Departments of 1Psychiatry, 2Physiology, and 3Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143
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ABSTRACT |
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Hjelmstad, Gregory O.,
John T. R. Isaac,
Roger A. Nicoll, and
Robert C. Malenka.
Lack of AMPA receptor desensitization during basal synaptic
transmission in the hippocampal slice. Excitatory postsynaptic currents in the CA1 region of rat hippocampal slices are mediated primarily by -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in response to synaptically released glutamate. Outside-out patches from pyramidal cells in this region have shown that
AMPA receptors are desensitized by short (1 ms) pulses of glutamate. We
have taken a number of approaches to ask whether synaptic receptors
desensitize in response to synaptically released glutamate in the
slice. Recordings with paired pulses and minimal stimulation conditions
that are presumably activating only a single release site do not show
evidence for desensitization. Furthermore, cyclothiazide, a drug that
blocks desensitization, does not alter paired-pulse ratios even under
conditions of high probability of release, which should maximize
desensitization. These results suggest that synaptic receptors do not
desensitize in response to synaptically released glutamate during basal
synaptic transmission.
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INTRODUCTION |
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In area CA1 of the hippocampus, it is now
generally thought that the decay of
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptor-mediated excitatory postsynaptic currents (EPSCs) is due to
receptor deactivation, that is, the closing of the receptor and
subsequent unbinding of the ligand, as opposed to receptor
desensitization (i.e., the ligand remains bound to the receptor in a
long-lasting nonconducting state) (Colquhoun et al.
1992
). Despite this, outside-out patches pulled from CA1 cells
clearly show desensitization in response to brief pulses of glutamate.
In other words, when two pulses are given in rapid succession, the
response to the second pulse is smaller than the first (Arai and
Lynch 1996
; Colquhoun et al. 1992
).
Surprisingly, recent experiments with paired pulses and minimal
stimulation protocols in the hippocampal slice have not seen evidence
for desensitization (Hjelmstad et al. 1997;
Stevens and Wang 1995
). On the other hand, protocols
using drugs that block receptor desensitization have led to mixed
conclusions (Arai and Lynch 1998
; Debanne et al.
1996
; Kullmann 1994
; Pananceau et al. 1998
; Wang and Kelly 1996
). To resolve this
discrepancy, we have taken a number of approaches to specifically
address whether AMPA receptors desensitize in response to synaptically
released glutamate in the hippocampal slice.
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METHODS |
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Methods for slice preparation, whole cell experiments, and
minimal stimulation criteria are the same as previously described (Hjelmstad et al. 1997; Isaac et al.
1996
) using 2- to 3-wk-old Sprague-Dawley rats. For the
paired-pulse recordings in Fig. 2, single and paired pulses were
interleaved, and the tail from the first pulse was subtracted before
calculating the ratio.
Exogenous AMPA was applied by ionophoresis from a glass microelectrode containing 10 mM AMPA placed in stratum radiatum. Current pulses (200-500 nA, 20-60 ms) were controlled with a WPI constant-current generator. A small holding current was used to prevent leak from the electrode. AMPA was used instead of glutamate to negate the effects of reuptake through glutamate transporters.
The expected effects of desensitization (Fig. 2E) were
based on values from outside-out patch experiments from the CA1 region of hippocampus where the recovery from desensitization was calculated as
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RESULTS |
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We initially reanalyzed paired-pulse data from a number of
previous minimal stimulation experiments (Hjelmstad et al.
1997; Isaac et al. 1996
) to specifically address
whether desensitization occurs in response to synaptic stimulation.
Only using cells that met criteria for single-site recordings
(Isaac et al. 1996
), we segregated responses on whether
the response to the first of two stimuli was a success or a failure. If
desensitization occurs, responses after a success should show
desensitization and have smaller amplitudes than responses after a
failure. To assay this, we compared the potency (Stevens and
Wang 1994
), or the mean amplitude of successes only, for the
second response (Fig. 1A). The
average ratio of the potency after a success to that after a failure
for 10 cells was 1.01 ± 0.04 (Fig. 1B), suggesting
that no desensitization occurred. Furthermore, there was no correlation
between the paired-pulse interval and the potency ratio (interval range
30-70 ms). These data suggest that desensitization is not occurring in
response to synaptic stimulation.
