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Effects of synaptic antagonists on perforant path paired-pulse plasticity: differentiation of pre- and postsynaptic antagonism
348
t3r,m Reseat'ci~ ~~,4{ i~'.~5"~ "..~.~.- ~5"
Elsc~ i~::r BRE 20792
Effects of synaptic antagonists on perforant path paired.pulse plasticity: differentiation of pre- and postsynaptic antagonism E. W. HARRIS and C. W. COTMAN Department of Psychobiology, University of California, Irvine, CA 92717 (U.S.A.)
(Accepted December 18th, 1984) Key words: perforant path - - synaptic transmission - - synaptic plasticity- - synaptic antagonism
The effects of different synaptic antagonists on paired-pulse plasticity of medial perforant path responses were studied in rat hippocampal slices. Baclofen reduces the response to activation of the perforant path, but does not have the same net effect on the first and second responses to paired stimulation: bactofen lessens the percent paired-pulse depression of medial perforant path responses. Furthermore, at doses that reduced the control medial perforant path response by half, paired-pulse plasticity changed from paired-pulse depression to paired-pulse potentiation. A similar effect on medial perforant path paired-pulse plasticity is produced by decreasing extracellular calcium concentration. Kynurenic acid reduces the first and second responses to paired stimulation proportionately the same, and, therefore, has no effect on the percent paired-pulse depression. These results suggest that baclofen acts presynaptically to reduce the synaptic response, whereas kynurenate acts postsynaptically. Adenosine was also found to be a potent antagonist of medial perforant path responses, with effects on paired-pulse plasticity similar to baclofen; a new synaptic antagonist, N-p-chtorobenzoylpiperazine-2,3-dicarboxylate,was found to have effects like kynurenate, suggesting that it is also a postsynaptic receptor blocker. Acidic amino acids are believed to be the neurotransmitters at a m a j o r i t y of excitatory synapses in the m a m m a l i a n central nervous system 2s, and m a n y acidic amino acid analogs act as agonists and antagonists in the CNS. Several c o m p o u n d s have been found to be p o t e n t synaptic antagonists in the hippocampus9,13,15, 25 and o t h e r central nervous system regions2,3,6, 9 but their mechanisms of action, specifically whether they act pre- or postsynaptically, are not known. The mechanism of action of one of these antagonists, 2 - a m i n o - 4 - p h o s p h o n o b u t y r a t e ( A P B ) , a potent antagonist of lateral p e r f o r a n t path responses 13, was previously investigated on the basis of interactions with paired-pulse p o t e n t i a t i o n of the synaptic response. This a p p r o a c h was taken because synaptic plasticity resulting from paired-pulse stimulation a p p e a r s to occur presynapticaUy in those systems in which it has been e x a m i n e d in detail 4,14A9,2°, and paired-pulse plasticity of the p e r f o r a n t path synaptic potential (as distinct from the postsynaptic population spike) also a p p e a r s to be a presynaptic phenomenon. Paired-pulse plasticity of the synaptic response is not p r o d u c e d by pairing stimulation of dif-
ferent afferents to the dentate gyrus, even if the pathways project to overlapping populations of target cells 12,19. It therefore does not result from generalized changes in the postsynaptic cells via. for example, interneuronal effects. All of the data pertaining to perforant path paired-pulse plasticity is consistent with a presynaptic mechanism 12.is.j927. APB interacts with lateral p e r f o r a n t path synaptic plasticity in a m a n n e r suggesting that it is a presynaptic antagonist 10. The present study concerns several antagonists of medial perforant path, and reports striking effects of several synaptic antagonists on pairedpulse depression along the medial portion o f the perforant path: k y n u r e n a t e 9,25, adenosine 5,2~ and a new c o m p o u n d that has recently been shown to block synaptic transmission and response to applied agonists in the hippocampus, N-(p-chlorobenzoyl)-piperazine2,3-dicarboxylate (pCB-PzDA)S. These results support the validity of paired-pulse analysis for investigating mechanisms of action of synaptic antagonists. A preliminary report of part of this work has appeared elsewhere~l. H i p p o c a m p a l slices were p r e p a r e d from 4 0 - 6 0 day
Correspondence." E. W. Harris, Department of Psychobiology, University of California, Irvine, CA 92717. U.S.A.
