Developmental changes in glutamate receptor-activated translocation of protein kinase C in cerebellar granule neurons

DEVELOPMENTAL BRAIN RESEARCH

ELSEVIER

Developmental Brain Research 94 (1996) 22-30

Research report

Developmental changes in glutamate receptor-activated translocation of protein kinase C in cerebellar granule neurons Manuel Barrios l, Sture Liljequist * Department of Clinical Neuroscience, Division of Drug Dependence Research, Karolinska Hospital, S-17176 Stockholm, Sweden

Accepted 23 January 1996

Abstract Developmental changes in glutamate receptor agonist-produced enhancement of 4-/3-[3H]phorbol- 12,13-dibutyrate binding ([ 3H]-PDBu binding), indicative of an intracellular translocation of protein kinase C (PKC), were investigated in cerebellar granule cells. Our observations demonstrate that the magnitude of glutamate-, NMDA-, and kainate-produced enhancement of PKC translocation was dramatically decreased between 2 and 12 DIV, whereas there was only a minor reduction in the corresponding response caused by the non-NMDA receptor agonist, AMPA, The maximally enhanced stimulation of PKC translocation caused by glutamate and NMDA was significantly reduced already at 4 DIV, whereas a significant reduction of the kainate-induced enhancement of [3H]PDBu binding was not observed until 8 DIV. Glutamate- and NMDA-induced responses were effectively blocked by the specific NMDA receptor antagonists MK-801 (1 /zM) and APV (100 /xM) as well as by the addition of Mg 2÷ into assay media. In contrast, the non-NMDA receptor antagonist, CNQX (10 /zM), effectively blocked the kainate-induced enhancement of [3H]PDBu binding, but had no effect on the NMDA- and glutamate-induced stimulation of PKC translocation, The metabotropic glutamate receptor agonist, ACPD (up to 250/zM), had no effect on the translocation of PKC. Taken together, our data support the working hypothesis that the rapidly occurring changes in the glutamate receptor agonist-produced translocation of PKC are most likely due to a differential maturation of glutamate ionotropic receptor subtypes and/or to development-dependent alterations in mechanisms responsible for the coupling between the glutamate receptor subtypes and the activation of PKC translocation in cerebellar granule neurons. Keywords: NMDA and non-NMDA receptors; Phorbol ester binding; PKC translocation; Developmental change; Cerebellar granule cells

1. Introduction The excitatory amine acid glutamate plays an important role in the differentiation of morphological, biochemical, and functional properties in brain neurons during their postnatal development [29]. For example, activation of glutamate receptors enh~mces the outgrowth of dendrites as well as initiates the migration of certain types of neurons [11,24,27,28,33]. Furthermore, glutamate exerts trophic actions by promoting cell survival in at least cerebellar granule, cortical, and spinal neurons [3,9,37]. The ability of glutamate to activate cation-specific ion channels via stimulation of various glutamate receptor subtypes, named NMDA, AMPA, and kainate according to their preferential

* Corresponding author. Fax: (46) (8) 729 5231; e-maih [email protected] l Present address: Department of Pharmacology(School of Medicine), University of Granada, E- 18012 Granada, Spain. 0165-3806/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S0165-3806(96)00039-9

agonist selectivity (see e.g. [21]), is considered to play a key role in these processes. Support for this idea derives from studies showing that glutamate- and NMDA-produced depolarization, accompanied by dramatic increases in Ca 2+ fluxes and intracellular Ca 2+ levels ([Ca2+]i), alters the morphology and the developmental characteristics of biochemical events in various types of brain neurons [3,18]. However, it is becoming increasingly more clear that the glutamate receptor complex not only regulates the differentiation of cellular responses in many neurons, but that the functional and pharmacological properties of the receptor complex themselves undergo dynamic postnatal changes during early cell maturation [20]. A selective decrease in the response to N M D A with increasing age was first reported by Garthwaite et al. [19] and has been confirmed in later studies. Furthermore, glutamate- and NMDA-induced functional Ca 2÷ responses and excitotoxicity are greatly enhanced during early postnatal development [16,17,35]. Ontogenic changes in the

