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The Journal of Neuroscience, June 1, 1999, 19(11):4609-4615
GABAB Receptor Antagonism: Facilitatory Effects on
Memory Parallel Those on LTP Induced by TBS but Not HFS
Ursula
Stäubli,
Joey
Scafidi, and
Daniel
Chun
Center for Neural Science, New York University, New York, New York
10003
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ABSTRACT |
The present experiments used CGP 35348, a selective
GABAB receptor antagonist with a significantly higher
affinity for post- versus presynaptic receptors, to dissociate the role
of antagonist concentration versus stimulation mode in determining
whether GABAB receptor blockade facilitates or suppresses
long-term potentiation (LTP). The antagonist was applied by pressure
ejection to one of two recording sites in area CA1 of hippocampal
slices before LTP was induced at both sites with either theta burst or
high-frequency stimulation (TBS or HFS). TBS produced a dose-dependent
facilitation of potentiation that turned into depression at the highest
concentration tested, a result reflecting the dose-dependent balance
between the drug's postsynaptic disinhibitory effect and its action on presynaptic autoreceptors regulating the release of GABA. In contrast, HFS-induced LTP increased monotonically with drug concentration, suggesting that blockade of postsynaptic GABAB receptors is
the only factor contributing to HFS-induced LTP. To test the relevance of the two sets of LTP results, we performed behavioral studies examining the effect of different dosages of antagonist on spatial retention and found that memory was enhanced at intermediate dosages but not at very low and high concentrations, reminiscent of the bell-shaped dose-response curve obtained for TBS-induced LTP. These
findings are consistent with the notion that LTP induced by electrical
stimulation modeled after endogenous theta-modulated activity patterns
bears more relevance to behavior than does potentiation induced by
arbitrary tetanic trains.
Key words:
LTP; memory; hippocampus; theta; GABAB; autoreceptors; postsynaptic; facilitation; impairment; dose-response curve
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INTRODUCTION |
GABAergic interneurons are known to
exert a powerful influence on the occurrence of long-term potentiation
(LTP) by regulating the degree of local depolarization in the
activity-receiving target areas. There are two main classes of GABA
receptors, the GABAA and the GABAB type, each
of which mediates inhibition via distinctly different cellular
mechanisms. The present study focused on the GABAB receptor
that modulates synaptic transmission by presynaptic inhibition of
transmitter release via auto- and heteroreceptors and by increasing
K+ conductance responsible for long-lasting (late)
IPSPs. Our interest in the GABAB receptor emerged from
previous findings that selective 5-HT3 receptor antagonism
in behaving rats causes a reduction in the firing rate of a subset of
hippocampal interneurons, thereby facilitating both the induction of
LTP and the retention of memory (Stäubli and Xu, 1995 ; Reznic and
Stäubli, 1997). The final trigger in this cascade of effects
appeared to be a reduction in postsynaptic GABAB
receptor-mediated currents. Thus, we sought to test this by
selectively blocking postsynaptic GABAB receptors and
determining whether such manipulation mimics the effect of the
serotonergic antagonist on LTP and memory.
There is evidence that pre- and postsynaptic GABAB
receptors exhibit differential responses with respect to time course
and affinity to ligands (Olpe et al., 1990, 1993a,b; Davies et al., 1991; Isaacson et al., 1993). The complexity of mechanisms of GABAB action is further underlined by results that suggest
both facilitation and suppression of potentiation, depending on the mode of LTP induction. Specifically, long tetanic trains enhanced LTP
irrespective of antagonist concentration (Olpe and Karlsson, 1990; Olpe
et al., 1993b), whereas brief theta pattern stimulation had no effect
at low antagonist level (Davies et al., 1991) but suppressed LTP at
high dosage (Davies et al., 1991; Mott and Lewis, 1991; Olpe et al.,
1993b). Behavioral work on the effect of GABAB receptor
blockade has produced results ranging from memory facilitation to
impairment (e.g., Bianchi and Panerai, 1993; Carletti et al., 1993;
Mondadori et al., 1993; Brucato et al., 1996; Getova et al., 1996).
