Department of Physiology, Faculty of Medicine, Toyama Medical and
Pharmaceutical University, Toyama, Japan
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Introduction |
The
decline of memory and other cognitive functions in patients with
Alzheimer's disease or cerebrovascular diseases is of great clinical
and social concern. (1R)-1-benzo [b]
thiophen-5-yl-2-[2-(diethylamino) ethoxy] ethan-1-ol hydrochloride,
or T-588, was developed for treatment of these neurodegenerative
diseases. T-588, characterized by high permeability through the
blood-brain barrier (Ono et al., 1993
), has been shown to improve
learning and memory deficits in rats. For example, T-588 significantly
improved learning by rats in the three-panel runway task (Ono et al.,
1994) and spatial learning task (Nakada et al., 1995) following
scopolamine treatment and transient forebrain ischemia, respectively.
T-588 also ameliorated the impaired performance of rats in active or
passive avoidance tasks following cerebral embolization and basal
forebrain lesion (Ono et al., 1995b).
To evaluate T-588's action in these findings, in vitro and in vivo
studies have been performed. In a microdialysis study, single oral
administration (3-30 mg/kg) of T-588 significantly increased the
amount of acetylcholine (ACh) and noradrenaline (NA) in the hippocampus
and neocortex (Ono et al., 1995a). T-588 was also found to increase NA
release in hippocampal slice preparation (Ono et al., 1995a). The
action of T-588 to increase neurotransmitter (or neuromodulator)
release could alleviate the cognitive dysfunction observed in patients
with neurodegenerative disorders, since several kinds of
neurotransmitters in the brain, especially in the hippocampus, declined
in these patients (Adolfsson et al., 1979; Whitehouse et al., 1981).
Furthermore, T-588 produced slow and long-lasting depolarization,
different from the mechanism of ACh, by affecting resting membrane
potential and conductance in CA1 pyramidal cells of rat hippocampal
slice preparation, probably mediated by suppression of potassium
currents (Kimura et al., 1999). This action may increase cell
excitability and may be involved in beneficial effects of T-588 on
cognitive dysfunction (Ghelardini et al., 1998). Since T-588 has such
effects on hippocampal slice preparation, it may influence synaptic
efficacy in vivo when it is applied systemically.
Changes in synaptic efficacy have been proposed as a cellular basis for
learning and memory. Long-term potentiation (LTP) is known as a
long-lasting enhancement of synaptic efficacy (Bliss and Collingridge,
1993). Previous studies have shown LTP induction in several kinds of
learning and memory tasks in vivo (Laroche et al., 1989; Bramham et
al., 1994; Doyère and Laroche, 1996; Rogan et al., 1997).
Furthermore, blocking the
N-methyl-D-aspartate receptor prevents
not only LTP induction, but also spatial learning in the water maze and
radial-arm maze tasks (Abraham and Mason, 1988; Butelman, 1989; Shapiro
and Caramanos, 1990). On the other hand, different forms of
physiological control for synaptic function related to learning and
memory have been characterized, for example, paired-pulse facilitation
and inhibition. Indeed, using paired-pulse stimulation, short-term
synaptic efficacy is likely to change when rats are exploring a novel
environment (Moser, 1996). Rapid adjustment of such temporal synaptic
processing has been assumed for another form of synaptic plasticity,
i.e., short-term synaptic plasticity.
As mentioned above, the increase of ACh and NA release by T-588 could
be a reason for the amelioration of the learning and memory deficits
observed in the previous in vivo studies. However, little is known
concerning the effects of T-588 on the electrophysiological parameters
for synaptic plasticity. Considering the alleviatory effect of T-588 in
the previous studies, we believe it is important to test whether T-588
affects synaptic plasticity (short-term, long-term, or both) in vivo.
Therefore, in the present study, we investigated the effects of T-588
on induction and maintenance of LTP as indices of long-term synaptic
plasticity, and responses to paired-pulse stimulation as an index of
short-term synaptic plasticity in the dentate gyrus (DG) of freely
moving rats. Our results showed that T-588 did not affect the
short-term synaptic plasticity, but enhanced long-term synaptic
plasticity in the DG-perforant pathway. A preliminary report has
already been published in abstract form (Yamaguchi et al., 1999).
