Patterns of Intracellular Calcium Fluctuation in Precursor Cells of the Neocortical Ventricular Zone

Abstract

Changes in intracellular free calcium concentration ([Ca2+]i) are known to influence a variety of events in developing neurons. Although spontaneous changes of [Ca2+]i have been examined in immature cortical neurons, the calcium dynamics of cortical precursor cells have received less attention. Using an intact cortical mantle and confocal laser microscopy, we examined the spatiotemporal patterns of spontaneous [Ca2+]i fluctuations in neocortical ventricular zone (VZ) cells in situ. The majority of activity consisted of single cells that displayed independent [Ca2+]i fluctuations. These events occurred in cells throughout the depth of the VZ. Immunohistochemical staining confirmed that these events occurred primarily in precursor cells rather than in postmitotic neurons. When imaging near the ventricular surface, synchronous spontaneous [Ca2+]i increases were frequently observed in pairs of adjacent cells. Cellular morphology, time-lapse imaging, and nuclear staining demonstrated that this activity occurred in mitotically active cells. A third and infrequently encountered pattern of activity consisted of coordinated spontaneous increases in [Ca2+]i in groups of neighboring VZ cells. The morphological characteristics of these cells and immunohistochemical staining suggested that the coordinated events occurred in gap junction-coupled precursor cells. All three patterns of activity were dependent on the release of Ca2+ from intracellular stores. These results demonstrate distinct patterns of spontaneous [Ca2+]i change in cortical precursor cells and raise the possibility that these dynamics may contribute to the regulation of neurogenesis.

Full text

Patterns of Intracellular Calcium Fluctuation in Precursor Cells of
the Neocortical Ventricular Zone
David F. Owens and Arnold R. Kriegstein
Department of Neurology and The Center for Neurobiology and Behavior, College of Physicians and Surgeons of
Columbia University, New York, New York 10032
Changes in intracellular free calcium concentration ([Ca
21
]
i
)are
known to influence a variety of events in developing neurons.
Although spontaneous changes of [Ca
21
]
i
have been examined
in immature cortical neurons, the calcium dynamics of cortical
precursor cells have received less attention. Using an intact
cortical mantle and confocal laser microscopy, we examined
the spatiotemporal patterns of spontaneous [Ca
21
]
i
fluctua-
tions in neocortical ventricular zone (VZ) cells in situ. The
majority of activity consisted of single cells that displayed
independent [Ca
21
]
i
fluctuations. These events occurred in
cells throughout the depth of the VZ. Immunohistochemical
staining confirmed that these events occurred primarily in pre-
cursor cells rather than in postmitotic neurons. When imaging
near the ventricular surface, synchronous spontaneous [Ca
21
]
i
increases were frequently observed in pairs of adjacent cells.
Cellular morphology, time-lapse imaging, and nuclear staining
demonstrated that this activity occurred in mitotically active
cells. A third and infrequently encountered pattern of activity
consisted of coordinated spontaneous increases in [Ca
21
]
i
in
groups of neighboring VZ cells. The morphological character-
istics of these cells and immunohistochemical staining sug-
gested that the coordinated events occurred in gap junction-
coupled precursor cells. All three patterns of activity were
dependent on the release of Ca
21
from intracellular stores.
These results demonstrate distinct patterns of spontaneous
[Ca
21
]
i
change in cortical precursor cells and raise the possi-
bility that these dynamics may contribute to the regulation of
neurogenesis.
Key words: neurogenesis; intracellular calcium; cell cycle;
corticogenesis; ventricular zone; embryonic cortex; calcium
imaging
Most neurons of the neocortex arise from precursor cells in the
ventricular zone (VZ), a pseudostratified proliferative epithelium
that lines the lateral ventricles (Berry and Rogers, 1965; Boulder
Committee, 1970). In the rat, neurogenesis proceeds from ap-
proximately embryonic day 13 (E13) to E21 (Bayer and Altman,
1995). During this time, precursor cells undergo interkinetic
nuclear migration (Seymour and Berry, 1975) in which cells in the
DNA synthetic S phase have their nuclei in the upper third of
the VZ. When cells pass from S to G
2
, the nuclei migrate toward
the VZ surface where mitosis occurs. After mitosis, daughter cells
can either remain proliferative and re-enter the cell cycle or
become terminally postmitotic and migrate out of the VZ (Mc-
Connell, 1995). Glial cells are primarily produced in a second
germinal zone, the subventricular zone that is located superfi-
cially to the VZ. Glial cell production increases as neurogenesis
declines, peaking during the early postnatal period (Bayer and
Altman, 1991).
Both intrinsic and extrinsic signals are likely to influence the
proliferative potential and eventual fates of precursor cells within
the VZ. For example, groups of adjacent precursor cells in dif-
ferent stages of the cell cycle are coupled within the VZ into
discrete columnar cell clusters by gap junction channels (LoTurco
and Kriegstein, 1991; Bittman et al., 1997). This allows for the
spread of electrical and chemical signals to cells within a defined
radial compartment within the VZ. In addition, regulation of gap
junction coupling seems to influence progression through the cell
cycle (Bittman et al., 1997). Ventricular zone cells have also been
shown to respond to local environmental signals through peptide
and neurotransmitter receptors that in turn can regulate the rate
of DNA synthesis in these cells (L oT urco et al., 1995; L u and
DiCicco-Bloom, 1997). Finally, transplantation experiments have
demonstrated that the laminar fate of early generated neurons is
influenced by environmental cues that commit cells to a specific
deep layer fate just before their final mitosis (McConnell and
Kaznowski, 1991; Bohner et al., 1997). However, upper layer
neurons seem to be fated for superficial layers independent of
environmental signals (Frantz and McConnell, 1996).
Modulation of intracellular free calcium concentration
([Ca
21
]
i
) may be part of the signaling pathway by which both
local environmental factors and cell autonomous developmental
programs influence corticogenesis. Calcium is a ubiquitous sec-
ond messenger that has been implicated in the regulation of a
variety of events in developing neurons, including differentiation
(Spitzer and Gu, 1997), migration (Komuro and Rakic, 1992,
1996), and circuit formation (Yuste et al., 1992; Wong et al., 1995;
Feller et al., 1996). Previous studies have demonstrated that
spontaneous [Ca
21
]
i
fluctuations occur in immature postmigra-
tory neurons in the postnatal cortex (Yuste et al., 1992, 1995;
Received Jan. 21, 1998; revised March 23, 1998; accepted April 24, 1998.
This work was supported in part by Grant FY95– 0879 from the March of Dimes
Birth Defects Foundation and by Grant NS 21223 from National Institutes of Health.
The confocal facility was established by National Institutes of Health Shared Instru-
mentation Grant 1S10 RR10506 and is supported by National Institutes of Health
Grant 5 P30 CA13696 as part of the Herbert Irving Cancer Center at C olumbia
University. We thank Theresa Swayne for technical assistance with the confocal
microscope, Drs. A. Frankf urter and J. E. Goldman for generously providing the
primary antibodies to T uJ1 and vimentin, Eric Kriegstein for help with the illus-
trations, and Dr. Raphael Yuste, Alexander Flint, Dr. Xiaolin Liu, and Dr. Joseph
LoT urco for helpf ul comments on the manuscript.
Correspondence should be addressed to Dr. A rnold R. Kriegstein, Department of
Neurology, College of Physicians and Surgeons of Columbia University, 630 West
168th Street, Box 31, New York, NY 10032.
Copyright © 1998 Society for Neuroscience 0270-6474/98/185374-15$05.00/0
The Journal of Neuroscience, July 15, 1998, 18(14):5374–5388

