Brain Reseatrh, 587 (1992) 102-108 ~t~ It)t)2 Elsevier Science Publishers B.V. Ail rights reserved t1006-8993/92/$05.00

102

BRES 17944

Epidermal growth factor selectively enhances NMDA receptor-mediated increase of intracellular C a 2 + concentration in rat hippocampal neurons K a z u h o A b e and Hiroshi Saito Deparmwnt of ('heroical Plumnacology. Faculty of Pharmaceutical Sciences, Unit'ersityof Tokyo, Tok~.o (Japan) (Accepted lO March It)92)

Key words: Epidermal growth factor; Intracellular Ca 2 * concentration; Hippocampal neuron; N-Methyl-o-aspartate receptor; Non-N-methyI-D-aspartate receptor; Long-term potentiation

We have previously reported that recombinant human epidermal growth factor (hEGF) facilitates induction of hippocampal long-term ix)tenti.'ttion ILTP). In order to clarify the mechanism underlying the LTP-facilitating effect of hEGF, the influence of hEGF on intracellular ( , a : ' concentration ([C;r' ' ]t) of hippocampal neurons was invesligated using dissociated cell cultures. Changes in [Ca 2 + ]i were measured by microfluorometrically monitoring the flttorescence intensities from individual neurons loaded with fura-2, Application of hEGF 10.6-211 nB/ml) alone did not affect the basal level of [Ca '~ ], in cultured hippocampal neurons, but significantly enhanced the [Ca -'+ ]i increase induced by i..glutamate (3 × 10 ~t, MI. The N-methyl-,)-:tspart:tte (NMDA) ( I 0 - '; and 3 x 10 - '~ M)-induced [Ca 2 ÷ ]i increase was also enhanced by hEGF, hut the quisqualate (11} ? and 3x 10 7 M).induced response was not affected by the presence of hEGF. These results suggest that hEGF selectively enhances Ihe NMDA receptor-mediated responses in hippocampal neurons. This action of hEGF may underlie the facilitation of hippocampal LTP.

INTRODUCTION Epidermal growth factor (EGF) is a single-chain polypeptide composed of 53 amino acids, which was originally isolated from male mouse submaxillary glands, and is well known as a potent mitogen for a variety of cell types ",'.32, Existence of EGF and its receptors in the brain has been confirmed It,~2.1~,2°,3~ but little is understood about the function of EGF on brain neurons. We have recently found that recombinant human epidermal growth factor (hEGF) enhances short-term potentiation of evoked potentials and facilitates induction of long-term potentiation (LTP) of evoked potentials in the CAI region of rat hippocampal slices4 and in the dentate gyrus of anesthetized rats ts, The LTP in the hippocampus is a form of synaptic plasticity and is considered to be the cellular basis of learning and memory 3.~. Therefore, this finding suggests that EGF may function in the brain as a physiological factor regulating synaptic plasticity, However, the mechanism underlying the effect of EGF is not known at all,

Based on extensive investigation on mechanisms underlying LTP, it has been shown that activation of the N-methyI-D-aspartate (NMDA) type of glutamate receptor following high-frequency stimulation of presynaptic affcrents and increase of intracellular Ca 2+ coflcentration ([Ca2+],) in postsynaptic cells are triggering events of LTP at least in Schaffer/commissural-CAI pyramidal cell synapses and in perforant path-dentate granule cell synapses t°,t4,'~,z!-23 Therefore, in the present study, we investigated the influences of EGF on basal [Ca 2*]~ and glutamate receptor agonists-induced responses in hippoeampal neurons to clarify the mechanism underlying the LTP-facilitating effect of EGF. For this purpose, we used primary cultures of dissociated hippocampal neurons because they are advanta. genus to measurement of [Ca2+]t at single cell level. MATERIALS AND METHODS

Cell ca/rares Procedure for cell culture was, for the most part, the same as described in our previous paper t, The hippocampi were isolated from embryonic day 20 Wistar rats and dissociated by incubation

Corrt'slmmh, nee: K, Abe, Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo ! 13, Japan.

