landsbury (1993) of "seeding" amyloid deposits in
vivo and to determine whether these deposits resulted in a
functional deficit in learning and/or alteration in synaptic
transmission and plasticity in the dentate gyrus. We used restricted
injections (two sites per hemisphere) in the vicinity of the dentate
gyrus where recording of the fEPSP was made. To test the specificity of
the combined fragments (A-c), other groups of rats were injected with either the same quantity of A40 (A40-s) as in the A-c or
double the quantity (A40-d) and the same quantity of A43 (A43-s) as in the A-c or double the quantity (A43-d).
Four weeks after injection of peptides, rats were tested on a spatial
learning task in the radial arm maze, in which their learning ability
was assessed by the total number of reference or working memory errors
they made per day. We found that the vehicle solution had no effect on
performance in the subgroup of control rats (n = 13)
compared with noninjected rats (n = 11) in the number
of reference (F < 1) and working
(F(1,22) = 1.69; p > 0.05) memory errors made, and these rats were pooled to form a single
control group. In the working memory element of the task, rats injected
with the combined fragments showed a modest but significant deficit in
learning (F(1,36) = 5.18;
p < 0.05), and in Figure
1a it can be seen that this
deficit was primarily restricted to the early part of learning. In
contrast, rats injected with either A40 or A43 at the same dose
showed learning equivalent to that of the control group
(F < 1 in both groups; Fig. 1b,c). Doubling
the dose of A40 similarly had no effect on learning (F < 1; Fig. 1b); however, when the dose of
A43 was doubled, rats showed a deficit
(F(1,33) = 5.31; p < 0.05; Fig. 1c) that was similar to that in rats injected
with the combined fragments. In terms of the reference memory type
errors, rats in all groups showed learning comparable with that of the
control group (all p values > 0.05; Fig.
1d-f).
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Figure 1.
Spatial learning on the radial arm maze, 4 weeks
after focal injections of amyloid peptides, shows the ability of rats
to learn the working memory element (a-c) or the
reference memory element (d-f).
a, Rats injected with combined fragments
(A-c) showed impairment in the working
memory aspect in the first few days of training (blocks of 8 trials/d)
compared with control rats (CT). This difference
was modest but significant. b, Rats injected with either
dose of A40 [single dose (A40-s);
double dose (A40-d)] were not
significantly impaired in working memory. c, Whereas
rats injected with the low dose of A43 were not impaired in
learning, those rats injected with the high dose of A43 showed an
overall impairment. d-f, All rats were able to learn
the reference memory aspect of the task to the same level as the
control rats. The y-axis denotes the total number of
reentries made in the working memory element (a-c) and
entries into nonbaited arms in the reference memory element
(d-f) of the task.
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In a subset of these animals, synaptic transmission and plasticity were
measured in the dentate gyrus. Basal synaptic function was assessed by
a range of stimulus intensities to generate I-O curves and analysis of
baseline responses before induction of LTP. There was no statistical
difference in fEPSP (F < 1) or baseline responses
(F < 1) in noninjected rats (n = 7)
and vehicle controls (n = 5), and so they were pooled
to form a single control group. With input-output curves, control rats
(n = 12) showed a normal increase in the fEPSP as the
intensity increased (Fig. 2a).
Rat that received the fragments alone (A40 or A43) and those that received the double dose of A40 showed no difference from control rats at each intensity (all p values > 0.05; Fig.
2b,c). There was a significant overall decrease in the fEPSP
slope at all intensities in those rats receiving double the dose of
A43 (F(1,15) = 4,97; p < 0.05; Fig. 2c), and a clear and
significant reduction occurred in the fEPSP in rats injected with the
combined fragments (F(1,20) = 15.46;
p > 0.01; Fig. 2a). The range of stimulus
intensities did not differ between groups (all p values > 0.05), and because the criteria for selecting the stimulus intensity
was to have a spike amplitude between 1 and 3 mV, there was no
significant difference in basal spike values (all p
values > 0.05). There was, however, a significant decrease in the
basal fEPSP in rats receiving combined injections
(F(1,19) = 16.81; p < 0.01) and double the dose of A43
(F(1,15) = 6,78; p < 0.05). No difference in the basal fEPSP was observed with the other
groups (all p values > 0.05). These results are in
accordance with the I-O curves and suggest an alteration in synaptic
transmission after injections of the combined fragments at a higher
quantity of A43.
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Figure 2.
