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Cultured cerebellar granule cells grown in medium containing 10 mM K+ (K10) underwent apoptosis after four to five days in vitro, unless they were rescued by the addition of insulin-like growth factor-I. The few GABAergic neurons present in the cultures were more resistant to apoptotic degeneration, as indicated by double fluorescent staining with the chromatin dye Hoechst 33258 and with glutamate decarboxylase-67 antibodies. As compared with sister cultures grown in 25 mM K+, K10 cultures showed an increased expression of the Egr-1 protein and a reduced expression of the Fos protein. The increase in Egr-1 was more substantial in granule cells than in GABAergic neurons, and was not observed in K10 cultures chronically exposed to insulin-like growth factor-I. To examine the temporal relationship between the increase in Egr-1 and the development of programmed cell death, we induced apoptosis in K25 cultures at six days in vitro by replacing their medium with serum-free K10 medium. A substantial, but transient, increase in Egr- expression was observed in granule cells 6 h after switching the medium, a time that preceded the appearance of the phenotypical markers of apoptotic death. An early reduction in the Fos protein was observed after switching the medium from K25 into serum-free K10, but also after switching the medium into serum-free K25, a condition which was not associated with the development of apoptosis nor with the increase in Egr-1. We suggest that a transient induction of Egr-1 contributes to the chain of events leading to the execution phase of neuronal apoptosis in culture.

Cultured cerebellar granule cells grown in medium containing 10 mM K+ undergo apoptosis after 4-5 days in vitro (DIV), and, at that time, the activity of metabotropic glutamate (mGlu) receptors coupled to polyphosphoinositide (PI) hydrolysis begins to decline. In granule cells at 4 DIV, the mGlu receptor subtype mGlu5 was expressed at high levels. The expression of another PI-coupled mGlu receptor, the mGlu1a, was low at 4 DIV but increased during the following days. In cultures at 4-5 DIV, the few cells that already showed an apoptotic phenotype were devoid of mGlu5 receptors, but they all expressed mGlu1a receptors. The development of apoptosis was accelerated after treating the cultures with: (i) mGlu5 antisense oligonucleotides; (ii) the mixed mGlu receptor antagonist, (+)-alpha-methyl-4-carboxyphenylglycine; or (iii) the glutamate depleting enzyme, alanine aminotransferase. In contrast, an induced overexpression of mGlu5 receptors protected cultured granule cells against apoptotic death. We suggest that the activity of mGlu5 receptors supports cell survival, and a decline in the expression of mGlu5 receptors gives access to programmed cell death in cerebellar granule cells developing in primary cultures.

Reciprocal developmental patterns of expression for BDNF and NT-3 have been observed in several neuronal types, including cerebellar granule neurons: NT3 mRNA level decreased and BDNF mRNA increased in granule cells concomitantly with their migration and maturation. In the present study we analysed cultured cerebellar granule neurons prepared from postnatal rat cerebellum, a model system widely used for studies on the maturation and survival of these neurons. We show that chronic depolarization, induced by 25 mM K+ in the culture medium, is able to sustain a persistent increase of BDNF expression in cerebellar granule neurons. It has been suggested that chronic depolarization in vitro mimics the effect of the earliest afferent inputs received by granule cells in vivo: on this basis we suggest that the beginning of neuronal activity in differentiated granule neurons may represent one of the signals that trigger the developmental increase in BDNF expression. Interestingly, we observed that up-regulation of BDNF expression in vitro is accompanied by a dramatic decrease of NT-3 expression: a differential regulation that is highly reminiscent of the reciprocal developmental patterns of expression observed in vivo for BDNF and NT-3. Another point raised by the present results is the possible role of BDNF, acting in an autocrine or paracrine manner, in the trophic effect of high potassium concentration. Indeed, repeated additions of BDNF to the culture medium have a trophic effect on cerebellar granule neurons but reproduce only partially the survival effect observed with 25 mM K+ conditions, suggesting that the increased expression of BDNF is not the only mechanism responsible for the trophic effects of high potassium. In conclusion we show the existence of a reciprocal regulation of BDNF and NT-3 expression in cultured cerebellar granule neurons and we propose that this culture system could represent an in vitro model for the study of the molecular mechanisms underlying the developmental regulation of these neurotrophins in cerebellum.

