Flow Cytometric

Strategies
to Study CNS Development
Dragan

Marie,

I, Introduction

lrina Marie,

and leffery

1. Barker

to Flow Cytometry

The technique of flow cytometry was initially developed to

count and size particles. However, it has progressively evolved
into a sophisticated analytic tool for rapidly quantifying multiple
properties of individual cells or cellular constituents in suspended
nonhomogeneous populations. All flow cytometry instruments
share a common feature: single cells or particles are pressured to
flow through a sensing region in which their electrical resistance
or optical properties are recorded. Most commonly, these properties are visualized with fluorescent molecules that bind specifically to the biological constituent(s) to be measured. Typically,
these fluorescent molecules are excited by laser beam(s) tuned at
specific wavelenghtts) and their emission(s) collected with an array
of appropriate filters that convey the signals to photomultiplier
tubes and ultimately to a computer.
Flow cytometry complements other optical and electrical
recording strategies that have recently evolved and offers clear
advantages, including the acquisition of multiple parameters at
very high rates (1000-3000 events/s), objectivity, and powerful
sorting capabilities. Over the last 25 yr, it has become widely used
in the fields of hematology, immunology, oncology. and microbiology. Cell counting, identification and classification, cell cycle
studies, measurements of DNA content and cell proliferation, chromosomal karyotyping, and studies of cellular physiology are
among the most widespread research and clinical applications of
flow cytometry (Melamed et al., 1990).
From
Neuromethods,
vol
Eds A A Boulton,
C B Baker,

33

Cell

Neurohology

and A N. Bateson

287

Techques

0 Humana

Press Inc

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Marie,

Marie,

and Barker

In the field of developmental

neurobiology,
however, flow
cytometry has not been extensively used so far. In this chapter,
we demonstrate several possible applications of flow cytometry
in the studies of CNS development: rapid identification of specific cell populations in the developing CNS using multiple surface
and cytoplasmic markers putatively specific for neuroepithelial,
neuronal, and glial cell lineages; analysis of cells in specific stages
of cell cycle and apoptosis; physiological recordings of membrane
potential and cytosolic calcium and pharmacological discovery
of functional receptors and ion channels; and precise isolation and
sorting of distinct cell populations, based on a specific epitope
expression or a functional response.
2. Cell Preparation
One of the most crucial steps in using flow cytometry to investigate physiological and pharmacological properties of developing CNS at a single-cell level is cell preparation. Cells composing
the CNS during embryonic (E) and early postnatal (P) periods can
be most completely dissociated into single cell suspensions by
enzymatic digestion with papain (Huettner and Baughman, 1986,
Marie et al., 1997). Other commonly used dissociation protocols,
including mechanical (Mandler et al., 1988), trypsin (Schaffner and
Daniels, 1982), and collagenase (Johnson and Argiro, 1983) can
lead to highly variable cell recoveries, which are associated with
up to 50% reduction in cell yield, together with a markedly
decreased cell viability (Marie et al., 1997).
In our study, the papain dissociation protocol was as follows
Embryonic (Eli-22) and early postnatal (PO-7) CNS tissues were
quickly dissected into telencephalic (Eli-14) and neocortical
(E15-P7), olfactory bulb, hippocampal, thalamic, hypothalamic,
mesencephalic, rhombencephalic, and spinal cord regions (Hebel
and Stromberg, 1986; Altman and Bayer, 1995) and immediately
placed in ice-cold saline to retard further developmental changes.
Tissues were cleaned, minced with forceps, and then completely
dissociated into single-cell suspensions by the enzymatic action
of papain (20 U/mL) for 30-45 min at 37C, and gentle trituration
as described (Huettner and Baughman, 1986). In some experiments, 350~pm thick coronal sections of late embryonic neocortex
were first microdissected along the incipient white matter into
cortical plate/subplate (CP/SP) zone, including layer I cells, and

CNS Development

Studies

289

ventricular/subventricular
(VZ/SVZ) zone, including lower intermediate zone (IZ) cells, and then dissociated as described. This
protocol routinely yielded single-cell suspensions with greater
than 95% vitality as determined by trypan blue exclusion on the
microscope stage and confirmed using vital (acridine orange) and
nonvital (propidium
iodide) dye staining of cell suspensions
analyzed by flow cytometry.
After isolation, the cells were labeled with fluorescent antibodies and/or indicator dyes, and passed through the laser-based fluorescence activated cell sorter (FACS), where up to five different
parameters of each single cell (including cell size and complexity,
and immunocytochemical, membrane potential and calcium fluorescence signals) were measured simultaneously, at the rate of
several thousand cells per second. In some experiments, precise
sorting of different cell subpopulations then followed, based on
any one or a combination of these different cell parameters. A schematic outline of the method and some of the cell properties that
can be quantified with different indicators by flow cytometry are
depicted in Fig. 1.
All recordings were carried out with a FACSTAR flow cytometer (Becton Dickinson, Mountain View, CA). Cells were excited
using an argon ion laser (Spectra Physics, Model 2016, Mountain
View, CA) operated at 500 mW and tuned to 488 nm. Forward
angle light scatter (FALS), a property related to cell size, and different fluorescence emissions of individual
elements were
randomly recorded at 1000-2000 events/s. This rate of data
acquisition allowed profiling the properties of approx 10,000 cells
in 5-10 s. FALS data were collected in a linear mode using a combination of 488 + 10 nm bandpass and neutral density filters,
whereas fluorescence emissions were logarithmically
amplified
and filtered at appropriate wavelengths. In multiple labeling
experiments, fluorescence emissions were corrected for color crossover by using electronic compensation. FALS properties and fluorescence intensities were each resolved into 1024 channels. The data
were analyzed using Cell Quest Analysis software operating on a
FACStation Macintosh-based computer platform (Becton Dickinson).

