N+!uron,
Vol. 7, 787-796,
November,
1991, Copyright
0 1991 by Cell Press
Calcium-Induced
Release of Calcium Regulates
Differentiation
of Cultured Spinal Neurons
Janet Holliday,*+
Richard J. Adams,*
Terrence J. Sejnowski,**§ and Nicholas
C. Spitzer*
*Department
of Biology
and Center
for Molecular
Genetics
University
of California,
San Diego
La Jolla,
California
92093
*L.aboratory
for Computational
Neurobiology
§Howard
Hughes
Medical
Institute
Salk Institute
La Jolla,
California
92138
Summary
calcium-sequestering
pumps
have
been observed
in
other
systems
(Leberer
et al., 1986; Brandt
et al., 1987;
Braun
et al., 1988; Gishan
and Arab,
1988; Cimino
etal.,
1990).
Changes
in calcium
buffering,
pumping,
and
release
from
intracellular
stores
could
contribute
to
the final
level
achieved
by stimulation
and thereby
regulate
neuronal
development.
We describe
depolarization-evoked
calcium
elevations
in differentiating
Xenopus
spinal
neurons
using
the calcium
indicator
fluo3AM
and high time resolution
confocal
microscopy.
We observe
a rapid
increase
in fluorescence
in the nucleus
and cytoplasm
upon
depolarization.
Insensitivity
of the peak
elevation to a range of extracellular
calcium
concentrations
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.
and reduction
of the response
upon
depletion
of intracellular
calcium
stores
indicate
that the fluorescence
signal
is produced
by calcium
release
from
stores
in addition
to calcium
influx.
We also find that the peak fluorescence
change
produced
by stimulation
is decreased
in mature
neurons
relative
to young
cells
as a result
of developmental
changes
in calcium
stores
and sequestration.
Under
conditions
allowing
spontaneous
calcium
influx,
neurite extension
and the differentiation
of neurotransmitter
phenotype
are both altered
by selective
depletion
of stores
at early
stages
of development.
The
results
indicate
that the contribution
of calcium
release to the elevation
of intracellular
calcium
is necessary to support
standard
differentiation.
introduction
Results
Action
potentials
often
involve
larger
calcium
currents
in embryonic
neurons
than
in mature
cells
(Spitzer,
1985).
Since
calcium
levels
can regulate
numerous
cellular
processes,
early elevations
of calcium
are likely
to be important
in the regulation
of subsequent
development.
This view is supported
by observations
of altered
patterns
of differentiation
under
conditions
that prevent
early
calcium
influx
(Walicke
and Patterson,
1981; Bixby
and Spitzer,
1984; Cohan
et al., 1987; Vidal
et al., 1989;
Holliday
and Spitzer,
1490). Furthermore,
spontaneous
transient
calcium
elevations
are observed
only at early
stages
of development
in cultured
embryonic
amphibian
spinal
neurons,
when
they
normally
generate
long-duration,
calcium-dependent
action
potentials
(Holliday
and
Spitzer,
1990).
While
developmental
changes
in calcium
influx
may
be expected
to produce
parallel
changes
in levelsof
intracellularcalcium,
theseexpectations
will be reinforced
or confounded
by concomitant changes
in calcium-handling
mechanisms.
Develoomental
increases
in calcium-buffering
proteins
and
Confocal Microscopy Enables Spatial and Temporal
Analysis of Calcium Elevation in Response
to Depolarization
+ i’resent address:
The Scripps
Research
Institute,
T(lrrey Pines Road, La Jolla, California
92037.
1066 North
Fluorescent
calcium
indicator
reveals
elevation
of calcium
in cultured
Xenopus
neurons
in response
to
depolarization.
Application
of 100 mM
KCI mimics
long-duration,
calcium-dependent
action
potentials
at early
stages
of neuronal
differentiation
(Baccaglini
and
Spitzer,
1977;
Spitzer
and
Lamborghini,
1976).
Confocal
microscopy
in the
image
mode
demonstrates
substantial
elevation
of intracellular
fluorescence
throughout
the nucleus
and cytoplasm,
with
detailed
spatial
resolution
(Figure
I). Little or no fluorescence
elevation
is associated
with
yolk
platelets
or lipid granules.
A l-pm
thick
optical
section
passes
through
the center
of the nucleus
(5-wrn
diameter;
Lamborghini
et al., 1979), demonstrating
that the increase
in calcium
levels
is not restricted
to the cytoplasm
and to the perinuclea;
region,
but appears
to
occur
within
the nucleus
itself in both young
and mature
neurons
(Figure
2).
Application
of confocal
microscopy
in a line-scanning
mode,
to provide
greater
temporal
resolution,
reveals
that calcium
elevations
following
depolarization reach their peakvaluesquickly
in bothyoungand
Neuron
788
Figure
1. Confocal
Imaging
of Changes
in Spatial
Distribution
of Flue-3
Fluorescence
upon
Depolarization
with
Transmission
image (A) and fluorescence
image (6) of a young cultured
neuron loaded with flue-3AM.
The plane
was chosen to visualize
the soma and details of the process.
Differential
dye filling of subcellular
compartments
and yolk granules
is apparent
(B). The pipette used to deliver a 500ms puff of 100 mM KCI was positioned
10 f.tm
the neuronal
process.
Images of the neuron were collected
before and after depolarization
at 3.5-s intervals.
(C) Fluorescence
levels change upon depolarization
(occurring
between
the first and second
frame), then start
(D) Images of(C) are normalized
to the resting fluorescence
level. Elevations
of fluorescence
persist longest in the
cone (left), whereas
little or no elevation
is associated
with yolk granules.
neurons,
within
500 ms of the first detectable
increase
(see Figures
2,3,5,
and 6). Single
2-ms scans
were
made
across
the nerve
cell body,
including
the
nucleus.
The maximum
rates of fluorescence
rise in
both nucleus
and cytoplasm
are substantially
faster
in
young
than in mature
neurons
(Table
1). Furthermore,
the rate of rise is 3-fold
greater
in the cytosol
than
in
the nucleus
of young
cells,
whereas
in mature
neurons
no difference
between
nuclear
and
cytosolic
rates is apparent.
These
differences
are not attributable to differences
in delays,
although
delays
between
cytosolic
and nuclear
fluorescence
elevations
consistent with
diffusion
were
observed.
However,
irregularity
in the three-dimensional
geometry
of neuronal
cytoplasm
and noise
in the fluorescence
signal
precluded
more
detailed
analysis.