0006-8993/85/$03.30 © 1985 Elsevier Science PuNishe rs B.V. (Biomedical Division)
349 old male S p r a g u e - D a w l e y rats using conventional procedures 1°,13. Slices were initially stored at 33 °C under a 95% 02/5% CO 2 atmosphere, partially submerged in medium consisting of 127 m M NaC1, 2.0 mM KCI, 26 mM NaHCO3, 1.3 mM KHzPO4, 10 mM glucose, 2.0 mM MgSO4, and, except for low calcium experiments, 2.0 mM CaCI 2. Slices were then transferred individually to a small chamber in which the solution bathing the slice could be changed quickly. Adenosine and kynurenate were purchased from Sigma (St. Louis, MO). Baclofen (Lioresal) was a gift from the Ciba-Geigy Corp. p C B - P z D A was supplied by Dr. J. C. Watkins, Bristol, U.K. Hippocampal fibers were stimulated with bipolar constant voltage pulses (2-35 V, 50-200 kts) using a bipolar stimulating electrode made of twisted 60/~m insulated stainless steel wires placed in the molecular layer of the dentate gyrus (Fig. 1A). Extracellular evoked synaptic potentials were recorded with 2 MNaCI filled glass micropipettes (2-15 Mr2) also placed in the appropriate portion of the molecular layer (Fig. 1A). Activating the medial perforant path produces in the middle molecular layer a negative
B
Control
wave that reflects the summed extracellular synaptic potentials from many granule cells, as shown in Figs. 1 B - C and 2. The slope and amplitude of this synaptic response were sampled electronically at a fixed time after the stimulus and representative responses were averaged and stored using a microcomputer. Stimulus intensity was kept below threshold for evoking a prominent granule cell population spike to avoid distortion of the synaptic potential. In order to obtain responses resulting from the activation of medial rather than lateral perforant path fibers, evoked potentials were screened with respect to their response to paired stimulation. The criteria for identifying a medial perforant path response were: (1) paired-pulse depression at 80 and 800 ms interpulse interval18; (2) 4 0 - 6 0 % reduction by 4 - 5 ~tM baclofen15; and (3) less than 10% antagonism by 12.5~M APB 13. Paired stimulation of the medial perforant path in vitro produces a response decrementlS (Fig. 1B, left). When baclofen is added, the first and second responses are reduced in amplitude, but there is a change in the paired-pulse plasticity. The percent
5uM Baclofen
Control (low stim. intensity)
A f \-
1 1
C
Control
25uM APB
+5uM Baclofen
~--C/A
"...
1
1
Fig. 1. Paired-pulse plasticity of medial perforant path responses and effects of baclofen. A: hippocampal slice preparation. EC, entorhinal cortex; DG, dentate gyrus; hil, hilus of dentate gyrus; hf, hippocampal fissure; CA3, CA3 region of hippocampus. Single letters indicate location of stimulating electrodes for activating lateral entorhinal fibers (L), medial entorhinal fibers (M) and commissural/associational fibers (C). Enlargement shows termination pattern of the lateral entorhinal cortex (LEC), the medial entorhinal cortex (MEC) and the commissural/associational system (C/A) along the dentate granule cell dendrites; oml, outer molecular layer; mini, middle molecular layer; im, inner molecular layer; gcl, granule cell layer. B and C: pairs of averaged (n = 3) response pairs (1 = first response, 2 = second response) recorded under the conditions indicated above are superimposed for comparison of the relative change in response amplitude with paired stimulation. Interstimulus interval was 80 ms, inter-pair interval was 20 s. B: effects on response amplitude and paired-pulse plasticity of 5 ktM baclofen (center), and comparison with a matched amplitude control first response (right). C: effect of adding 25 ~uM _+APB (center) on the effects of subsequent addition of 5 .uM baclofen (right). Calibration bars = 0.5 mV, 4 ms.
350 paired-pulse depression decreases as the antagonism is increased, and with greater than 50% antagonism by baclofen, the second response is larger than the first (Fig. 1B, center). This change in paired-pulse plasticity reverses as the drug is washed out and the response amplitude returns to control levels. The effect on paired-pulse plasticity is not simply due to reducing response amplitude, since decreasing the response by lowering stimulus intensity did not noticeably change the paired-pulse depression (Fig. 1B, right). The shift from depression toward potentiation is also not due to unmasking baclofen-resistant 15 lateral perforant path responses, which exhibit pairedpulse potentiation 18, because if 25/~M + APB was added beforehand, a shift from depression to potentiation still occurred during baclofen application (Fig. 1C, right). Baclofen appears to reduce neurotransmitter release in other systems and it is likely that its effects on medial perforant path responses are via a presynaptic mechanism, possibly one related to calcium ion availability. Therefore, pairs of evoked responses were collected in normal medium and in medium in which calcium was reduced to 1.0-0.75 mM, to reduce the evoked responses by 50-75%, corresponding to the amount of baclofen antagonism at which a clear shift in the paired-pulse plasticity was seen. At this calcium concentration, medial perforant path responses exhibited paired-pulse potentiation (see Fig. 2A). Kynurenic acid is also a medial perforant path antagonist, but does not change the paired-pulse plasticity (Fig. 2B). Kynurenic acid at 200-800 ~tM did not lessen the paired-pulse depression in any of the 10 slices examined, even though these doses reduced medial perforant path responses by as much as 75%. Medial perforant path responses are also antagonized by adenosine. Application of 25 ktM adenosine reduced medial perforant path responses by 42 + 5% (5 slices) and caused the medial perforant path paired-pulse response to shift from depression to potentiation (Fig. 2C). The effects of adenosine reached a plateau within minutes and reversed upon washing with control medium. It is unlikely that the shift produced by adenosine results from changes in response amplitude or from contamination by lateral perforant path or commissural/associational (C/A) responses, because addition of 25 ktM adenosine reduced lateral perforant path synaptic responses by
Control
Treatmenz
A 2
Low Ca++
1
B
1
C
1
D : ...'
"...:
%..'