I-O curves of the field EPSP (fEPSP) in
rats with focal injections of amyloid fragments. a, Rats
injected with the combined fragments
(A-c) show a significant reduction in
the size of the fEPSP at all intensities compared with control
(CT) rats. b, In rats injected
with a single dose (A40-s) or a double
dose (A40-d) of A40, there was no
effect of the peptide at any intensity compared with the
CT group. c, The single dose of A43
had no effect on I-O curves, but rats injected with the double dose
showed a significant reduction in fEPSP, although not as severe as that
of rats injected with the combined peptides. The y-axis
denotes the slope of the fEPSP measured in millivolts per millisecond,
and the x-axis indicates the lowest and highest
intensities used in microamperes. Increments were an average of 60 µA.
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We next examined LTP induced by tetanic stimulation of the perforant
path. In control rats, LTP of the fEPSP was induced and maintained for
the duration of recording (Fig. 3). In
contrast, in rats injected with the combined fragments (Fig.
3a) and A43-d (Fig. 3c), very little LTP could
be induced (F(1,18) = 6.73;
p < 0.05; and F(1,14) = 7.91; p < 0.05, compared with controls, respectively). Although the rats injected with both concentrations of
A40 (Fig. 3b) appeared to show a slightly smaller
magnitude of LTP throughout the recording period, these groups were not significantly different from the control group either 30 min after the
induction of LTP or at the end of the recording period. Similarly, there was no difference in the magnitude of LTP measured at the two
different time points in the group receiving A43-s (all p values > 0.05; Fig. 3c).
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Figure 3.
LTP in the dentate gyrus measured for 3 hr after
the induction of LTP in rats injected with amyloid peptides.
a, In rats injected with the combined fragments
(A-c), no LTP was induced after the
tetanus. b, A40 injected at either a single dose
(A40-s) or double dose
(A40-d) had no significant effect on
either induction or maintenance of LTP compared with control rats
(CT), although there was a slight reduction
throughout the recording period. c, The single dose of
A43 had no significant effect on induction or maintenance of LTP,
but at double the dose, there was a blockade of induction of LTP in a
manner similar to that of the A-c
group, and this was also significantly reduced compared with the
control group. The y-axis denotes the percentage of
change in slope of the fEPSP after induction of LTP (indicated by
arrow), and the x-axis denotes the time
of recording.
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After electrophysiological recordings, histological examination of the
brains (~7 weeks after injection) was performed on a sample number of
rats in each group to assess the presence of aggregated material in the
dentate gyrus. In all rats injected with the combined fragments that
were observed (six of six), there was a strong Thioflavin S-positive
staining near and between the sites of injection. In contrast rats
injected with the low dose of A40 showed no staining of aggregated
material (zero of four), and in those injected with the low dose of
A43, very weak staining in two of four rats was observed, suggesting
that the fragments alone were not capable of forming aggregated
material. The double dose of A40 showed a profile similar to that of
the low doses with no evidence of aggregated material, whereas the
double dose of A43 showed a level of aggregated material (five of
six) similar to that observed with the combined fragments. Lesions were
induced in the dentate gyrus, and the extent of damage induced by
injections of combined peptides was ~3.4 mm, which included a lesion
containing a lot of debris and cell loss in the dentate gyrus, in the
hilus, and, to a lesser extent, in CA3 and CA1. The lesions were
observed in all injected rats, including those injected with the
vehicle solution, suggesting that they alone could not account for the impairment in learning and synaptic transmission and plasticity that
was observed. The lesions, presumably in part because of mechanical
pressure, appeared, however, to be exacerbated in rats injected with
the combined fragment and double the dose of A43. In addition the
lesions were surrounded by aggregated A stained material, suggesting
a potential interactive effect (Fig.
4).
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Figure 4.
Nissl-stained sections
(a-f) show the amount of damage to the dentate
gyrus and cell loss in sample rats in each group, and Thioflavin S
staining (g-j) shows where amyloid material has
aggregated in rats in which focal injections were made.
a, Nissl staining in a control rat injected with the
vehicle solution shows a small amount of damage and loss of cells that
is likely to be caused by mechanical damage. g,
Thioflavin S staining around the site of injection in vehicle-injected
rats does not fluoresce, indicating the absence of aggregate amyloid.
b, c, Nissl staining in rats injected with A40-s
(b) and A43-s (c) shows
that there is no greater level of cell loss than that observed with the
vehicle-injected rats. d, h, Nissl staining in a rat
injected with A40-d (d) shows a small lesion
that is likely to be caused by the increased volume injected, because
there is no accompanying presence of aggregated amyloid detected with
Thioflavin S staining (h). e, f,
In rats injected with A43-d (e) or the
combined injections (f), there is the presence of
a lesion that is exacerbated by aggregated material. The level of cell
loss and damage made in the dentate gyrus and the amount of aggregated
material observed in and around the lesion were similar in these two
groups. i, An example of Thioflavin S positively stained
aggregated material outside of the lesion in a rat injected with
A43-d is shown. j, An example of aggregated material
in and around the site of the lesion in a rat injected with the
combined peptides is shown. Scale bars: a-f, 500 µm;
g-j, 200 µm.
