Developmental Brain Research, 46 (1989) 97-105 97 Elsevier

BRD50875

Effect of potassium depolarization on phosphate-activated glutaminase activity in primary cultures of cerebellar

granule neurons and astroglial cells during development

Julio Moran and Ambrish J. Patel Physiological and Neural Mechanisms Group, National Institute for Medical Research, Mill Hill, London (U. K.)

(Accepted 18 October 1988)

Key words: Depolarization; High K+i Phosphate-activated glutaminase; Cultured granule neuron; Cultured astrocyte; Development; Transmembrane Ca 2+ entry

The cerebellar granule cells are believed to be glutamatergic neurons. During the normal development of granule cells grown in a chemically defined medium, the specific activity of phosphate-activated glutaminase increased from 60 at 3 days to 150 (nmol/h/mg protein) at 15 days in vitro. Treatment with 25 mM K ÷ for the last 2 days elevated glutaminase activity in an age-dependent manner: about 100% at 3 and 6 days, 75% at 10 days, and 40% at 15 days in vitro. The enhancement of glutaminase in granule cells was dose- dependent. The half-maximal effect was obtained at about 20 mM K ÷, whereas the maximum concentration, which produced about a 2.5-fold increase in 3-day-old cultures was about 40 mM K ÷. The voltage-sensitive Na ÷ channel inhibitor tetrodotoxin had no effect on the depolarization-induced activity in granule cells. However, the increase in glutaminase by 25 mM K ÷ was significantly blocked by both organic (nifedipine) and inorganic (Ni 2÷ and Mg 2÷) calcium antagonists, indicating that elevation in activity may be mediated through transmembrane Ca 2+ entry into granule cells. In contrast to neurons, in cultured cerebellar astrocytes, the activity of glutami- nase slightly decreased during development, and treatment with 25 mM K + had no significant effect on this enzyme activity. The pres- ent findings, together with previous observations, would indicate that depolarization with K +, which is believed to mimic in vivo pre- synaptic stimulation, could be one of the mechanisms that selectively controls the development and function of neurons, when mea- sured in terms of the activity of the enzymes involved in the synthesis of cell-specific neurotransmitters.

INTRODUCTION

Granule cells in the cerebel lum are bel ieved to be

glutamatergic neurons (for refs. see refs. 14, 16). It

has been demons t ra ted that in prepara t ions enriched

in separa ted or in cul tured granule cells, phosphate-

act ivated glutaminase activity is several-fold greater

than in similar prepara t ions enriched in astroglial cells 13'2°'33'36. The relat ively high glutaminase activity

in neurons is consistent with early subcellular frac-

t ionat ion studies showing that a large propor t ion of

this enzyme is located in the synaptosomal frac- tion 8'24'3°, and that the depolar izing stimulus evoked

a preferent ia l release of the [t4C]glutamate newly

formed from [14C]glutamine9,tT. In addit ion, recent

immunocytochemical studies suggest that a very

large percentage of neurons that use an excitatory

amino acid t ransmit ter also shows pronounced gluta-

minase-l ike immunoreact ivi ty 1,46. However , even

though phosphate-ac t iva ted glutaminase is a good

marker for glutamatergic neurons (see ref. 37), the

enzyme is not entirely confined to nerve cells; a rela-

tively low but significant amount of enzyme activity has been detected in cultured astroglial cells 24,36,4°.

There is a growing body of information suggesting

that presynapt ic impulses exert a crucial control over

the deve lopment of postsynaptic neurons (see ref. 6),

and in cultured nerve cells K ÷ depolar izat ion can mimic such an effect 25,31,39. Also, it has been shown

that in conventional serum-containing medium, the

Correspondence: A.J. Patel, Division of Neurophysiology and Neuropharmacology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.

0165-3806/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)

98

survival of nerve cells depends on K+-induced depo- larization 5'22'25. However, it is now possible to main-

tain neuronal cultures for a reasonable length of time in a chemically defined medium that contains a physi- ological concentration of K ÷ (about 5 mM) 22. This

has made it possible to study the effect of depolariz- ing concentrations of K ÷ on the activity of glutami- nase in glutamatergic neurons and in astrocytes dur- ing development. In the present study, we have used two such cultures derived from the cerebellum of 8- day-old rats, namely granule cell and astrocyte cul- tures. The former, when grown in a chemically de- fined medium, comprised over 90% glutamatergic granule neurons, while the latter culture contained the same proportion of astroglial cells 22'34,35. The re-

sults show that treatment with depolarizing concen- trations of K + increases glutaminase activity only in granule neurons, but not in astroglial cells, and that

the increase in enzyme activity appeared to be me- diated through transmembrane Ca 2÷ entry into cere- bellar neurons.