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Materials and Methods |
Subjects.
Forty-four male Fischer/344 rats (Japan SLC, Inc.,
Hamamatsu, Japan), weighing 211-259 g at the time of surgery, were
used for the experiment. Rats were housed individually in a
temperature-regulated room (24 ± 1°C) and maintained on a 12-h
light/dark cycle with food and water available ad libitum. They were
handled and weighed daily at around 9:00 AM.
Surgery.
Rats were anesthetized with sodium pentobarbital
(50 mg/kg i.p., supplemented as necessary during surgery) and placed
into a stereotaxic apparatus. The scalp was incised at midline and the
skull was exposed. Lambda and bregma were positioned in the horizontal
plane and 1.5-mm diameter holes were drilled to allow for placement of
the electrodes. Recording and stimulating electrodes were constructed
from pairs of twisted Teflon-coated, 125-µm diameter stainless steel
wires (A-M Systems, Inc., Everett, WA). The recording electrode
consisted of wires cut flat at different lengths with a 2-mm vertical
distance between the tips. The bipolar stimulating electrode had 0.5-mm
tip separation and was uninsulated with 0.5 mm at the tip. The
recording electrode was inserted into the right-side DG (4.2 mm
posterior to bregma and 2.5 mm lateral to the midline). The stimulating
electrode was inserted into the perforant-path fibers on the same side
as the recording electrode (7.8 mm posterior to bregma and 4.4 mm
lateral to the midline). The electrodes were fixed at the location in
which the population spike (PS) amplitude and field excitatory
postsynaptic potential (fEPSP) slope recorded in the DG in response to
the perforant-path stimulation were maximized. Two screws with a
stainless steel wire were implanted into the skull over the cerebellum
as the ground electrodes. All electrodes were connected to pins of a
lightweight plastic miniature socket as a head-stage and fixed to the
skull with dental acrylic. Rats were given at least 8 days for recovery
before electrophysiological testing. Animals were treated in strict
compliance with the National Institutes of Health Standards for
Treatment of Laboratory Animals and the Guiding Principle for the Care
and Use of Animals approved by the Council of the Physiological Society
of Japan.
Electrophysiological Recording Apparatus.
All the recordings
were performed after the rat had been transferred from the home cage to
a recording box (30 × 30 × 30 cm) with woodchip bedding.
The head-stage of the rat was connected to a flexible, shielded cable
containing field-effect transistors (2SK389; Toshiba, Tokyo, Japan).
The cable allowed the rat to move freely in the recording box. Evoked
responses were collected while the rat was motionless and awake with
its eyes open. The electroencepharography signal through the recording
electrode was monitored on an oscilloscope (VC-10, Nihon Kohden, Tokyo, Japan). To confirm that the rat was awake, sensory stimuli (tapping sidewall of the recording box, etc.) were sometimes presented.
Evoked responses were generated by single biphasic square pulses of
100-µs duration per half-wave using a constant current isolation unit
(SS202J; Nihon Kohden, Tokyo, Japan). The signals from the recording
electrode were amplified (×100), filtered (bandpass from 0.1-10 kHz)
and digitized at 50 kHz through an A/D converter (DAQcard-1200,
National Instruments, Austin, TX). Waveforms, displayed on a PC screen
(MN-370-X20; Sharp, Tokyo, Japan), were stored in the hard disk online.
The amplified and filtered signals were also monitored on an
oscilloscope and stored on a digital audiotape by a digital audiotape
recorder (RD-125T; TEAC, Tokyo, Japan) for off-line analysis.
Drug Treatment.
T-588 (Fig. 1)
was obtained from Toyama Chemical Co., Ltd. (Tokyo, Japan) and
dissolved in distilled water. T-588 solution was given orally to each
animal at concentrations of 0.3 and 3 mg/kg at a volume of 5 ml/kg
b.wt.
Recording Protocol: Acclimatization and Input/Output (I/O) Curve
Generation.
