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Potassium-induced long-term potentiation in rat hippocampal slices
100
Brain Research, 580 (1992) 100-105 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17709
Potassium-induced long-term potentiation in rat hippocampal slices Mark W. Fleck a, Alan M. Palmer b'c and German Barrionuevo a'b Departments of aBehavioral Neuroscience, bpsychiatry, and Cpharmacology, University of Pittsburgh, Pittsburgh, PA 15260 (USA) (Accepted 24 December 1991) Key words: Long-term potentiation; Hippocampus; CA1; Potassium
We observed that a transient increase in extracellular potassium concentration (50 mM for 40 s) was sufficient to induce long-term potentiation (LTP) of synaptic transmission in area CA1 of the hippocampal slice. Potassium-induced potentiation of the Schaffer collateral/commissural synapses demonstrated several features characteristic of tetanus-induced LTP: (1) population excitatory post-synaptic potential (EPSP) amplitudes were enhanced to a similar magnitude (on average 70% above baseline) which (2) lasted for more than 20 min; (3) induction was blocked by bath application of the specific N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovalerate (D-APV), and (4) was attenuated by reduction of the concentration of calcium in the extracellular medium. Induction of either potassiuminduced LTP or tetanus-induced LTP occluded the subsequent expression of the other. Finally, exposure to high potassium in the absence of electrical stimulation was sufficient to induce LTP. Taken together, these data indicate that brief depolarizing stimuli other than tetanus can induce LTP. Because potassium-induced LTP is not restricted to the subset of afferents examined electrophysiologically, such a method could facilitate analyses of the biochemical events underlying both the induction and expression of LTP. INTRODUCTION Long-term potentiation (LTP) is a persistent enhancement of synaptic transmission that is induced by brief, high-frequency (tetanic) stimulation of excitatory afferent fibers. Such long-term synaptic modification, lasting for hours or longer, has received considerable attention, largely because it has been implicated in the natural mechanisms of m e m o r y and learning. In area CA1 of the hippocampus, the induction of LTP follows a Hebbian rule; that is, induction is critically dependent on the concurrent activation of pre- and post-synaptic elements. Post-synaptic manipulations alone, such as weak tetanus or depolarizing current injection, are insufficient to produce LTP. Post-synaptic hyperpolarization during strong tetanus can prevent LTP ~8'23, whereas post-synaptic depolarization can facilitate LTP induction by weak tetanic stimulation 28. The requirement for concurrent pre- and post-synaptic depolarization is largely attributed to the voltage-dependent properties of the N-methyl-D-aspartate ( N M D A ) receptor-channel complex 8'13-15'21. Indeed, LTP induction can be blocked by both competitive 9'~4 and noncompetitive 7'26 N M D A receptor antagonists. In addition, induction can be prevented by reducing extracellular calcium concentration 1°, or by injecting calcium chelators into the post-synaptic ceU 19'22, suggesting that
the requirement for calcium influx is post-synaptic. These data are consistent since N M D A receptors mediate a calcium influx 3'2°'25. The above model for LTP induction, which emphasizes concurrent pre- and post-synaptic depolarization, predicts that LTP should also be produced by depolarizing manipulations other than tetanic stimulation, such as elevation of extracellular potassium concentration. The association between elevated extracellular potassium concentration and LTP induction is not without precedent. Several groups have demonstrated that tetanic stimulation produces a transient, intensity-dependent increase in extracellular potassium concentration 1'5. This increased extracellular potassium would tend to depolarize nearby elements and thus increase their excitability. Such changes in extracellular potassium concentration may contribute to LTP induction. It has been reported that elevation of extracellular potassium relieves the magnesium block of N M D A receptors 6, and more recently, a cooperative interaction between extraceUular potassium and tetanic stimulation has been reported 4. In the present study, we describe LTP induced by transient elevation of extracellular potassium concentration and compare this potentiation with LTP induced by tetanic stimulation. Portions of this work have been previously reported 11.
Correspondence: G. Barrionuevo, 446 Crawford Hall, Department of Behavioral Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA. Fax: (1) (412) 624-9198.
101 MATERIALS AND METHODS
Preparation and maintenance of hippocampal slices Hippocampal slices were prepared in a conventional manner from male, Sprague-Dawley rats (125-250 g; from Zivic-Miller, PA). Briefly, both hippocampi were used to obtain 500/~m thick slices cut perpendicular to the longitudinal axis of the hippocampus using a vibratome. Slices were incubated in vials of continually oxygenated artificial cerebro-spinal fluid (ACSF) at 34 + I°C before use. Slices of area CA1 were prepared by subsequently removing most of CA3 and dentate regions. Normal ACSF consisted of (in mM): NaC1 125, KC1 5, NaH2PO 4 1.25, MgC12 1.5, CaC12 2, NaHCO 3 26, glucose 10 and was saturated with 95% 02/5% CO 2 to obtain pH 7.3-7.4. High-potassium medium contained 50 mM KCI, with an equimolar reduction in NaC1 concentration.
Extracellular electrophysiology After a 1-h recovery period, slices were individually transferred as needed to a submerged recording chamber (with an approximate vol. of 250 ~1). Within the recording chamber, slices were continuously perfused at a rate of 800 /A/min with fresh, oxygenated ACSF warmed to 32 _+ 1°C. Extracellular recording micropipettes were pulled from 1.5 mm o.d. borosilicate glass tubing, filled with 2 M NaC1, and selected for tip resistances of 1-3 Mfl measured in normal saline. Bipolar stimulating electrodes were fashioned with 62/zm diameter, insulated nichrome wire. Extracellular recordings of population excitatory post-synaptic potentials (EPSPs) were obtained from stratum radiatum of the CA1 subregion. A bipolar stimulating electrode was placed in stratum radiatum of CA1 for orthodromic stimulation of the Schaffer collateral/commissural afferents at 0.2 Hz. Pulse duration was fixed at 100/ts. Stimulus intensity was set to elicit population EPSPs which were approximately half of that required for the elicitation of a population spike. Sampled extracellular field potentials were displayed on a Nicolet oscilloscope, digitized and stored on IBM-386 system hard drive for subsequent, off-line analysis. LTP-inducing stimuli consisted of either a 40-s pulse of high-potassium medium (50 mM) or tetanic stimulation (10 bursts of stimuli at 1 Hz, with each burst consisting of 10 stimuli at 100 Hz). LTP was operationally defined as an increase of greater than 15% in the amplitude of the population EPSP lasting for at least 20 min.
for 5 min. After a 20-min washout period, a new baseline population amplitude was recorded for 5 min, and another 40-s pulse of high-potassium medium was perfused. The magnitude of potentiation was measured 20 min after each exposure to high-potassium.