Owens et al., 1996). Less understood are the Ca
21
dynamics of
cortical precursor cells in the proliferative zone. The possibility
that Ca
21
-dependent signaling mechanisms in precursor cells
might influence neurogenesis led us to investigate the endoge-
nous Ca
21
dynamics of cells within the intact neocortical VZ.
MATERIALS AND METHODS
Tissue preparation. Results were obtained from slices and slabs of telen-
cephalic hemispheres obtained from litters of rat pups ranging in age
from E15 to E20. Gravid Sprague Dawley rats (Taconic, Germantown,
NY) were anesthetized with an intraperitoneal injection of ketamine (50
mg/kg) and xylazine (10 mg/kg), and embryos were exposed by cesarean
section. Embryos were decapitated, and heads were immediately placed
in ice-cold artificial CSF (ACSF) (124 mMNaCl, 5 mMKCl, 1.25 mM
NaH
2
PO
4
,1mMMgSO
4
,2mMCaCl
2
,26mMNaHCO
3
, and 10 mM
glucose) oxygenated with 95% O
2
/5% CO
2
, pH 7.4. Cerebral hemi-
spheres were prepared as slabs of neocortex by trimming off the hip-
pocampus and striatal anlage (see Fig. 1E). For experiments requiring
brain slices, whole embryonic brains were removed and embedded in
warm (28–30°C) 3–4% low-melting agarose (Fisher Scientific, Houston,
TX) in AC SF, were hardened on ice, and were sliced into coronal
sections (300–400
m
m) with a vibratome.
Calcium imaging. Neocortical slabs and slices were loaded in the dark
with the Ca
21
indicator dye fluo-3 by immersion for at least 30 min in
ACSF containing fluo-3 AM (10
m
M) followed by a brief ACSF wash.
Tissue was placed in a imaging chamber continuously perfused with
oxygenated ACSF, on the stage of a Zeiss Axiovert microscope (403;
numerical aperture, 0.75 objective). I llumination was provided with
either a Bio-Rad MRC-600 argon laser scanning confocal attachment or
a Zeiss argon crypton laser scanning confocal attachment. E xcitation
was at 488 nm, and emissions were collected using a 515 nm long-pass
emission filter. Neutral density filters were used to filter the argon laser
light to 1% to minimize photobleaching. Generally, one image was
acquired every 2.88–7 sec, with each image consisting of an average of
two to four frames. In some experiments, we used faster (up to one
image every 0.2 sec without frame averaging) or slower (down to one
image every 11 sec to average up to 16 frames per image) image acquisition.
Images were acquired on an IBM-compatible computer running either
Comos (Bio-Rad, Hercules, CA) or LSM (Zeiss) acquisition software.
Fluorescence micrographs were digitized, and relative changes in
[Ca
21
]
i
were measured in selected cells using the public domain National
Institutes of Health Image program (written by Wayne Rasband at the
National Institutes of Health) on a Macintosh 7200 computer. Data are
expressed as a change in fluorescence over baseline fluorescence (DF/F).
Depending on the number of images acquired per experimental trial, the
baseline F was defined as the image with the minimum level of fluores-
cence or as an average of the five minimum images for each trial.
Intracellular Ca
21
transient durations were estimated by measuring the
time from the initial deviation from baseline to return (Ferrari et al.,
1996). Average values are expressed as mean 6SEM. Statistical analysis
was performed with a two-tailed Student’s ttest, and pvalues of #0.05
were considered statistically significant. Unless otherwise stated, all
experiments were performed at room temperature (RT; 21–25°C).
Ca
21
imaging and subsequent cell identification. In some experiments,
tissue was processed for immunohistochemistry after C a
21
imaging. In
these experiments, maps were made of anatomical landmarks, and the
orientation of tissue in the imaging chamber was recorded so that the
same areas could be visualized subsequently for neuronal-specific immu-
noreactivity. After we performed live C a
21
imaging, the tissue was fixed
in 4% paraformaldehyde and stored overnight at 4°C. Tissue was washed
in PBS and then permeabilized and blocked in PBS with 0.5% Triton
X-100 and 10% normal goat serum (NGS) for 1 hr at RT. Tissue was then
incubated overnight at 4°C with anti-T uJ1 primary antibody (1:500
dilution; generously provided by Dr. A. Frankf urter, University of Vir-
ginia) in PBS with 0.1% Triton X-100 and 3% NGS. Tissue was washed
and then incubated for 1 hr at RT with rhodamine-conjugated anti-
mouse secondary antibody (1:200 dilution; ICN Biomedicals, Cleveland,
OH) in PBS with 0.1% Triton X-100 and 3% NGS. After being washed,
the tissue was viewed on the confocal microscope as described above;
however, excitation was at 568 nm, and emissions were collected using a
590 nm long-pass emission filter. Once the T uJ1-stained tissue was
oriented correctly in the imaging chamber, a Z series of up to 60 serial
1–2
m
m sections was taken through the tissue beginning at the most
superficial tissue plane. This was done to facilitate recovery of the same
optical section that had been viewed during Ca
21
imaging, even if tissue
distortion had occurred during processing. Z-series sections were then
digitally superimposed for each tissue field viewed during the Ca
21
imaging experiments with the aid of National Institutes of Health Image
and Freehand (Macromedia) programs running on a Macintosh 7200
computer. Cells that were active during the Ca
21
imaging period were
then checked for corresponding TuJ1 immunoreactivity. In many cases
we were able to positively identif y individual cells in the same areas using
these methods.
In some experiments, Ca
21
imaging was followed by incubation of
cortical slabs in oxygenated AC SF containing 5
m
Msyto-11 (Chenn and
McConnell, 1995) for 5 min. Tissue was then rinsed, transferred to the
imaging chamber, and reimaged with the laser confocal microscope as
described above.
Filling of VZ cells with the cell-impermeant potassium salt of fluo-3. We
used the “blind” whole-cell patch-clamp recording method (Blanton et
al., 1989) to fill gap junction-coupled clusters of VZ cells (L oTurco and
Kriegstein, 1991). Briefly, 8–12 MVelectrodes were filled with a record-
ing solution containing 130 mMKC l, 5 mMNaC l, 1 mMMgC l
2
,10mM
HEPES, and the impermeant K
1
salt of fluo-3 (100
m
M; Molecular
Probes, Eugene, OR). Fluo-3 is a molecule small enough to pass through
gap junction channels (960 molecular weight). We identified cells as
being members of clusters because of their low membrane resistances
(LoTurco and Kriegstein, 1991). After dye filling, the injected slab was
transferred from the electrophysiological recording chamber to the im-
aging chamber and visualized with the laser confocal microscope as
described above.
Pharmacolog ical agents and drug application. Bicuculline methiodide
(BMI), lanthanum (La
31
), EGTA, and tetrodotoxin (TTX) were ob-
tained from Sigma (St. Louis, MO); 6-cyano-7-nitroquinoxaline-2,3-
dione (CNQX), 2-amino-5-phosphonopentanoic acid (AP-5), caffeine,
and thapsigargin were obtained from Research Biochemicals (Natick,
MA); and fluo-3 AM and syto-11 were obtained from Molecular Probes.
All drugs were bath applied. Drugs were either added directly to solu-
tions or kept as concentrated stock solutions at 220°C (BMI, CNQX,
AP-5, syto-11, and thapsigargin) or 4°C (La
31
and TTX) and diluted to
the desired concentration on the day of the experiment. Fluo-3 AM
solution was made on the day of the experiment from aliquots stored
at 220°C.
Immunohistochemistry. Embryos were transcardially perfused with 4%
paraformaldehyde; heads were removed, post-fixed in 4% paraformalde-
hyde, and stored overnight at 4°C. Heads were washed in PBS and placed
in 30% sucrose for 24 hr. Whole brains were removed from the heads,
placed in embedding medium (Tissue-tek, OCT, Sakura Fine Tek, Tor-
rance, CA), and frozen. Frozen coronal sections (15–20
m
m) were cut on
a cryostat and air dried. Sections were washed in PBS and then perme-
abilized and blocked in PBS with 0.5% Triton X-100 and 10% NGS for
1 hr at RT. Next, tissue was incubated for 2 hr at RT with primary
antibody [anti-TuJ1, 1:500 dilution; anti-vimentin, 1:6 dilution (gener-
ously provided by Dr. J. E. Goldman, Columbia University)] in PBS with
0.1% Triton X-100 and 3% NGS. Tissue was washed and then incubated
for 1 hr at RT with rhodamine-conjugated anti-mouse secondary anti-
body (1:200 dilution; ICN Pharmaceuticals) in PBS with 0.1% Triton
X-100 and 3% NGS. After being washed, the tissue was viewed with laser
confocal microscopy as described above or by epifluorescence using a
Zeiss Axioscope.
5-Bromo-29-deoxyuridine labeling. Embryonic brains were isolated as
described above and placed in 20
m
M5-bromo-29-deoxyuridine (BrDU)
in oxygenated AC SF at 37°C for 1, 4, 6, and 8 hr. These times were
selected to label cells primarily in S; S and G
2
;S,G
2
, and M; and S, G
2
,
M, and G
1
phases of the cell cycle, respectively (Takahashi et al., 1995).
Tissue was fixed overnight in 4% paraformaldehyde at 4°C and then
washed in PBS. Frozen coronal sections were made as described above.
Sections were rinsed in PBS and then incubated in 2N HCl for 30 min at
37°C. After being washed, sections were incubated in 0.1 Mborax for 10
min at RT, washed, permeabilized, and blocked in PBS with 0.5% Triton
X-100 and 10% NGS for 1 hr at RT. Sections were then incubated in
anti-BrDU primary antibody (1:200 dilution; Vector Laboratories, Bur-
lingame, CA) in 0.1% Triton X-100 and 3% NGS in PBS for 2 hr at
RT, washed in PBS, and then incubated in C y3-conjugated goat anti-
mouse secondary antibody (1:200 dilution; Jackson Immuno-
Research, West Grove, PA) in 0.1% Triton X-100 and 3% NGS in PBS
for 1 hr at RT. After being washed, the tissue was visualized with laser
confocal microscopy as described above or by epifluorescence.
Owens and Kriegstein •Ca
21
Fluctuations in Neocortical VZ Cells J. Neurosci., July 15, 1998, 18(14):5374–5388 5375