MATERIALS AND METHODS

Materials Radiochemicals were obtained from Amersham

International, Amersham. Dowex AG-1 (X8, ace- tate form) was from Bio-Rad, Richmond. The heat- inactivated foetal calf serum and culture media were purchased from Flow Laboratories, Irvine, or from Imperial Laboratories, Salisbury. Other materials and chemicals used were of the highest quality and purchased from BDH Chemicals, Enfield, or Sigma (London), Poole, or other usual sources.

Cell cultures Primary cultures, greatly enriched in neurons

(more than 90% granule cells) were obtained by plat- ing dissociated cells isolated from the cerebellum of 8-day-old rats, at a density of 265 × 103 cells/cm 2 in plastic dishes (60 ram) coated with poly-L-lysine (5 #g/ml; tool. wt. >300,000) 22. The chemically defined culture medium contained 3 parts of Dulbecco's modified Eagle's to one part Ham's F12 media, and was supplemented with 100 ktg/mi transferrin, 5/~g/ml insulin, 100 #M putrescine, 30 nM selenium, 20 nM progesterone, 1 mM pyruvate, 50 U/ml penicillin and 50/~g/ml streptomycin. The culture dishes were incu-

bated at 37 °C in 7% CO 2 in air saturated with water vapour. Primary cultures, greatly enriched in astro- glial cells (more than 95%) were obtained by plating dissociated cells isolated from 8-day-old cerebellum, at a density of 212 × 103 cells/cm 2 in plastic dishes (60 ram) 35. The culture medium contained basal Eagle's

medium supplemented with 10% heat-inactivated foetal calf serum, 50 U/ml penicillin and 50 #g/ml streptomycin. The culture dishes were incubated at 37 °C in 5% CO 2 in air saturated with water and the medium was changed every 3-4 days. Both granule cell and astrocyte cultures were grown at a physiolog- ical concentration (4.75 raM) of K +. In experimental groups, the cultures were treated with appropriate concentrations of one or more compounds for the last 2 days before they were viewed by phase-contrast mi- croscopy or harvested for biochemical analysis.

Biochemical analysis At the indicated ages, the cells were washed twice

with prewarmed Krebs salt solution and once with sa- line at 37 °C. The cells were scraped into 10 mM imi- dazole buffer, pH 7.4, at 0 °C with a rubber police- man and homogenized using an all-glass homogeniz- er. Whole homogenate was used for the estimation of phosphate-activated glutaminase and lactate dehy- drogenase (LDH) activities and protein 37'38. The glu-

taminase was estimated by a slight modification of the sensitive radioisotopic method of Kvamme and Olsen 23 at a phosphate concentration of 20 mM 36'37.

The results are expressed as enzyme activity per mg protein.

Statistical analysis The data were analyzed either by Student's t-test

or by analysis of variance 42. The results are given as mean values + S.E.M.

RESULTS

Effect of high K + and of Ca 2+ antagonist on morpho- logical features

When dissociated cerebellar cells were seeded in a. chemically defined medium, they soon attached to the substratum and started emitting fibres (Fig. 1). In comparison with granule cells grown in serum-con- taining medium, cell migration, the formation of clumps and the fasciculation of fibres were limited in

Fig. 1. Phase-contrast micrographs of primary cultures of cerebellar granule cells grown in a chemically defined medium. Control cul- tures (about 5 mM K +) grown for (a) 3 DIV and (e) 6 DIV. The 3-DIV granule cells were treated for the last 2 days with (b) 25 mM K +, (c) 25 mM K + plus 0.1/zM nifedipine and (d) 25 mM K+plus 10 mM Mg 2+. The 6-DIV granule cells were treated for the last 2 days with (f) 25 mM K +, (g) 25 mM K + plus 0.5/zM nifedipine and (h) 25 mM K + plus 10 mM Mg 2+. Also the treatments with TTN (1/~M), nifedipine (0.5/zM), or Mg 2+ (10 mM) alone had no significant effect on the appearance in phase contrast of cultured granule cells (5 mM K+), these results are not shown in the Figure. Bar = 100~um.