Given that an unfamiliar environment or motor activity
may affect the PS and fEPSP recordings (Hargreaves et al., 1990;
Bramham et al., 1994; Kitchigina et al., 1997), rats were acclimatized to the recording box for 30 min/day for a 3-day period. Furthermore, rats were allowed 
30 min to habituate in the recording box before data collection on each day.
On the last day of acclimatization, I/O curves were generated with the
use of varying stimulating current intensities (25-1500 µA) to
establish the test intensity used in the subsequent experiment. In the
LTP experiment, constant test stimulus intensity was adjusted to give
an fEPSP slope of 60 to 70% of the maximum response calculated by the
probit (probability unit) method for each individual rat (PS amplitude
was approximately 1 mV). In the paired-pulse experiment, the stimulus
intensity was set at PS amplitude to produce a level 50 to 60% of the
maximum calculated by the same method as for the LTP experiment. Rats
were also habituated to the oral administration by giving vehicle
solution during the acclimatizing period.
LTP Experiment.
In this experiment, the rats were assigned
to one of the following three experimental groups: 1) vehicle only
(N = 8), 2) low-dose (0.3 mg/kg) T-588
(N = 8), and 3) high-dose (3 mg/kg) T-588
(N = 8).
Response stability was monitored for 60 min, 2 days after the I/O curve
measurement (day 
1). The next day (day 0), evoked potentials in the DG of each rat were collected for 240 min after a
30-min habituation period with a 15-min inter-recording interval (IRI)
in the first 120 min and with a 30-min IRI in the following 120 min.
Test solution (vehicle or T-588) was given orally 30 min after the
onset of the recording. High-frequency stimulation (HFS) was applied to
the perforant path 30 min after the administration of test solution
because the concentration of T-588 in the brain peaks at 30 to 60 min
when administered orally (Ono et al., 1993), and its time course is
consistent with that of the increase in ACh and NA release in
microdialysates (Ono et al., 1995a). Ten trains of 10 stimuli
[interpulse interval, 2.5 ms (400 Hz); intertrain interval, 10 s]
were used as HFS at the same current intensity as the test pulse.
Twenty-four hours (day 1) and 48 h (day 2) later, evoked
potentials were further collected for 60 min each, with a 15-min IRI
after the 30-min habituation period.
As the control for the LTP series described above, non-LTP series was
also performed using 15 rats (five rats each). The procedure was the
same as that of the LTP series, except for the absence of HFS.
Paired-Pulse Experiment.
Five rats were used in this
experiment. After the rats were acclimatized, an I/O curve was
generated for each rat as described above, and we recorded responses to
paired-pulse stimulation. Pairs of stimulus pulses were delivered at
interpulse intervals (IPIs) of 20, 25, 30, 40, 50, 70, 100, 200, 300, 500, and 1000 ms. Responses to paired-pulse stimulation were recorded
just before and 30 min after high-dose T-588 administration.
Histology.
At the end of the experiments, each rat was given
an overdose of sodium pentobarbital (i.p.). The electrode tip
placements were marked by passing 20 µA of d.c. through the tips.
Each rat was then perfused intracardially with 0.9% saline containing
heparin, followed by 10% buffered formalin. The brain was removed and
fixed in the same solution, and placed in 30% sucrose and 10%
formalin solution overnight before section. The brain was cut into
60-µm-thick slices on a freezing microtome (coronal section)
and stained with cresyl violet to verify electrode positioning (Fig.
2).
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Fig. 2.
Photomicrographs of a 60-µm-thick coronal section
indicating the sites of stimulating (A) and recording (B) electrodes.
Note that the stimulating electrode was implanted in layer II of the
entorhinal cortex (A), and the recording electrode was implanted in the
granule cell layer of the dentate gyrus (B). Scale bars: 500 µm.
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Data Analysis.
The extracted parameters (PS amplitude, fEPSP
slope, and PS onset latency) were analyzed as follows (Fig.
