Stimulation of the N-methyl-D-aspartate receptor promotes the biochemical differentiation of cerebellar granule neurons and not astrocytes

Cerebellar granule cells are believed to be glutamatergic, but, as they receive excitatory amino acidergic input from mossy fibers, they also possess N-methyl-D-aspartate (NMDA) receptors. The possible involvement of these NMDA receptors in the biochemical differentiation of cultured granule neurons was studied in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When the partially depolarized cells were treated with NMDA for the last 3 days (i.e. between 2 and 5 days in vitro), it elevated specific activity of glutaminase in the dose- and time-dependent manners. The half-maximal effect was obtained at about 10 microM NMDA, whereas the maximum concentration, which produced about a 2.7-fold increase in 5-day-old cultures, was about 50 microM NMDA. This increase in glutaminase was completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and by the NMDA receptor-linked Ca2+ ion channel blockers, MK 801 and Mg2+. The effect of NMDA was not related to the survival of the granule cells, as the experiments were carried out before the dependence on high K+ for the survival of granule cells develops in culture, and during the period of investigation none of the compounds used compromised the survival of these cells. The enhancement of glutaminase activity was due to an induction in enzyme protein, since it was completely blocked by cycloheximide and actinomycin D. In contrast to granule neurons, the treatment with NMDA had no significant effect on the activity of glutaminase and glutamine synthetase in cultured cerebellar astroglial cells. Our present results on glutaminase enzyme would indicate that an increase in the cellular concentration of free Ca2+ mediated through the NMDA induced increase in Ca2+ conductance, leads to long term changes in differentiating cerebellar granule neurons, and it is possible that this kind of physiological stimulation of granule cells is normally provided in vivo by the presynaptic glutamatergic mossy fibers.     

Treatment with excitatory amino acids or high K+ and NMDA receptors in cerebellar granule cells.

N-methyl-D-aspartate (NMDA) receptor activity was examined, in terms of 45Ca2+ influx, during the development of cerebellar granule cells grown under 'non-trophic' [10 mM potassium (K10)] or 'trophic' conditions [25 mM potassium (K25), NMDA and kainate (KA)]. NMDA receptor activity increased sharply between 2 and 4 days in vitro (DIV) irrespective of growth conditions which upon further cultivation exerted a powerful influence, the NMDA response increasing progressively in K25 and NMDA grown cells, while remaining at a constant level in KA treated cells. In contrast, in K10 grown cells the NMDA response declined by 7 DIV to about 20% of the estimates in K25 at 9 DIV. Trophic conditions are, therefore, essential for the proper functional expression of NMDA receptors.     

Agonist-induced Down-regulation of NMDA Receptors in Cerebellar Granule Cells in Culture

In contrast to the acute toxic effect of NMDA on mature cerebellar granule cells, chronic treatment with NMDA (140 μM from 1 to 9 days in vitro ) did not compromise cell survival. Such treatment markedly suppressed NMDA receptor activity: at 8 days in vitro NMDA-induced ⁴⁵Ca²⁺ influx was reduced by -60% and acute exposure to NMDA (highest concentration tested, 1 mM) at 9 days in vitro did not cause detectable toxicity. The reduction in NMDA receptor activity was accompanied by a significant decrease (±80% at 9 days in vitro ) in the level of the NR1 and the NR2A NMDA receptor subunit protein, detected using the selective photoaffinity ligand [¹²⁵I]CGP55802A. It seems, therefore, that the agonist-induced decrease in NMDA receptor activity is due to receptor down-regulation. In contrast to the marked influence of chronic NMDA exposure on the cellular content of the NMDA receptor subunit proteins, mRNA levels of the different subunits (NR1, NR2A, NR2B and NR2C) were not significantly affected. It seems, therefore, that agonist-induced down-regulation of the NMDA receptor involves critically mRNA translation and/or post-translational regulation.     

