Intracellular Ca2+ regulation in rat motoneurons during development

Changes in intracellular Ca2+ concentration ([Ca2+]i) control the setting up of the neuro-muscular synapse in vitro and probably in vivo. Dissociated cultures of purified embryonic (E15) rat motoneurons were used to explore the molecular mechanisms by which endoplasmic reticulum Ca2+ stores, via both ryanodine-sensitive and IP3-sensitive intracellular Ca2+ channels control [Ca2+]i homeostasis in these neurons during ontogenesis. Fura-2 microspectrofluorimetry monitorings in single neurons showed that caffeine-induced responses of [Ca2+]i increased progressively from days 1–7 in culture. These responses were blocked by ryanodine and nicardipine but not by ω-conotoxin-GVIA or ω-conotoxin-MVIIC suggesting a close functional relationship between ryanodine-sensitive and L-type Cav1 Ca2+ channels. Moreover, after 6 days in vitro, neurons exhibited spontaneous or caffeine-induced Ca2+ oscillations that were attenuated by nicardipine. In 1-day-old neurons, both thapsigargin or CPA, which deplete Ca2+ stores from the endoplasmic reticulum, induced an increase in [Ca2+]i in 75% of the neurons tested. The number of responding motoneurons declined to 25% at 5–6 days in vitro. Xestospongin-C, a membrane-permeable IP3 receptor inhibitor blocked the CPA-induced [Ca2+]i response in all stages. RT-PCR studies investigating the expression pattern of RYR and IP3 Ca2+ channels isoforms confirmed the presence of their different isoforms and provided evidence for a specific pattern of development for RYR channels during the first week in vitro. Taken together, present results show that the control of motoneuronal [Ca2+]i homeostasis is developmentally regulated and suggest the presence of an intracellular ryanodine-sensitive Ca2+ channel responsible for a Ca2+-induced Ca2+ release in embryonic motoneurons following voltage-dependent Ca2+ entry via L-type Ca2+ channels.