M. Barrios, S. Liljequist/ Developmental Brain Research 94 (1996) 22-30

sensitivity of NMDA/glutamate receptors to the modulatory actions produced by Mg 2+, glycine, and polyamines have also been recently reported [8,22,23,32,44]. The underlying mechanisms for these phenomena are currently not fully understood but results from recent molecular biology studies indicate that the differentiation of pharmacological and functional properties of glutamate receptors may be governed by developmentally determined specific alterations in the assembly of glutamate receptor subunits [1,30,31,35,36,42]. Activation of glutamate receptors produces not only a marked enhancement of Ca 2+ fluxes and accumulation of [Ca2+]i, but also strongly stimulates the translocation of protein kinase C (PKC) at least in striatal, cortical, and cerebellar granule neurons, an event which has been suggested to be largely Ca 2+-dependent and thus secondary to the activation of the glutamate-produced stimulation of Ca 2+ responses in these neurons [2,14,26,38-40,43]. Since both Ca2+-dependent and Ca2+-independent PKC isoenzymes exist (see e.g. [2,38]), available observations seem to implicate that the effects of glutamate are mediated through an activation of Ca2+-sensitive isoforms of PKC. The activity of PKC is considered to play a critical role in the regulation of various developmental processes and neuronal plasticity in the brain [6,13,41]. For example, PKC appears to be an important factor for long-term potentiation (LTP), a phenomenon which is associated with the establishment of learning and memory responses [5,7]. Given this information, we considered it of interest to examine the ontogeny of glutamate agonist-stimulated 4-/3[3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding, indicative of a translocation of the enzyme PKC from the cytosol to the inner neuronal membrane [4], during the in vitro maturation of cerebellar granule neurons. These neurons are known to represent a powerful model system for examining the functional properties of glutamate receptors [10]. In order to differentiate between the PKC translocation caused by stimulation of various glutamate receptor subtypes at various stages of neuronal development, we have compared the effects induced by the specific ionotropic glutamate receptor agonists, glutamate, NMDA, AMPA, and kainate postnatally at 2, 4, 6, 8, 10, and 12 days in vitro (DIV).

23

solution, and dispersed by trituration in a DNase and soybean trypsin inhibitor-containing solution (0.01% and 0.05%, respectively). Cells were plated (106 cells/2 ml/dish) onto 35-ram Nunc dishes (Nunc AC, Denmark) coated with 5 /xg/ml of poly-L-lysine (M w = 7 0 0 0 0 150 000), and cultured for 8 days at 37°C in an atmosphere of 5% CO2/95% air in a basal Eagle's medium (Gibco; T~iby, Sweden) supplemented with 10% heat-inactivated fetal calf serum (Gibco), 25 mM KC1, 2 mM glutamine, and 100 /zg/ml gentamicin (Gibco). The addition of cytosine-/3-arabinofuranoside (10 /zM; Sigma) 24 h after plating limited the number of glia cells to less than 5% [39]. The medium was not changed until the cultures were used in the binding experiments. 2.2. [ 3H]Phorbol ester binding to intact cells

2. Material and methods

A detailed description of assay conditions and binding characteristics of [3H]PDBu can be found in previous studies [26,39] in which evidence was also presented indicating that [3H]PDBu binding can be used as a measure of PKC translocation in cerebellar granule cells. Briefly, monolayer cultures were grown for 2, 4, 6, 8, 10, or 12 DIV. To start the experiments the cultures were washed once with 2 ml of Mg2+-free Locke's solution (154 mM NaC1, 5.5 mM KC1, 3.6 mM CaC12, 3.6 mM NaHCO 3, 5.6 mM glucose, 5 mM HEPES; pH 7.4) and incubated in 1 ml of Locke's solution containing 10 nM [3H]PDBu (spec. act. 20 Ci/mmol; Du Pont Scandinavia AB, Stockholm, Sweden) in 0.5% fatty acid-free bovine serum albumin (Sigma). Monolayers were incubated at 22°C for 15 min, after which they were rapidly washed three times with ice-cold, Mg2+-free, Locke's solution and suspended overnight with 0.1 M NaOH. Aliquots of the suspension were then used for protein determination (100 /zl) and determination of radioactivity (500 /zl) by liquid scintillation counting (LKB Rackbeta 1217; Uppsala, Sweden). Nonspecific binding was determined in the presence of 10 /~M 12-O-tetradecanoylphorbol 13-acetate (TPA; Sigma) and represented about 20% of the total binding. TPA was added to the incubation medium as a 100-fold concentrated solution in 10% dimethyl sulfoxide (DMSO). Other drugs of interest were dissolved either in Locke's solution, slightly warmed if necessary, or in 10% DMSO and premixed to the incubation medium before the start of the cell incubation. These concentrations of DMSO had no effects by themselves on basal [3H]PDBu binding.

