Department of Psychology and the Neuroscience Research Centre, University of Otago, Dunedin, New Zealand
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ABSTRACT |
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Cohen, Akiva S.,
Christine M. Coussens,
Clarke R. Raymond, and
Wickliffe C. Abraham.
Long-Lasting Increase in Cellular Excitability Associated With
the Priming of LTP Induction in Rat Hippocampus.
J. Neurophysiol. 82: 3139-3148, 1999.
The mechanisms
underlying the facilitation (priming) of long-term potentiation (LTP)
by prior activation of metabotropic glutamate receptors (mGluRs) were
investigated in area CA1 of rat hippocampal slices. In particular, we
focused on whether a long-lasting increase in postsynaptic excitability
could account for the facilitated LTP. Administration of the mGluR
agonist 1S,3R-aminocyclopentanedicarboxylic acid (ACPD) produced rapid
decreases in the amplitude of both the slow spike
afterhyperpolarization (AHPslow) and spike frequency adaptation recorded intracellularly from CA1 pyramidal cells. These
changes persisted after drug washout, showing only a slow decay over 20 min. ACPD also caused a leftward shift of the field EPSP-population
spike relation and an overall increase in population spike amplitude,
but this effect was not as persistent as the intracellularly measured
alterations in cell excitability. ACPD-treated cells showed increased
spike discharges during LTP-inducing tetanic stimulation, and the
amplitude of the AHPslow was negatively correlated with the
degree of initial LTP induction. The -adrenergic agonist isoproterenol also caused excitability changes as recorded
intracellularly, whereas in extracellular experiments it weakly primed
the induction but not the persistence of LTP. ACPD primed both LTP
measures. Isoproterenol administration during the tetanus occluded the
priming effect of ACPD on initial LTP induction but not its effect on LTP persistence. We conclude that the persistent excitability changes
elicited by ACPD contributes to the priming of LTP induction but that
other ACPD-triggered mechanisms must account for the facilitated
persistence of LTP in the priming paradigm.
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INTRODUCTION |
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The ability to produce activity-dependent synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD) is not a fixed property of individual synapses but rather a highly regulated function. External signals such as hormones and modulatory neurotransmitters that affect cellular excitability or signalling in biochemical pathways can exert a profound influence on the degree of synaptic plasticity induced by a particular stimulus protocol. Such changes in cellular or synaptic "state" can be induced either directly, by activation of postsynaptic receptors, or indirectly by modulating the activity of afferent principal cells and interneurons.
Another factor contributing to the synaptic state, and pertinent to
synaptic plasticity, is the history of pre- and postsynaptic activity.
Prior synaptic activity has been shown in some cases to inhibit
subsequent LTP and facilitate LTD, or in other cases to facilitate LTP
(see Abraham and Tate 1997 for a review). This family of
effects has been collectively referred to as "metaplasticity" (Abraham 1996
; Abraham and Bear
1996
). An early report of facilitated LTP showed that prior
activation of metabotropic glutamate receptors (mGluRs) can set a
"molecular switch" that enables the establishment of a late phase
of LTP by subsequent tetanization (Bortolotto et al.
1994
). Although the existence of this mGluR-mediated molecular switch is controversial (Thomas and O'Dell 1995
), we
recently confirmed that transient activation of mGluRs by the agonist
(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD), which by
itself had no long-lasting effect on the slope of the field excitatory
postsynaptic potential (fEPSP), nonetheless facilitated the induction
and stability of LTP induced by a weak tetanus delivered 20-60 min
later (Cohen and Abraham 1996
). This LTP "priming"
effect did not require coactivation of
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
(AMPA) or N-methyl-D-aspartate (NMDA)
receptors, but was blocked by the mGluR antagonist
(+)-2-amino-3-phosphonopropionic acid (L-AP3).
Whereas the mechanism underlying the ACPD-induced priming effect has
yet to be elucidated, protein kinases may be involved because the
general kinase inhibitor K-252b blocked the setting of the molecular
switch (Bortolotto et al. 1994). mGluRs belong to the
large superfamily of G-protein coupled receptors that activate several
biochemical cascades, but a recent study from our lab has limited the
potential mechanisms to those activated by Group I mGluRs that couple
to phospholipase C (PLC) (Cohen et al. 1998
). Activation
of this PLC pathway leads to the liberation of the second messengers
inositol trisphosphate (IP3) and diacylglycerol following
hydrolysis of phosphatidylinositol. It also increases basal cAMP
accumulation by an L-AP3-sensitive mechanism (Winder and Conn
1992
). Thus both protein kinase C (PKC) and protein kinase A
(PKA) are putative candidate kinases for mediating the priming of LTP
by mGluR activation.