3). PS amplitude (mV) was measured as the
distance of a vertical line from the negative peak to a tangent line
drawn between spike onset and offset. The slope of fEPSP (mV/ms) was
measured as the inclination between 20 and 80% of the first positive
peak amplitude. PS onset latency (ms) was defined as the time from
stimulatory artifact to first positive peak. For each time point during
the experiment, average and S.E.M. were calculated from the data on
eight (for LTP and basal synaptic response experiments) or five (for
I/O curve recordings and paired-pulse experiments) successive evoked responses (0.033 Hz). A mean value of responses at three time points on
day 0 (0, 15, and 30 min after the onset of recordings) was defined as
the baseline (0%). Subsequent data were expressed as the percent
change from the baseline.
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Fig. 3.
Evoked potential parameters. A typical waveform of
evoked potential and definition of analyzed parameters; PS amplitude,
PS onset latency, and fEPSP slope in the dentate gyrus of freely moving
rats. PS amplitude (mV) was measured as the distance of a vertical line
from the negative peak to a tangent line drawn between spike onset and
offset (c). Field EPSP slope (mV/ms) was measured as the inclination
between 20 and 80% value of the first positive peak amplitude (b/a).
PS onset latency (ms) was defined as the time from stimulatory artifact
to first positive peak (d).
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Statistical analysis was performed as follows. In the LTP and basal
synaptic response experiments, to confirm whether there are differences
in the absolute values of each parameter at baseline and stimulus
intensity for the three groups, one-way analysis of variance (ANOVA)
was performed. Two-way ANOVA was used to determine changes from the
baseline after HFS and statistically significant differences were
evaluated further by a two-tailed Dunnett's post hoc test. In the
paired-pulse experiment, data on each IPI before and after T-588
administration were analyzed using one-way ANOVA. Statistically
significant differences were evaluated at P < 0.05.
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Results |
LTP Experiment
Stimulus Intensity and the Mean Absolute Value of Baseline.
Prior to evaluating the effect of T-588, we attempted to determine
whether differences were present in the stimulus intensities defined by
I/O responses and mean absolute values of baseline for each parameter
of evoked potentials between groups (see Data Analysis).
Figure 4 shows the summarized results. No
significant differences in stimulus intensity were observed among the
three groups either in the absence of LTP [F(2,12) = 0.082, P > 0.05] or in the presence of LTP
[F(2,21) = 0.035, P > 0.05] (Fig.
4A). No significant differences were noted among the three groups in the mean absolute value of PS amplitude (Fig. 4B), the fEPSP slope (Fig. 4C), and PS onset latency (Fig. 4D), either in the absence or
presence of LTP.
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Fig. 4.
Stimulus intensity for each rat calculated by
input-output response (A) and absolute basal values of PS amplitude
(B), fEPSP slope (C) and PS onset latency (D). Open columns,
vehicle-treated group; hatched columns, low-dose T-588-treated group;
filled columns, high-dose T-588-treated group.
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Effect of T-588 on Basal Synaptic Response.
In the non-LTP
experimental series, we tested the effect of T-588 on basal synaptic
response. As shown in Fig. 6A-b, there was a significant effect of
group [F(2,120) = 3.842, P < 0.05] on PS amplitude. Two-tailed Dunnett's post hoc comparison test revealed that PS amplitude in the high-dose (3 mg/kg) T-588 group was
slightly but significantly higher than that of the vehicle group on day
0 (P < 0.05). There was also a significant group effect for the fEPSP slope (Fig. 6B-b) on day 0 [F(2,120) = 11.251, P < 0.05] and
day 1 [F(2,60) = 4.938, P < 0.05].
The fEPSP slope of the high-dose T-588 group was significantly higher
than that of the vehicle group both on day 0 and day 1 (P < 0.05). No significant group effect was observed
for PS onset latency (Fig. 6C-b).
Effect of T-588 on Induction of LTP.
