Glutamate acting on NMDA receptors stimulates neurite outgrowth from'cerebellar granule cells

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1 Volume 223, number 1, FEB October 1987 Glutamate actng on NMDA receptors stmulates neurte outgrowth from'cerebellar granule cells Ian A. Pearce, Martn A. Cambray-Deakn and Robert D. Burgoyne MRC Secretory Control Group, The Physologcal Laboratory, The Unversty of Lverpool, PO Box 147, Lverpool L69 3B.Y, England Receved 2 September 1987 The effect of endogenous glutamate on neurte outgrowth from cerebellar granule cells n culture was examned. Neurte outgrowth was nhbted by enzymatc removal of endogenous glutamate from the culture medum. The broad-spectrum glutamate receptor antagonst kynurenate also nhbted neurte outgrowth from granule cells n serum-contanng and serum-free cultures; the nhbton by kynurenate was reversed by exogenous glutamate. Neurte outgrowth was nhbted to the same extent by the NMDA receptor antagonst APV. These results ndcate that endogenous glutamate, possbly released by granule cells themselves, stmulated neurte outgrowth through actvaton of the NMDA class of glutamate receptors. Actvaton of NMDA receptors on developng neurons may be an mportant mechansm for the regulaton of neuronal growth and dfferentaton. Neurte; Glutamate; N-Methyl-D-aspartate; Neuronal dfferentaton; (Cerebellum) 1. INTRODUCTION A great deal of attenton has been pad to the solaton of factors that regulate survval and dfferentaton of CNS neurons [1] n the way that NGF acts on perpheral sympathetc and sensory neurons [2]. So far, lmted progress has been made n the precse dentfcaton of such molecules. One possblty s that conventonal neurotransmtters could regulate neuronal dfferentaton. Glutamate s a major exctatory neurotransmtter n the CNS whch acts on three receptor types classfed on the bass of ther agonsts as qusqualate, kanate or N-methyl-D-aspartate (NMDA) receptors [3,4]. Actvaton of NMDA but not the other two classes of glutamate receptors results n the openng of channels that allow Ca 2+ Correspondence address: R.D. Burgoyne, MRC Secretory Control Group, The Physologcal Laboratory, Unversty of Lverpool, PO Box 147, Lverpool L69 3BX, England nflux [5] and leads to a rse n cytosolc free Ca 2+ [6,7]. It has been suggested that the growth of neurtes from solated neurons s controlled by the level of cytosolc calcum n the growng tp of the neurte [8-10] and therefore actvaton of NMDA receptors s a potental mechansm for the control of neurte growth. Immature cerebellar granule cells n culture release glutamate n both a basal- and depolarsaton-dependent fashon [11,12]. In addton, these cells possess NMDA receptors [13] whch are lnked to channels wth a conductance of 50 ps [14,15]. We have examned the possblty that endogenous glutamate actng through NMDA receptors stmulates neurte outgrowth from cerebellar granule cells n culture. 2. MATERIALS AND METHODS Cerebellar granule cells were dssocated from the cerebella of 4 or 5-day-old rats by trypsn treatment [16,17], resuspended n culture medum and Publshed by Elsever Scence Publshers B.V. (Bomedcal Dvson) /87/$ Federaton of European Bochemcal Socetes 143

2 Volume 223, number 1 FEBS LETTERS October 1987 plated at a cell densty of 1 103/mm 2 onto poly- L-lysne-coated glass coverslps n the wells of a 24-well tray. Culture medum was ether serum + (Eagle's mnmal essental medum contanng fetal calf serum, chck embryo extract, 100/zg/ml gentamycn, 33 mm glucose, 290/zg/ml glutamne, 25mM KCI) or serum- (Eagle's mnmal essental medum contanng 30/zg/ml nsuln, 30 nm sodum selente, 33 mm glucose, 290/~g/ml glutamne, 25 U/ml penclln, 25/zg/ml streptomycn, 25 mm KCI). In the case of drug addtons the drugs were present throughout the perod of culture. After 8 h the cells were fxed wth 4070 formaldehyde n PBS, examned on an Olympu s nverted IMT mcroscope and the percentage of granule cells wth processes scored. In each well 350 cells were counted. 3. RESULTS Cultures of dssocated cells of the early postnatal rat cerebellum consst predomnantly [19,20] of granule cells ( of the cell populaton), whch can easly be dstngushed from the other cell types by ther small sze (dameter #m). Granule cells were grown n sparsely seeded cultures to mnmse cellular nteractons and to faoltate assessment of neurte outgrowth. When placed nto culture, some granule cells begn to extend neurtes wthn mnutes [21]. By 8 h after platng 23_ 1 70 (n = 35) of cells had extended neurtes (fg. 1) and were examned at ths tme n order to assess the rate of ntaton of neurte outgrowth rather than long term neurte growth and cell survval. By 1 day n culture most granule cells have extended neurtes. The neurtes are axonal n nature snce after 1 or more days n culture they contan synaptc vescles [22]. Addton of low concentratons (30/zM) of NMDA to the cultures had lttle effect on neurte outgrowth and hgher concentratons were neurotoxc (not shown). Snce granule cells n culture release glutamate the possblty that endogenous glutamate was suffcent to stmulate neurte outgrowth was tested. In serum contanng cultures n whch glutamate receptors were blocked by the broad-spectrum glutamate receptor antagonst kynurenate [23] neurte outgrowth was nhbted Fg. 1. Phase-contrast mcrographs of granule cells fxed after 8 h n culture n a serum* medum. The three panels show the range of morphologes of granule cell neurtes (arrows). The granule cells can easly be dstngushed from nonneuronal cells (arrowheads). Bar, 10/~m. 144