Possible reasons for these inconsistencies include the use of tasks
with unknown relationships to hippocampal processes and the failure to
establish dose-response relationships between the drug and the
behavior tested.
The goal of this study was (1) to disentangle the role of drug
concentration versus LTP induction mode in determining whether GABAB antagonism facilitates or suppresses potentiation and
(2) to assess the relevance of the observed physiological results to
cognitive processes. All experiments involved CGP 35348, an antagonist
with significantly higher affinity for post- versus presynaptic
receptors (Olpe et al., 1990, 1993b; Davies et al., 1991) that crosses
the blood-brain barrier (Olpe et al., 1993a). A technique was used in
which potentiation could be simultaneously induced at two sites in the
same hippocampal slice, only one of which was exposed to the
antagonist, allowing for within-slice and same-time comparisons of test
and control LTP (see Fig. 1). The drug was applied at increasing
concentrations, and LTP was induced with either pairs of brief theta
bursts or prolonged high-frequency trains. Behavioral testing involved
increasing concentrations of CGP 35348 administered to rats before
performing a radial maze task.
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MATERIALS AND METHODS |
In vitro hippocampal physiology
Rat hippocampal slices were prepared from 2- to 3-month-old
Sprague Dawley rats and maintained in an interface chamber using standard conditions, as described in previous work (Stäubli et al., 1998). The rats were decapitated, and their brains were rapidly removed and placed in 0°C oxygenated (95% O2/5%
CO2) artificial CSF (aCSF) of the following
composition (in mM): NaCl 124, KCl 3, KH2PO4 1.25, MgSO4 2.5, CaCl2 3.4, NaHCO3 36, D-glucose 10, and L-ascorbate 2. The hippocampi were quickly dissected
free in ice-cold aCSF, placed on a McIlwain tissue chopper, cut into 400 µm sections, and collected into a Petri dish containing ice-cold aCSF. The slices were then immediately placed on a nylon net in an
interface chamber and maintained at a temperature of 31 ± 1°C. They were continuously perfused with preheated aCSF at a rate of 75 ml/hr while their upper surface was exposed to warm, humidified 95%
O2/5% CO2.
Recording and stimulating began after an incubation time of at least 1 hr. All experiments involved local drug application via pressure
ejection in combination with two extracellular recording sites (glass
micropipettes filled with 2 mM NaCl) in the apical dendrites of fields CA1a and CA1c, with one of the two sites randomly selected for drug application and the other serving as control (Fig.
1). Stimulation pulses were delivered to
the Schaffer-commissural axons passing through the stratum radiatum
using a bipolar-stimulating electrode (twisted nichrome wires, 65 µm)
centered between the two recording electrodes, ~500 µm from each.
The stimulus strength was adjusted to produce two field EPSPs whose
amplitudes were ~60% of the maximum spike-free response.
Experiments were initiated after establishing a stable baseline
recording for at least 20 min.
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Figure 1.
Hippocampal setup for within-slice
and same time-frame comparisons of control and test LTP.
A, LTP is induced and recorded simultaneously in a
control and test population of synapses at equidistant positions from
the stimulating electrode in field CA1. Drug is applied using a
pressure ejection system (Picospritzer) through a pipette placed within
~150 µm of and at the same depth (~50-100 µm) as the test
recording electrode. B, Superimposed waveforms show
representative baseline and potentiated responses recorded 45 min after
LTP induction at the test and control site. The GABAB
receptor antagonist CGP 35348 was ejected at the test site starting 15 min before LTP induction with two pairs of theta bursts separated by 30 sec. Calibration: 1 mV, 10 msec.