3. lmmunocytochemistry
One of the major difficulties encountered when studying the
development of the CNS is the inability to readily identify specific

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Development

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291

cell lineages at distinct phases of proliferation and differentiation.

One of the reasons IS the lack of availability of uniquely specific
cell markers. There is at present a rapidly growing number of commercially available polyclonal and monoclonal antibodies that can
be used to detect specific cell surface, cytoplasmic, or nuclear
epitopes in CNS cells. However, many of these epitopes are shared
among neuroepithelial,
neuronal, and glial cell types at some
stages of their development. Therefore, there is an increasing need
for using double- and triple-immunostaining
procedures, in order
to obtain a more precise identification of specific cell populations
under mvestigation. A flow cytometer equipped with dual and
triple emission filter sets is ideally suited to access this complexity and diversity of specific CNS populations in a very rapid and
precise manner. In the following sections, we describe the identification of putative neuroepithelial, nerve- and glia-specific markers on several populations of acutely dissociated embryonic and
early postnatal CNS cells using flow cytometry and double- or
triple-immunolabeling
protocols with specific antibodies against
cytoplasmic and plasma membrane epitopes.
3.1. lmmunocharacterization

by Cytoplasmic

Markers

Different cytoplasmic markers tagged with fluorochrome-conjugated antibodies can be identified by flow cytometry, but prior
cell fixation and membrane permeabilization are necessary. Laserbased flow cytometry is more sensitive in detection of immunoFig. 1. ~~WZVOUS
page)Accessing CNS development by flow cytometry
(A) In order to study the biological properties of developing neuroeplthehal, neuronal, and glial cell lineages during CNS development, the cells
first have to be dissociated into uniform single-cell suspensions.
(B) The
cells are then immunoreacted,
stained or loaded with reagents that target
their distinct phenotyprc or physiological
properties
The labeled cells are
passed through a nozzle tip (with an aperture of 70 pm> and Illuminated
one at a time with a laser set at a desired excitation wavelength.
Then
light-scattering
and fluorescence
emission properties
are collected with
an array of specific filters connected to their respective photomultiplier

tubes, which convey the signals to the computer. (C) By vibrating the
nozzle tip at high frequencies
(typically
24,000 Hz) and electronically
charging the mdivrdual droplets of salme in which each cell is suspended,
It is possible to sort specific populations of cells based on a distmct combrnation of then light scattering and fluorescence emission properties.

292

Mar/c,

Marie,

and Barker

fluorescence signals than conventional lamp-based fluorescence

microscopy and offers the advantage of precise and objective electronic quantification of different fluorescence intensities in tens
of thousands of cells virtually all at the same time. This is particularly important
in studies on the developmental
appearance
and
disappearance
of different cell markers, since it is often difficult
to distinguish
precisely
between
background
and very low
immunopositive
signals with conventional
methods. Figure 2 represents the results obtained
after immunostaining
acutely disso-

ciated and ethanol-fixed

El9 neocortical cells with a rabbit
polyclonal class IgG anti-nestin antibody, an intermediate filament
protein associated with neuroepithelium-derived
progenitor cells
(Hockfield
Bethesda,

and McKay,
1985) (a gift from R. McKay,
NIH,
MD), and a mouse monoclonal
class IgG anti-MAP2

antlbody, a neuronal cytoskeletal marker (Sigma, St. Louis, MO).

For flow cytometry,
these immunoreactions
were respectively
visualized with a phycoerythrin (PE)-conjugated goat anti-rabbit
IgG and biotinylated
goat anti-mouse
IgG (Jackson ImmunoResearch

Laboratories

Inc.,

West

Grove,

PA),

followed

by

Fig. 2. (opposzte page) Double immunolabeling

of cytoskeletal markers
in fixed cell preparations.
(A) Flow cytometric assessment of antmestm
and anti-MAP2
immunostaming
of El9 neocortlcal cells reveals four
distinct subpopulatlons.
Nestin- /MAP2-,
Nestm+/MAP2-,
Nestin-/
MAP2+, and Nestin+/MAl??
Whereas most of the Nestin+/MAP2and
Nestm/MAP2+
cells are located m the VZ/SVZ and CP/SP, respectively,
both regions contain Nestin+/MAlY
subpopulations.
However, there
are marked region-specific
fluorescence
intensity
differences
in both
cytoskeletal
markers
between these two subpopulations
Nestmhgh/
MAP2 expressors
are located in the VZ/SVZ and Nestinow/MAP2t~h
expressors
appear m the CP/SP (B) Immunostainmg
of acutely plated
CP/SP and VZ/SVZ
cells with the same antibodies
clearly reveals
MAl2hgh immunopositive
cells in the CP/SP and Nestinhgh cells m the

VZ/SVZ,

whereas the quantification

of Nestn+

and MAP2

subpopu-

lations IS somewhat
ambiguous using the light microscope
(C) Immunostaining
of the El9 coronal sections of the cortex under identical
conditions used for flow cytometry confirms that nestin-lmmunoposltlve
cell bodies are for the most part located in the VZ/SVZ, whereas MAP2-

lmmunopositlve

cells are present mainly

sections can not resolve the intensity

marker m individual
cells.

m the CP/SP

differences

However,

tissue

of either cytoskeletal

Anti-Nestin-PE

Anti-Nestin

Anti-Nestin

Anti-MAP2h..