Data analyses and statistics Data are presented as means _+ S.E.M. Population EPSP amphtudes were compared pre- and post-stimulation (repeated measures). To determine whether any observed enhancement of synaptic efficacy was significant (P < 0.05) one-tailed Student's t-tests were employed. To determine whether there were any significant differences between the magnitudes of enhancement produced by different stimulation protocols, two-tailed, independent t-tests were employed.
RESULTS
Potassium-induced L T P occludes tetanus-induced L T P T r a n s i e n t p e r f u s i o n o f slices w i t h h i g h - p o t a s s i u m m e d i u m (50 m M f o r 40 s) a l w a y s t e m p o r a r i l y a b o l i s h e d CA1 population EPSPs evoked by Schaffer collateral/ c o m m i s s u r a l s t i m u l a t i o n . W h e n d e t e c t a b l e , t h e f i b e r volley, p r e s u m a b l y r e p r e s e n t i n g t h e s y n c h r o n o u s a c t i v a t i o n o f a f f e r e n t fibers, w a s also a b o l i s h e d b y h i g h - p o t a s s i u m . U p o n r e c o v e r y , p o p u l a t i o n E P S P a m p l i t u d e s o f t e n disp l a y e d a s u s t a i n e d e n h a n c e m e n t w i t h n o c h a n g e in d u r a t i o n , t i m e t o p e a k o r in t h e a m p l i t u d e o f f i b e r volleys. P o t a s s i u m - i n d u c e d LTP, like t e t a n u s - i n d u c e d LTP, w a s
A.
]3°
1.5
Potassium-induced LTP and occlusion of tetanus-induced LTP. Population EPSPs were recorded in CA1 in response to test stimulation of the Schaffer collateral/commissurals. After a stable amplitude baseline response had been recorded for at least 5 rain, a 40-s pulse of ACSF containing 50 mM KCl was perfused. The magnitude of potassium-induced LTP was measured 20 rain after the potassium pulse as the percent increase in the amplitude of the population EPSP above baseline. In some of these experiments, a tetanic stimulus was subsequently delivered to the test input to determine whether potassium-induced LTP occluded the expression of tetanus-induced LTP. In other experiments, LTP of the test input was saturated with 3 tetanic stimuli before exposure to high-potassium to determine whether tetanus-induced LTP occluded the subsequent expression of potassium-induced LTP. Data were retained if either potassium-induced or tetanus-induced LTP was observed. Input-specificity of potassium-induced LTP For some experiments, test stimulation was paused for 10 min, beginning 1 min before high-potassium perfusion, to determine whether the LTP induced by potassium does not require concurrent electrical stimulation of the Schaffer collateral/commissural afferents. Any potentiation resulting from exposure to high-potassium alone could not be restricted to the specific subset of Schaffer collateral/commissural inputs being tested with electrical stimulation. APV blockade of potassium-induced LTP. Slices were perfused with medium containing 20 #M t)-2-amino-5-phosphonovalerate D-APV (Cambridge Research Biochemicals) which was allowed to act for at least 15 rain before beginning to record baseline responses. A 40-s pulse of APV-medium containing 50 mM KC1 was then perfused after a stable baseline response had been recorded
1.0 m
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Time (minutes)
Fig. 1. Transient exposure to artificial cerebrospinal fluid (ACSF) containing 50 mM KC1 induces long-term potentiation (LTP) of population excitatory post-synaptic potential (EPSP) in CA1 (see also Table I). A: population EPSPs from a single experiment are shown (1) before, and (2) 20 rain after 40-s exposure to isotonic medium containing 50 mM KCI. Both the initial slope and peak amplitude of the population EPSP were increased 20 min after high-potassium exposure. Each waveform represents the average of 5 consecutive responses (Bars = 0.25 mV, 5 ms). B: plot shows the time course of potentiation from the same experiment. In this and subsequent figures, horizontal bar indicates the timing of the 40-s perfusion of high-potassium containing medium and numbers refer to the times at which the above waveforms were recorded. No population EPSP could be evoked for several minutes after highpotassium exposure. Upon recovery, stimulation of the test input at the pre-set intensity evoked larger amplitude population EPSPs.
102 A.
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Time {minutes)
Fig. 2. Potassium-induced LTP prevents the subsequent expression of tetanus-induced LTP (see also Table I). A: population EPSPs from a representative experiment are shown (1) before exposure to 50 mM KCI, (2) after high-potassium exposure, and (3) after tetanization of the test input. Each waveform represents the average of 5 consecutive responses (Bars = 0.25 mV, 5 ms). The amplitude of the population EPSP increased 93% above baseline, as measured 90 min after high-potassium exposure. No further increase was seen after tetanic stimulation. B: plot shows the time course of the same experiment. Vertical bar indicates the time at which tetanic-stimulus was delivered to the test input.
