We quantified cytoplasmic Ca2+ (Ca2+c) levels in cells dissociated from the embryonic (E) rat cortex during neurogenesis. Dualrecordings by flow cytometry using calcium and voltage-sensitive dyes revealed that, at the beginning of cortical development (E11–12), precursor cells exhibited either low (<100 nM), moderate (~250 nM) or high (>1 μM) resting Ca2+c levels and well-polarized (–70 mV) or less-polarized (–40 mV) resting membrane potentials which reflected postmitotic or proliferative stages of the cell cycle. Ca2+c levels of all cells included a Ca2+o entry component, which was also Mn2+-permeant in actively proliferating precursors. Postmitotic, but not premitotic, precursors exhibited thapsigargin-sensitive intracellular Ca2+ (Ca2+i) stores, which had similar capacities throughout neuronal lineage development. Differentiating neurons, but not precursors expressed Ca2+i stores with ryanodine and caffeine sensitivity and baseline Ca2+c levels that depended on Na+–Ca2+ exchange activity. Voltage-dependent Ca2+o entry was not detected in precursors, but emerged during neuronal differentiation, with most of the neurons expressing functional L-type Ca2+ channels. Ca2+ imaging of individually immunoidentified cells acutely recovered in culture confirmed that precursors differentiate into neurons which stereotypically exhibit Ca2+o entry at the level of the membrane with increased Ca2+i release mechanisms on Ca2+i stores, Na+–Ca2+ exchange activity and expression of voltagedependent Ca2+ channels.
Fluctuations in cytosolic Ca2+ (Ca2+c) levels are critical to the formation of multicellular organisms beginning with fertilization of the oocyte by sperm (Steinhardt et al., 1977). Ca2+c transients occurring during the mitotic cell cycles following fertilization (Wilding, 1996; Santella, 1998) involve cyclical generation of Ca2+ release from intracellular Ca2+ (Ca2+i) stores via autonomous oscillations in inositol trisphosphate at Ca2+ release channels on Ca2+i stores (Ciapa et al., 1994). Extracellular Ca2+ (Ca2+o) entry via different types of voltage-dependent Ca2+ channels in the plasma membrane coincides with different phases of the cell cycle (Kuga et al., 1996). Mitogens elevate Ca2+c levels by activating plasma membrane channels with varying degrees of Ca2+ selectivity (Munaron et al., 1997). In the developing central nervous (CNS), Ca2+i store-derived fluctuations in Ca2+c occur spontaneously in actively dividing, electrically coupled and uncoupled precursor cells (Owens and Kriegstein, 1998). Ca2+c fluctuations underlie migration of postnatal cerebellar neuroblasts (Komuro and Rakic, 1996) and mediate postmitotic embryonic cortical neuroblast migration to GABA and brain-derived neurotrophic factor (BDNF) (Behar et al., 1996, 1998). Ca2+-induced Ca2+i release is critical to the differentiation of cultured embryonic spinal cord neurons (Holliday and Spitzer, 1990; Holliday et al., 1991; Spitzer, 1995), while the capacitative Ca2+o influx and Ca2+i mobilization mechanisms in neuro-epithelial cells have been shown to accompany neurogenesis of the embryonic vertebrate retina (Sakaki et al., 1997). Most recently, voltage-dependent T-type Ca2+ channels have been recorded in the neuroepithelial cells composing the pre-and postnatal floor plate (Frischknecht and Randall, 1998). Thus, the development of homeostatic mechanisms to regulate Ca2+o entry, Ca2+i stores and Ca2+c levels is undoubtedly critical to the complex process of CNS morphogenesis.
In this study, we investigated the cellular distribution of mechanisms involved in Ca2+o entry and Ca2+i homeostasis during neurogenesis in the embryonic (E) rat cortex. At the begining of cortical neurogenesis (E11–22), the tissue progressively transforms from an intensely proliferative neuroepithelium into a complex and laminated cortical tissue, largely dominated by differentiating neurons during last several days before birth (Maric et al., 1997). In order to obtain a complete account of the cellular distribution of physiological properties relevant to Ca2+i homeostasis and Ca2+o entry, we optimized the cell preparation protocol to dissociate the cortical tissue completely into single-cell suspensions devoid of dead cells and cell clusters, then utilized flow cytometry in conjunction with Ca2+-indicator dye to record Ca2+c levels of individual cells. This random and rapid recording strategy allowed us to compile statistically complete data in thousands of cells in seconds. The results complement the recently published study of resting potential and development of membrane excitability in the cortex using potentiometry and flow cytometry (Maric et al., 1998a).
