Disassembly of Subplasmalemmal Actin Filaments Induces Cytosolic Ca2+ Increases in Astropecten aranciacus Eggs

Background/Aims: Eggs of all animal species display intense cytoplasmic Ca2+ increases at fertilization. Previously, we reported that unfertilized eggs of Astropecten aranciacus exposed to an actin drug latrunculin A (LAT-A) exhibit similar Ca2+ waves and cortical flashes after 5-10 min time lag. Here, we have explored the molecular mechanisms underlying this unique phenomenon. Methods: Starfish eggs were pretreated with various agents such as other actin drugs or inhibitors of phospholipase C (PLC), and the changes of the intracellular Ca2+ levels were monitored by use of Calcium Green in the presence or absence of LAT-A. The concomitant changes of the actin cytoskeleton were visualized with fluorescent F-actin probes in confocal microscopy. Results: We have shown that the LAT-A-induced Ca2+ increases are related to the disassembly of actin flaments: i) not only LAT-A but also other agents depolymerizing F-actin (i.e. cytochalasin B and mycalolide B) induced similar Ca2+ increases, albeit with slightly lower efficiency; ii) drugs stabilizing F-actin (i.e. phalloidin and jasplakinolide) either blocked or significantly delayed the LAT-A-induced Ca2+ increases. Further studies utilizing pharmacological inhibitors of PLC (U-73122 and neomycin), dominant negative mutant of PLC-ɣ, specific sequestration of PIP2 (RFP-PH), InsP3 uncaging, and quantitation of endogenous InsP3 all indicated that LAT-A induces Ca2+ increases by stimulating PLC rather than sensitizing InsP3 receptors. In support of the idea, it bears emphasis that LAT-A timely increased intracellular contents of InsP3 with concomitant decrease of PIP2 levels in the plasma membrane. Conclusion: Taken together, our results suggest that suboolemmal actin filaments may serve as a scaffold for cell signaling and modulate the activity of the key enzyme involved in intracellular Ca2+ signaling.

Differential Involvement of Projection Neurons During Emergence of Spontaneous Activity in the Developing Avian Hindbrain

To better characterize the emergence of spontaneous neuronal activity in the developing hindbrain, spontaneous activity was recorded optically from defined projection neuron populations in isolated preparations of the brain stem of the chicken embryo. Ipsilaterally projecting reticulospinal (RS) neurons and several groups of vestibuloocular (VO) neurons were labeled retrogradely with Calcium Green-1 dextran amine and spontaneous calcium transients were recorded using a charge-coupled-device camera mounted on a fluorescence microscope. Simultaneous extracellular recordings were made from one of the trigeminal motor nerves (nV) to register the occurrence of spontaneous synchronous bursts of activity. Two types of spontaneous activity were observed: synchronous events (SEs), which occurred in register with spontaneous bursts in nV once every few minutes and were tetrodotoxin (TTX) dependent, and asynchronous events (AEs), which occurred in the intervals between SEs and were TTX resistant. AEs occurred developmentally before SEs and were in general smaller and more variable in amplitude than SEs. SEs appeared at the same stage as nV bursts early on embryonic day 4, first in RS neurons and then in VO neurons. All RS neurons participated equally in SEs from the outset, whereas different subpopulations of VO neurons participated differentially, both in terms of the proportion of neurons that exhibited SEs, the fidelity with which the SEs in individual neurons followed the nV bursts, and the developmental stage at which SEs appeared and matured. The results show that spontaneous activity is expressed heterogeneously among hindbrain projection neuron populations, suggesting its differential involvement in the formation of different functional neuronal circuits.