Role of brain-derived neurotrophic factor in the protective action of N-methyl-D-aspartate in the apoptotic death of cerebellar granule neurons induced by low potassium

Several neurotrophic factors, including brain-derived neurotrophic factor (BDNF), and neurotransmitters, such as glutamate, may influence neuronal apoptotic death. Rat cerebellar granule neurons (CGN) cultured in low potassium (5 or 10 mM KCl) for more than 5 days in vitro (DIV) die apoptotically. These cells survive in the presence of high potassium (25 mM KCl, K25) or N-methyl-D-aspartate (NMDA), an agonist of glutamatergic receptors. CGN transferred from high to low potassium die apoptotically. Here, we characterized the effect of BDNF and NMDA on the apoptotic death induced by low potassium in CGN. Cell death of CGN by culturing in low potassium for 6 DIV was inhibited by BDNF and NMDA. When CGN were cultured in K25 and transferred to a low-potassium medium, 65% of neurons died after 48 hr. Under these conditions, BDNF, NMDA, or BDNF + NMDA increased CGN survival. Both BDNF and NMDA decreased caspase-9 activity and mRNA caspase-3 levels and activity induced by low potassium. CGN survival induced by BDNF is mediated by TrkB activation, whereas that induced by NMDA is mediated by NMDA receptor and TrkB activation. NMDA, but not BDNF, raised [Ca(2+)](i), which was reduced by low-potassium treatment. These results suggest that NMDA receptor stimulation induces CGN survival through the influx of extracellular Ca(2+) that may evoke the release of BDNF and the activation of TrkB. Complementary mechanisms induced by depolarization and changes in Ca(2+) levels would also contribute to the neuroprotection exerted by NMDA and potassium.     

Stimulation of the N-methyl-d-aspartate receptor promotes the biochemical differentiation of cerebellar granule neurons and not astrocytes

Cerebellar granule cells are believed to be glutamatergic, but, as they receive excitatory amino acidergic input from mossy fibers, they also possess N-methyl-d-aspartate (NMDA) receptors. The possible involvement of these NMDA receptors in the biochemical differentiation of cultured granule neurons was studied in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When the partially depolarized cells were treated with NMDA for the last 3 days (i.e. between 2 and 5 days in vitro), it elevated specific activity of glutaminase in the dose- and time-dependent manners. The half-maximal effect was obtained at about 10 μM NMDA, whereas the maximum concentration, which produced about a 2.7-fold increase in 5-day-old cultures, was about 50 μM NMDA. This increase in glutaminase was completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and by the NMDA receptor-linked Ca²⁺ ion channel blockers, MK 801 and Mg²⁺. The effect of NMDA was not related to the survival of the granule cells, as the experiments were carried out before the dependence on high K⁺ for the survival of granule cells develops in culture, and during the period of investigation none of the compounds used compromised the survival of these cells. The enhancement of glutaminase activity was due to an induction in enzyme protein, since it was completely blocked by cycloheximide and actinomycin D. In contrast to granule neurons, the treatment with NMDA had no significant effect on the activity of glutaminase and glutamine synthetase in cultured cerebellar astroglial cells. Our present results on glutaminase enzyme would indicate that an increase in the cellular concentration of free Ca²⁺ mediated through the NMDA induced increase in Ca²⁺ conductance, leads to long term changes in differentiating cerebellar granule neurons, and it is possible that this kind of physiological stimulation of granule cells is normally provided in vivo by the presynaptic glutamatergic mossy fibers.     

Rapid, sensitive, and simple method for quantification of both neurotoxic and neurotrophic effects of NMDA on cultured cerebellar granule cells

A simple and sensitive method adapted from the staining of living cells with fluorescein diacetate was developed to rapidly estimate the number of living cells remaining in a culture dish 24 hr after a few min of NMDA treatment of cerebellar neurons. This method consists of the measurement, after cell lysis, of the total amount of fluorescein produced from fluorescein diacetate by the living granule cells present in each culture dish. We show that this method can also be used to quantify the survival effect of chronic exposure of granule cells to either K+ or NMDA. In both cases, the fluorescence measured was found to be proportional to the number of fluorescein-labelled cells counted under a fluorescence microscope, indicating that the present method can be used to quantify both toxic and trophic effects of NMDA on cerebellar granule cells. This study confirms that these two NMDA effects occur at the same NMDA concentration, and both are inhibited by MK 801 in the same concentration range. We showed, moreover, that granule neurons developed in the presence of NMDA are much less sensitive to NMDA toxicity than neurons developed in K(+)-enriched medium.     