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CGP 35348 (a generous gift from Dr. W. Froestl, Novartis, Basel,
Switzerland) is a selective antagonist for GABAB receptors with a half-maximal IC50 of 34 ± 5 µM
(Bittiger et al., 1990). In slices, the drug has been shown to block
postsynaptic GABAB receptor-mediated late IPSPs completely
at a concentration of 100 µM (Davies et al., 1991; Olpe
et al., 1990, 1993a). Presynaptic GABAB autoreceptors, on
the other hand, were found to be fully antagonized at 1 mM,
in accordance with the notion that CGP 35348 has a 10 times lower
affinity for pre- compared with postsynaptic receptors (Davies et al.,
1991). On the basis of the above information, the drug was tested at
four concentrations: 100, 250, 500, and 1000 µM. It was
diluted to its final concentration with aCSF and applied locally by
pressure ejection (Picospritzer; General Valve, Fairfield, NJ) from a
glass micropipette placed next to (within 150 µm) and at the same
depth (~50-100 µm) as the test recording electrode. Pipette
ejection pressure was set at 8-12 psi (pulse duration, 10 msec) to
supply ~3 nl of drug every 5 sec. Drug application started 15 min
before LTP induction and was terminated immediately thereafter. Control
experiments involved local application of aCSF starting 15 min before
LTP induction.
LTP was induced by either of two stimulation modes, both of which
involved 100 Hz episodes but differed in duration and pattern of
delivery. The first induction paradigm was called theta burst stimulation (TBS) and consisted of two four-pulse bursts at 100 Hz
separated at theta frequency (i.e., 200 msec) and repeated once at 30 sec. This stimulation pattern mimics the typical firing mode of
hippocampal pyramidal cells during learning, which consists of two to
four sequential high-frequency bursts (100-400 Hz) paced at theta
frequency intervals, with three to five spikes per burst (Otto et al.,
1991; O'Keefe and Recce, 1993; Skaggs et al., 1996). The other
induction paradigm, called high-frequency stimulation (HFS), consisted
of a 200 msec long train of 21 pulses at 100 Hz that was repeated once
at 30 sec. This second stimulation pattern, although not modeled after
endogenous hippocampal activity, had been used by others testing the
effect of GABAB receptor blockade on LTP (Olpe and
Karlsson, 1990; Olpe et al., 1993b). The stimulation intensity was not
increased during TBS or HFS. Within-slice comparisons between
potentiation at the site receiving the injection of the antagonist
(test response) versus potentiation at the site that did not (control
response) were used to determine the effect of the drug on LTP.
Behavioral protocol
Animals. Behavioral training was conducted with a
group of eight male Long-Evans rats that were 24-26 months of age at
the time of testing. They were housed individually and kept on a 12:12 hr light/dark cycle, with the experiments being conducted during the
light phase. The animals had restricted access to food to maintain
their body weight at 90% of that of the same-age controls, while water
was available ad libitum.
Spatial delayed nonmatch-to-sample task. An elevated
eight-arm radial maze made of painted wood was used. It was kept in a well-lit room containing numerous distinct extramaze cues, such as
posters, bookshelves, cabinets, and lamps, etc.. The center platform
was 38 cm in diameter with eight arms radiating from it (12 cm wide, 56 cm long, and 3.5 cm high enclosing walls), separated from the central
platform by Plexiglas guillotine-type doors that could be raised and
lowered by the experimenter via a system of pulleys and strings.
Training involved one sample and one test trial per day. In the sample
trial, three of the eight arms were blocked, and the animals were
allowed to enter the open arms to retrieve a chocolate chip reward
hidden in a recessed food well at the end of each arm. After a delay,
they were returned to the maze for the test trial in which all eight
arms were open, but rewards were contained only in the previously
blocked arms (blocked arms were randomized across days). The number of
incorrect entries (i.e., arms entered more than once) until all rewards
were collected and the number of correct choices made before a reentry
occurred were recorded. Rats trained extensively in this task generally
perform accurately at delays up to 2 hr, but they begin to commit
errors that increase in number the longer the intertrial interval. The
present study involved rats with extensive previous experience in this
task and used a delay of 8 hr between the sample and test trial.