103 with 0.25% trypsin and 0.01% DNase !, followed by pipetting, The dissociated cells were suspended in modified Eagle's medium supplemented with 10% fetal bovine serum and were plated on poly-Liysine-coated glass coverslips with silicon-rubber wall at a cell density of 2× lOS/cm 2. They were cultured at 3TC in a humidified 5% CO~-05% air atmosphere, After 24 h, the medium was changed to serum-free D F / T I P medium I and the neurons were grown for 6-8 days further,

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20ng/ml hEGF

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Measurement of /Ca" ]i Changes of [Ca -~+ ]i were detected by using a microfluorometrical technique with a Ca~-*-sensitive fluorescent dye, fura-213. The solution for loading the neuronal cells with fura-2 was prepared by adding 5 /zM fura-2 acetoxymethyl ester (fura-2/AM) and 0.02% Pluronic FI27 dissolved in dimethylsulfoxide to HEPES-buffered artificial cerehrospinal fluid (ACSF). Pluronic F127 is a non-cytotoxic detergent that helps to solubilize fura-2/AM in physiological media"~l. The composition of ACSF was as follows (raM): NaCI 130,0, KCI 5,0, CaCl.~ 2,4, MgSO,~ 1.3, KH.,PO,I 1,24, glucose 10.0, HEPES-NaOH 20 (pH 7.4), After the culture medium was removed, the cells were exposed to the loading solution and incubated at 37°C for 90 rain. The cells were rinsed with fresh ACSF after the loading. The coverslips with silicon-rubber wall containing the fura-2loaded cells were then perfused continuously with fresh ACSF (32°0 at a rate of 3 ml/min. The volume of the well was always maintained at 0.2 ml. The cells were allowed to equilibrate for 20 rain prior to fluorescence measurement in order to thoroughly wash out the remaining extracellular dye and to ensure stable responsiveness of the cells. For measurement of fura-2 fluorescence we used an inverted fluorescence microscope (Nikon, DIAPHOT-TMD.EF2) and image analysis system (Mitsubishi Kasei FC-200). Light from a Xenon-lamp was filtered by either of two different band-pass filters (340 nm or 360 nm) in the excitation path and conducted to the specimen on the microscope stage through a dichroic mirror and an epifluorescence objective (40 × UV). The excitation wavelength was usually set 340 nm, but was switched to 360 nm only at the beginning and the end of each observation. The fluorescence emitted from the cells was passed through a hand-pass filter (510 nm). The video images were obtained using a silicon.intensified target camera. A digital video analyzer resolved the images into 512x512 plxcls and assigned each point a value from 0 to 255 depending on the fluorescence intensity. The diBitized imatles were color.coded and visualized on a color display, Multiple video I~ames were sequentially added to memory to increase the signal-to-noise ratio in the averaged image. 5 ×5 pixels located in the cell body area of single neurons were selected, and the averaged values in the block of pixels were calculated every 0,5 s and forwarded to a personal computer. The fluorescence intensity at 34() nm excitation (F340) was divided by that at 360 nm excitation (F3f~0), We employed the fluorescence ratio, F340/F360, as an index of intracellular Ca-"+ level.

Dr.gs hEGF (a generous gift from Wakunaga Pharmaceutical Co., Ltd., Osaka, Japan) was first diluted to a concentration of l 0 / z g / m l in distilled water supplemented with I mg/ml bovine serum albumin to prevent non-specific binding of the protein, Small aliquots were stored at -20°C until use, The stored drug was further diluted to the desired concentration by adding ACSF just before use. Dt.-2-Amino5-phosphonovalerate (APV), N.methyI-D-aspartate (NMDA) and quisqualate were purchased from Sigma Chemical Co. (St. Louis, Me, USA). MK-801 was obtained from Research Biochemicals Inc. (Natick, MA, USA), L-Glutamate and other chemicals were purchased from Wake Pure Chemical Industries, Ltd. (Osaka, Japan), RESULTS First we investigated the i n f l u e n c e o f h E G F on basal level of [Ca2+]i. T h e typical result is showl~ in

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Fig. 1, Effects of hEGF (20 ng/ml) alone on basal level of [Ca 2+ ], in hippocampal neurons. For comparison, the effect of depolarizing stimulation by high concentration of K ÷ (50 raM) on [Ca "-+ ]i was subsequently observed. The data shown in this figure were simultaneously recorded from 4 neurons in the same culture. Open and closed bars under the tracing indicate the time during which each agent was perfused.