Effect of depolarization on the maturation of cerebellar granule cells in culture

The effect of depolarization on the maturation of granule cells derived from cerebella of 8-day-old rats can be studied in cultures in chemically defined media because their survival is not dependent on elevated K+ as it is when they are grown in serum-containing media. As an index of maturation, stimulus-coupled transmitter release was examined. This was chosen because it is closely associated with the neuronal phenotype and, in contrast to granule cells grown under depolarizing conditions in serum-containing media, it has not been known whether this property is expressed during the development of serum-free cells in culture. Veratrine-induced release of preloaded D-[3H]aspartate (Asp), an analogue of glutamate (Glu; the transmitter of the granule cells), was not detectable in the serum-free cells at a time (8-12 days in vitro) when this property was fully developed in cells grown in a medium containing serum and 25 mM K+ (reference cultures). This finding may be related to the failure of the expression of voltage-sensitive calcium channels in the serum-free granule cells. However, in comparison with reference cultures, voltage-sensitive 45Ca2+ entry was only transiently retarded in the serum-free cells. Furthermore, in contrast to the exogenous D-[3H]Asp, stimulated release of endogenous Glu was detectable, although it was substantially lower than in the reference cultures. Autoradiographic studies indicated that the failure to elicit evoked release of the exogenous amino acid was due to a severe retardation of the expression of the acidic amino acid carrier in the serum-free granule cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of potassium depolarization on phosphate-activated glutaminase activity in primary cultures of cerebellar granule neurons and astroglial cells during development

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 (Ni2+ and Mg2+) calcium antagonists, indicating that elevation in activity may be mediated through transmembrane Ca2+ entry into granule cells. In contrast to neurons, in cultured cerebellar astrocytes, the activity of glutaminase slightly decreased during development, and treatment with 25 mM K+ had no significant effect on this enzyme activity. The present findings, together with previous observations, would indicate that depolarization with K+, which is believed to mimic in vivo presynaptic stimulation, could be one of the mechanisms that selectively controls the development and function of neurons, when measured in terms of the activity of the enzymes involved in the synthesis of cell-specific neurotransmitters.     

Plasticity of NMDA Receptor Expression During Mouse Cerebellar Granule Cell Development

A period of hypersensitivity to N -methyl- d -aspartate (NMDA) has been described during the early development of different types of neuron. Since activation of NMDA receptors can also induce rapid neuron death, the hypersensitivity to NMDA may be tightly controlled. In the present study we show that mouse cerebellar granule neurons become transiently hypersensitive to NMDA between days 10 and 14 after plating in a culture medium containing 30 mM K⁺. The NMDA sensitivity is higher when cells are cultured in the presence of an NMDA receptor antagonist [30 mM K⁺ plus 100 μM 3-((±)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP)], and no hypersensitivity is observed when cells are cultured in the continuous presence of NMDA (12.5 mM K⁺ plus 100 μM NMDA). The high NMDA sensitivity in control cells is associated with a higher density of NMDA receptors than that measured in NMDA-treated cells, suggesting that the sensitivity to NMDA may be partly controlled by activity-dependent NMDA receptor down-regulation. We also examined the level of NMDA-ζ1 mRNA and found no correlation between this parameter and the transient pattern of NMDA sensitivity. Such NMDA receptor plasticity may be of importance in the central nervous system, protecting developing cells from excitotoxicity at critical developmental stages.     

Phosphoproteins of Cultured Cerebellar Granule Cells and Response to the Differentiation-promoting Stimuli NMDA, High K+ and Ionomycin