Figure
5 shows examples of changes in evoked
potentials before and after LTP induction in rats treated with vehicle
(A) or high-dose (3 mg/kg) T-588 (B). High-frequency stimulation
produced a moderate potentiation of evoked potentials in the vehicle
group. PS amplitude increased by 257.4% 15 min after HFS, which was
then decreased to 171.3% at 90 min and 133.8% at 180 min. Field EPSP slope increased by 22.4% 15 min after LTP induction and returned to
4.9% at 180 min. PS onset latency shortened by 28.2% 15 min after HFS
and 23.4% at 180 min. In contrast to this moderate potentiation seen
in the vehicle-treated rat, HFS produced a prominent potentiation in
the high-dose T-588-treated rat. PS amplitude increased by 442.8% at
15 min, 335.9% at 90 min, and 382.5% at 180 min after LTP induction.
Field EPSP slope also increased by 18.5% after 15 min and 4.9% after
180 min. PS onset latency at 15 and 180 min after HFS shortened by 28.1 and 29.1%, respectively.
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Fig. 5.
Examples of waveforms recorded preinduction and at
15, 45, 90, and 180 min, and at 24 and 48 h postinduction of LTP
in (A) vehicle-treated (stimulus intensity 170 µA) and (B) high-dose
T-588 (3 mg/kg p.o.)-treated rats (stimulus intensity 80 µA). Filled
circles indicate stimulus artifact. Arrows indicate the time of HFS.
Calibration marks indicate 5 ms and 2 mV.
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Figure 6A-a shows the effect of T-588 on
the induction of LTP as population data (means ± S.E.M.) on day
0. T-588 significantly augmented the induction of LTP for PS amplitude
in a dose-dependent manner on day 0. ANOVA indicated a significant
group effect [F(2,168) = 19.218, P < 0.05]. Post hoc testing revealed that PS amplitude in both the
low-dose (P < 0.05) and high-dose (P < 0.05) T-588 groups was significantly higher than that of the vehicle
group.
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Fig. 6.
Effects of T-588 on basal synaptic responses and LTP
recorded from the dentate gyrus of freely moving rats. The changes in
PS amplitude (A), fEPSP slope (B), and PS onset latency (C) in LTP (a;
n = 8) and basal response (b; n = 5) are shown (see Materials and Methods). Open
circles, vehicle-treated group; gray triangles, low-dose T-588 (0.3 mg/kg p.o.)-treated group; filled diamonds, high-dose T-588 (3 mg/kg
p.o.)-treated group. *Significant difference between the high-dose
T-588-treated group and the vehicle-treated group
(P < 0.05). #Significant difference between the
low-dose T-588-treated group and the vehicle-treated group
(P < 0.05).
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Administration of low-dose and high-dose T-588 (0.3, 3 mg/kg p.o.) also
significantly enhanced the increase in the fEPSP slope induced by HFS
[F(2,168) = 7.707, P < 0.05] (Fig.
6B-a), although it was not as prominent as that seen for PS amplitude.
Decreases in PS onset latency induced by HFS were not different between groups [F(2,168) = 0.868, P > 0.05]
on day 0 (Fig. 6C-a).
Effect of T-588 on Retention of LTP.
To determine the effect
of T-588 on the maintenance of LTP, we also recorded the field-evoked
potentials on the subsequent 2 days (day 1 and day 2) after LTP
induction (day 0). In the vehicle group, although the increase in PS
amplitude induced by HFS was 200.7 ± 59.9% at 15 min after LTP
induction, it decayed to 80.1 ± 37.8% on day 1 and 7.5 ± 25.2% on day 2, which was almost the same as the pre-HFS level. In the
low-dose T-588 group, the augmented PS amplitude (290.9 ± 51.0%
at 15 min after HFS on day 0) (57.0 ± 30.7% on day 1 and
26.4 ± 14.6% on day 2) decreased, following a time course
identical with that observed in the vehicle group. In the high-dose
T-588 group, a higher increase of PS amplitude generated by HFS was
maintained until day 2, compared with the low-dose group. In the
high-dose T-588 group, the augmented PS amplitude (388.8 ± 84.3%) also decreased during the subsequent 2 days (173.2 ± 40.5% on day 1 and 90.6 ± 40.2% on day 2). However, ANOVA
indicated significant group differences in PS amplitude on day 1 [F(2,105) = 10.529, P < 0.05] and
day 2 [F(2,105) = 8.905, P < 0.05].