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In summary, focal injections of a combination of A peptides induced
an impairment in working memory and deficits in both synaptic
transmission and plasticity. The same quantity of each peptide and
double the quantity of A40 alone did not induce these deficits,
whereas double the quantity of A43 had a similar profile of effect.
Importantly, it was only in rats receiving the combined injections or
the high dose of A43 that we observed the presence of aggregated
amyloid deposits, suggesting that the presence of these amyloid
deposits induced a functional deficit. The data, therefore, support the
theory postulated by Jarrett and Landsbury (1993) that small quantities
of A43 can accelerate the formation of aggregated amyloid. The fact
that the high dose of A43 produced a profile similar to that of the
combined injections but the high dose of A40 was ineffectual
confirms the dose-dependent self-aggregating properties of A43, of
which A40 is not capable (Snyder et al., 1994), and suggests that
A43 is necessary for the nucleation of the amyloid material.
Although the focal injections induced small lesions at injection sites,
probably because of the pressure of the injections or the volume of
fluid, our data suggest that the lesion alone could not account for the
functional deficits because these deficits were not observed in
vehicle-injected rats compared with noninjected rats. Importantly, the
lesions appeared to be exacerbated and stained positive for aggregated
amyloid material in those rats injected with combination of peptides
and the high dose of A43. This suggests a potential aggravated
effect of lesion and aggregated deposits and agrees with experimental
data showing that small ibotenic acid lesions and injections of
A25-35 in combination induce behavioral deficits in rats that do
not occur when each is given individually (Dornan et al., 1993).
Distributed injections of amyloid peptides in the dorsal
dentate gyrus
The next set of experiments were designed to characterize further
the behavioral and electrophysiological effects induced by aggregated
-amyloid deposits in the dentate gyrus. For this, we selected the
combined A40-A43 peptide injection protocol because it reliably
induced aggregated -amyloid deposits with low doses of the peptides
that, alone, were ineffective. In this specific experiment, we used
distributed injections along the dorsal part of the dentate gyrus with
the same overall quantity of peptides as in the previous experiment but
a reduced volume per site (see Materials and Methods), in an attempt to
minimize the mechanical lesion produced by injections and to generate
aggregated -amyloid deposits over a wider rostrocaudal portion of
the dentate gyrus to assess the magnitude of the behavioral deficit. In
the first learning experiment using a mixed task, we found that
aggregated -amyloid deposits in the dentate gyrus were associated
with a deficit in learning the working but not the reference memory
component. Previous studies that have used colchicine injections or
adrenalectomy to investigate the specific contribution of the dentate
gyrus to spatial learning have in general shown rather mixed results, with deficits in both reference (Sutherland et al., 1983; Wishaw, 1987;
Barone et al., 1991; Conrad and Roy, 1993) and working (Jarrard et al.,
1984; Vaher et al., 1994) memory. We therefore extended the repertoire
of spatial tasks to characterize the learning deficits produced by the
presence of aggregated -amyloid deposits.
Spatial learning on the radial arm maze
We first tested rats on the same radial arm maze task used in the
previous experiment, 4 weeks after injections, to determine whether the
same deficit in learning could be reproduced using distributed
injections of either the combined fragments (n = 27) or
the vehicle solution (n = 25). In the working memory
element of the task, rats injected with the vehicle solution learned
the task in much the same manner as did the control group in the
previous experiment. Rats injected with the combined fragments showed, once again, a modest but significant deficit in learning compared with
the vehicle control group (F(1,52) = 6.33; p < 0.05; Fig. 5a). Importantly, the deficit
shown by the rats injected with the fragments had a pattern very
similar to that of the group with focal injections of the combined
fragments, where the major deficit was in the early phases of learning,
and in fact there was no difference in the performance level in these
two groups (F < 1). As with the focal injections, rats
injected with the fragments showed no significant deficit in learning
the reference memory element when compared with vehicle-injected
controls (F(1,52) = 3,42;
p > 0.05; Fig. 5b). These data suggest that
the specific deficit in working memory induced by amyloid deposits in
the dentate gyrus, although modest, is highly replicable regardless of
whether the injections are made in a focal or distributed manner.
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Figure 5.