RESULTS
Distinct patterns of spontaneous [Ca
21
]
i
fluctuation in VZ cells
During cortical neurogenesis, the VZ contains neural and glial
precursor cells, radial glia, and postmitotic neurons. Because
fluo-3 appears to label most cells in the in vitro embryonic cortex,
we performed immunohistochemistry using specific markers to
help identify imaged cells as proliferative cells, radial glia, or
postmitotic neurons. Incubation in fluo-3 AM leads to loading of
a majority of the cells in the VZ (Fig. 1A). Proliferating cells in
S, G
2
, and M phases of the cell cycle were labeled by exposing the
cortex to BrDU for 6 hr in vitro (Fig. 1B). The majority of the
cells in the VZ are positively stained for this marker. In contrast,
Figure 1Cdemonstrates that very few VZ cells were positively
stained by the TuJ1 antibody, a marker of postmitotic neurons
(Lee et al., 1990). Figure 1 Dshows vimentin staining at E17,
which labels radial glia (Rakic, 1995). Vimentin-stained fibers can
be seen coursing around negatively labeled cell bodies within the
VZ. These experiments indicate that at E16 and E17, the major-
ity of cells imaged in the VZ are proliferating precursor cells in
different phases of the cell cycle.
To visualize the spatiotemporal pattern of activity in neocor-
tical VZ cells in situ, we used an experimental preparation that
kept the cortical mantle intact. The embryonic cerebral cortex was
removed, and after being loaded with fluo-3 AM, the intact
cortical mantle (cortical slab) was placed ventricular surface down
on the stage of a confocal microscope (Fig. 1E). Cells within the
VZ were then visualized and imaged (Fig. 1F). This preparation
differs from conventional brain slices because it enables observa-
tions of spontaneously active VZ cells within a large sheet of
intact embryonic cortex. Using the slab preparation, we were able
to image cells in an optical section parallel to the ventricular
surface to depths up to 40
m
m. We observed three distinct
patterns of spontaneous [Ca
21
]
i
fluctuations in VZ cells; individ-
ual cells undergoing independent [Ca
21
]
i
fluctuations, pairs of
adjacent cells undergoing synchronous [Ca
21
]
i
fluctuations, and
clusters of neighboring cells undergoing coordinated [Ca
21
]
i
fluctuations. In each case the [Ca
21
]
i
increase was localized to
the cell soma.
Single-cell behavior
The most common pattern of [Ca
21
]
i
fluctuation consisted of
individual cells displaying intermittent [Ca
21
]
i
transients (Fig. 2).
Spontaneously active single cells were present at all ages exam-
ined (E15–E20). These cells were distributed throughout the slab
and could be in close proximity to one another but were generally
not in contact. In Figure 2 A
1
, a representative microscopic field
from an E17 slab is shown with all of the cells active during the
20 min imaging period circled. Figure 2A
2
shows a single cell
before, during, and after an event. The temporal patterns of
Figure 1. Most imaged cells in the VZ are proliferative neocortical precursor cells. A, Single optical section of a fluo-3 AM-loaded coronal E16 brain
slice. Large numbers of cells load with the C a
21
indicator. B, Single optical section of an E16 coronal brain slice pulsed for 6 hr with BrDU to label cells
in S, G
2
, and M phases of the cell cycle. C, Single optical section of a coronal E17 brain slice stained for the neuronal marker TuJ1. Scale bar: A–C,
50
m
m. D, Single optical section of an E17 brain slice stained for the radial glia marker vimentin. E, Schematic representation of the experimental
preparation. A section of an intact neocortical hemisphere (cor tical slab) is removed, loaded with fluo-3 AM, and placed ventricular surface down in an
imaging chamber attached to the stage of an inverted confocal microscope. F, Representative view of the VZ from an E17 cortical slab.
5376 J. Neurosci., July 15, 1998, 18(14):5374–5388 Owens and Kriegstein •Ca
21
Fluctuations in Neocortical VZ Cells

[Ca
21
]
i
increase for four of the cells are shown in Figure 2 A
3
and
illustrate the range of transient durations encountered (32, 95,
and 14 sec for cells 1–3, respectively) as well as the occurrence of
recurrent transients in the same cell (cell 4). The mean duration
for events recorded from a sample of 63 cells at E17 was 37.1 6
4.3 sec (range, 11.5–121 sec), and the majority of cells (82.5%)
demonstrated a single transient in the course of 20 min. Further-
more, these events seemed to occur randomly throughout each
field with no obvious intercellular synchrony. Similar behavior
was observed in experiments conducted at 32–34°C. In a sample
of 29 active cells at 32–34°C, the average transient duration was
33 63.6 sec, and 81% of the cells displayed a single transient over
the imaging period.
Considering the sampling rate of ;3 sec/image used in these
experiments, we determined that the fastest event that could be
resolved was ;6 sec. To investigate whether we were missing
faster events, we performed several experiments in which images
were acquired at faster sampling rates. Figure 2B
1
shows a tran-
sient from a cell sampled every second; the duration of the event
was 11 sec, and the interval from transient onset to peak spanned
several images, indicating that the time-to-peak of the transient is
in the range of seconds. In 15 active cells sampled at 0.2–1
Figure 2. Individual cells display intermittent [Ca
21
]
i
transients. A
1
, A microscopic field from an E17 slab imaged in an optical plane ;20
m
m from the
ventricular surface. Circles indicate cells that were active over a continuous imaging period of ;20 min. A
2
, A cell before (Rest), during (Peak), and after
(Return) a spontaneous [Ca
21
]
i
increase. A
3
, Activity graphs of the numbered cells (1– 4 ) shown in A
1
. Calcium transients ranged from relatively fast
events (cell 3) to slow events (cell 2). The inset for cell 3 is an expanded plot of the event with measured values indicated by filled circles. Scale bar in inset,
10 sec. A minority of cells showed multiple transients (e.g., cell 4 ) over the imaging period. B, Faster sampling showing that the single-cell events generally
occurred over many seconds. B
1
, A cell sampled every second. B
2
, A cell sampled every 0.215 sec. C, A developmental increase in the number and
frequency of single-cell events but no change in duration. C
1
, The similar mean durations of [C a
21
]
i
transients at E15 and E19. C
2
, A significant increase
in the percentage of active cells/field/trial at E19. Asterisk indicates a significant difference. C
3
, At E19, a larger percentage of cells with multiple
transients than at E15. The inset displays the mean frequency per trial of all cells analyzed at the two ages. There was an increase in mean frequency
at E19. Asterisk indicates a significant difference. D, Spontaneous [Ca
21
]
i
fluctuations observed in cells throughout the VZ. D
1
, A fluo-3-loaded coronal
brain slice at E16 with the area imaged indicated by a box.D
2
, Higher magnification image with active cells indicated by circles.
Owens and Kriegstein •Ca
21
Fluctuations in Neocortical VZ Cells J. Neurosci., July 15, 1998, 18(14):5374–5388 5377