One consistent downstream effect of both PKA and PKC activation is a
general increase in hippocampal pyramidal cell excitability, evidenced
by resting membrane depolarization, an increase in input resistance
(Rin), a decrease in several potassium conductances, including the transient A current and the slow spike
afterhyperpolarization (AHPslow), and a decrease in spike
frequency adaptation, i.e., the decrease in spike firing rate during a
prolonged depolarizing current pulse (Hoffman and Johnston
1998; Nicoll 1988
). These changes can be
engendered by activation of a variety of G-protein coupled receptors
including mGluRs of the Group I type (Charpak et al.
1990
; Desai and Conn 1991
; Gereau and
Conn 1995
; Goh and Ballyk 1993
), muscarinic
(Cole and Nicoll 1983
),
-adrenergic (Dunwiddie
et al. 1992
; Madison and Nicoll 1986
), and
dopamine receptors (Gribkoff and Ashe 1984
;
Pedarzani and Storm 1995
). In the case of
-adrenergic
receptor activation, the increased cell excitability decays very slowly
after drug washout and it has been suggested that such changes might
promote the subsequent induction of LTP, which is a voltage-dependent
process (Dunwiddie et al. 1992
). If the mGluR-mediated
excitability changes are equally long-lasting, it is possible that they
could contribute to the priming of LTP by ACPD.
The present study was undertaken to investigate whether the same
moderate dose of ACPD that primes LTP (Cohen and Abraham 1996) also produces a persistent aftereffect on postsynaptic
cell excitability that would account for its facilitation of subsequent LTP. The effects of ACPD on cell excitability and the induction of LTP
were compared with those generated by the
-adrenergic agonist
isoproterenol. Our findings indicate that increased postsynaptic excitability can account for part of the LTP facilitation effect elicited by mGluR priming, i.e., the enhanced initial induction, but
not for the enhanced persistence of LTP. A preliminary report of some
of these results has appeared (Cohen and Abraham 1997
).
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METHODS |
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Tissue preparation
Transverse hippocampal slices (400 µm thick) were prepared
from young adult male Sprague-Dawley rats (2-3 mo old, 200-300g) as
previously described (Kerr and Abraham 1995). All
procedures were performed in accord with New Zealand animal welfare
legislation and the experiments and procedures were approved by the
University of Otago Committee on Ethics in the Care and Use of
Laboratory Animals. Rats were either anesthetized with halothane or
injected with ketamine (100 mg/kg, i.p.) and decapitated. The brain was quickly removed and the hippocampi were dissected free. To reduce potential hyperexcitability, area CA3 was routinely removed by a manual
knife cut. This procedure also prevents the slow-onset potentiation
that can otherwise arise following mGluR activation (Bortolotto
and Collingridge 1993
). Slices were transferred to a slice
chamber and allowed to equilibrate for 2 h while being continually
superfused (2-3 ml/min) by an artificial cerebrospinal fluid (ACSF)
with the following composition (in mM) 124 NaCl, 3.2 KCl, 1.25 NaH2P04, 26.0 NaHCO3, 2.5 CaCl2, 1.3 MgCl2, and 10 glucose (equilibrated with 95%
O2-5% CO2 at 32.5°C).
Recording and data analysis
Intracellular recording microelectrodes were fabricated from
borosilicate glass on a Flaming/Brown model P-97 micropipette puller
(Sutter Instruments). The electrodes had resistances ranging from
60-100 M when filled with 4 M potassium acetate. Standard current
clamp recordings were made with an Axoclamp-2A amplifier (Axon
Instruments). Hippocampal CA1 pyramidal neurons were impaled in the
bridge mode, and when the impalement stabilized, the recording configuration was switched to the discontinuous current clamp mode
(sample rate about 5 KHz, filtered at 3 KHz). Current and voltage
signals were stored on a microcomputer for offline analysis.
The input resistance (Rin) of the neuron was
monitored by measuring the amplitude of the voltage transient in
response to a 0.5 nA hyperpolarizing current step (100 ms duration).
AHPslow was induced by a train of four action
potentials and elicited by four separate depolarizing current pulses (3 nA, 2 ms duration, 5 ms interpulse interval). Adaptation of spike
firing was tested using a 0.5 nA depolarizing current step (250 ms
duration). EPSPs were evoked by stimulation of the Schaffer
collateral/commissural pathway in area CA1 by a 75 µm monopolar
teflon-coated stainless steel electrode (100 µs pulse duration).