Diffusion
from
the
edge of the nucleus
toward
the center
is evident
(Figure 2), suggesting
that the source
of nuclear
calcium
may not be intrinsic
to the nucleus.
Neuronal
calcium
elevations
persist
for 5-20 s (Figure 3). The rate of decay
of fluorescence
following
stimulation
is slow
in neurons
compared
with
myocytes
under
the same conditions,
in which
elevations
mature
High
Potassium
of focus for this cell
such as the nucleus
to the left and below
to recover.
nucleus
and growth
last only slightly
longer
than the duration
of the stimulus (Figure
3B, inset).
The decline
of neuronal
fluorescence
was fitted
by two exponentials
(seeTable
1). The
more
rapid time constant
does not differ
significantly
between
young
and mature
neurons,
nor between
nucleus
and cytosol.
Theother
time constant
is significantly
slower
in the nucleus
and cytosol
of young
cells.
The relative
contributions
of time constants
to
the decline
are variable
and do not depend
obviously
upon
peak
levels
of intracellular
calcium
attained.
Thus
a developmental
change
occurs
in calcium
buffering
and/or
resequestration,
and calcium
homeostasis
becomes
more
efficient
with
maturation.
Developmental
Changes
in the Magnitude
Fluorescence
Elevation
The peak. elevation
in fluorescence
with
tion
is significantly
larger
in young
than
neurons.
During
neuronal
development,
decrease
by one-third
in the nucleus
and
in thecytoplasm
(Figure3).
A500-msapplication
reliably
produces
a fluorescence
elevation
600%
that
is comparable
to peak
values
of
depolarizain mature
mean values
by one-half
of KCI
of about
of calcium
ntracellular
‘89
Ca*+ Release
1000
in Neuronal
Differentiation
-
800
-
600
-
400
-
.I
9s
l
.
iL
Nucleus
6
cytosol
.
o!
.
0.0
I
-
0.2
I
-
0.4
I.
I
0.6
0.8
-
,
-
1.0
I
1.2
Time (sets)
!‘igure
2. Temporal
Resolution
of Fluorescence
Changes
Achieved
with
Single
Line Scans
That
Also
Provide
Spatial
Information
Young neuron
loaded with flue-3AM
and depolarized
as in the previous
figure. (A) A single scan was made across the nerve cell body,
Including
the nucleus.
Transmission
image (left); fluorescence
image of loaded cell (right). (B) This measurement
was repeated
every
2 ms, and sequential
intensities
are displayed
from the top to the bottom.
Fluorescence
levels increase throughout
the scanned
region
upon depolarization
with high KCI for 500 ms. Fluorescence
elevations
reach the center of the nucleus
more slowly than the edges,
suggesting
a diffusion
driven mechanism.
(C) Fluorescence
changes
in the cytoplasm
and nucleus (left and right carats in [A] and [B])
were normalized
to their individual
resting levels and plotted. Smoothing
of noise from these rapid scans was accomplished
by spatial
averaging.
A column
(l-urn wide in [B]) was averaged
and smoothed
with a lO-ms wide, cosine-weighted
kernel.
greater
than
1 uM (Holliday
and Spitzer,
1990). This
response
has not saturated
the dye, since
longer
stimulation
produces
larger
peak elevation.
Saturation
is
generally
achieved
with
stimulus
durations
greater
than 1 s. Changes
in both calcium
influx
and intracellular
release
could
contribute
to the developmental
differences
in peak
elevations
and rates of rise.
Table
1. Kinetics
of Fluorescence
Changes
Maximum
in Young
and Mature
Rise Rates
(% Change/ms)
Contribution
of Calcium
Peak calcium
the magnitudeof
of fluorescence
are not seen
and presence
lar calcium
elevations
are relatively
insensitive
to
calcium
influx.
However,
elevations
are dependent
upon influx,
since they
in the absence
of extracellular
calcium
of 1 mM EGTA.
Reduction
of extracelluis expected
to reduce
calcium
influx
Influx
Neurons
Decline
Time
Constants
(s)
Young
Mature
Young
Mature
Nucleus
3.1 f 1.3 (6)
0.8 * 0.4 (4)
(:ytosol
9.5 f
0.6 f 0.3 (4)
Nucleus/DTBHQ
CytosollDTBHQ
0.4 (2)
0.4 (2)
6.0 * 1.1 (4)
0.7 + 0.52
5.0 f 1.7 (4)
0.2 * 0.04
24.5 (2)
16.5 (2)
2.1
0.4
1.6
0.2
27.2
22.2
5.7 (6)
0.09 (2)
0.07 (2)
f 0.5 (4)
f 0.16
f 0.1 (4)
& 0.07
II)
II)
The maximum
rate of rise in fluorescence
in response
to depolarization
is significantly
faster in young cytosol
than in mature cytosol
(055/o, Fisher’s PLSD). The decline
in fluorescence
was fitted with two time constants.
There are no significant
differences
in the faster
time constant.
The slower time constant
appears to change with maturation,
in the nucleus and cytosol (p <0.009 and 0.096, respectively).
Both time constants
are slow compared
with those of myocytes,
which are about 0.02 and 0.1 s (data not shown).
Values are mean *
SEM for the number
of observations
noted in parentheses.
Neuron
790
B
A
YOUNG
766
766-
766-
+ Nucleus
6W-
0
MATURE NEURON
NEURON
:
666
Myocyte
C
IJ Young
Cl Mature
neurons
neurons
0
0
2
4
8
6
Time (set)
Figure
3. Amplitudes
of Fluorescence
Elevations
in Young
and
Mature
Neurons
(A and B) Fluorescence
elevations
upon depolarization
are larger in the nucleus
than in the cytoplasm
and larger in young than in
mature neurons.
Records are from two representative
neurons.
The signal in a similarly
stimulated
myocyte
is displayed
for comparison
(inset, B). (C) Peak amplitudes
of fluorescence
elevation
decrease
with neuron age. Values are mean f SEM, normalized
to resting levels,
for the number of observations
noted in parentheses.
Paired comparisons
between different
ages but within compartments
are different
(p < 0.05).
der these conditions,
the amplitude
of depolarization-evoked
calcium elevations is largely unchanged
in the nucleus, despite the large reduction in extracellular calcium. Peak elevations may decrease less than
2-fold in the nucleus and less than 5-fold in the cytosol
(Figure4). Changes in influxaresomewhat
reflected in
cytosolic fluorescence
measurements,
but contribute
less to the change in fluorescence
in the nucleus.