1
-PzDA
____.j
1
Fig. 2. Effects of different synaptic antagonists on medial per-
forant path paired-pulse plasticity. Pairs of responses (80 ms interstimulus interval, 20 s interpair interval)were averaged (n = 3) before (left) and during (right) antagonism by the treatment indicated to the right. Identical results have been obtained in at least 3 other slices for each treatment: Calibration bars = 0.5 mV, 5 ms. greater than 50% (3 slices) and C/A responses by 40 + 3.5% (n = 3). N-(p-chlorobenzoyl)-piperazine2,3-dicarboxylic acid (pCB-PzDA) has been shown to be a potent antagonist of medial perforant path responses in the hippocampus 8. Addition of up to 250 ~tM pCB-PzDA resulted in a 78 ___ 6% reduction of medial perforant path responses, but produced no noticeable change in the percent paired-pulse depression. The effects of the various antagonists were examined at several interpulse intervals in an effort to further differentiate between treatments and better characterize the effects on paired-pulse plasticity. The similarity of effect of baclofen, adenosine and low calcium medium on paired-pulse plasticity, and the lack of effect of kynurenate and pCB-PzDA on
351 A Control Q Low Ca • Baclofen • Adenosine O Kynurenate [~ CB-PzDA
1.2
1.0 2nd Response 1St Response
0.8
0.6
4'0
160
640
Interstimutus Interval (msec)
Fig. 3. Time course of effects on paired-pulse plasticity. The ratio of the 2nd/lst response (fractional change) in averaged (n = 3) response pairs is plotted at 3 interstimulus intervals (ISis) (for clarity, data from 80,320 and 1200 ms ISI are not plotted). Each point represents the mean of ratios obtained from 3 slices. Antagonism of the control first response of 45-65% was produced by each treatment. the time course of paired-pulse plasticity are shown in Fig. 3. These results indicate a fundamental difference in the mechanisms of synaptic antagonism of several potent antagonists of medial perforant path synaptic responses. Baclofen and adenosine antagonism are associated with a shift in paired-pulse plasticity from depression towards potentiation. This shift does not result simply from reducing evoked response amplitude, since decreasing the stimulus intensity does not cause a shift in medial perforant path paired-pulse plasticity (Fig. 1B, right), and kynurenic acid and pCBPzDA, which reduce the response amplitude, do not change paired-pulse plasticity. The effects of baclofen and adenosine also do not result from contamination by other responses that exhibit potentiation and exhibit differential sensitivity to these antagonists. The shift from paired-pulse depression to pairedpulse potentiation reflects an interaction between baclofen and the processes underlying paired-pulse plasticify, which appears to occur presynaptically. Baclofen 3,22, and adenosine 7 have been shown to decrease presynaptic neurotransmitter release in several systems, and it is likely that the process whereby these compounds decrease release involves presynaptic calcium, although based on an indirect analysis, Dunwiddie5 has suggested that adenosine may not work via a calcium-related mechanism. Decreasing calcium availability has been found to increase or unmask potentiation in other systems ]6,17,24, and superfusing hippocampal slices with medium containing no calcium causes changes in paired-pulse plasticity along the medial perforant path is. Less drastic reduc-
tions in calcium and evoked responses also produce a shift from depression to potentiation, especially at short interstimulus intervals (Figs. 2A, 3). The similarities between the effects of baclofen, adenosine and lowering calcium on medial perforant path responses - - causing a shift from depression to potentiation, and the temporal profile of this shift - - suggest a common mechanism of action: decreasing presynaptic transmitter release by reducing presynaptic calcium influx. The shift in paired-pulse plasticity then follows as an expected consequence of reducing release17,24. Although believed to act primarily as presynaptic antagonists, both baclofen and adenosine have also been reported to have direct effects on postsynaptic cells. Baclofen causes hyperpolarization and an increase in conductancet, 20, although at higher doses than were used in the present study. Adenosine also produces a slight hyperpolarization as well as a reduction in calcium spike amplitude in postsynaptic cells 23. However, it is unlikely that these postsynaptic effects are involved in the mechanism of primary synaptic antagonism, or would have any effect on synaptic paired-pulse plasticity. It may be that the mechanism of inhibition of postsynaptic calcium spikes by adenosine is similar to its mechanism of reducing presynaptic calcium, however. Kynurenate and pCB-PzDA have effects unlike those elicited by baclofen, adenosine or lowering extracellular calcium concentration. Kynurenate blocks synaptic responses as well as responses to ionophoretically applied agonists in the hippocampus '~ and other central nervous system structures 2,21. Kynurenate also reduces glutamate binding to synaptic membranes prepared from whole rat brains, as well as from sites in the dentate gyrus molecular layer revealed by [3H]glutamate autoradiography (D. T. Monaghan and C. W, Cotman, unpublished observations). These data indicate that kynurenate is a competitive blocker of postsynaptic acidic amino acid receptors. It follows that synaptic antagonism by kynurenate will not be accompanied by a change in paired-pulse plasticity. If the action of kynurenate is postsynaptic, it will not interact with the processes underlying paired-pulse plasticity of the synaptic response. The paired responses should be reduced proportionately the same, and the time course of pairedpulse plasticity should be unaffected. The lack of ef-