m a r k e d by an increase in the initial slope and p e a k amplitude of the population EPSP as shown in Fig. 1A. Of the 30 slices tested for potassium-induced LTP, 10 were discarded for failing to express either potassium-induced LTP or tetanus-induced LTP. Of the remaining 20 slices, 18 expressed potassium-induced LTP (t = 6.96, P < 0.001) with an average increase in population E P S P amplitude of 76 + 19% when m e a s u r e d after 20 min. Initial slopes of the population EPSPs (between 10% and 90% of the p e a k amplitude) were also m e a s u r e d to control for changes in feedforward inhibitory responses. E P S P initial slopes were significantly increased from 0.26 + 0.03 V/s during baseline to 0.41 + 0.03 V/s after high-potassium (t = 7.18, P < 0.001). Two slices did not demonstrate potassium-induced LTP (enhancements of 0% and 2%) but did subsequently express tetanus-induced LTP (enhancements of 30% and 36%). The magnitude of potassium-induced LTP was not significantly different from these two slices and in slices r e p e a t e d l y tetanized to induce LTP (t = 0,89, P > 0.05). In several experiments (not shown), population EPSPs were r e c o r d e d for m o r e than 2 h after high-potassium exposure and displayed little decrement. Of the 18 slices expressing potassium-induced LTP, 14 were presented with tetanic stimulation m o r e than 20 min later to d e t e r m i n e whether potassium-induced LTP occluded the subsequent induction of tetanus-induced
i 0
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30
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40
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Time {minutes)
Fig. 3. Tetanus-induced LTP prevents the subsequent expression of potassium-induced LTP (see also Table I). A: population EPSPs from a representative experiment are shown (1) before, and (2) after repeated tetanization of the test input, and again (3) after subsequent high-potassium exposure. Each waveform represents the average of 5 consecutive responses (Bars = 0.25 mV, 5 ms). The amplitude of the population EPSP increased 42% above baseline, as measured 20 min after the initial tetanus. No further increase was seen after high-potassium exposure. B: plot shows the time course of the same experiment. Vertical bars indicate times at which tetanic-stimuli were delivered to the test input.
LTP (shown in Fig. 2). Only two of these slices expressed tetanus-induced LTP (enhancements of 15% and 21%). On average, the increase in population E P S P amplitude resulting from subsequent tetanic stimulation (2 + 3%) was not significant (t = 1.24, P > 0.05). E v e n the two slices that expressed subsequent tetanus-induced LTP showed partial occlusion as the average 18% enh a n c e m e n t observed was significantly lower than was observed with tetanus alone (t = 3.77, P < 0.05). A n o t h e r 5 slices were tested for occlusion in the reverse order. A representative experiment is shown in Fig. 3. Presentation of 3 tetanic stimuli to the test input resulted in a significant e n h a n c e m e n t averaging 63 + 11% as m e a s u r e d 20 min after tetanus (t = 5.53, P < 0.005). Subsequent exposure to high-potassium induced LTP in only one of these slices although the magnitude of that e n h a n c e m e n t (20%) was much r e d u c e d from what is typically seen with high-potassium alone. High-potassium exposure resulted in a change in the amplitude of the population EPSP averaging - 1 + 6% in these previously p o t e n t i a t e d slices (t = 0.28, P > 0.05).
Potassium-induced LTP is not restricted to the subset of Schaffer collateral/commissural afferents sampled with electrical stimulation Four slices were tested for potassium-induced LTP
103 TABLE I
/L
Characteristics of potassium-induced LTP Values are population EPSP amplitudes in area CA1 as percent of baseline control + S.E.M. as measured under various conditions. Control values were measured during a baseline recording period. Post-stimulus values were measured 20 min after stimulation either by tetanus or by 40-s exposure to 50 mM KC1. n represents the number of slices used for analysis (not rejected)/total number tested in a given set of experiments.
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Fig. 4. Potassium-induced LTP is blocked by bath application of the competitive N-methyl-o-aspartate ( N M D A ) receptor antagonist
o-2-amino-5-phosphonovalerate (D-APV) (see also Table I). A: population EPSPs from a single experiment are shown (1) before, and (2) 20 rain after high-potassium exposure in the presence of 20 /~M D-APV. No lasting changes in population EPSP amplitude were observed. Perfusion medium was then changed to normal medium for 20 min to allow the APV to washout. Population EPSPs were again recorded (3) before, and (4) 20 min after high-potassium exposure. In this experiment, a marked potentiation of population EPSP amplitude was observed 20-min after the second exposure to high-potassium. Each waveform represents the average of 5 consecutive responses (Bars = 0.25 mV, 5 ms). B: plot shows the time course of the same experiment.
with electrical stimulation p a u s e d for 10 min, beginning 1-min before exposure to high-potassium. W h e n electrical test stimulation of the Schaffer collateral/commissurals was restarted, these slices expressed an increase in p o p u l a t i o n E P S P amplitude averaging 55 ___ 24% (t = 3.23, P < 0.05). T h e r e was no significant difference between these slices and slices that were electrically stimulated during high-potassium exposure (t = 0.92, P > 0.05). The remaining slice did not express potassium-induced LTP, but did subsequently express tetanus-induced LTP (30% enhancement).
A P V blocks potassium-induced LTP In s e p a r a t e experiments, 8 slices were e x p o s e d to high-potassium during bath application of 20/~M D-APV (see Fig. 4); none exhibited potassium-induced LTP (t = 0.25, P > 0.05). A f t e r 20-rain washout, transient highpotassium exposure p r o d u c e d an increase in p o p u l a t i o n E P S P a m p l i t u d e averaging 20 + 7% when all 8 slices were considered (t = 2.80, P < 0.05). O f these 8 slices, 3 expressed potassium-induced LTP (greater than 15%) which a v e r a g e d 34 _ 2% as m e a s u r e d m o r e than 20 min after high-potassium exposure.
KC1 stimulation KC1 - - no electrical stim. Tetanus after KCI KC1 in 20/,M D-APV KC1 after APV washout KC1 in low calcium KC1 after calcium wash Tetanic stimulation KCI after tetanus
Control
Post-stimulus
n
100 100 100 100 100 100 100 100 100
176 155 102 102 134 136 121 163 99
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* Increased from baseline control P < 0.05, **P < 0.005.
