The role of intracellular free calcium and phosphorylation in survival and differentiation of cultured cerebellar granule cells

Abstract

Cultures of cerebellar granule cells from neonatal rats can be maintained in culture for several days provided certain requirements are met. These are a depolarizing concentration of K' [ l ] or the presence of the glutamate receptor agonist N-methyl-D-aspartate (NMDA) [2]. Granule cells develop a sensitivity to these factors between 2-4 days in vitro @IV); if they are not provided cell death begins and is substantial by 7-10 DIV (31. Both factors cause a rise in intracellular free Ca" ([Ca2+],), K' via voltage sensitive Ca2+ channels and NMDA via a receptor gated ion channel 141. We have investigated the possibility of stimulating survival by raising [CaZ+], in the absence of depolarisation or NMDA receptor activation. This was done by using the CaZ+ ionophore ionomycin, to allow a nonspecific Ca2' influx across the plasma membrane, or by mobilising intracellular Ca2+ pools. Measurements of [CaZ+], by fluorescent spectroscopy were made, together with assessment of cell survival. In addition we have investigated the phosphorylation of proteins following the rise in [Ca"], induced by survival stimulating agents.For this cells were incubated with "P orthophosphate, and labelled phosphoproteins were resolved by two-dimensional (2-D) electrophoresis and visualised by autoradiography .Primary cultures of granule cells were prepared by dissociation of 6-8 day old rat cerebella [5]. Cells were plated at a density of either 3 X lo6 cells/dish (for [Ca'+); measurements) or 6 X 106 cells/dish (for I2P incorporation) and grown in 3ml of media (Eagle's modified minimum essential medium plus Earle's salt$, 10% foetal calf serum, 2mM glutamine, 33mM glucose, 25Ulml penicillin, 25pglml streptomycin (plus 12.5mM KCI for "P incorporation experiments). All additions and experiments were performed at 3'DIV.[Caz+], measurements were determined by loading cells (grown on poly-lysine coated round glass coverslips) with 2pM fura 2-AM in buffer A (in mM: 145 NaCI, 5KC1, 1.3 MgCI,, 1.2NaH2PO,, 10 glucose, 20 Hepes, pH 7.4) at room temperature for 40min followed by washing and incubation for a further 60 min in buffer A. The coverslips were inserted into a stirred thermostated cuvette at 3 7 T in buffer A and challenged by the addition of drugs to the indicated final concentration. A PC was used to drive the fluorimeter alternatively between excitatory wavelengths of 340nM and 380nM to produce a ratio every 3.8 sec. [Ca'+], was calculated from the ratio using a K, of 225nM (determined experimentally) [6].Survival was assessed in response to additions of IOOpM NMDA, 25pM 2,5-di-(m-hutyl)-l ,4-benzo-hydroquinone (tuBHQ), 150pM 1 aminocyclopentane-l,3-dicarboxylic acid (ACPD) or various concentrations of ionomycin. At 7 DIV cells were tixed in 4% formaldehyde in PBS. An assessment of survival was made by counting perikarya in randomly selected microscope fields on covers1 ips.For 3zP incorporation experiments cultures were incuhated in fresh media containing 167pCi '*Plml for 4h at 37°C. After washing the challenging agent was added in Earl...

High K+ and IGF‐1 protect cerebellar granule neurons via distinct signaling pathways

“…In granule neuron cultures, K + depolarization is thought to mimic the arrival of the first synaptic afferents arising from the mossy fibers in vivo (Pearson et al, 1992; Graham et al, 1993). As the synapses start to form, appropriate neuronal activities or K + ‐induced depolarization in granule neurons play an essential role for neuronal survival.…”