In order to investigate signalling pathways involved in the control of granule cell differentiation, survival and other functions by depolarization or activation of NMDA receptors we have characterized protein phosphorylation in cerebellar granule cells. Cultures of cerebellar granule cells were incubated with ³²P orthophosphate and then challenged with NMDA, K⁺ or the Ca²⁺ ionophore ionomycin, agents which raise [Ca²⁺]_i and stimulate differentiation and survival. Upon separation of labelled phosphoproteins by two-dimensional gel electrophoresis three differences were found in response to all of these agents. These were an increase in acidity of two phosphoproteins of 87 and 48 kDa (p87 and p48) and increased ³²P-incorporation into a phosphoprotein of 120 kDa (p120). Treatment with PMA which stimulates neurite outgrowth but not survival affected p87 (increased its acidity) but not p48. The acidic shift of p87, therefore, is not sufficient to stimulate granule cell survival. The identification of p87 as the actin-binding MARCKS protein and the demonstration of its presence in neurites and growth cones of granule cells suggests that it may be involved in NMDA-stimulated neurite outgrowth. The phosphoproteins p120 and p48 may potentially be involved in events linking the rise in [Ca²⁺]_i to increased granule cell survival or other aspects of granule cell differentiation.     

The role of depolarization in the survival and differentiation of cerebellar granule cells in culture

Cultures greatly enriched in granule cells from early postnatal cerebellum (P8) were grown in a medium containing fetal calf serum. Under the conditions used, nerve cells died, usually within a week, unless the K+ concentration in the medium was greater than or equal to 20 mM. The requirement for elevated [K+]e was manifested by about 3 d in vitro, and after this time continuous exposure to high [K+]e was essential for the survival of the granule cells. The initial morphological and biochemical maturation of the granule cells was similar in the presence and the absence of elevated [K+]e, suggesting that the dependence on depolarizing conditions develops in parallel with the expression of the differentiated characteristics of the cells. The positive effect of elevated [K+]e on granule cell survival was not influenced by preventing bioelectric activity in the cultures with TTX and xylocaine. On the other hand, depolarization-induced transmembrane Ca2+ flux was essential in securing the maintenance of the granule cells. Depolarized nerve cells were compromised when Ca2+ entry was blocked by elevated Mg2+, EGTA, or organic Ca2+ antagonists, while dihydropyridine Ca2+ agonists [BAY K 8644, (+)-(S)-202 79 1 and CGP 28392] were potent agents preventing nerve cell loss in the presence of 15 mM [K+]e, which was ineffective on its own. Calmodulin inhibitors (1 microM trifluoperazine or calmidazolium) blocked the beneficial effect of K+-induced depolarization on granule cells. The comparison of the timing of the differentiation and innervation of the postmitotic granule cells in vivo with the development of the K+ dependence in vitro would indicate that depolarization of the granule neurons in culture mimics the influence of the physiological stimulation in vivo through excitatory amino acid receptors, including N-methyl-D-aspartate receptors, involving Ca2+ entry and the activation of a Ca2+/calmodulin-dependent protein kinase.     

NMDA receptors contribute to the survival promoting effect of high potassium in cultured cerebellar granule cells

The present report further analyzes the survival promoting effect of high potassium, a condition that mimics neural activity in cultured cerebellar granule cells, an excellent model to study trophic mechanisms induced by depolarization and trophic factors. We found that the survival promoting effect measured at 7 days in vitro (DIV 7) of depolarizing potassium concentrations (25 mM KCl), added at DIV2, is partially prevented by adding at DIV 2 the non-competitive NMDA blocker MK801 (10 microM). The concentration of MK801 used blocks completely the survival promoting effect of a supramaximal effective concentration of NMDA (100 microM). The addition at DIV 2 of anti-brain derived neurotrophic factor (anti-BDNF) antibody, failed to modify the effect of high potassium. The present report provides evidences that in cultured cerebellar granule cells, high potassium-induced survival promoting effect is due in part by the activation of NMDA receptors. The effect does not require the presence of BDNF.     

Characterization of GABAergic neurons in cerebellar primary cultures and selective neurotoxic effects of a serum fraction