sec/image, we captured the entire transient and found that the
mean duration was 11.1 61.6 sec and that the fastest event was
;4–5 sec (Fig. 2B
2
, sampled at 0.215 sec/image). Collectively
these results suggest that single-cell transients have relatively slow
onsets, last many seconds, and repeat at low frequency. Also, we
would be likely to detect most, if not all, of these events by
sampling every 5–10 seconds.
Developmental change in single-cell behavior
To investigate developmental differences, we imaged multiple
areas from slabs at E15 and E19, ages that correspond to early
and late neurogenesis, respectively (Bayer and Altman, 1991). In
these experiments, we sampled tissue fields every 7 sec over a
period of 3.5 min; each of these samples was considered one trial.
Comparing multiple cells at the two different ages demonstrated
that the transient durations of single-cell events did not change
with age (Fig. 2C
1
). The mean duration at E15 was 42.5 64.4 sec
(n548), and the mean duration at E19 was 38.6 63.0 sec (n5
78); these values were not significantly different.
Although the single-cell transient durations were similar be-
tween the two ages analyzed, there was a tendency for cells from
older embryos to be more active in terms of the number of cells
demonstrating [Ca
21
]
i
fluctuations and of the frequency of
[Ca
21
]
i
fluctuations in individual cells. To quantify these trends,
we counted the number of active cells for a defined area and the
number of transients per cell during a single imaging trial. Figure
2C
2
shows the percentage of active cells in multiple fields from
slabs at E15 and E19. The mean percentage of active cells per
trial at E15 was 6.64 60.92% (n56 fields from three slabs),
whereas at E19 it was 12.5 61.2% (n55 fields from two slabs).
This indicates an increase of 53.2% in active cells with age ( p,
0.004). Figure 2C
3
shows that at E15 20% of the cells showed
more than one transient, whereas at E19 this value increased to
;35%. The inset of Figure 2C
3
displays the mean frequency of all
cells analyzed at the two ages. At E15 there was a mean frequency
of 1.24 60.1 transients per imaging trial (n550), and at E19
there was a mean frequency of 1.64 60.1 transients per imaging
trial (n556). The difference between these values was statisti-
cally significant ( p,0.01).
Spontaneous [Ca
21
]
i
fluctuations occur
throughout the VZ
To examine the spatial distribution of VZ cells demonstrating
spontaneous [Ca
21
]
i
fluctuations, we performed experiments in
brain slices. In multiple experiments, we observed spontaneous
[Ca
21
]
i
fluctuations in cells that spanned the entire depth of the
VZ. The kinetics of the [Ca
21
]
i
transients was similar to that
observed in the cortical slab preparation. Figure 2D
1
shows an
example of a coronal brain slice from an E16 embryo with the
area imaged indicated by a box. Figure 2 D
2
shows the imaged
area at higher magnification and the spontaneously active cells
outlined (circles). In the 26 active cells seen over ;15 min of
imaging in this example, the mean transient duration was 36.8 6
5.3 sec, and 79% of the cells had a single transient during the
imaging period. These values from cortical slices are similar to
those obtained from cortical slabs and indicate that single-cell
behavior is similar in both tissue preparations.
Mechanisms of spontaneous [Ca
21
]
i
fluctuations in VZ cells
Cells within the VZ express functional amino acid transmitter
receptors that, when activated, lead to membrane depolarization
and increases in [C a
21
]
i
(LoTurco et al., 1995). There are several
neuronal populations in the developing cortex that could be
sources of endogenous transmitter release, including neurons in
the intermediate zone (I Z), subplate, and cortical plate (C P)
(Kim et al., 1991; McConnell et al., 1994; Behar et al., 1996;
Anderson et al., 1997). We therefore examined whether sponta-
neous [Ca
21
]
i
fluctuations in VZ cells were mediated by action
potential-dependent transmitter release. Images of cortical slabs
(E19) were taken to establish a basal level of spontaneous activ-
ity. Slabs were subsequently preincubated in a solution containing
TTX (2
m
M), to block sodium-dependent action potentials, for a
minimum of 3 min and then reimaged in the continued presence
of TTX. Similar levels of activity were present before and after
immersion in TTX (data not shown). A second series of experi-
ments was performed using brain slices to circumvent the possi-
bility that the intact ventricular surface provided a barrier to drug
access. Cells near the ventricular surface of the slice were mon-
itored, and activity was still present in the TTX-containing solu-
tion (data not shown). We also found that activity persisted in
solutions that contained, in addition to TTX, the nonspecific
voltage-gated Ca
21
channel (VGCC) blocker lanthanum (50
m
M),
the GABA
A
receptor blocker BMI (20
m
M), the non-NMDA
glutamate receptor blocker CNQX (20
m
M), and the NMDA
receptor blocker AP-5 (100
m
M). Figure 3Adepicts the levels of
activity for three cells in an E19 slice before and after the
addition of the inhibitors. From these data, we conclude that
neural activity, VGCC activation, and amino acid neurotransmit-
ter receptor activation are not required for the spontaneous
[Ca
21
]
i
increases in individual VZ cells.
To test whether the [Ca
21
]
i
increases were dependent on
extracellular Ca
21
, we performed experiments in C a
21
-
containing and Ca
21
-free (0 Ca
21
and2mMEGTA) ACSF
solutions. Embryonic cortical slabs were preincubated in normal
(2 mMCa
21
)orCa
21
-free ACSF for at least 30 min before
imaging, and comparisons were made of the activity in both
conditions. In some experiments, slabs and slices were imaged
first in normal ACSF, followed by imaging of the same area
;20–30 min after exchange of normal ACSF for Ca
21
-free
ACSF. Results from several experiments showed that in all cases
similar levels of activity were present in VZ cells in both C a
21
and Ca
21
-free conditions. Figure 3Bshows graphs of [Ca
21
]
i
changes from three cells in an E16 slice recorded in both normal
and Ca
21
-free ACSF bath solutions. There were no obvious
differences in the behavior of the [C a
21
]
i
transients under the
two imaging conditions. In experiments using E19 slabs, we found
the mean transient duration was 45.4 63.6 sec (n580) in
Ca
21
-free conditions, and the average frequency was 1.60 60.1
transients per imaging trial (n565). Comparison of these pa-
rameters with those obtained at E19 under conditions of standard
extracellular Ca
21
(2 mM) showed no significant differences
(mean duration at E19 was 38.6 63.0 sec; mean frequency was
1.64 60.1 transients per imaging trial).
These results suggest that the majority of spontaneous [C a
21
]
i
fluctuations in VZ cells may be mediated by C a
21
released from
intracellular stores. To test this possibility directly, we examined
cortical slabs before and after incubation in an ACSF solution
containing thapsigargin (5
m
M),aCa
21
-ATPase inhibitor that
depletes intracellular C a
21
stores (Thastrup et al., 1990). Fluo-
3-loaded cortical slabs were first imaged to establish baseline
levels of activity. The slabs were subsequently incubated in thap-
sigargin for at least 15 min and then reimaged in the continued
presence of thapsigargin. After treatment with thapsigargin,
spontaneous activity in VZ cells was almost completely abol-
5378 J. Neurosci., July 15, 1998, 18(14):5374–5388 Owens and Kriegstein •Ca
21
Fluctuations in Neocortical VZ Cells