CGP 35348 has been shown to penetrate the blood-brain barrier rapidly
and to exhibit activity in vivo (Bittiger at al., 1990; Olpe
et al., 1990; Froestl et al., 1996). In vivo experiments in
rats have demonstrated that CGP 35348 given intraperitoneally has
moderate effects on postsynaptic GABAB receptors at 30 mg/kg but causes an almost complete antagonism at 100 mg/kg. Side
effects such as ataxia were observed at dosages >300 mg/kg (Olpe et
al., 1990). The present study tested CGP 35348 at dosages between 12.5 and 300 mg/kg. Each concentration was administered over a period of 2 consecutive weeks in the following sequence: 50, 25, 12.5, 100, 200, and 300 mg/kg. The drug was dissolved in saline and administered
intraperitoneally 30 min before the sample trial. On the first day of
each week, all rats were tested with vehicle. To allow for
within-animal comparisons of the drug's effect on memory, we randomly
divided the rats into two groups and administered either drug or
vehicle in a counterbalanced manner on 4 consecutive days of the week,
starting on the second of 5 weekly test days. Data analysis involved
all drug and vehicle days except the first vehicle day of the week
(i.e., 4 vehicle and 4 drug days per concentration and animal).
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RESULTS |
Local pressure ejection of the artificial CSF carrier vehicle used
in these experiments had no detectable effect on slice physiology or
potentiation. Figure 2 summarizes
experiments in which the GABAB antagonist CGP 35348 was
tested at four concentrations (100, 250, 500, and 1000 µM) for its effect on LTP induced by two different
stimulation modes, TBS and HFS. Within-slice comparisons (paired
t test) between potentiation at the test site receiving the
injection of GABAB antagonist versus potentiation at the
control site were used to determine the effect of the drug on LTP. When TBS was used, the potentiation measured at the test site between 45 and
50 min after induction was significantly facilitated at 100 µM [t(5) = 5.1; p < 0.01] and 250 µM [t(4) = 4.53; p < 0.01] but was only marginally facilitated
at 500 µM [t(3) = 1.79;
p < 0.08] and was significantly suppressed at 1000 µM [t(4) = 3.01; p < 0.02]. The average within-slice difference (± SEM) between control and test sites during the last 5 min of recording
was 16.5 ± 3.2 for 100 µM, 22.2 ± 5.5 for 250 µM, 12.5 ± 6.9 for 500 µM, and
 14.4 ± 4.8 for 1000 µM. In brief, a bell-shaped
dose-response curve characterized the interaction between the drug and
TBS-induced LTP. This point is also illustrated in a later figure (see
Fig. 4C), which displays the mean of the paired differences
between the control and test site for each concentration.
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Figure 2.
Dose-dependent facilitatory and suppressive
effects of the GABAB antagonist CGP 35348 on LTP induced by
TBS versus HFS. Left, Comparisons of the amount of
control and test LTP induced by TBS in the presence of increasing
concentrations of CGP 35348, using the experimental setup illustrated
in Figure 1. Right, Comparisons the same as those on the
left, except that LTP was induced by HFS. The
n values indicate the number of slices (1 slice per
animal) tested per concentration, and each data point represents the
group mean of one response per animal (± SEM).
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In contrast, when HFS was used to induce LTP, the potentiation measured
at the test site between 45 and 50 min after induction was facilitated
at all but the lowest drug concentrations [100 µM,
t(3) = 1.49; p > 0.1; 250 µM, t(4) = 3.0; p < 0.05; 500 µM, t(4) = 2.93;
p < 0.05; and 1000 µM,
t(4) = 4.79; p < 0.01]. The average within-slice difference (± SEM) between the control and test
sites during the last 5 min of recording grew steadily with increasing
concentration and was 8.6 ± 5.8 at 100 µM,
17.3 ± 5.7 at 250 µM, 26.4 ± 9.0 at 500 µM, and 26.0 ± 6.2 at 1000 µM. Thus, a linear dose-response curve that leveled off at the high end characterized the interaction between the GABAB receptor
blocker and LTP induced by HFS (see Fig. 4C). It may be
speculated that concentrations >1 mM might have revealed
inhibitory effects on HFS-induced LTP. However, as noted previously,
CGP 35348 has an affinity of 34 µM for the postsynaptic
GABAB receptor (Bittiger et al., 1990) and an ~10 times
lower affinity for the presynaptic site (Davies et al., 1991; Olpe et
al., 1993b). According to standard calculations used in
receptor-binding chemistry, a compound with these affinities can be
expected to produce a virtually complete receptor-ligand saturation at
1 mM, both with respect to post- and presynaptic
GABAB receptors. Thus, it is unlikely that testing concentrations >1 mM would have produced any additional
changes other than side effects mediated by non-GABAB receptors.