The d e p e n d e n c e of potassium-induced LTP on extracellular calcium concentration was also tested in 5 separate slices. O f these, two slices d e m o n s t r a t e d potassium-induced LTP even when high-potassium exposure occurred in 0.5 m M calcium (enhancements of 51% and 85%). Two others failed to d e m o n s t r a t e potassium-induced LTP in low calcium but did express LTP after subsequent potassium-exposure in normal calcium (enhancements of 44% and 38%). The remaining slice failed to d e m o n s t r a t e potassium-induced LTP altogether. A further reduction of extracellular calcium concentration below 0.5 m M might have clarified these results, but could not be accomplished without abolishing synaptic transmission. Table I summarizes the observed characteristics of potassium-induced LTP. DISCUSSION
The present results d e m o n s t r a t e that the depolarization caused by transient elevation of extracellular potassium concentration is sufficient to induce LTP of the Schaffer collateral/commissural synapses. This potassium-induced LTP displays m a n y of the features that define LTP induced by tetanic stimulation. Specifically, (1) they display similar magnitudes of e n h a n c e m e n t , (2) they both persist for m o r e than 20 min, (3) both are blocked by bath application of the N M D A r e c e p t o r antagonist, D - A P V 9'14, and (4) both are sensitive to the extracellular concentration of calcium 1°. In addition, induction of either form of LTP prevents the subsequent induction of the other, which strongly argues that the two types of
104 LTP are expressed via a common mechanism. Similar LTP-like events in area CA1 have been previously reported. Izumi et al) 6 observed that LTP could be induced using conditioning solutions which contained high-potassium (up to 15 mM), low-magnesium, and either quisqualate or L-glutamate. Such potentiation was not observed in the presence of APV. Because high-potassium exposure itself evokes the release of endogenous excitatory amino acids (EAAs), such addition of exogenous EAAs in the medium should not be necessary. In preliminary experiments, we also observed that perfusion of 15 mM KCI medium alone did not produce any lasting changes in population EPSP amplitude and failed to evoke any detectable release of endogenous aspartate or glutamate, although it did cause transient epileptiform activity. It is possible that 15 mM KC1 does not depolarize pre- and post-synaptic fibers sufficiently to fulfill the Hebbian requirement for LTP induction without the addition of exogenous glutamate or quisqualate. Another similar potentiation has been reported by May et al. 24, except that the potentiation they observed occurred only when elevated potassium concentrations were accompanied by reduced calcium concentrations. Such a result is difficult to reconcile with previous studies which suggest that elevation of free calcium in the post-synaptic cell is both necessary 1°'19'22 and sufficient 12' 22,27 for the induction of LTP, and contrasts with our results which indicate that LTP induction with high-potassium is facilitated in normal calcium concentrations. The apparent discrepancy may have resulted from differences in the duration of high-potassium exposure, as the study of May et al. 24 utilized a substantially longer exposure time (3 min). They observed that such prolonged exposure tended to depress subsequent evoked population EPSPs in normal calcium. One interpretation of this could be that the number of cells contributing to the population response was reduced after high-potassium which could mask whatever potentiation was produced. Indeed, others have reported that high-potassium exposure produces a lasting depression of hippocampal synaptic transmission in a calcium and NMDA-receptor-de-
REFERENCES 1 Aitken, P.G. and Somjen, G.G., The sources of extracellular potassium accumulation in the region of hippocampal slices, Brain Res., 369 (1986) 163-167. 2 Aniksztejn, L. and Ben Ari, Y., Novel form of LTP produced by a K÷ channel blocker in the hippocampus, Nature, 349 (1991) 67-69. 3 Ascher, P. and Nowak, L., The role of divalent cations in the N-methyl-D-aspartate responses of mouse central neurones in culture, J. Physiol., 399 (1988) 247-266. 4 Ballyk, B.A. and Goh, J.W., Role of extraceUular K÷ in the induction of hippocampal LTP, IBRO, Abstr. (1991) 45.2.
pendent manner 17. Reduced extracellular calcium may have protected cells from excitotoxicity while still allowing LTP to occur. Several studies suggest that elevation of free calcium in the post-synaptic cell is not only necessary but sufficient for LTP induction. Transient perfusion of ACSF containing tetraethylammonium 2 or high concentrations of extracellular calcium 12'27 has been shown to produce LTP in area CA1. LTP induced by these manipulations is resistant to APV, but is reduced or prevented by blockade of voltage-sensitive calcium channels. This implies that the intracellular rise in calcium necessary for LTP induction need not be mediated by NMDA receptor-gated channels. Indeed, Malenka et al. 22 demonstrated that direct, transient elevation of post-synaptic calcium using the photo-labile calcium chelator Nitr-5 is sufficient to produce an enhancement similar to LTP. It should be noted that the LTP we observed after high-potassium perfusion was blocked by APV and so is induced by E A A transmitter release rather than by direct activation of voltage-sensitive calcium channels. The present observation that high-potassium exposure is sufficient to induce LTP in hippocampal area CA1, although novel, is not entirely unexpected. The currently accepted model for LTP induction which emphasizes concurrent pre- and post-synaptic depolarization predicts that depolarizing agents such as potassium should induce LTP. Unlike tetanus-induced LTP, potassium-induced LTP is not restricted to a small subset of afferents. Thus, inducing LTP with high-potassium could sufficiently enhance the biochemical signals associated with LTP to enable us to examine the underlying changes. Experiments utilizing potassium-induced LTP to establish the roles of excitatory amino acids and their receptors in LTP are currently in progress.
Acknowledgements. We are grateful to Drs. Jon Johnson and Dave Wood for their editorial comments. This work was supported in part by Grants NIMH MH30915 (seed money Grant R203), NINDS NS01196 and NS24288, and Andrew Mellon and NIMH predoctoral fellowships.
5 Benninger, C., Kadis, J. and Prince, D.A., Extracellular calcium and potassium changes in hippocampal slices, Brain Res., 187 (1980) 165-182. 6 Carter, C.J., Noel, F. and Scatton, B., Raised extracellular potassium relieves the blockade by magnesium of NMDA-induced cerebellar cyclic GMP production, Neurosci. Left., 82 (1987) 201-205. 7 Coan, E.J., Saywood, W. and Collingridge, G.L., MK-801 blocks NMDA receptor-mediated synaptic transmission and long-term potentiation in rat hippocampal slices, Neurosci. Len., 80 (1987) 111-114. 8 Collingridge, G.L. and Bliss, T.V.P., NMDA receptors -- their role in LTP, Trends Neurosci., 10 (1987) 288-293.