2.1. Cell cultures

2.3. Drugs

Primary cultures of cerebellar granule cells were obtained from 8-day-old Sprague-Dawley rat cerebellum as previously described by Levi et al. [25]. Briefly, after dissection, eight cerebelli were pooled, and sliced with a McIlwain tissue chopper in two orthogonal directions (slices were 0.4 mm thick), incubated in 0.25% trypsin

The glutamate receptor agonists glutamic acid, Nmethyl-I)-aspartic acid (NMDA), and kainic acid were all purchased from Sigma, whereas (RS)-a-amino-3-hydroxy5-methyl-4-isoxazolepropionic acid (AMPA), (1S,3R)-Iaminocyclopentane-l,3-dicarboxylic acid (ACPD), 2amino-5-phosphonopentanoic acid (APV), 6-cyano-7-

M. Barrios, S. Liljequist / Developmental Brain Research 94 (1996) 22-30

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Fig. i. The leftpanel shows the effects of increasing concentrations of glutamate (I-25 /zM) on [3H]PDBu (10 nM) binding to monolayers of intact cerebellar granule cells after 2, 4, 6, 8, I0, and 12 DIV. The fight panel shows the gradual decrease in the maximal stimulation of [3H]PDBu binding following the application of 2 5 / z M glutamate after various DIV. Shown are the means + S.E.M. from 4-6 determinations performed in two separate cell culture preparations. Statisticaldifferences were calculated with A N O V A followed by Bonfcrroni's test for multiple comparisons. * * P < 0.01 as c o m p a r e d to values at 2 DIV.

nitroquinoxaline-2,3-dione (CNQX) were purchased from Tocris Neuramin. (+)-MK-801 hydrogen maleate was a generous gift from Merck Sharp&Dohme (UK).

3. R e s u l t s

2.4. Statistics

Data displayed in Fig. 1 show the effects of increasing concentrations of glutamate (1-25 /xM) on the binding of [3H]PDBu to monolayers of intact cerebellar granule cells at 2, 4, 6, 8, 10, and 12 DIV. As shown, increasing concentrations of glutamate produced a concentration-dependent enhancement of [3H]PDBu binding, an effect which was fully blocked in the presence of 1 mM Mg 2÷ (data not shown, but see Fig. 5). The glutamate-induced enhancement of [3H]PDBu binding was most pronounced in cells after 2 DIV (325.2 _+ 18.1% stimulation over basal binding), after which time the effects of glutamate gradu-

3.1. NMDA- and glutamate-induced translocation of PKC

The computer program GraphPad Inplot (Graphpad Software Inc., USA) for PC was used for the estimation of ECs0 values and to construct the best-fit dose-response curves. The statistical calculations when several groups were compared was performed using one-way analysis of variance (ANOVA), followed by Bonferroni's test for multiple comparisons. When only two groups of data were compared, a non-paired Student's t-test was used. In all cases, P < 0.05 was considered statistically significant.

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ally decreased, reaching its lowest level, that is 90.2 + 11.4% stimulation of [3H]PDBu binding, after 12 DIV (90.2 _+ 11.4%; Fig. 1, fight panel). The potency of glutamate to stimulate [3H]PDBu binding was not altered during the development, with a maximal ECs0 value of 3.36 p~M (range 1.76-6.42 /~M; 95% confidence interval) at 12 DIV, and with a lowest ECs0 value of 2.24 ~M (range 1.44-3.48 /xM) at 4 DIV. Neither was there any change (ANOVA; P > 0.10) in the amount of basal binding between 2 and 12 DIV (with the lowest, that is 371.4 + 21.4 fmol/mg protein, and the highest, that is 443.1 _+33.5 fmol/mg protein observed at 4 and 6 DIV). Results depicted in Fig. 2 show the findings of similar experiments following the application of increasing concentrations of NMDA (5-250 ~M). As with glutamate, NMDA produced a concentration-dependent increase of [3H]PDBu binding with the maximal response obtained at 2 DIV (328.0 + 27.2% stimulation). In comparison to the 100