Cultured cerebellar granule cells underwent apoptotic degeneration when grown in medium containing 10 instead of 25 mM K+. Knowing that apoptosis is associated with changes in the expression of primary response genes, we have measured c-fos, zif/268, and c-jun mRNA levels during maturation of cultured granule cells grown in 10 or 25 mM K+. The constitutive expression of c-fos and zif/268 was differentially regulated by extracellular K+ concentration at 5 days of maturation in vitro (DIV), when cells grown under suboptimal conditions (i.e. in 10 mM K+) are committed to degenerate. At this stage, c-fos mRNA levels were detectable only in cultures grown in 25 mM K+, whereas zif/268 mRNA levels were dramatically elevated in cultures grown in 10 mM K+. This provides one of the few conditions in which c-fos and zif/268 are differentially regulated in nerve cells. Substantial changes in c-jun, or beta-actin mRNA levels were detectable only at 7 DIV, when the percentage of apoptotic cells had already reached a plateau in cultures grown in 10 mM K+. We speculate that changes in the expression of zif/268 are important in the gene program associated with the induction of apoptosis by trophic deprivation in cultured neurons.

We have studied the expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits in cultured cerebellar granule cells [7 days in vitro (DIV)] grown in medium containing different concentrations of K+ (10, 25, or 40 mM) with or without 100 microM N-methyl-D-aspartate (NMDA; added once after 2 DIV). All these conditions are known to influence maturation and survival of granule cells, as well as the functional expression of NMDA receptors during development in culture. The expression of both glutamate receptor (GluR) subunit 1 mRNA and receptor protein was low in cultures grown in 10 mM K+ (K10) and increased dramatically in cultures grown in 25 mM K+ (K25), with intermediate levels found in cultures grown in K10 and chronically exposed to NMDA (K10 + NMDA). In cultures grown in 40 mM K+ (K40), the expression of GluR1 mRNA and receptor protein was lower than in K25 but still higher than in K10. GluR2 and -3 subunits were differently regulated by growth conditions, with their expression being higher in K10 and progressively reduced to the lowest levels in K40 (both mRNA and receptor proteins). GluR4 mRNA levels did not differ between K10 and K25, although they were reduced by chronic exposure to NMDA. To test how the differential expression of the various subunits affects the functional activity of AMPA receptors, we have measured AMPA-stimulated 45Ca2+ influx and 4 beta-[3H]phorbol 12,13-dibutyrate binding in intact cells. Both functional parameters increased along with the K+ concentration and were maximal in K40, in coincidence with the lowest expression of the GluR2 subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamate receptors coupled to polyphosphoinositide (PPI) hydrolysis (metabotropic glutamate receptors, mGluR), are highly efficient during the early stages of postnatal life and are thought to be involved in developmental plasticity. The dramatic decrease with age in mGluR activity suggests the existence of mechanisms that down-regulate this receptor after a certain stage of neuronal maturation. In cultured cerebellar granule neurons grown under conditions that promote the survival and maturation of cells (serum-containing medium with 25 mM K+), enzymatic depletion of extracellular glutamate prevented the age-dependent decrease in mGluR agonist-stimulated PPI hydrolysis that normally occurs after 4 days of maturation in vitro, suggesting that mGluR activity declines as a result of developmental changes affecting homologous desensitization. This was borne out by the observation that glutamate at low concentrations (1-10 microM) readily desensitized mGluR at 7 days but not at 4 days in culture. Furthermore, the critical period during which the high sensitivity to agonist-induced desensitization of mGluR developed coincided with the period when phorbol ester-activated protein kinase C acquired the ability to suppress mGluR activity. The developmental pattern of mGluR agonist-induced PPI hydrolysis was similar in granule cells grown under "trophic" and "nontrophic" conditions (in cultures in 25 mM K+ and in a medium containing "low" K+, in this study, 10 mM, respectively). However, the developmental decline in the response to mGluR stimulation after 4 days in vitro was not prevented in cells grown in 10 mM K+ by the removal of extracellular glutamate; rather, it could be counteracted by treatment with N-methyl-D-aspartate (NMDA) (EC50, approximately 4 microM), which blocked the development of mGluR desensitization. The effect was NMDA receptor mediated and required DNA transcription and protein synthesis. However, NMDA exerted a different effect in cells grown in 25 mM K+, inducing a substantial decrease rather than an increase in mGluR activity. The effect of growth conditions was also examined on mGluR mRNA levels, which were not always correlated with mGluR activity. In general, either increases in the medium K+ concentrations or NMDA supplementation of the cultures resulted in a decrease in mGluR mRNA levels. It is noteworthy that NMDA could also restore mGluR activity after the metabotropic response had reached its peak. This implies that NMDA receptor activation may be involved in the increase in mGluR activity in adult life under conditions that elicit plastic changes in the nervous system.