These results suggest that the fluorescence
signal is
mainly determined
by other sources, such as calciumdependent
release from intracellular
stores (Lipscombe et al., 1988; Milani et al., 1990). A small calcium
influx may be sufficient
to trigger calcium release
from internal
stores, whereas influx makes only a
modest contribution
to the total signal.
through voltage-dependent
channels during depolarization. A 5-fold reduction
to 2 mM does not significantly affect average peak fluorescence
values in any
compartment
at any age (p > 0.1 for n = 4-7). Moreover, calcium elevations are often observed in young
neurons, even when extracellular
calcium has been
decreased IOO-fold, to 0.1 mM. At this concentration,
the macroscopic
calcium current is generally so small
as to be undetected
by whole-cell
voltage clamp,
although
a sufficient
calcium elevation
is achieved
during
a depolarizing
pulse to activate calciumdependent
potassium channels (Ribera and Spitzer,
1987; O’Dowd etal., 1988). Computer simulations
indicate that influx may also be reduced by as much as
IOO-fold (Lockery and Spitzer, unpublished
data). Un-
10 mM
Figure4.
Similar Peak Elevations
of Fluorescence
Elicited
under
Conditions
Expected
to Produce
Either Large or Small
Inward
Calcium
Currents
in the Same
Neuron
Calcium
(A) Responseof
ayoungcell
observed
upon
stimulation
by a 500-ms puff of 100 mM KCI
in 10 mM extracellular
calcium.
(B) Resting
and peak levels of fluorescence
decrease
slightly when the medium is changed
from
10 too.1 mM calcium,
and there is no elevation of fluorescence
in the absence
of extracellularcalcium
(data not shown). All fluorescence
values are normalized
to resting
levels in 10 mM extracellularcalcium.
Peak
fluorescence
elevations
change
less than
z-fold in the nucleus
and less than 5-fold in
the cytosol.
5
0
Time
(set)
5
Intracellular
Cab Release
in Neuronal
Differentiation
791
A
Caffeine
0
2
B
response
4
6
Depolarization
8
C
Depolarization
After
Caffeine Treatment
0
2
4
6
8
Time (set)
Figure
5. Caffeine
Causes
Fluorescence
Elevations
in Mature
Neurons
by Release
of Calcium
from
Intracellular
Stores
(A) Caffeine
(20 mM, 500 ms) elicits fluorescence
elevations
in both the cytosol
and nucleus of a mature
neuron.
Young neurons
fail
to respond
(inset) even with multiple
applications
of caffeine (5 in this example).
(B and C) A mature neuron that responds
to depolarization displays
reduced
elevations
of calcium
when intracellular
stores are depleted
by IO-min bath application
of caffeine.
Elevations of calcium are never evoked from mature
=ells in 0.1 mM extracellular
calcium. This is in conrrast to young cells, in which elevations
are unihanged or slightly reduced. The degree of variability
n peak elevations of calcium in young cells suggests
?:hat the small amount of calcium influx achieved by
depolarization
in 0.1 mM extracellular
calcium may
3e near the threshold
for release from stores. The
difference
between young and mature neurons may
be due to developmental
changes in this threshold.
fiowever, the variability of the response from neuron
:o neuron in low calcium, even when controlled
for
*age, prevented
construction
of a reliable calcium
dose-response
curve using these methods.
Contribution
of Release
from
Stores
Cultured
neurons contain intracellular
stores of calcium. Caffeinecan
be used todeplete
some intracellular calcium pools and is a useful probe with which to
examine calcium-handling
mechanisms
(Lipscombe
et al., 1988). It has been proposed to enhance the release of calcium from the endoplasmic
reticulum (Sitsapesan and Williams, 1990). Mature neurons consistently respond to a brief application
of caffeine (20
mM) with relatively slow elevations
in fluorescence,
in both the nucleus and cytoplasm,
even in the absence of extracellular
calcium (Figure 5). Young neurons appear to be relatively insensitive.
Eighty-eight
percent of mature neurons responded
whereas only
8% of young cells exhibited elevation of fluorescence
(n = 9 and 12), although all cells responded to depolarization with KCI. Calcium stores in young neurons
are clearly different from those in mature cells with
respect to the pharmacology
of release. The lack of
a response to caffeine does not indicate that young
neurons lack calcium stores, since neurons treated
with the calcium ionophore
ionomycin
(IO PM), in the
absence of extracellular
calcium and presence
of
’ mM EGTA, display substantial elevations in fluores-
cence (data not shown). lonomycin
can make intracellular membranes,
as well as the plasma membrane,
permeable to calcium and therefore effects release of
calcium from membrane-bound
intracellular
stores.
Calcium-induced
releaseof calcium from intracellular stores can be prevented
by depletion
of the stores
with caffeine prior to stimulation
(Lipscombe
et al.,
1988). Bath application
of 20 mM caffeine has no observable effect upon the depolarization-evoked
signal
in young cells, consistent with the general absence of
a response to caffeine alone (Figure 5A, inset). Depolarization of mature neurons pretreated with caffeine
produces
elevations
that are reduced
by approximately two-thirds
(Figures 56 and 5C). Peak values are
of similar magnitude
in both the cytosol and nucleus.
DTBHQ (2,5-di(tert-butyl)-l+benzohydroquinone)
(IO PM), which blocks the calcium ATPase associated
with the endoplasmic
reticulum
(Moore et al., 1987;
Kass et al., 1989), is effective in releasing calcium from
a subset of internal stores. This agent produces slow
elevations of neuronal fluorescence
upon addition to
the bath solution. A similar effect is observed in both
young and mature cells. The application
of DTBHQ
for as long as 6 hr has no detectable
effect on the
amplitude
of voltage-dependent
calcium currents (X.
Gu, unpublisheddata).
Similartreatment
has noeffect
on the amplitude
of the delayed rectifier potassium
current (M. Desarmenien,
unpublished
data). Thus
the slow elevation is likely to be the result of calcium
leak from intracellular
pools (Kass et al., 1989). Not all
calcium stores are depleted, since treated cells display
further elevation in fluorescence
when ionomycin
is
applied in calcium-free
medium (data not shown). Mitochondria
contain substantial calcium stores and are
likely to be unaffected by DTBHQ. We expect that the
endoplasmic
reticulum is depleted while other pools
are spared.
