Neuroscience Letters, 149 (1993) 185-188 1993 Elsevier Scientific Publishers Ireland Ltd.

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High concentrations of glycine induce long-lasting changes in synaptic efficacy in rat hippocampal slices
Kavian Shahi, Juan-Carlos Marvizon and Michel Baudry
Neuroscience Program, University of Southern California, Los Angeles, CA 90089-2520 (USA)
(Received 6 August 1992; Revised version received 14 October 1992; Accepted 14 October 1992)

Key words." Hippocampus; NMDA receptor; Plasticity; Long-term potentiation; Long-term depression; Epilepsy
Brief perfusion of adult rat hippocampal slices with high concentrations of glycine results in a slowly developing, long-lasting increase in synaptic responses in field CA1. Two observations indicated that the effect requires the activation of NMDA receptors by glycine. First, the glycine-induced potentiation is reduced by ketamine, an NMDA receptor channel blocker. Second, glycine potentiates the NMDA receptor-mediated epileptiform activity recorded in the presence oflow magnesium concentration andpicrotoxin. Inslices prepared from rat pups(5 8 postnatalday),perfusion with glycine results in a slowly developing, long-lasting depression of EPSP amplitude. These results provide a new way of producing potentiation of synaptic efficacy and suggest new properties of NMDA receptors.

Several manipulations have been found to produce a long-lasting potentiation of synaptic efficacy at excitatory synapses. These include the well described high frequency electrical stimulation that results in what is now generally defined as long-term potentiation (LTP) [6] and which requires the influx of calcium in postsynaptic structures following the activation of N M D A receptors [18]. Variants of this form of potentiation involve repetitive pairing of presynaptic stimulation with postsynaptic depolarization [12]. Another form of potentiation does not require N M D A receptor activation, but rather the activation of voltage-dependent calcium channels and can be produced by electrical stimulation at frequencies higher than that required for LTP [10] or by blockade of potassium channels with tetraethylammonium (TEA) [1]. More recently, another form of potentiation has been described which involves activation of metabotropic glutamate receptors following N M D A receptor activation [20]. In this case, the potentiation develops slowly over a period of tens of minutes, as compared to the few minutes that are required to fully establish stable LTP [2, 11]. It is yet unclear whether or not these different forms of potentiation simply reflect the activation of a common sequence of intracellular events. Although it has been shown that glycine acts as a coagonist of glutamate at the N M D A receptor, the physioCorrespondence: K. Shahi, Neuroscience Program, HNB 311, USC, Los Angeles, Ca 90089-2520, USA. Fax: (1) (213) 746-2863.

logical significance of the glycine site still remains puzzling as the affinity of the site for glycine is so high that it is likely to be always saturated [13, 24]. Indeed, most attempts to find direct effects of exogenous glycine or glycine agonists on N M D A receptor-mediated physiological events have proven unsuccessful. Only when glycine antagonists are used is it possible to observe blockade of N M D A receptor function which is then reversed by the addition of exogenous glycine [4, 19, 23]. We postulated that there might exist glycine sites with an affinity low enough that they had not been detected by ligand binding techniques or that they had not been stimulated by the relatively low concentrations of glycine used in previous studies. In the present work, we report that high concentrations of glycine produce a slowly developing, long-lasting potentiation of synaptic transmission and present evidence that the effects require the activation of N M D A receptors. Experiments were performed on hippocampal slices (400/lm thick) prepared from adult (150-200 g) and neonatal (5-8 postnatal days) Sprague-Dawley rats. Slices were placed in an interface chamber and subfused at a rate of 1 ml/min with medium containing (in mM): NaC1 124, KCI 3, KH2PO4 1.25, CaCI2 3, MgCI2 1, NaHCO3 26, L-glucose 10, L-ascorbate 2. In experiments analyzing rhythmic bursting activity, picrotoxin (50 /IM) was added and the concentration of Mg 2+ was lowered to 50 ~tM. Slices were maintained at 35C and oxygenated with a mixture O2:CO2 (95:5). After at least 1 h ofincuba-