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glutamate-induced response, a significantly lower enhancement of [3H]PDBu was observed at 4 DIV, followed by a further gradual decrease in the amount of NMDA-enhanced [3H]PDBu binding reaching its minimum at 12 DIV (54.4_ 5.4% stimulation). The ECs0 value for the NMDA-induced stimulation of [3H]PDBu binding did not change significantly during the observation period. However, NMDA was considerably less potent than glutamate to increase the binding of [3H]PDBu to the intact cerebellar granule cells shown by the ECs0 values ranging between 36.8 /xM (range 22.0-61.5 /xM) at 12 DIV, and 26.5 /zM (range 17.4-40.2 /xM) at 8 DIV. 3.2. Kainate- and AMPA-induced translocation of PKC

The effects of the non-NMDA receptor agonists, kainate (5-100 /xM) and AMPA (1-100 /xM), were also investigated. As demonstrated in Fig. 3 (left panel), kainate z

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Fig. 5. The left panel shows the inhibitory effects of Mg 2+ (1 mM) on the maximalenhancementof [3H]PDBu(10 nM) bindingproducedby kainate(50 /zM; diamonds), glutamate (10/xM; circles), and NMDA (100/~M; squares) in the absence of Mg 2+ after various periods of DIV (2-12 days). The right panel shows the effects of increasing concentrations of Mg 2+ (0.1-1 mM) on kainate-, glutamate-, and NMDA-stimulated [ 3H]PDBu) binding after 2 and 8 DIV, respectively. Shown are the means + S.E.M. from 4 - 6 determinations performed in two separate cell culture preparations. Statistical differences were calculated with ANOVA followed by Bonferroni's test for multiple comparisons. * * P < 0.01 as compared to control values without the addition of Mg ~+.

produced a concentration-dependent enhancement of [3H]PDBu binding, although its maximal effect (217.8 + 16.3% at 2 DIV) was significantly lower than that observed with glutamate and NMDA. As with glutamate and NMDA, the kainate-produced enhancement of [3H]PDBu binding decreased with increasing number of DIV. However, in contrast to the observations with glutamate and NMDA, which showed a significant decrease in the agonist-enhanced [3H]PDBu binding already after 4 DIV, there was no signific~at reduction in the kainate-induced maximal stimulation of [3H]PDBu binding until 6 DIV, after which the amount of binding rapidly decreased reaching a steady-state level between 6 and 12 DIV (59.4 _ 7.7% stimulation; Fig. 3, right panel). There was a slight tendency for the potency of kainate to increase with the maturation of the cells, ECs0 values being 19.8 /zM (range 12.7-31.0 /xM) at 2 [)IV, and 6.1 /xM (range 4.4-12.4 /xM) at 10 DIV. The application of AMPA into assay media also produced a concentration-dependent increase of [3H]PDBu binding (Fig. 4, left panel), although the pattern of

AMPA-induced enhancement of [3H]PDBu binding clearly differed from those obtained with the other glutamate receptor agonists. First, and as demonstrated in Fig. 4, the AMPA-induced maximal enhancement of [3H]PDBu binding was considerably lower than that obtained with glutamate, NMDA, and kainate. Furthermore, although there was a slight reduction in the AMPA-produced stimulation of [ 3H]PDBu binding over time, these changes were smaller than for the other glutamate receptor agonists. However, a significant difference was obtained when data from 2 DIV were compared with results from the other treatment groups. Neither was there any change in the potency of AMPA over time, the ECs0 values ranging from 1.9 /xM (range 0.21-17.7/xM) at 6 DIV, to 7.7/xM (range 4.8-12.4 /zM) at 12 DIV.

3.3. 1S,3R-ACPD-induced translocation of PKC In the experiments where we examined the effects of the glutamate metabotropic receptor agonist 1S,3R-ACPD on [3H]PDBu binding in cerebellar granule cells, we found

Table 1 Effect of NMDA (MK-801, APV) and non-NMDA (CNQX) glutamate receptor antagonists on the percentage of [3H]PDBu binding induced by glutamate, NMDA, and kainate at two different stages of development (2 and 8 DIV) in cerebellar granule cells

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Control

MK-801 (1 /xM)

APV (100/xM)

CNQX (I0/J.M)

100 (4.8) 100 (7.4) 100 (5.8) 100 (2.5) 100 (6.5) 100 (7.5)

