Glycine-induced changes in synaptic efficacy in hippocampal slices involve changes in AMPA receptors

261

Bram Research, 627 (1993) 261-266 © 1993 Elsevier Soence Pubhshers B V All rights reserved 0006-8993/93/$06 00

BRES 19386

Glycine-induced changes in synaptic efficacy in hippocampal slices involve changes in AMPA receptors Kawan Shahl, Michel Baudry

*

Neuroscwnce Program, USC, Los Angeles, CA 90089-2520, USA (Accepted 15 June 1993)

Key words Hlppocampus, Glutamate, Receptor, Glycme, Long-term potentmtlon (LTP), Long-term depresston (LTD), Plasticity

Brtef apphcatlons of htgh glyclne concentrations to hlppocampal shces have been shown to produce long-lasting changes m synaptlc efficacy In the present study, we show that glycme apphcatlon transiently and reverstbly increases the amphtude and prolongs the duration of synaptlc potentmls medmted by N-methyl-o-aspartate (NMDA) receptors The long-lasting changes m synapttc potentmls mediated by AMPA receptors are correlated with changes m the binding of [3H]a-ammo-3-hydroxy-5-methylisoxazole-4-proprlomc acid ([3H]AMPA) to membranes prepared from glycme-treated slices The changes in binding properties of AMPA receptors in adult slices are due to an increase m affimty of the agonist for the receptor Furthermore, glycme-mduced increases in [3HIAMPA binding and in synaptic potentmls in adult htppocampal shces are markedly reduced m the presence of low extracellular calcmm or of the phosphohpase mhlbttor bromophenacylbromlde Finally, glyclne-mduced potentmtlon of synaptlc potentials is assocmted w~th an increased potency of the glutamate receptor antagonist, 6,7-dimtroquinoxahne (DNQX), to inhibit synaptlc potentmls The results mdscate that glyclne-lnduced changes m synaptlc efficacy are hkely triggered by the activation of NMDA receptors and expressed by changes m the properties of AMPA receptors As stmdar events underly long-term potentiation (LTP), this phenomenon might prowde important clues to elucidate the molecular mechanisms involved m LTP maintenance

INTRODUCTION Several mechanisms of synaptlc plasticity have been identified in different brain circuits and have been extensively studied as they potentially represent mechanisms of information storage 4 None has been more investigated than the long-term potentiation (LTP) of synaptlc efficacy elicited m several hlppocampal and cortical pathways by brief bursts of high frequency electrical stimulation 3'5 The mechanisms underlying LTP are best understood at the exotatory glutamaterglc synapses in the CA1 field of hlppocampus LTP is triggered by the actwatlon of the N M D A subtype of glutamate receptors and the resulting influx of calcium in postsynaptlc structures I4'I6'17 The exact cascade of reactions stimulated by the rise in intracellular calcium that leads to LTP expression is still a matter of debate, although several arguments support the Idea that a change m properties of the A M P A subtype of glutamate receptors is involved 8'11'I8'25'28'3° In particular, LTP in the perforant path to dentate gyrus is associ-

* Corresponding author Fax (1) (213) 746-2863

ated with an mcrease in A M P A receptor blndmg in the molecular layer 31 Interestingly, under some conditions and in particular in neonatal slices, actwatlon of N M D A receptors, instead of producing LTP, elicits a long-term decrease in synaptlc efficacy often referred to as long-term depression (LTD) 9'24 We previously reported that a brief apphcatlon of a high concentration of glyclne to hlppocampal shces resulted in slowly developing, long-lasting changes in synaptic efficacy at excitatory synapses in field CA1 of hlppocampus 29 Slices prepared from adult rats exhibited an increase m synaptlc efficacy whereas shces prepared from neonatal rats exhibited a decrease in synaptlc efficacy Glyclne is a reqmred co-agonlst of the N M D A receptors 12 and we presented some evidence that, under the conditions we used, glyclne might be potentiating N M D A receptor-mediated functions 29 In the present study, we further expand these results and directly show that (1) glycme apphcatlon increases the amplitude and prolongs the duration of synaptlc potentials mediated by N M D A receptors, (n) the

262 glycme-mduced changes in synaptlc efficacy are correlated with changes in the binding and pharmacological propertxes of the AMPA receptors and (in) blocking the glycme-mduced changes m synaptlc efficacy by low calcium or the phosphohpase A2 inhibitor, bromophenacylbromlde, prevents the changes in AMPA receptor binding Th~s para&gm might thus prowde an interesting tool to evaluate the molecular mechamsms

involved in the long-term maintenance of the modxflcatxons of synaptlc efficacy MATERIALS AND METHODS Preparation of htppocampal shces Experiments were performed on hippocampal shces (400 ~ m thnck) prepared from adult (150-300 g) and neonatal (PND 5-8) Sprague-Dawley rats as previously described zI Shces were kept at