If the hypothesis that LTP plays a role in the encoding of information
in tasks requiring the hippocampus is correct, then we would expect
that one of the above sets of LTP data is predictive of the drug's
behavioral effects. To test this, we compared the ability to retain
spatial memory in rats treated with increasing concentrations of
GABAB receptor antagonist (12.5, 25, 50, 100, 200, and 300 mg/kg) versus performance on vehicle days. The drug caused no obvious
behavioral side effects at any dosage, consistent with a study by Olpe
et al. (1990) who found no measurable effects on motor performance at
concentrations up to 1000 mg/kg. Average scores on vehicle versus drug
days for each dosage are illustrated in Figure
3, A and B. There
was no overall significant difference in performance over time on
vehicle days during the 12 weeks of behavioral testing, as shown by the
results of a repeated measures ANOVA [F(5,35) = 0.152; p > 0.05]. Therefore, vehicle scores were pooled for further data analysis. A repeated measures ANOVA revealed a
significant main effect for dosage on the number of correct choices
before the first error [F(6,42) = 3.725;
p < 0.01] and reentry errors
[F(6,42) = 2.636; p < 0.05].
Subsequent within-animal comparisons (paired t test) for
correct choices before the first error indicated significant
improvements on drug versus vehicle days at 25 mg/kg
[t(7) = 2.11; p < 0.05], 50 mg/kg [t(7) = 6.15; p < 0.001], and 100 mg/kg [t(7) = 3.06;
p < 0.01]. However, memory facilitation was absent at
the lowest and the two highest dosages tested (see Fig. 3A;
i.e., at 12.5, 200, and 300 mg/kg [t(7) = 0.55, 1.66, and 0.36; p > 0.05, respectively]). The average
within-animal increment in correct choices between drug and vehicle
days was 0.06 ± 0.11 for 12.5 mg/kg, 0.34 ± 0.16 for 25 mg/kg, 0.56 ± 0.09 for 50 mg/kg, 0.25 ± 0.08 for 100 mg/kg,
0.19 ± 0.11 for 200 mg/kg, and 0.03 ± 0.09 for 300 mg/kg.
Thus, as illustrated in Figure 4A, a bell-shaped
dose-response curve characterized the interaction between the drug and
the improvement in correct choices before the first error.
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Figure 3.
Dose-dependent facilitatory effects of the
GABAB antagonist CGP 35348 on retention in a spatial
delayed nonmatch-to-sample task. A, Mean number of
correct choices (± SEM) made by a group of eight rats before
reentering any of the five arms visited in the sample trial 8 hr
earlier. Each of the concentrations (12.5, 25, 50, 100, 200, and 300 mg/kg) was tested over a period of 2 weeks with drug and vehicle days
counterbalanced, allowing us to use each rat as its own control.
B, Experiment the same as that in A but
for the number of reentries into arms visited previously
(*p < 0.05; **p < 0.01;
***p < 0.001, paired t test).
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Figure 4.
Comparison of mean within-rat differences in
memory retention versus mean within-slice differences in TBS- and
HFS-induced LTP as a function of drug dosage. A, Average
within-rat improvement in the number of correct choices between the
drug and vehicle condition. B, Average within-rat
decrement in the number of reentry errors between the drug and vehicle
condition. C, Average within-slice increment and
decrement in the amount of potentiation between the test and control
recording site for LTP induced by HFS versus TBS.