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Fig, 2. Concentration.dependent effects of t.-glutamale, NMDA and quisqualate on the [Ca'* ]j level in hippocampal neurons. As shown in A, the effects of each agonist at different concentrations were observed stepwise in the same neurons and the concentration-response curves were obtained. The results observed in many cells are summarized in B. The abscissa indicates the molar concentration of agonists and the ordinate indicates the peak increase of F340/F360. Symbols and vertical bars represent the mean ± S,E.M. of n observations, e, L-glutamate, n = 2 5 ; A, NMDA, n = 3 0 ; II, quisqualate. n = 37.

104 Fig. 1. When hEGF (0.6-20 ng/ml) was applied by perfusion for 15 rain, the basal level of [CaZ+], did not change in any neuron observed (n = 25). Depolarizing ~timulation with 50 mM K* caused a large increase of [Ca-"+]i in all neurons. Next we attempted to investigate the influences of hEGF on the responses induced by glutamate receptor agonists. Fig. 2 shows the effects of an excitatory neurotransmitter, L-glutamate, and selective agonists for glutamate receptor, NMDA and quisqualate, on the [CaZ+]i of cultured hippocampal neurons under the present experimental condition. Application of Lglutamate increased the [Ca:÷]i of cultured hippocampal neurons. The effect of L-glutamate was concentra-

tion-dependent in the range of 10-" to 3 × I0 --~ M. Application of NMDA (3 × 1 0 - ~ ' - 1 0 - 4 M ) and quisqualate ( 3 × 10-~-3 × 10 -" M) also induced a rapid increase of [Ca z+ ]i in a concentration-dependent manner. The maximum increase of [Ca2+]i induced by NMDA was a little smaller than that by L-glutamate and quisqualate. We also observed the effect of NMDA in MgZ+-free ACSF, because it is known that Mg 2+ blocks Ca z+ influx through the cation channel linked to the NMDA receptor in a voltage-dependent manner z4"z~. Application of NMDA clearly increased [CaZ+]i in the absence of Mg z+, but both potency and efficacy of NMDA were almost the same as those in MgZ+-containing, normal ACSF (data not shown). A

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i , ,o. Fig. 3. Effects of APV, a NMDA receptor antagonist, on the L-glutamate-, NMDA. and quisqualate-induced [Ca ~" ]i increase in hippocampal neurons. A: a typical result showing th~ effect of APV ~)n NMDA-induced response, NMDA and APV were applied by perfusion during the time indicated by closed and open bars, respectively, The [Ca z * ], increase induced by application of NMDA alone was first observed in normal ACSF and 0hen the effect of the same concentration of NMDA in the p~sence of APV was ob~rved in the same neuron. After washing out APV, the response to NMDA in nolmal ACSF was again observed to verify the stability of the responsiveness, in the same way, the effects of 10- s M APV on the responses induced by different concentrations of agonlsts were observed in many different cells and summarized in B. The peak increase of [Ca"" ], level induced by each agonist in the presence of APV (o) were compared with thai in the absence of APV (e). Abscissas and ordinates are as in Fig. 2. The data are represented as the mean ± S,E.M. of 13-2"/observations. Asterisks indicate significant differences from the data without APV (e): * P < 0.f)5: * * P < O.Ol: *** P < {).0(}1; Student's t-test,

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Fig, 4. Typical results showing the effects of hEGF (20 n g / m l ) on [Ca z'~ ]i increase induced by NMDA (a,b) or quisqualate (c,d) in hippocampal neurons, The effects of NMDA or quisqualate alone were first observed in normal ACSF as controls and then the effects of the same stimulation in the presence of hEGF were' observed in the same neurons, After washing ol,t hEGF, the responses to each agonist were again observed in

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Fig, 5. Collected data of effects of hEGF (20 ng/ml) on [Ca: + ]l increase induced by i.-glutamate (3 X 10-r' M), NMDA (1.0- s or 3 × I0- s M) or quisqualate (lO -T or 3 x 10 -7 M) in hippocampal neurons, The procedure of the experiment was as in Fig. 4. The responses to each agonist in the absence (filled columns) or presence (stippled columns) of 20 ng/ml hEGF were evaluated in terms of peak increase of F340/F36D (upper panels) and area of F340/F360 increase (lower panels). The data are represented as the mean + $.E.M, of 16-27 observations, Asterisks indicate significant differences from the respective control data (filled columns): * P < 0.05; * * P < 0.01; * * * P < 0.001; Student°s t-test.

