Glycine facilitates induction of long-term potentiation of evoked potential in rat hippocampus

Neuroscience Letters, 117 (1990) 87-92 Elsevier Scientific Publishers Ireland Ltd.

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Glycine facilitates induction of long-term potentiation of evoked potential in rat hippocampus K a z u h o A b e , Fei-jun Xie, Y a s u m a s a W a t a n a b e a n d H i r o s h i Saito Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo (Japan)

(Received 13 April 1990; Revised version received2 May 1990; Accepted 7 May 1990) Key words: Glycine;N-Methyl-D-aspartatereceptor; Long-term potentiation; Evoked potential; Rat hippocampus; CA 1 pyramidal neuron

Effects of glycine were investigated in Schaffer/commissural-CA1 pyramidal cell synapsesof the rat hippocampal slices. Perfusion of glycine (0.05 raM) did not change baseline population spikes evoked by test stimulation but significantly enhanced short-term potentiation induced by a single shorter tetanus (100 Hz, I 1 impulses); the effects resulted in production of long-term potentiation (LTP). LTP produced by a longer tetanus (100 Hz, I00 impulses, 2 trains) was not significantly influenced. Higher concentration (0.5 mM) of glycine increased the baseline spike amplitude. All these effectsof glycine were not observed in the presence of 10-5 M 2-amino-5-phosphonovalerate,an N-methyl-D-aspartate(NMDA) antagonist. These results demonstrate that glycine can facilitate induction of LTP probably by activating NMDA receptor. It is well known that glycine functions as an inhibitory transmitter in the central nervous system [1]. However, it has recently been found that glycine potentiates Nmethyl-D-aspartate ( N M D A ) receptor-mediated responses in cultured brain neurons [7]. This finding has been supported by several electrophysiological studies or radioligand binding studies [16]. The modulation of N M D A receptor by glycine is very interesting because N M D A receptor plays important roles in m a n y plastic and pathological processes in the brain [4]. Long-term potentiation (LTP) of the excitatory synaptic transmission in the hippocampus is a form of synaptic plasticity and induction of L T P requires the activation of N M D A receptor [3]. Therefore, we attempted to investigate effects of glycine on LTP in the rat hippocampal slices. Very recently, Tauck and Ashbeck [15] have reported that addition of 1/2M glycine increased L T P induced by tetanic stimulation (100 impulses at 100 Hz) in the rat hippocampal slice. However, their data are a little questionable because m a n y investigators have reported that exogenous glycine at the micromolar level had no effect on N M D A evoked responses in the slice preparations [5, 9, 12] unlike in dissociated cells [6, 7]. Correspondence: K. Abe, Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo 113, Japan.

0304-3940/90/$ 03.50 © 1990 ElsevierScientific Publishers Ireland Ltd.

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In order to elucidate the influence of exogenous glycine on LTP, we compared the effects of higher concentrations of glycine on short-term potentiation (STP) and LTP. The hippocampi were quickly isolated from male Wistar rats (250--300 g) and cut into transverse slices of 400-500/zm thickness manually. The slices were allowed to recover for more than 1 hr in the incubation chamber containing artificial cerebrospinal fluid (ACSF) which was maintained at 35°C and continuously oxygenated with 95 % O ~ 5 % CO2. The composition of ACSF was as follows (raM): NaCI 124,0, KC1 5.0, CaC12 2.4, MgSO4 1.3, KHzPO4 1.24, NaHCO3 26.0, glucose 10.0. Each slice was transferred into a recording chamber (1.0 ml) in which warmed (35' C) and oxygenated (95% O~5% CO2) ACSF was continuously perfused at a speed of 1 ml/min. A bipolar tungsten electrode was placed in the stratum radiatum to stimulate Schaffer collateral-commissural afferents. The evoked potential (population spike) was extracellularly recorded from pyramidal cell layer of CAI subfield with a glass capillary microelectrode filled with 0.9% NaC1 (tip resistance 5-10 MI2). Single test stimulation (0.05 ms duration) was given at intervals of 10-30 s. The stimulus intensity was always adjusted so that the amplitude of population spike was 50% of the maximum. The responses were allowed to equilibrate enough until stable baseline values were obtained at least for 10 rain before application of drugs or tetanic stimulation. LTP was considered to have occurred if the potentiated response maintained more than 20% higher level than baseline 30 min after tetanic stimulation. Perfusion of the slices with the ACSF containing 0.01 mM (data not shown; n = 4) or 0.05 mM (Fig. 1) glycine for 90 min did not change the baseline responses evoked by test stimulation at all. However, perfusion of 0.5 mM glycine increased slightly the spike amplitude (Fig. 1). When perfusion solution was changed into normal ACSF without glycine, the increased response recovered gradually.