Anti-MAP2

I
CP
SP

IZ
I.

svz
vz
Fig. 2(A-C)
293

i,,

I
!
i

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Max,

Mar/c,

and Barker

streptavidin-Red670
(Life Technologies Inc., Gaithersburg, MD).
Cell immunofluorescence
characteristics were acquired using a
488 nm laser excitation and fluorescence filters set at 575 rt 25 and
670 _+ 20 nm to detect PE and Red670 emissions, respectively.
Reactions in acutely plated cells were visualized with appropriate blotinylated secondary antibodies, followed by streptavidinperoxidase (Jackson ImmunoResearch
Laboratories Inc., West
Grove, PA) and the development of peroxidase reaction product
in 3-amino-9-ethylcarbazole
(AEC) containing 0 001% HzO,.
Quantitative flow cytometric assessment of logarithimlcallyamplified anti-nestin and anti-MAP2 immunofluorescence intensities revealed different levels of nestin and MAP2 expression in
neocortlcal cells, as some transformed from progenitor stages in
the VZ/SVZ to more differentiated neuronal stages in the CP/SP
(Fig 2A). We akl y expressing nestin- and MAP2-immunoposltive
cells comprised distinct subpopulations in the flow cytometric
recordings, although they could not be easily accounted for under
the microscope (Fig. 2B), despite the fact that the percent of highexpressing immunopositive cells obtained with both methods was
quite similar. For example, it was very difficult to precisely quantify the large population (approx 50%) of nesti@ positive cells in
the CP/SP dissociates without a flow cytometer, even when the
antibody reaction in acutely plated cells was visualized with a
much more sensitive enzymatic endpoint, instead of a fluorescent
endpoint. Because of this objective, extremely sensitive and rapid
data acquisition, the results obtained with flow cytometry are often
more complete compared to the results obtained with conventional
microscopy techniques.
3.2. lmmunocharacterization

by Cell Surface Markers

Living CNS cells in different stages of neuronal and glial lineage progression can be identified using antibodies against distinct cell-surface markers. A variety of monoclonal antibodies are
now available that recognize specific ganghosides and other
epitopes on the plasma membranes of developing CNS cells. In
our studies, we have used a mixture of tetanus toxin fragment C
(TnTx) and a mouse monoclonal class IgG anti-TnTx antibody, a
marker of terminally postmitotic developing neurons (Koulakoff
et al., 19831, a mouse monoclonal class IgM anti-A2B5 antibody, a
neuronal and O-2A progenitor marker (Abney et al., 1983), a mouse
monoclonal class IgM anti-04, and a mouse monoclonal class IgG

CNS Development

295

Studies

anti-galacto-cerebroside
(GalC) antibodies (Boehringer Mannheim
Biochemicals, Indianapolis, IN), two markers of early and late
stages of oligodendrocyte lineage development (Raff et al., 1978,
Schachner et al., 1981). Acutely dissociated cells were double
labeled with different combinations of these antibodies and primary immunoreactions were then visualized by immunostaininmg
with PE-conjugated goat anti-mouse IgM antibody and a biotinylated goat anti-mouse IgG (Fey fragment specific) antibody
(Jackson ImmunoResearch
Laboratories, West Grove, PA) followed by streptavidin-Red670
(Life Technologies, Gaithersburg,
MD). Results of TnTx/A2B5
and GalC/04
double-immunostaining reactions at several ages and regions during CNS development revealing qualitative and quantitative
differences in
expressions and coexpressions of these surface epitopes are presented in Fig 3.
4. Assay of Proliferative
and Apoptotic
of Neocortical
Subpopulations

Potentials

It is well accepted that development of the CNS system involves

both cell proliferation
and naturally occurring cell death, or
apoptosis (Naruse and Keino, 1995). Here we show that these processes can be expeditiously
detected and quantified by flow
cytometry using fluorescently labeled antibodies against thymidine analog bromodeoxyuridine
(BrdU), a marker of S-phase cells
(Gratzner, 19821, annexin V, an anticoagulant protein that preferentially binds to phosphatidyl serine phospholipids exposed on
the outer leaflet of the cytoplasmic membrane early in apoptosis
(Koopman et al., 1994; Martin et al., 1995), and propidium iodide
(PI), a fluorescent dye that binds to all double-stranded
nucleic
acids and can be used to measure total DNA content (Dolbeare
et al., 1983).
4.1. Detection of BrdU Incorporation
by DNA -replicating
Cells
Timed pregnant dams at embryonic day 16 were given a single
intraperitoneal injection of BrdU (50 ug/g body weight) (Sigma)
and sacrificed 60 min later. The pups were removed and several
regions of the developing CNS acutely dissociated as previously
described. Detection of BrdU incorporation
was conducted by
permeabilizing the ethanol-fixed cells with 2 N HC1/0.5% Triton

296

Marie,
Neocortex K!P/SP)

Marie,

and Barker

Neocortex NZISVZI

I 1
32.3

Spinal Cords
PI

Anti-A2BS-PE
Neocortex

Fluorescence

Olfactory

Cerebellum

Bulb

Hippocampus

Rhombencephalon
1.7

hl

I7

Anti-040PE

Fluorescence

Fig. 3(A,B)

CNS Development

297

Studies

X-100 and immunoreacting the exposed DNA with FITC-conjugated

mouse anti-BrdU monoclonal antibody (Becton Dickinson). Finally,
the immunoreacted nuclei were counter-stained for total DNA content by resuspending the cells in PBS containing 5 ug/mL PI.
Bivariate distributions of BrdU incorporation and total DNA content were then assessed on a single-cell level by flow cytometry
(Fig. 4). Upon excitation at 488 nm, the green (FITC-conjugated
anti-BrdU) and red (PI) fluorescence intensities emitted by each
cell were acquired using bandpass filters set at 530 + 30 and
575 + 20 nm, respectively. Electronic gating was used to exclude
any residual cellular aggregates, which consistently accounted
for ~5% of the total number of events. The percentages of BrdU+
(S-phase cells) and BrdU- subpopulations with diploid or tetraploid DNA content (reflecting cells in GJG, and GJM stages of
the cell cycle, respectively) were quantified using a Cell Quest
data analysis system.
4.2. Detection
and Nuclear