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Similarly, paired within-animal comparisons for the number of reentry
errors showed a marginally significant reduction on drug versus vehicle
days at 25 mg/kg [t(7) = 1.76;
p = 0.06] and significant reductions at 50 mg/kg
[t(7) = 3.55; p < 0.01], 100 mg/kg [t(7) = 2.39; p < 0.05], and 200 mg/kg [t(7) = 2.39;
p < 0.05]. No facilitatory effects were apparent at
the lowest and highest concentrations tested (see Fig. 3B;
i.e., at 12.5 and 300 mg/kg [t(7) = 0.55 and
1.43; p > 0.05, respectively]). The average
within-animal decrement in reentry errors between drug and vehicle days
was 0.06 ± 0.11 for 12.5 mg/kg, 0.25 ± 0.14 for 25 mg/kg, 0.38 ± 0.11 for 50 mg/kg, 0.19 ± 0.08 for 100 mg/kg, 0.19 ± 0.08 for 200 mg/kg, and 0.09 ± 0.07 for
300 mg/kg. Thus, a bell-shaped dose-response curve characterized the
interaction between the drug and the reduction in reentry errors, as
illustrated in Figure 4B.
However, one caveat to keep in mind with respect to the above
behavioral results is that, because the drug was administered systemically, we cannot be certain that the results are attributable to
actions on hippocampal GABAB receptors only.
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DISCUSSION |
The present LTP experiments were prompted by previous work
demonstrating that GABAB receptor blockade suppresses LTP
in response to brief high-frequency bursts patterned at theta frequency
(i.e., TBS) but facilitates LTP in response to long trains of
high-frequency stimulation (i.e., HFS) (Olpe and Karlsson, 1990; Davies
et al., 1991; Mott and Lewis, 1991; Olpe et al., 1993b). In an attempt to reconcile these apparently contradictory results, we conducted a
parametrical study comparing the effects of TBS versus HFS on LTP
induced under variable levels of GABAB receptor blockade. Our results indicate that the mode of induction is not the sole factor
in determining whether GABAB receptor blockade suppresses or facilitates LTP but that the antagonist concentration is equally crucial. Thus, although HFS consistently enhanced LTP in a manner approximately proportional to the antagonist concentration, we found
that a bell-shaped dose-response curve characterized the interaction
between the drug and TBS-induced LTP. That is, TBS suppressed LTP at
the highest dosage, in accordance with the results of others (Davies et
al., 1991; Olpe et al., 1993b), but enhanced it at low and intermediate
concentrations. These results provide the first demonstration that
GABAB receptor antagonism is capable of facilitating
TBS-induced LTP under certain conditions.
The question arises as to how this complex interaction between drug
level and LTP induction mode may be reconciled with our current
understanding of the role of pre- and postsynaptic GABAB receptors in hippocampal physiology. It is well known that
GABAB autoreceptors, located on interneuron terminals that
synapse on pyramidal cells via GABAA receptors, mediate an
IPSP refractory period that reaches its maximum at ~200 msec (i.e.,
the theta period), leading to a prolongation of EPSPs occurring at this interval, thereby creating conditions needed for activation of the NMDA
receptor currents that trigger LTP (Larson and Lynch, 1986). The exact
mechanism underlying this feedforward IPSP suppression has been
identified as transient hyperpolarization of the terminal, a process
that peaks between 150 and 250 msec after autoreceptor activation,
causing a suppression of GABA release to a subsequent input that is
maximal if it occurs at the period of the theta rhythm (Larson and
Lynch, 1986; Pacelli et al., 1989; Davies et al., 1991; Mott and Lewis,
1991). In contrast, activation of postsynaptic GABAB
receptors on pyramidal cells triggers a slowly rising, long-lasting IPSP that has little inhibitory effect on fast AMPA receptor-mediated currents but is highly effective at counteracting responses mediated by
NMDA receptors. From the above points it can be predicted that blockade
of post- versus presynaptic GABAB receptors will have opposite effects on LTP. That is, a reduction in postsynaptic GABAB current is expected to increase the duration of
dendritic NMDA receptor-mediated currents and thereby facilitate the
induction of LTP, whereas blockade of presynaptic receptors is likely
to counteract the autoinhibition of GABA release, thereby making it
more difficult for LTP to occur.