105 9 Collingridge, G.L., Kehl, S.J. and McLellan, H., Excitatory amino acids in synaptic transmission in the Schaffer collateral/ commissural pathway of the rat hippocampus, J. Physiol., 334 (1983) 33-46. 10 Dunwiddie, T. and Lynch, G., The relationship between extracellular calcium concentrations and the induction of hippocampal long-term potentiation, Brain Res., 169 (1979) 103-110. 11 Fleck, M.W., Palmer, A.M. and Barrionuevo, G., Characterization of an LTP-like phenomenon induced by a transient increase in extracellular potassium concentration, Soc. Neurosci. Abstr., 16 (1990) 212.7. 12 Grover, L.M. and Teyler, T.J., Differential effects of NMDA receptor antagonist APV on tetanic stimulation induced and calcium induced potentiation, Neurosci. Lett., 113 (1990) 309314. 13 Gustafsson, B. and Wigstrom, H., Physiological mechanisms underlying long-term potentiation, Trends Neurosci., 11 (1988) 156-16Z 14 Harris, E.W., Ganong, A.H. and Cotman, C.W., Long-term potentiation in the hippocampus involves activation of N-methyl-o-aspartate receptors, Brain Res., 323 (1984) 132-137. 15 Herron, C.E., Lester, R.A., Coan, E.J. and Collingridge, G.L., Frequency-dependent involvement of NMDA receptors in the hippocampus: a novel synaptic mechanism, Nature, 322 (1986) 265-268. 16 Izumi, Y., Miyakawa, H., Ito, K. and Kato, H., Quisqualate and N-methyl-v-aspartate (NMDA) receptors in induction of hippocampal long-term facilitation using conditioning solution, Neurosci. Lett., 83 (1987) 201-206. 17 Jing, J., Aitken, EG. and Somjen, G.G., Lasting neuron depression induced by high potassium and its prevention by low calcium and NMDA receptor blockade, Brain Res., 557 (1991) 177-183. 18 Kelso, S., Gonong, A.H. and Brown, T.H., Hebbian synapses in hippocampus, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 53265330.
19 Lynch, G., Larson, J., Kelso, S., Barrionuevo, G. and Schottier, E, Intracellular injections of EGTA block induction of hippocampal long-term potentiation, Nature, 305 (1983) 719-721. 20 MacDermott, A.B., Mayer, M.L., Westbrook, G.L., Smith, S.J. and Barker, J.L., NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones, Nature, 321 (1986) 519-522. 21 MacDonald, J.F., Porietis, A.V. and Wojtowicz, J.M., L-Aspartic acid induces a region of negative slope conductance in the current-voltage relationship of cultured spinal cord neurons, Brain Res., 237 (1982) 248-253. 22 Malenka, R.C., Kauer, J.A., Zucker, R.S. and Nicoll, R.A., Postsynaptic calcium is sufficient for potentiation of hippocampal synaptic transmission, Science, 242 (1988) 81-84. 23 Malinow, R. and Miller, J.P., Postsynaptic hyperpolarization during conditioning reversibly blocks induction of long-term potentiation, Nature, 320 (1986) 529-530. 24 May, EB.Y., Goh, J.W. and Sastry, B.R., Induction of hippocampal long-term potentiation in the absence of extracellular Ca2+, Synapse, 1 (1987) 273-278. 25 Mayer, M.L., MacDermott, A.B., Westbrook, G.L., Smith, S.J. and Barker, J.L., Agonist- and voltage-gated calcium entry in cultured mouse spinal cord neurons under voltage clamp measured using Arsenazo III, J. Neurosci., 7 (1987) 3230-3244. 26 Stringer, J.L. and Guyenet, EG., Elimination of long-term potentiation in the hippocampus by phencyclidine and ketamine, Brain Res., 258 (1983) 159-164. 27 Turner, R.W., Bainbridge, K.G. and Miller, J.J., Calcium-induced long-term potentiation in the hippocampus, Neuroscience, 7 (1982) 411-416. 28 Wigstrom, H. and Gutstafsson, B., Hippocampal long-term potentiation is induced by pairing single afferent volleys with intracellularly injected depolarizing current pulses, Acta Physiol. Scand., 126 (1986) 317-319.
Brain Research, 580 (1992) 100-105 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17709
Potassium-induced long-term potentiation in rat hippocampal slices Mark W. Fleck a, Alan M. Palmer b'c and German Barrionuevo a'b Departments of aBehavioral Neuroscience, bpsychiatry, and Cpharmacology, University of Pittsburgh, Pittsburgh, PA 15260 (USA) (Accepted 24 December 1991) Key words: Long-term potentiation; Hippocampus; CA1; Potassium
We observed that a transient increase in extracellular potassium concentration (50 mM for 40 s) was sufficient to induce long-term potentiation (LTP) of synaptic transmission in area CA1 of the hippocampal slice. Potassium-induced potentiation of the Schaffer collateral/commissural synapses demonstrated several features characteristic of tetanus-induced LTP: (1) population excitatory post-synaptic potential (EPSP) amplitudes were enhanced to a similar magnitude (on average 70% above baseline) which (2) lasted for more than 20 min; (3) induction was blocked by bath application of the specific N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovalerate (D-APV), and (4) was attenuated by reduction of the concentration of calcium in the extracellular medium. Induction of either potassiuminduced LTP or tetanus-induced LTP occluded the subsequent expression of the other. Finally, exposure to high potassium in the absence of electrical stimulation was sufficient to induce LTP. Taken together, these data indicate that brief depolarizing stimuli other than tetanus can induce LTP. Because potassium-induced LTP is not restricted to the subset of afferents examined electrophysiologically, such a method could facilitate analyses of the biochemical events underlying both the induction and expression of LTP. INTRODUCTION Long-term potentiation (LTP) is a persistent enhancement of synaptic transmission that is induced by brief, high-frequency (tetanic) stimulation of excitatory afferent fibers. Such long-term synaptic modification, lasting for hours or longer, has received considerable attention, largely because it has been implicated in the natural mechanisms of m e m o r y and learning. In area CA1 of the hippocampus, the induction of LTP follows a Hebbian rule; that is, induction is critically dependent on the concurrent activation of pre- and post-synaptic elements. Post-synaptic manipulations alone, such as weak tetanus or depolarizing current injection, are insufficient to produce LTP. Post-synaptic hyperpolarization during strong tetanus can prevent LTP ~8'23, whereas post-synaptic depolarization can facilitate LTP induction by weak tetanic stimulation 28. The requirement for concurrent pre- and post-synaptic depolarization is largely attributed to the voltage-dependent properties of the N-methyl-D-aspartate ( N M D A ) receptor-channel complex 8'13-15'21. Indeed, LTP induction can be blocked by both competitive 9'~4 and noncompetitive 7'26 N M D A receptor antagonists. In addition, induction can be prevented by reducing extracellular calcium concentration 1°, or by injecting calcium chelators into the post-synaptic ceU 19'22, suggesting that
the requirement for calcium influx is post-synaptic. These data are consistent since N M D A receptors mediate a calcium influx 3'2°'25. The above model for LTP induction, which emphasizes concurrent pre- and post-synaptic depolarization, predicts that LTP should also be produced by depolarizing manipulations other than tetanic stimulation, such as elevation of extracellular potassium concentration. The association between elevated extracellular potassium concentration and LTP induction is not without precedent. Several groups have demonstrated that tetanic stimulation produces a transient, intensity-dependent increase in extracellular potassium concentration 1'5. This increased extracellular potassium would tend to depolarize nearby elements and thus increase their excitability. Such changes in extracellular potassium concentration may contribute to LTP induction. It has been reported that elevation of extracellular potassium relieves the magnesium block of N M D A receptors 6, and more recently, a cooperative interaction between extraceUular potassium and tetanic stimulation has been reported 4. In the present study, we describe LTP induced by transient elevation of extracellular potassium concentration and compare this potentiation with LTP induced by tetanic stimulation. Portions of this work have been previously reported 11.