352 fect of p C B - P z D A on paired-pulse plasticity suggests that it too acts postsynaptically to antagonize synaptic responses. In fact, p C B - P z D A has been shown to block the responses to applied acidic amino acids in the hippocampus 8. The finding that the effect of calcium is more pronounced at shorter interpair intervals suggests that there are two components underlying medial pairedpulse plasticity, one earlier component that is more sensitive to changes in calcium, and a later component that is less sensitive to changes in calcium, it may be that McNaughton 19 observed potentiation only after profound reduction of the evoked response because he used a relatively long interstimulus interval (150 ms), while the effects of presynaptic antagonism are most pronounced at shorter interstimulus intervals (40-80 ms). Paired-pulse analysis is a simple tool for investigating mechanisms of synaptic antagonism. Although it does not rule out additional postsynaptic effects, antagonism that is associated with a change in synaptic paired-pulse plasticity almost certainly involves a presynaptic effect, one probably related to presynaptic calcium. It is possible, however, that an antagonist could reduce presynaptic release via a mechanism unrelated to paired-pulse plasticity, for example, by being taken up into a presynaptic terminal and serving as a false transmitter. In this case, paired responses would be reduced proportionately the same, and paired-pulse analysis would reveal no evidence of presynaptic effects. Thus, paired-pulse analysis could yield a false negative concerning presynaptic antagonism. Nonetheless, in systems in which
1 Brady, R. J. and Swann, J. W., Intracellular studies of the effects of baclofen on bicuculline-induced epileptiform activity in CA3 pyramidal cells, Soc. Neurosci. Abstr., 9 (1983) 397. 2 Cochran, S. L., Pharmacological antagonism of climbing fiber-, parallel fiber-, and acidic amino acid-induced excitation of frog cerebellar purkinje cells, Soc. Neurosci. Abstr., 9 (1983) 1142. 3 Collins, G. G. S., Anson, J. and Kelly, E. P., Baclofen: effects on evoked field potentials and amino acid neurotransmitter release in the rat olfactory cortex slice, Brain Research, 238 (1982) 371-383. 4 DelCastillo, J. and Katz, B., Statistical factors involved in neuromuscular facilitation and depression, J. PhysioL (Lond.), 124 (1954) 574-585. 5 Dunwiddie, T. V., Interaction between the effects of aden-
changes in synaptic efficacy can be ascribed witi,. some confidence to presynaptic mechamsms, pairedpulse analysis may, in lieu of definitive mtraceltular quantal analysis, provide clues as to probable mechanisms of synaptic antagonism. Paired-puise atlalysis can also suggest than an antagonist acts postsynaptically. A more positive indication of postsynaptic antagonism might be found by examining the effectiveness of a drug at blocking applied agonists, such a test requires at least a partial identification of the neurotransmitter and its receptors for the system in question, however, and does not rule oul additional presynaptic effects. Paired-pulse analysis is a simple way of identifying possible postsynaptic antagonists and can give a strong indication of presynaptic amagonism. These results support for the idea that baclofen and adenosine are central nervous system presynaptic release antagonists, and also that kynurenic acid is a central nervous system acidic amino acid receptor blocker. Since kynurenic acid2.9, 25 and adenosine 7 are potent antagonists of many central nervous system pathways, these two compounds may be useful for locating mechanisms underlying synaptic plasticity, for example long-term potentiation, by permitting selective pre- and postsynaptic experimental intervention. Thanks to Alan Ganong and Tim Hearn for helpful suggestions, to Ciba Geigy for baclofen, and to Dr. J. C. Watkins for pCB-PzDA. Supported by D A M D 17-83-C-3189 and N1H Postdoctoral Grant NS06480 (E.W.H.).
osine and calcium on synaptic responses in rat hippocampus in vitro, J. Physiol. (Lond.), 350 (1984) 545-559: 6 Evans, A. H., Francis, A. A., Jones, A. W., Smith, D. A. S. and Watkins, J. C., The effects of a series of c~-phosphonic-ct-carboxylic amino acids on electrically evoked and excitant amino acid-induced responses in isolated spinal cord preparation, Brit. J. Pharmacol., 75 (1982) 65-75. 7 Fredholm, B. B. and Hedqvist, P., Modulation of neurotransmission by purine nucleotides and nucleosides, Biochem. Pharmacol., 29 (1980) 1635-1643. 8 Ganong, A. H., Cotman, C. W., Jones, A. W., Watkins, J. C., Analogues of piperazine-2,3-dicarboxylic acid inhibit excitatory synaptic transmission in rat hippocampus, Soc Neurosci. Abstr., in press. 9 Ganong, A. H., Lanthorn, T. H. and Cotmam C. W., Kynurenic acid inhibits synaptic and acidic amino acid-in-
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17
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19
duced responses in the rat hippocampus and spinal cord, Brain Research, 273 (1983) 170-174. Harris, E. W. and Cotman, C. W., Effects of acidic amino acid antagonists on potentiation along the lateral perforant path, Exp. Brain Research, 52 (1983) 455-460. Harris, E. W. and Cotman, C. W., Antagonism of medial perforant path responses by baclofen, kynurenic acid and adenosine: Differentiation of pre- and postsynaptic antagonism, Soc. Neurosci. Abstr., 9 (1983) 1141. Harris, E. W., Lasher, S. S. and Steward, O., Analysis of habituation-like decrements in transmission along the temporodentate pathway of the rat, Brain Research, 162 (1979) 21-32. Koerner, J. F. and Cotman, C. W., Microlomar L-2-amino4-phosphonobutyric acid selectively inhibits perforant path synapses from lateral entorhinal cortex, Brain Research, 216 (1981) 192-198. Kuno, M., Mechanism of facilitation and depression of the excitatory synaptic potential in spinal motoneurons, J. Physiol. (Lond.), 175 (1964) 100-112. Lanthorn, T. H. and Cotman, C. W., Baclofen selectively inhibits excitatory synaptic transmission in the hippocampus, Brain Research, 225 (1981) 171-178. Lundberg, A. and Quilish, H., On the effect of calcium on presynaptic potentiation and depression at the neuro-muscular junction, ActaphysioL scand., Suppl. 30, 111 (1953) 121-129. Mallart, A. and Martin, A. R., The relation between quantum content and facilitation at the neuromuscular junction of the frog, J. Physiol. (Lond.), 196 (1968) 593-604. McNaughton, B. L., Evidence for two physiologically distinct perforant pathways to the fascia dentata, Brain Research. 199 (1980) 1-19. McNaughton, B. L. and Barnes, C. L., Physiological iden-