After treatment
with DTBHQ to deplete calcium
stores, depolarization
of neurons elicits a decreased
Neuron
792
A
B
Depolarization
Young
DeDolarization
After
D?BHQ
Treatment
C
DeDolarization
. Fecovery
After
neuron
+ Nucleus
J Cytosol
0
0
0
2
4
6
a
0
2
4
Time
Figure
6. Depletion
of Intracellular
(A) A young neuron
depolarized
cytosol.
(B) DTBHQ was added
the nucleus and l.bfold
in the
nucleus.
The inset displays
the
occur for 25 min. The fluorescence
levels in the nucleus.
Values in
of changing
dye concentrations
Stores
with
DTBHQ
Decreases
6
6
0
2
4
6
T
(set)
Fluorescence
Elevations
in Response
to Standard
Depolarization
in 10 mM extracellular
calcium
displays
a large elevation
of fluorescence
in both the nucleus
and
to the medium
to a final concentration
of 10 PM, causing
a small elevation
of fluorescence
@fold in
cytosol).
After 15 min, the fluorescence
response
to depolarization
is reduced
in both the cytosol and
same data on an expanded
scale. (C) DTBHQ
medium
was washed
out, and recovery
was allowed to
response
to depolarization
returns
to levels originally
achieved
in the cytosol
and exceeds
original
each panel are normalized
to the resting level of fluorescence
in that panel to minimize
contributions
over the long duration
of the experiment.
fluorescence
response
and
reduction
of the difference between
the nucleus
and cytosol
(Figure
6). Both
young
and mature
neurons
were affected
in a manner
similar
to depolarization
of mature
neurons
treated
with
caffeine
(see Figure
5C). Peak elevations
in depleted
neurons
are onlytwice
the resting
level in both
the nucleus
and cytosol
at both
ages. This result
suggests that changes
in influx
during
maturation
are not
the basis of changes
in peak
fluorescence,
since
the
remaining
signal
does not change
with age. While
the
resting
fluorescence
of both the nuclear
and cytosolic
signals
is increased
by calcium
release,
the difference
between
peak
evoked
fluorescence
in the nucleus
and the cytoplasm
is less apparent
when
the fluorescence
signal
is produced
by influx
alone
(Figure
6B),
implying
that the nuclear
signal
is especially
amplified
by calcium
release.
These
data further
indicate
that
calcium
release
from
stores
accounts
for the greater
part of the calcium
elevation.
The effect
of DTBHQ
is
reversible
within
0.5 hr by washing
out the drug
(Figure 6C).
The maximum
rate of rise in fluorescence
is approximately
IO-fold
slower
in DTBHQ-depleted
cells than
in untreated
cells in all compartments
at any age (Table 1). Elevations
of fluorescence
in depleted
neurons
in the presence
of DTBHQ
follow
the duration
of the
depolarizing
stimulus,
i.e., slow
elevations
continue
to increase
for the duration
of the stimulus
and then
decline
following
its termination.
These
observations
showthatthe
signal
in depleted
cells reflects
primarily
influx
rather
than
release
from
intracellular
stores.
The rate of decline
is also slowed
considerably,
and
in one case
achieved
a time
constant
of approximately
20 5.
Developmental
Significance
of Stores
Depletion
of calcium
stores
through
the application
of DTBHQaffects
neuronal
differentiation
in the same
manner
as inhibition
of calcium
influx,
during
a particular period
of development.
Previous
work
has shown
that cultures
grown
in the absence
of extracellular
calcium
display
a decreased
incidence
of neurons
with y-aminobutyric
acid (GABA)-like
immunoreactivity and increased
incidence
of neurons
with long processes
(Bixby
and Spitzer,
1984; Holliday
and Spitzer,
1990; Spitzer
et al., unpublished
data).
Furthermore,
theseeffectsareproduced
by intervention
specifically
at early
times
in culture
(Holliday
and Spitzer,
1990;
Spitzer
et al., unpublished
data).
Neurons
grown
in
the presence
of DTBHQ
for hours
6-12 of culture
also
display
increased
neurite
length
and decreased
GABA.like immunoreactivity
(Figure
7). The effect
is reduced
when
DTBHQ
is applied
during
other
6 hr periods.
The small
steady-state
elevation
of intracellular
calcium
due to calcium
ATPase
inhibition
does not support
standard
differentiation,
and elevations
due to
spontaneous
influx
(Holliday
and Spitzer,
1990) in the
absence
of amplification
by calcium-induced
calcium
release
are also insufficient.
Discussion
Calcium-dependent
release
of calcium
from
intracellular
stores
makes
a large
contribution
to the total
elevation
stimulated
by depolarization
of differentiating Xenopus
spinal
neurons.
Calcium
influx
is required
since depolarization
in theabsenceof
extracellular calcium
and presence
of ECTA is insufficient
to
elicit the standard
response.
However,
the response
ntracellular
‘93
Ca2+ Release
in Neuronal
Differentiation
80
Figure 7. Effects of DTBHQ
Neuronal
Differentiation
Mature
neurons
neurons
‘ercent
3f neurons
rith a neurite
‘onger
than
150pm
lfter 24 hr
60
10mMCa
Control
B
40
Percent
o! neurons
30
with GABA-like
immunoreactivity
after 24 hr
20
OCa
conditions
1-6
6-I 2
m
Period of DTBHQ
in 10 mM calcium
12-18
18-24 hr
Treatment
on
DTBHQ
treatment
of young
neurons
for
hours 6-12 of culture
alters neurite length
(A) and expression
of GABA-like
immuioreactivity(B)
in a manner similar to that produced by growth
in the absence
of extracellular calcium
during this period. A 6-hr
treatment
at other tirnes produces
smaller
alterations
in these two developmental
parameters.
Values are mean + SEM for 3 separate experiments;
typically
50-100
neurons were assessed for each condition.
The
columns
marked with stars aresignificantly
different
from standard
values in the presence of calcium
(95% level using Fisher’s
PLSD).
treatment
medium
10
-
0
I mM&
Control
OCa
conditions
16
3-l 2
Period of DTBHQ
in 10 mM calcium
s insensitive
to variation
of the external
calcium
con1:entration
over a wide range.
Furthermore,
depletion
of internal
calcium
stores
in the endoplasmic
reticuurn greatly
reduces
elevations
of calcium
produced
)y depolarization.