ished; only a few cells were seen to produce spontaneous [Ca
21
]
i
fluctuations in multiple imaging trials from two separate slabs
(Fig. 3C). This effect was not caused by cell injury or death
because VZ cells still produce [C a
21
]
i
increases in the presence
of thapsigargin when exposed to agents that depolarize the cells
(data not shown).
Most single-cell transients occur in non-neuronal cells
Although immunohistochemical analysis suggests that the major-
ity of imaged cells are precursor cells (see Fig. 1), similar spon-
taneous [Ca
21
]
i
fluctuations have been observed in postmitotic
neurons, including migrating cerebellar granule cells (Komuro
and Rakic, 1996), immature spinal cord neurons (Gu et al., 1994;
Gu and Spitzer, 1995), and neonatal cortical neurons (Yuste et
al., 1992; Owens et al., 1996). We therefore combined Ca
21
imaging and TuJ1 labeling to confirm the identity of spontane-
ously active VZ cells. The neuronal marker T uJ1 has been used
previously to identif y immature neurons in the neocortical VZ
(Menezes and Luskin, 1994; O’Rourke et al., 1997). We first
imaged both slabs and slices of embryonic cortex to observe cells
that displayed spontaneous [Ca
21
]
i
fluctuations. The tissue was
subsequently fixed and stained for TuJ1 immunoreactivity, and
the same regions were reimaged (see Materials and Methods).
Consistent with results reported in the mouse (Menezes and
Luskin, 1994), we found little or no T uJ1 immunoreactivity in the
VZ on E15 (approximately E13 in the mouse); however, in these
same slices, many cells throughout the depth of the VZ had
spontaneous [Ca
21
]
i
fluctuations (Fig. 4A). Figure 4 Ashows a
coronal slice from an E15 embryo that was imaged for spontane-
ous [Ca
21
]
i
increases and then for TuJ1 immunoreactivity. The
cells outlined with circles were active during Ca
21
imaging (Fig.
4A,left), and the corresponding cell locations are indicated in the
TuJ1-stained section (Fig. 4A,right). In only one case was a clear
TuJ1-positive cell body present where an active cell was seen
during Ca
21
imaging (Fig. 4 A,arrow on right). Furthermore,
there were no TuJ1-stained cell bodies near the VZ surface
where the active cells in tissue slab experiments were seen. These
results suggest that the majority of spontaneous single-cell activ-
ity is mediated by precursor cells that do not express the TuJ1
antigen.
As neurogenesis proceeds, there is an increase in the number
of postmitotic neurons that are TuJ1-positive in the VZ (Menezes
and Luskin, 1994). Therefore, it is possible that at later develop-
mental periods (e.g., E19) the VZ contains both neurons and
precursor cells that both display spontaneous [Ca
21
]
i
fluctua-
tions. This may be reflected in the greater number of active cells
seen in the VZ at E19 compared with E15 (Fig. 2C). To address
this, we imaged E19 slabs and subsequently stained them for TuJ1
immunoreactivity. Many more fibers and cell bodies were stained
with TuJ1 in the VZ at E19 than at E15, and a greater number of
spontaneously active cells was found in regions in which there was
positive TuJ1 staining (Fig. 4B,arrows on right). This result
suggests that at later stages of neurogenesis the VZ can contain
both precursor cells and neurons that display spontaneous
[Ca
21
]
i
fluctuations.
Synchronous [Ca
21
]
i
fluctuations in VZ cell pairs
A second distinctive form of spontaneous [Ca
21
]
i
behavior was
observed in VZ cells at the ventricular surface. Intracellular
Ca
21
fluctuations were seen in pairs of adjacent cells whose
nuclei often protruded from the surface of the slab. Figure 5A
shows one such doublet in an E19 cortical slab before, during, and
after the [Ca
21
]
i
increase. Figure 5Bdisplays a line graph of
three doublet events from the same slab shown in Figure 5A
(arrows indicate transients shown in Fig. 5A). These events were
highly synchronized; the peak of the transients occurred at the
same time point, and the duration of the events were nearly
identical in both cells (Fig. 5). For example, in a sample of 15 of
these doublet events, the average duration of one cell of the pair
was 36.7 63.1 sec, whereas the other was 37.1 63.7 sec. Doublet
events were observed in tissue slabs at or near the ventricular
surface at all ages examined (E15–E20).
The rounded morphology of these cells and their location at
the ventricular surface suggested that they might be M-phase cells
in the process of cytokinesis. By using the vital stain syto-11 to
label cellular DNA (Chenn and McConnell, 1995), we found that
cell doublets were in the same tissue plane as pairs of daughter
cells in various stages of mitosis. For example, Figure 6 Ashows
an optical section of the ventricular surface of an E16 cortical slab
stained with fluo-3. Many of the stained cells were in adjacent
Figure 3. Mechanisms of spontaneous [Ca
21
]
i
fluctuation in VZ cells.
A, Three cells (solid,dashed, and dotted lines) near the ventricular surface
of a coronal slice at E19. Activity persisted in the presence (solid hori-
zontal bar)ofTTX(2
m
M), La
31
(50
m
M), BMI (20
m
M), CNQX (20
m
M),
and AP-5 (100
m
M). B, Three cells at E16 recorded in Ca
21
(2 mM)ACSF
(control ) and after ;20 min of perfusion with C a
21
-free/2 mMEGTA
ACSF. There were no obvious differences in the behavior of the [Ca
21
]
i
transients. C, Representative examples of activity in three cells (solid
lines) under control conditions and three cells after exposure to thapsi-
gargin (5
m
M). Spontaneous activity in VZ cells was abolished after
exposure to thapsigargin.
Owens and Kriegstein •Ca
21
Fluctuations in Neocortical VZ Cells J. Neurosci., July 15, 1998, 18(14):5374–5388 5379

pairs, had rounded morphology, and displayed synchronized
spontaneous [Ca
21
]
i
fluctuations. Imaging in the same focal
plane after incubation in syto-11 revealed multiple pairs of
daughter cells with patterns of condensed chromatin characteris-
tic of mitotically active cells (Fig. 6B). This pattern of staining
was only observed at the ventricular surface of the slab. When
focusing deeper into the VZ, only a diffuse nuclear staining was
seen, a result similar to that found in slices of ferret cortex
(Chenn and McConnell, 1995). Furthermore, when focusing at
this deeper level, doublet events were not observed. In addition,
negatively stained condensed chromatin could sometimes be seen
in the nuclei of fluo-3-labeled cell doublets, as shown in the inset
Figure 4. Most active single cells in the VZ are not neurons. A, Coronal slice at E15 that was imaged for spontaneous [C a
21
]
i
increases and subsequently
for TuJ1 immunoreactivity. Lef t, A single optical section with cells active during the imaging period circled.Right, An average of 50 serial 1
m
m sections
of the same area after processing for TuJ1 immunoreactivity. In only one case was a T uJ1-positive cell body present where an active cell was seen during
Ca
21
imaging (arrow). Dashed lines approximate the boundary of the VZ. B, An E19 slab imaged for spontaneous [C a
21
]
i
increases and subsequently
for TuJ1 immunoreactivity. Lef t, A single optical section ;15
m
m from the ventricular surface with cells that were active during the imaging period
circled.Right, An average of 50 serial 1
m
m sections of the same area after processing for TuJ1 immunoreactivity. There were many more TuJ1-labeled
cells at E19 than at E15, and in several instances cells active during Ca
21
imaging were TuJ1-positive (ar rows).
5380 J. Neurosci., July 15, 1998, 18(14):5374–5388 Owens and Kriegstein •Ca
21
Fluctuations in Neocortical VZ Cells