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tification and analysis of dentate granule cell responses to stimulation of the medial and lateral perforant pathways in the rat, J. comp. Neurol., 175 (1977) 439-454. Newberry, N. R. and Nicoll, R. A., Direct inhibitory action of baclofen on hippocampal pyramidal cells, Soc. Neurosci. Abstr., 9 (1983) 457. Perkins, M. N. and Stone, T. W., An ionophoretic investigation of convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid, Brain Research, 247 (1982) 184-187. Potashner, S. J., Baclofen: effects on amino acid release and metabolism in slices of guinea pig cerebral cortex, J. Neurochem., 32 (1979) 103-109. Proctor, W. R. and Dunwiddie, T. V., Adenosine inhibits calcium spikes in hippocampal pyramidal neurons in vitro, Neurosci. Lett., 35 (1983) 197-200. Rahamimoff, R., A dual effect of calcium ions on neuromuscular facilitation, J. Physiol. (Lond.), 195 (1968) 471-480. Robinson, M. E., Anderson, K. D. and Koerner, J. F., Kynurenate antagonism of hippocampal synaptic filed potentials, Soc.Neurosci. Abstr., 9 (1983) 264. Schubert, P. and Mitzdorf, U., Analysis and quantitative evaluation of the depressive effect of adenosine on evoked potentials in hippocampal slices, Brain Research, 172 (1979) 186-190. Steward, O., White, W. F. and Cotman, C. W., Potentiation of the excitatory synaptic action of commissural, associational and entorhinal afferents to dentate granule cells, Brain Research, 134 (1977) 551-560. Watkins, J. C. and Evans, R. H., Excitatory amino acid neurotransmitters, Ann. Rev. Pharmacol. Toxicol.. 21 (1981) 165-204.
t3r,m Reseat'ci~ ~~,4{ i~'.~5"~ "..~.~.- ~5"
Elsc~ i~::r BRE 20792
Effects of synaptic antagonists on perforant path paired.pulse plasticity: differentiation of pre- and postsynaptic antagonism E. W. HARRIS and C. W. COTMAN Department of Psychobiology, University of California, Irvine, CA 92717 (U.S.A.)
(Accepted December 18th, 1984) Key words: perforant path - - synaptic transmission - - synaptic plasticity- - synaptic antagonism
The effects of different synaptic antagonists on paired-pulse plasticity of medial perforant path responses were studied in rat hippocampal slices. Baclofen reduces the response to activation of the perforant path, but does not have the same net effect on the first and second responses to paired stimulation: bactofen lessens the percent paired-pulse depression of medial perforant path responses. Furthermore, at doses that reduced the control medial perforant path response by half, paired-pulse plasticity changed from paired-pulse depression to paired-pulse potentiation. A similar effect on medial perforant path paired-pulse plasticity is produced by decreasing extracellular calcium concentration. Kynurenic acid reduces the first and second responses to paired stimulation proportionately the same, and, therefore, has no effect on the percent paired-pulse depression. These results suggest that baclofen acts presynaptically to reduce the synaptic response, whereas kynurenate acts postsynaptically. Adenosine was also found to be a potent antagonist of medial perforant path responses, with effects on paired-pulse plasticity similar to baclofen; a new synaptic antagonist, N-p-chtorobenzoylpiperazine-2,3-dicarboxylate,was found to have effects like kynurenate, suggesting that it is also a postsynaptic receptor blocker. Acidic amino acids are believed to be the neurotransmitters at a m a j o r i t y of excitatory synapses in the m a m m a l i a n central nervous system 2s, and m a n y acidic amino acid analogs act as agonists and antagonists in the CNS. Several c o m p o u n d s have been found to be p o t e n t synaptic antagonists in the hippocampus9,13,15, 25 and o t h e r central nervous system regions2,3,6, 9 but their mechanisms of action, specifically whether they act pre- or postsynaptically, are not known. The mechanism of action of one of these antagonists, 2 - a m i n o - 4 - p h o s p h o n o b u t y r a t e ( A P B ) , a potent antagonist of lateral p e r f o r a n t path responses 13, was previously investigated on the basis of interactions with paired-pulse p o t e n t i a t i o n of the synaptic response. This a p p r o a c h was taken because synaptic plasticity resulting from paired-pulse stimulation a p p e a r s to occur presynapticaUy in those systems in which it has been e x a m i n e d in detail 4,14A9,2°, and paired-pulse plasticity of the p e r f o r a n t path synaptic potential (as distinct from the postsynaptic population spike) also a p p e a r s to be a presynaptic phenomenon. Paired-pulse plasticity of the synaptic response is not p r o d u c e d by pairing stimulation of dif-
ferent afferents to the dentate gyrus, even if the pathways project to overlapping populations of target cells 12,19. It therefore does not result from generalized changes in the postsynaptic cells via. for example, interneuronal effects. All of the data pertaining to perforant path paired-pulse plasticity is consistent with a presynaptic mechanism 12.is.j927. APB interacts with lateral p e r f o r a n t path synaptic plasticity in a m a n n e r suggesting that it is a presynaptic antagonist 10. The present study concerns several antagonists of medial perforant path, and reports striking effects of several synaptic antagonists on pairedpulse depression along the medial portion o f the perforant path: k y n u r e n a t e 9,25, adenosine 5,2~ and a new c o m p o u n d that has recently been shown to block synaptic transmission and response to applied agonists in the hippocampus, N-(p-chlorobenzoyl)-piperazine2,3-dicarboxylate (pCB-PzDA)S. These results support the validity of paired-pulse analysis for investigating mechanisms of action of synaptic antagonists. A preliminary report of part of this work has appeared elsewhere~l. H i p p o c a m p a l slices were p r e p a r e d from 4 0 - 6 0 day
Correspondence." E. W. Harris, Department of Psychobiology, University of California, Irvine, CA 92717. U.S.A.