of Plasma Membrane
Markers
of Apoptotic

Cells

Apoptotic cells can be first detected at the level of plasma membrane using annexin V (Koopman et al., 1994; Martin et al., 1995).
Fig. 3 fprevzous page) Double immunolabeling
of surface markers on
viable cell preparations
quantified by flow cytometry. (A) Anti-A2B5
and anti-TnTx immunostaining
of El9 neocortical cells reveals four distinct subpopulations.
A2B5-/TnTx-,
A2B5+/TnTx;
A2B5-/TnTx+,
and
A2B5+/TnTx+. Whereas all four subpopulations
can be found in the proliferative and early differentiating
VZ/SVZ regions of the El9 neocortex, the differentiating
CP/SP region is for the most part composed of
A2B5-/TnTx+ cells, which we independently
identified as a vrrtually pure
neuronal population using cytoskeletal markers and expressed morphological characterrstics m short-term cultures (see Fig. 2). Other mvestigated CNS regions at El9 reveal a variable presence of all of the above
populatrons with the exception of the hippocampus, which notably lacks
A2B5-immunopositive
cells. (8) Anti-04
and anti-GalC
immunoreactions of P6 neocortical cells also reveal 4 distinct subpopulations.
04-/GalC-, 04+/GalC-, 04-/GalC+, and 04*/GalC+. Whereas 04+/GalCsubpopulation
1s detected in all CNS regions tested at P6, the rhombencephalic and spinal cord regions exhibit the greatest abundance of
04+/GalC+
cells, with the former also showing the greatest percentage of 04-/GalC+
cells.

298

Marie,

Marie,

and Barker

HYPOTHALAMUS

CORTEX
Km
II n-. _<nLlm

RHOMBENCEPHALON
W-0

$4
e!

GdGv91.8%

SPINAL CORD
lumbosacral (SC 1)

bl:

9.4%

ag

;
43
\ +*,

SPINAL CORD
thoracic (SC t )
Gn/G1:86.0%

Fig. 4. Three-dimensronal
histograms of bivariate DNA data acquired
by flow cytometry illustrate an anatomical gradient of cellular prohferatron and differentiation
throughout
the neuroaxis at Elk Relatively
few cells are actively syntheslzmg new DNA in the cervical spinal cord
and rhombencephalon,
whereas cerebral cortrcal and lumbosacral
spinal cord populations
exhibit the greatest percentages of cells m S-phase

CNS Development

299

Studies

In our studies we have triple-stained acutely dissociated El9 neocortical cells with anti-A2B5-PE, anti-TnTx-Red670, and annexin
V-FITC and analyzed them by flow cytometry (Figs. 5A and B).
Green (FITC), orange (PE), and red (Red6701 fluorescence emissions were simultaneously
measured with bandpass filters at
530 _+30,575 A 20, and 670 + 20 nm, respectively. Percentages of
single-, double- and triple-positive and negative cells were quantified with the Cell Quest Analysis software using a logical gating
strategy.
Late stages of apoptosis were assessed by measuring total DNA
content of fixed cells stained with PI (Fig. 50, Cells doubleimmunoreacted
with A2B5-PE and TnTx-Red670 were sorted
based on their surface epitopes into A2B5-/TnTx-, A285+/TnTx-,
A2B5-/TnTx+, and A2B5+/TnTx+ subpopulations (seeSection 7.1.)
and fixed in 70% ethanol. The A2B5 and TnTx immunoreactions
were then stripped off the cell membranes in Triton/HCl
solution and the cells further processed for detection of their total DNA
content (seeSection 4.1.). Late apoptotic or A,, cells were identified as cells with hypodiploid DNA content with respect to that
of G,/G, cells, which have diploid DNA. This cytometric property, also referred to as sub-G,/G, peaks, depicting cells undergoing DNA fragmentation, has been shown to be a reliable marker
of cell death by apoptosis (Telford et al., 1991; Darzynkiewicz et
al., 1992; McCloskey et al., 1994).
5. Potentiometric
of Dissociated

Signals
Embryonic

Neocortical

Cells

One of the most crucial processes in CNS cytogenesis is the

development of membrane excitability. Although classical electrophysiological techniques have been extensively used to characterize membrane receptor/channel
and ion properties of cells,
technical difficulties can be encountered in recording small, proliferating, and immature cells, which constitute the majority of
cytoarchitecture during the earliest stages of CNS development
(reviewed by Barry and Lynch, 1991). In addition, microelectrode
techniques can be invasive to the cell membrane and only a limited number of cells can be recorded at any one time under the
same experimental conditions. One way of overcoming these difficulties is to use noninvasive techniques with fluorescent voltage-sensitive indicator dyes. Several studies have already reported

300

Marie,

Marie,

and Barker
A2BS+iTnTx+

A2B5PE

Immunofluorescence

Annexin V-FITC

Fluorescence

A2BS+fhTf

Propidium

Iodide Fluorescence

Fig. 5. Flow cytometric assessment of early and late apoptotic neocortical cells at E19: (Al Live, unfixed cells were triple stained with antiA2B5 and anti-TnTx antibodies
and annexin V-FITC. We have used
electronic gates (depicted by the cross-hairs) on A2B5 and TnTx doubleimmunoreacted
cells to reveal their expression of annexin V. (B) Annexin
V-FITC binding to the plasma membranes reveals a differential
presence of annexin V and annexin Vhigh-positive cells in the four immuno-

CNS Development

301

Stud/es

the utility of voltage-sensitive dyes in investigations of potentiometric signals of developing CNS tissues using digital video
microscopy (Walton et al., 1993) and flow cytometry (Mandler et
al., 1988; Krieger et al., 1991; di Porzio et al., 1993; Fiszman et al.,
1993). The advantage of these methods is that they allow experimental access to the physiological properties of entire populations
of intact cells regardless of their size or morphology, and can provide a statistically complete account of potentiometric signals randomly acquired from hundreds to thousands of individual cells
within a matter of seconds.
In our studies, we have used flow cytometry and oxonol, an
anionic voltage-sensitive indicator dye that partitions into the cells
according to membrane potential (Petit et al., 1993), to investigate
the development of membrane excitability throughout the embryonic CNS. After papain dissociation, cells were resuspended in a
physiological saline (in n-&I): 145 NaCl, 5 KCl, 1.8 CaCl,, 0.8 MgCl,,
10 HEPES,