Thus,
most
of the stimulated
fluo‘escence
elevation
is due to amplification
of the influx
;ignal
by calcium
release.
The parallel
reduction
in
.ate of fluorescence
elevation
further
indicates
that
he endoplasmic
reticulum
is a major
contributor
to
he control
of calcium
levels.
Since
release
is depenjent upon
calcium
influx,
a calcium-sensitive
calcium
store
is likely
to be of primary
significance,
rather
han a calcium-insensitive
mitochondrial
pool
or an
nositol
trisphosphate-sensitive
store,
although
this
‘ias not been
directly
evaluated
(Verma
et al., 1990a,
1990b).
The large
reduction
in rate of fluorescence
decline
in the presence
of DTBHQ
suggests
that the
:alcium
ATPase
plays a major
role in calcium
homeo;tasis
through
rapid
resequestration.
Mitochondria
in these
cells
possess
sequestered
:alcium
stores.
The direct
investigation
of mitochonllrial contribution
to calcium
handling
using
standard
Jncoupling
agents
presented
difficulties.
In other
3 ‘1; 6-24 hr
treatment
medium
neuronal
systems,
application
of mitochondrial
poisons
causes
small
transient
elevations
of calcium
(Thayer
and Miller,
1990).
However,
FCCP
(carbonyl
cyanide
p-(trifluoromethoxy)phenylhydrazone)
caused
a large,
persistent
elevation
of calcium
that resulted
in cell lysis (data
not shown).
Examination
of mitochondrial
profiles
in neurons
at early stages
of development
reveals
putative
calcium
precipitates
in young
mitochondria
that
decrease
during
differentiation
(Lamborghini
et al., 1979).
Such
precipitates
suggest
that immature
Xenopus
neurons
possess
large
mitochondrial
calcium
stores,
which
may explain
the difference
in response
to mitochondrial
poisons
between
embryonic
Xenopus
neurons
and neonatal
rat
dorsal
root ganglion
cells.
Other
cellular
organelles
can contribute
to the observed
fluorescence
values.
Inclusion
of large
yolk
platelets
and lipid granules
in the analysis
was avoided.
Movements
of these
organelles
were
rare, and experiments
were
terminated
when
such
movements
were
observed.
However,
smaller
organelles
may contribute to averaged
fluorescence
measurements.
The
magnitude
of their
contribution
will depend
upon
Neuron
794
several
factors,
including
the occupied
fractional
volume, extent
of dye loading,
level of free calcium,
and
degree
of organelle
movement.
Large elevations
of calcium
indicator
fluorescence
have
been observed
in neuronal
nuclei
and may be
due to release
of calcium
from
the nuclear
envelope
(Hernandez-Cruz
et al., 1990;
Przywara
et al., 1991).
Both nuclear
and cytosolic
elevations
are sensitive
to
depletion
of an endoplasmic
reticulum
store that may
be contiguous
with the nuclear
envelope.
Elevations
observed
in the cytosol
are influenced
to a greater
extent
by influx
than
those
associated
with
the nucleus.
Nuclei
are thought
to possess
nonselective
pores,
although
some
recent
evidence
suggests
that
gradients
of ions
can be maintained.
A potassiumselective
permeability
has been
reported
for the nucleus
(Mazzanti
et al., 1990), and other
channels
may
be present
(Matzke
et al., 1990; Wozniak
et al., 1989).
The number
of nuclear
pores
depends
on the metabolic
state of the cell (Johnson
and Sears,
1989; Perez
et al., 1991). Isolated
hepatic
nuclei
have been shown
to regulate
intranuclear
calcium
through
calcium
pumping
and release
from
nuclear
stores
(Nicotera
et
al., 1989). Interestingly,
stores
associated
with isolated
hepatic
nuclei
are insensitive
to DTBHQ,
perhaps
as
a result
of physical
separation
of the endoplasmic
reticulum
from
the nuclear
envelope.
The nucleus
responds
differently
from
the cytosol
under
standard
culture
conditions.
The difference
between
nuclear
and cytosolic
elevations
collapses
when
intracellular
stores
are depleted,
suggesting
that
release
from
stores
selectively
amplifies
the nuclear
signal;
however, the magnitude
of the nuclear
elevation
may be
artificially
exaggerated
as a result
of different
dye behavior
in this compartment.
Furthermore,
the differences
in neither
rise nor decline
rates
between
nucleus
and cytoplasm
can be explained
by differences
in linear
scaling
of the fluorescence
signal.
The apparently
preferential
release
of calcium
associated
with
the nucleus
may decrease
with
development as a result
of ultrastructural
changes.
Early in the
differentiation
of Xenopus
neurons,
the nucleus
is
lobulated
with
cytoplasmic
intrusions
in the crosssectional
profile
examined
by transmission
electron
microscopy
(Lamborghini
et al., 1979). As differentiation proceeds,
the nuclear
profile
becomes
more
regular and spherical.
This early
topology
may facilitate
vectorial
release
of calcium
from
the endoplasmic
reticulum
and nuclear
envelope
to the nuclear
compartment,
as well as an increase
in nuclear
export
of RNA
at this time (Newport
and Forbes,
1987).
The decrease
in calcium
release
from
stores
during
differentiation
has several
possiblecauses.
The sizeor
subcellular
location
of calcium-releasable
pools
may
change
with
maturation,
and the number
of release
channels
may also decrease.
Our evidence
suggests
that the calcium
threshold
for calcium-dependent
release from
stores
increases
as cells mature.
Depolarization
in low extracellular
calcium
is less likely
to
produce
an amplified
response
in mature
neurons
than
in young
cells.
Changes
in the structure
of the
releasechannel
occurringwith
differentiation
mayaccount
for changes
in sensitivity
to calcium
and pharmacological
agents
such
as caffeine
(Sutko
et al.,
1991).
Decreased
influx
does
not appear
to be involved.
However,
increased
calcium
buffering,
sequestration,
and pumping
are likely
to play a role,
since
maturational
changes
were
revealed
in analysis
of the time constants
of fluorescence
decay.
The efficiency
of homeostatic
mechanisms
appears
to increase
with
age and may prevent
attainment
of the
large calcium
elevations
seen in immature
neurons.