The performance of rats with distributed
injections of the combined fragments
(A-c) or vehicle solution in the
radial arm maze task. a, Rats injected with the
fragments were impaired in the working memory aspect of the task, in
much the same manner as were the rats with focal injections of the
combined peptides. b, Also similar to the rats with
focal injection, those with distributed injections of the combined
fragments were not impaired in the reference memory task. The
y-axis denotes the total number of reentries made in the
working memory element (a) and entries into
nonbaited arms in the reference memory element
(b) of the task. CT,
Control.
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Spatial navigation in the open-field water maze
To confirm further whether reference memory was unaffected by the
injection of fragments, a subgroup of the rats tested on the radial arm
maze was then tested in a spatial navigation task in the water maze (7 weeks after injection). By the use of a protocol in which rats were not
overtrained (two trials a day for 7 d), there was no significant
difference between rats injected with the combined peptides
(n = 10) and the vehicle-injected rats
(n = 8) in the latency to escape the water
(F < 1; Fig. 6). On the probe trial, both groups spent significantly more time in the training
quadrant (controls, F(3,24) = 3.06;
p < 0.01; combined fragments,
F(3,36) = 3.40; p < 0.05), and there was no difference in the amount of time spent in the
training quadrant between groups (F < 1; Fig. 6). When
the escape platform was repositioned to the opposite quadrant, both
groups learned the new location of the platform equally well (data not
shown).
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Figure 6.
Spatial navigation in the water maze. No
impairment was observed in rats injected with combined fragments
(A-c; open circles or
bars) compared with the control group receiving
vehicle (VH; closed circles or
bars) in either the acquisition phase
(a) or the probe trial measured 24 hr later
(b). Both groups spent significantly
(double asterisks) more time in the training
quadrant than in the others. Ar, Adjacent right;
Tg, target; Al, adjacent left; Opp,
opposite.
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To pursue further the nature of the working memory deficit, rats were
tested either on a classical working memory task or on a delayed
match-to-place task on the radial arm maze between 10 and 12 weeks
after injection.
Working memory on the radial arm maze
Rats were tested on a strict working memory task on the radial arm
maze using a standard elimination task that places a greater demand on
spatial working memory. Vehicle-injected rats (n = 11) learned the task rapidly, reaching asymptote after approximately six
trials (Fig. 7). In contrast, rats
injected with the combined fragments (n = 8) were
slower to learn the task but eventually attained the same level of
learning as the controls with overtraining. ANOVA confirmed this
learning deficit by the number of reentries made
(F(1,17) = 15.51; p < 0.01; Fig. 7). As anticipated, the deficit in performance on this task
was more severe than that shown in the working memory element of the
spatial learning task, but once again the deficit was restricted mainly
to the early phases of training.
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Figure 7.
Working memory in the eight-arm elimination task.
Rats injected with the combined fragments
(A-c) were impaired
(asterisks) at the beginning of training
compared with control rats (CT) but attained a
similar level of performance with overtraining. The
x-axis denotes the number of trials, and the
y-axis denotes the number of reentries.
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Delayed match-to-place on the radial arm maze
Because the behavioral impairment observed in rats injected with
combined peptides is of a working memory nature, we tested whether it
was a general impairment or whether it was subject to a temporal
component. We interposed different times between the forced, sample
trial and the choice trial. Rats injected with the fragments
(n = 9) made significantly more errors than did vehicle-injected rats (n = 9) at the short time delays
(zero delay, F(1,57) = 4.41;
p < 0.01; and a 10 min delay,
F(1,88) = 9.16; p < 0.01; Fig. 8). At the longer time delays
of 20, 60, or 180 min, there was an increasing number of errors made by
the control rats, resulting in no further difference in performance
between the two groups (all F values > 1; Fig. 8). We
also observed a strong correlation with the increasing number of errors
made by the control group with the increasing delay (r = 0.93; p < 0.01). This was not the case with rats
injected with fragments, who tended to make as many errors at short
delays as they did with the long delays except with a 180 min delay in
which they made more. Analysis of the level of accuracy in the choice
trial showed that the angular deviation from the goal arm increased
from 45 to 135° in a delay-dependent manner in control rats. Both
angular deviation and the number of arms visited before a correct
choice was made were increased in rats injected with the combined
fragments at the 10 min delay, suggesting a deficit in retaining
information about the recently visited arm.
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Figure 8.
Delayed match-to-place on the radial arm maze.
Rats injected with combined fragments
(A-c) were impaired at a 0 and 10 min
delay (asterisks) compared with the control
group (CT). When the delay was extended (20-180
min), a similar increase in the number of errors was observed in both
the CT group and rats injected with the combined
peptides.
