Michel Didier

Cerebellar granule cell survival and maturation induced by K+ and NMDA correlate with c-fos proto-oncogene expression

Persistent depolarization with a high K+ concentration (30 mM) or sustained activation of N-methyl-D-aspartate (NMDA) receptors (12.5 mM K+ plus 100 microM NMDA) enhance both survival and maturation of mouse cerebellar granule neurons in vitro taking as criteria the amount of protein and DNA and the release of endogenous glutamate respectively. K+ and NMDA neurotrophic effects are associated with c-fos protein expression in the nucleus of these cells suggesting that c-fos protein could play a role in the survival and/or maturation of granule neurons.

Characterization of nicotinic acetylcholine receptors expressed in primary cultures of cerebellar granule cells

Nicotinic acetylcholine receptors (nAChRs), like other calcium permeable channel receptors, may play a crucial role during neuronal development. We have characterized nAChRs in developing mouse cerebellar granule cells in primary culture. L-[3H]Nicotine, [3H]cytisine and [125I]alpha-bungarotoxin binding experiments revealed the presence of a single class of saturable and specific high affinity binding sites for each ligand. The expression of these nicotinic binding sites followed a developmental pattern reaching a maximum during the establishment of excitatory amino acid synaptic contacts. Immunolabeling with monoclonal antibodies to nAChR subunits revealed the presence of alpha 4 and beta 2 subunits in most neurons. Moreover, some neuronal cells displayed a somatic as well as a neuritic localization for the alpha 7 subunit as shown by [125I]alpha-bungarotoxin autoradiography. The reverse transcription-polymerase chain reaction (RT-PCR) detected the presence of mRNAs for alpha 3, alpha 4, alpha 5, alpha 7, beta 2 and beta 4 nAChR subunits. Non-neuronal cells did not express nAChRs, as shown by [3H]nicotine and [125I]alpha-bungarotoxin binding, immunocytochemistry and PCR. Maximum Ca2+ influx elicited by nicotine, and partly sensitive to alpha-bungarotoxin, was observed around 10-14 days after plating. This correlated with the time period at which the highest number of nicotine binding sites was detected. Sensitivity to several NMDA receptor antagonists as well as to removal of endogenous glutamate by pyruvate transaminase treatment revealed a glutamatergic component in the nicotine stimulated calcium influx. The time-dependent specific nAChR expression and the potential association between nAChRs and NMDA receptor activation suggest that nAChRs may regulate glutamatergic activity during synaptogenesis in cerebellar granule cells.

Acute rise in the concentration of free cytoplasmic calcium leads to dephosphorylation of the microtubule-associated protein tau.

The objective of this study was to asses the response of the microtubule-associated protein tau to acute rise in the concentration of free cytoplasmic calcium ([Ca2+]i) in rat cortical neurons and mouse cerebellar granule cells in culture. One-hour exposure to glutamate (100 microM), N-methyl-D-aspartate (100 microM), KCl (50 mM), and ionomycin (5 microM) led to tau protein dephosphorylation as indicated by an appearance of additional faster moving bands on Western immunoblots with a phosphorylation-independent antibody and an increase in the tau-1 immunoreactivity associated with the appearance of an additional faster moving band. Lowering the extracellular concentration of Ca2+ to less than 1 microM fully prevented the drug-induced tau protein dephosphorylation indicating a dependence on Ca2+ influx from the extracellular environment. Administration of okadaic acid (inhibitor of phosphatase 1/2A) simultaneously with the above mentioned drugs decreased the drug-mediated dephosphorylation. Pre-incubation with okadaic acid fully prevented the dephosphorylation. Treatment with cypermethrin (inhibitor of phosphatase 2B) was without effect when administered either alone, simultaneously with the drugs, or pre-incubated. These findings indicate that, independently of the influx pathway, [Ca2+]i elevation leads to dephosphorylation of the microtubule-associated protein tau and implicate phosphatase 1 and/or 2A in the process.