Materials and Methods
Experiments were carried out on embryos recovered from timed-pregnant Sprague–Dawley rats (Taconic Farms, Germantown, NY) during the last half of gestation. The embryonic (E) age was determined by comparing the crown–rump lengths of embryos with previously published values (Hebel and Stromberg, 1986). The day of conception was taken as E1. All of the research was performed in compliance with the Animal Welfare Act and the Public Health Service policy on Humane Care and Use of Laboratory Animals and was approved by the National Institute of Neurological Disorders and Stroke Animal Care and Use Committee.
Single-cell suspensions were isolated from rat telencephalic (E11–13) and cortical tissues (E14–22), as detailed previously (Maric et al., 1997). Highly reproducible optimal yields of cells were obtained using papain digestion and gentle trituration. Other enzymatic and mechanical methods consistently led to variable yields of significantly fewer vital cells (Maric et al., 1997). Tissue dissociates were finally resuspended at a density of 2 × 106 cells/ml in a normal physiological medium (NPM: 145 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 10 mM HEPES and 10 mM glucose, pH 7.3 with osmolarity adjusted to 290 mOsm) supplemented with 1 mg/ml of fatty acid-free bovine serum albumin (Sigma, St Louis, MO) before being subjected to quantitative flow cytometry (see below).
In a parallel series of experiments, aliquots of E12 and E18 cells were acutely plated at a density of 5 × 104 cells/cm2 on poly-d-lysine-coated coverslips, which were photo-etched with an alpha-numeric grid (Bellco Glass Inc., Vineland, NJ) and pre-glued to 35 mm tissue culture dishes (MatTek Corp., Ashland, MA). The cells were incubated in NPM for 2 h at 37°C before Ca2+ imaging by videomicroscopy (see below) or were allowed to recover their morphology by a short-term (24 h) culture in Neurobasal Medium supplemented with B27 and G5 additives (Life Technologies Inc., Frederick, MD) before immunoidentification.
Immunocytochemical Identification of Developmentally Relevant Epitopes
We quantified the numbers of proliferating cells using the thymidine analogue 5-bromo-2´-deoxyuridine (BrdU; Sigma), which identifies cells in the S-phase of the cell cycle (Gratzner, 1982). S-phase cells in vivo were labeled using a single i.p. injection of BrdU (50 μg/g body wt) into timed-pregnant dams, followed by killing the animals 2 h later. S-phase cells in vitro were labeled using 10 μM BrdU added to the culture medium 2 h before Ca2+ imaging or termination of culture. BrdU-labeled cells were processed for immunocytochemistry as previously described (Maric et al., 1997) and visualized using fluorescein isothiocyanate (FITC)-conjugated monoclonal class IgG1 anti-BrdU antibody (Becton Dickinson, Mountain View, CA). The percentage of BrdU+ cells in suspensions was quantified with flow cytometry using 488 nm excitation, 530 ± 30 nm emission and Cell Quest analysis software (Becton Dickinson). The BrdU+ cells in culture were detected using standard fluorescence microscopy (see below).
Other relevant markers used to identify cell phenotypes in acutely plated and short-term cultured cells included surface labeling with tetanus toxin fragment C (TnTx) and a mouse monoclonal class IgG1 anti-TnTx antibody (Boehringer Mannheim Biochemicals, Indianapolis, IN), which labels postmitotic neurons (Koulakoff et al., 1983), or cytoplasmic labeling of tubulin β III (TuJ1) with mouse monoclonal class IgG2a anti-TuJ1 antibody (Berkeley Antibody Company, Richmond, CA), which labels both neuronal precursors and postmitotic neurons (Huber and Matus, 1984; Tucker et al., 1988; Lee et al., 1990; Menezes and Luskin, 1994). In addition, rabbit polyclonal anti-nestin (Rat 401) antibody (a gift of Dr Ron McKay, NIH, Bethesda, MD) was used to identify neuroepithelial and immature cells (Hockfield and McKay, 1985). The primary immunoreactions were visualized with appropriate secondary antibodies conjugated with tetramethyl rhodamine isothiocyanate (TRITC) (Southern Biotechnology Associates Inc., Birmingham, AL) or amino-methylcoumarin (AMCA) (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). The cells were quantified for BrdU, nestin, TuJ1 and/or TnTx expression using the Axiovert 135 fluorescence microscope (Carl Zeiss Inc., Thornwood, NY) equipped with standard FITC/ AMCA/RHOD filter sets (Omega Optical, Brattleboro, VT).