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Thus, considering the data as a whole, injections of the combined
peptides appear to impair short-term or working-type memory processes,
leaving long-term or reference memory processes intact. The deficit is
restricted to the early phases of learning because the rats injected
with peptides tend to recover their ability to learn with additional
training. Although previous data have shown that rats with damage to
the dentate gyrus are impaired in reference memory, particularly
spatial navigation in the water maze (Sutherland et al., 1983; Wishaw,
1987; Barone et al., 1991; Conrad and Roy, 1993), others have shown
working memory deficits (Jarrard et al., 1984; Vaher et al., 1994) or
evidence of dentate granule cell activation during learning of a
working memory task (Friedman and Goldman-Rakic, 1988; Vann et
al., 2000). We do not know what cellular or molecular mechanisms may be
rendered dysfunctional with injections of -amyloid peptides, but it
is conceivable that normal processing of information in the dentate
gyrus is perturbed but not disrupted totally.
Synaptic transmission and plasticity
After behavioral testing, electrophysiological analysis was
undertaken in a subset of the rats to verify whether the distributed injections of the combined fragments induced a correlated deficit in
synaptic transmission and plasticity. Analysis of input-output curves
showed no overall significant difference in fEPSP at the different
intensities between rats injected with the combined fragments
(n = 8) and the control group (n = 5;
F(1,12) = 2.18; p > 0.05); however, there was a significant
interaction (p < 0.01), and clearly rats
injected with fragments showed consistently a smaller fEPSP for each
intensity after the fourth intensity (Fig. 9a). These results thus
replicate the alteration in synaptic transmission found in the first
experiment, although to a more limited extent. The population spike was
slightly reduced, but there was no significant difference at any
intensity (all p values > 0.05; data not shown). There
was no difference in the intensities used for generating the I-O
curves (range for A-c, 64 ± 11 to 726 ± 16 µA; for
vehicle, 76 ± 9 to 720 ± 40 µA; F < 1).
Tests of paired-pulse facilitation and inhibition in a sample of
animals did not reveal any alteration in short-term presynaptic
plasticity or inhibitory processes (data not shown).
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Figure 9.
Synaptic transmission and LTP in rats with
distributed injections of vehicle (Vh) or combined
peptides (A-c). a, I-O
curves of the fEPSP show that rats injected with
A-c have a significantly smaller
increase in fEPSP when compared with Vh-injected rats.
This effect is similar to that observed with focal injections. The
y-axis denotes the change in the slope of the fEPSP in
millivolts per millisecond, and the x-axis denotes
increasing intensities (increments of 60 µA). b,
Sample responses from rats injected with the vehicle solution and
combined peptides before and after (arrowhead) induction
of LTP are shown. The left-hand panel shows potentiation
30 min after induction of LTP, and the right-hand panel
shows potentiation 3 hr after induction of LTP. c, Rats
injected with the combined peptides show normal induction of LTP of the
fEPSP, but this declined back to basal levels 3 hr after the tetanus.
The y-axis denotes the percentage of change in fEPSP
slope. d, In contrast, the spike amplitude was
potentiated, although significantly less than in the vehicle-injected
rats, and the level of potentiation was maintained for the duration of
recording. The y-axis denotes the change in spike
amplitude in millivolts, and the arrow indicates when
the tetanus was delivered.
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In the first experiment, we found that LTP was greatly reduced after
the combined fragments were injected, but it was difficult to determine
whether this was entirely caused by an alteration in the mechanistic
properties of LTP or, at least in part, by the alteration in synaptic
transmission. In an attempt to dissociate these possibilities in the
present LTP experiment, and because there was much less difference in
the I-O curves, we were in a position to select stimulus intensities
that were comparable between groups and that gave rise to similar
response amplitudes. Thus, in this experiment, neither the baseline
fEPSP slope (F(1,12) = 1.78;
p > 0.05) and spike amplitude (F < 1)
nor the stimulus intensity used to generate the responses
(F(1,12) = 4.21; p > 0.05) was significantly different. After the tetanus, we found that LTP
could be induced in rats injected with combined fragments to more or
less the same extent as in the control rats, with no significant
difference in the magnitude of LTP between the two groups
(F(1,12) = 4.29; p > 0.05). Although LTP was induced in the rats injected with the combined
peptides, it was not long lasting and had decayed essentially to basal
levels by the end of recording (Fig. 9c). We also observed
potentiation of the population spike in rats injected with the
peptides. This was significantly less than that observed in the control
rats (F(1,12) = 5.25;
p < 0.5), but it remained stable throughout the
recording period (Fig. 9c), an effect that has also been
observed in APP transgenic mice (Chapman et al., 1999). Analysis of the
input-output curves allowed examination of the EPSP-spike (E-S)
relationship, before and after LTP. In both groups of rats there was a
shift to the left in the E-S curves after LTP (data not shown) that is
a classic indication of E-S potentiation (Abraham et al., 1985). Thus,
despite the decline in potentiation of the EPSP, the maintenance of a certain amount of spike potentiation may reflect intact E-S
potentiation in the rats injected with peptides.