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An assumption of this experiment is that we are recording from a single release site. If we were recording from multiple synapses, we would expect to see less evidence for desensitization because two subsequent EPSCs may not be from the same release site. Nevertheless, we would still expect to see some evidence for desensitization. For the example in Fig. 1, based on desensitization rates from outside-out patches (see METHODS), a single release site should show a potency ratio of 0.73. A simple binomial model of release with two release sites predicts a potency ratio of 0.85 and with three sites predicts a ratio of 0.89.
Because of the provocative nature of these results, we sought to obtain
additional evidence by addressing this issue in other ways. If
desensitization is occurring in response to synaptic stimulation, then
drugs that block AMPA receptor desensitization, such as cyclothiazide
(CTZ), should alter the magnitude of paired-pulse facilitation (PPF).
Previous attempts to address this issue in the hippocampus (Arai
and Lynch 1998; Debanne et al. 1996
;
Kullmann 1994
; Pananceau et al. 1998
;
Wang and Kelly 1996
) have provided inconsistent results.
One possible explanation for this is that these previous experiments
were all conducted under normal conditions in which the basal
probability of release (pr) is low. This will tend to underestimate the importance of desensitization because a given
synapse will only occasionally respond to both stimuli, as illustrated
in Fig. 2E. Therefore, to
maximize the potential effects of desensitization on the paired-pulse
ratio, we increased pr by raising
Ca2+ to 5 mM and by adding 50 µM 4AP, a protocol that
more than doubles pr (Hjelmstad et al.
1997
; Hsia et al. 1998
). Additionally, this protocol should occlude any presynaptic effects of CTZ on
pr (Diamond and Jahr 1995
). Under
these recording conditions, EPSCs in response to paired stimuli (40 ms)
showed no facilitation, and in some cases we observed paired-pulse
depression (Fig. 2A). Addition of 100 µM CTZ had no effect
on the paired-pulse ratio (1.01 ± 0.14 Control; 1.02 ± 0.12 CTZ, n = 6; Fig. 2B). The drug did slow the
decay of the EPSCs (Fig. 2B) consistent with the effects of CTZ on receptor deactivation (Patneau et al. 1993
).
Analogous experiments with aniracetam, which also blocks AMPA receptor
desensitization, likewise failed to show an increase in the
paired-pulse ratio (Fig. 2D; 92.7 ± 4.3%,
n = 3).
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An alternate method that has been used to show evidence for
desensitization in the chick nucleus magnocellularis (Otis et al. 1996a) makes use of the voltage dependence of
desensitization (Thio et al. 1991
), the magnitude of
which decreases with depolarization. Again, under conditions of
increased pr, the paired-pulse ratio was
measured at both hyperpolarized (
70 mv) and depolarized (+60) conditions (Fig. 2C1) in the presence of 100 µM D-APV to isolate the AMPA receptor-mediated current.
Scaling the two sets of synaptic responses to the amplitude of the
first EPSC showed that there was no change in the paired-pulse ratio
(
4.5 ± 4.7%, n = 7; Fig. 2, C and
D).
The absence of desensitization in these assays raises the question
whether AMPA receptors in the intact hippocampal slice are capable of
desensitization at all. To address this, we measured the currents
elicited by the ionophoretic application of AMPA to the dendrites of a
CA1 pyramidal cell. After the bath application of 100 µM CTZ, the
amplitude of the ionophoresis response increased dramatically (Fig.