The morphological and functional differentiation of GABAergic interneurons present in cerebellar primary cultures has been examined by means of [3H]gamma-aminobutyric acid (GABA) autoradiography and [3H]GABA depolarization-evoked release. At 2 days in vitro these neurons showed scarce accumulation of radioactivity and no Ca2+-dependent K+-evoked or veratridine-induced release of [3H]GABA. At 5 days in vitro GABAergic interneurons appeared more intensely labeled and had grown out long and often branched neuritic processes; a large Ca2+-dependent release of [3H] GABA could be evoked by high K+. At later stages the progressive increase in labeling and branching of the neuritic processes was paralleled by a further increase in the amount and Ca2+ dependence of [3H]GABA release; a tetrodotoxin-sensitive, veratridine-stimulated release was also demonstrated. The [3H]GABA-accumulating stellate astrocytes present in the culture were not responsible for the observed release of the amino acid. GABAergic neurons were also identified by indirect immunofluorescence, using antibodies to the specific marker glutamic acid decarboxylase. Total renewal of the culture medium at 7 days in vitro caused a drastic (90%) reduction in the number of GABAergic neurons and a concomitant decrease in the amount of [3H]GABA uptake and release in the cultures. The disappearance of GABAergic neurons was caused by a low molecular weight (Mr less than 1000) fraction of the serum used to supplement the basal culture medium. This serum component did not significantly influence the survival of the major neuronal population of the culture (the granule cells) and appeared to be selectively toxic for GABAergic neurons only after they had reached a quite advanced degree of morphological and functional differentiation in vitro. The toxic activity was no longer present in neuronal or glial conditioned media.     

Brain-derived neurotrophic factor mediates the anti-apoptotic effect of NMDA in cerebellar granule neurons: signal transduction cascades and site of ethanol action.

Cerebellar granule neurons cultured in medium containing a physiological concentration of KCl (5 mM) undergo apoptosis. The cells can be rescued by the in vitro addition of NMDA. The protective effect of NMDA is thought to reflect the in vivo innervation of developing cerebellar granule neurons by glutamatergic afferents. In the current work, we investigated the mechanism of the anti-apoptotic (protective) effect of NMDA. NMDA treatment reduced caspase-3-like activity in cerebellar granule neurons, and the time course and concentration dependence of the protective effect of NMDA mirrored the ability of NMDA to induce brain-derived neurotrophic factor (BDNF) expression. Furthermore, a Trk receptor antagonist, K252a, as well as a blocking antibody to BDNF, attenuated the protective effects of both NMDA and BDNF. These results suggest that NMDA-induced BDNF expression mediates the anti-apoptotic effect of NMDA. The protective effects of NMDA and BDNF were reduced by inhibitors of the phosphatidylinositol 3'-OH kinase (PI 3-kinase) signal transduction cascade (wortmannin and LY29004) but not by a MAP kinase kinase (MEK) inhibitor (PD98059) or a protein kinase A inhibitor (Rp-cAMPS). BDNF increased phosphorylation of Akt, a target of PI 3-kinase, and NMDA also induced Akt phosphorylation, but only after an exposure that was long enough to induce BDNF expression. Furthermore, ethanol, which interferes with NMDA receptor function, inhibited the NMDA-induced increase in BDNF levels but did not block the protective effect of BDNF. These findings further support the role of BDNF in the anti-apoptotic effect of NMDA in cerebellar granule neurons and suggest that the NMDA-BDNF interaction may play a key role in in vivo cerebellar granule neuron development, as well as in the deleterious effects of ethanol on the developing cerebellum.     

Agmatine induces glutamate release and cell death in cultured rat cerebellar granule neurons

We investigated the effect of agmatine on cell viability of rat cerebellar granule neurons in a high-K+ (27.5 mM) medium. Exposure of cultured rat cerebellar granule neurons to agmatine (200-800 microM) resulted in a significant decrease in cell viability. Agmatine-induced neuronal death began to occur 6-12 h after addition, and gradually progressed. The agmatine neurotoxicity was attenuated by N-methyl-D-aspartate (NMDA) receptor antagonists and by enzymatic degradation of L-glutamate with glutamic pyruvic transaminase. Furthermore, a significant increase in extracellular L-glutamate concentration was detected before cell death occurred. In addition, agmatine-induced glutamate release and cell death were both blocked by pretreatment with botulinum toxin C, which is known to specifically inhibit the exocytosis. The agmatine neurotoxicity was not observed when extracellular K+ concentration was lower (10 mM). These results suggest that agmatine induces glutamate release through the exocytosis and thereby causes NMDA receptor-mediated neuronal death in conditions in which extracellular K+ concentrations are elevated.     