8.6 30.9 7.4 7.9 84.1

31.7 (2.5) 53.8 (10.5) 12.4 (2.3) * * 11.2 (3.3) * * 95.7 (4.2)

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(1.4) (3,2) (2.1) (2.1) (4.8)

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(18.6) (13.4) (18.8) * (18.8) *

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M. Barrios, S. Liljequist/ DevelopmentalBrain Research 94 (1996) 22-30

no evidence for the assumption that 1S,3R-ACPD (100250 /xM) would alter the binding of [3H]PDBu in cells maintained for 2, 4 or 8 DIV (data not shown). 3.4. Effects of Mg e+ on agonist-induced stimulation of [ 3H]PDBu binding

In order to test whether the observed development-dependent sensitivity to glutamate receptor agonist-enhanced [3]PDBu binding could be due to a shift in the responsiveness to Mg 2+, the effects of Mg 2-- was investigated at different time periods, after 2, 4, 6, 10, and 12 DIV. As indicated by results in Fig. 5 (left panel), the effects of Mg 2+ on the stimulation of [3H]PDBu binding produced by glutamate (10/xM), NMDA (100/xM), and kainate (50 ~M) remained unaltered between 2 and 12 DIV. Thus the glutamate- and NMDA-induced enhancement was consistently inhibited by 1 mM Mg 2÷ throughout the entire incubation period, whereas it had no influence on the kainate-produced increase of [3H]PDBu binding. As could be expected, the efficacy of Mg 2÷ to block the effects of NMDA was higher as compared to its actions on glutamate-potentiated [3H]PDBu binding. Again, various concentrations of Mg 2+ were without effects on AMPA- and kainate-induced potentiation of [3H]PDBu binding. 3.5. Blockade of glutamate receptor agonist-induced enhancement of PKC translocation

Results in Table 1 show the effects of specific NMDAand non-NMDA receptor antagonists on the enhancement of [3H]PDBu binding produced by glutamate, NMDA, and kainate. As can be seen, the effects of both glutamate and NMDA were effectively blocked by the specific NMDA receptor blocking agents MK-801 (1 /xM) and APV (100 /xM), whereas the specific non-NMDA receptor antagonist CNQX (10 /xM) was without effect in this situation. In contrast, CNQX produced a marked reduction of the kainate-induced enhancement of [3H]PDBu binding, whereas MK-801 and APV had no influence on the kainate-induced response.

4. Discussion

Results from previous studies indicate that the functional and pharmacological properties of glutamate receptor systems are altered in many types of brain neurons during their postnatal development. For example, it has been shown that NMDA- and glutamate-induced potentiation of Ca 2÷ fluxes are rather weak immediately after birth, but that the magnitude of these responses increases dramatically already during the first to second postnatal week [15,16,35]. Furthermore, during this period of cell maturation the increasingly enhanced stimulation of glutamate-induced Ca 2+ fluxes is accompanied by a concomi-

27

tant alleviation of glutamate-produced neurotoxic actions [17]. Given these observations, the most striking finding in our present study is the demonstration that the magnitude of the glutamate-produced stimulation of PKC translocation in cerebellar granule cells follows an opposite developmental pattern as compared to glutamate-stimulated Ca 2+ fluxes and neurotoxicity. Thus we found, and at least to our knowledge for the first time, that the glutamate-produced PKC activity is maximally enhanced during the early phase of cerebellar granule cell maturation, whereafter especially the NMDA- and glutamate-produced translocation of the enzyme is rapidly attenuated already after a few days in vitro. These data suggest that the markedly enhanced translocation of PKC may represent an important regulatory mechanism for trophic actions of glutamate which most likely are involved in the glutamate-induced promotion of cell maturation and differentiation seen during this early period of development, whereas a similar, although significantly lower, activation of PKC may be an important link in the chain of biochemical events associated with the marked increase of glutamate-produced cell death typically seen during later postnatal periods. Our current findings that [3H]PDBu binding is stimulated by various NMDA and non-NMDA glutamate receptor agonists, indicative of an enhanced translocation of PKC from cytosolic stores to the cellular membrane, are in excellent agreement with previous data obtained in 8 DIV cultures of cerebellar granule [14,26,39] and cortical neurons [40]. However, we now provide additional information showing that the developmental pattern for glutamate-produced enhancement of PKC translocation is not uniform for all glutamate receptor subtypes. For instance, activation of NMDA specific glutamate receptor subtypes produced a maximal enhancement of PKC translocation at 2 DIV with a significantly attenuated response seen already at 4 DIV. In contrast, the magnitude of the kainate-produced stimulation of PKC translocation remained essentially unaltered until 6 DIV, at which time a significant decrease was observed. It should also be noted that in comparison to the marked enhancement of PKC translocation seen after the application of glutamate, NMDA, and kainate, respectively, the amount of PKC stimulation produced by the non-NMDA receptor agonist, AMPA, was consistently lower during the whole observation period (2-12 DIV). These findings suggest that the relative importance of various glutamate receptor subtypes for the enhancement of PKC translocation differs during the development of cerebellar granule neurons. Currently we are not able to provide any direct experimental support for this working hypothesis but a rapidly increasing number of recent observations from several molecular biology laboratories have shown that both the production of various glutamate receptor subunit mRNAs and their expression undergo considerable absolute and relative changes in various types of brain neurons during approximately the