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tion, a glass recording electrode (1 5 MD, filled with 2 M NaC1) was positioned in the dendritic layer of field CA1 to record extracellular field potentials. Schaffer-commissural fibers were activated by bipolar stimulating electrodes positioned in stratum radiatum. When recording spontaneous bursting activity, no stimulation was given. A concentrated stock solution of glycine was adjusted to pH 7.4 with NaOH, and was added to the perfused buffer for the indicated periods of time. Population EPSPs were recorded, analyzed, and stored on computer. EPSP amplitudes and slopes were calculated and amplitude data are presented in the results section (similar results were obtained using slope data). Perfusion of 10 mM glycine for 10 rain in a Mg 2+containing buffer did not produce any significant alteration in the amplitude or slope of the EPSP (Fig. 1A), nor did it affect the fiber volley, an index of afferent recruitment. Upon washout of glycine, a slowly developing increase in EPSP was observed; the increase reached a plateau after about 1 h when it represented a 94 + 17% increase in EPSP amplitude (mean + S.E.M. of 6 experiments) and lasted up to 3 h. The gtycine-induced potentiation was markedly reduced in slices incubated in the presence of ketamine (250 pM) (Fig. IA); under these conditions the maximum increase in EPSP amplitude represented a 34 + 11% increase (mean + S.E.M. of 9 experiments). We compared the effect of ketamine on glycine-induced potentiation to that observed on LTP elicited by theta burst stimulation (Fig. 1B). Ketamine similarly reduced the extent of LTP, reducing the increase in EPSP amplitude from 52 + 12% to 16 + 5% (means + S.E.M. of 4-5 experiments). Glycine-induced potentiation was not accompanied by a change in the extent of paired-pulse facilitation, an index of transmitter release (data not shown). While no effects of glycine on the amplitude and slope of EPSPs were observed during its application in normal Mg 2+containing buffer, a small depression of these parameters was present during the application in low Mg2+-contain ing buffer. To further document the possibility that high concentrations of glycine could activate N M D A receptors, we developed a model of spontaneous epileptic activity. In the presence of picrotoxin (50/IM) and low Mg 2+ concentration (50/~M), extracellular recordings of field potentials in the dendritic layer of field CA1 revealed spontaneous bursting activity (Fig. 2A). This bursting activity was dependent on N M D A receptor activation as it was totally blocked by ketamine (100/~M) (Fig. 2C) or by Mg 2+ ( 1 4 mM) (data not shown). Perfusion of high concentrations of glycine (5-10 mM) resulted in a significant increase in the frequency of the bursting activity (Fig. 2B), even when CA3 afferents to CA1 were interrupted; this effect was reversible upon

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Fig. 1. Effect of ketamine on glycine-induced (A) or TBS-induced (B) potentiation of synaptic responses in CAl. Hippocampal slices were prepared and population EPSPs were evoked and recorded as described under Methods in the absence (open squares) or presence (closed squares) of ketamine (250 yM). A: glycine (10 mM) was added to the perfusion buffer during the period indicated by the horizontal bar. B: at the time indicated by the arrow, theta burst stimulation (TBS) was delivered to the stimulating electrode. Results represent the amplitude of the population EPSP (similar results were obtained on EPSP slopes) and are expressed as percentage of the mean base-line value obtained during 10 min before glycine or TBS application. Means _+ S.E.M. of 4 9 experiments.

washout of glycine. Moreover, no effect of glycine was observed when slices were pretreated with ketamine (Fig. 2D). Several studies have shown that LTP is absent in slices prepared from rat pups before postnatal day 10-12 [5, 21]. Perfusion of 10 mM glycine in slices prepared from 5 to 8-day-old rat pups initially produced a depolarization as reflected by the decrease in EPSP slope and amplitude during the perfusion in low Mg2+-containing buffer (Fig. 3). Upon washout of glycine, the parameters of the evoked responses returned to pre-glycine values before exhibiting a slowly developing and long-lasting depression. Like the glycine-induced potentiation seen in slices from adult animals, the glycine-induced depression reached a plateau at about 1 h following glycine applica-

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Fig. 2. Effect of various compounds on spontaneous bursting activity. Extracellular recording electrodes were placed in stratum radiatum of area CA 1 of hippocampal slices maintained in a medium containing 50 /IM Mg > and 50/IM picrotoxin. Field potentials were recorded in the absence of any stimulation and under control conditions (A), in the presence of 10 m M glycine (B), in the presence of ketamine (100/aM) (C), and in the presence of glycine and ketamine (D). Bar = 1 mV, 1 s. The experiment was repeated 3 4 times with essentially similar results.

tion, at which point it represented a 30 + 6% decrease in amplitude of the EPSPs. The present results indicate that perfusion of slices with millimolar concentrations of glycine produces slowly developing, long-lasting and opposite changes in synaptic efficacy in adult and neonatal rats. While potentiation followed glycine applications in adults, the same conditions induced depression in neonatal rats. Several

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Fig. 3. Glycine-induced depression in neonatal slices. Hippocampal slices from neonatal rats (PND 5 8) were prepared and maintained in a medium containing low Mg > concentration (50/IM) and population EPSPs were recorded as described under Methods. Glycine (10 mM) was applied during the period indicated by an horizontal bar. Results represent the amplitude of the EPSPs (similar results were obtained on EPSP slopes) and are expressed as percentage of the mean base-line value recorded during 10 rain before glycine application. Means + S.E.M. of 5 experiments.