Fluorescent Physiological Indicator Dye Staining
To record cytoplasmic Ca2+ (Ca2+c) levels of cells in suspension, the dissociates were loaded with a mixture of 1 μM fluo-3/AM and 25% (w/v) Pluronic F-127, a nonionic solubilizing detergent (Molecular Probes, Eugene, OR) for 30–45 min at 37°C. The cells were then washed to remove the unincorporated dye and resuspended in NPM at a final density of 2 × 106 cells/ml. In experiments designed to record Ca2+c and membrane potential of the same cells simultaneously, fluo-3-loaded cells were stained with 200 nM red oxonol (DiBAC4(5); Molecular Probes), a fluorescent potentiometric dye that is negatively charged at physiological pH, for 2–10 min before the beginning of the recordings. To record Ca2+c levels in acutely plated cells, the cells were loaded with a mixture of 2 μM fura-2/AM and 25% (w/v) Pluronic F-127 for 60 min at 37°C.
Flow Cytometric Recordings of Physiological Properties
Fluo-3 and oxonol fluorescence signals of single cells in suspension were analyzed using a FACSTAR+ dual-laser flow cytometer (Becton Dickinson), as previously described (Maric et al., 1998a,b, 1999). Briefly, fluo-3 dye was excited by an argon ion laser (Model 2016, Spectra Physics, Mountain View, CA) tuned to obtain 500 mW power at 488 nm, and the resulting fluorescence emissions detected with a bandpass filter set at 525 ± 15 nm. In experiments where cells double-labeled with fluo-3 and red oxonol were recorded, the oxonol dye was excited by the second, dye laser (Model 375B, Spectra Physics) tuned to obtain 200 mW power at 568 nm, and the resulting fluorescence emissions detected with a bandpass filter set at 625 ± 35 nm.
Fluo-3 (FL) and oxonol (OX) distributions of fluorescence intensities were quantified and illustrated as single-parameter frequency histograms using the Cell Quest data acquisition and analysis software (Becton Dickinson), with which modes, coefficients of variation and peak amplitudes could be calculated. Each histogram consisted of 10 000 individual cell fluorescence emissions, which were randomly sampled at the rate of ~2000 cells/s. Overlays of respective control and experimental FL or OX histograms permitted quantitative calibrated analysis of the Ca2+c and membrane potential changes to a given test condition (Maric et al., 1998a,b, 1999). Unless otherwise stated, all experiments were performed at 24°C.
In a separate series of experiments, fluo-3-loaded cells were stained with propidium iodide (PI) to discriminate between vital (PI–) cells and dead or dying (PI+) cells that have lost membrane integrity. The PI+ cells, which accounted for <10% of E12 dissociates and <5% of E18 dissociates, also exibited low forward-angle light scatter (a property related to cell size) of <300 arbitrary light scatter channels (out of 1024 channels maximally resolved on a FACStar+ flow cytometer). This property was then routinely used to electronically exclude compromized cells from the rest of the population. Therefore, all potentiometric and Ca2+c measurements were obtained from vital PI– cells.
Digital Videomicroscopic Imaging of Ca2+c
In a separate series of experiments, developing E12 and E18 cortical cells were allowed to adhere onto poly-d-lysine-coated coverslips for 2 h in NPM at 37°C. The cells were pulse-labeled with BrdU at the time of plating and fura-2-loaded for 1 h before Ca2+c imaging. Fields of 30–50 individual fura-2-loaded cells were then recorded using the Attofluor RatioVision workstation (Atto Instruments, Rockville, MD) equipped with an Axiovert 135 inverted microscope (Carl Zeiss) and an ICCD camera (Atto Instruments). Fura-2 was sequentially excited at 1 s intervals with a 100 W mercury arc lamp filtered at 334 ± 5 and 380 ± 5 nm. Fluorescence emissions were acquired through a 510-nm dichroic mirror and 520-nm long-pass filter set (Chroma Technology). Regions of interest (ROIs) were drawn electronically around individual cell bodies and indicator dye fluorescence signals of each ROI were digitized with a Matrox image processing board, then plotted as line graphs using Attograph for Windows analysis software (Atto Instruments). Throughout the course of the experiment, the cells were supefused with NPM or altered salines, which were delivered to the 150 μl recording chamber using gravity-driven perfusion at ~2 ml/min.