Long-term expression of the c-fos protein during the in vitro differentiation of cerebellar granule cells induced by potassium or NMDA

Levels of the c-fos protein were assayed in mouse cerebellar granule cells during their in vitro development under different culture conditions. When grown in media favoring both their survival and differentiation, i.e. in the presence of 30 mM K+ or 12.5 mM K+ plus 100 microM N-methyl-D-aspartate (NMDA), the c-fos protein becomes detectable in the nucleus of granule cells on and after 6 days and persists to high levels until the culture begins to decline. The protein c-fos appears therefore after the critical period described for the survival effect of K+ depolarization or NMDA receptor stimulation which corresponds to days 2-5 after plating. The c-fos protein remains however scarcely detectable or undetectable throughout the life-span of cells cultured under conditions providing poor survival and differentiation, i.e. in the presence of low K+ (5 or 12.5 mM) alone or when the effect of NMDA is blocked by the NMDA receptor antagonist MK-801. Interestingly, in cortical and striatal neurons, the survival and differentiation of which being not affected by depolarizing media, no c-fos protein is detected whatever the culture conditions tested at least during the first 18 days in vitro. This suggests that long-term expression of the c-fos gene might be related to some aspect of the late in vitro differentiation process of cerebellar granule cells.

Differential expression and co-assembly of NMDA zeta 1 and epsilon subunits in the mouse cerebellum during postnatal development.

The differential distribution of NMDA receptor subunit mRNAs in the developing mammalian cerebellum has been previously described. In this study, we investigated the temporal expression of NMDA receptor proteins in the postnatal murine cerebellum using antibodies specific for the NMDA zeta 1 and NMDA epsilon subunits. Our results showed a gradual increase during the first three weeks of life in the relative amount of NMDA zeta 1 and NMDA epsilon 1 proteins. In contrast, NMDA epsilon 2 increased transiently during this period, reaching a maximum around postnatal day 9 and decreasing thereafter to nearly undetectable levels by the end of the third week of life. The level of NMDA epsilon 3 increased dramatically between postnatal days 9 and 15 and thereafter remained constant. Immunoprecipitation of native proteins revealed that a large fraction of NMDA epsilon 2 was associated with NMDA zeta 1 and epsilon 1. At later developmental stages, NMDA epsilon 3 was predominantly assembled with NMDA zeta 1 but not with NMDA epsilon 1 or NMDA epsilon 2. These results demonstrate that NMDA receptor subtypes, formed by the assembly of different NMDA epsilon subunits with NMDA zeta 1, are sequentially expressed in the developing mouse cerebellum. The time course of their expression suggest, that these NMDA receptor subtypes may contribute to specific aspects of granule cell differentiation in the cerebellum.

Developmental regulation of the recovery process following glutamate-induced calcium rise in rodent primary neuronal cultures

CNS neurons exhibit a profound, maturation-dependent increase in the vulnerability to injury. Little is, however, known about the cellular mechanisms involved. This study investigated the developmental influence on the ability to recover the resting concentration of free cytoplasmic Ca2+ ([Ca2+]i) following stimulation with 100 microM glutamate in hippocampal and cerebellar granule cells in culture. Primary neurons were exposed to glutamate for either 1 min or 10 min. Hippocampal neurons were evaluated at 7, 12-14, and 17-19 days in vitro (DIV), and cerebellar granule cells were tested at 8-9 or 15-16 DIV. In hippocampal neurons, either an increased age in culture or longer drug exposure were both associated with less efficient [Ca2+]i recovery. Additionally, for both 1-min and 10-min drug exposure, increased age in culture was the primary determinant of the development of secondary [Ca2+]i destabilization followed by a very variable recovery patterns. Similar to hippocampal neurons, older cerebellar granule cells also recovered less efficiently from glutamate-mediated [Ca2+]i rise. The difference in the extent of recovery was not directly influenced by the magnitude of the [Ca2+]i rise, since cerebellar granule cells recovered from both high or low [Ca2+]i rise with similar kinetic profiles. Overall, the results presented in this study implicate the age in culture as an important influencing factor of both the less efficient recovery from glutamate-induced Ca2+ load and the development of secondary [Ca2+]i destabilizations. The progressive, maturation-dependent, decrease in the ability to recover from Ca2+ load might represent a potentially important mechanism contributing to the increased vulnerability of fully developed neurons to injury.

Expression of the α4 neuronal nicotinic acetylcholine receptor subunit in the developing mouse hippocampus