Blockade
of the high affinity
calcium
ATPase
associated with
the endoplasmic
reticulum
increases
the
resting
concentration
of intracellular
calcium,
as expected
(Kass et al., 1989). The steady-state
concentration of calcium
is then
likely
to be determined
by
other
homeostatic
mechanisms,
such as mitochondrial
buffering,
plasma
membrane
calcium
ATPase
and the
sodium/calcium
exchanger
(Rasmussen
and Barrett,
1984).
However,
this
new,
elevated,
and sustained
level of calcium
is not sufficient
to enable
standard
differentiation.
Higher
elevations
of calcium
achieved
through
internal
release
under
standard
conditions
are apparently
required.
The release
of calcium
from
stores,
which
greatly
elevates
calcium
levels,
is required
for standard
neuronal differentiation
in culture.
The period
of sensitivity to depletion
corresponds
to the period
of sensitivity to the removal
of extracellular
calcium
and to the
period
of most frequent
spontaneous
calcium
elevations (Hollidayand
Spitzer,
1990; Spitzer
et al., unpublished
data).
Since
calcium
and potassium
currents
are not inhibited
by DTBHQ,
spontaneous
calcium
influx
alone
appears
insufficient
to support
standard
differentiation.
Calcium
influx
was previously
shown
to be necessary
for differentiation
and is now shown
to be required
to trigger
the critical
release
of calcium
from
stores.
The calcium
concentration
of the culture
medium
may cause
increased
loading
of neuronal
calcium
stores
relative
to those
of neurons
in vivo.
The
contributions
of both influx
and release
to patterns
of
differentiation
in situ have yet to be investigated.
A prominent
theme
in differentiation
of spinal
neurons is the decrease
in calcium
influx
during
maturation of the action
potential
(Spitzer,
1985). Since spontaneous
calcium
elevations
have an important
role in
differentiation
(Holliday
and Spitzer,
1990), depolarization-induced
elevations
may be maximized
during
a developmentally
critical
period,
when
epigenetic
mechanisms
are shaping
differentiation.
These
elevations
may be minimized
upon
maturation,
when
the
function
of action
potentials
is largely
for rapid signaling.
Developmental
changes
in calcium
handling
through
influx,
release,
and buffering
may be redundant or interactive
mechanisms
to accommodate
the
changing
role of impulse
activity
in these
neurons.
We hypothesize
that
spontaneous
production
of
long,
calcium-dependent
action
potentials
triggers
the release
of calcium
from
intracellular
stores
that
Intracellular
795
CaZ+ Release
in Neuronal
Differentiation
supports
standard
differentiation.
In contrast,
short,
sodium-dependent
action
potentials
may fail to trigger release
from
stores,
or may trigger
only small elecations.ThesefactorscouIdcontributetotheobservation
of high
frequencies
of spontaneous
calcium
elevations
occurring
during
the period
of time when
calcium-dependent
action
potentials
can
be produced.
Simultaneous
tions
of cells
in vivo
impulse
activity
with
environment.
Experimental
electrical
and optical
observawill extend
the correlations
of
calcium
handling
in the normal
Procedures
Neurons
were cultured
from Xenopus
embryos
at neural plate
stage as previously
described
(Holliday
and Spitzer, 1990). Standard culture
medium
contained
10 mM calcium.
Neurons
were
recognized
by neurite
extension,
after about 6 hr in culture.
Neurons
were considered
“young”
at this time, when
longduration
action potentials
can be elicited.
Neurons
were considered “mature”
at 1 day in culture,
when action potentials
are
brief (Spitzer, 1985). Cells were loaded with thecalcium
indicator
flue-3AM
(Minta et al., 1989), at a concentration
of 3 uM for 30
min. They were then washed and examined
within 2 hr. Intracellular
concentration
of deesterified
dye was estimated
to be
5-10 uM by comparison
with standards
of known concentration
contained
in 20-pm thick microslides
(Vitro Dynamics,
Rockaway, NJ). Calcium
buffers
(O-10 uM free calcium)
were included
irr these standards
and were imaged under the same conditions
used to estimate
experimental
calcium
concentrations.
Calcium
concentrations
were calculated
from stability
constants
(Martell
and Smith, 1974; Robertson
and Potter, 1984). Cultures
were
maintained
and experiments
were performed
at room temperature (20°C-220C).
Calcium
influx was evoked
by depolarization
of neurons,
using a picospritzer
(General
Valve Corp.) to apply a pulse of standardculturemediumcontaining100mMKCI.Thestimuluspulse
was 500 ms in duration,
and its decay was determined
with fluorescent
tracer.
Decay of potassium
concentration
to a level at
which the membrane
potential
is subthreshold
for activation
of
high voltage-activated
calcium
current
occurred
within 100-200
ms.Thestimulusdurationissimilartothatofcalciumdependent
action potentials
and reliably elicited a response
from neurons
when the pipette was positioned
between
5 and 10 urn from the
soma. Release of calcium
from intracellular
stores was evoked
bybathapplicationofvariousagentsorbystimulationofcalcium
influx
by picospritzer.
Caffeine
was obtained
from
Sigma,
DTBHQ
from Aldrich,
ionomycin
from Calbiochem,
and Fluo3AM from Molecular
Probes.
Images were acquired
with a Bio-Rad MRC-600 confocal
imaging system
attached
to a standard
Zeiss upright
microscope
using a Zeiss 40x 0.75 NA water-immersion
objective.
The depth
of focus of the optical slice was approximately
1 pm. Most data
were collected
in a line-scanning
mode (Hernandez-Cruz
et al.,
1990, and Figure 21, then transferred
to a Macintosh
computer.
The Image program
(W. Rasband,
National
Institutes
of Health)
was used to extract fluorescence
intensity
values over time. Are.as analyzed
from line scans (see Figure 2B) were chosen to best
avoid large organelles
and were about 1 x 0.125 urn. Thus the
fluorescence
measured
in these experiments
is equivalent
to a
volume
of about 0.125 pm3 within the cell. A plane of focus that
passed through
the approximate
center of the nucleus
of linescanned
cells was chosen.
To minimize
contributions
of spatial
v,uiations
in dye concentration
to fluorescence
changes
resulting from calcium
transients,
fluorescence
intensities
in diffr,rent
regions
of the cell were normalized
as a percentage
of
p-estimulated
levels.
Absolute
calcium
concentrations
reflected
by elevations
of
fl.do-3 fluorescence
were derived
using the method of Kao et al.
(1988). However,
treatment
of neurons
with the calcium
iono-
phore ionomycin
causes large calcium
elevations
and rapid leakage of dye, preventing
meaningful
calculation
of calcium
concentrations
with this method.