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2 rasec Fig 1 Effects of glycine on NMDA receptor- and AMPA receptor-mediated synaptxc responses in CA1 radIatum A amphtude of populahon epsps evoked m CA1 radiatum in normal ACSF (filled circles) and ACSF containing 50 p,M Mg 2+ and 10 # M DNQX Slices were peffused with 10 mM glycine for the period indicated by the horizontal bar The NMDA receptor-mediated responses were primed after the initial 5 rain to minimize contribuhons from GABA A receptors B average traces taken at times indicated by the arrows m A Left traces show the unprIrned (1) and the primed (2) NMDA receptor-mediated epsp Right traces show the primed NMDA receptor-mediated epsp before (2), during (3), and after washout (4) of glycine The lower three traces represent the AMPA receptor-mediated epsp before (A), during (B), and 1 h after washout (C) of glycine

263 35°C in an interface chamber and subfused at a rate of 1 m l / m i n with artificial cerebrosplnal fluid (ACSF) containing (m mM) NaCl 124, KCl 3, KHEPO 4 1 25, CaCl 2 3, MgCl 2 l, NaHCO 3 26, D-glucose 10, L-ascorbate 2, continuously gased with a mixture of 0 2 CO 2 (95% 5%) A set of slices was kept under static conditions and were used as control for binding experiments as preliminary experiments m&cated no slgmficant difference in AMPA binding between membranes prepared from such shces or shces kept under continuous perfuslon for 90-120 mln Recordings were made from one slice (out of a total of four in the perfuslon chamber) to represent that group when hgand binding was performed Following 1 h of equilibration, a glass recording electrode (R ~ 1-5 M J2, filled with 2 M NaCl) was positioned in CA1 stratum radmtum to record extracellular field potentials Schaffer collateral-commissural fibers were stimulated every 30 s with bipolar stimulating electrodes For primed responses, two stimulating electrodes were placed on both sides of the recording electrode to trigger two independent inputs to the same dendritic field One Input served to prime the other by delivering two stlmuh, 15 ms apart, 200 ms prior to dehvermg the test stimulus to the second input, this procedure has been shown to effectively ehmmate GABAa receptor contribution I3 In experiments using neonatal hlppocampal slices, Mg 2+ concentration was lowered to 50 /~M In experiments analyzing NMDA receptor-medmted responses, Mg 2+ was lowered to 50/zM, and 10/.LM DNQX was added to the ACSF, addition of the NMDA receptor antagonist APV (50 ~M) totally eliminated the evoked epsp, indicating that it represents N M D A receptor-medmted synaptic responses A concentrated stock of glycine was added to the perfuslon line for the indicated periods Population epsps were recorded, analyzed, and stored on computer For the experiments comparing the effect of DNQX on control and glyclne-treated slices, a slice was transferred from the static bath to the perfusion bath after the glycine treatment, in order to ehmlnate the problem of perfuslon rate or other difference between recording chambers

Membrane preparatton and bmdtng assay Sixty minutes after glycine application, hlppocampal slices (3-5) from control and glyclne-treated groups were homogenized in 100 mM Tris/acetate buffer (pH 7 4) containing 1 mM EGTA and 100 /xM leupeptln first with a teflon-glass homogenizer then a glass-glass dounce homogenizer Homogenates were centrifuged at 40,000× g for 20 mln The supernatant was discarded and the pellet was resuspended by sonication in 100 mM Trls/acetate buffer (pH 7 4) containing 100 ~ M EGTA For binding assays, allquots of membrane suspension were incubated for 45 mln at 0°C with 50 nM [3H]AMPA in the presence of 50 mM KSCN Incubation was terminated by centrffugation at 40,000× g for 15 rain Pellets were superficially washed with 100 mM Trls/acetate buffer (pH 7 4) containing 100 ~ M E G T A and 50 mM KSCN Non-specific binding was defined as the binding measured in the presence of 2 mM L-glutamate Proteins were determined with the method of Bradford 7 with bovine serum albumin as standard

RESULTS Previous results showed that a 10-mln bath application of glyclne (10 mM) produced a slowly developing increase m the slope and amplitude of field epsps evoked in CA1 radlatum by Schaffer-commissural fiber stimulation 29 Fig 1A illustrates the time course of glycine effects on A M P A receptor- and N M D A receptor-mediated epsps The A M P A receptor-mediated epsps were recorded in normal ACSF and display the typtcal rise in slope and amplitude previously described The N M D A receptor-mediated epsps were recorded In ACSF containing 5 0 / x M Mg 2+ and 10/.~M