Post hoc testing revealed that PS amplitudes of the high-dose T-588
group were significantly higher than those of vehicle group on day 1 (P < 0.05) and day 2 (P < 0.05).
Paired-Pulse Experiment.
Figure
7 shows typical waveforms of evoked
potentials by paired-pulse stimulation with the IPI at 25 (A), 100 (B),
and 300 ms (C). On the second evoked potential, a strong depression of PS amplitude was observed compared with the first evoked potential at
25-ms IPI (Fig. 7A, early paired-pulse depression). In contrast, a
clear augmentation of PS amplitude was observed on the second evoked
potential at 100-ms IPI (Fig. 7B, paired-pulse facilitation). Paired-pulse depression was again observed at 300-ms IPI (Fig. 7C, late
paired-pulse depression). Figure 8 shows
averages of data collected from the five rats for each parameter. With
increases in IPIs from 20 to 1000 ms, the typical triphasic response
pattern (early depression, facilitation, and late depression) was again observed in PS amplitude (Fig. 8A). There were no significant effects
of T-588 administration on the paired-pulse stimulation for the early
depression (at 25-ms IPI: preT-588, 61.1 ± 10.8%; postT-588,
53.9 ± 15.1%; P = 0.710), facilitation (at
100-ms IPI: preT-588, 56.9 ± 34.0%; postT-588, 54.9 ± 32.8%; P = 0.968) and late depression (at 300-ms IPI:
preT-588, 18.9 ± 7.5%; post-T-588, 28.8 ± 7.6%;
P = 0.993).
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Fig. 7.
Representative recordings elicited by paired-pulse
stimulation of the perforant path. Interpulse intervals were 25 (A),
100 (B), and 300 ms (C). Arrows indicate stimulus artifact. Calibration
marks indicate 5 ms and 5 mV.
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Fig. 8.
Effects of T-588 on short-term synaptic plasticity.
PS amplitude (A), fEPSP slope (B), and PS onset latency (PSOL; C) in
response to paired-pulse stimulation were obtained in the dentate gyrus
at varying interpulse intervals of 20 to 1000 ms. Values are expressed
as the means ± S.E.M. ratio of the second evoked potential
parameters relative to that of the first evoked potential parameters
just before (open diamonds) and 30 min after (filled diamonds)
high-dose T-588 (3 mg/kg) administration.
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In contrast to PS amplitude, only a small depression was observed in
fEPSP at shorter IPIs (20-100 ms), but facilitation of the second
evoked potential did not occur (Fig. 8B). Population spike onset
latency of the second evoked potential was longer than that of the
first evoked responses from 20- to 300-ms IPI (Fig. 8C). Neither
paired-pulse depression of the fEPSP slope nor the increase in PS onset
latency in the second evoked potential was significantly changed by
T-588.
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Discussion |
Effect of T-588 on Long-Term Synaptic Plasticity: Effect of T-588
on the Induction of LTP.
In the present study, we demonstrated
that an oral administration of T-588 potentiated the amplitude of
evoked potentials by perforant-path stimulation after HFS in the DG of
freely moving rats. The effective doses (0.3, 3 mg/kg) for this action
were the same as the doses that produced alleviatory effects on
learning and memory deficits observed in previous studies (Ono et al., 1994, 1995b; Nakada et al., 1995).
In rat brain slice preparation, T-588 enhances NA release due to
suppression of NA uptake in a calcium-independent manner (Miyazaki et
al., 1997; Maekawa et al., 1998). Moreover, T-588 (3-30 mg/kg p.o.)
significantly increases NA and ACh content in the hippocampus of freely
moving rats (Ono et al., 1995a). Anatomically, the DG receives
noradrenergic and cholinergic inputs through the projection of fibers
from the medial septum, diagonal band, and the nucleus locus coeruleus
(Loy et al., 1980; Frotscher and Leranth, 1985). Activation of
cholinergic or noradrenergic afferents enhances LTP induction at
perforant path-DG granule cell synapses (Burgard and Sarvey, 1990).
