previously shown (10), however, this manipulation by itself does not reliably result in the development of a large

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 85, pp , December 1988 Neurobiology Long-term potentiation differentially affects two components of synaptic responses in hippocampus (plasticity/n-methyl-d-aspartate/d-2-amino-5-phosphonovglerate/facilitation) DOMINIQUE MULLER*t AND GARY LYNCH Center for the Neurobiology of Learning and Memory, University of California, Irvine, CA Communicated by Leon N Cooper, September 6, 1988 (received for review June 20, 1988) ABSTRACT We have used low magnesium concentrations and the specific antagonist D-2-amino-5-phosphonopentanoate (D-AP5) to estimate the effects of long-term potentiation (LTP) on the N-methyl-D-aspartate (NMDA) and non-nmda receptor-mediated components of postsynaptic responses. LTP induction resulted in a considerably larger potentiation of non- NMDA as opposed to NMDA receptor-related currents. Increasing the size of postsynaptic potentials with greater stimulation currents or with paired-pulse facilitation produced opposite effects; i.e., those aspects of the response dependent on NMDA receptor's increased to a greater degree than did those components mediated by non-nmda receptors. These results pose new constraints on hypotheses about the locus and nature of LTP and strongly suggest that postsynaptic modifications ate part of the effect. Long-term potentiation (LTP), a long-lasting increase in synaptic efficacy observed in hippocampus (1) and elsewhere in forebrain (2), has attracted interest as a possible substrate of behavioral memory (3). While considerable progress has been made in identifying the cellular events that trigger LTP (4-7), the final and stable modifications that underlie the increased synaptic potency remain to be resolved. In an effort to restrict the list of proposed mechanisms, we have tested the possibility that LTP has selective effects on different components of the postsynaptic response. Recent work has identified conditions under which a sizable portion of the field excitatory postsynaptic potential (EPSP) elicited by afferent stimulation is blocked by antagonists of the N- methyl-d-aspartate (NMDA) receptor (8-10). Here we report that the NMDA and non-nmda components of synaptic responses in hippocampus are differently affected by LTP, the pattern of results being opposite that observed with paired-pulse facilitation, an effect attributed to increased transmitter release (11-13). MATERIAL AND METHODS Hippocampal slices (450,m thick) were prepared and maintained as described elsewhere in a surface recording chamber and continuously perfused with a medium containing in mm: NaCl, 124; KCl, 3; KH2PO4, 1.25; CaC12, 3; MgCI2, 1; NaHCO3, 26; glucose, 10; L-ascorbate, 2 (ph 7.4). The slices were kept at 350C and oxygenated with 95% 02/5% Co2. After an hour of recovery, the perfusion medium was switched to a solution containing only 10 or 20 tlm magnesium, which was perfused for another hour before the experiments were started. Responses were evoked by stimulation of the Schaffercommissural projections in the stratum radiatum offield CA1 and recorded with glass micropipettes (1-5 Mfl). The record- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. ing electrode was positioned in field CAlb between two stimulating electrodes placed in fields CAla and CAlc; this allowed us to activate separate inputs to a common pool of target cells. Stimulation voltages were adjusted to produce field EPSPs of -1.5 mv and did not elicit population spikes in any of the responses included for data analysis. Paired-pulse facilitation was produced by applying two stimulation pulses separated by 30 or 50 ms to the same stimulating electrode and LTP was induced by patterned burst stimulation-i.e., 10 bursts delivered at 5 Hz, each burst being composed of four pulses at 100 Hz (see ref. 5). Suppression of Inhibitory Potentials with "Priming" Stimulation. The NMDA receptor ionophore is blocked in a voltage-dependent fashion by magnesium ions (14, 15). As a consequence, antagonists of the receptor [e.g., D-2-amino-5- phosphonopentanoate (D-AP5)] have little effect on field EPSPs elicited by single-stimulation pulses in slices maintained in normal medium. Two procedures were used in the present experiments to reduce the blockade of the receptor