Coding of neuronal differentiation
by calcium transients
Nicholas C. Spitzer,* Nathan J. Lautermilch, Raymond D. Smith,
and Timothy M. Gomez
Summary
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 pro-
cesses of differentiation have been identified only more
recently. Voltage-dependent and transmitter-activated
channels are often expressed at early stages of devel-
opment prior to synaptogenesis, and allow influx of
Ca
2
. Here we examine the functions of spontaneous
transient elevations of intracellular Ca
2
in embryonic
neurons. These Ca
2
transients abruptly raise levels of
Ca
2
as much as tenfold, for brief periods, repeatedly,
and can be highly localized. Like cloudbursts on the
developing landscape, Ca
2
transients modulate
growth and stimulate differentiation, in a frequency-
dependent manner, probably by changes in phosphor-
ylation or proteolysis of regulatory and structural
proteins in local regions. We review the mechanisms
by which Ca
2
transients are generated and their effects
in regulating motility via the cytoskeleton and differ-
entiation via transcription. BioEssays 22:811±817,
2000. ß 2000 John Wiley & Sons, Inc.
Introduction
Embryonic amphibian spinal neurons exhibit spontaneous
elevations of free intracellular Ca
2
concentration ([Ca
2
]
i
)
both in culture and in vivo during an early period of
differentiation, revealed by imaging the fluorescence of
Ca
2
indicator dyes.
(1±3)
These spontaneous elevations
are eliminated by removal of extracellular Ca
2
or applica-
tion of general Ca
2
channel blockers and thus depend on
Ca
2
influx. In contrast, no significant changes in steady-
state levels of intracellular Ca
2
are observed.
(1)
Imaging
fluctuations in [Ca
2
]
i
in these embryonic neurons for
extended periods in vivo and in culture reveals that neuronal
cell bodies and growth cones can exhibit multiple sponta-
neous elevations of [Ca
2
]
i
per hr, each of which lasts for
10±30 seconds.
These studies reveal the existence of two types of intra-
cellular Ca
2
transients, Ca
2
spikes and Ca
2
waves,
which differ in significant ways (Fig. 1A). Spikes depend on
Ca
2
influx through voltage-gated channels and Ca
2
release from intracellular stores (Ca
2
-induced Ca
2
release via ryanodine receptors), and achieve a mean
[Ca
2
]
i
of ~500 nM.
(1,3±5)
Spikes have relatively rapid
kinetics (mean duration approximately 10 s), and since they
involve Ca
2
-dependent action potentials they are propa-
gated thoughout a neuron. The frequency of spontaneous
Ca
2
spikes in cultured neurons varies from 1-10/hr and is
temporally regulated, rising to a mean of 3/hr for a 5 hr period
starting 5 hr after plating and then declining; a similar change
in frequency is observed during the corresponding neural
tube stages in vivo.
In contrast to spikes, Ca
2
waves are kinetically slower
(mean duration 30 s in growth cones) and do not involve
action potentials for their generation. Waves occur locally
and decay with distance from their site of initiation. Ca
2
waves appear to depend on Ca
2
influx through a Ca
2
entry pathway that is active at the resting potential, are
amplified by Ca
2
release from intracellular stores,
(4)
and
raise [Ca
2
]
i
to a lower level than attained by spikes. The
frequency of spontaneous Ca
2
waves in cultured neurons
is spatially regulated: after axonogenesis they are generated
in growth cones at a mean frequency of 8±9/hr and in the cell
body at approximately 1/hr. Ca
2
waves are smaller in
amplitude than Ca
2
spikes, probably due both to differ-
ences in the amount of Ca
2
entry across the plasma
membrane and the extent of Ca
2
release from stores.
Remarkably, spikes and waves are each required for
specific aspects of differentiation. Their elimination by
removal of extracellular Ca
2
or suppression with intracel-
lular BAPTA prevents normal development (Fig. 1B). More-
over, reimposition of Ca
2
transients at these low
frequencies rescues normal development, demonstrating
that they are sufficient to encode these aspects of
differentiation (Fig. 1C). Coding of differentiation appears to
be achieved through the different frequencies with which
Ca
2
spikes and waves are spontaneously produced, since
the amplitude, duration and time integral of the imposed
Ca
2
transients are invariant with frequency.
(5)
There
BioEssays 22:811±817, ß 2000 John Wiley & Sons, Inc. BioEssays 22.9 811
Department of Biology and Center for Molecular Genetics, UCSD,
La Jolla, California.
Funding agency: NINDS NIH Grant Nos: RO1 NS15918 and NS37002
*Correspondence to: Dr. Nicholas C. Spitzer, Department of Biology
and Center for Molecular Genetics, UCSD, La Jolla, CA 92093-0357.
E-mail: nspitzer@ucsd.edu
Abbreviations: BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N
0
,N
0
-
tetraacetic acid; Ca
2
, calcium; cAMP, cyclic adenosine monopho-
sphate; GABA, gamma-aminobutyric acid; GAP-43, 43 kilodalton
growth-associated protein; PCR, polymerase chain reaction; mRNA,
messenger ribonucleic acid; TTX, tetrodotoxin.
Review articles