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Fig. 1. Effects of glycine (O, 0.05 mM, n=8; &, 0.5 mM, n=8) on amplitude (mV) of the population spike evoked by test stimulation. The spike amplitude was defined as the average of the amplitude from the first positive peak to the succeeding negative peak and the amplitude from the negative peak to the second positive peak. Perfusion of glycine was started at 0 min and continued for 90 min. The data were expressed as the mean + S.E.M. *Significant difference from the data at 0 min: P < 0.05; Student's t-test.

89 When a single shorter tetanic stimulation (11 impulses at 100 Hz) was given, the following responses were rapidly potentiated and then declined gradually. In 13 out of 51 slices tested, the potentiation resulted in LTP: the spike amplitude 30 rain after tetanic stimulation was 134.6 ___5.9 % (mean ___ S.E.M.; n = 13) of the baseline values. In 38 other slices, the potentiated responses returned to baseline level completely within 30 min after tetanus. This change was regarded as STP but not LTP. The STP could be observed repeatedly in the same slice. Furthermore, STP induced by second tetanus was identical to first STP in terms of both initial maximum potentiation and decay time course (Fig. 2A). This result enables effects of drugs on STP to be examined with one slice. In the following experiments, the second STP in the presence of drugs was compared with the control STP induced by first tetanus. As shown in Fig. 2B, STP was blocked by 10 -5 M 2-amino-5-phosphonovalerate (APV), a selective N M D A antagonist, suggesting that the STP involves activation of N M D A receptor. The component resistant to APV may reflect a transient increase in transmitter release, probably analogous to the post-tetanic potentiation in peripheral synapses [2]. A

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Fig. 2. A: reproducibility of STP induced by single shorter tetanus (11 impulses at 100 Hz). After the STP induced by first tetanus ( a ) was observed for 50~0 min, the STP induced by second tetanus (©) was observed in the same slice (n=8). B-D: effects of APV (10 5 M, B, n=6), glycine (0.05 mM, C, n=5; 0.5 mM, D, n=6) on STP. After the control STP ( 0 ) was observed for 30 min, perfusion of APV or glycine was started. The second tetanus was given 30 min later and the STP in the presence of the drugs (©) was observed. The following is common to A-D. Abscissas: the time after tetanic stimulation in min. Ordinates: spike amplitude expressed as the percentage of baseline values at 0 min (just before tetanic stimulation). The data were expressed as the mean _ S.E.M. Significant differences from control values (0): *P<0.05; **P<0.01; Student's t-test.

90 Glycine at the concentration of 0.01 mM had no effects on STP (data not shown: n = 5) but at the concentration of 0.05 and 0.5 mM significantly potentiated STP (Fig. 2C,D). Particularly the decay of STP became slower and the LTP occurred in 2 out of 5 slices in the presence of 0.05 mM glycine and in 6 out of 6 slices in the presence of 0.5 mM glycine. However, these effects of glycine were not observed in the presence of 10 -5 M APV in 5 slices tested (data not shown). The STP observed in the present study is thought to be, at least in part, coupled to LTP induction mechanisms in the following respects: (1) the decay was slower than the post-tetanic potentiation; (2) it involved activation of N M D A receptor; (3) it sometimes resulted in LTP. Therefore, the present results suggest that glycine can facilitate induction of LTP, probably by enhancing activation of N M D A receptor. Significant effects of glycine on STP cannot be explained by changes of baseline responses since 0.05 mM glycine never influenced the baseline responses (Fig. I ). To generate LTP completely, tetanic stimulation (100 impulses at 100 Hz) was given twice at an interval of 30 s. This tetanic stimulation produced LTP in all slices tested and no greater LTP could be produced by any pattern of tetanus, indicating that LTP mechanisms had been saturated under this condition. Since the LTP lasted throughout the experiment ( > 3 h), pairs of adjacent slices from the same hippocampus were used to evaluate effects of glycine. LTP in the slice perfused with 0.01 mM (data not shown; n = 5) or 0.05 mM (Fig. 3A) glycine was not significantly different from LTP in normal ACSF. This result suggests that exogenous glycine does not affect the magnitude of saturated LTP. The result that the magnitude of LTP was significantly increased in the presence of 0.5 mM glycine (Fig. 3B) may be partly due to an increase of baseline response shown in Fig. 1. Glycine was absolutely required for activation of N M D A receptor expressed in Xenopus oocytes [10]. In tissues endogenous glycine must exist at a concentration enough to activate N M D A receptor normally. Although sub-synaptic concentration of endogenous glycine in the slice preparations is unknown, it has been reported that B

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Fig. 3. Effectsof glycine(0.05 mM, A, n=6; 0.5 mM, B, n=6) on LTP induced by longer tetanic stimulation (100 impulses at 100 Hz, 2 trains at 30 s interval). LTP in the presence of glycine((3) was compared with LTP in the absence of glycine(0) using pairs of adjacent slices from the same hippocampus. Perfusion of glycine was started 30 rain before tetanic stimulation. Ordinates and abscissas are as in Fig. 2. Significant differences from the data without glycine(Q): *P < 0.05; Student's t-test.