100

these cultures 22. However, because of their limited maturation, the cultures serve as an excellent system for the study of factors affecting the differentiation of cerebellar granule cells. The extensive studies car- ried out by Balazs and his co-workers 3 have shown

that non-neuronal cells do not survive in cultures grown in a chemically defined medium, and that over 90% of the cells in these cultures are granule neu- rons 22. In comparison with the respective 3-day and

6-day-old control (5 mM K ÷) cultures (Fig. la and e), the morphological appearance of cultures grown in either 25 mM K ÷ (Fig. lb and f), or 25 mM K ÷ plus nifedipine (Fig. lc and g), or 25 mM K ÷ plus 10 mM Mg 2÷ (Fig. ld and h) was not significantly different.

In contrast to neuronal cultures, the astrocyte cul- tures contained mainly polygonal epitheloid cells (Fig. 2). We have previously reported that in these cultures more than 90% of the cells were immuno- cytochemically positive to glial fibrillary acidic pro- tein and thus were astroglial cells 34'35. Treatment

with 25 mM K ÷ had no significant effect on the ap- pearance of these cultured astrocytes at the light-mi-

croscope level.

Effect of depolarizing concentrations of K + on gluta- minase activity in granule neurons

In primary cultures of cerebellar granule cells, the specific activity of glutaminase increased by about 2.5-fold between 3 and 15 days in vitro (DIV; Table 1) 13 . The elevation in enzyme activity was gradual, about 50% between 3 and 6 DIV, 15% between 6 and 10 DIV and 45% between 10 and 15 DIV. On the other hand, during development a significant in- crease in LDH activity was observed only between 3 and 6 DIV, and thereafter the specific activity values for LDH changed little up to 15 DIV (Table I).

The addition of 25 mM K ÷ to the culture medium for the last 2 days markedly stimulated the specific activity of glutaminase at all experimental ages stud- ied (Table I). The effect on enzyme activity was age-

dependent; a high K+-mediated increase of about 100% was observed at 3 and 6 DIV, 75% at 10 DIV and 40% at 15 DIV. Also, the effect of K ÷ on gluta- minase activity in granule cells was dose-dependent (Fig. 3). The specific activity of glutaminase in- creased up to 40 mM K ÷, and treatment with higher concentrations of K ÷ did not induce enzyme activity

Fig. 2. Appearance in phase contrast of astroglial cell primary cultures derived from rat cerebellum. The 15-DIV astrocytes (a) control and (b) treated for the last 2 days with 25 mM K ÷. Bar = 100,um.

101

TABLE I

Effect of a high concentration of K + on glutaminase and LDH activities and protein content in cultured cerebellar granule cells during de-

velopment

Primary cultures of granule cells derived from the cerebellum of 8-day-old rats were grown in a chemically defined medium. In the ex- perimental groups, the cells were treated with an additional 20 mM KC1 for the last 48 h. At the indicated days in vitro cells were washed and homogenized in 10 mM imidazole buffer, pH 7.4. Whole homogenate was used for the estimation of enzyme activities and protein. The values are means -+ S.E.M. Significant differences between mean values of the two successive ages in the controls: +P < 0.05, ++P < 0.01. Significant differences between mean values of the control (5 mM K +) and experimental (25 mM K +) groups:

**P < 0.01.

Days in K + Number of Protein ( l ~ g / 6 0 Glutaminase LDH (l~mol/h/mg vitro (raM) observations mm dish) (nmol/h/mg protein) protein)

5 (control) 9 239 + 11.6 59 + 3.7 53 + 2.4 3 25 11 212 + 12.1 121 + 8.7** 79 + 3.3**

5 (control) 5 292 + 12.8 + 87 + 5.4 ++ 94 _+ 6.0 ++ 6 25 5 286 + 10.6 176 + 14.1"* 105 -+ 9.8

5 (control) 5 186 + 19.5 ++ 100 + 7.6 88 + 5.5 10 25 5 214 + 6.1 177 + 7.7** 107 + 7.2

5 (control) 4 139 _+ 12.9 143 + 5.8 ++ 82 +_- 7.0 15 25 4 166 + 4.6 200 + 5.5** 111 + 9.0