0006-8993/85/$03.30 © 1985 Elsevier Science PuNishe rs B.V. (Biomedical Division)
349 old male S p r a g u e - D a w l e y rats using conventional procedures 1°,13. Slices were initially stored at 33 °C under a 95% 02/5% CO 2 atmosphere, partially submerged in medium consisting of 127 m M NaC1, 2.0 mM KCI, 26 mM NaHCO3, 1.3 mM KHzPO4, 10 mM glucose, 2.0 mM MgSO4, and, except for low calcium experiments, 2.0 mM CaCI 2. Slices were then transferred individually to a small chamber in which the solution bathing the slice could be changed quickly. Adenosine and kynurenate were purchased from Sigma (St. Louis, MO). Baclofen (Lioresal) was a gift from the Ciba-Geigy Corp. p C B - P z D A was supplied by Dr. J. C. Watkins, Bristol, U.K. Hippocampal fibers were stimulated with bipolar constant voltage pulses (2-35 V, 50-200 kts) using a bipolar stimulating electrode made of twisted 60/~m insulated stainless steel wires placed in the molecular layer of the dentate gyrus (Fig. 1A). Extracellular evoked synaptic potentials were recorded with 2 MNaCI filled glass micropipettes (2-15 Mr2) also placed in the appropriate portion of the molecular layer (Fig. 1A). Activating the medial perforant path produces in the middle molecular layer a negative
B
Control
wave that reflects the summed extracellular synaptic potentials from many granule cells, as shown in Figs. 1 B - C and 2. The slope and amplitude of this synaptic response were sampled electronically at a fixed time after the stimulus and representative responses were averaged and stored using a microcomputer. Stimulus intensity was kept below threshold for evoking a prominent granule cell population spike to avoid distortion of the synaptic potential. In order to obtain responses resulting from the activation of medial rather than lateral perforant path fibers, evoked potentials were screened with respect to their response to paired stimulation. The criteria for identifying a medial perforant path response were: (1) paired-pulse depression at 80 and 800 ms interpulse interval18; (2) 4 0 - 6 0 % reduction by 4 - 5 ~tM baclofen15; and (3) less than 10% antagonism by 12.5~M APB 13. Paired stimulation of the medial perforant path in vitro produces a response decrementlS (Fig. 1B, left). When baclofen is added, the first and second responses are reduced in amplitude, but there is a change in the paired-pulse plasticity. The percent
5uM Baclofen
Control (low stim. intensity)
A f \-
1 1
C
Control
25uM APB
+5uM Baclofen
~--C/A
"...
1
1
Fig. 1. Paired-pulse plasticity of medial perforant path responses and effects of baclofen. A: hippocampal slice preparation. EC, entorhinal cortex; DG, dentate gyrus; hil, hilus of dentate gyrus; hf, hippocampal fissure; CA3, CA3 region of hippocampus. Single letters indicate location of stimulating electrodes for activating lateral entorhinal fibers (L), medial entorhinal fibers (M) and commissural/associational fibers (C). Enlargement shows termination pattern of the lateral entorhinal cortex (LEC), the medial entorhinal cortex (MEC) and the commissural/associational system (C/A) along the dentate granule cell dendrites; oml, outer molecular layer; mini, middle molecular layer; im, inner molecular layer; gcl, granule cell layer. B and C: pairs of averaged (n = 3) response pairs (1 = first response, 2 = second response) recorded under the conditions indicated above are superimposed for comparison of the relative change in response amplitude with paired stimulation. Interstimulus interval was 80 ms, inter-pair interval was 20 s. B: effects on response amplitude and paired-pulse plasticity of 5 ktM baclofen (center), and comparison with a matched amplitude control first response (right). C: effect of adding 25 ~uM _+APB (center) on the effects of subsequent addition of 5 .uM baclofen (right). Calibration bars = 0.5 mV, 4 ms.