DNQX, and display a transient and reversible increase in amplitude These epsps were totally eliminated in the presence of the N M D A receptor antagonist APV (50 ~ M ) Representatwe traces of these epsps are depicted in Fig 1B The N M D A receptor-mediated potentials display a typical longer time-course than the fast A M P A receptor-mediated potentials The left two traces show the unprtmed (1) and primed (2) N M D A receptor-mediated epsps After the first 5 mm, all subsequent N M D A receptor-mediated epsps were primed to mlnimtze contributions from G A B A A receptors, which are reflected by a large hyperpolanzation in trace 1 and mask the effect of glycine The right three traces show the primed N M D A receptor-mediated epsp before (2), during (3) and after washout (4) of glyclne Glycine produced a transient and reversible increase m amplitude and duration of the epsps with no increase in the inltml slope The bottom three traces show the A M P A receptor-mediated epsps before (A), during (B), and 1 h after washout (C) of glyclne The rise m A M P A receptor-mediated synaptic potentials started approxtmately at the end of the glycine apphcation To elucidate the mechantsms involved m the glycine-mduced mcrease in A M P A receptor-mediated epsps, we performed hgand-binding assays usmg [3H]AMPA and membranes prepared from hippocampal slices treated wtth glycine and from control slices kept under non-perfused condttions Membranes prepared from glycine-treated slices exhibited an Increase in [3H]AMPA binding that was well correlated with the observed change in epsp amplitudes (Fig 2) The effects of glycme on both physiological responses and [3H]AMPA bmding were markedly reduced when slices were incubated in the presence of a low Ca 2+ concentration m the ACSF (1 mM) or of the phosphohpase Inhibitor BPB (100 IzM) Previous results showed that perfusmg shces with 10 mM glycine produced a slowly developing depression of evoked epsps in hlppocampal slices prepared from 5- to 8-day-old rat pups z9 Fig 2 also illustrates that there is a good correlation between the decrease in epsp amphtude and m [3H]AMPA binding to membranes prepared from neonatal hippocampal slices Saturation binding at equilibrium was performed using membranes from glycme-treated and control adult hippocampal slices to determine the nature of the observed increase in [3H]AMPA binding The affinity of the A M P A receptor for ItS agonIst was higher in slices treated with glycine as compared to control ( K d = 235 _+ 48 nM vs 358 + 33 nM, mean _+ S E M of 3 expertments, P < 0 05), whereas the Bmax was slightly decreased (4 32 + 0 13 vs 5 29 + 0 04) We further investigated the changes m A M P A receptor

264 properties ehclted by glyclne apphcatlon by determining the effects of the antagonist D N Q X (5 /~M) on synaptic potentials Slices pretreated with glyclne were more sensitive to D N Q X than control shces (Fig 3), as the time required to observe half-maximal inhibition of the synaptic potentials was decreased while the magnitude of the inhibition was increased One hour followlng the onset of D N Q X application, the initial slopes of the evoked epsps were significantly more decreased in glycine-treated slices as compared to control (79 _+

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Fig 3 Differential effects of D N Q X on control and glyclne-potentlated synaptlc responses The Initial slope of population epsps evoked in CA1 radlatum of control (filled circles) and glycine-treated (open circles) slices was determined before and during the perfuslon of D N Q X (5 /~M) for the period indicated by the horizontal bar Results are expressed as percentage of the mean baseline value obtained prior to D N Q X application, and are m e a n s + S E M of 6 experiments

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Fig 2 Correlation between glyclne-lnduced changes in epsp amphtude and in [3H]AMPA binding A each point represents a single experiment where glyclne-mduced changes In epsp amphtude in adult (open circles) or neonates (filled circles) were measured in a single slice (out of four) and plotted against the average change in [3H]AMPA binding measured in membranes prepared from those shces 60 mzn following glyctne treatment Binding assays were performed in triplicate and the error on each point is less than 5% of the value Results of experiments performed with adult slices in the presence of 1 mM Ca 2+ (hatched square) or 100/~M BPB (hatched triangle) are represented as means_+S E M of 5 experiments B means_+ S E M values of the glyclne-mduced changes in epsp amplitude and [3H]AMPA binding in adult hippocampal slices (* P < 0 05)