3 Volume 223, number l FEBS LETTERS October 1987 by (fg.2). Kynurenate also nhbted neurte outgrowth n serum- cultures by 54o7o (fg.2) showng that serum was not the sole source of glutamate n the cultures. These results suggest that endogenous glutamate released by granule cells n culture s suffcent to stmulate neurte outgrowth. In order to exclude the possblty that the nhbtory effect of kynurenate was a non-specfc effect of the drug rather than an effect on glutamate receptors we examned the effect of addton of exogenous glutamate on the kynurenate nhbton. As shown n fg.3 exogenous glutamate prevented the nhbton of neurte outgrowth by kynurenate consstent wth a drect effect of the antagonst on glutamate receptors. The role of NMDA receptors n the stmulaton of neurte outgrowth by endogenous glutamate was examned by growng the cells n the presence of the selectve NMDA receptor antagonst [3,4] D-2-amno-5-phosphonovalerate (APV). APV nhbted neurte outgrowth to the same extent as kynurenate (fg.3) and the nhbton by APV was y~ 5G- e-.= 25" a b c d e Fg.3. Effect of kynurenate and APV on neurte outgrowth from granule cells n serum + cultures. (a) 3 mm kynurenate; (b) 3 mm kynurenate + 10/~M glutamate; (c) 3 mm kynurenate + 100/~M glutamate; (d) 3 mm kynurenate + 250/zM glutamate; (e) 3 mm kynurenate + 1 mm glutamate; (f) 30 mm DL-APV; (g) 300/zM DL-APV; (h) 1 mm DL-APV. The data shown are expressed as percentage nhbton of neurte outgrowth and are taken from 4-10 wells per treatment. In control wells o (n = 16) of granule cells had neurtes. * p<0.001; ** p<0.02 compared to control. g h ; J W.=.=- 0 30" r _T_. F--]Control vrtually maxmal at 30/~M DL-APV. The specfcty of the nhbtory effect of APV was demonstrated n a separate seres of experments n whch 30/~M DL-APV nhbted neurte outgrowth by 43 +_ 1307o (n -- 4) but the nhbton was reduced to (n = 4) when 30/~M NMDA was also present. The requrement for endogenous glutamate for normal neurte outgrowth n culture was also demonstrated by enzymatc removal of glutamate from the culture medum. Serum + cultures were mantaned n the presence of 2 mm pyruvate and 1 unt/ml glutamate pyruvate transamnase whch should result n converson of endogenous glutamate to c~-ketoglutarate. Ths treatment resulted n a (n = 7) nhbton of neurte outgrowth. '~ "= Serum Fg.2. Effect of kynurenate on neurte outgrowth from granule cells n serum + and serum- cultures. Granule cells were grown n the ndcated medum n the presence or absence of 3 mm kynurenate. Data shown are the percentage of cells wth neurtes from counts of 350 cells n 4 wells per treatment. * p<0.001 compared to the respectve control. 4. DISCUSSION The results presented here suggest that endogenous glutamate released by granule cells n culture stmulates neurte outgrowth. Ths acton of glutamate appears to be medated by the NMDA class of glutamate receptors. Glutamate analogues have been shown to stmulate producton of cgmp [24,25] and nostol phosphates [20] 145