arguments indicate that the glycine-induced increase in synaptic efficacy in adult slices requires the activation of N M D A receptors. First, the glycine-induced potentiation was considerably reduced when slices were pretreated with the N M D A channel blocker ketamine and the extent of the reduction was the same as the reduction of LTP elicited by theta burst stimulation. In the absence of Mg 2 and with inhibition blocked by picrotoxin, epileptiform activity characterized by repetitive bursting activity spontaneously developed in field CAI of hippocampal slices even when the connections between CA3 and CA1 were interrupted. This bursting activity was due to the activation of N M D A receptor as it was totally prevented by non-competitive N M D A receptor antagonists, such as ketamine and Mg >. Under these conditions, glycine increased the bursting frequency suggesting that high concentrations of glycine potentiated NMDA receptor activation. Finally, binding experiments using the binding of [3H]MK-801 to well-washed membranes as a functional index of channel opening also showed that high concentrations of glycine stimulated [3H]MK-801 binding even in the absence of glutamate [15]. However, while these arguments support the idea that glycine is capable of activating N M D A receptors, there are marked differences between the effects of glycine reported here and those produced by the direct application of an NMDA receptor agonist such as NMDA. Under normal ionic conditions, perfusion of slices with moderate concentrations of N M D A (100/IM) produced depolarization [8] and seizure activity, probably as a result of the regenerative property of the NMDA receptors (i.e. NMDA receptor activation leads to depolarization making neighboring N M D A receptors less sensitive to blockade by Mg 2+ and therefore more prone to activation). Furthermore, treatment of slices with N M D A alone (either by bath application or by iontophoresis) does not produce a long-lasting potentiation of synaptic response but rather a short-lived potentiation lasting about 30 min [3, 14]. We thus have to conclude that if glycine activates NMDA receptors, it does it very differently than a full agonist of the receptors. Perhaps, glycine acts as a weak or partial agonist which produces enough activation to trigger a long-lasting change in synaptic efficacy but does not generate a significant depolarization. Interestingly, we noted that under depolarizing conditions produced by theta burst stimulation, glycineinduced potentiation developed faster and at a lower glycine concentration than in the absence of high frequency stimulation (data not shown). In neonatal rats, high frequency stimulation does not induce LTP at least not before postnatal day (PND) 1012 [5, 21]. However, long-lasting depression of synaptic transmission is much more readily observed in neonatal