350 paired-pulse depression decreases as the antagonism is increased, and with greater than 50% antagonism by baclofen, the second response is larger than the first (Fig. 1B, center). This change in paired-pulse plasticity reverses as the drug is washed out and the response amplitude returns to control levels. The effect on paired-pulse plasticity is not simply due to reducing response amplitude, since decreasing the response by lowering stimulus intensity did not noticeably change the paired-pulse depression (Fig. 1B, right). The shift from depression toward potentiation is also not due to unmasking baclofen-resistant 15 lateral perforant path responses, which exhibit pairedpulse potentiation 18, because if 25/~M + APB was added beforehand, a shift from depression to potentiation still occurred during baclofen application (Fig. 1C, right). Baclofen appears to reduce neurotransmitter release in other systems and it is likely that its effects on medial perforant path responses are via a presynaptic mechanism, possibly one related to calcium ion availability. Therefore, pairs of evoked responses were collected in normal medium and in medium in which calcium was reduced to 1.0-0.75 mM, to reduce the evoked responses by 50-75%, corresponding to the amount of baclofen antagonism at which a clear shift in the paired-pulse plasticity was seen. At this calcium concentration, medial perforant path responses exhibited paired-pulse potentiation (see Fig. 2A). Kynurenic acid is also a medial perforant path antagonist, but does not change the paired-pulse plasticity (Fig. 2B). Kynurenic acid at 200-800 ~tM did not lessen the paired-pulse depression in any of the 10 slices examined, even though these doses reduced medial perforant path responses by as much as 75%. Medial perforant path responses are also antagonized by adenosine. Application of 25 ktM adenosine reduced medial perforant path responses by 42 + 5% (5 slices) and caused the medial perforant path paired-pulse response to shift from depression to potentiation (Fig. 2C). The effects of adenosine reached a plateau within minutes and reversed upon washing with control medium. It is unlikely that the shift produced by adenosine results from changes in response amplitude or from contamination by lateral perforant path or commissural/associational (C/A) responses, because addition of 25 ktM adenosine reduced lateral perforant path synaptic responses by
Control
Treatmenz
A 2
Low Ca++
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B
1
C
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D : ...'
"...:
%..'
1
-PzDA
____.j
1
Fig. 2. Effects of different synaptic antagonists on medial per-
forant path paired-pulse plasticity. Pairs of responses (80 ms interstimulus interval, 20 s interpair interval)were averaged (n = 3) before (left) and during (right) antagonism by the treatment indicated to the right. Identical results have been obtained in at least 3 other slices for each treatment: Calibration bars = 0.5 mV, 5 ms. greater than 50% (3 slices) and C/A responses by 40 + 3.5% (n = 3). N-(p-chlorobenzoyl)-piperazine2,3-dicarboxylic acid (pCB-PzDA) has been shown to be a potent antagonist of medial perforant path responses in the hippocampus 8. Addition of up to 250 ~tM pCB-PzDA resulted in a 78 ___ 6% reduction of medial perforant path responses, but produced no noticeable change in the percent paired-pulse depression. The effects of the various antagonists were examined at several interpulse intervals in an effort to further differentiate between treatments and better characterize the effects on paired-pulse plasticity. The similarity of effect of baclofen, adenosine and low calcium medium on paired-pulse plasticity, and the lack of effect of kynurenate and pCB-PzDA on
351 A Control Q Low Ca • Baclofen • Adenosine O Kynurenate [~ CB-PzDA
1.2
1.0 2nd Response 1St Response
0.8
0.6
4'0
160
640
Interstimutus Interval (msec)
Fig. 3. Time course of effects on paired-pulse plasticity. The ratio of the 2nd/lst response (fractional change) in averaged (n = 3) response pairs is plotted at 3 interstimulus intervals (ISis) (for clarity, data from 80,320 and 1200 ms ISI are not plotted). Each point represents the mean of ratios obtained from 3 slices. Antagonism of the control first response of 45-65% was produced by each treatment. the time course of paired-pulse plasticity are shown in Fig. 3. These results indicate a fundamental difference in the mechanisms of synaptic antagonism of several potent antagonists of medial perforant path synaptic responses. Baclofen and adenosine antagonism are associated with a shift in paired-pulse plasticity from depression towards potentiation. This shift does not result simply from reducing evoked response amplitude, since decreasing the stimulus intensity does not cause a shift in medial perforant path paired-pulse plasticity (Fig. 1B, right), and kynurenic acid and pCBPzDA, which reduce the response amplitude, do not change paired-pulse plasticity. The effects of baclofen and adenosine also do not result from contamination by other responses that exhibit potentiation and exhibit differential sensitivity to these antagonists. The shift from paired-pulse depression to pairedpulse potentiation reflects an interaction between baclofen and the processes underlying paired-pulse plasticify, which appears to occur presynaptically. Baclofen 3,22, and adenosine 7 have been shown to decrease presynaptic neurotransmitter release in several systems, and it is likely that the process whereby these compounds decrease release involves presynaptic calcium, although based on an indirect analysis, Dunwiddie5 has suggested that adenosine may not work via a calcium-related mechanism. Decreasing calcium availability has been found to increase or unmask potentiation in other systems ]6,17,24, and superfusing hippocampal slices with medium containing no calcium causes changes in paired-pulse plasticity along the medial perforant path is. Less drastic reduc-