Progressive Changes in Ca2+c Levels Parallel Neurogenesis
Cells were profiled daily during the last half of gestation (E11– 22), when neurogenesis occurs. Simultaneous dual-recordings of Ca2+c and membrane potential in highly proliferative dissociates revealed a complex relationship between cellular Ca2+ concentration and resting membrane potential (Fig. 1A). At the onset of neurogenesis (E11–12), the fluo-3 fluorescence distribution of proliferating and newly committed precursors was always tri-modal (Fig. 1A), depicting cell populations with low (<100 nM), moderate (~250 nM) and high (>1 μM) average resting Ca2+c levels. Cells with moderate Ca2+c levels (FLDIM) were well-polarized at ~–70 mV (OXDIM), while cells with low (FLVERY DIM) and high (FLBRIGHT) Ca2+c levels were depolarized ~30 mV relative to FLDIM cells, each resting at near –40 mV (OXBRIGHT). Sorting of OXDIM and OXBRIGHT cells by flow cytometry at the onset of neurogenesis has previously revealed that the former population was predominantly composed of premitotic/proliferating (BrdU+) cells, while the latter population consisted mainly of postmitotic/differentiating (BrdU–) cells (Maric et al., 1998a), implying a close relationship between membrane potential and Ca2+c and the stage of the cell cycle during neuronal lineage progression.
Correspondingly, the complexity of conjoint FL and OX fluorescence signal distributions progressively simplified (Fig. 1B) as proliferation decreased and neurons started to differentiate (Fig. 2C1,2). During this period, the percentage of cells in the FLBRIGHT and OXBRIGHT population rapidly decreased, highly correlating with the disappearance of BrdU+ cells (r = 0.99, P < 0.001; Fig. 2C1), with the great majority of cells becoming FLDIM and OXDIM by E15–16 (Fig. 2A1,B1) and exhibiting low to moderate (100–200 nM) Ca2+c levels (Fig. 2A2) together with progressively increasing well-polarized (ranging from –75 to –90 mV) membrane potentials (Fig. 2B2). Interestingly, while the absolute Ca2+c levels of the remaining FLBRIGHT cells decreased even more sharply with neurogenesis (Fig. 2A2) compared to FLDIM cells, their resting membrane potential remained relatively constant basing at ~–40 mV (Fig. 2B2). Previous studies (Maric et al., 1998a) have demonstrated that the dominant well-polarized populations at early and late embryonic periods exhibit membrane potentials that are dependent on K+o in a Nernstian manner, and that the more negative potential in late embryonic OXDIM cells reflects a developmental increase in K+i from ~100 to ~150 mM, while OXBRIGHT cells remain polarized near –40 mV throughout embryonic development in a Cl–-dependent manner.
Immunocharacterization of Short-term Cultured Cortical Cells
To further immunophenotype the cells isolated at the begining and the end of cortical neurogenesis, acutely plated E12 and E18 cortical cells were pulse-labeled in vitro for 2 h before the end of a 24 h culture, then fixed in 70% ethanol and triple-immunostained for BrdU incorporation and nestin expression to identify proliferating precursors, and TuJ1 expression to identify differentiating neurons (Fig. 3). The data revealed that ~75% of E12 cells were BrdU+, comparable to percentage of BrdU+ cells labeled in vivo (Fig. 2C1), and most of these were also nestin+, comparable to those observed in acutely isolated cells (Fig. 2C2). By contrast, in the E18 preparation, >75% of cells were postmitotic (BrdU–) and TuJ1+ after 24 h culture, comparable to the percentage of postmitotic neurons observed in acutely prepared dissociates (Fig. 2C1,2). Hence, the primarily proliferating E12 telencephalon composed of premitotic, putatively uncommitted stem/precursor cells becomes transformed into the differentiating cortex by E18 populated largely by postmitotic neuronal cells. We have therefore chosen to compare the various mechanisms that regulate Ca2+o, Ca2+c and Ca2+i exhibited by cells at E12 and E18 in the experiments that follow, using them as a model to characterize the developmental changes in calcium homeostasis as putative stem/precursor cells commit to the neuronal lineage.
Ca2+c Dependency on Ca2+o Decreases as Precursors Differentiate into Neurons
We studied the sources of Ca2+ contributing to steady-state Ca2+c levels in early and late embryonic cells by resuspending them in altered salines. Participation of Na+–Ca2+ exchange activity in Ca2+ homeostasis was examined by resuspending the cells in salines with altered [Na+]o, using N-methylglucamine to replace Na+ in an isosmolar manner. These experiments were conducted in the presence or absence of extracellular Ca2+, with results being recorded several times over a 10 min period. Resuspension of proliferating precursor cells in Na+o-free saline did not affect the trimodal distribution of their Ca2+c signals (Fig. 4A1). However, resuspension in Ca2+o-free media lowered Ca2+c significantly in all precursors (Fig. 4B1,3), indicating that Ca2+c levels in all subpopulations were immediately and directly dependent on Ca2+o but insensitive to Na+o. In contrast, resuspension of differentiating neurons in Na+o-free saline, which by itself did not affect resting membrane potential (Maric et al., 1998a), rapidly led to a several-fold increase in Ca2+c in the majority of cells that was dependent on Ca2+o entry and remained sustained for ~10 min (Fig. 4A2,3). There was typically an inverse relationship between modal Ca2+c values of E18 cells and [Na+]o, with steeply increasing Ca2+c elevation occurring below ~25 mM Na+o (Fig. 4A3). The increase in Ca2+c recorded upon lowering Na+o was eliminated in Ca2+o-free saline, identifying the extracellular source of Ca2+ in the phenomenology (Fig. 4A3). In fact, the average Ca2+c level decreased modestly but significantly in virtually all late embryonic cells in Ca2+o-free saline (Fig. 4B2,3). These results indicate that all cells exhibit steady-state Ca2+c levels whose Ca2+o entry component is most evident in precursors, but still present in differentiating neurons.