140

IZO

c

m'

i

!E g

2o

/

0 I I I I I I I !

0 10 20 30 40 50 60 70 ~1 (~)

Fig. 3. Dose- response curve of the effect of K + on the activity of glutaminase in cerebellar granule cells cultured in a chemi- cally defined medium. The granule neurons were treated with indicated concentrations of K + for the last 2 days. At 3 DIV the activity of phosphate-activated glutaminase was measured using whole homogenate, for further details, see Table I and Materials and Methods. Each point is the mean of 3 experi- ments, with S.E.M. indicated by the bars.

further. All enzyme activity values after treatment with 15 mM K + or more were significantly elevated compared to controls. The maximum concentration, which produced about a 2.5-fold increase in 3-DIV cultures, was about 40 mM K ÷, while the half-maxi- mal effect was obtained at about 20 mM K ÷ (Fig. 3).

Effect of high K + on glutaminase activity in astrocytes In cultured cerebellar astrocytes the specific activi-

ty of glutaminase decreased, while that of LDH ac- tivity and the amount of protein increased during de- velopment (Table II). Between 3 and 15 DIV the re- duction in glutaminase activity was about 35%, whereas the increase in LDH activity and the amount of protein was about 2- to 3-fold. In contrast to gran- ule neurons (Table I), treatment with 25 mM K + had no significant effect on the specific activity of gluta- minase in cultured astrocytes (Table II). Also, con- sistent with earlier findings 2°,36, at 15 DIV the rel-

ative values for the specific activity of glutaminase in

astrocytes were only 30% and 20%, in comparison with the values for granule cells cultured in medium containing 5 mM K ÷ or 25 mM K ÷, respectively. At all ages studied, the values for LDH activity and the amount of protein differed little between astrocyte groups treated with 5 mM and 25 mM K ÷ (Table II).

102

TABLE II

Effect of 25 mM K + on glutaminase and LDH activities and pro- tein content in cultured cerebellar astrocytes during development

Primary cultures of astrocytes derived from the cerebellum of 8-day-old rats were grown in a basal Eagle's medium contain- ing 10% foetal calf serum. In the experimental groups, the cells were treated with an additional 20 mM KCI for the last 48 h. For further experimental details and statistical analysis, see Table I. The values are means + S.E.M. of 3 experiments. Sig- nificant differences between the mean values of the two succes- sive ages in the controls: +P < 0.05, ++P < 0.01. At all ages the mean values in the experimental groups were not signifi- cantly different from the respective values in the control groups.

Days K + Protein Glutaminase LDH (/~mol/ in (mM) (~g/60 (nmol/h/mg h/mg vitro mm dish) protein) protein)

5 (control) 168 + 2.6 63 _+ 2.2 63 _+ 5.9 3 25 187 _+ 17.1 64 _+ 5.7 57 _+ 3.7

5 (control) 201 _+ 11.3 59 _+ 3.8 80 _+ 8.5 6 25 209 _+ 8.4 64 _+ 1.6 104 _+ 5.1

5 (control) 493 + 23.5 ++ 42 _+ 2.3 + 15 25 494 + 26.8 43 _+ 1.2

167 _+ 9.5 ++ 174 __+ 11.7

Effect o f Ca 2+ antagonists on depolarization-induced

glutaminase activity in granule neurons

It has b e e n shown that e leva t ion of K ÷ in the medi -

um or incubat ion fluid leads to an increase in Ca 2÷

up take in d i f ferent p repara t ions of bra in tissue 15'26'a4.

T h e r e f o r e , to ascer ta in whe the r the s t imula tory ef-

fect on g lu taminase act ivi ty was due to a depolar iza-

t i o n - d e p e n d e n t Ca 2÷ influx into granule cells, the

cu l tured cells were t r ea ted with 25 m M K ÷ alone, or

in combina t ion with c o m p o u n d s that inhibi t Ca 2+ in-

flux into cells (see ref. 21). The addi t ion of the organ-

ic antagonis t , n i fedip ine , to the m e d i u m significantly

inhib i ted the increase of g lu taminase by 25 m M K ÷ in

bo th 3- and 6 - D I V granule cell cul tures (Tab le III) .

Similar ly, a high concen t r a t ion of Ni 2÷ or Mg 2÷ is

also known to b lock the Ca 2÷ influx into the cells.