28

M. Barrios, S. Liljequist / Developmental Brain Research 94 (1996)22-30

same time period of cell maturation as examined in this study [1,31,34,36,42,44]. In cerebellar granule cells, the perhaps most consistent finding so far is the shift in the relative composition of NMDA/glutamate receptor subunits, seen as a marked increase in the occurrence of NR2A subunits concomitantly with a corresponding marked disappearance in the expression of the subunit NR2D [34,42]. Although the:re have been some preliminary attempts to correlate the relative composition of various specific NMDA receptor subunits a n d / o r their expression to the magnitude of NMDA/glutamate receptor activated Ca 2+ responses, further studies are clearly needed to clarify this issue. Furthermore, at least to our knowledge, there are currently no data available concerning the relative importance of specific NMDA receptor subunits a n d / o r their combinations for the glutamate-induced enhancement of PKC translocation in these neurons. There was a clear difference in the developmental profiles for the activation of PKC translocation produced by NMDA and non-NMDA receptor agonists, that is AMPA and kainate, respectively. Furthermore, of the non-NMDA receptor agonists, kainate produced a much stronger enhancement of PKC translocation as compared to AMPA. The reason for this discrepancy between AMPA and kainate is not clear, especially since it has been suggested that the effects of these agonists may be largely mediated through the same non-NMDA receptor subunits (for discussion, see [21]). Thus, results from molecular cloning studies show that AMPA receptor,; are comprised of the subunits GluR1-GluR4, through which many functional responses of kainate also are mediated. In contrast to AMPA, kainate also displays affinity for GluR5-GluR6 as well as KA1KA2 subunits although the functional significance of these subunits a n d / o r their combinations for kainate-induced responses still remains to be established. Furthermore, only a few studies are available where the developmental patterns of the glutamate receptor subtypes GluR1-GluR4 in cerebellar granule cells have been described [14]. Moreover, we have not been able to find any data concerning the developmental characteristics for GIuR5-GluR6 and KA1-KA2 subunits. Although several investigators have shown that the production and relative frequency of nonNMDA receptor subunits most likely are of importance for the functional effects of various glutamate receptor agonists [14], the relative importance and the developmental characteristics of various glutamate receptor subunits with regard to their involvement in the coupling between glutamate receptors and the activation of PKC translocation has, at least to our knowledge, not been investigated. Thus further studies are needed to analyze whether glutamate receptor subunits display differential patterns in the development of their functional characteristics and if such differences can explain the discrepancy in AMPA- and kainate-produced transilocation of PKC observed in our current series of expe:fiments. However, in this context another, perhaps more plausible, explanation should also