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concentration of glycine in dorsal spinal cord extracellular fluid was of a micromolar level [13]. It is currently controversial whether high-affinity glycine receptor is saturated by endogenous glycine. However, our results and others that exogenous glycine could potentiate synaptic or NMDA-evoked responses in slice preparations [12, 17] or strychnine-induced convulsions in vivo [11] suggest that the concentration of endogenous glycine in tissues was submaximal, at least close to synaptic receptors. The concentration of exogenous glycine required for showing significant effects on LTP in the present study is very consistent with that required for potentiating NMDA-evoked responses in the rat hippocampal slices [12]. This consistency supports that LTP facilitating effect of glycine is mediated by activation of NMDA receptor. Some reasons why a higher concentration of glycine is necessary for showing significant effects in slice preparations than in the dissociated cells could be: (1) the drugs may have some difficulty to penetrate evenly throughout the tissue; (2) subsynaptic concentration of glycine may be regulated by active uptake mechanisms [8]; (3) excitatory action of glycine may be possibly masked by its inhibitory action; etc. In conclusion, we demonstrated that exogenous glycine could facilitate induction of LTP in the rat hippocampal slice. Recently it has been reported that regulation of NMDA receptor by glycine was reduced in Alzheimer's disease [14]. Further investigations will be focused on the role of glycine in the process of synaptic plasticity.

1 Alger, B.E., GABA and glycine: postsynaptic actions. In M.A. Rogawski and J.L. Barker (Eds.), Neurotransmitter Actions in the Vertebrate Nervous System, Plenum, New York, 1985, pp. 33~9. 2 Anwyl, R., Mulkeen, D. and Rowan, M.J., The role of N-methyl-D-aspartate receptor in the generation of short-term potentiation in the rat hippocampus, Brain Res., 503 (1989) 148-151. 3 Collingridge, G.L., Kehl, S.J. and McLennan, H., Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus, J. Physiol., 334 (1983) 33-46. 4 Engelsen, B., Neurotransmitter glutamate: its clinical importance, Acta Neurol. Scand., 74 (1986) 337355. 5 Fletcher, E.J. and Lodge, D., Glycine reverses antagonism of N-methyl-D-aspartate (NMDA) by 1hydroxy-3-aminopyrrolidone-2 (HA-966) but not by D-2-amino-5-phosphonovalerate (D-AP5) on rat cortical slices, Eur. J. Pharmacol., 151 (1988) 161-162. 6 Forsythe, I.D., Westbrook, G.L. and Mayer, M.L., Modulation of excitatory synaptic transmission by glycine and zinc in cultures of mouse hippocampal neurons, J. Neurosci., 8 (1988) 3733-3741. 7 Johnson, J.W. and Ascher, P., Glycine potentiates the NMDA response in cultured mouse brain neurons, Nature, 325 (1987) 529-533. 8 Johnston, G.A.R. and Iversen, L.L., Glycine uptake in rat central nervous system slices and homogenates: evidence for different uptake system in spinal cord and cerebral cortex, J. Neurochem., 18 (1971) 1951-1961. 9 Kemp, J.A., Foster, A.C., Leeson, P.D., Priestley, T., Tridgett, R., Iversen, L.L. and Woodruff, G.N., 7-Chlorokynurenic acid is a selective antagonist at the glycine modulatory site of the N-methyl-Daspartate receptor complex, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 6547~550. 10 Kleckner, N.W. and Dingledine, R., Requirement for glycine in activation of NMDA-receptors expressed in Xenopusoocytes, Science, 241 (1988) 835-837. 11 Larson, A.A. and Beitz, A.J., Glycine potentiates strychnine-induced convulsions: role of NMDA receptors, J. Neurosci., 8 (1988) 3822-3826. 12 Minota, S., Miyazaki, T., Wang, M.Y., Read, H.L. and Dunn, N.J., Glycine potentiates NMDA responses in rat hippocampal CA1 neurons, Neurosci. Lett., 100 (1989) 237-242.

~2 13 Skilling, S.R., Smullin, D.H., Beitz, A.J. and Larson, A,A., Extracellular amino acid concentrations in the dorsal spinal cord of freely moving rats following veratridine and nociceptive stimulation, ,1, Neurochem., 51 (1988) 127 132. 14 Steele, J,E., Palmer, A.M., Stratmann, G.C. and Bowen, D.M., The N-methyl-l)-aspartate receptor complex in Alzheimer's disease: reduced regulation by glycine but not zinc. Brain Res., 500 (1989) 369 373, 15 Tauck, D.L. and Ashbeck, G.A., Positive modulation of long-term potentiation by glycine, Soc. Neurosci. Abstr., 15 (1989) 400. 16 Thomson, A.M,, Glycine modulation of the NMDA receptor/channel complex, Trends Neurosci,, 12 (1989) 349 353. 17 Thomson, A.M., Walker, V.E. and Flynn, D.M., Glycine enhances NMDA-receptor mediated synaptic potentials in neocortical slices, Nature, 338 (1989) 422 424.