As mentioned previously, pre- and postsynaptic GABAB
receptors differ not only with respect to their physiological role but also in affinity for ligands; that is, postsynaptic receptors are
completely blocked by 100 µM CGP 35348, whereas an
~10-fold higher concentration is needed to antagonize presynaptic
autoreceptor function fully (Davies et al., 1991; Olpe et al.,
1993b).
How does the above set of information explain our finding that low drug
levels facilitate LTP irrespective of stimulation protocol, whereas
high levels have opposite effects depending on the induction mode? It
seems reasonable to suggest that both TBS and HFS facilitated LTP at
low to intermediate levels of CGP 35348 because postsynaptic receptors
were the main target in this concentration range, producing an increase
in the amount of dendritic depolarization during both induction
paradigms. However, it is likely that TBS caused LTP suppression when
the antagonist concentration was raised, because blockade of
presynaptic GABAB receptors at high drug levels prevented
the interneurons from entering their refractory period, thereby
facilitating the release of GABA that is normally suppressed during
TBS. HFS, on the other hand, still enhanced potentiation at high dosage
because the physiological timing patterns and chemistries that regulate
LTP induced with theta activity are likely to be of reduced importance
when sustained pulse trains are used, as suggested by Arai and Lynch
(1992); that is, LTP induced by HFS is less sensitive to the
autoreceptor-mediated reduction in GABA release, leaving the
disinhibitory effect of blocking postsynaptic GABAB
receptors as the drug's main contributing factor.
A second goal of this project was to compare the drug's effect on LTP
and learning. Assuming that LTP-like mechanisms are engaged during
learning, it seemed reasonable to expect that GABAB blockers might be effective means to manipulate memory. Previous work
by others examining the contribution of GABAB receptors to learning demonstrated a facilitatory action of an orally administered GABAB antagonist (CGP 36742) in various behavioral
paradigms including shock avoidance tests, social learning, and operant
conditioning (Bianchi and Panerai, 1993; Carletti et al., 1993;
Mondadori et al., 1993; Getova et al., 1996). Although these studies
suggested a role for GABAB receptors in memory formation,
they failed to demonstrate that the compound promoted both the
occurrence of synaptic plasticity in a given brain site and the
formation of memory known to be subserved by that same site. Because of
our LTP results, we tested CGP 35348 at increasing concentrations for
its effect on retention in the radial maze, a task well known to
involve hippocampal circuitries. Although low and intermediate dosages
were expected to enhance memory, it was conceivable that higher dosages
might produce either facilitation or impairment, depending on whether
HFS- or TBS-induced LTP better mimics the process by which synapses
normally change their strength during learning. We found that the drug
enhanced hippocampal memory moderately at low dosage and substantially
at intermediate dosages and had no effect at high concentration,
reminiscent of the bell-shaped dose-response curve that describes the
interaction between the drug and TBS-induced LTP. However, the question
arises whether the drug concentrations used in the behavioral study
reached the brain in the concentration range in which selective effects
on pre- and postsynaptic receptors occur? As mentioned above, in vivo experiments by others have shown that the dosage range for modest to almost complete postsynaptic GABAB receptor
blockade spans 30-100 mg/kg (Olpe et al., 1990). On the basis of these observations and given that the affinity of CGP 35348 for pre- versus
postsynaptic receptors is ~10 times lower, we can assume autoreceptor
blockade to be absent or moderate at 100 mg/kg but substantial at 500 mg/kg and complete at 1000 mg/kg. Our memory performance results
correspond well with the above notion. We found memory facilitation at
dosages between 25 and 100-200 mg/kg, drug levels at which
postsynaptic blockade is dominant. The peak of enhancement occurred at
50 mg/kg, with facilitation monotonically decreasing at increasingly
higher and lower dosages. Thus, the effect of the drug on behavior best
mimics the effect of the drug on TBS-induced LTP, because both
facilitation of LTP and memory occur as long as the antagonist
predominantly blocks postsynaptic receptors (between 100 and 500 µM in slices and 25 and 200 mg/kg in vivo) but
diminishes progressively as the proportion of receptors that is
presynaptic increases. This is not the case for HFS-induced LTP, which
was increasingly facilitated the larger the dosage and, in fact, shows
the largest enhancement at 1 mM at which both pre- and
postsynaptic receptors are known to be blocked.