be considered. Thus, there is increasing evidence suggesting that a typical feature of kainate is its ability to produce non-desensitizing currents. In contrast, AMPA-induced responses are in many situations rapidly reduced due to a fast desensitization of AMPA receptors [45,46]. To which extent differences in the rate a n d / o r magnitude of nonNMDA receptor desensitization can explain the significantly lower enhancement of PKC translocation seen after the application of AMPA, as compared to kainate, remains to be further elucidated. In this context it is of interest to note that we recently have found that cyclothiazide, an agent which acts by blocking the desensitization of AMPA receptors, caused a stronger potentiation of AMPA-produced Ca 2÷ responses in cerebellar granule cells as compared to corresponding actions of kainate in 8 days old cerebellar granule neurons [12]. Since then we have observed that cyclothiazide potentiates also AMPA- and kainate-induced stimulation of PKC translocation in these cell cultures (Liljequist, in preparation). Consequently, it seems reasonable to assume that the developmentally determined changes in the ability of AMPA and kainate, respectively, to enhance PKC translocation may be due to a complex interplay between changes in the composition of various non-NMDA subunits a n d / o r fluctuations in AMPA- and kainate-induced desensitization of certain non-NMDA receptor subunits at various stages of cell maturation. Our results showing that the developmental pattern for glutamate-produced activation of PKC translocation, an initially strong enhancement followed by an attenuation of the response, is quite opposite to the ontogenic profile seen with regard to glutamate-induced Ca 2÷ fluxes and neurotoxicity, where an initially weak response is accompanied by a subsequent marked potentiation of the actions of glutamate. This negative correlation between the increased responsiveness to the neurotoxic actions of glutamate as reviewed by McDonald and Johnston [29] and the decreased enhancement of glutamate-induced PKC translocation found in this study shows a remarkable coincidence in time. Since the effects of glutamate on Ca 2+ fluxes follows a similar time pattern as glutamate-produced neurotoxicity [16,17,35], these observations raise some intriguing questions regarding the role of Ca 2÷ for the glutamate-induced translocation of PKC at various time points of cellular maturation. Initially it was thought that PKC activity and its translocation from intracellular stores to the inner side of the neuronal cell membrane was fully dependent on the presence of Ca 2+, evidenced by the fact that removal of extracellular Ca 2÷ fully blocked the translocation of PKC produced by glutamate at least in more mature cerebellar granule neurons [39]. Today we know that there is a family of various PKC isoforms, some of which are not Ca2÷-dependent [2,38]. Although we currently are unable to provide any experimental support for such hypothesis, our data may suggest that there might be a switch from the involvement of Ca 2+-non-dependent

M. Barrios, S. Liljequist / Developmental Brain Research 94 (1996) 22-30

to Ca2+-dependent PKC isoforms in glutamate-produced PKC translocation during early cerebellar granule cell maturation. Developmentally determined changes in the properties of NMDA receptors have also been reported with regard to their sensitivity to voltage-dependent blockade by Mg 2÷, altered kinetics of EPSPs, and to the modulatory properties of glycine, polyamines and ifenprodil [8,22,23,32,44]. In the present investigation we examined the modulatory effects of Mg 2+ on glutamate- and NMDA-induced potentiation at various periods of DIV and at various drug concentrations of Mg 2÷. As expected, Mg 2÷ (1 mM) fully blocked the translocation of PKC induced by NMDA with a weaker and no inhibition obtained following the concomitant application with glutamate and kainate, respectively. These observations taken together with the findings that NMDA-induced responses were fully blocked by the NMDA receptor antagonists MK-801 as well as APV, thereby providing convincing evidence for the assumption that the effects of NMDA were mediated through a specific activation of NMDA receptors. The probably most interesting findings described in this study are the observations that glutamate receptor agonists differ in their ability to enhance the translocation of PKC in cerebellar granule neurons. Furthermore, the magnitude of this response is largely dependent on the developmental stage of these brain neurons, the general consensus being that all ionotropic glutamate receptor agonists, perhaps with exception of AMPA, produce a marked enhancement of PKC translocation in immature neurons, whereas metabotropic glutamate receptors seem not to be involved in these phenomena. Additional studies are now in progress in this laboratory in order to elucidate to which extent the obtained differences can be explained by developmentally determined differences in the assembly of various NMDAand non-NMDA receptor subunits a n d / o r differences in their functional properties at various time points of cell maturation.

Acknowledgements This study was supported by the Swedish Medical Research Council (Project No. 7688), Sven and EbbaChristina Ha~bergs Foundation, Hans and Loo Ostermans Foundation, Ake Wibergs Foundation, and funds from the Karolinska Institute. M.B. was a Visiting Fellow from the Department of Pharmacology, University of Granada, Spain, and was supported by fellowships from the Ministerio de Asuntos Exteriores in Spain, the European Science Foundation, and the Human Frontier Science Program. References [1] Akazawa, C., Shigemoto, R., Bessho, Y., Nakanishi, S and Mizuno, N., Differential expression of five N-methyl-i>aspartate receptor

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