10 glucose,

and 1 mg/mL

fatty-acid-free

bovine

serum

albumin (Sigma), the pH and osmolarity of which was adjusted

to 7.3 and 290 mOsm, respectively. Cells were then stained with
bis(l,3-dibutyl
barbituric acid) trimethine oxonol (Molecular
Probes, Eugene, OR), a potentiometric
dye that is negatively
charged at physiological pH. Since virtually all living cells exhibit
negative potentials, the dyes negative charge opposes its cellular
accumulation at resting potentials, whereas depolarized cells stain
lo-fold

or more

relative

to cells at rest. All of the potentiometric

results were recorded using 200 nM oxonol, since this concentration effectively stains cells well above autofluorescence levels.
Staining with oxonol requires approx 2 min to equilibrate at room
temperature, after which the mode and distribution of fluorescence signals remain stable for at least the duration of the typical
(Frg. 5, contznuedfvom prevtous page) identified subpopulatlons
Separate
experiments
using unfixed cells stained with annexm V-FITC and PI
revealed that only a few of annexin V and the majority of annexin
Vh~hcells were PI positive, demonstrating
that membrane permeability
of most annexm Vi cells is not significantly
compromised,
thus mdicatmg the early phase of apoptosis. (Cl PI staming of sorted and fixed
A2B5-ITnTx-,
A2B5+/TnTx-,
A2B5-/TnTx+,
and A2B5+/TnTx+ subpopulations reveals a percentage of hypodiploid
cells that positively
correlates with the percentage
of annexin V-positive
cells m the same
subpopulations

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and Barker

recording period (5-15 min). Oxonol-stained cells were passed

through a flow cytometer at a rate of 2000 cells/s, excited one at a
time by 488 nm and the resulting emissions detected with a single
filter set at 530 + 30 nm. A typical recording involved the acquisition of oxonol fluorescence intensities of 10,000 randomly sampled
cells recorded over 5 s, the distribution of which was then plotted
as a single-parameter frequency histogram (Fig 6).
5.1. Calibration

of Membrane

Potential

The relationship between oxonol fluorescence intensity and cell

membrane potential can be calibrated by recording the oxonol fluorescence intensity profile under resting conditions and again after
exposing cells in the same test tube to gramicidin, a monovalent
cationophore previously used to relate oxonol fluorescence to theoretical membrane potential in spectrophotometric (Breuer et al.,
1988; MacDougall et al., 1988; Cruciani et al., 1991; Brent et al.,
1993) and flow cytometric studies (di Porzio et al., 1993). In electrophysiological recordings, 1 PM gramicidin depolarizes cells to
0 mV in physiological saline. In our recordings, we have empuitally determined that saturating concentrations (21 PM) of gramicidin increase the fluorescence modes of oxonol-stained cortical
cells in a [Na+lO-dependent manner (Fig. 6). These results allowed
us to use a Goldman-Hodgkin-Katz
formulation to relate oxonol
fluorescence to membrane potential and to calibrate the signals
Assuming that after gramicidin permeabilization total intracellular concentration of permeant Na and K cations remains constant at approx 150 mM during the 10-s recording period, then
oxonol fluorescence modes of the signals in altered Na+O salines
can be related to membrane potential using a simplified GoldmanHodgkin-Katz equation in which the membrane potential is E, E0 = RT/ZF log [Na + K+ll/[Na+ + K+lO,where R, T, Z, and F have
their usual meanings. Modes of oxonol fluorescence can be calibrated in terms of membrane potential over much of the physiological range, i.e., -90 mV-0 mV (Fig. 6B).
5.2. Survey of Membrane
in Developing
Neurons

Excitability

We have investigated the chemosensitivity of acutely dissociated El9 CP/SP neurons to saturating concentrations of various
neuroactive agents including acetylcholine, y-aminobutyric acid

CNS Development

303

Studies

Oxonohained
pmicidin-treated
[Na+l,bM)
0

145
+I0

V,,,= 0307FL,,-228
R = 0.99

0
gB

145

-10

7257

'i;j -20
P
$ -30
2

207

-40

if! -50
aa

69

P -60
0
3 -70

RMP

-80

/
-loll~-~
400

Oxonol

Fluorescence

Intensity

(Channels)

450

Oxonol

500

550

Fluorescence

600

650

Intensity

700

750

4
800

(Channels)

Fig 6. Calibration of oxonol fluorescence signals in terms of estrmated

membrane potential values. (A) Oxonol-stained
neurons isolated from
the El9 CP/SP were resuspended in salines containing varying iNa+&
(145, 72.5, 20.7,6.9, and 0 n-M), which was replaced by equimolar concentrations of membrane impermeable
N-methyl-D-glucamine.
The cells
were then treated with 1 uM gramicidin
and their fluorescence levels
recorded (B) The modal fluorescence values of the oxonol fluorescence
distributions
(FL,,) m different [Na+10 are plotted against theoretical
membrane
potentials
(V,,,) as calculated
by a simplified
GoldmanHodgkin-Katz
equation, The slope of this relationship
reveals a relatively constant conversion factor, which defined that a change in approx
3 fluorescence channel units in oxonol intensity is equivalent to 1 mV
change in membrane potential
By substituting
the modal oxonol fluorescence value of El9 CP/SP neurons under our control resting conditions for FL,,, we estimated the modal resting membrane potential of
these cells at -85 mV.