Steady-state Ca2+ entry via putative receptor-operated or Ca2+-release channels on the plasma membrane was tested by profiling cells under control conditions, then exposing them to 2 mM MnCl2 for 5–10 min and recording their fluo-3 fluorescence intensity distribution. Mn2+ ions readily pass through activated types of these Ca2+ channels and effectively displace Ca2+ ions from fluo-3, thereby quenching Ca2+-dependent fluorescence signals (Omann and Harter, 1991). Inclusion of Mn2+ rapidly and selectively reduced the fluo-3 fluorescence of all FLBRIGHT cells in dissociates of proliferating precursors, which redistributed to the lowest fluorescence levels and superimposed with the FLVERY DIM population (Fig. 4C1). The fluorescence levels of FLBRIGHT cells after the addition of Mn2+ were lower than those recorded after their resuspension in Ca2+o-free saline (Fig. 4B1), suggesting that Mn2+ was not simply blocking Ca2+o entry, otherwise similar responses would have been obtained in both experiments. Rather, the results indicate that Mn2+ permeated Ca2+o entry pathway(s) to displace Ca2+ from, and bind to fluo-3, thereby quenching its emission and resulting in levels of fluorescence intensity identical to those recorded after adding Mn2+ to ionomycin-treated cells (data not shown). These selective effects of Mn2+ on FLBRIGHT cells also revealed the upper limb of the approximately normal distribution composed by the FLDIM cells, which were unaffected by Mn2+ (Fig. 4C1,3). The fluo-3 fluorescence of FLBRIGHT cells present in dissociates of late embryonic cortex, which accounted for <5% of the total cells, was also quenched by Mn2+, again redistributing to the lowest fluorescence levels, while the remaining ~95% of the cells were unaffected (Fig. 4C2,3). These results demonstrate a Ca2+o entry pathway with significant Mn2+ permeability expressed by precursors that is not expressed by differentiating neurons.
Voltage-dependent Ca2+ Channels Appear during Neurogenesis
Voltage-dependent Ca2+o entry was investigated by exposing the cells to altered media containing 1–150 mM [K+]o, which, over the 5–150 mM range, depolarized the great majority of all telencephalic and cortical cells according to a Nernstian relationship between K+o and modal membrane potential (Maric et al., 1998a). Resuspension of cells in 150 mM KCl saline, in which Na+o was replaced by K+o in an equimolar manner, or simply adding an extra 145 mM K+o to normal saline with 5 mM K+o, which controlled for possible contributions of Na+-Ca2+ exchange, did not alter the trimodal Ca2+c signal distribution of E12 precursors (Fig. 5A1), despite the fact that 150 mM K+o depolarized all cells, including the FLBRIGHT population, near 0 mV (Maric et al., 1998a). In contrast, addition of an extra 5–145 mM K+o to E18 differentiating neurons suspended in 5 mM K+o induced a K+o-dependent increase in their Ca2+c levels, with the great majority of cells responding at 150 mM K+o (Fig. 5A2). This increase in Ca2+c was dependent on Ca2+o which also contributed to steady-state Ca2+c levels in cells resuspended in 1–5 mM K+o (Fig. 5B1). Addition of an extra 5–145 mM Na+o to normal saline with 145 mM Na+o, to mimic the hyperosmolar changes induced by adding [K+]o to saline without isotonic substitution, had little or no effect on Ca2+c over the ~5–10 min recording period. These results demonstrate a widespread expression of voltage-dependent Ca2+o entry mechanisms expressed in many differentiating neurons but not in proliferating precursors.