4 Volume 223, number 1 FEBS LETTERS October 1987 n granule cell cultures. The producton of both of these messengers was not restrcted to NMDA but was seen after actvaton of all three glutamate receptor types. However, a major dfference between NMDA and non-nmda receptor agonsts s the specfc ablty of NMDA to open channels whch allow Ca 2 nflux [5] and a rse n cytosolc free Ca 2 [6,7]. We have found that NMDA elevates cytosolc free calcum n cerebellar granule cells n culture (our unpublshed observatons). Cytosolc free calcum has been suggested to be nvolved n the control of neurte outgrowth [8-10] and so t s lkely that the mode of acton of glutamate n stmulatng neurte outgrowth nvolves the NMDA-receptor medated rse n cytosolc calcum. Consstent wth ths we found (unpublshed) that addton of 3 mm EGTA to granule cell cultures nhbted neurte outgrowth by 80o7o. Ths effect, however, could also nvolve nhbton of cellular adheson. The NMDA receptor s partally nhbted at physologcal concentratons (1 mm) of Mg 2+ and ths block s overcome by depolarsaton. In our standard culture condtons wth 0.8 mm Mg 2 n the medum the Mg2+-block would be at least partally overcome by depolarsaton by the 25 mm K present. It s also possble that mmature granule cells may respond to NMDA wthout addtonal depolarsaton snce t has recently been shown that whle the restng membrane potental of mature granule cells n culture s -60 mv that of mmature granule cels s - 30 to - 35 mv [27]. NMDA receptors are also regulated by glycne [28] whch ncreases ther senstvty to agonst. The culture medum used n ths study dd not contan added glycne but the possblty does exst that the acton of glutamate on NMDA receptors n the cultures could have been potentated by glycne released from granule cells. The fndngs of ths study suggest that, at least for the cerebellar granule cell, glutamate could have a role n the stmulaton of neurte outgrowth durng development. The condtons under whch glutamate stmulates neurte outgrowth n vvo reman to be determned as does the senstvty of other developng neurons to ths wdespread neurotransmtter. It s of nterest that hgher levels of responsveness to NMDA have been found for mmature cerebellar granule cells, Purknje cells [13] and vsual cortex neurons [29] compared to these cell types n the adult bran. Whether these developmental changes are related to an mportant role of the NMDA receptor at crtcal stages of bran development s as yet unknown. ACKNOWLEDGEMENTS Ths work was supported by the Wellcome Trust. We thank Dr J. Garthwate for hs helpful comments durng the course of ths project and the revewers for suggestng the use of glutamate pyruvate transamnase. REFERENCES [11 Thoenen, H. and Edgar, D. (1985) Scence 229, [2] Lev-Montalcn, R. (1982) Annu. Rev. Neurosc. 5, [3] Watkns, J. and Evans, R.H. (1981) Annu. Rev. Pharmacol. Toxcol. 21, [4] Foster, A.C. and Fagg, G.E. (1984) Bran Res. Rev. 7, [5] Jahr, C.E. and Stevens, C.F. (1987) Nature 325, [6] MacDermott, A.B., Mayer, M.L., Westbrook, G.L., Smth, S.J. and Barker, J.L. (1986) Nature 321, [7] Mller, R.J. (1987) Scence 235, [8] Anglster, L., Farber, I.C., Shahar, A. and Grnvald, A. (1982) Dev. Bol. 94, [9] Bolsover, S.R. and Spector, I. (1986) J. Neurosc. 6, [10] Connor, J.A. (1986) Proc. Natl. Acad. Sc. USA 83, [11] Pearce, B.R. and Dutton, G.R. (1981) FEBS Lett. 135, [12] Gallo, V., Cott, M.T., Colett, A., Alos, F. and Lev, G. (1982) Proc. Natl. Acad. Sc. USA 79, [13] Garthwate, J., Garthwate, G. and Hajos, F. (1986) Neuroscence 18, [14] Cull-Candy, S.G. and Ogden, D.C. (1985) Proc. R. Soc. Lond. B. 224, [15] Cull-Candy, S.G., Howe, J.R. and Ogden, D.C. (1987) J. Physol., n press. [16] Curre, D.N., Dutton, G.R. and Cohen, J. (1979) Experenta 35, [17] Dutton, G.R., Curre, D.N. and Tear, K. (1981) J. Neurosc. Methods 3, [18] Curre, D.N. (1980) n: Tssue Culture n Neurobology (Gacobn, E. et al. eds) pp , Raven, New York. 146

5 Volume 223, number 1 FEBS LETTERS October 1987 [19] Ncolett, F., Wroblewsk, J.T., Novell, A., Alho, H., Gudott, A. and Costa, E. (1986) J. Neurosc. 6, [20] Cambray-Deakn, M.A., Morgan, A. and Burgoyne, R.D. (1987) Dev. Bran Res., n press. [21] Cambray-Deakn, M.A., Norman, K.M. and Burgoyne, R.D. (1987) Dev. Bran Res. 34, 1-7. [22] Qunlan, J.E. and Davs, J. (1985) Neurosc. Lett. 66, [23] Garthwate, J. and Garthwate, G. (1987) J. Neurochem. 48, [24] Novell, A., Ncolett, F., Wroblewsk, J.T., Alho, H., Costa, E. and Gudott, A. (1987) J. Neurosc. 7, [25] Nowak, L., Bregostovsk, P., Ascher, P., Herbet, A. and Prochantz, A. (1984) Nature 307, [26] Mayer, M.L., Westbrook, G.L. and Guthre, P.B. (1984) Nature 309, [27] Hoekberger, P.E., Tseng, H.Y. and Connor, J.A. (1987) J. Neurosc. 7, [281 Johnson, J.W. and Ascher, P. (1987) Nature 325, [29] Tsumoto, T., Haghara, K., Sato, H. and Hata, Y. (1987) Nature 327,