Thus, we confirm that the injection of combined peptides induced an
alteration in synaptic transmission and plasticity in the dentate
gyrus. With small quantities and distributed injections, the alteration
in synaptic transmission was less severe. We believe that the
alteration of LTP maintenance observed here cannot be accounted for by
the decrease in synaptic transmission that was also observed, because
the fEPSP and spike were of similar magnitude and LTP was induced to a
level similar to that of controls. Presumably, the more detrimental
effects found with focal injections were at least in part caused by the
magnitude of damage induced in the dentate gyrus around the site of
recording. We conclude that the injection of the combined fragments
affected both synaptic transmission and some, as yet unknown, mechanism
underlying the maintenance of LTP.
Anatomical assessment
Immunocytochemical procedures were performed on a sample of brains
from rats injected with the peptides (n = 19) or the
vehicle solution (n = 15) ~16 weeks after injections.
Within each group all rats showed a similar profile. As with the rats
given focal injections of the peptides, we found strong Thioflavin
S-positive staining (Fig.
10g). Deposits of aggregated
material were observed near and between the injection sites; whereas
the deposits were relatively restricted with focal injections, they
covered a wider extent over the complete dorsal dentate gyrus.
Immunohistochemical analysis showed A immunoreactivity (Fig.
10f,h) confined mainly to the cell layer, whereas no
staining was observed at the injection site in control rats (Fig.
10a-c).
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Figure 10.
Representative examples of pathological markers
of AD in the dentate gyrus from a rat injected with the vehicle
solution (a-c) or with the combined peptides
(d-j). a-c, Serial sections from a
vehicle-injected rat show the site of injection
(arrowhead) marked with Nissl staining
(a), minimal cell loss restricted to the site of
injection (arrowhead) with NeuN
(b), and no A immunoreactivity
(c). d, e, Serial sections from a
rat injected with the combined fragments and stained with Nissl
(d) or NeuN (e) show cell
loss near the injection site (arrowhead) that is more
extensive than that observed with vehicle-injected rats. Note the
thinning of the cell body layer in the upper blade of the dentate
gyrus, indicating additional cell loss away from the site of injection.
f, h, A immunoreactivity shows the presence of
amyloid material in the dentate gyrus corresponding with the site of
injection observed with Nissl and NeuN (f) and
shows amyloid material further from the site of injection
(h). g, i, j, Adjacent sections to
f show Thioflavin S-positive staining, suggesting that
the amyloid material is aggregated (g), and
inflammation reactivity around the site of injection (i,
j). i, Extensive GFAP-positive staining forming
a virtual wall around the site of the injection is shown.
Inset, An astrocyte with swollen processes, magnified
1000×, is shown. j, OX-42 immunostaining in the
adjacent section shows strong microglial reactivity in the site of the
injection. Inset, An example of a microglia magnified
1000× is shown. Scale bars: h, 100 µm; a-g,
i, j, 200 µm.
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Because a common feature of Alzheimer pathology is inflammation
(Akiyama et al., 2000), we used GFAP and OX-42 immunostaining to detect
signs of astrocytosis and activation of macrophages and microglia. We
observed a GFAP- and OX-42-immunopositive reaction in the
vehicle-treated rats, which, in general, was restricted to the
injection site and is likely to have been caused by the mechanical
damage. There was, however, extended reaction in the hippocampal tissue
in rats injected with the peptides (Fig. 10i,j) showing
intense astrogliosis and microgliosis, characterized by activated
astrocytes with swollen processes observed around the amyloid deposits,
forming a virtual wall around the deposits. We found no discernible
reduction in synaptophysin immunostaining, suggesting that presynaptic
terminals were intact in rats injected with fragments.
Importantly, with distributed injections we found no overt presence of
a lesion as observed with the focal injections; however there was a
greater extent of cell loss distributed across the dorsal dentate gyrus
(Fig. 10d,e), extending 100-500 µm. We suspect that this
extent of cell loss is caused by the nature of the distributed injections rather than a spreading of the amyloid material because we
observed no loss of cells in regions CA3 and CA1. The fact that lesions
were observed in rats with focal injections of the combined peptides
and the high dose of A43 may well account for the difference in
synaptic transmission and plasticity observed with focal and
distributed injections. With focal injections, the damage was
restricted over a relatively smaller region, and this may well have
sufficiently altered the local cellular environment to impair synaptic
transmission and both induction and maintenance of LTP more severely.