3). Because CTZ affects deactivation in
addition to desensitization, we also monitored miniature EPSCs from the same cells because it is thought that the time course of individual mEPSCs are not influenced by desensitization (Hestrin
1992). Consistent with data from hippocampal cultures
(Diamond and Jahr 1995
), CTZ caused an increase in both
the mEPSC charge transfer (Fig. 3B) and the mEPSC frequency
(not shown). More importantly, the increase in the charge transfer of
the evoked responses was significantly larger than that of the minis
(Fig. 3, B and C), suggesting that desensitization was occurring in response to exogenous application of
AMPA.
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In a final set of experiments, we bath applied a low concentration of AMPA (50 nM) and monitored the holding current of a voltage-clamped pyramidal cell. The application of AMPA had a negligible effect on the holding current, but after application of CTZ the holding current increased dramatically (Fig. 3D). Therefore under conditions of prolonged agonist application AMPA receptors in the intact slice exhibit marked desensitization.
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DISCUSSION |
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We have shown that, although AMPA receptors in the intact
hippocampal slice are capable of desensitization, there is no
detectable desensitization in response to the synaptic release of
glutamate. These results stand in contrast to data from the chick
nucleus magnocellularis, where there is strong evidence showing AMPA
receptor desensitization in response to synaptically released glutamate (Otis et al. 1996a,b
).
There are a number of possible explanations for why desensitization was
not detected in response to synaptic stimulation as opposed to the
desensitization that occurs with glutamate application to outside-out
patches. First, the magnitude of desensitization will depend on the
degree of receptor occupancy at the synapse. If, for instance, the
synapse is far from saturated by a single vesicle of glutamate, then a
large pool of available receptors will obscure any desensitization
caused by previous activity. Although a 1-ms pulse of 1 mM glutamate
has been argued to mimic the time course of glutamate in the synaptic
cleft, recent estimates indicate that the time course is dramatically
faster (Clements 1996). Thus the disparity between the
results from the patch and the synapse may be due to the slower
clearance of glutamate from the outside-out patch.
It is also possible that the dynamics of desensitization are different
between receptors in outside-out patches and at intact synapses. There
are two possible scenarios by which this might occur. First, synaptic
receptors may exhibit different properties than extrasynaptic
receptors, perhaps because of some posttranslational modification.
Alternatively, a consequence of pulling outside-out patches may be to
change some aspects of receptor function, as is the case for the NMDA
receptor (Sather et al. 1992).
Our results differ from those of some previous studies (Arai and
Lynch 1998; Wang and Kelly 1996
), which reported
a change in PPF after application of CTZ. Although the reason
underlying these differences is not clear, the previous experiments
measured either extracellular field responses or EPSCs with
intracellular sharp electrode recordings. The synaptic responses that
are recorded with these methods may be subject to nonlinear summation.
In summary, in this study we have presented data suggesting that a single EPSC does not cause significant AMPA receptor desensitization. It is possible, however, that during repetitive synaptic activity the accumulation of desensitized receptors may have significant influences on excitatory synaptic responses.
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ACKNOWLEDGMENTS |
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We thank M. Frerking for helpful discussions during the course of these experiments.
R. C. Malenka, a member of the Center for the Neurobiology of Addiction and the Center for Neurobiology and Psychiatry, was supported by grants from the National Institutes of Health, an Investigator Award from the McKnight Endowment Fund for Neuroscience, and a grant from the Human Frontier Science Program. R. A. Nicoll, a member of the Keck Center for Integrative Neuroscience and the Silvio Comte Center for Neuroscience Research, was supported by grants from the National Institutes of Health. J.T.R. Isaac was supported by the Welcome Trust.
Present address of J.T.R. Isaac: Dept. of Anatomy, University of Bristol, School of Medical sciences, University Walk, Bristoll BS8 1TD, U. K.
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FOOTNOTES |
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Address for reprint requests: R. C. Malenka, Dept. of Psychiatry, University of California, LPPI, Box 0984, 401 Parnassus Ave., San Francisco, CA 94143.
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 14 December 1998; accepted in final form 3 March 1999.
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REFERENCES |
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