Correspondence: G. Barrionuevo, 446 Crawford Hall, Department of Behavioral Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA. Fax: (1) (412) 624-9198.
101 MATERIALS AND METHODS
Preparation and maintenance of hippocampal slices Hippocampal slices were prepared in a conventional manner from male, Sprague-Dawley rats (125-250 g; from Zivic-Miller, PA). Briefly, both hippocampi were used to obtain 500/~m thick slices cut perpendicular to the longitudinal axis of the hippocampus using a vibratome. Slices were incubated in vials of continually oxygenated artificial cerebro-spinal fluid (ACSF) at 34 + I°C before use. Slices of area CA1 were prepared by subsequently removing most of CA3 and dentate regions. Normal ACSF consisted of (in mM): NaC1 125, KC1 5, NaH2PO 4 1.25, MgC12 1.5, CaC12 2, NaHCO 3 26, glucose 10 and was saturated with 95% 02/5% CO 2 to obtain pH 7.3-7.4. High-potassium medium contained 50 mM KCI, with an equimolar reduction in NaC1 concentration.
Extracellular electrophysiology After a 1-h recovery period, slices were individually transferred as needed to a submerged recording chamber (with an approximate vol. of 250 ~1). Within the recording chamber, slices were continuously perfused at a rate of 800 /A/min with fresh, oxygenated ACSF warmed to 32 _+ 1°C. Extracellular recording micropipettes were pulled from 1.5 mm o.d. borosilicate glass tubing, filled with 2 M NaC1, and selected for tip resistances of 1-3 Mfl measured in normal saline. Bipolar stimulating electrodes were fashioned with 62/zm diameter, insulated nichrome wire. Extracellular recordings of population excitatory post-synaptic potentials (EPSPs) were obtained from stratum radiatum of the CA1 subregion. A bipolar stimulating electrode was placed in stratum radiatum of CA1 for orthodromic stimulation of the Schaffer collateral/commissural afferents at 0.2 Hz. Pulse duration was fixed at 100/ts. Stimulus intensity was set to elicit population EPSPs which were approximately half of that required for the elicitation of a population spike. Sampled extracellular field potentials were displayed on a Nicolet oscilloscope, digitized and stored on IBM-386 system hard drive for subsequent, off-line analysis. LTP-inducing stimuli consisted of either a 40-s pulse of high-potassium medium (50 mM) or tetanic stimulation (10 bursts of stimuli at 1 Hz, with each burst consisting of 10 stimuli at 100 Hz). LTP was operationally defined as an increase of greater than 15% in the amplitude of the population EPSP lasting for at least 20 min.
for 5 min. After a 20-min washout period, a new baseline population amplitude was recorded for 5 min, and another 40-s pulse of high-potassium medium was perfused. The magnitude of potentiation was measured 20 min after each exposure to high-potassium.
Data analyses and statistics Data are presented as means _+ S.E.M. Population EPSP amphtudes were compared pre- and post-stimulation (repeated measures). To determine whether any observed enhancement of synaptic efficacy was significant (P < 0.05) one-tailed Student's t-tests were employed. To determine whether there were any significant differences between the magnitudes of enhancement produced by different stimulation protocols, two-tailed, independent t-tests were employed.