Contributions of voltage-dependent Ca2+-channels to K+odepolarized cells at E18 were further evaluated by exposure to 50 mM [K+]o, which depolarized virtually all cells to ~–35 mV and increased their Ca2+c levels ~4-fold (Fig. 5B1,2). Cells were recorded under control conditions and in the presence of either 2 mM CoCl2, CdCl2, ZnCl2 or MnCl2, which block most of the differentiated types of low-and high-voltage-activated Ca2+ channels, or in the presence of 100 μM NiCl2 (blocker of T-type Ca2+ channels), 10 μM nitrendipine (blocker of L-type Ca2+ channels), 100 nM ω-conotoxin GVIA (blocker of N-type Ca2+ channels), or 10 or 100 nM ω-agatoxin VIA (blocker of P- and Q-type Ca2+ channels).
Inclusion of 10 μM nitrendipine revealed that only a fraction (<10%) of cells still responded to 50 mM K+o, with those that responded exhibiting minimally elevated Ca2+c levels (Fig. 5B2). These results show that most of the differentiating neurons expressed functional L-type Ca2+ channels. K+o-elevated Ca2+c levels were practically unaffected by inclusion of 10 nM ω-agatoxin VIA, implying that few cells expressed functional P-type Ca2+ channels. Many cells (>60%) still responded to 50 mM K+o in either 100 nM ω-agatoxin VIA, 100 μM Ni+ or 100 nM ω-conotoxin GVIA, but the average Ca2+c increase in this population was significantly less than in control (Fig. 5B2). Therefore, differentiating neurons may also exhibit functional Q-, N- and T-type Ca2+ channels, although their contribution to the Ca2+c response appears to be considerably less than that of L-type Ca2+ channels. Inclusion of a cocktail of voltage- dependent Ca2+ channel antagonists (ZnCl2, NiCl2, nitrendipine, ω-conotoxin GVIA, ω-agatoxin VIA) led to results that were statistically similar to those obtained with nitrendipine alone. Taken together, these findings suggest that the great majority of differentiating neurons express functional L-type Ca2+ channels, which generate most of the Ca2+ entry.
The Emergence of Ca2+i Stores in Developing Cortical Cells
We studied the appearance of intracellular store components involved in Ca2+ homeostasis at early and late stages of embryonic cortical neurogenesis. We used thapsigargin, which blocks Ca2+-ATPase activity associated with endoplasmic reticular membranes, and ryanodine and caffeine, modulators of Ca2+ release channels, which are known to be expressed by organelles storing Ca2+ in CNS neurons (Berridge, 1998).
Thapsigargin transiently elevated Ca2+c levels in almost all of the E12 FLDIM cells without affecting steady-state Ca2+c levels in the other two subpopulations of proliferating precursors (Fig. 6A1). This shift in Ca2+c peaked at ~5 min, then completely relaxed by ~10 min (not shown). All of the thapsigargin-evoked effects were due to unloading of Ca2+i stores, since the same transient elevation in Ca2+c was recorded in Ca2+o-free saline (Fig. 6B1). Thapsigargin also triggered transient elevations in Ca2+c of all E18 differentiating neurons, the amplitudes of which were similar in the presence (Fig. 6A2) or absence of Ca2+o (Fig. 6B2), indicating that the mechanism involved a Ca2+i store depletion. Thapsigargin-depleted Ca2+i store capacities of E18 cells were comparable to those expressed by E12 FLDIM precursors (Fig. 6A3,B3).
The effects of saturating concentrations of ryanodine (1 μM) and caffeine (10 mM) on E12 FLDIM cells were marginal (Fig. 6B1,3). By contrast, many E18 cells transiently released Ca2+i in response to each agonist (Fig. 6B2). However, the peak rise in Ca2+c to caffeine was several-fold greater compared to ryanodine (Fig. 6B2,3). The data suggest that, even though both of these modulators may tap the same Ca2+i store compartments (Berridge, 1998), their potency in activating Ca2+ release channels in differentiating E18 neurons is quite different.
Exposure to a saturating concentration of ionomycin (10 μM) in Ca2+o-free saline unloaded all available Ca2+i stores in all E18 cells, generating a transient elevation in Ca2+c that was greater than either of those induced by other agents (Fig. 6B2,3). The ionomycin effect was similar in FLDIM cells at E12 (Fig. 6B1,3). The Ca2+c responses induced by each of the above-mentioned agonists did not relax in Na+o-free saline, indicating that Na+–Ca2+ exchange mechanisms were activated by Ca2+c elevations resulting from Ca2+i-store depletion (not shown).
Interestingly, resuspension of E12 FLBRIGHT cells in Ca2+o-free saline, which lowered their micromolar Ca2+c levels so that they superimposed with those of FLDIM cells (Fig. 6B1), revealed no thapsigargin-, ryanodine- or caffeine-releasable Ca2+i stores. Stimulation with ionomycin did produce a just-detectable modal increase in Ca2+c in these cells (Fig. 6B1), implying almost nonexistent capacity of Ca2+i stores in the FLBRIGHT population.