Analysis of the intracellular electrophysiological data were performed
with pClamp 6.0 software (Axon Instruments). Neurons were accepted for
study if they had a stable resting membrane potential (<60 mV)
throughout the experiment, an action potential overshoot >15 mV, an
Rin >25 M
, and showed typical properties of
pyramidal cells such as spike frequency adaptation and
AHPslow. The following measures were routinely obtained throughout the recording period at 2-4 min intervals: Rin, the holding current required to keep the
resting membrane potential at about
70 mV, the number of spikes per
depolarizing current pulse, peak amplitude of the
AHPslow, and EPSP slope. It should be noted that
the holding current was manually adjusted throughout the experiment to
keep the resting membrane potential constant (nominally at
70 mV).
The resting membrane potential was later corrected for the tip
potential recorded after exiting from the cell, and the actual resting
membrane potential was found to vary between
65 mV and
72 mV.
In some experiments, fEPSPs and population spikes were recorded
extracellularly in the stratum radiatum and stratum pyramidale, respectively. Recordings were made with glass microelectrodes (1-2
M) filled with 2 M NaCl. LTP (for both intracellular and extracellular experiments) was induced by theta-burst stimulation (TBS), which consisted of 5 × 100 Hz bursts (5 diphasic
pulses/burst, 200 ms interburst interval). During intracellular
recordings, TBS was given at a stimulus intensity that was raised to
just above threshold for postsynaptic action potentials. For
extracellular recordings, TBS was given at baseline stimulation
strength. Responses were followed for 30 min posttetanus in
intracellular experiments and for 60 min in extracellular experiments.
The slope of the EPSP was expressed as percent change from baseline
values. LTP was measured as the mean of the last 5 min of values during
the posttetanus recording period. Student's t-tests were
performed to determine significance at the P < 0.05 confidence level, and data are presented as group means ± SE.
To examine the effects of mGluR activation on the field EPSP-population spike (E-S) relationship, mini-input/output (I/O) curves were obtained by continuously rotating the stimulus strength (100 µs duration, 0.67 Hz) across four amplitudes that during the baseline recording period generated population spikes that were just at threshold, or were 0.5, 1.0, and 1.5-2.0 mV in amplitude. Recordings were made for 30 min before and after a 10 min application of ACPD.
Reagents
Reagents were procured from the following vendors: all salts from BDH Laboratory Supplies, ACPD from Tocris Cookson, and isoproterenol from Sigma. All drugs were made as stock solutions and diluted to their final concentration in the bathing medium. Control cells received sham drug administration, i.e., the flow line was switched for 10 min to a reservoir identical to that used in the drug experiments, but in this case the reservoir contained only normal ACSF. This procedure controlled for any switching-induced pressure artifacts that may have affected cell membrane parameters in addition to those caused by the drugs. Switching artifacts were rarely apparent.
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RESULTS |
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mGluR activation causes a long-lasting increase in postsynaptic excitability
Putative CA1 pyramidal cells were impaled with sharp
microelectrodes and a 10 min bath application of ACPD (20 µM) was
used to produce several changes in postsynaptic membrane parameters, as
previously reported (see INTRODUCTION). These changes
included membrane depolarization (as inferred by the amount of holding current required to keep the cell at about 70 mV), an increase in
input resistance, a reduction in spike frequency adaptation, a decrease
in the peak amplitude and duration of the
AHPslow, and a decrease in the EPSP slope. Many
of these changes however, occurred in a variable fashion with only the
depression of the AHPslow (
83 ± 6%,
n = 10, P < 0.001) and the reduction
in spike frequency adaptation (6.7 ± 1.5 additional spikes per
current pulse, n = 5, P < 0.01)
reaching statistical significance (Fig. 1, A-D). Control cells
exposed to a sham solution change did not show such changes
(AHPslow:
16 ± 4%, n = 5; spike adaptation: 0.4 ± 0.3 spikes, n = 5;
both measures P < 0.01 compared with ACPD-treated
cells).