RESULTS
Potassium-induced L T P occludes tetanus-induced L T P T r a n s i e n t p e r f u s i o n o f slices w i t h h i g h - p o t a s s i u m m e d i u m (50 m M f o r 40 s) a l w a y s t e m p o r a r i l y a b o l i s h e d CA1 population EPSPs evoked by Schaffer collateral/ c o m m i s s u r a l s t i m u l a t i o n . W h e n d e t e c t a b l e , t h e f i b e r volley, p r e s u m a b l y r e p r e s e n t i n g t h e s y n c h r o n o u s a c t i v a t i o n o f a f f e r e n t fibers, w a s also a b o l i s h e d b y h i g h - p o t a s s i u m . U p o n r e c o v e r y , p o p u l a t i o n E P S P a m p l i t u d e s o f t e n disp l a y e d a s u s t a i n e d e n h a n c e m e n t w i t h n o c h a n g e in d u r a t i o n , t i m e t o p e a k o r in t h e a m p l i t u d e o f f i b e r volleys. P o t a s s i u m - i n d u c e d LTP, like t e t a n u s - i n d u c e d LTP, w a s
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Potassium-induced LTP and occlusion of tetanus-induced LTP. Population EPSPs were recorded in CA1 in response to test stimulation of the Schaffer collateral/commissurals. After a stable amplitude baseline response had been recorded for at least 5 rain, a 40-s pulse of ACSF containing 50 mM KCl was perfused. The magnitude of potassium-induced LTP was measured 20 rain after the potassium pulse as the percent increase in the amplitude of the population EPSP above baseline. In some of these experiments, a tetanic stimulus was subsequently delivered to the test input to determine whether potassium-induced LTP occluded the expression of tetanus-induced LTP. In other experiments, LTP of the test input was saturated with 3 tetanic stimuli before exposure to high-potassium to determine whether tetanus-induced LTP occluded the subsequent expression of potassium-induced LTP. Data were retained if either potassium-induced or tetanus-induced LTP was observed. Input-specificity of potassium-induced LTP For some experiments, test stimulation was paused for 10 min, beginning 1 min before high-potassium perfusion, to determine whether the LTP induced by potassium does not require concurrent electrical stimulation of the Schaffer collateral/commissural afferents. Any potentiation resulting from exposure to high-potassium alone could not be restricted to the specific subset of Schaffer collateral/commissural inputs being tested with electrical stimulation. APV blockade of potassium-induced LTP. Slices were perfused with medium containing 20 #M t)-2-amino-5-phosphonovalerate D-APV (Cambridge Research Biochemicals) which was allowed to act for at least 15 rain before beginning to record baseline responses. A 40-s pulse of APV-medium containing 50 mM KC1 was then perfused after a stable baseline response had been recorded
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Fig. 1. Transient exposure to artificial cerebrospinal fluid (ACSF) containing 50 mM KC1 induces long-term potentiation (LTP) of population excitatory post-synaptic potential (EPSP) in CA1 (see also Table I). A: population EPSPs from a single experiment are shown (1) before, and (2) 20 rain after 40-s exposure to isotonic medium containing 50 mM KCI. Both the initial slope and peak amplitude of the population EPSP were increased 20 min after high-potassium exposure. Each waveform represents the average of 5 consecutive responses (Bars = 0.25 mV, 5 ms). B: plot shows the time course of potentiation from the same experiment. In this and subsequent figures, horizontal bar indicates the timing of the 40-s perfusion of high-potassium containing medium and numbers refer to the times at which the above waveforms were recorded. No population EPSP could be evoked for several minutes after highpotassium exposure. Upon recovery, stimulation of the test input at the pre-set intensity evoked larger amplitude population EPSPs.
102 A.
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Fig. 2. Potassium-induced LTP prevents the subsequent expression of tetanus-induced LTP (see also Table I). A: population EPSPs from a representative experiment are shown (1) before exposure to 50 mM KCI, (2) after high-potassium exposure, and (3) after tetanization of the test input. Each waveform represents the average of 5 consecutive responses (Bars = 0.25 mV, 5 ms). The amplitude of the population EPSP increased 93% above baseline, as measured 90 min after high-potassium exposure. No further increase was seen after tetanic stimulation. B: plot shows the time course of the same experiment. Vertical bar indicates the time at which tetanic-stimulus was delivered to the test input.
m a r k e d by an increase in the initial slope and p e a k amplitude of the population EPSP as shown in Fig. 1A. Of the 30 slices tested for potassium-induced LTP, 10 were discarded for failing to express either potassium-induced LTP or tetanus-induced LTP. Of the remaining 20 slices, 18 expressed potassium-induced LTP (t = 6.96, P < 0.001) with an average increase in population E P S P amplitude of 76 + 19% when m e a s u r e d after 20 min. Initial slopes of the population EPSPs (between 10% and 90% of the p e a k amplitude) were also m e a s u r e d to control for changes in feedforward inhibitory responses. E P S P initial slopes were significantly increased from 0.26 + 0.03 V/s during baseline to 0.41 + 0.03 V/s after high-potassium (t = 7.18, P < 0.001). Two slices did not demonstrate potassium-induced LTP (enhancements of 0% and 2%) but did subsequently express tetanus-induced LTP (enhancements of 30% and 36%). The magnitude of potassium-induced LTP was not significantly different from these two slices and in slices r e p e a t e d l y tetanized to induce LTP (t = 0,89, P > 0.05). In several experiments (not shown), population EPSPs were r e c o r d e d for m o r e than 2 h after high-potassium exposure and displayed little decrement. Of the 18 slices expressing potassium-induced LTP, 14 were presented with tetanic stimulation m o r e than 20 min later to d e t e r m i n e whether potassium-induced LTP occluded the subsequent induction of tetanus-induced
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Fig. 3. Tetanus-induced LTP prevents the subsequent expression of potassium-induced LTP (see also Table I). A: population EPSPs from a representative experiment are shown (1) before, and (2) after repeated tetanization of the test input, and again (3) after subsequent high-potassium exposure. Each waveform represents the average of 5 consecutive responses (Bars = 0.25 mV, 5 ms). The amplitude of the population EPSP increased 42% above baseline, as measured 20 min after the initial tetanus. No further increase was seen after high-potassium exposure. B: plot shows the time course of the same experiment. Vertical bars indicate times at which tetanic-stimuli were delivered to the test input.
LTP (shown in Fig. 2). Only two of these slices expressed tetanus-induced LTP (enhancements of 15% and 21%). On average, the increase in population E P S P amplitude resulting from subsequent tetanic stimulation (2 + 3%) was not significant (t = 1.24, P > 0.05). E v e n the two slices that expressed subsequent tetanus-induced LTP showed partial occlusion as the average 18% enh a n c e m e n t observed was significantly lower than was observed with tetanus alone (t = 3.77, P < 0.05). A n o t h e r 5 slices were tested for occlusion in the reverse order. A representative experiment is shown in Fig. 3. Presentation of 3 tetanic stimuli to the test input resulted in a significant e n h a n c e m e n t averaging 63 + 11% as m e a s u r e d 20 min after tetanus (t = 5.53, P < 0.005). Subsequent exposure to high-potassium induced LTP in only one of these slices although the magnitude of that e n h a n c e m e n t (20%) was much r e d u c e d from what is typically seen with high-potassium alone. High-potassium exposure resulted in a change in the amplitude of the population EPSP averaging - 1 + 6% in these previously p o t e n t i a t e d slices (t = 0.28, P > 0.05).