Cell lysis eventually
ensued.
Given the difficulty
of direct calibration
of calcium concentration, the results were expressed
in terms of relative fluorescence
that is proportional
to the actual calcium concentration
(CornellBell et al., 1990). Consistent
percent
changes
in fluorescence
were achieved
when values resulting
from stimulation
were normalized to those at rest. Based on previous
furameasurements
(Holliday
and Spitzer, 1990), resting levels (100%) were estimated
to lie between
200 and 400 nM calcium
and the average resting
level did not change
during
development.
This high estimate
could be due to culture
conditions
in high extracellular
calcium,
although
developing
neurons
may have generally
high resting
levels of calcium
(Bentley
et al., 1991). Transients
reaching
an
elevation
of 600% are thus >I PM intracellular
calcium.
Normalization of intensity
changes
to initial fluorescence
levels reflects
changes
in free intracellular
calcium
in a semiquantitative
manner. The maximum
rate of rise was calculated
from
data
smoothed
using a cosine-weighted
kernel. Rates were calculated
as the difference
between
neighboring
points
according
to
It+1 - 11-1
2dt
where I = fluorescence
intensity.
Decay rates were fitted to two
exponentials
using the SIMPLEX algorithm
(Nelder
and Mead,
1965).
The developmental
effects of depletion
of calcium
from intracellular stores were assessed
by treatment
for 6hr periods with
DTBHQ,
which stimulates
release of calcium from the endoplasmic reticulum
(Moore
et al., 1987). Washout
was followed
by
analysis
of neurite
length and GABA-like
immunoreactivity
at
24 hr in vitro (Bixby and Spitzer, 1984; Spitzer et al., unpublished
data).
Acknowledgments
We thank Rosario C. de Baca for technical
assistance
and Beverly
Clendening
for review of the manuscript.
J. H. is a fellow of the
USPHS. Grants ONR N00014-89-J-1766
and NSF BNS-9006600
to
T. 1. S. and NIH 15918 to N. C. S. are acknowledged.
N. C. S. is
a Fellow of the J. S. Guggenheim
Foundation.
The costs of publication
of this article were defrayed
in part
by the payment
of page charges.
This article must therefore
be
hereby marked “advertiseme&
in accordance
with 18 USC Section 1734 solely to indicate this fact.
Received
June
4, 1991; revised
August
6, 1991.
References
Baccaglini,
P. I., and Spitzer, N. C. (1977). Developmental
changes
in the inward
current
of the action potential
of Rohon-Beard
neurones.
J. Physiol. 277, 93-117.
Bentley,
D., Cuthrie,
P. B., and Kater, S. B. (1991). Calcium
ion
distribution
in nascent
pioneer
axons and coupled
preaxonogenesis
neurons
in situ. J. Neurosci.
77, 1300-1308.
Bixby, J. L., and Spitzer, N. C. (1984). Early differentiation
brate spinal neurons
in the absence
of voltage-dependent
and Na+ influx. Dev. Biol. 706, 89-96.
of verteCa2+
Brandt, C. J., deleon,
S., Martin,
D. R., and MacLennan,
D. H.
(1987). Adult forms of the Ca2+ ATPase of sarcoplasmic
reticulum
expression
in developing
skeletal
muscle.
I. Biol. Chem. 262,
3768-3774.
Braun, K., Scheich,
H., Zuschratter,
W., Heizmann.
C. W., Matute, C., and Streit, P. (1988). Postnatal
development
of parvalbumin-,
calbindin-,
and adult GABA-immunoreactivity
in two visual nuclei of zebra finches.
Brain Res. 475, 205-217.
Cimino,
M., Chen,
J. F., and Weiss,
B. 11990). Ontogenetic
devel-
NeUrOn
796
opment of calmodulin
tion histochemistry.
mRNA in rat brain using
Dev. Brain Res. 54, 43-49.
in situ hybridiza-
Cohan, C. S., Connor,
J. A., and Kater, S. B. (1987).
and chemically
mediated
increases
in intracellular
neuronal
growth
cones. J. Neurosci.
7, 3588-3599.
Electrically
calcium
in
Cornell-Bell,
A. H., Finkbeiner,
S. M., Cooper,
M. S., and Smith,
S. J. (1990). Glutamate
induces calcium
waves in cultured
astrocytes: long-range
glial signaling.
Science 247, 470-473.
Gishan,
F. K., and Arab, N. (1988). Active
intestinalendoplasmicreticulumduringmaturation.Am.J.
iol. 254, C74-80.
calcium
transport
by
Phys-
Henderson,
L. P., Smith, M. A., and Spitzer,
N. C. (1984). The
absence of calcium blocks impulse-evoked
release of acetylcholine but not de nova formation
of functional
neuromuscular
synaptic
contacts
in culture.
J. Neurosci.
4, 3140-3150.
Hernandez-Cruz,
A., Sala, F., and Adams, P. R. (1990). Subcellular
calcium
transients
visualized
by confocal
microscopy
in a voltage-clamped
vertebrate
neuron.
Science 247, 858-862.
Holliday, J., and Spitzer, N. C. (1990). Spontaneous
and its roles in differentiation
of spinal neurons
Biol. 747, 13-23.
Johnson,
I. P., and Sears, T. A. (1989).
thoracic
alpha- and gamma-motoneurons
Brain Res. 489, 400-405.
calcium
in culture.
influx
Dev.
Organelle
changes
in cat
following
axotomy.
Kao, J. P. Y., Harootunian,
A. T., and Tsien, R. Y. (1989).
chemically
generated
cytosolic
calcium
pulses and their
tion by flue-3. J. Biol. Chem. 264, 8179-8184.
Photodetec-
Kass, G. E. N., Duddy, S. K., Moore, CA., and Orrenius,
S. (1989).
2,5-Di-(tert-butyl)-l,&benzohydroquinone
rapidly elevates cytosolic Ca*+ concentration
by mobilizing
the inositol 1,4,5-triphosphate-sensitive
CaZt pool. J. Biol. Chem. 264, 15192-15198.
Lamborghini,
J. E., Revenaugh,M.,and
Spitzer, N. C. (1979). Ultrastructural
development
of Rohon-Beard
neurons:
loss of intramitochondrial
granules
parallels loss of calcium
action potentials.
J. Comp. Neural.
783, 741-752.
