Nicholas C. Spitzer

Electrical activity in early neuronal development

The construction of the brain during embryonic development was thought to be largely independent of its electrical activity. In this view, proliferation, migration and differentiation of neurons are driven entirely by genetic programs and activity is important only at later stages in refinement of connections. However, recent findings demonstrate that activity plays essential roles in early development of the nervous system. Activity has similar roles in the incorporation of newly born neurons in the adult nervous system, suggesting that there are general rules underlying activity-dependent development. The extensive involvement of activity makes it likely that it is required at all developmental stages as a necessary partner with genetic programs.

In vivo regulation of axon extension and pathfinding by growth-cone calcium transients

Growth cones at the tips of extending neurites migrate through complex environments in the developing nervous system and guide axons to appropriate target regions using local cues,. The intracellular calcium concentration ([Ca2+]i) of growth cones correlates with motility in vitro, but the physiological links between environmental cues and axon growth in vivo are unknown. Here we report that growth cones generate transient elevations of [Ca2+]i as they migrate within the embryonic spinal cord and that the rate of axon outgrowth is inversely proportional to the frequency of transients. Suppressing Ca2+ transients by photorelease of a Ca2+ chelator accelerates axon extension, whereas mimicking transients with photorelease of Ca2+ slows otherwise rapid axonal growth. The frequency of Ca2+ transients is cell-type specific and depends on the position of growth cones along their pathway. Furthermore, growth-cone stalling and axon retraction, which are two important aspects of pathfinding, are associated with high frequencies of Ca2+ transients. Our results indicate that environmentally regulated growth-cone Ca2+ transients control axon growth in the developing spinal cord.

Adaptation in the chemotactic guidance of nerve growth cones

Pathfinding by growing axons in the developing nervous system may be guided by gradients of extracellular guidance factors. Analogous to the process of chemotaxis in microorganisms, we found that axonal growth cones of cultured Xenopus spinal neurons exhibit adaptation during chemotactic migration, undergoing consecutive phases of desensitization and resensitization in the presence of increasing basal concentrations of the guidance factor netrin-1 or brain-derived neurotrophic factor. The desensitization is specific to the guidance factor and is accompanied by a reduction of Ca2+ signalling, whereas resensitization requires activation of mitogen-associated protein kinase and local protein synthesis. Such adaptive behaviour allows the growth cone to re-adjust its sensitivity over a wide range of concentrations of the guidance factor, an essential feature for long-range chemotaxis.

Activity-dependent homeostatic specification of transmitter expression in embryonic neurons

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.

Dynamic interactions of cyclic AMP transients and spontaneous Ca(2+) spikes.

Transient increases of intracellular Ca2+ drive many cellular processes, ranging from membrane channel kinetics to transcriptional regulation, and links of Ca2+ to other second messengers should activate signalling networks. However, real-time kinetic interactions have been difficult to investigate. Here we report observations of spontaneous increases in concentration of cyclic AMP (cAMP) in embryonic spinal neurons, and their dynamic interactions with Ca2+ oscillations. Blocking the production of these cAMP transients decreases the intrinsic frequency of spontaneous Ca2+ spikes, whereas inducing cAMP increases causes spike frequency to increase. Transients of cAMP in turn are absent when Ca2+ spikes are blocked, and are generated only in response to specific patterns of stimulated spikes that mimic endogenous Ca2+ kinetics. We present a mathematical model of Ca2+–cAMP reciprocity that generates the slow cAMP oscillations and reproduces the dynamics of Ca2+–cAMP interactions observed experimentally. The model predicts that this module of coupled second messengers is tuned to optimize production of cAMP transients, and that simultaneous stimulation of Ca2+ and cAMP systems produces distinct temporal patterns of oscillations of both messengers. Our findings may prove useful in the investigation of the regulation of gene expression by second-messenger transients.

Coding of neuronal differentiation by calcium transients

Excitability has long been recognized as the basis for rapid signaling in the mature nervous system, but roles of channels and receptors in controlling slower processes of differentiation have been identified only more recently. Voltage‐dependent and transmitter‐activated channels are often expressed at early stages of development prior to synaptogenesis, and allow influx of Ca2+. Here we examine the functions of spontaneous transient elevations of intracellular Ca2+ in embryonic neurons. These Ca2+ transients abruptly raise levels of Ca2+ as much as tenfold, for brief periods, repeatedly, and can be highly localized. Like cloudbursts on the developing landscape, Ca2+ transients modulate growth and stimulate differentiation, in a frequency‐dependent manner, probably by changes in phosphorylation or proteolysis of regulatory and structural proteins in local regions. We review the mechanisms by which Ca2+ transients are generated and their effects in regulating motility via the cytoskeleton and differentiation via transcription. BioEssays 22:811–817, 2000.

Calcium-induced release of calcium regulates differentiation of cultured spinal neurons

Voltage-dependent calcium influx has been shown to regulate the differentiation of cultured amphibian spinal neurons. We have examined the transient elevation of intracellular calcium induced by depolarization, using calcium indicators and confocal microscopy with high temporal and spatial resolution. Rapid calcium elevations in both the nucleus and the cytosol are primarily due to calcium-dependent release of calcium from intracellular stores. Depletion of stores associated with the endoplasmic reticulum reduces all transients. Elevations diminish with neuronal maturation. Depletion of stores of intracellular calcium at early times affects neuronal differentiation in a manner similar to the prevention of influx. The results indicate that both influx and release are necessary to promote neuronal differentiation.

Developmental changes in the inward current of the action potential of Rohon‐Beard neurones

1. Rohon‐Beard cells in the spinal cord of Xenopus tadpoles have been studied in animals from early neural tube to free‐swimming larval stages. The onset and further development of electrical excitability of these neurones has been investigated in different ionic environments, to determine the ionic species carrying the inward current of the action potential.

Regulation of Growth Cone Behavior by Calcium: New Dynamics to Earlier Perspectives

Guidance of axons to their targets is in part due to theability of motile growth cones to integrate signalsgenerated through coincident interactions with multi-ple extracellular cues and to translate those signalsinto proper behavioral changes. Although many newaxon guidance cues and their receptors have recentlybeen discovered (for review see Goodman andTessier-Lavigne, 1997), signaling through traditionalsecond-messenger systems continues to emerge as acrucial intermediary for guidance. For example,changes in intracellular calcium concentration([Ca

Spontaneous Ca2+ spikes and waves in embryonic neurons: signaling systems for differentiation

Many excitable cells are specialized to promote Ca2+ influx at early stages of development. This article focuses on spontaneous fluctuations of intracellular Ca2+ that are observed during this period. Removal of Ca2+ or suppression of influx alters subsequent differentiation. Thus these signals appear to regulate aspects of early maturation.