A detailed pharmacological characterization of metabotropic glutamate receptors (mGluR) was performed in primary cultures of cerebellar granule cells at 6 days in vitro (DIV). The rank order of agonists induced polyphosphoinositide (PPI) hydrolysis (after correcting for the ionotropic component in the response) was as follows: in terms of efficiency, Glu > quisqualate (quis) = ibotenate (ibo) > (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) > beta-methyl-amino-L-alanine (BMAA) and in terms of potency, quis > ACPD > Glu > ibo = BMAA. Ionotropic excitatory amino acid (EAA) receptor agonists, such as alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) were relatively inactive (in the presence of Mg2+). Quis and ACPD-induced PPI hydrolysis was unaffected by ionotropic Glu receptor antagonists, but was inhibited, in part by L-2-amino-3-phosphonopropionate (AP3). In contrast, Glu-or ibo- induced PPI hydrolysis was reduced, in part, by both AP3 and NMDA receptor antagonists. Characteristic interactions involving different transmitter receptors were noted. PPI hydrolysis evoked by quis and 1S,3R-ACPD was not additive. In contrast, PPI hydrolysis stimulated by quis/ACPD and carbamylcholine was additive (indicating different receptors/transduction pathways). In the presence of Mg2+, the metabotropic response to quis/AMPA and NMDA was synergistic (this being consistent with AMPA receptor-induced depolarization activating NMDA receptor). On the other hand, in Mg(2+)-free buffer the effects of quis and NMDA, at concentrations causing maximal PPI hydrolysis, were additive (indicating that PPI hydrolysis was effected by two different mechanisms).(ABSTRACT TRUNCATED AT 250 WORDS)

Excitatory amino acid (EAA)-induced polyphosphoinositide (PPI) hydrolysis was studied during the development in culture of cerebellar granule cells. The developmental pattern was similar using metabotropic glutamate (Glu) receptor (mGluR) agonists, including L-Glu, quisqualate, and trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid: The stimulation of [3H]inositol monophosphate ([3H]-InsP) formation was low at 2 days in vitro (DIV), but the response increased steeply, reaching a peak at 4 DIV, followed by a progressive decline. In contrast, carbamylcholine-induced PPI hydrolysis exhibited a plateau after a pronounced increase during the first week in vitro. At 6 DIV, but not at 4 DIV, when the activity peaked, PPI hydrolysis elicited by Glu was reduced by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, indicating that in cultured granule cells, NMDA receptors contribute to [3H]-InsP formation and that this component of the response develops relatively late. Accordingly, NMDA-induced [3H]-InsP formation, estimated under Mg(2+)-free conditions, increased markedly from very low values at 2 DIV to a plateau at 8-10 DIV. The developmental pattern of EAA-induced PPI hydrolysis was paralleled by changes in the level of an mRNA for a specific mGluR subtype (mGluR1 mRNA). RNA blot analysis performed with the pmGR1 cDNA probe revealed that the hybridization signal in RNA extracts from cultures at 1 DIV was very weak, but mGluR mRNA levels increased dramatically between 1 and 3 DIV, followed by a progressive decrease, so that by 15 DIV the mRNA levels were only approximately 10% of the values at 3 DIV.(ABSTRACT TRUNCATED AT 250 WORDS)