(GABA), glycine, kainic acid (agonist of a subtype of glutamate

receptors), and veratridine (agonist of voltage-dependent
Na+
channels). Potentiometric responses were quantified in terms of
percentage of responsive cells and the amplitude of the responses,
which were, after calibration, converted into mV. After recording
a control profile, cells were exposed to different ligands and change
in oxonol fluorescence recorded after 2 min. All recordings were
performed at room temperature. The heterogeneity of excitatory
responses obtained in recordings of these cells (Fig. 7) indicate

304

Mar/c,

f;
a

1
:9

B 60
z

Mar/c,

and Barker

40

c2
20

80
60
I

loo

40

20

.
(;*:*?,
\:j::
.J
:

Membrane

Potential

(mV)

Fig. 7. Excitatory
membrane
potential
responses
of El9 CP/SP
neurons.
Approx
90-95% of cells depolarize
to either 10 PM GABA
or 100 pM veratridine,
whereas less than 5% depolarize
to 10 FM acetylcholme.
Veratrldme
depolarizes
cells to +20 mV, whereas
GABA

CM

Development

30.5

Studies

that FACS potentrometry

can serve as a powerful
tool for the
investigation
of the cellular distribution
of functional
receptor/ion
channels in different subpopulations
of developing
CNS tissues.

6. Intracellular
Calcium Signals
of Dissociated Embryonic Neocortical

Cells

Measurement
of cytoplasmic
calcium
([Ca],)
concentrations
in living cells is of great interest to many investigators,
since Ca*+
is a ubiquitous
second messenger throughout
the CNS. Cazcc signals have been implicated
in the development
of many neuronal
and glial cell functions.
Cell survival
(Spitzer, 1994) and death
(Franklin
and Johnson,
1994), neurotransmitter
release (Hille,
1992), growth cone motility
and neurite elongation
(Mattson and
Kater, 1987), cell migration
(Komuro
and Rakic, 1992), synaptic
plasticity
(Bear and Malenka,
1994), and regulation
of gene
expression
(Gallin and Greenberg,
1995) are only some of the Ca2+related processes that have been described.
Investigation
of the
mechanisms
that regulate intracellular
Ca2+ during histogenesis
of the CNS is therefore of crucial importance
in understanding
the physiology
of cell proliferation,
migration,
differentiation,
death, and cell-to-cell
communication.
Initially,
electrophysiological
methods were used to study Ca2+
homeostasis
and plasma membrane
expression
of Ca2+-selective
channels. However, recent development
of a growing family of Ca*+sensitive fluorescence
indicator
probes has led to alternative
optical and confocal microscopic
strategies of recording
Ca*+signals at
cellular and subcellular
levels. One such probe is l-[2-amino-5-(2,7dichlor-6-hydroxy-3-oxy-9-xanthenyl)
phenoxyl-2-(2-amino-5methyl
phenoxy)
ethanel-N,N,N,N-tetra-acetic
acid or Fluo-3.
Flu03 is a fluorescein-derived
Ca*+-sensitive
dye that produces a
40-fold increase in fluorescence
intensity
upon bmding
with free
(Fzg 7, confinuedfvom previous page) depolarizes virtually all cells to -40
mV. Only 50% of cells depolarize to -40 mV after exposure to 100 PM
glycme, whereas 100 PM kainic acid depolarizes approx 70% of cells
mainly to approx 0 mV. Gramicidin,
which chemically clamps all cells
at 0 mV by permeabilrzing
their plasma membranes with monovalent
cation-selective
channels, is always used as a control at the end of each
experiment
to reveal the fluorescence mtensrty and distribution
of
potentrometrlc
srgnals corresponding
to 0 mV

306

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Marie,

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cytosolic calcium (Minta et al., 1989). Although other types of Ca*+sensitive dyes have been described, e.g., quin-2, fura- and indo-l
(reviewed by Tsien, 1980; Grynkiewicz et al., 19851, the major advantage of Fluo-3 lies in its high signal-to-noise resolution, low cytotoxic
and mitogenic properties as well as in optimal excitation properties,
at wavelengths in the visible as opposed to UV range (Tsien, 1989).
In our studies, we have used Flu03 in conjunction with flow
cytometry in order to reveal and characterize, on qualitative and
quantitative levels, the presence of functional intracellular Ca*+stores,
Na+-Ca*+ exchange mechanisms, and several voltage- and ligandstimulated Ca2 channels in acutely isolated El9 CP/SP neurons. The
cells were loaded with 1 PM Flu03 for 45 mm at room temperature,
then washed and resuspended in physiological saline, passed through
a flow cytometer, excited one at a time by 488 nm and the resulting
emissions detected with a single filter set at 530 + 30 nm.
6.1. Calibration

of [Ca2+lc

The fluorescence of Fluo-3-loaded cells is measured in arbitrary

intensity units, i.e., fluorescence channels, which can be converted
into estimated [Ca], values after the calibration procedure is performed (Kao et al., 1989) at the end of each experiment (Fig. 8). Fluo3-loaded cells were acutely treated with lo-20 PM ionomycin, a Ca*+
ionophore, and the resulting saturated levels of Flu03 fluorescence
then maximally quenched by the addition of 2 mM MnCl,. [Ca2+lclevels under resting and experimental conditions were calculated
according to the following equation: [Ca*+], = Kd x [F - F,,,] / [FmaX- F].
Kd is defined as the dissociation constant for Ca2+-bound Flu03 and
is 400 nM at room temperature (Minta et al., 1989). Fmaxrepresents
the maximum Flu03 fluorescence value, whereas F represents the
fluorescence value of cells under resting or experimental conditions.
FmInis defined as the minimum Flu03 fluorescence in the presence
of saturating concentrations of MnCl,. Since Mn*+ ions readily displace Ca*+ ions from Flu03 (Hesketh et al., 1983) and the Mn*+/Fluo3 complex is only one fifth as fluorescent as Ca*+ /Flu03 complex
(Kao et al., 1989; Minta et al., 19891, F,,, is then calculated as follows
(Vandenberghe and Ceuppens, 1990): F,,, = F,,, - (FmuX- F,,,,,,) x
1.25. In our flow cytometric experiments, the fluorescence values of
F, Lx~ and h4na2 were defined as the arbitrary channel number of
the mode of Flu03 fluorescence distributions recorded from 10,000
randomly sampled cells under appropriate resting and experimental conditions (Fig. 8).