Therefore, the facilitatory effect of T-588 on LTP induction observed
in the present study could partly be generated by activation of NA
and/or the ACh pathway.
Although we observed the enhancement of LTP both in PS amplitude and
the fEPSP slope, the increase in PS amplitude was larger than that in
the fEPSP slope. PS amplitude reflects the number of granule cell
discharges excited by a given afferent input (Andersen et al., 1971),
whereas the fEPSP slope (or amplitude) is regarded as the efficacy of
the synaptic process through neurotransmitter release from presynaptic
and/or postsynaptic receptor properties (Bliss and Gardner-Medwin,
1973). It has been reported that
m1-m4 muscarinic ACh
receptor subtypes are differentially expressed in the presynaptic and
postsynaptic regions of the DG (Rouse et al., 1998). Due to the
different localization of muscarinic ACh receptors at presynaptic and
postsynaptic sites, ACh decreases the fEPSP amplitude by decreasing
glutamate release from perforant path terminals in behaving rats
(Foster and Deadwyler, 1992), whereas it increases granule cell
excitability postsynaptically (Fantie and Goddard, 1982). This
differential influence of ACh could be the differential effects of
T-588 on PS amplitude and the fEPSP slope. However, the differential
influence of ACh on presynaptic and postsynaptic (or cell excitability)
components is not consistent with the present observation of an
increase in basal EPSP responses to T-588. To explain this, it is
necessary to postulate some other mechanism, such as a common
excitatory action of T-588 on presynaptic and postsynaptic components.
It is also possible that the differential action of T-588 on fEPSP and
PS is specific for long-term synaptic plasticity, but not true for
basal synaptic responses.
Although basal synaptic response of PS amplitude and the fEPSP slope
increased slightly after T-588 oral administration, these changes were
not remarkable compared with those after LTP induction. Hirata et al.
(1999) have reported that synaptic vesicular endocytosis (vesicular
uptake) was reduced by T-588 during tetanic stimulation in
glutamatergic crayfish neuromuscular junction, whereas no apparent changes in the amplitude of single endplate potential and endplate current (i.e., single stimulation) were observed by T-588
application. These findings show that T-588 produces significant
changes in responses when a neuron receives high-frequency inputs,
compared with low-frequency inputs. Similar effects (i.e., a difference between single and tetanic stimulations) have been reported in studies
on different compounds: agonists of the phosphatidylinositol hydrolysis-coupled receptor, such as subtypes of
m1, m3 ACh receptors, or
metabotropic glutamate receptors 1 and 5 do not change basal synaptic
responses, but facilitate LTP induction in the rat hippocampus (Burgard
and Sarvey, 1990; Riedel et al., 1995). It is intriguing for us to
investigate the effects of T-588 on activity-dependent synaptic
efficacy, including long-term depression and depotentiation. Further
studies are necessary to address this issue.
In the present study, there were no significant differences for the
decrease of PS onset latency between the T-588 and vehicle-treated groups, except for day 2. This time course was different from those of
T-588 effects on PS amplitude and the fEPSP slope after HFS. At
present, there are no data to explain this discrepancy; a further study
is necessary to elucidate this point.
Effects of T-588 on Maintenance of LTP.
Maintenance of LTP is
known to be important for retention of associative memory (Doyère
and Laroche, 1992). The cellular processes in maintenance of LTP are
known to be distinct from those involved in its induction. It has two
physiological components: synaptic LTP and E-S (EPSP-spike)
potentiation. EPSPs are increased after LTP due to synaptic LTP and
EPSPs more readily elicit postsynaptic spike firing due to E-S
potentiation (Chavez-Noriega et al., 1989; Evans and Viola-McCabe,
1996).
We observed that the PS amplitude in the high-dose T-588 group was
significantly higher than that in the vehicle-treated group. This
indicates that T-588 contributes to the maintenance of LTP, at least
through E-S potentiation. T-588 may modify LTP maintenance postsynaptically, because E-S potentiation is thought to be produced by
an increase in postsynaptic excitability (Bliss and Gardner-Medwin, 1973) and/or a reduction in postsynaptic inhibition. Abraham et al.