The implications of the above observation are two-fold. First, it
underscores the notion that LTP induced by electrical stimulation that
mimics endogenous theta-modulated neural activity patterns typically
present when the rat is in learning mode bears more relevance to memory
than does LTP induced with arbitrary tetanic trains. Second, it
supports the hypothesis that both learning and LTP make use of the same
underlying cellular mechanism and that LTP-like enhancements in
synaptic efficacy might actually occur during learning. It is of
interest in this regard that LTP in area CA1 of the awake animal is
best elicited by a few brief theta bursts, whereas prolonged tetanic
episodes frequently cause nonspecific neuronal depression and loss of
response (Stäubli and Scafidi, 1997).
The highest behavioral dosage examined (300 mg/kg) did not produce
memory impairment but simply lacked facilitatory effects, in contrast
to our LTP study in which TBS suppressed potentiation at the highest
dosage (1 mM) tested. This incongruity may, at least
partly, be explained by the choice of our paradigm that involved highly
trained rats committing an average of 1.5-2 errors on control days,
thus leaving little room for further impairments. Another reason may be
that 1 mM CGP 35348 in slices is a much higher dosage than
the concentration that reaches the brain after systemic injection of
300 mg/kg (animals injected with dosages larger than 300 mg/kg
manifested mild signs of physical discomfort, which led us to stop the
experiment at 300 mg/kg). That both of these explanations may apply is
suggested by results from another study (Brucato et al., 1996) that
involved CGP 46381, an antagonist with a several times higher affinity
for the GABAB site than has CGP 35348 [IC50 of
CGP 46381 is 4.9 vs 34 µM for CGP 35348 (Olpe et al.,
1993a)]. The drug was tested at 100 mg/kg, a dosage demonstrated to be
sufficient for blockade of presynaptic autoreceptors in vivo, and, as expected, was found to suppress both TBS-induced LTP
in anesthetized rats and acquisition in the Morris water maze, a task
known to be relatively stressful and therefore prone to revealing
impairments, especially in naive rats.
The present results would gain additional strength if LTP was recorded
in the freely moving rat and it was demonstrated that the threshold
dosage for memory enhancement and LTP facilitation in the awake brain
is similar. In any event, the present study has benefited from the use
of CGP 35348 in that it enabled us to distinguish between the
usefulness of two distinct LTP induction paradigms for identifying the
basic physiologies and chemistries active during learning. It is clear
that further developments of GABAB antagonists that
selectively interact with the postsynaptic site are likely to be of
immense therapeutic value.
| |
FOOTNOTES |
Received Jan. 14, 1999; revised March 4, 1999; accepted March 10, 1999.
This work was supported in part by the Whitehall Foundation Grant
M97R05 to U.S. and by the National Science Foundation Grant IBN-9726779
to U.S. We thank Dr. Wolfgang Froestl (Novartis, Basel, Switzerland)
for the generous supply of CGP 35348 and Sherman Wiebe for critical
comments on this manuscript.
Correspondence should be addressed to Dr. Ursula Stäubli, Cortex
Pharmaceuticals, Inc., 15231 Barranca Parkway, Irvine, CA 92618.
| |
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ABSTRACT
INTRODUCTION
Compound via MeSH