channel and allow response components mediated by NMDA receptors to appear. First, the concentration of magnesium in the medium was reduced from 1 mm to um. As previously shown (10), however, this manipulation by itself does not reliably result in the development of a large postsynaptic response component sensitive to D-AP5. Presumably, because of the small amount of magnesium still present in the medium, the degree and duration of the depolarization produced by a single field EPSP are not sufficient to counteract the voltage-dependent blockade of the receptor channel. However, when the feedforward inhibitory postsynaptic potentials (IPSPs) that normally accompany and truncate synaptic responses in hippocampus (16) are removed, then a sizable portion of the field EPSP is blocked by NMDA receptor antagonists (8, 10). Accordingly, we used a technique referred to as priming (see ref. 5 and Fig. la for an illustration) to suppress feedforward IPSPs. Feedforward IPSPs exhibit a short (-0.5 s) refractory period once having been activated (5, 6, 16), an effect that is readily apparent in experiments using two separate inputs (Schaffercommissural fibers) to a common pool of intermeurons and pyramidal cells. If one collection of afferents (the "priming input") is used to trigger the excitatory as well as inhibitory potentials in the target region, then the responses to the second (or "test") input activated 200 ms later are largely free of IPSPs. The intracellularly recorded afterhyperpolarization produced by feedforward IPSPs is suppressed (Fig. lb), while the repolarization phase of the field EPSP measured with extracellular electrodes is prolonged (Fig. lc). As Abbreviations: LTP, long-term potentiation; NMDA, N-methyl- D-aspartate; EPSP, excitatory postsynaptic potential; IPSP, inhibitory postsynaptic potential; D-AP5, D-2-amino-5-phosphonopentanoate. *Present address: Department of Pharmacology, Geneva School of Medicine, CMU, 1206 Geneva, Switzerland. tto whom reprint requests should be addressed. 9346

2 Neurobiology: Muller and Lynch shown in Fig. ic, priming has a comparable effect on control responses as it does on field potentials that had been increased either by induction of LTP or with paired-pulse facilitation. The results show that after suppression of the fast IPSPs, the primed responses evoked in all three conditions are characterized by a similar time course, since they can be superimposed after normalization of their amplitude. Effect of D-AP5 on Responses Evoked with Paired-Pulse Stimulation, Increased Stimulation Intensity, or Following LTP Induction. Paired-pulse facilitation was produced by applying two pulses separated by 30 or 50 ms to a group of afferents terminating in a dendritic field that had been primed by stimulation of a second input 200 ms earlier (Fig. 1). The area of the postsynaptic responses evoked by the first and second pulses to the test input was measured before and after application of D-AP5 (50-125,uM) to the medium. In some cases, the drug was washed out of the slices and the same experiment was repeated. Comparisons were thus made of the degree of paired-pulse facilitation found in the presence and absence of the NMDA receptor antagonists. To assess the effect of D-AP5 on responses of different sizes, primed EPSPs were evoked by applying alternatively stimulation pulses of different intensities to the same stimulation electrode. The area of both responses was then measured before and after application of the drug. Two types of experiments were conducted to determine the effect of D-AP5 on potentiated responses. In nine cases, we tested for the effect of the drug on control responses, and then, following a washout period (40-60 min), LTP was induced and D-AP5 was reintroduced at the same concentration. In some cases, the control responses were evoked by using a paired-pulse paradigm, thus allowing for a direct comparison of the effect of D-AP5 on facilitated and potentiated responses (see Fig. 3). In five other experiments, we used two independent, equal-sized test inputs to the same dendritic field in addition to the priming input (i.e., three stimulation electrodes activating converging afferents were used). LTP was then induced on one of the test inputs and the effect of a single application of D-AP5 (50,4M) was measured on potentiated and control responses. In some cases, control