In summary, the anatomical observations made here suggest that
injections of the combined fragments appear to mimic some of the
pathology of AD that includes hippocampal atrophy (Fox et al., 1996),
the presence of aggregated material (Selkoe, 1991), and ongoing
inflammation of neuronal tissue (Akiyama et al., 2000). In our model,
these neuropathological signs were stable and observed between 7 and 16 weeks after injections, and this appears to induce dysfunction in both
synaptic transmission and plasticity and impairment in the early phases
of learning requiring spatial working memory.
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DISCUSSION |
The aim of these experiments was to assess how formation of senile
plaques may specifically contribute to memory deficits associated with
AD pathology. To this end, we tested the ability of rats to learn a
variety of hippocampal-dependent learning tasks and measured synaptic
transmission and plasticity in the dentate gyrus, using a model of
plaque formation proposed by Jarrett and Landsbury (1993). We injected
amyloid peptides into the dentate gyrus because it is one of the first
regions to show plaques in humans with AD (Duyckaerts et al., 1997). We
found that a single quantity of either the short (A40) or the long
(A43) form of the peptide had negligible effects on the development
of neuropathological signs of AD, spatial learning or synaptic
transmission and plasticity in the dentate gyrus. In combination,
however, peptides induced modest but reliable deficits in spatial
working memory, impaired both synaptic transmission and plasticity, and
caused clear neuropathological signs of AD, characterized by the
presence of aggregated amyloid material, loss of cells, and activation
of macrophages and glia. Double quantities of A40 had a profile
similar to that of the single quantity, inducing no discernible
effects, whereas double quantities of A43 had a profile similar to
that shown with the combination of fragments as expected, based on its
aggregation kinetics. Thus we show, for the first time, that the
hypothesis proposed by Jarrett and Landsbury (1993) that A43 can
promote the formation of amyloid deposits can be demonstrated in
vivo and has functional consequences. Although promotion of plaque formation has been demonstrated using TGF (Frautschy et al., 1996),
heparin sulfonate proteoglycan (Snow et al., 1994), and apolipoprotein E (Wisniewski et al., 1994) in the
presence of A40, it seems particularly relevant to use A43
because it constitutes the nidus of plaque formation, being present in
the brain at relatively high concentrations before the onset of
Alzheimer pathology (Walker et al., 2000).
As with most studies using infusion or injection of amyloid fragments,
most of the damage observed with combined peptides was restricted to
the sites of injection (Frautschy et al., 1996), with a presence of
reactive glia and macrophages routinely observed around amyloid
deposits (Selkoe, 1991). In addition, we found either no staining with
both doses of A40 or very weak staining with the low dose of A43.
This is not surprising because all peptides, whether injected
individually or in combination, were in a soluble condition in our
experiment, and it has been shown that A40 is rapidly cleared from
the brain (Games et al., 1992; Greenberg, 1995). In contrast, positive
staining of aggregated material has been observed with A40 when
injected in a preformed state (Frautschy et al., 1991; Giovanelli et
al., 1995; Weldon et al., 1998). Although mechanisms of aggregation and
toxicity appear to be similar for both A40 and A43 (May et al.,
1992; Pike et al., 1993), A43 is more susceptible to aggregation
than is A40 (Yankner et al., 1990; Pike et al., 1991) and is
deposited in the brain before A40 (Iwatsubo et al., 1994). Thus, in
our study, whereas A43 at the low dose was weakly stained,
suggesting the presence of diffuse material, the high dose was
sufficient to form aggregates, as was expected (see Snyder et al.,
1994).
Relatively few studies have reported behavioral effects of injections
of A40 or A43 into the brain, but the results have varied
dramatically, showing deficits in the acquisition of reference memory
tasks (Nitta et al., 1994, 1997), consolidation of learning (McDonald
et al., 1994, 1996), object recognition (Giovanelli et al., 1995), and
working memory (O'Hare et al., 1999; Yamada et al., 1999). This is not
surprising because of the variation in choice of peptide length used,
mode of delivery, vehicle solution, delay between injection and
behavioral testing, and state of aggregation of peptides at the time of
injection. In our experiments, we tested rats at least 1 month after
injections and found that the combined fragments, regardless of whether
they were focal or distributed, induced behavioral deficits that were
specifically related to working memory, leaving reference memory
intact. It is worthy of note that in those experiments showing working
memory deficits, they were induced by injections of A42 (O'Hare et
al., 1999; Yamada et al., 1999) and not A40 (McDonald et al., 1994;
Cleary et al., 1995). More important, however, the type of deficits we observed is in keeping with those observed in the early stages of AD,
which mainly concern short-term and working memory (Folstein and
Whitehouse, 1983; Hyman et al., 1986; McDonald and Overmier, 1998).