of Figure 6B. Not surprisingly, synchronous cell pairs were also
found to be TuJ1-negative (Fig. 6C). Furthermore, during long
imaging trials, we were able to observe cell division (Fig. 6D). In
the example illustrated in Figure 6 D
1
, the dividing cell first
appeared rounded, but over the next 20 min, an equatorial con-
striction and cleavage plane appeared in the cell, and condensed
chromatin separated in a polarized manner. Changes in [C a
21
]
i
in both daughter cells were synchronized (Fig. 6D
2
). These
observations confirmed that at least some, if not all, cells exhib-
iting synchronized [C a
21
]
i
fluctuations were mitotically active
daughter cells. Finally, as with the single-cell events seen in VZ
cells, doublets could occur in Ca
21
-free ACSF (Fig. 6E), sug-
gesting that these events are also mediated by the release of Ca
21
from intracellular stores.
Coordinated [Ca
21
]
i
fluctuations in groups of VZ cells
The third pattern of spontaneous [Ca
21
]
i
fluctuation consisted of
coordinated [Ca
21
]
i
increases in groups of neighboring VZ cells.
An example of such an event from an E17 neocortical slab is
illustrated in Figure 7A. This example shows nine sequential
pseudocolored images before, during, and after the coordinated
event. The activity seems to originate with 1 or 2 cells (arrow) and
spreads outward to 14 –16 neighboring cells. Figure 7Bshows the
time course of [Ca
21
]
i
change in eight of these cells and the
combined mean value for all cells together (inset). There was a
close spatiotemporal coupling of [Ca
21
]
i
change within the cell
group. In this example, the propagation rate of [C a
21
]
i
spread
from the first cell to neighboring cells was ;8
m
m/sec (see below).
These events were found to occur infrequently; over the course of
our experiments, we observed 21 such events under standard
imaging conditions, and in only two instances did the same cell
cluster demonstrate a second coordinated [Ca
21
]
i
increase. We
observed up to four spontaneous coordinated [C a
21
]
i
events in a
single cortical slab, and in all cases the events were spatially
distinct. In clusters in which all or most participating cells were
captured in the field of view (n513), the number of cells ranged
from 4 to 20, with an average of 9.5 61.33 cells. In cases in which
the entire event duration was captured (n510), the average
duration of the [Ca
21
]
i
increase was 50.1 64.37 sec. It should be
noted that the cell numbers reported here are based on optical
sections that cut through the radially oriented clusters at right
angles (see Fig. 8A). Because the optical section samples only a
portion of participating cells, the f ull number of cells per cluster
is presumably larger. Furthermore, we observed several of these
events while imaging the VZ in brain slices (Fig. 8C
2
). They
involved groups of cells oriented radially that spanned several cell
diameters within the VZ.
These events resemble previously described coordinated
[Ca
21
]
i
fluctuations termed “neuronal domains” observed in neo-
natal cortical neurons in which Ca
21
or a related second messen-
ger is thought to propagate the [Ca
21
]
i
signal by passing through
gap junction channels and triggering release of [Ca
21
]
i
from
intracellular stores (Yuste et al., 1992, 1995). Coordinated events
in the VZ also depend on Ca
21
release from intracellular stores.
We observed several cluster events in tissue slabs from experi-
ments with 0 Ca
21
/2 mMEGTA in the bathing solution. These
events were similar to those seen under standard conditions. The
number of cells per cluster ranged from 5 to 23 with an average of
11 64.1 cells (n54), and in clusters in which we resolved the
entire event, the average duration was 55.7 68.2 sec (n53). In
three clusters in Ca
21
-free solution, images were captured fast
enough to estimate the rate of [Ca
21
]
i
propagation. Figure 7C
1
shows a cluster event (approximately nine cells total) with the
putative trigger cell (cell 1) and two follower cells (cells 2 and 3)
labeled. Figure 7C
2
shows the activity graph for all of the cells in
the cluster. By measuring the distance of each follower cell from
the trigger cell and the time of onset of [Ca
21
]
i
increase in each
of these cells, we could estimate the rate of signal propagation
(Fig. 7C
2
,inset). Using this method, we found that these events
propagated at an average rate of 6.8 62.0
m
m/sec (range, 2.4–
15.2
m
m/sec). This is in the range of speeds found for diffusion of
Ca
21
or related second messengers through gap junction-coupled
cells (Cornell-Bell and Finkbeiner, 1991; Meyer, 1991; Yuste et
al., 1995; Newman and Zahs, 1997). These results suggest that
cluster events are mediated by intracellular C a
21
release and
most likely propagate by diffusion of Ca
21
or other messengers
through gap junction channels.
Because of their infrequent occurrence, we performed several
manipulations to determine whether we could trigger cluster
events or increase their frequency. We lowered the temperature
of the bath solution by several degrees, a technique that has been
used to trigger neuronal domains (Yuste et al., 1995). Decreasing
the bath ACSF from 21 to 16°C by adding chilled ACSF at the
standard perfusion rate or by adding a bolus of chilled ACSF did
not produce spontaneous cluster activity. Imaging trials per-
formed in ACSF warmed to 32–34°C also produced no increase in
cluster behavior. Attempts to trigger the cluster events by incu-
bating tissue in low concentrations of caffeine (100–500
m
M) also
did not trigger cluster events. We removed extracellular Mg
21
Figure 5. Pairs of VZ cells at or near the ventricular surface show
synchronized increases in [C a
21
]
i
.A, An example of a doublet event from
an E19 cortical slab before (A
1
), during (A
2
), and after (A
3
)a[Ca
21
]
i
increase. B, A three-dimensional graphic representation of three highly
synchronized doublet events. The ar rows indicate transients shown in A.
All cells are from the same slab.
Owens and Kriegstein •Ca
21
Fluctuations in Neocortical VZ Cells J. Neurosci., July 15, 1998, 18(14):5374–5388 5381

3
Figure 7. Top. Coordinated increases in [Ca
21
]
i
occur in clusters of neighboring VZ cells. A, A spontaneously active VZ cluster at E17. This example
shows nine sequential pseudocolored images taken every 4 sec before, during, and after a cluster event. B, The time course of the [C a
21
]
i
change plotted
for eight of the cells from the cluster shown in A.Arrow indicates putative trigger cell. The time course of the mean value for all of the cells is shown
in the inset.C, Coordinated cell activity occurring in Ca
21
-free ACSF and propagating at ;10
m
m/sec. C
1
, A cluster event in Ca
21
-free/2 mMEGTA
ACSF with a putative trigger cell (cell 1) and two follower cells (cells 2 and 3) labeled. C
2
, Activity graph for all of the cells in the cluster with the onset
of the Ca
21
transients for the three labeled cells displayed in the inset. Measuring the distance of each follower cell from the trigger cell and the time
of onset of the [Ca
21
]
i
increase in each cell allowed estimation of the rate of signal propagation.
Figure 8. Bottom. Cells demonstrating coordinated [C a
21
]
i
increases correspond to groups of gap junction-coupled VZ cells. A, A schematic diagram
of the radial arrangement of a coupled VZ cell cluster. The focal plane of the confocal microscope is indicated by shading. The recorded cell is shown
with a schematic electrode. B, A confocal image of a section through a dye-stained (pseudocolored) cell cluster at E17. Multiple cells are dye-filled after
injection of a single VZ cell (arrow). C, Spontaneously active cell clusters that do not include T uJ1-positive cells. C
1
, An average of 20 serial 2
m
m
sections through an E16 coronal slice stained for T uJ1 after Ca
21
imaging. A coordinated C a
21
increase was observed in a cluster of cells located within
the box.C
2
, Fluo-3-stained image showing the peak [C a
21
]
i
increase for the spontaneously active cell cluster from the area highlighted by the box in C
1
.
C
3
, An average of three serial 2
m
m sections of the area shown in C
2
after TuJ1 staining. There was no obvious correspondence between T uJ1-stained
cells and cells participating in the coordinated [Ca
21
]
i
increase.
Figure 6. Synchronously active
cell pairs are M-phase cells in the
process of cell division. A,A
fluo-3-loaded E16 slab imaged at
the ventricular surface. Notice
the presence of a great many
cells apparently in the state of
mitosis. B, Syto-11 staining and
imaging of the VZ surface dis-
playing patterns of condensed
chromatin. The inset shows con-
densed chromatin visible with
fluo-3 loading in the doublet in-
dicated by arrows in A.C, Dou-
blet event (arrows) in an E19 slab
during Ca
21
imaging (C
1
) and
after subsequent TuJ1 staining
(C
2
), demonstrating that dou-
blets are not neurons. D
1
, A di-
viding E17 VZ cell observed over
a 20 min period. Each image
(1– 4) is separated by ;5min.
D
2
, Spontaneous fluctuations in
[Ca
21
]
i
that were synchronized
in both daughter cells. E, Dou-
blets occurring in Ca
21
-free
ACSF, suggesting that these
events are mediated by the re-
lease of Ca
21
from intracellular
stores. Inset shows images before
(1), during (2), and after (3) the
doublet event.
5382 J. Neurosci., July 15, 1998, 18(14):5374–5388 Owens and Kriegstein •Ca
21
Fluctuations in Neocortical VZ Cells