responses were also evoked using a paired-pulse paradigm, allowing for a direct comparison of the effect of the receptor antagonist on control, facilitated, and potentiated responses. The two types of LTP experiments (i.e., single vs. sequential application of D-AP5) generated similar results. RESULTS As anticipated, the combination of priming and low magnesium medium resulted in field EPSPs that contained a significant component that was blocked by D-AP5 (Fig. 2a); this effect was considerably smaller when using primed responses in 1 mm magnesium. In 19 experiments conducted in the presence of 10-20,uM magnesium, we found that 50 or 125 AtM D-AP5 reduced the area of the primed field potential by 28.0o ± 0.9% (mean ± SEM), whereas the reduction in area was only 7.8% ± 1.5% in six experiments carried out in 1 mm magnesium. Fig. 2b shows that D-AP5 reduced in a similar way the size of the dendritic responses recorded intracellularly by the same paradigm. The effects of D-AP5 observed in a typical experiment on control, facilitated, and potentiated responses evoked on the same pathway are illustrated in Fig. 2c. To quantify the effects of LTP on the D-AP5 sensitive and insensitive components of synaptic responses, we subtracted the area of the averaged field EPSPs recorded in the presence of the drug from that obtained in its absence. This was done for control, potentiated, and facilitated responses in each slice, and the results are compared. Fig. 3 illustrates the method. Fig. 3a (Left) shows a control response in the a b c Priming paradigm Intracellular EPSPs Extracellular EPSPs Proc. Natl. Acad. Sci. USA 85 (1988) mu 20 ms 2 mu Control Potentiation Facilitation Superimposed 1 mu 40 ms 10 ms FIG. 1. Priming paradigm. (a) Illustration of the paradigm used to suppress feedforward inhibitory responses. The records show the field potentials collected by one recording electrode and elicited on priming and test inputs by two different stimulating electrodes. The responses to a pair of priming pulses are shown at the beginning of the trace, while those elicited by paired-pulse (50-ms interpulse interval) stimulation of the test input 200 ms later are illustrated at the end of the record. Recordings with (arrowhead) and without priming are superimposed. (b) Effect of the priming paradigm on intracellular EPSPs. The afterhyperpolarization that reflects synaptically mediated IPSPs and that normally shortens the dendritic EPSP to single-stimulation pulses is eliminated in the primed condition. Primed (arrowhead) and nonprimed potentials are superimposed to illustrate the difference in their decay phase. (c) Effect of the priming paradigm on extracellular field potentials. The records show superimposed primed and nonprimed control, potentiated, and facilitated responses. In the three conditions, priming (and thus suppression of IPSPs) results in a significant prolongation of the repolarization phase, and all three primed responses (arrowheads) are then characterized by a similar time course when superimposed at normalized scales. All responses were recorded in the presence of 1 mm magnesium; records are mean of three or four individual traces. presence and absence of D-AP5. Subtraction of the two potentials (Fig. 3b) yields the NMDA receptor-mediated component of the EPSP, whereas the response recorded in the presence of D-AP5 reflects current through non-nmda receptors (Fig. 3c). Note that, as expected, the D-AP5 sensitive component has a slower time course than the D-AP5 insensitive response. The next sets of traces are, respectively, potentiated (middle column) and paired-pulse facilitated (right column) responses from the same slice in the

3 9348 Neurobiology: Muller and Lynch Proc. Natl. Acad. Sci. USA 85 (1988) a b 1 mu 5 mu 10 ms 20 Ms C o< o- Time, min FIG. 2. Effect of D-AP5 on primed synaptic responses. (a) Superimposed field potentials recorded in the absence and presence of 50 AM D-AP5. The drug reduces the size of the EPSP by affecting primarily the amplitude and the rate of decay of the response. (b) Primed intracellular EPSPs recorded in the absence and presence of 50,M D-AP5. The time course of responses is slower in intracellular recording but the effect of the drug is very similar. (c) Changes in area produced by 125,uM D-AP5 (a 10-min application beginning at the 10-min time point) on the primed responses evoked by