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It was of particular interest in these experiments to see whether the
ACPD-induced increase in cell excitability was persistent enough to
account for the previously reported LTP priming effect by this same
dose of ACPD (Cohen and Abraham 1996). Thus recordings were continued for
20 min washout period in normal ACSF following ACPD application, a washout time during which priming of LTP is robustly observed. The ACPD-treated slices showed only a partial recovery over the first 20 min of washout, with both the
AHPslow (
54 ± 7%, n = 10) and spike frequency adaptation (3.9 ± 1.1 spikes) still
significantly reduced at this time point (P < 0.05 compared with control cells; Fig. 1, A-D). In two cells
with stable impalements, the AHPslow remained
reduced by 45 and 50%, respectively, 30-45 min post-ACPD. These
persistent effects were unlikely to be caused by a rundown of the
relevant Ca2+ or K+
channels because control cells held for 20 min postsham treatment showed no deterioration of the response over this time period (n = 5; Fig. 1, A and C).
mGluR activation produces short-lasting E-S potentiation
One common way to test for changes in cell excitability is to
examine the E-S ratio over a range of stimulus intensities. An increase
in postsynaptic excitability is often expressed as an increase in the
E-S ratio, termed E-S potentiation. To assess whether ACPD would cause
E-S potentiation in our CA1 mini-slices, as was reported for CA3-intact
slices (Breakwell et al. 1996), field potential
recordings were made in both stratum pyramidale, to record the
population spike and in stratum radiatum to record the fEPSP. Single
pulse stimuli were continuously rotated across four strengths for 30 min before and for 30 min following a 10 min application of the
broad-spectrum metabotropic receptor agonist, ACPD (20 µM). This dose
and postdrug time period are effective parameters for priming LTP
(Cohen and Abraham 1996
). Synaptic stimulation was
halted during the period of drug administration. The fEPSPs and
population spikes were normalized to the maximum response obtained
during the baseline recording and I/O curves constructed
from averages of the potentials at each stimulus strength. In the first
1-6 min post-ACPD there was a depression of the fEPSPs and a
significant facilitation of the population spikes [1-way analysis of
variance (ANOVA), P < 0.05], thus producing a marked reduction in the population spike threshold and an overall shift to the
left of I/O curve (n = 10, Fig.
2). This effect was not entirely caused
by a decrease in synaptic inhibition because E-S potentiation was also
observed in picrotoxin-treated slices (n = 5; data not
shown). The facilitation of the population spikes did not persist
however, because the increase in the population spikes was no longer
statistically significant 20-30 min post-ACPD (1-way ANOVA,
P > 0.05), despite a trend for persistently larger spikes at the higher stimulus intensities. Thus, whereas ACPD did
produce changes in the E-S ratio indicative of changes in postsynaptic
membrane excitability, their persistence was more transient and
variable than the ability to prime LTP. It is possible, therefore that
the large initial E-S changes were a function of the other membrane
effects of ACPD, such as the transient membrane depolarization and
input resistance increases which were observed to occur in some of the
neurons recorded from intracellularly.
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Relation between heightened cell excitability and priming of LTP
It is reasonable to hypothesize that the excitability increase
produced by ACPD and recorded intracellularly may be an important mechanism contributing to the priming of LTP by this mGluR agonist (Cohen and Abraham 1996). To test this hypothesis more
directly however, we first addressed whether priming of LTP could be
observed for intracellularly recorded EPSPs. Four of the ACPD-treated
cells described above in Fig. 2 were given mild TBS (5 bursts) 20-min post-ACPD application and the amount of LTP generated was compared with
that obtained in five new control cells. The stimulus intensity during
the tetanus was increased for all cells so that a single test shock was
just above threshold for consistently firing an action potential. The
magnitude of response change measured 5-min post-TBS was significantly
greater in the ACPD group (107 ± 20%) relative to the control
group (32 ± 10%, P < 0.01; Fig.
3A). LTP, measured 25-30-min
post-TBS, also occurred robustly in the ACPD group (35 ± 11%)
but was absent in the control group (
2 ± 7%; P < 0.01; Fig. 3A). Thus intracellularly recorded EPSPs
showed an mGluR-mediated priming effect similar to that previously
observed for field EPSPs (Cohen and Abraham 1996
).
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We then addressed whether the ACPD-induced change in membrane
parameters in these cells related to the facilitated LTP induction by
carrying out two further analyses. First, the number of action potentials generated during the LTP-inducing TBS was compared between
the primed and control cells. As predicted by the ACPD-induced reduction in spike frequency adaptation, there was a significant increase in the total number of action potentials occurring during the
TBS for the ACPD-treated cells (15.8 ± 3.0 spikes,
n = 4) relative to the control cells (9.2 ± 0.7 spikes, n = 5; P < 0.05; Fig. 3,
B and C). Second, we considered whether the
amplitude of the AHPslow, measured just before
the TBS, was predictive of the amount of LTP. A linear regression
analysis indicated that there was a statistically significant
correlation between the degree of potentiation 5 min post-TBS and the
amplitude of the pretetanus AHPslow
(r = 0.80; P < 0.01; Fig.