Legend continues.
Owens and Kriegstein •Ca
21
Fluctuations in Neocortical VZ Cells J. Neurosci., July 15, 1998, 18(14):5374–5388 5383

from the bathing solution, another manipulation that has been
reported to increase the frequency of neuronal domains in the
neocortex (Yuste et al., 1995). Although we did observe several
cluster events while imaging under this condition, clusters still
occurred infrequently and at random. Overall, we found no ma-
nipulations that could trigger cluster events in the VZ.
Based on the resemblance of these cell clusters to clusters of
gap junction-coupled cells described previously in the embryonic
rat VZ (LoTurco and Kriegstein, 1991), we suspected that the
coordinated [Ca
21
]
i
fluctuations were occurring in precursor
cells coupled by gap junction channels. Because of the random
and infrequent occurrence of [Ca
21
]
i
transients in clusters and
our inability to evoke them, we were unable to examine the effect
of gap junction channel-blocking agents on cluster transients.
Instead, the relationship between clusters of cells demonstrating
spontaneous [Ca
21
]
i
transients and gap junction-coupled cell
clusters was examined indirectly by comparing their morpholog-
ical features. Single E17 VZ cells were dye-filled using microelec-
trodes filled with the impermeant K
1
-salt of fluo-3 (Fig. 8A).
Clusters of adjacent stained cells could be visualized from the VZ
surface (Fig. 8 B;n54), indicating the passage of the dye through
gap junction channels. Although these filled clusters were not
spontaneously active, the number of cells and their spatial ar-
rangement were very similar to that of the clusters of cells
participating in spontaneous coordinated [C a
21
]
i
transients
(compare Fig. 7Awith Fig. 8 B).
Gap junction-coupled cell clusters in the VZ have been shown
to be composed of proliferating neuroepithelial cells in all phases
of the cell cycle except M (Bittman et al., 1997). In addition, the
majority of VZ clusters include at least one radial glia cell but not
TuJ1-positive neurons (Bittman et al., 1997). If the spontaneously
active clusters seen in this study correspond to gap junction-
coupled clusters, then we would predict that the cells are TuJ1-
negative. There were only a few cluster events in experiments in
which Ca
21
imaging was followed by T uJ1 staining, and in only
one case were we confident that the same anatomical areas could
be compared. Figure 8C
1
shows an E16 slice stained for T uJ1 with
the area in which the cluster event occurred highlighted by a box.
Figure 8C
2
shows the cluster event near the peak of the Ca
21
transient, with the extent of the event outlined. The event ex-
tended radially for several cell diameters. Figure 8C
3
shows the
corresponding area stained for T uJ1. When overlaid, we found no
obvious correspondence between the spontaneously active cells
and TuJ1-positive cells. In addition, in single optical sections of
TuJ1-stained slabs, there were no cases of multiple adjacently
labeled neurons in the VZ, as would be expected for cells belong-
ing to spontaneously active clusters (data not shown). Within the
VZ, only proliferating precursor cells have multiple closely ap-
posed somata that could comprise an active cell cluster (see Fig.
1B). These events therefore most likely occur in gap junction-
coupled precursor cells in the VZ.
Spontaneous [Ca
21
]
i
fluctuation in immature neurons
To address the issue of whether patterns of spontaneous [Ca
21
]
i
fluctuation change after exit from the cell cycle, we also imaged
cells in the I Z, marginal zone (MZ), and CP of the embryonic
cortex, regions that contain mostly postmitotic neurons. In addi-
tion to single cells displaying spontaneous [Ca
21
]
i
events with
similar kinetics to those described in the VZ, we found that many
cells in these regions produced transients at much higher frequen-
cies. Consistent with previous findings (Menezes and L uskin,
1994), we found that these regions show high levels of TuJ1
staining. Figure 9A
1
shows the MZ of an E15 cortical slice during
Ca
21
imaging (lef t) and after TuJ1 staining (right). The MZ was
intensely stained for T uJ1. Many cells in this region were highly
active, showing multiple spontaneous [Ca
21
]
i
fluctuations over a
5 min imaging period. An example of one such cell is shown in
Figure 9A
2
. In the MZ, many of the active cells had elongated
horizontally oriented cell bodies that were bipolar (Fig. 9A
1
,
arrows on lef t). These features are reminiscent of Cajal-Retzius
cells, a population of early generated neurons commonly found in
the MZ of the developing neocortex (Bayer and Altman, 1991).
Likewise, radially oriented presumptive neurons in the C P (data
not shown) were highly active. Figure 9Bshows the activity plot
of an E16 CP cell demonstrating multiple [C a
21
]
i
transients. In a
number of cases, we also observed cells in the IZ that displayed
multiple spontaneous [Ca
21
]
i
fluctuations. Figure 9C
1
shows an
example of one such cell both during Ca
21
imaging and after
TuJ1 staining; the corresponding activity graph for this cell is
shown in Figure 9C
2
. These experiments suggest that as cells
become terminally postmitotic and migrate away from the VZ,
they continue to undergo spontaneous [Ca
21
]
i
fluctuations; how-
ever, the fluctuations are often more frequent.
Previous results have shown that individual neurons in the
early postnatal cortex display [C a
21
]
i
fluctuations either sponta-
neously (Owens et al., 1996) or when exposed to very low con-
centrations of glutamate receptor agonists (Yuste and Katz,
1991). These events can be sensitive to TTX and blockers of
VGCCs (Yuste and Katz, 1991; Owens et al., 1996), suggesting
mediation via neuronal activity and VGCC activation. Consistent
with these findings, we observed that the spontaneous [Ca
21
]
i
fluctuations seen in some cells of the embryonic CP and MZ can
be either blocked entirely or significantly reduced after removing
extracellular Ca
21
(Fig. 9D). These observations suggest that
after exit from the cell cycle, some neurons undergo a develop-
mental change in the mechanism as well as the dynamics of their
spontaneous [Ca
21
]
i
fluctuations.
DISCUSSION
This study describes patterns of spontaneous [Ca
21
]
i
fluctuation
in neocortical VZ cells in situ. A schematic diagram of the cell
types demonstrating the different patterns of spontaneous
[Ca
21
]
i
fluctuations is shown in Figure 10 (see figure legend for
details). Studies of cellular behavior in situ are particularly im-
portant to help unravel signaling mechanisms in a spatially com-
plex structure such as the VZ that is both stratified and composed
of columnar compartments. For example, coupling of VZ cells
into columnar clusters seems to be necessary for cells to progress
through the cell cycle in situ (Bittman et al., 1997), and the
orientation of the cleavage plane of M-phase cells, a feature that
can only be observed in sit u, seems to be important for determin-
ing whether cells re-enter the cell cycle or become terminally
postmitotic (Chenn and McConnell, 1995).
Spontaneous [Ca
21
]
i
fluctuation in VZ cells
Fluctuations in [C a
21
]
i
in cortical precursor cells could have
several potential roles including regulation of cell cycle progres-
sion. Transient increases in [Ca
21
]
i
are associated with nuclear
envelope breakdown, chromatin condensation, and the onset of
anaphase in sea urchin eggs (Poenie et al., 1985) and cultured
animal cells (Keith et al., 1985; Kao et al., 1990). Cells make a
commitment to divide by crossing from G
1
to S phase and initi-
ating DNA synthesis, a transition that is often environmentally
regulated (Murray and Hunt, 1993) and associated with [Ca
21
]
i
5384 J. Neurosci., July 15, 1998, 18(14):5374–5388 Owens and Kriegstein •Ca
21
Fluctuations in Neocortical VZ Cells

transients (Lu and Means, 1993). Beyond the critical G
1
to S
transition, cell cycle progression is presumably autonomous, and
no further external signals are required (Reddy, 1994). Most of
the cells reported here are within several cell diameters of the
ventricular surface, and as a result of interkinetic nuclear migra-
tion, they are in G
2
, M, or early G
1
. Our results show that [Ca
21
]
i
fluctuations in these cells are not regulated by neuronal activity,
ionotropic GABA and glutamate receptor activation, or activa-
tion of VGCCs but do not eliminate the possibility that [Ca
21
]
i
fluctuations in late G
1
to S-phase cells are regulated and /or
induced by these signals. In addition, contact-dependent signals
and short-range diff usible factors such as neurotrophins may also
influence [C a
21
]
i
. For example, proliferation of cortical precursor
cells can be stimulated by basic fibroblast growth factor (bFGF)
(Ghosh and Greenberg, 1995), and many growth factors including
bFGF act via tyrosine kinase receptors that in turn can lead to
release of Ca
21
from intracellular stores (Ullrich and Schless-
inger, 1990; Pende et al., 1997).
Figure 9. Spontaneous [Ca
21
]
i
fluctuations in developing neurons. A
1
,MZ of an E15 cortical slice during C a
21
imaging (lef t) and after T uJ1 staining
(right). The MZ contained many active cells, some of which had the morphological features of C ajal-Retzius neurons (arrows), and contained a high
density of TuJ1-stained cells. A
2
, Activity graph of an MZ cell shown in A
1
.B, Activity graph of an E16 CP cell. C
1
, Presumptive migrating neuron in
the IZ (arrows) of an E16 coronal brain slice during Ca
21
imaging (lef t) and after T uJ1 staining (right). Pial surface is to the top right-hand corner.C
2
,
Activity graph of the cell shown in C
1
.D,AnE17MZ cell recorded in Ca
21
(2 mM) ACSF and after ;30 min of perfusion with C a
21
-free ACSF.
Transients failed to appear in the Ca
21
-free condition but returned once normal ACSF was reperf used (Wash).
Owens and Kriegstein •Ca
21
Fluctuations in Neocortical VZ Cells J. Neurosci., July 15, 1998, 18(14):5374–5388 5385

