Many forms of electrical excitability expressed in the embryonic nervous system depend on Ca(2+) influx. This discovery has stimulated investigation of the functions of spontaneous elevations of intracellular Ca(2+) and their roles in neuronal development. We present a protocol for imaging different classes of intracellular Ca(2+) transients in embryonic Xenopus (amphibian) spinal neurons grown in dissociated cell culture and in the intact neural tube (the developing spinal cord), focusing on early stages of neuronal differentiation around the time of neural tube closure. The protocol describes methods for gain-of-function and loss-of-function experiments to reveal the functions of these Ca(2+) transients. The methods can also be applied to explant and organotypic cultures. The procedures are sufficiently simple that they can be further adapted for dissociated neuronal cell cultures from other developing embryos, embryonic spinal cords of vertebrates such as zebrafish, and ganglia in the developing nervous systems of invertebrates.
[Show abstract][Hide abstract] ABSTRACT: Neurotransmitters are essential for interneuronal signalling, and the specification of appropriate transmitters in differentiating neurons has been related to intrinsic neuronal identity and to extrinsic signalling proteins. Here we show that altering the distinct patterns of Ca2+ spike activity spontaneously generated by different classes of embryonic spinal neurons in vivo changes the transmitter that neurons express without affecting the expression of markers of cell identity. Regulation seems to be homeostatic: suppression of activity leads to an increased number of neurons expressing excitatory transmitters and a decreased number of neurons expressing inhibitory transmitters; the reverse occurs when activity is enhanced. The imposition of specific spike frequencies in vitro does not affect labels of cell identity but again specifies the expression of transmitters that are inappropriate for the markers they express, during an early critical period. The results identify a new role of patterned activity in development of the central nervous system.
[Show abstract][Hide abstract] ABSTRACT: Cell adhesion is crucial for migration of cells during development, and cell-substrate adhesion of motile cells is accomplished through the formation and removal of focal complexes that are sites of cell-substrate contact. Because Ca2+ signaling regulates the rate of axon outgrowth and growth cone turning, we investigated the potential role of Ca2+ in focal complex dynamics. We describe a novel class of localized, spontaneous transient elevations of cytosolic Ca2+ observed both in Xenopus neuronal growth cones and fibroblasts that are 2-6 mum in spatial extent and 2-4 s in duration. They are distributed throughout growth cone lamellipodia and at the periphery of fibroblast pseudopodia, which are regions of high motility. In both cell types, these Ca2+ transients lead to disappearance of phosphorylated focal adhesion kinase (pFAK) and deadhesion from the substrate as assessed by confocal and internal reflection microscopy, respectively. The loss of pFAK is inhibited by cyclosporin A, suggesting that these Ca2+ transients exert their effects via calcineurin. These results identify an intrinsic mechanism for local cell detachment that may be modulated by agents that regulate motility.
[Show abstract][Hide abstract] ABSTRACT: The finding that astrocytes possess glutamate-sensitive ion channels hinted at a previously unrecognized signaling role for
these cells. Now it is reported that cultured hippocampal astrocytes can respond to glutamate with a prompt and oscillatory
elevation of cytoplasmic free calcium, visible through use of the fluorescent calcium indicator fluo-3. Two types of glutamate
receptor--one preferring quisqualate and releasing calcium from intracellular stores and the other preferring kainate and
promoting surface-membrane calcium influx--appear to be involved. Moreover, glutamate-induced increases in cytoplasmic free
calcium frequently propagate as waves within the cytoplasm of individual astrocytes and between adjacent astrocytes in confluent
cultures. These propagating waves of calcium suggest that networks of astrocytes may constitute a long-range signaling system
within the brain.
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