Leberer,
E., Seedorf,
U., and Pette,
gene expression
in skeletal muscle.
teins in developing
and chronically
muscles.
Biochem.
J. 239, 295-300.
D. (1986). Neural control
of
Calcium-sequestering
prostimulated
rabbit skeletal
Lipscombe,
D., Madison,
D. V., Poenie, M., Reuter, H., Tsien,
R. W., and Tsien, R. Y. (1988). Imaging of cytosolic
Ca*+ transients
arising from Ca *+ stores and Ca2+ channels
in sympathetic
neurons. Neuron
7, 355-365.
Martell,
Volumes
A. E., and Smith, R. M. (1974). Critical
l-4 (New York: Plenum Publishing
Stability
Corp.).
Constants,
Matzke,
A. J., Weiger, T. M., and Matzke,
M. A. (1990). Detection
of a large cation-selective
channel
in nuclear envelopes
of avian
erythrocytes.
FEBS Lett. 277, 161-164.
Mazzanti,
channels
M., DeFelice,
in the nuclear
L. J., Cohn,
envelope.
J., and Malter, H. (1990).
Nature 343, 764-767.
Ion
Milani,
D., Malgaroli,
A., Guidolin,
D., Fasolato,
C., Skraper,
S. D., Meldolesi,
j., and Pozzan,
T. (1990). Ca2+ channels
and
intracellular
Ca2+ stores in neuronal
and neuroendocrine
cells.
Cell Calcium
77, 191-199.
Minta, A., Kao, J. P. Y., and Tsien, R. Y. (1989). Fluorescent
indicators for cytosolic
calcium
based on rhodamine
and fluorescein
chromophores.
J. Biol. Chem. 264, 8171-8178.
Moore, G. A., McConkey,
D. J., Kass, G. E. N., O’Brien,
Orrenius,
S. (1987). 2,5-Di(tert-butyl)-I,4benzohydroquinone-a
novel inhibitor
of liver microsomal
CaZ+ sequestration.
224, 331-336.
Nelder,
J. A., and Mead, R. (1965). A geometric
optimisation.
Computer
J. 7, 308-327.
Newport,
function,
P. J., and
FEBS Lett.
technique
for
J. W., and Forbes, D. J. (1987). The nucleus:
structure,
and dynamics.
Annu. Rev. Biochem.
56, 535-565.
Nicotera,
P., McConkey,
D. J., Jones, D. P., and Orrenius,
S.
(1989). ATP stimulates
Ca*+ uptake and increases
the free Cal’
concentration
in isolated
rat liver nuclei. Proc. Natl. Acad. Sci.
USA 86, 453-457.
Q’Dowd,
D. K., Ribera, A. B., and Spitzer, N. C. (1988). Developmentof
voltage-dependent
calcium,
sodium, and potassium
currents in Xenopus
spinal neurons.
J. Neurosci.
8, 792-805.
Perez, J., Hernandez,
P., and Garcia-Segura,
L. M. (1991). Estradiol
increases
the number
of nuclear
pores in the arcuate
neurons
of the rat hypothalamus.
J. Comp. Neural.
303, 225-232.
Przywara,
D.A., Bhave, S. V., Bhave, A., Wakade,
T. D., and Wakade, A. R. (1991). Stimulated
rise in neuronal
calcium
is faster
and greater in the nucleus than in the cytosol.
FASEB J. 5, 217222.
Rasmussen,
H., and
system:
an integrated
Barrett,
view.
P. Q. (1984). Calcium
messenger
Physiol. Rev. 64, 938-983.
Ribera, A. B., and Spitzer, N. C. (1987). Both
activate neuronal
potassium
currents.
Proc.
84, 6577-6581.
barium and calcium
Natl. Acad. Sci. USA
Robertson,
S., and Potter, J. D. (1984). The regulation
of free Cal+
ion concentration
by metal chelators.
In Methods
in Pharmacology, Volume 5, A. Schwartz,
ed. (New York: Plenum Publishing
Corp.),
pp. 63-75.
Sitsapesan,
R., and Williams,
A. J. (1990). Mechanisms
of caffeine
activation
of single calcium-release
channels
of sheep cardiac
sarcoplasmic
reticulum.
J. Physiol. 423, 425-439.
Spitzer,
N. C. (1985). The control
of development
of neuronal
excitability.
In Molecular
Bases of Neural Development,
C. M.
Edelman, W. E. Gall, and W. M. Cowan, eds. (New York: Rockefeller University
Press), pp. 67-88.
Spitzer, N. C., and Lamborghini,
J. E. (1976). The development
of
the action potential
mechanism
of amphibian
neurons
isolated
in culture.
Proc. Natl. Acad. Sci. USA 73, 1641-1645.
Sutko, J. L., Airey, J. A., Murakami,
K., Takeda,
M., Beck, C.,
Deerinck,
T., and Ellisman,
M. A. (1991). Foot protein
isoforms
are expressed
at different
times during embryonic
chick skeletal
muscle development.
J. Cell Biol. 773, 793-803.
Thayer, S. A., and Miller, R. J. (1990). Regulation
of the intracellular free calcium concentration
in single rat dorsal root ganglion
neurones
in vitro. J. Physiol. 425, 85-115.
Verma,A.,
Hirsch, D. J., Hanley, M. R.,Thastrup,
O., Chistensen,
S. B., and Snyder, S. H. (1990a). lnositol triphosphate
and thapsigargin discriminate
endoplasmic
reticulum
stores of calcium
in
rat brain. Biochem.
Biophys.
Res. Commun.
772, 811-816.
Verma, A., Ross, C. A., Verma, D., Supattapone,
S., and Snyder,
S. H. (1990b). Rat brain endoplasmic
reticulum
calcium
pools are
anatomically
and functionally
segregated.
Cell Reg. 7, 781-790.
Vidal, S., Raynaud,
B., and Weber,
M. J. (1989). The
channels
of the L type in neurotransmitter
plasticity
sympathetic
neurons.
Mol. Brain Res. 6, 187-196.
Walicke,
P. A., and Patterson,
the transmitter
choice made
J. Neurosci.
7, 343-350.
role of Ca2+
of cultured
P. H. (1981). On the role of Ca*+ in
by cultured
sympathetic
neurons.
Wozniak,
R. W., Bartnik,
E., and Blobel,
ture analysis
of an integral membrane
clear pore. J. Cell Biol. 72, 183-195.
C. (1989). Primary strucglycoprotein
of the nu-