stimulation of the same afferents in control conditions (o), using a paired-pulse paradigm at 30-ms intervals (facilitated response: *), and following LTP induction (e). Results are expressed as percent of the response area measured before D-AP5 application. The onset of the drug action was rapid and it affected a larger fraction of facilitated than potentiated responses. Each point represents the mean area of two field EPSPs. presence and absence of the drug. The results of the subtraction are shown underneath, with the size of the component observed in control responses superimposed (dotted trace). As is evident, LTP had a much greater effect on the non-nmda aspect of the postsynaptic response than it did on the NMDA-mediated component, a result that contrasts with that observed following facilitation (see below). Fig. 4 summarizes the data for all 14 LTP experiments. For each slice, we measured the drug-sensitive and druginsensitive portions of field EPSP for control and potentiated responses. The figure expresses the increase in response size induced by high-frequency stimulation on, respectively, the NMDA and non-nmda receptor-mediated components of the synaptic responses. For all 14 experiments, LTP had a much greater effect on the D-AP5 insensitive aspects of the response than it did on the NMDA component (63% + 3% vs. 19% ± 6%, respectively; P < ). The more than 3-fold difference in the effects of LTP on the NMDA vs. non-nmda components of the postsynaptic responses was not reproduced by simply increasing the size of the field EPSP with greater stimulation voltages. In five experiments, we tested the effects of D-AP5 on responses that were 161% ± 5% of control values (i.e., slightly greater than potentiated responses). In each of these cases the NMDA component of the field potential increased at least as much as and on the average more than the non-nmda component (58% ± 4% for the non-nmda and 75% ± 6% for the NMDA component; P < 0.05; see Fig. 4). A similar pattern of results was also obtained when analyzing the effect of paired-pulse facilitation on the relative 30 balance of NMDA and non-nmda components of field EPSPs. When two stimulation pulses are given in rapid succession (<200 ms apart), the response to the second pulse is considerably larger than that to the first. This effect has been analyzed in detail at peripheral synapses and shown to reflect an increase in transmitter release (11, 12, 13). Pairedpulse facilitation in hippocampus is characterized by a time course and a sensitivity to extracellular calcium levels that are very similar to those described at neuromuscular junctions, suggesting strongly that it reflects the operation of the same presynaptic variables. The area of the second response in the paired-pulse experiments was found to be 65% and 57% larger than that recorded after the first pulse at, respectively, 30- and 50-ms interpulse intervals. Figs. 2c and 3 illustrate the results of a typical experiment in which the effect of D-AP5 was tested on control, facilitated, and potentiated responses evoked by stimulation of the same afferents. As shown in Fig. 3 (Right), subtracting the responses collected in the presence of the drug from those obtained in its absence (Fig. 3a) again yields NMDA (Fig. 3b) and non-nmda (Fig. 3c) receptor-mediated aspects of the field EPSP. For comparison, the components observed in the first responses evoked in the paired-pulse paradigm (Left) are shown superimposed (dotted traces). In marked contrast to the effect observed with LTP, the D-AP5 sensitive component increased to a greater degree than did the D-AP5 insensitive response. In 18 of 19 experiments in which we used a paired-pulse paradigm at 30- or 50-ms intervals, we found that the increase in the D-AP5 sensitive component was larger than that of the D-AP5 insensitive