3D). The correlation between the degree of LTP at 25-30 min
posttetanus and the amplitude of the AHPslow was
weaker (r =
0.62, P < 0.08).
-adrenergic receptor activation increases cell excitability and
primes LTP induction
If the ACPD-induced increase in neuronal excitability contributes
to the LTP priming effect, it follows that activation of other
neurotransmitter receptors known to heighten pyramidal cell excitability should also prime LTP. Therefore we investigated whether
activation of -adrenergic receptors, which has been reported to
result in a persistent decrease in the AHPslow
and a persistent increase in cell excitability, measured as a
long-lasting increase in population spike amplitude (Dunwiddie
et al. 1992
), would prime LTP.
For these experiments, it was essential to first confirm the ability of
the -adrenergic receptor agonist isoproterenol to induce persistent
changes in membrane parameters consistent with an increase in cell
excitability. A 10 min application of 0.5 µM isoproterenol strongly
depressed the AHPslow (
63 ± 14%,
n = 7; P < 0.01) and reduced spike
frequency adaptation (4.4 ± 1.1 spikes per 250 ms current pulse,
n = 7, P < 0.01; Fig.
4, A and B).
Isoproterenol also caused membrane depolarization and an increase in
Rin, but as for ACPD these changes were variable
and not statistically significant (data not shown). Four cells were
held for
20 min after drug washout, and whereas the drug-induced
depression of both the AHPslow and spike
frequency adaptation dissipated during this period, they were still
reduced compared with baseline values (AHPslow:
39 ± 14%; P < 0.05; spike adaptation:
2.5 ± 1.3; n.s.; Fig. 4, C and D).
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Having confirmed that -adrenergic activation leads to persistent
membrane changes consistent with an increase in pyramidal cell
excitability, we used field potential recordings to test whether the
same dose of isoproterenol would prime LTP. In correspondence with the
protocols used for the ACPD experiments, 0.5 µM isoproterenol was
bath-applied for 10 min and 0.5 TBS was administered 20 min after drug
washout. The initial induction of LTP (taken as an average of 6 data
points around 5 min post-TBS) was weakly enhanced by isoproterenol
(56 ± 10%, n = 6) compared with a group of
interleaved control slices (33 ± 7%, n = 6;
P < 0.05, one-tailed Student's t-test;
Fig. 5, A and B).
The degree of LTP measured at 60 min post-TBS for isoproterenol treated
slices (27 ± 6%) was larger than, but not significantly
different from, that for control slices (20 ± 4%). To
characterize the persistence of LTP in these slices independently of
the initial induction, a decay curve for each slice was fit by the sum
of two negative exponential functions, and the decay time constant
(
= 1/rate) for the second, slower exponential was extracted as
an estimate of LTP persistence. There was no significant difference
between the two groups on this measure, indicating that although
isoproterenol enhanced the initial induction of LTP, this was not
associated with a concomitant increase in its stability (Fig. 5,
B and C). These findings contrast with the
effects of ACPD, which enhanced both the initial induction and the
persistence of LTP (Fig. 5, D and E), confirming
our previous report (Cohen and Abraham 1996
).
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Partial occlusion of ACPD-induced LTP priming by isoproterenol
We predicted that if isoproterenol and ACPD both facilitate the level of initial LTP induction through common mechanisms, such as enhanced postsynaptic cell excitability, isoproterenol treatment should occlude the ability of ACPD to prime the induction of LTP. To test this, the LTP generated in isoproterenol-treated slices was compared against that for slices treated with both isoproterenol and ACPD and against control slices. In these experiments, ACPD was administered 20 min before TBS, whereas isoproterenol was applied for 10 min before and during the TBS to elicit maximal excitability changes at the time of the TBS. Because isoproterenol did not facilitate LTP persistence (Fig. 5C), we predicted that it would not occlude ACPD's facilitation of the decay time constant.
In control slices, 0.5 TBS elicited a typical moderate induction of a
decaying form of LTP (5 min: 43 ± 6%; 60 min: 18 ± 3%, n = 8). Isoproterenol (0.5 µM) given during the
tetanus, whereas having no effect on baseline synaptic transmission,
produced a substantial increase in LTP induction (5 min: 72 ± 11%, n = 9; P < 0.05 compared with
controls; Fig. 6, A and
B). In contrast, but as expected, the second exponential
decay constant of this isoproterenol-enhanced LTP was not affected
(isoproterenol = 80.2 min; control
= 82.5 min; Fig.