T r e a t m e n t with 10 m M Mg 2+ p r e v e n t e d the increase

of g lu taminase act ivi ty by high K ÷ in both 3- and 6-

D I V granule cells (Table III) . The high concent ra -

t ion of Ni 2÷ was toxic to the cells, but a less toxic con-

cen t ra t ion of 0.1 m M Ni 2+ also significantly inhibi ted

the depo la r i za t ion - induced increase in g lu taminase

act ivi ty in 3 - D I V granule cells. In contras t to the ef-

fect of Ca 2+ antagonists , t e t rodo tox in , an inhibi tor of '

the vol tage-sens i t ive Na + channel , had no effect on

the depo la r i za t ion- induced specific activity of gluta-

minase in granule neurons (Table III) . N o n e of these

c o m p o u n d s caused a significant effect on L D H activi-

ty.

DISCUSSION

It has long been recognized that b ioe lec t r ic activity

inf luences the d e v e l o p m e n t of ne rve cells. For exam-

ple, Black and his co-workers 6'v have shown that pre-

gangl ionic input is essent ial for the normal deve lop-

m e n t of a u t o n o m i c ne rve cells. As one of the conse-

quences of synapt ic neuro t ransmiss ion is m e m b r a n e

depola r iza t ion , the t rans-synapt ic regula t ion of neu-

ronal p roper t ies is be l i eved to be med ia t ed through a

depola r iza t ion of postsynapt ic neurons . In tissue cul-

TABLE III

Effect of Ca d+ antagonists and tetrodotoxin (TTN) on high K +- mediated elevation of glutaminase activity in cultured cerebellar granule cells

Primary cultures of granule neurons grown in a chemically de- fined medium were treated for the last 48 h with the indicated concentration of one or more compounds. Whole homogenate was used for the estimation of enzyme activities. For further ex- perimental details, see Table I and Materials and Methods. The values are means + S.E.M. of 3 experiments. Since the treatments with TTN, nifedipine, or Mg 2+ alone had no signifi- cant effect on the activities of glutaminase and LDH in the con- trol (5 mM K +) cultures, these results are not shown in the Table. Significant differences between 25 mM K + and other groups: *P < 0.05, **P < 0.01.

Experimental Glutaminase LDH (nmol/h/mg (ktmol/h/ protein) mg protein)

3 Days in vitro 25 mM K + 117 + 5.0 70 + 5.2 25 mM K+ plus 1 pM TTN 119_+4.6 82_+2.9 25 mM K + plus 0.1 ~M

Nifedipine 81 _+ 5.4** 62 + 4.2 25 mM K+ ptus 0.1raM Ni z+ 88+7.6* 68_+4.3 25 mM K + plus 10 mM Mg 2+ 63 _+ 3.5"* 49 _+ 3.2* Control (5 mM K +) 58 + 3.9** 52 _+ 3.1" 6 Days in vitro 25 mM K + 153 + 4.0 74 _+ 9.2 25 mM K + plus 0.5 ktM

Nifedipine 108 _+ 5.7** 77 + 7.9 25 mM K + plus 10 mM M f + 96 + 4.3** 64 + 9.1 Control (5 mM K +) 84 + 1.3"* 62 _+ 2.1

103

ture, a chronic depolarization of nerve cells can be achieved by increasing the extraceUular concentra- tion of K ÷ ions. Such a depolarization is found to en- hance the survival of a number of nerve cell types in culture, including cerebellar neurons 15,25, sympa- thetic neurons 39, ciliary ganglion cells 5m and dorsal root ganglion neurons nm. High K ÷ can also influ- ence the development of nerve cells; for example, it guides sympathetic neurons towards completing ad- renergic differentiation 45, and it increases the activ- ities of choline acetyltransferase (CHAT), tyrosine hydroxylase and glutamate decarboxylase in mixed cultures containing either peripheral or central neu- rons 18'19'31. In the present study we examined the ef-

fects of K ÷ ions on the biochemical maturation of cul- tured cerebellar granule cells. When these cells are grown in a chemically defined medium, the culture predominantly contains a single class of neurons with very few glial cells, and the cultured cells do not re- quire depolarizing concentrations of K + for their sur- vival 22. Thus under these conditions, the influence of factors derived from non-neuronal cells and of selec- tive cell survival on the final outcome is greatly di- minished.