4 Neurobiology: Muller and Lynch Proc. Natl. Acad. Sci. USA 85 (1988) 9349 CONTROL POTENTIATION FACILITATION a) EPSP before and after D-AP5 b) D-AP5 sensitive component c) D-AP5 insensitive component FIG. 3. Typical experiment illustrating the effect of LTP and paired-pulse facilitation on the D-AP5 sensitive and D-AP5 insensitive components of synaptic responses. (a) Representative field potentials recorded in each condition before and after application of 125,AtM D-AP5. All responses were evoked by stimulation of the same afferents. (b) Subtraction of the responses recorded after D-AP5 application from those obtained before yields the D-AP5 sensitive component of the synaptic response. The component observed in control conditions is superimposed as a dotted trace on the results of the subtraction obtained following LTP induction and facilitation. (c) EPSPs recorded after D-AP5 application. Again the control response has been superimposed as a dotted trace on the potentiated and facilitated potentials. Each record represents the mean of four individual EPSPs; scales are 10 ms and 1 mv in a and c and 0.5 mv in b. component (average increase at, respectively, 30 or 50 ms interpulse intervals: 96% ± 13% and 86% ± 9% for the NMDA and 55% ± 3% and 47% ± 3% for the non-nmda receptor-mediated aspects of the EPSP; see Fig. 4). DISCUSSION Much evidence indicates that the postsynaptic NMDA receptors play a major role in the induction of LTP in area CA1 of hippocampal slices (6, 7). In the present experiments, however, we analyzed the contribution of these receptors to the responses recorded before and after LTP induction to obtain information about the processes that are responsible for the maintenance of the potentiation effect. Measurements of NMDA receptor-mediated components of synaptic responses were obtained after suppression of IPSPs using a priming technique. As illustrated in Fig. 1, we did not obtain * NMDA non-nmda LTP Facilitation Facilitation Increased (30 ms) (50 ms) stimulation FIG. 4. Changes in NMDA and non-nmda receptor-mediated components of synaptic responses observed in the different conditions tested. Results are expressed as the increase in size of the D-AP5 sensitive (solid bar) and insensitive (hatched bar) components of field potentials measured by the subtraction procedure in each condition. Results are mean ± SEM of 5-14 experiments. evidence that this paradigm differentially affected control, facilitated, or potentiated responses. We assume that the size of the D-AP5 sensitive aspect of the synaptic responses is controlled by two major factors: (i) the amount of transmitter released and hence the number of NMDA receptors activated and (ii) the depolarization of the dendritic spines, which results from the effects of the released transmitter on the non-nmda receptors. Depolarization can be expected to affect currents through NMDA receptors since a substantial amount of magnesium was left in the medium (14). In the case of the paired-pulse experiments, one would expect these two factors to be operative, if, as shown in peripheral systems, the facilitation effect results from an increase in transmitter release. The fact that we observed in 18 of 19 experiments a larger increase in D-AP5 sensitive than in D-AP5 insensitive aspects of field potentials supports the interpretation that both increased release and a greater depolarization of the spines were involved. It cannot be excluded, however, that other more subtle effects may also have participated (e.g., a delayed repolarization of thle spines or a residual activation of NMDA receptor channels). It should be noted, however, that the repolarization of synaptic responses as well as of the D-AP5 sensitive component were both completed by 50 ms (see Fig. 2b), making it unlikely that delayed influence of the first response would have contributed significantly to the larger D-AP5 sensitive component observed on the second response. The major result of the present experiments is that LTP has markedly different effects on components of the postsynaptic responses mediated by different transmitter receptors. This finding places new constraints on hypotheses concerning the locus and nature of the LTP effect and in particular suggests that it is not due to a simple increase in release (7) or to the addition of new contacts with properties like those found before potentiation. Facilitated release would have been expected to affect those aspects of the field EPSP mediated by the NMDA receptors at least to the same degree that it did non-nmda receptors, something that was observed with paired-pulse facilitation but clearly did not occur following