6C). Slices primed with ACPD and then treated with
isoproterenol during the TBS showed no additional LTP induction (5 min:
74 ± 7%, n = 7) beyond that seen in slices treated with isoproterenol alone (Fig. 6, A and
B). However, the slices primed with ACPD did show a decay
constant (
= 145.0 min) significantly greater than either
control slices or slices treated with isoproterenol alone
(P < 0.05 for both comparisons). The occlusion of the
ACPD-induced facilitation of LTP induction by isoproterenol supports
the view that common mechanisms triggered by the two receptor agonists
are responsible for the priming of LTP induction. Some other mechanism
unique to the ACPD treatment appears to be responsible for facilitating
LTP persistence.
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DISCUSSION |
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Activation of mGluRs produces a persistent increase in cellular excitability
In our experiments, ACPD produced an acute depression of synaptic
responses accompanied by an increase in postsynaptic cellular excitability, confirming many previous studies of the effects of mGluR
activation on the electrophysiological properties of hippocampal
neurons (e.g., Charpak et al. 1990; Desai and
Conn 1991
). In this study, two measures of postsynaptic
excitability, spike frequency adaptation and the amplitude of the
AHPslow, were persistently reduced by ACPD. These
persistent changes were not observed in some previous studies, probably
because of the use of a different ACPD isomer (1S,3S-ACPD) which is not
as an effective agonist at Group I mGluRs (Gereau and Conn
1994
), the use of a lower concentration of the active isomer
(Hu and Storm 1991
), or the application of the ACPD for
a shorter length of time than that used here (Nouranifar et al.
1998
). The fact that the membrane depolarization,
Rin, and EPSP slope changes were not elicited reliably enough to reach statistical significance probably reflects our
use of a relatively low dose of ACPD (20 µM). Because the excitability increases that were observed might be expected to facilitate the induction of LTP, and thereby contribute to the priming
of LTP by ACPD (Cohen and Abraham 1996
), their
persistence was followed for
20 min and up to 45 min following ACPD
washout. We confirmed that the changes in these two parameters did
indeed last long enough to serve as candidate mechanisms underpinning LTP priming. It is interesting to note that the present findings are
similar to those of a previous study which found that a large dose of
quisqualate (100 µM), a potent agonist of the Group I mGluRs, caused
a reduction of the AHPslow and a decrease in
positive holding current lasting for ~60 min following drug washout
(Baskys 1992
). Selective activation of AMPA receptors,
which are also activated by quisqualate, did not affect the AHP
confirming a role of mGluRs in mediating the quisqualate effect. In
accordance with these observations, we have observed that selective
activation of AMPA receptors does not prime LTP (Cohen et al.
1998
).
It could be hypothesized that the prolonged excitability changes were
because of incomplete washout of the drug. However, there are several
good reasons why this was not the case. First, the acute effects of
ACPD on the fEPSP slope washed out extremely quickly on return to
drug-free solution (e.g., Fig. 6A), whereas the depression
of the AHPslow and spike frequency adaptation
were observed to last up to 45 min after drug washout. The rapid
recovery of the EPSP depression is consistent with our use of submerged slices and a high flow rate (2-3 ml/min) for the superfusion bathing medium. Second, we have shown previously that application of an mGluR
antagonist during ACPD delivery, but not shortly after, blocked the
priming of LTP (Cohen and Abraham 1996). Similar results have been reported by Bortolotto et al. (1994)
. These
findings clearly demonstrate an efficient washout of the drug. Finally, it is noteworthy that agonists of other neurotransmitter receptors, such as
-adrenergic receptors (Dunwiddie et al. 1992
;
present results), dopamine receptors (Gribkoff and Ashe
1984
) and histamine receptors (Selbach et al.
1997
) also have been reported to exert long-lasting effects on
cellular excitability. Thus the prolonged excitability increase induced
by mGluR activation may be characteristic of the responsible potassium
channels (e.g., IAHP,
IM, and/or IA), for which there appears to be a
slow return to the baseline state following the presumed
phosphorylation events that lead to their initial down-regulation.
An increase in cell excitability can also be detected, in principle, as an E-S potentiation in field potential recordings. Indeed, in the present experiments ACPD induced a significant potentiation of the population spike and E-S relation, but these effects largely decayed over the 20-30 min wash period. Thus the E-S potentiation did not correlate well with the long-lasting decreases in the AHPslow and spike frequency adaptation or with the priming of LTP. The E-S potentiation, therefore may be caused by other actions of ACPD such as reduced GABAergic inhibition or a increased input resistance and membrane depolarization.