(1987) have reported that E-S potentiation is due primarily to a
long-lasting GABA-mediated inhibition (Abraham et al., 1987). Enhancing
GABAA receptor function, which may disturb memory
formation, reduced E-S potentiation. In addition, a contribution of the
dopaminergic system in E-S potentiation is indicated (Yanagihashi and
Ishikawa, 1992). Therefore, changes in the GABAergic and/or
dopaminergic system could be involved in the effects of T-588 on E-S
potentiation observed in the present study.
In the present study, both low-dose and high-dose T-588 treatments
slightly, but significantly, enhanced the fEPSP slope on day 1, whereas
vehicle treatment did not. This indicates that T-588 contributes to the
maintenance of LTP through synaptic LTP as well. Ono et al. (1995a)
have reported that T-588 (3-10 µM) potentiates cAMP accumulation
when applied with isoproterenol (0.3 µM) to bath medium in rat
hippocampal slices. Since protein kinase A signal transduction pathway
activated by cAMP is reported to be critical for LTP maintenance
(Nguyen and Kandel, 1996), we speculate that this amplification of cAMP
accumulation is one possible mechanism for the maintenance of LTP.
Effect of T-588 on Short-Term Synaptic Plasticity.
Consistent
with the results in previous in vivo studies (Joy and Albertson, 1993;
Moser, 1996), by varying IPIs from 20 to 1000 ms, we identified a
typical triphasic pattern of paired-pulse early depression,
facilitation, and late depression for the PS amplitude of second evoked
potentials. The early depression of PS is known to be due to the
activation of interneurons, which feed back on the somatic region in
the DG (Halasy and Somogyi, 1993; Moser, 1996) and produce
GABA-mediated inhibition (Albertson and Joy, 1987). The late depression
seems to be mediated by Ca2+-dependent
K+ channels opening (Thalmann and Ayala, 1982).
PS paired-pulse facilitation results from a selective increase in an
N-methyl-D-aspartate-mediated synaptic
response (Joy and Albertson, 1993). Furthermore, field EPSP reportedly
depends upon the increase of neurotransmitter (glutamate) release
response to second pulse stimulation (Joy and Albertson, 1993). In the
present study, administration of T-588 had no significant effects on
the triphasic response of PS amplitude, the inhibitory process of
fEPSP, and the increase of PS onset latency in any IPIs. Therefore, the
cellular and synaptic mechanisms for the short-term synaptic plasticity
may be less involved in the alleviatory effects of T-588 seen in the
previous behavioral experiment (Ono et al., 1993, 1994, 1995b; Nakada
et al., 1995) in which the same doses of T-588 as those in the present study were used. However, this does not rule out a possibility that
T-588 has some effects on short-term synaptic plasticity. The present
results only indicated that T-588 does not show significant effects on
short-term synaptic plasticity at a considerably low concentration
range (0.3 or 3.0 mg/kg). Indeed, Hirata and coworkers (1999) have
reported that, at a higher concentration range, T-588 reduces
paired-pulse and repetitive-pulse facilitation at mouse and crustacean
neuromuscular junctions.
Conclusion.
In the present study, we showed that T-588
enhanced and maintained long-term synaptic plasticity (E-S potentiation
and synaptic LTP), but did not affect short-term synaptic plasticity,
in the DG-perforant pathway of freely moving rats. Since maintenance of
LTP is known to be important for learning and memory (Doyère and
Laroche, 1992), the effects of T-588 on long-term synaptic plasticity
could contribute to the alleviation of cognitive dysfunction in senile dementia.
This work was partly supported by Grants-in-Aid for Scientific
Research on Priority Areas (A)-Research for Comprehensive Promotion of
Study of Brain (12050220), Priority Areas (C)-Advanced Brain Science
Project (12210009), and Scientific Research (11308033 and
11680805)-from Ministry of Education, Culture, Sports, Science and
Technology, Japan.
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Abstract
Introduction
-aminobutyric acid.