Thus, during neurogenesis the majority of neuronal precursors differentiate into neurons that express Ca2+i homeostatic mechanisms to maintain low Ca2+c levels and filled Ca2+i stores, which are regulated via both enzymatic Ca2+ pumps and Ca2+- release channel mechanisms. In addition, the differentiating neurons exhibit Na+–Ca2+ exchange activity as well as voltage- independent and voltage-dependent Ca2+o entry mechanisms.
Ca2+ Homeostatic Mechanisms in Single Cell Recordings
We have also carried out Ca2+ imaging on individual cells acutely plated from E12 and E18 cortical dissociates. The same cells were then processed for triple-staining immunoreactions and relocated in the recording field to identify cells at proliferative (BrdU+), immature (nestin+) or committed stages (TuJ1+) of neuronal lineage development. The results revealed that >75% of actively proliferating precursor cells (nestin+BrdU+TuJ1–) at E12 and E18 stereotypically exhibited relatively high (>250 nM) and Ca2+o-dependent baseline Ca2+c levels, just-detectable Na+o–Ca2+c exchange activity and caffeine-sensitive Ca2+i stores, and thapsigargin-sensitive Ca2+ ATPase activity, but neither voltage-dependent Ca2+ channels nor ryanodine-sensitive Ca2+i stores (Fig. 7A). Baseline Ca2+c levels in all of the recorded dividing neuronal progenitors (nestin+BrdU+TuJ1+; n = 4) were 50–100 nM lower relative to BrdU+ precursors, but still showed a sustained Ca2+o entry component (Fig. 7B). These cells also stereotypically exhibited increased Na+o–Ca2+c exchange and thapsigargin-sensitive Ca2+ ATPase activity, greater capacity of caffeine- and ryanodine-sensitive Ca2+i stores, and the appearance of voltage-dependent L-type Ca2+ channels (Fig. 7B). In contrast, the differentiating neurons (nestin–BrdU–TUJ1+) averaged baseline Ca2+c levels of 85 ± 25 nM (mean ± SEM), some of which depended on Ca2+o entry (Fig. 7C), and >70% of them showed an even more enhanced Na+o–Ca2+c exchange, voltage- dependent L-type Ca2+ channel and thapsigargin-sensitive Ca2+ ATPase activity, compared to nestin+BrdU+TuJ1+ neuronal progenitors. Caffeine-sensitive Ca2+i stores typically remained quite similar between these two populations of cells, while the capacity at ryanodine-sensitive Ca2+i stores were increased in differentiating neurons (Fig. 7C).
We used flow cytometry in conjunction with Ca2+-sensitive fluorescent indicator dye to explore the development of Ca2+ homeostasis mechanisms in embryonic cortical cells during neurogenesis. Precursors at early stages of cortical development were composed of three subpopulations of cells with low (<100 nM), moderate (~250 nM) and high (>1 μM) baseline Ca2+c levels, while one Ca2+c level (100–200 nM) predominated as these precursors differentiated into neurons. Baseline Ca2+c levels of all cells included a Ca2+ entry component whose contribution decreased during differentiation. Actively proliferating (BrdU+) E12 precursors, which were polarized near –40 mV in a Cl–-dependent manner (Maric et al., 1998a), expressed micromolar levels of Ca2+c, which depended on Ca2+o entry via a Mn2+-permeable pathway. These cells did not express significant Na+–Ca2+ exchange activity, voltage-dependent Ca2+o entry mechanisms or effective Ca2+i stores. By contrast, postmitotic (BrdU–) E12 precursors, which were polarized at near –70 mV in a K+-dependent manner (Maric et al., 1998a), exhibited submicromolar levels of Ca2+c, which were dependent on Ca2+o entry via Mn2+-insensitive pathway, along with Ca2+i stores, which were sensitive to thapsigargin and ionomycin but not to ryanodine or caffeine. Like their premitotic siblings, the postmitotic E12 precursors lacked significant Na+–Ca2+ exchange activity and voltage-dependent Ca2+o entry.
With differentiation, E18 cortical neurons exhibited lower baseline Ca2+c levels than their E12 precursors, with a reduced voltage-independent Ca2+o entry component, which was insensitive to Mn2+. Their baseline Ca2+c levels were sensitive to Na+o, reflecting dynamic Na+–Ca2+ exchange mechanisms, and exhibited voltage-dependent Ca2+ entry, predominantly through L-type Ca2+ channels. The Ca2+i stores in these cells exhibited several compartments which were regulated via both enzymatic Ca2+ pumps and Ca2+-elease channel mechanisms. Their steady-state membrane potentials were determined by K+ ions according to a Nernstian relationship (Maric et al., 1998a). Ca2+ imaging of individual immunoidentified cells retreived near the beginning and the end of cortical neurogenesis confirmed the developmental pattern of emergent Ca2+ homeostatic mechanisms observed in populations of cells at different stages of neuronal lineage progression obtained by flow cytometry.