106 resting membrane potential of cultured hippocampal neurons in the present study may be partly depolarized, so that the Ca 2+ influx is not blocked by the presence of Mg -'+. Burgoyne et al. 7 have also observed that cultured cercbellar granule cells from neonatal rats responded to NMDA stimulation even in the presence of Mg 2÷. In the following experiments, the effects of all drugs were observed in normal ACSF. Fig. 3 shows the effect of APV, an NMDA receptor antagonist, on the responses induced by L-glutamate, NMDA and quisqualate. The L-glutamate-induced [Ca2+]~ increase was partly blocked by 10 -~ M APV. The NMDA-induced [Ca"*]i increase was greatly blocked by APV, while the quisqualatc-induced responses were not affected by the presence of APV at all. Furthermore, the [Ca-'*]i increase induced by 3 × 10 -~ M NMDA was completely blocked by 10 -s M MK-801, a non-competitive NMDA receptor art~agonist (n = 8, data not shown). These results ensure that the NMDAand quisqualate-induced responses were mediated by NMDA and non-NMDA receptors respectively. The responses to glutamate receptor agonists in the presence of hEGF were compared with those in normal ACSF using the same cell cultures. The typical results are shown in Fig. 4. In the case of Fig. 4a, 10 -'~ M NMDA had no effect on [Ca"*] i in normal ACSF, but applic~,tion of I() ~°'~ M NMDA in the presence of 20 ng/ml hEGF produced an apparent increase of [Ca"*]i. In the case of Fig. 4h, application of 3 × 1(}°'~ M NMDA alone produced a large increase of [Ca:* ]i in normal ACSF, and th~ peak of the NMDA-induccd [Ca"*]l increase was not ft.rther enhanced but the decay time cours~ became much slower in th~ presence of 20 ng/ml hEOF. After washing out hEGF, the NMDA-induced response was restored to the original, indicating that the effect of hEGF was reversible. On the other hand, the quisqualate (10 -~ and 3 × 10-~ M).induced responses were not affected by the presence of hEGF in any ease. The influences of hEGF on the responses induced by glutamate receptor agonists were observed in many neurons and the results are summarized in terms of the peak increase of F340/F360 and the area, which corresponds to the integral of F340/F350 increase (Fig. 5), The employmcnt of the area as an index makes it possible to evaluate such changes as observed in Fig, 4b, The collected data shown in Fig, 5 clearly suggest that hEGF enhances selectively NMDA receptor-mediated responses. The effect of hEGF on the NMDA (3 x 10 --~ M)-induced response was observed at different concentrations (0,6 and 6 ng/ml), hEGF at the concentration of 0.6 ng/ml did not significantly influence the NMDA-