5 9350 Neurobiology: Muller and Lynch LTP induction. A possible explanation for this result would be that NMDA receptors are only present in limited quantities and thus are unaffected by greater release. Two very different observations argue against this. First, binding studies indicate that NMDA receptors are present in at least as great numbers as the non-nmda amino acid receptors, including the "quisqualate type" thought to mediate transmission (17). Second, paired-pulse facilitation, which very probably involves increased release, had greater effects on the NMDA component of the response than it did on the non-nmda components. Electron microscopic studies have shown that LTP is accompanied by an apparent increase in two types of contacts, sessile and shaft synapses (18-20). A simple increase in synapse number would be expected to affect equally both NMDA and non-nmda components of the field EPSP and indeed we found that the percentage reduction caused by D-AP5 increased slightly when postsynaptic responses were increased by delivering stronger afferent stimulation pulses (i.e., when more synapses were activated; see Fig. 4). Therefore, if increased numbers of synapses were to account for LTP, one would have to assume that the added contacts contained an abnormally low density of NMDA receptors. There is also ultrastructural evidence that modifications in the shape of dendritic spines occur when LTP is induced (18-21). In fact, the added contacts described above could be due to some type of spine transformation effect. Two possibilities relevant to LTP might be considered here. First, the anatomical changes could alter the surface chemistry of the spine. Analyses ofthe calcium dependency of transmission in hippocampus and of paired-pulse facilitation suggest that a simple increase in receptor number cannot account for LTP (22), but it remains possible that changes in the properties of the non-nmda receptors (e.g., increased coupling with ionic channels, changes in the biophysical properties of the channel) participate in the LTP effect. Second, morphological adjustments should change the biophysics of the spine, and Rall and others (22) have proposed effects of this kind as the substrate of synaptic potentiation. In light of our results, these biophysical changes would have to favor rapidly developing synaptic potentials (i.e., the non-nmda component) and have little effect on more slowly evolving currents (i.e., the NMDA component). Computer simulations would be useful in asking if such effects are plausible and if so whether the necessary changes in spine and spine neck Proc. Natl. Acad. Sci. USA 85 (1988) geometry accord with the structural modifications that correlate with LTP. Information ofthis type will clearly be of use in further localizing the changes responsible for LTP. We thank M. Baudry, J. Turnbull, and J. Larson for helpful discussion, and J. Porter and M. Lay for secretarial help. This work was supported by Air Force Office of Scientific Research Grant to G.L. D.M. holds a fellowship from the Swiss National Science Foundation, Grant Bliss, T. V. P. & Lomo, T. (1973) J. Physiol. (London) 232, Racine, R. J., Milgram, N. W. & Hafner, S. (1983) Brain Res. 260, Morris, R. G. M., Anderson, E., Lynch, G. & Baudry, M. (1986) Nature (London) 319, Collingridge, G. L., Kehl, S. J. & McLennan, H. (1983) J. Physiol. (London) 334, Larson, J. & Lynch, G. (1986) Science 232, Larson, J. & Lynch, G. (1988) Brain Res. 441, Collingridge, G. L. & Bliss, T. V. P. (1987) Trends Neurosci. 10, Wigstrom, H., Gustafsson, B. & Huang, Y.-Y. (1985) Acta Physiol. Scand. 124, Collingridge, G. L., Herron, C. E. & Lester, R. A. J. (1988) J. Physiol. (London) 399, Muller, D. & Lynch, G. (1988) Synapse, in press. 11. Katz, B. & Miledi, R. (1968) J. Physiol. (London) 195, Wernig, A. (1972) J. Physiol. (London) 226, Zucker, R. S. (1973) J. Physiol. (London) 229, Nowak, L., Bregestovski, P., Asher, P., Herbet, A. & Prochiantz, A. (1984) Nature (London) 307, Mayer, M. L., Westbrook, G. L. & Guthrie, P. B. (1984) Nature (London) 309, Alger, B. E. & Nicoll, R. A. (1982) J. Physiol. (London) 328, Cotman, C. W., Monaghan, D. T., Ottersen, 0. P. & Storm- Mathisen, J. (1987) Trends Neurosci. 10, Lee, K., Oliver, M., Schottler, F. & Lynch, G. (1981) Electrical Activity in Isolated Mammalian CNS Preparations (Academic, New York), pp Lee, K., Schottler, F., Oliver, M. & Lynch, G. (1980) J. Neurophysiol. 44, Chang, F. L. & Greenough, W. T. (1984) Brain Res. 309, Fifkova, E. & Anderson, C. L. (1981) Exp. Neurol. 74, Rall, W. (1967) in Studies in Neurophysiology (Cambridge Univ. Press, Cambridge, U.K.), pp