tions in calcium and evoked responses also produce a shift from depression to potentiation, especially at short interstimulus intervals (Figs. 2A, 3). The similarities between the effects of baclofen, adenosine and lowering calcium on medial perforant path responses - - causing a shift from depression to potentiation, and the temporal profile of this shift - - suggest a common mechanism of action: decreasing presynaptic transmitter release by reducing presynaptic calcium influx. The shift in paired-pulse plasticity then follows as an expected consequence of reducing release17,24. Although believed to act primarily as presynaptic antagonists, both baclofen and adenosine have also been reported to have direct effects on postsynaptic cells. Baclofen causes hyperpolarization and an increase in conductancet, 20, although at higher doses than were used in the present study. Adenosine also produces a slight hyperpolarization as well as a reduction in calcium spike amplitude in postsynaptic cells 23. However, it is unlikely that these postsynaptic effects are involved in the mechanism of primary synaptic antagonism, or would have any effect on synaptic paired-pulse plasticity. It may be that the mechanism of inhibition of postsynaptic calcium spikes by adenosine is similar to its mechanism of reducing presynaptic calcium, however. Kynurenate and pCB-PzDA have effects unlike those elicited by baclofen, adenosine or lowering extracellular calcium concentration. Kynurenate blocks synaptic responses as well as responses to ionophoretically applied agonists in the hippocampus '~ and other central nervous system structures 2,21. Kynurenate also reduces glutamate binding to synaptic membranes prepared from whole rat brains, as well as from sites in the dentate gyrus molecular layer revealed by [3H]glutamate autoradiography (D. T. Monaghan and C. W, Cotman, unpublished observations). These data indicate that kynurenate is a competitive blocker of postsynaptic acidic amino acid receptors. It follows that synaptic antagonism by kynurenate will not be accompanied by a change in paired-pulse plasticity. If the action of kynurenate is postsynaptic, it will not interact with the processes underlying paired-pulse plasticity of the synaptic response. The paired responses should be reduced proportionately the same, and the time course of pairedpulse plasticity should be unaffected. The lack of ef-
352 fect of p C B - P z D A on paired-pulse plasticity suggests that it too acts postsynaptically to antagonize synaptic responses. In fact, p C B - P z D A has been shown to block the responses to applied acidic amino acids in the hippocampus 8. The finding that the effect of calcium is more pronounced at shorter interpair intervals suggests that there are two components underlying medial pairedpulse plasticity, one earlier component that is more sensitive to changes in calcium, and a later component that is less sensitive to changes in calcium, it may be that McNaughton 19 observed potentiation only after profound reduction of the evoked response because he used a relatively long interstimulus interval (150 ms), while the effects of presynaptic antagonism are most pronounced at shorter interstimulus intervals (40-80 ms). Paired-pulse analysis is a simple tool for investigating mechanisms of synaptic antagonism. Although it does not rule out additional postsynaptic effects, antagonism that is associated with a change in synaptic paired-pulse plasticity almost certainly involves a presynaptic effect, one probably related to presynaptic calcium. It is possible, however, that an antagonist could reduce presynaptic release via a mechanism unrelated to paired-pulse plasticity, for example, by being taken up into a presynaptic terminal and serving as a false transmitter. In this case, paired responses would be reduced proportionately the same, and paired-pulse analysis would reveal no evidence of presynaptic effects. Thus, paired-pulse analysis could yield a false negative concerning presynaptic antagonism. Nonetheless, in systems in which
1 Brady, R. J. and Swann, J. W., Intracellular studies of the effects of baclofen on bicuculline-induced epileptiform activity in CA3 pyramidal cells, Soc. Neurosci. Abstr., 9 (1983) 397. 2 Cochran, S. L., Pharmacological antagonism of climbing fiber-, parallel fiber-, and acidic amino acid-induced excitation of frog cerebellar purkinje cells, Soc. Neurosci. Abstr., 9 (1983) 1142. 3 Collins, G. G. S., Anson, J. and Kelly, E. P., Baclofen: effects on evoked field potentials and amino acid neurotransmitter release in the rat olfactory cortex slice, Brain Research, 238 (1982) 371-383. 4 DelCastillo, J. and Katz, B., Statistical factors involved in neuromuscular facilitation and depression, J. PhysioL (Lond.), 124 (1954) 574-585. 5 Dunwiddie, T. V., Interaction between the effects of aden-
changes in synaptic efficacy can be ascribed witi,. some confidence to presynaptic mechamsms, pairedpulse analysis may, in lieu of definitive mtraceltular quantal analysis, provide clues as to probable mechanisms of synaptic antagonism. Paired-puise atlalysis can also suggest than an antagonist acts postsynaptically. A more positive indication of postsynaptic antagonism might be found by examining the effectiveness of a drug at blocking applied agonists, such a test requires at least a partial identification of the neurotransmitter and its receptors for the system in question, however, and does not rule oul additional presynaptic effects. Paired-pulse analysis is a simple way of identifying possible postsynaptic antagonists and can give a strong indication of presynaptic amagonism. These results support for the idea that baclofen and adenosine are central nervous system presynaptic release antagonists, and also that kynurenic acid is a central nervous system acidic amino acid receptor blocker. Since kynurenic acid2.9, 25 and adenosine 7 are potent antagonists of many central nervous system pathways, these two compounds may be useful for locating mechanisms underlying synaptic plasticity, for example long-term potentiation, by permitting selective pre- and postsynaptic experimental intervention. Thanks to Alan Ganong and Tim Hearn for helpful suggestions, to Ciba Geigy for baclofen, and to Dr. J. C. Watkins for pCB-PzDA. Supported by D A M D 17-83-C-3189 and N1H Postdoctoral Grant NS06480 (E.W.H.).
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