Superficially, our E-S findings conflict with those of Breakwell
et al. (1996), who demonstrated that transient ACPD application led to a long-lasting E-S potentiation accompanied by a potentiation of
the fEPSP. This potentiation, however, developed slowly and was
prevented by NMDA receptor antagonists and removal of area CA3. These
features suggest that such ACPD-induced E-S potentiation is induced by
hyperactivity among the CA3 afferents to area CA1 (Chinestra et
al. 1994
). Our findings indicated that there is no significant
long-lasting E-S potentiation caused by ACPD application in isolated
CA1 slices that lack area CA3 and provide further evidence that ACPD is
cleared from the slices during the wash period.
mGluR-mediated changes in cellular excitability and the facilitation of LTP
The primary rationale for the present experiments was to
investigate possible mechanisms underlying the priming of LTP by prior
synaptic or pharmacological activation of mGluRs (Bortolotto et
al. 1994; Christie et al. 1995
; Cohen and
Abraham 1996
). Because the priming stimulation generally did
not affect synaptic transmission directly, we reasoned that changes in
cell excitability not apparent in field potential recordings might
underlie the facilitation of LTP. Indeed, our data confirm that
activation of mGluRs leads to an increased cellular excitability that
correlates in at least three ways with the facilitation of LTP
induction. First, both effects are induced by pharmacological
activation of mGluRs. Second, the excitability changes are persistent
enough to span the period of time between the priming stimulus and the
subsequent induction of LTP, although it was not feasible to hold the
cells long enough to determine whether the excitability changes
recovers over the same 1-3 h period as does the LTP priming effect
(Cohen and Abraham 1996
). Finally, we found a
significant correlation between the degree of
AHPslow suppression and the degree of LTP
initially induced by the tetanus. Taken together, these findings
indicate that raised postsynaptic excitability is an important
regulator of LTP induction, confirming previous studies showing that
suppression of the AHPslow by either
pharmacological or tetanic synaptic activation leads to facilitated LTP
(Blitzer et al. 1995
; Sah and Bekkers 1996
). The latter study, however, concentrated on the role that activation of noradrenergic receptors may play in modulating
concurrently induced LTP. In contrast, the present study
emphasizes the role that mGluRs, as well as
-adrenergic receptors,
play in regulating cell excitability and LTP over periods of time well
after the mGluR activation. Thus these experiments have identified one
set of potential "metaplasticity" (Abraham and Bear
1996
) mechanisms that may mediate priming of LTP (Cohen
and Abraham 1996
) and the mGluR-controlled "molecular
switch" proposed by Bortolotto et al. (1994)
.
-adrenergic priming of LTP
If enhanced postsynaptic membrane excitability induced by mGluR
activation is important for the associated priming of LTP, one would
expect that excitability increases elicited by other means should prime
LTP. We chose to use activation of -adrenergic receptors to test
this hypothesis, because the agonist isoproterenol is known to decrease
the AHPslow and increase excitability in hippocampal pyramidal cells (Madison and Nicoll 1986
).
Furthermore, these effects have been reported to outlast the period of
drug application for
45 min, and it has been proposed that there may be a corresponding persistently enhanced ability to elicit LTP (Dunwiddie et al. 1992
). In the present experiments, we
confirmed that a low dose of isoproterenol produces a large and durable increase in pyramidal cell excitability and that in this altered state
LTP was more readily induced. However, the increase in excitability and
the priming of LTP appeared to be slightly weaker for isoproterenol than for ACPD. More importantly, we found that the decay of LTP remained unaffected by isoproterenol, in contrast to the more prolonged
LTP following ACPD exposure.
To test whether -adrenergic receptor and mGluR activation
prime at least the initial induction of LTP through common mechanisms, we undertook an occlusion experiment, using ACPD as a priming stimulus
while applying isoproterenol during the tetanus to maximize its
effects. It is notable that under this protocol isoproterenol alone
induced a pronounced facilitation of LTP induction, as previously reported (Sah and Bekkers 1996
), but still without
effect on the persistence of LTP. Wheras isoproterenol successfully did
occlude the ability of ACPD to further increase LTP induction, it did not prevent ACPD from slowing the decay of the isoproterenol-enhanced LTP.