induced response (n = 13, data not shown), while 6 ng/ml hEGF significantly enhanced the NMDA-induced response (n = 16, data not shown). The threshold effective concentration of hEGF seemed to be between 0.6 and 6 ng/ml. DISCUSSION it has been reported that stimulation by EGF alone increases [Ca'-+]i in A431 cells, NIH 3T3 cells and cultured porcine thyroid cells 15"2s'2~''~4. Furthermore, it has been found that stimulation by EGF causes a long-lasting hyperpolarization of cell membrane in NIH 3T3 cells 3°. These events may underlie the stimulation of cell proliferation by EGF. However, in our previous study, hEGF did not influence normal synaptic transmission in the Schaffer/commissuraI-CAl pyramidal cell synapses of the rat hippocampal s l i c e s 4 and in the perforant path-dentate granule cell synapses of anesthetized rats ~s. Furthermore, in the present study, hE(iF alone had no effect on [Ca2+]i of rat hippocampal neurons. It is likely that hEGF alone has little effect on the cell membrane excitability of hippocampal neurons, although it remains possible that the hEGF slightly changes the membrane potential to a degree so that the voltage-gated Ca 2+ influx is not influenced, it is probable that the action of EGF on neuronal cells is considerably different from that on non-neuronal cells in terms of the underlying mechanisms. The novel finding in the present study was that hEGF selectively enhanced NMDA receptor-mediated increase of [Ca"*]t in hippocampal neurons. Normal glutamatcrgic neurotransmission in the hippocampus is mainly mediated by non-NMDA receptors Iq~.The present result that hEOF did not affect the quisqualate-indueed response is very consistent with our previous observation that hEGF did not affect excitatory synaptic response normally evoked by low frequency stimulation of presynaptic fibers in the hippocampus 4a". On the other hand, the NMDA receptors are involved in the process of short,term potentiation and induction of LTP "~'¢''Hu~'l~'. In our electrophysiological studies previously reported, hEGF enhanced short-term potentiation and facilitated the induction of LTP in the Schaffcr/commissuraI-CAI pyramidal cell synapses of rat hippocampal slices~ and in the perforant path-dentate granule cell synapses of anesthetized rats I~. The threshold effective concentration of hEGF in enhancing NMDA receptor-mediated response in the present study was between 0,6 and 6 ng/ml, which was well consistent with the concentration of hEGF effective in facilitating the induction of LTP 4, Therefore, the selec-

107 tive enhancement of NMDA receptor-mediated response by hEGF can consistently account for the LTPfacilitating effect of hEGF, The mechanisms by which hEGF selectively enhances the NMDA receptor-mediated responses are not yet known from the present data alone, but some possibilities can be proposed. Although the effect of hEGF on the membrane potential of hippocampal neurons is not yet known, possible changes in curtcnt-voltage relationship by hEGF may affect the Ca 2+ influx through the NMDA receptor/channel complex. In addition, it is known that the NMDA receptor/channel complex contains several modulatory sites, i.e., glycine site vh3x3~', polyamine site "~3''~'~, etc. It is possible that hEGF enhances the affinity of N M D A receptors for agonist or the efficiency of coupling to its channel through these known allosteric sites or some unknown modulatory site. We have very recently found that hEGF promotes the survival of cultured brain neurons through activation of protein kinase(s)"~. Furthermore, Urushihara et al. 37 have reported that treatment with a protein kinase C activator, 12-o-tetradecanoylphorbol- 13-acetate (TPA), selectively potentiated the NMDA-induced currents in Xenopusoocytes injected with rat brain mRNA. Therefore, it is also possible that hEGF indirectly modulates the NMDA receptor/channel complex through protein kinase response(s). While it is well known that EGF receptor has intrinsic tyrosine kinas¢ activity -'~, the evidence that tyrosine kinases are involved in hippocampal LTP has been reported very recently :7. We are proceeding with further ¢lectrophysiological and pharmacological experiments to elucidate these points. In the present study, the cells were pretreated with hEGF for a longer time than in the case with NMDA receptor antagonists, since the mode of'the action of hEGF is not known at all. Investigating the time course of the action of hEGF may also give useful information about the underlying mechanisms. Morrison et al. 2"s have found that EGF promotes the survival of primary cultured neurons from the cerebral cortex and striatum of neonatal rats. Furthermore, we have reported that EGF has survival-promoting effects on cultured neurons from various brain regions including the hippocampus 2. These findings suggest the possibility that EGF functions as a neurotrophic factor in the brain. On the other hand, the present results show that short-term application of hEGF influences the function of brain neurons, like a neuromodulator. The possibility that EGF has such dual functions, i.e., ncurotrophic action and neuromodulator-like action, is also interesting in relation to development and plasticity of neural connections in the brain.

In conclusion, we have found that hEGF selectively enhances the N M D A receptor-mediated increase of [Ca2+]i in hippocampal neurons, It is probable that hEGF facilitates induction of hippocampal LTP by directly or indirectly regulating the NMDA receptor/ channel complex on postsynaptic membrane. In addition, further investigation of the mechanisms underlying modulation of N M D A receptor-mediated response by hEGF will provide a new understanding of the regulatory mechanism of the N M D A receptor/channel complex. Acknowledgenwnts. The authors are grateful to Wakunaga Pharmaceutical Co. for the generous gift of hEGF.

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