CNS Development

Studies

9000

8000

F,,= 814
Fbfna2= 451
Fmln= 360

7000

6000

ct-"

5000

cl

4000

ICa2+lc = 400

3000

Flue-3Fluorescence

Intensity

Khannels)

2000

1000

0
400

440

400

520

560

Flue-3 Fluorescence

600

640

Intensity

l""I"T'~l~~~~l600

720

760

(Channels)

Fig. 8 Calibration
of Fluo-3 signals. Flu03 loaded El9 CP/SP neurons were treated wrth 10 pM ionomycin
for 2 min followed by 2 mM
MnCl, for an additional
1 min. F,,, and FMvlnCIz
were measured from the
modal values of the resulting Fluo-3 fluorescence distributions.
By substituting the modal Fluo-3 fluorescence value of El9 CP/SP neurons
under our control resting conditions for F, we estimated the modal resting lCaz+lc levels of these cells at approx 140 nM
6.2. Survey

of the Contributors

to Calcium

Homeostasis

Regulation
of Ca 2+ levels in most cells is achieved
through
interactions
of Ca2+ &ansport
mechanisms
in the plasma
and
endo(sarco)plasmic
reticulum membranes
and Ca*+ buffering mechanisms in the cytoplasm (Kostyuk and Verkhratsky,
1994). Ca2+ transport in transmembranes
is regulated by different Ca*+ channels, Ca*+
pumps, and Ca*+ exchangers, whereas intracellular
Ca2+ stores and
Ca*+-binding
molecules serve as a Ca2+-buffering
system.
In our studies, we have surveyed the developmental
expression of several mechanisms
involved
in Ca2+ homeostasis
of El9
CP/SP neurons. intracellular
calcium stores, Na+-Ca*+-exchange,
and voltage-sensitive
calcium channels (VSCC) (Fig 9). Intracel-

rl
1100

308

Marie,

Max,

and Barker

Voltaee-Sensitive

Cytosolic Calcium Concentration

control

Calcium Channels

(CLM)

1
.=I1

I *sa

Fig. 9. Survey of several mecharusms

involved in Ca2+ homeostasis
in El9 CP/SP neurons
(A) All cells exhibit
micromolar
levels of calcium mobilization
after treatment
with 10 uA4 ionomycin,
a Ca2+ ionophore.
Whereas
thapsigargin
elevates [Ca2+lc to submicromolar
levels m virtually
every cell recorded,
only 64% and 35% of cells
respond to caffeine and ryanodine stimulation,
respectively.
(Bl Resuspension
in Na+O-free saline produces approx
100 nM increase of [Ca2+lc in the majority of cells, and a micromolar
increase of [Ca2+Jc in the remaining
5% of the
cells. Subsequent exposure of the same cells to 10 mM caffeine produces nucromolar
levels of [Ca], in many cells,
which are sustamed even after 5 min of continuous
recording
(right-most
panel) This response is in sharp contrast
to the response of the cells suspended
in regular physiological
saline (145 n-&I Na+,), where caffeine produces
a
transient increase in [Ca*+lc that recovers to resting levels wlthm 5 min of stimulation
in most cells These results
suggest that Na+-Ca*+-exchange
may play an important
role in regulatron
of resting levels of [Ca2+lE. (Cl Stimulation of El9 CP/SP neurons with 40 mM K+O produces a significant calcium entry in 65% of the population,
which is
totally abolished by repeating the experiment
in Ca2+0- free saline. A complete block of calcium entry induced with
40 mM K0 is also achieved by pre-exposing
the cells to 100 uM nitrendrpine,
implying that virtually
all cells have
L-type VSCC. Prestimulation
of cells with 100 nM o-conotoxin
GVIA or 100 nM o-agatoxin
VIA reveals that 18% of
the cells lack N-type and 23% lack P-type VSCC, respectively.

loo

310

Marie,

Marie,

and Barker

lular calcium stores were studied by resuspending the cells in Ca2+free saline and then stimulating them separately with 10 mM caffeine, 10 pM ryanodine, 10 pM thapsigargin, or 10 uM ionomycin
(Fig. 9A). Na+-C a*+-exchange mechanisms revealed under resting
conditions and after exposure to caffeine were studied by resuspending the cells u-t Na+O-free salines (Fig. 9B). Functional L-type,
N-type, and P-type VSCC were individually
revealed by exposing the cells suspended in physiological salme to 40 mM K*O in
the presence of nitrendipine, o-conotoxin GVIA, or cu-agatoxin
VIA, respectively (Fig. 90. Due to the transient nature (lo-300 s
range) of the Ca2+cresponses obtained with some of the above conditions, these recordings involved the acquisition of Ca2+csignals
at higher rates (3000 cells/s) than used in other experiments. In
addition, the dead time between application of the stimulus and
the recording was reduced to approx 2 s by using a Time Zero
module equipped with an injector system (Cytek Development,
Fremont, CA).
6.3. Caztc responses to neurotransmitter

ligands

We tested Ca2+cresponses in El9 CP/SP neurons to asymptotic

concentrations of several neuroactive agents, including acetylcholine, GABA, glycine, kainic acid, and veratridine. The responses
were recorded using a Time Zero module as described above. After
recording a control profile, cells were exposed to different ligands
and changes in Fluo-3 fluorescence recorded after approx 2 s. Typical peak Ca2+cresponses, recorded at room temperature, are illustrated in Fig. 10.