If the observed [Ca
21
]
i
transients are influencing cell cycle
events, it is possible that the developmental changes in the be-
havior of the [Ca
21
]
i
transients may reflect developmental
changes in the cell cycle. In mouse, the neocortical neurogenic
interval has been well characterized (Caviness et al., 1995).
During neurogenesis, there is a progressive increase in the dura-
tion of the cell cycle and an increase in the number of cells exiting
the cell cycle. The observed increase in the number of active cells
and the frequency of spontaneous [Ca
21
]
i
fluctuations between
E15 and E19 may reflect these developmental changes in cell
cycle parameters.
The close match between the location and appearance of cell
pairs demonstrating synchronized [Ca
21
]
i
fluctuations and cells
in mitosis, as demonstrated by syto-11 staining, indicates that
doublet cells are near the end of the cell division cycle. This was
confirmed in time-lapse studies of doublet cells undergoing divi-
sion. Transient increases in [Ca
21
]
i
have been associated with the
onset of cytokinesis and with the activation of actomyosin fila-
ments that serve to separate daughter cells at the end of telophase
(Ratan et al., 1988; Whitfield et al., 1995). The [Ca
21
]
i
increases
observed in doublets could be associated with either of these cell
cycle events. Furthermore, recent data from studies of dividing
cells in the rat VZ have demonstrated that mitotic spindles are
highly motile (Adams, 1996), and the [Ca
21
]
i
transients seen
during doublet events could possibly serve to influence these
movements. The finding that [C a
21
]
i
oscillations in doublets are
always synchronous is probably because of the passage of [Ca
21
]
i
or second messengers through the relatively large cytoplasmic
bridges that couple dividing daughter cells.
Another potential role of [C a
21
]
i
fluctuations in VZ cells could
be to regulate gap junction coupling. Cell coupling in the VZ is
dynamic; proliferative cells in the VZ uncouple from clusters
before M phase and recouple in G
1
or S phase to progress through
the cell cycle (Bittman et al., 1997). The permeability of gap
junction channels is also dynamic and can be regulated by intra-
cellular Ca
21
levels (Turin and Warner, 1977; Spray et al., 1981).
Fluctuations in [C a
21
]
i
could therefore be involved in the regu-
lation of gap junction permeability and could underlie the dy-
namic changes observed in VZ cell coupling.
Coordinated [Ca
21
]
i
increase in VZ cell clusters
The observations presented here that VZ cell clusters undergo
spontaneous coordinated fluctuations in [Ca
21
]
i
suggest that gap
junction-coupled clusters in the VZ may act as functional units
and that synchronized [C a
21
]
i
increases may coordinate intercel-
lular signaling among cluster members. A possible role of such
coordinated transients might be to synchronize cell cycle events.
Experiments based on clonal analysis and birthdate labeling also
indicate that small groups of adjacent cortical precursor cells,
similar in size to the gap junction-coupled cell clusters, pass
through the cell cycle in relative synchrony (Reznikov and van der
Kooy, 1995; Cai et al., 1997). We hypothesize that these synchro-
nously cycling cells may belong to individual gap junction-
coupled VZ cell clusters. If true, this would support a role for
coupling and possibly coordinated [C a
21
]
i
increases in cell cycle
synchronization.
Coordinated changes in [Ca
21
]
i
in groups of adjacent cells
have been described in a variety of intact tissue preparations. In
addition to the cluster events described in this study, spontaneous
increases in [Ca
21
]
i
have been reported in groups of gap junction
coupled neurons in the developing postnatal cortex (Yuste et al.,
1992, 1995), and spontaneous waves of [Ca
21
]
i
increase have
been reported in neighboring neurons in the developing retina
(Wong et al., 1995; Feller et al., 1996). Coordinated [Ca
21
]
i
increases thus seem to be a general feature of developing post-
natal CNS neurons (Yuste, 1997). It has been proposed that
coordinated Ca
21
signaling via gap junction channels observed in
cortical neurons may be involved in synaptic circuit development
(Peinado et al., 1993b; Kandler and Katz, 1995; Katz and Shatz,
1996), but the role of gap junction coupling in precursor cells in
the VZ is likely to serve a completely different function possibly
more analogous to the role of coupling in other populations of
proliferating cells (Guthrie and Gilula, 1989). It is noteworthy
that after terminal mitosis, uncoupled neurons migrate to the
cortical plate, recouple perinatally, and once again undergo co-
ordinated [Ca
21
]
i
increases (Yuste et al., 1992; Peinado et al.,
1993a; Bittman et al., 1997). It is interesting to speculate that an
individual postnatal neuronal domain may consist of neurons
whose clonal antecedents were once coupled together within
the VZ.
Spontaneous [Ca
21
]
i
fluctuation in immature neurons
At least some of the cells exhibiting single-cell events in the VZ
at late embryonic ages are likely to be postmitotic neurons.
Distinct patterns of spontaneous [C a
21
]
i
fluctuations have been
described for postmitotic neurons during early stages of migration
and differentiation in other experimental systems. Intermittent
[Ca
21
]
i
increases associated with periods of migrational move-
Figure 10. Schematic of cell types demonstrating different patterns of
spontaneous [Ca
21
]
i
fluctuations. Numbers refer to cells demonstrating
single-cell events (1), double-cell events (2), and coordinated multicell
cluster events (3). Single active cells are primarily TuJ1-negative prolif-
erative cells but could also include members of clusters that display
independent [Ca
21
]
i
fluctuations (?) and postmitotic neurons in the
process of migration, particularly during the later stages of neurogenesis.
Single-cell activity might also include radial glia cells. Cell pairs demon-
strating synchronized activity are mitotically active precursor cells. Active
cell clusters correspond to groups of gap junction-coupled cells that
include precursor cells in G
1
,G
2
, and S and at least one radial glia cell.
Spontaneous [Ca
21
]
i
fluctuations are also seen in neurons of the IZ,CP,
and MZ (shaded cells).
5386 J. Neurosci., July 15, 1998, 18(14):5374–5388 Owens and Kriegstein •Ca
21
Fluctuations in Neocortical VZ Cells

ment have been observed in cerebellar granule cells (Komuro and
Rakic, 1996). Both Ca
21
influx and release of Ca
21
from intra-
cellular stores contribute to the [C a
21
]
i
fluctuation associated
with granule cell migration (Komuro and Rakic, 1996). Calcium
transients associated with cell movement have also been observed
in cultured cortical neurons (Behar et al., 1996). Some of the
single-cell [Ca
21
]
i
oscillations observed in the VZ could there-
fore be early [Ca
21
]
i
surges that act to propel cells during
migration.
After migration out of the VZ, postmitotic neurons in the MZ
and CP continue to exhibit spontaneous [C a
21
]
i
fluctuations.
These events could serve to influence the differentiation of im-
mature neurons. Calcium transients have been associated with
neurite outgrowth and growth cone motility (Mattson et al., 1988;
Rehder and Kater, 1992; Kater et al., 1994). Differentiating
amphibian spinal neurons generate waves, spikes, and clusters of
[Ca
21
]
i
increase (Spitzer and Gu, 1997). Calcium spikes promote
normal neurotransmitter expression and channel maturation,
whereas Ca
21
waves are associated with neurite extension (Gu
and Spitzer, 1995). The observations presented here of sponta-
neous [Ca
21
]
i
increases in both proliferative and postmitotic
cortical cells suggest that similar changes in [Ca
21
]
i
may underlie
different signaling events during distinct phases of neocortical
development.
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