Another candidate animal model of AD is the transgenic mouse
overexpressing APP that shows elevated levels of both A40 and A43. In studies in which these mice have been tested behaviorally, deficits have been reported in both working memory (Moran et al., 1995;
Chapman et al., 1999) and spatial navigation (Moran et al., 1995; Hsiao
et al., 1996; Nalbantoglu et al., 1997; Berger-Sweeney et al., 1999).
Although we find no deficit in spatial navigation, amyloid deposits in
our experiments were primarily restricted to the dentate gyrus, whereas
they are more widespread in transgenic mice, particularly in CA1, a
region involved in encoding elements of spatial orientation (McNaughton
et al., 1991). Thus in our case, the deficits observed in working
memory mimic those observed in early AD more closely.
In terms of synaptic transmission and plasticity, we found rats
injected with combined fragments were deficient in both. Importantly, we found that focal injections induced a deficit in the induction phase
of LTP, which was not the case with distributed injections. Focal
injections were used to concentrate pathology around the region of
recording, whereas distributed injections were used to maximize the
region of pathology over the rostrocaudal extent of the dorsal dentate
gyrus. This may well explain the more severe alteration in synaptic
transmission and the early phase of LTP with focal than with
distributed combined peptides.
We do not believe that the deficit observed in normal transmission and
plasticity is related to presynaptic malfunction because we found no
difference in the synaptophysin reactivity characteristic of
degenerating terminals; induction of LTP was normal using distributed injections, and preliminary evidence suggests that paired-pulse facilitation, a measure of presynaptic function, was not affected. To
date we know of no report describing effects of synthetic peptides on
synaptic transmission and LTP in the dentate gyrus. In a comparable study in CA1 in vivo, a reduction in synaptic transmission
with no deficit in synaptic plasticity was observed, but this was 24 and 48 hr after injections of A40 alone (Cullen et al., 1997).
In APP transgenic mice, a few studies in the CA1 slice have shown
contradictory results as to whether synaptic transmission (Hsia et al.,
1999; Larson et al., 1999) or synaptic plasticity is impaired
(Nalbantoglu et al., 1997; Chapman et al., 1999), discrepancies that in
all likelihood relate to variables such as strain difference, type of
mutation, and level and location of APP expression (Hsiao,
1998). In our experiments we found a clear deficit in the maintenance
of LTP, which gains support from in vivo studies in the
dentate gyrus showing similar decremental LTP in transgenic mice
(Chapman et al., 1999). Exactly how these peptides induce anomalies in
the circuitry and impair synaptic plasticity is not known, but several
proposals suggest malfunctioning of calcium homeostasis (Mattson et
al., 1992) or downstream kinase signaling or gene cascades (Chapman et
al., 1999).
AD is initiated in the entorhinal cortex, and as a result the first
circuit to become dysfunctional is the perforant path dentate gyrus
synapse (Braak and Braak, 1991; Gomez-Isla et al., 1996). A hypothesis
proposed by Mesulam (1999) suggests that deficits in plasticity precede
cognitive impairment. He suggests that the mutations in genes involved
in AD pathology and various environmental and risk factors that
contribute to the pathology lead to malfunctioning of the capacity of
the brain for plasticity. With age, cognitive and mnemonic-induced
modification places an excess burden on plasticity and, in essence,
exceeds the threshold of normal functioning, accelerating events
leading to brain pathology, and is responsible for the cognitive
deterioration. Our in vivo data support this hypothesis and
suggest that A toxicity induces deficits in synaptic transmission
and plasticity, giving rise to learning difficulties.
In summary, we show in vivo that small quantities of A43
promote formation of stable aggregated amyloid deposits and
inflammatory responses when injected in combination with A40 in the
hippocampus. In AD, plaques are first observed in the molecular layer
of the dentate gyrus. The behavioral deficits we observed are of the type observed in the early onset stage of AD, correlated with alteration in synaptic function and plasticity. Although aged transgenic mice mimic genetic forms of AD, single injections of combined peptides into the brains of young adult rats provide the
opportunity to investigate, without contamination of aging components,
specifically how aggregated amyloid may contribute to the cognitive
deficits and how cellular activity may be altered in the prevalent,
sporadic form of AD. Thus, it prese |
TOP
ABSTRACT
INTRODUCTION