Peroxynitrite and Nitric Oxide Donors Induce Neuronal Apoptosis by Eliciting Autocrine Excitotoxicity

Endogenous generation of nitric oxide and its congeners, including peroxynitrite (ONOO^-), has been implicated in the mechanism of neuron loss in neurodegenerative diseases. Accordingly, nitric oxide donors and ONOO^-can elicit both apoptosis and necrosis in neuron cultures. Here we show that nitric oxide donors and ONOO^- are each able to trigger apoptosis of mouse cerebellar granule cells by an excitotoxic mechanism requiring exocytosis and NMDA receptor-mediated intracellular Ca²⁺ overload. This conclusion is supported by the following findings. Apoptosis was induced by various nitric oxide donors or by direct addition of ONOO^- to differentiated cerebellar granule cell cultures that were sensitive to NMDA toxicity, but not in cerebellar granule cells that did not display NMDA-induced cell death (i.e. early days in culture) or in various glial cell populations. Donors of ONOO^- or nitric oxide stimulated a sustained increase in intracellular Ca²⁺, which was prevented by inhibitors of NMDA receptors, such as MK-801 and 5-phospho-aminovaleric acid, or by dampening neuronal electrical activity with high concentrations of extracellular Mg²⁺. Moreover, these treatments and the exposure of cerebellar granule cells in nominally Ca²⁺-free media prevented apoptotic cell death. Both the intracellular Ca²⁺ increase and apoptosis elicited by ONOO^- or the nitric oxide donors were prevented by blocking exocytosis with tetanus toxin or botulinum neurotoxin C.     

Relationship Between Intracellular Free Calcium Concentration and NMDA-induced Cerebellar Granule Cell Survival In Vitro

The survival of cerebellar granule cells in culture is stimulated by activation of the N -methyl- d -aspartate (NMDA) class of glutamate receptors. Activation of these receptors at the key period for cell survival in vitro (3 days; 3DIV) resulted in a sustained elevation of intracellular free calcium concentration [Ca²⁺]_i over the same concentration range of NMDA that led to granule cell survival. Agents that release Ca²⁺ from intracellular stores led to only small, transient elevations of [Ca²⁺]_i and were unable to stimulate granule cell survival. Addition of the Ca²⁺ ionophore ionomycin to granule cell cultures at 3DIV resulted in increased granule cell number at 7DIV. The ability of ionomycin to stimulate granule cell survival was related to the [Ca²⁺]_i elicited, indicating that a rise in [Ca²⁺]_i is sufficient to activate the processes leading to granule cell survival and that the extent of the elevation in [Ca²⁺]_i is crucially important in determining granule cell fate.     

Characterization of the activation of glutaminase induced by N-methyl-D-aspartate and potassium in cerebellar granule cells

Chronic stimulation of cerebellar granule cells with N-methyl-D-aspartate (NMDA) or KCl induces a specific activation of the enzymes directly involved in glutamate neurotransmitter synthesis. Phosphate-activated glutaminase (PAG) activity is enhanced in cultured granule neurons incubated with 150 μM NMDA or 25 mM KCl. Other enzymes are not affected by this treatment like lactate dehydrogenase (LDH) and glutamate dehydrogenase (GLDH), which is also a mitochondrial enzyme but not directly involved in neurotransmitter synthesis. This effect is dependent on protein synthesis and is induced after 12 hr of NMDA or KCl stimulation. Kinetics of PAG activity showed that K_m values were unaffected, in contrast to V_max values that were increased approximately 70% and 215% over control by NMDA and KCl treatment, respectively. For GLDH, we found two isoforms that were affected differentially by the experimental conditions. Western blot analysis clearly evidenced an increase of approximately 120–180% in the amount of PAG in NMDA- and KCl-treated cells, whereas GLDH was not significantly modified. These results demonstrate that the NMDA- and KCl-induced activation of PAG are not due to the modification of the preexisting enzyme, but to an increase in the synthesis of this enzyme. This suggests that NMDA receptor stimulation during critical periods of the cerebellar granule cell development leads to the activation of gene expression involved in the process of cell differentiation. © 1996 Wiley-Liss, Inc.