It has been proposed that in glutamatergic neu- rons, such as cerebellar granule cells, an interaction between these cells and astrocytes is required for normal glutamate-mediated neurotransmission (for references, see refs. 2, 33, 40). The two enzymes in- volved in this metabolic cooperation are glutaminase and glutamine synthetase, which are selectively en- riched in neurons and astrocytes, respectively 2°,32,36.

However, as discussed above, glutaminase is not ab- solutely confined to neurons 24,36. In the present

study, we now show that exposure of glutamatergic neurons to depolarizing concentrations of K + markedly increases the activity of glutaminase (Table I). A similar elevation was not observed in the activity of lactate dehydrogenase in granule neuron cultures after 3 DIV, nor in glutaminase or in lactate dehydrogenase in cerebellar astroglial cultures of any age (Tables I and II). Moreover, the enhance- ment in glutaminase activity may be due to an in- crease in enzyme protein rather than to the activation of an existing enzyme, because the effect was ob- served only several hours after high K ÷ treatment and it was dose- and age-dependent (Table I and Fig. 3). Also, in partially depolarized granule cells the el-

fect of high K ÷ could be mimicked by the addition of N-methyl-D-aspartate to the serum-containing cul- ture medium, and this increase in glutaminase activi- ty was completely blocked by protein synthesis inhib- itors, cycloheximide and actinomycin D 29. The pres- ent findings, together with earlier observations on ChAT activity in cultures from the chick ciliary gan- glion, mouse spinal cord and rat septal-diagonal band regions, on tyrosine hydroxylase in cultures from the rat superior cervical ganglia, and on glutamate decar- boxylase activity in cultures from the rat septal re- gion, would demonstrate that depolarizing agents act only on nerve ceils and enhance the maturation of these neurons when measured in terms of the expres- sion of transmitter enzymes (A.J. Patel and A. Hunt, unpublished observations on cultures derived from the rat septal-diagonal band regions) 1s,19,31.

High K ÷ concentrations in the culture medium may induce biochemical events that are normally elicited either by bioelectric activity or by the depo- larization of neurons, or both. However, the inhibi- tion of bioelectric activity by tetrodotoxin had no ef- fect on the K+-mediated increase in the activity of glutaminase in the cultured granule cells (Table III). These findings are consistent with previous observa- tions on the survival of cultured granule cells grown in serum-containing medium 15 and on the develop- ment of synaptic networks in explant cultures derived from the cerebral cortex or spinal cord 27. The present results would indicate that in contrast to the earlier observations on ChAT 19,31, the effect of high K + on glutaminase activity is not mediated through the volt- age-sensitive Na ÷ influx.

Nerve cell depolarization by high K + is known to activate voltage-sensitive Ca 2÷ channels 26,47, and

such channels are detectable in cerebellar granule cells 1°A5'21. In the present study we have therefore

examined the possibility that the increase in glutami- nase activity by high K ÷ is mediated through a depo- larization-induced increase of Ca 2÷ influx. Both the organic and the inorganic Ca 2+ antagonists signifi- cantly diminished a high K+-mediated increase in the activity of glutaminase in cultured granule cells (Table III). This effect was not related to the survival of the granule cells, because neither the organic (ni- fedipine) nor the inorganic (Mg2+), Ca 2+ antagonists were cytotoxic (Fig. 1). Moreover, our more recent experiments now suggest that the effect of the depo-

104

larization-induced t ransmembrane Ca2+-flux is me-

diated through the activation of N-methyl-D-aspar-

tate receptors 2s'29. Earlier studies have indicated that

cerebeilar granule cells possess the N-methyl-D-as-

partate subtype of excitatory amino acid receptors ~2,

and the addition of N-methyl-D-aspartate to the se-

rum-containing culture medium promotes survival of

granule cells as do high K ÷ ions 4. Many of the cere-

bellar mossy fibres use acidic amino acids as their

neurotransmitters 43. It is therefore very probable

that, in our present experiments on the K ÷ depolar-

ization-mediated biochemical differentiation of cul-

tured granule cells, we may have mimicked the physi-

ological stimulation of granule cells normally pro-

vided in vivo by the presynaptic mossy fibres.

ACKNOWLEDGEMENT

Dr. J. Moran is from the Instituto de Fisiologia Ce-

lular, Universidad Nacional Autonoma de Mexico,

Mexico, and he gratefully acknowledges the award of

the British Council Scholarship.

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