The present results indicate that mllhmolar concentrations of glycine produce a transient increase in N M D A receptor-mediated epsps and a long-lasting increase in A M P A receptor-mediated epsps The observed increase m amplitude and prolonged duration of the primed N M D A receptor-mediated potentials with little effect on the initial slope fit well with the hypothesis that glyclne acts to reduce N M D A receptor desensltlzatlonlSZ2 This finding also suggests that the glycme site of the N M D A receptor is not saturated under our conditions Mllhmolar concentrations of glyclne may be required to overcome efficient uptake systems, which might explain why many reports have failed to observe direct effects of lower glyclne concentrations on N M D A receptor-mediated synaptlc responses in hlppocampal shces 2'26 This would also fit with results of studies showing that bath-apphed glutamate, even at concentrations up to 3 mM, does not reach synaptlc receptors 1 However, it is also possible that high glycme concentrations produce effects not related to N M D A receptors The time-course of the effect of glycme on N M D A receptor- and A M P A re-

265 ceptor-mediated epsps suggests that N M D A receptor activation precedes and may be causally related to the slower rise in A M P A receptor-mediated epsps This is consistent with our previous findings that ketamine, a non-competitive N M D A receptor channel blocker, significantly reduces glycine's effects 29 It is also interesting to note that the time-course of the effect of glyclne on synaptlc responses resembles that reported by Davies et al for the development of increased responsiveness to A M P A following L T P induction in hippocampal shces s as well as for the ACPD-lnduced potentiation 6 Several approaches were used to identify the possible mechantsms underlying the expression of glyclneinduced increase in synaptic efficacy The technique of radiolabeled hgand binding assays can be used to detect possible changes in the properties of the synaptlc receptors The good correlation between changes in epsp a m p h t u d e and [3HIAMPA binding to m e m b r a n e s prepared from glycine-treated hlppocampal shces suggests that changes in A M P A receptor properties play a critical role in the expression of plasticity The correlation spans a wide range of a m p h t u d e changes from a decrease, in the case of neonates, to an increase, in the case of adults The correlation is especmlly good when one considers that, from each group of glycme-treated shces, the value for the epsp a m p h t u d e was obtained from only one shce The effect of glycme application on synaptlc responses m adult shces was sigmficantly reduced when experiments were performed in low Ca 2+ concentration (1 mM), an effect consistent with the ketamlne results previously mentioned The effect was also reduced m the presence of BPB (100 /zM), a PLA2 inhibitor U n d e r both conditions, low Ca 2+ or BPB, the increase m synapt~c response as well as the associated increase in [3H]AMPA binding were reduced Since PLA2 is a calcmm-dependent enzyme which has been shown to be actwated following N M D A receptor stimulation 1°, the above results suggest that PLA2 plays an Important role in the m e c h a m s m responsible for the glyclne-mduced changes in A M P A receptor properties and functions We have previously shown that treatment of synaptlc m e m b r a n e s w~th PLA2 results m an increase m the affinity of the A M P A receptor for Its agonlst 19 This is consistent with the observation that the increase in [3H]AMPA binding reduced by glyclne is due to an Increase m the affinity of A M P A receptors We have also previously shown that increasing the affinity of the synaptlc receptors for their endogenous agonlst, glutamate, increases the slope and a m p h t u d e of synapUc responses, indicating that changing the binding properties of the receptors does change their

functional properties 28 Interestingly, PLA2 treatment of synaptlc m e m b r a n e s p r e p a r e d from neonatal rats (PND 5) results in a decrease m [3H]AMPA binding 2° As we also observed a decrease in the synaptlc response and A M P A binding in neonatal rats, it is tempting to implicate PLA2 in the mechanism responsible for A M P A receptor changes in neonates following glycine treatment Finally, the glycine-mduced changes in A M P A receptor-mediated potentials were associated with a change in pharmacological properties of the A M P A receptors The effect of D N Q X , a competltwe A M P A receptor blocker, on synaptic potentials was increased following glycine application This effect appears somewhat paradoxical as the results from the binding experiments indicated an increased affinity of agomsts for the A M P A receptors We have thus to assume that this increased affinity for agonist is also associated with at least an equal increase in affinity for the antagonist In any event, these results are all consistent with changes in A M P A receptor properties Glyclne produces opposite effects in synaptic responses as well as [3H]AMPA binding on adult and neonatal hlppocampus One important difference in adult and neonate rats is the differential expression of A M P A receptor subunlts (GluRs) 23'27 The presence of different subunits for the A M P A receptors may therefore be critical in determining the way synaptic responses change following N M D A receptor activation Future experiments will attempt to test this h~pothesis Acknowledgements

This work was supported by Grants BNS 911937 from the National Science Foundation and N00014-91-J-1796 from the Office of Naval Research to M B

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