Our interpretation of the above experiments is that the mGluR-mediated
priming of LTP has at least two components; enhanced initial induction
and facilitated persistence. Isoproterenol shares the ability to prime
LTP induction, and we propose that a facilitation of postsynaptic
pyramidal cell excitability is the common mechanism operating for both
receptor systems. Other shared effects such as upregulated NMDA
receptor function or downregulated synaptic inhibition could in
principle be common mechanisms facilitating LTP, but these two
possibilities have been ruled out as being involved in the mGluR
priming effect (Cohen and Abraham 1996). One prediction
arising from the above interpretation is that other neurotransmitters
which elicit a persistent increase in pyramidal cell excitability, such
as histamine (Selbach et al. 1997
), should also be able
to prime the induction of LTP. We previously have reported that the
muscarinic agonist carbachol, which is known to increase pyramidal cell
excitability through a PKC pathway (Cole and Nicoll
1983
; Worley et al. 1987
), can prime LTP to a similar extent as isoproterenol (Cohen et al. 1998
). It
is not known in this case, however, whether carbachol's membrane
effects are long-lasting enough to account for the LTP priming effect.
Mechanisms downstream of mGluR activation for facilitating LTP induction
Group I mGluR activation is transduced by diverse second-messenger
pathways beginning with the activation of PLC, which leads to the
liberation of IP3 and activation of PKC by
diacylglycerol and elevation of cAMP concentrations. Either pathway is
in principle capable of mediating increases in postsynaptic
excitability via final common regulation of potassium channels, such as
the AHPslow (Nicoll 1988). It has
been suggested that ACPD, at a higher dose (100 µM) than that used in
this study, produces an increase in pyramidal cell excitability through
stimulation of adenylyl cyclase and increased activation of protein
kinase A by cAMP (Goh and Ballyk 1993
). This finding is
consistent with studies showing that activation of other receptors
positively coupled to the cAMP signaling cascade can also induce
long-lasting changes in pyramidal cell excitability (Dunwiddie
et al. 1992
; Gribkoff and Ashe 1984
; Pedarzani and Storm 1995
) and that Group I mGluRs and
-adrenergic receptors can work synergistically to produce this same
effect (Gereau and Conn 1994
; Gereau et al.
1995
). Alternatively, it recently has been reported that the
ACPD-induced inhibition of the AHPslow in dentate
granule cells is mediated by tyrosine kinases activated following
release of Ca2+ from intracellular stores
(Abdul-Ghani et al. 1996
). Thus further work is required
to determine the relative roles of PKA, PKC and tyrosine kinases in
mediating the effects observed here.
mGluR activation and the persistence of LTP
Whereas the initial induction of LTP relies on posttranslational
mechanisms, the later phases of LTP require de novo protein synthesis.
It has been suggested that mGluR activation can play an important role
in establishing these later phases (Behnisch et al.
1991; Bortolotto et al. 1994
;
Manahan-Vaughan and Reymann 1996
). It is not obvious,
however, that changes in cellular excitability will contribute to late,
protein-synthesis dependent phases of LTP. Indeed, we found that
isoproterenol did not promote LTP persistence, despite profoundly
increasing cell excitability. We propose, therefore that mGluRs
activate a privileged alternative signaling pathway that modulates LTP
persistence. One possibility is the coupling of mGluR activation to
dendritic protein synthesis machinery (Weiler and Greenough
1993
; Weiler et al. 1996
). Indeed, other studies from our laboratory have shown that the ACPD-induced priming of LTP
persistence is blocked by protein synthesis inhibitors (Raymond et al.
submitted). However, identifying the signaling pathways leading to the
protein synthesis and the critical proteins themselves requires much
further exploration.
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ACKNOWLEDGMENTS |
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We thank Drs. R. Sayer and J. Wickens for helpful comments on previous versions of this manuscript. We also thank K. Atkinson for help with some of the field potential experiments.
This research was funded by the Health Research Council of New Zealand. A. S. Cohen, C. M. Coussens, C. R. Raymond, and W. C. Abraham contributed equally to this work.
Present addresses: A. S. Cohen, Abramson Research Center, Room 410, 3516 Civic Center Blvd., Philadelphia, PA 19104; C. R. Raymond, John Curtin School of Medical Research, Division of Neurosciences, The Australian National University, Canberra, ACT 2601, Australia.
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FOOTNOTES |
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Address for reprint requests: W. C. Abraham, Dept. of Psychology, University of Otago, Box 56, Dunedin, New Zealand.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 May 1999; accepted in final form 4 August 1999.
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REFERENCES |
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