188 t h a n in adult slices following brief periods (2-4 min) of low frequency s t i m u l a t i o n (Fitzpatrick et al, u n p u b l i s h e d data). Interestingly, glycine application in slices o f P N D 5-8 a n i m a l s produced a long-lasting depression which represented a m i r r o r image o f the p o t e n t i a t i o n observed in adult slices. F u r t h e r m o r e , we also observed opposite effects o f PLA2 t r e a t m e n t of synaptic m e m b r a n e s on the b i n d i n g properties of the A M P A receptors in n e o n a t a l a n d a d u l t rat b r a i n [17]. PLA2 t r e a t m e n t p r o d u c e d a decrease in [3H]AMPA b i n d i n g in n e o n a t a l m e m b r a n e s a n d a n increase in adult m e m b r a n e s . We have also argued that the PLA2-induced changes in receptor properties are part o f the m e c h a n i s m s u n d e r l y i n g LTP [16]. As N M D A receptor activation has been linked to P L A 2 activity [7], these results suggest the possibility that the glycine-induced changes in synaptic efficacy are mediated t h r o u g h PLA~ stimulation. Finally, o u r results provide an a d d i t i o n a l way of producing long-lasting changes in synaptic transmission. Thus, in a d d i t i o n to high frequency stimulation, it has been shown that increased calcium c o n c e n t r a t i o n [25], short period of K+-induced depolarization [9], T E A application [1], application of a c o m b i n a t i o n of N M D A , glycine, calcium a n d spermine [22], a n d more recently activation of N M D A a n d g l u t a m a t e m e t a b o t r o p i c receptors d u r i n g low frequency s t i m u l a t i o n [20], all p r o d u c e increased synaptic efficacy. W h e t h e r all these m a n i p u l a tions p r o d u c e the activation of a c o m m o n final p a t h w a y leading to a single modification of one element regulating synaptic t r a n s m i s s i o n or produce u n i q u e modifications of several regulatory elements remains to be determined. NMDA receptors activate the arachidonic acid cascade system in striatal neurons, Nature, 336 (1988) 69 70. 8 Fagni, L., Baudry, M. and Lynch, G., Classificationand properties of acidic amino acid receptors in hippocampus, J. Neurosci., 3 (1983) 1538 1546. 9 Fleck, M.W., Palmer, A.M. and Barrionuevo, G., Potassium-induced long-term potentiation in rat hippocampal slices, Brain Res., 580 (1992) 100 105. 10 Grover, L.M. and Teyler,T.. Two components of long-term potentiation induced by different patterns of afferent activation, Nature, 347 (1990) 477 479. tl Gustafsson, B. and Wigstr6m, H., Long-term potentiation in the hippocampal CA 1 region: its induction and early temporal development, Prog Brain Res., 83 (1990) 223 232. 12 Gustafsson, B., Wigstr6m, H.. Abraham, W.C. and Huang, Y.Y.. Long-term potentiation in the hippocampus using depolarizing current pulses as the conditioning stimulus to single volley synaptic potentials, J. Neurosci.. 7 (1987) 774- 780. 13 Kleckner, N.W. and Dingledine, R., Requirement for glycine in activation of NMDA receptors expressed in Xenopus oocytes, Science, 241 (1988) 835 837. 14 Manabe, T., Rennet. P. and Nicholl, R.A., Postsynaptic contribution to long-term potentiation revealed by the analysis of miniature synaptic currents, Nature. 355 (1992) 50- 55. 15 Marvizon. J.C. and Baudry, M., Glutamate and glycine act synergistically to stimulate [~H]MK-801 binding to NMDA receptors, Soc. Neurosci. Abstr., 18 (1992) 1154. 16 Massicotte. G. and Baudry, M., Triggers and substrates of hippocampal synaptic plasticity, Neuro. Biobehav. Rev., 15 (1991) 415 423. 17 Massicotte, G.. Bernard, J. and Baudry, M., Postnatal changes in AMPA receptor regulation by phospholipase A2 treatment of synaptic membranes: temporally differential affects on agonist and antagonist binding. Dev. Brain Res., 66 (1992) 203 208. 18 McDermott. A.B., Mayer, M.L., Westbrook, G.L., Smith, S.J. and Barker. J.L., NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurons, Nature, 321 il986) 519 522. 19 Oliver, M.W., Kessler. M., Larson, J., Schottler, F. and Lynch, G., Glycine site associated with the NMDA receptor modulates longterm potentiation. Synapse, 5 11990)265 270. 20 Radpour, S. and Thomson, A.M.. Synaptic enhancement induced by NMDA and Qp receptors and presynaptic activity, Neurosci. Len., 138(1992) 119 122. 21 Teyler, T.J., Perkins. A.T. and Harris, K.M., The development of long-term potentiation in hippocampus and neocortex, Neuropsychologia, 27 (1989) 31 39. 22 Thibault, O.. Joly, M., Muller. D.. Schottler, F., Dudek, S. and Lynch, G., Long-lasting physiological effects of bath applied Nmethyl-D-aspartate. Brain Res., 476 (1989) 170 173. 23 Thiels. E,, Weisz, D.J. and Berger, T.W., In vivo modulation of N-methyl-D-aspartatc receptor-dependent long-term potentiation by the glycine modulatory site, Neuroscience, 46 (1992) 501 509. 24 Thomson, A.M., Glycine is a coagonist at the NMDA receptor/ channel complex, Prog. Neurobiol., 35 (1990) 53 74. 25 Turner, R.W., Baimbridge, K.G. and Miller, J.J., Calcium-induced long-term potentiation in the hippocampus, Neuroscience, 7 (1982) 1411 1416.

1 Aniksztejn, L. and Ben-Art, Y., Novel form of long-term potentiation produced by a K* channel blocker in the hippocampus, Nature, 349 (1991) 67 69. 2 Arai, A., Larson, J. and Lynch, G., Anoxia reveals a vulnerable period in the development of long-term potentiation, Brain Res., 511 (1990) 353 357. 3 Asztely, F., Hanse, E., Wigstr6m, H. and Gustafsson, B., Synaptic potentiation in the hippocampal CA1 region induced by application of N-methyl-D-aspartate,Brain Res., 558 (1991) 153 156. 4 Bashir, Z.I., Tam, B. and Collingridge, G.L., Activation of the glycinesite in the NMDA receptor is necessary for the induction of LTE Neurosci. Lett., 180 (1990) 261 266. 5 Baudry, M., Arst, D., Oliver, M. and Lynch, G.. Development of glutamate binding sites and their regulation by calcium in rat hippocampus, Brain Res., 227 (1981) 37-48. 6 Baudry, M. and Davis, J.L., Long-term Potentiation: A Debate of Current Issues, MIT Press, Cambridge, 1991. 7 Dumuis, A., Sebben, M., Haynes, L., Pin, J.P. and Bockaert, J.,