Potassium-induced LTP is not restricted to the subset of Schaffer collateral/commissural afferents sampled with electrical stimulation Four slices were tested for potassium-induced LTP
103 TABLE I
/L
Characteristics of potassium-induced LTP Values are population EPSP amplitudes in area CA1 as percent of baseline control + S.E.M. as measured under various conditions. Control values were measured during a baseline recording period. Post-stimulus values were measured 20 min after stimulation either by tetanus or by 40-s exposure to 50 mM KC1. n represents the number of slices used for analysis (not rejected)/total number tested in a given set of experiments.
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Fig. 4. Potassium-induced LTP is blocked by bath application of the competitive N-methyl-o-aspartate ( N M D A ) receptor antagonist
o-2-amino-5-phosphonovalerate (D-APV) (see also Table I). A: population EPSPs from a single experiment are shown (1) before, and (2) 20 rain after high-potassium exposure in the presence of 20 /~M D-APV. No lasting changes in population EPSP amplitude were observed. Perfusion medium was then changed to normal medium for 20 min to allow the APV to washout. Population EPSPs were again recorded (3) before, and (4) 20 min after high-potassium exposure. In this experiment, a marked potentiation of population EPSP amplitude was observed 20-min after the second exposure to high-potassium. Each waveform represents the average of 5 consecutive responses (Bars = 0.25 mV, 5 ms). B: plot shows the time course of the same experiment.
with electrical stimulation p a u s e d for 10 min, beginning 1-min before exposure to high-potassium. W h e n electrical test stimulation of the Schaffer collateral/commissurals was restarted, these slices expressed an increase in p o p u l a t i o n E P S P amplitude averaging 55 ___ 24% (t = 3.23, P < 0.05). T h e r e was no significant difference between these slices and slices that were electrically stimulated during high-potassium exposure (t = 0.92, P > 0.05). The remaining slice did not express potassium-induced LTP, but did subsequently express tetanus-induced LTP (30% enhancement).
A P V blocks potassium-induced LTP In s e p a r a t e experiments, 8 slices were e x p o s e d to high-potassium during bath application of 20/~M D-APV (see Fig. 4); none exhibited potassium-induced LTP (t = 0.25, P > 0.05). A f t e r 20-rain washout, transient highpotassium exposure p r o d u c e d an increase in p o p u l a t i o n E P S P a m p l i t u d e averaging 20 + 7% when all 8 slices were considered (t = 2.80, P < 0.05). O f these 8 slices, 3 expressed potassium-induced LTP (greater than 15%) which a v e r a g e d 34 _ 2% as m e a s u r e d m o r e than 20 min after high-potassium exposure.
KC1 stimulation KC1 - - no electrical stim. Tetanus after KCI KC1 in 20/,M D-APV KC1 after APV washout KC1 in low calcium KC1 after calcium wash Tetanic stimulation KCI after tetanus
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The d e p e n d e n c e of potassium-induced LTP on extracellular calcium concentration was also tested in 5 separate slices. O f these, two slices d e m o n s t r a t e d potassium-induced LTP even when high-potassium exposure occurred in 0.5 m M calcium (enhancements of 51% and 85%). Two others failed to d e m o n s t r a t e potassium-induced LTP in low calcium but did express LTP after subsequent potassium-exposure in normal calcium (enhancements of 44% and 38%). The remaining slice failed to d e m o n s t r a t e potassium-induced LTP altogether. A further reduction of extracellular calcium concentration below 0.5 m M might have clarified these results, but could not be accomplished without abolishing synaptic transmission. Table I summarizes the observed characteristics of potassium-induced LTP. DISCUSSION
The present results d e m o n s t r a t e that the depolarization caused by transient elevation of extracellular potassium concentration is sufficient to induce LTP of the Schaffer collateral/commissural synapses. This potassium-induced LTP displays m a n y of the features that define LTP induced by tetanic stimulation. Specifically, (1) they display similar magnitudes of e n h a n c e m e n t , (2) they both persist for m o r e than 20 min, (3) both are blocked by bath application of the N M D A r e c e p t o r antagonist, D - A P V 9'14, and (4) both are sensitive to the extracellular concentration of calcium 1°. In addition, induction of either form of LTP prevents the subsequent induction of the other, which strongly argues that the two types of
104 LTP are expressed via a common mechanism. Similar LTP-like events in area CA1 have been previously reported. Izumi et al) 6 observed that LTP could be induced using conditioning solutions which contained high-potassium (up to 15 mM), low-magnesium, and either quisqualate or L-glutamate. Such potentiation was not observed in the presence of APV. Because high-potassium exposure itself evokes the release of endogenous excitatory amino acids (EAAs), such addition of exogenous EAAs in the medium should not be necessary. In preliminary experiments, we also observed that perfusion of 15 mM KCI medium alone did not produce any lasting changes in population EPSP amplitude and failed to evoke any detectable release of endogenous aspartate or glutamate, although it did cause transient epileptiform activity. It is possible that 15 mM KC1 does not depolarize pre- and post-synaptic fibers sufficiently to fulfill the Hebbian requirement for LTP induction without the addition of exogenous glutamate or quisqualate. Another similar potentiation has been reported by May et al. 24, except that the potentiation they observed occurred only when elevated potassium concentrations were accompanied by reduced calcium concentrations. Such a result is difficult to reconcile with previous studies which suggest that elevation of free calcium in the post-synaptic cell is both necessary 1°'19'22 and sufficient 12' 22,27 for the induction of LTP, and contrasts with our results which indicate that LTP induction with high-potassium is facilitated in normal calcium concentrations. The apparent discrepancy may have resulted from differences in the duration of high-potassium exposure, as the study of May et al. 24 utilized a substantially longer exposure time (3 min). They observed that such prolonged exposure tended to depress subsequent evoked population EPSPs in normal calcium. One interpretation of this could be that the number of cells contributing to the population response was reduced after high-potassium which could mask whatever potentiation was produced. Indeed, others have reported that high-potassium exposure produces a lasting depression of hippocampal synaptic transmission in a calcium and NMDA-receptor-de-
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Acknowledgements. We are grateful to Drs. Jon Johnson and Dave Wood for their editorial comments. This work was supported in part by Grants NIMH MH30915 (seed money Grant R203), NINDS NS01196 and NS24288, and Andrew Mellon and NIMH predoctoral fellowships.
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