7. Flow Cytometric
Sorting
of Embryonic Neocortical

Subpopulations

The studies of specific populations of the CNS in vitro are complicated by our limited abilities to unequivocally
identify and
expeditiously isolate pure cell types. Investigators commonly use
Fig. 10. (upposzte page) Survey of Ca2+c responses of El9 CP/SP neurons to several neuroactrve
ligands After the addrtion of 10 pM acetyl-

choline, approx 70% of the cells exhibit an immediate submicromolar

rise in [Ca2+Jc that recovers to resting levels within 2 min of stimulation
(kinetics data not shown). At peak response, GABA and kainic acid mduce a Ca2+c rise in approx 60% of neurons to submrcromolar
and micro-

CNS Development

Studies
Rcs(fllg
,f J

SO-

SO

40-

100

0 OS 3

1 ZSIO

I
60-

;!
,

40-

00512

Cytosolic

Calcium

Concentration

(PM)

molar levels, respectively,

whereas glycine affects 30% of the cells,
elevating their Ca 2+cby 400 nM. Veratridine affects approx 90% of cells,
increasing [Ca+] levels above 1 PM. Ionomycin typically mduces a maxImum rise in [Ca5+lc in all cells recorded.

312

Max,

Marie,

and Barker

selective culture conditions to isolate neurons, astrocytes, oligodendrocytes, and other cell populations. However, these methods usually require several days to weeks of culturing, during which
time cell properties may change and no longer reflect those
expressed in vivo. A variety of methods now exist that permit
enrichment of specific subpopulations based on surface epitopes (i.e.,
panning and complement lysis). However, these are complicated by
the fact that many antigenic epitopes are shared among different cell
types during development and hence a combination of markers is
required for the identification and isolation of specific cell subpopulations. Using a flow cytometer equipped for sorting, it is possible to
isolate very pure specific cell subpopulations based on the presence
of multiple phenotypic or functional cell markers.
7.1. Sorting Based on Surface Epitope Expression
El9 neocortical cells were double immunostained with anti-A2B5
and TnTx antibodies, as described previously (seeSection 3.2.) and
categorized into four populations (TnTx+/A2B5-, TnTx+/A2B5+,
TnTx-/A2B5+, and TnTx-/A2B5-) based on their fluorescence signatures determined by FACS electronic gates (Fig. 11, left most
panel). The four populations were sorted by means of electrically
charged saline droplets, which were deflected by charged plates
directly into appropriate test tubes (see Fig. 1). Sorted cells were
then washed twice in physiological saline and re-analyzed to test
for sorting purity, which was greater than 96% in all cases (Fig.
11, four right panels). After sorting, the viability of the cells
remained unchanged, with less than 5% trypan blue or PI-positive (dead or dying) cells in every sorted subpopulation.
7.2. Sorting Based on functional

Response

Flow cytometers equipped for sorting also have a unique capability of isolating purified responding and nonresponding
cell
populations based on sustained or transient functional responses
in different cells. El9 CP/SP neurons stained with oxonol or loaded
with Fluo-3 were stimulated with 100 pM kainic acid or 10 PM acetylcholine, respectively. Responding and nonresponding subpopulations were sorted based on a sustained membrane depolarization
induced by kainic acid or a transient calcium increase induced by
acetylcholine using electronic gates as shown in Fig. 12 (shaded
areas>. To test for the purity of kainic acid responding
and

CNS Development

Studies

I2
Y4
a,- - - - - - -, 2
I
I

314

Maw,

Maw,

and Barker

Before Sorting
loo .

Kainlc Acid
(59%)

Sorted-Responders
loo .

100
Acetylcholine

Kainic Acid
,I!

80-

t
, I
1 ,

60-

.
;

40-

Sorted-Non-Responders
l-

Membrane

Potential

(mV)
Fig. 12

[Cytosolic

Calcium]

(PM)

CNS Development

315

Studies

nonresponding populations after sorting, the cells were rinsed

twice in physiological saline, restained with oxonol and restimulated with 100 IAM kainic acid. Virtually all sorted responders
depolarized again after restimulation, confirming the purity and
functional viability of the sort. By contrast, none of sorted nonresponder cells depolarized to kainic acid after restimulation.
Similarly, acetylcholine restimulation of sorted acetylcholineresponding and nonresponbding subpopulations revealed >95%
purity of each sort. The results confirm that functional sorting
according to both membrane potential and Ca2+cresponses is very
effective, and provides the opportunity for further study of very
specific cell subpopulations in developing CNS.

8. Conclusion
In this chapter, we have described several strategies
fying

and studying

and physiological
cytometry.

different

phenotypic,

proliferative,

properties of developing

The sort capability

for identiapoptotic,

CNS cells using flow

of flow cytometers

further

allows

isolation and purification of subpopulations of CNS cells expressing specific epitopes or functional receptors for more detailed cellular and molecular
analyses in culture. With
have begun to map the biological
properties

these strategies, we
of CNS cells in the

context of lineage progression. In sum, the versatility,

objectivity

and sort capability

of flow cytometry
may be ideally suited for
confronting
the complexity
of CNS development,
providing
an
unparalleled
perspective
on the distribution
of physiologically
relevant properties
as the cells transform
from proliferative
to a
more differentiated
state.

Fig. 12. (previous page) Functional sorting of responding and nonresponding cells according to membrane potential and calcium signals.
El9 cells were loaded with either oxonol, a voltage-sensitive
dye (panel
A), or Fluo-3, a calcium-sensitive
dye (panel B) and sorted into responding and nonrespondmg
populations
after the addition of 100 ~JM kainic
acid to oxonol-loaded
cells or 10 PM acetylcholine to Fluo-3-loaded
cells
(sorting gates are shown as shaded areas). Reanalyses of sorted and
restimulated
subpopulations
revealed > 95% purity of functionally
responsive and nonresponsive cells.

316

Marie,

Marie,

and Barker

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