Neurotransmitters such as acetylcholine can control neuritogenesis of hippocampal cells. The timing of its receptors expression consequently may influence synaptogenesis and neuronal activity in the developing hippocampus. We investigated the mRNA expression of the nicotinic acetylcholine‐gated ion channel receptor (nAChR) α4 subunit in the embryonic and postnatal hippocampal formation. Although its expression level is low in the adult hippocampus, this protein consitutes the major nAChR subunit in the central nervous system. We carried out in‐situ hybridization experiments to determine whether or not the α4 AChR subunit mRNA distributions show evidence of regional and developmental regulation during hippocampal maturation. Our studies reveal that α4 AChR mRNA expression was low at the embryonic stage, but increased transiently during postnatal development reaching a maximum during the second week of life and decreasing thereafter, to a minimum at adulthood. In hippocampal regions, the peak values of α4 AChR expression were between 400 and 800% of adult α4 messenger levels. In the postnatal hippocampus, most of the cells from the pyramidal layer of the CA3 and CA2 areas displayed a strong hybridization signal for the α4 AChR subunit. In the hilus and the CA1 regions, the localization of the α4 transcripts seemed to be restricted to some interneurons and pyramidal cells, respectively. Moderate and uniform in‐situ hybridization signals were observed in granular cells from the dentate gyrus. The transient profile of α4 expression suggests that nAChRs may participate in the early postnatal maturation of hippocampal circuity.

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.

35 mM K+-stimulated 45Ca2+ uptake in cerebellar granule cell cultures mainly results from NMDA receptor activation

In primary cultures of cerebellar granule cells, the Ca2+ influx resulting from K+ depolarization (35 mM) was equal to one-third of that observed with 100 microM N-methyl-D-aspartate (NMDA) and was reduced in a major part (90%) by NMDA receptor antagonists. The rank order of potency of these competitive and non-competitive NMDA receptor antagonists was very close to their affinity for the NMDA and phencyclidine sites respectively. Granular cell depolarization with 35 mM K+ also induced a large increase in the extracellular glutamate concentration. Repeated washes of the culture wells, addition of glutamate pyruvate transaminase (+2 mM pyruvate), or pretreatment of the cells with tetanus toxin resulted in a parallel reduction of the extracellular glutamate concentration and 45Ca2+ uptake measured after a 35 mM K+ stimulation. Dihydropyridine (BAY K-8644) stimulated the release of glutamate in a nifedipine-sensitive manner in the presence of 15 mM K+. However, nifedipine (1 microM), which decreased by 60% the K(+)-induced 45Ca2+ uptake, did not reduce the 35 mM K(+)-evoked glutamate release. Taken together, these results demonstrated that in cerebellar granule cell cultures, 90% of the 35 mM K(+)-stimulated 45Ca2+ influx resulted from the release of glutamate and the consecutive activation of NMDA receptors. Activation of these glutamate receptors then allows Ca2+ influx to occur through L-type voltage-operated Ca2+ channels.

Involvement of three glutamate receptor ϵ subunits in the formation of N-methyl-d-aspartate receptors mediating excitotoxicity in primary cultures of mouse cerebellar granule cells

The N-methyl-D-aspartate receptors have been implicated in neuronal plasticity and their overactivation leads to neurotoxicity. Molecular cloning and co-expression of various glutamate receptor zeta and epsilon complementary DNAs support a heteromeric structural organization for N-methyl-D-aspartate receptors. In this study, we show that cerebellar granular neurons in primary culture of mouse express glutamate receptor zeta1 and at least three glutamate receptor epsilon (epsilon1, epsilon2, and epsilon3) protein subunits. In vitro, the temporal patterns of glutamate receptor epsilon1, epsilon2, and epsilon3 subunit expression depend on culture stages. By day 9, a somatic and neuritic immunolocalization for all N-methyl-D-aspartate subunits was clearly identified in most neuronal, but not glial cells. The role of particular subunits in N-methyl-D-aspartate-mediated excitotoxicity was probed by exposing the cerebellar granule cells to antisense oligodeoxynucleotides generated against specific N-methyl-D-aspartate receptor subunits. Antisense oligodeoxynucleotide treatments significantly down-regulated the amounts of the corresponding N-methyl-D-aspartate subunits. The decrease in N-methyl-D-aspartate subunit protein correlated with a reduction in N-methyl-D-aspartate-induced calcium influx and N-methyl-D-aspartate-mediated excitotoxicity in cerebellar cultures. In contrast, antisense oligodeoxynucleotide treatment failed to protect neurons from 1-methyl-4-phenylpyridinium-induced metabolic cell toxicity. Antisense oligodeoxynucleotide treatment targeted at N-methyl-D-aspartate glutamate receptor epsilon subunits demonstrate that glutamate receptor epsilon1, epsilon2, and epsilon3 proteins form N-methyl-D-aspartate receptors responsible for neurotoxic effects on cerebellar neurons. This study provides direct evidence for the existence of distinct N-methyl-D-aspartate receptor subunit proteins in cerebellar granule cells developing in vitro that may trigger N-methyl-D-aspartate-dependent excitotoxicity.