Ca2+o Entry Determines Baseline Ca2+ Levels and Membrane Potentials of Embryonic Cortical Cells
In our experiments, baseline Ca2+c levels in immature cortical cells included a significant Ca2+o entry component. Contribution of Ca2+o entry to baseline Ca2+c levels has been recently reported in acutely plated embryonic spinal cord cells (Liu et al., 1998) and has been confirmed in preliminary experiments on cultured embryonic cortical cells (Xian et al., 1996). These results indicate that Ca2+o entry also occurs in embryonic cells that have recovered from trauma of cell dissociation, and is not an artifact of our cell preparation. Ca2+o entry has also been detected at the level of the resting potential in CA1 pyramidal neurons recorded in acutely sliced adult hippocampal tissue, but this has been attributed to the activity of voltage-gated Ca2+ channels (Magee et al., 1996). In our previous study of baseline membrane potential mechanisms (Maric et al., 1998a), we found that resuspension of precursors in Ca2+o-free saline immediately hyperpolarized the majority of BrdU+ E12 cells and simultaneously depolarized BrdU– E12 cells from more negative potentials. Thus, baseline Ca2+c levels in pre- and postmitotic precursors include a significant Ca2+o entry component, which activates different ionic mechanisms to polarize cells at different membrane potentials. Ca2+o-dependent baseline potential of early embryonic postmitotic precursors may involve activation of transitional forms of Maxi-K+ channels, since patch-clamp recordings of embryonic cortical cells recorded in slice preparations revealed a widespread distribution of large conductance Ca2+-activated Maxi-K+ channels (Bulan et al., 1994). Ca2+o entry and activation of Maxi-K+ channels would be governed by the complex relationship between Ca2+o entry sites and K+ channels, the rates of Ca2+o entry and cytoplasmic buffering via Ca2+ ATPase- mediated sequestration into stores or export from the cell, by Na+o–Ca2+c exchange, and equilibria with Ca2+-binding proteins and other cellular components. A lower contribution to baseline membrane potential might be expected in physically coupled cortical cell clusters, which exhibit low input resistance, compromising the contribution of Maxi-K+ channel activity to the membrane potential of individual cells (LoTurco and Kriegstein, 1991; Owens and Kriegstein, 1998). However, coupled cells progressively decrease in cluster size as precursors differentiate into neurons (LoTurco and Kriegstein, 1991; Mienville et al., 1994). These developmental changes increase the possibility that tonic or transient Ca2+o entry could activate Maxi-K+ channels and contribute to membrane potential of differentiating cells in vivo.
Ca2+o Entry Occurs via Mn2+-permeant and Mn2+-impermeant Pathways
When Ca2+o entry was compared between proliferating, BrdU+ cells and postmitotic, BrdU– cells, the results revealed that Ca2+o entry in BrdU+ cells occurred (i) via a Mn2+-permeant pathway, (ii) generated several-fold greater Ca2+c levels and (iii) activated Cl–- rather than K+-dependent contributions to membrane potential (Maric et al., 1998a). Mn2+ permeation implicates Ca2+o entry pathways previously characterized as either receptor- or Ca2+i store-operated (Fasolato et al., 1994). A family of mammalian genes encoding Ca2+o entry channels has recently been described (Zhu et al., 1996). These genes are mammalian homologs of those encoding cation channel-forming proteins initially characterized in Drosophila mutants, which express transient photoreceptor potentials (trp) (Montell and Rubin, 1989). Structurally, trp and trp-like (trpl) (Phillips et al., 1992) proteins resemble voltage-gated Ca2+ channels without voltage-sensitive regions. Recombinant expression of trp-related proteins demonstrates variable degrees of Ca2+ selectivity, Mn2+ permeation, and stimulated or constitutive activity (Philipp et al., 1998). One subfamily, which is involved in store-depletion- activated Ca2+o entry, has been implicated in the movement of growth cones of embryonic neurons (Gomez et al., 1995). Ca2+o entry following ryanodine-triggered Ca2+i store depletion occurs at the level of the resting potential (Garaschuk et al., 1997) and may involve a trp/trpl-related protein. Thus, trp/trpl cation entry channels could provide a pathway for Ca2+o influx, which could activate K+ and Cl– ion conductances and thereby modulate cellular potential. Ca2+ activation of Cl– channels has already been described in cultured embryonic spinal and dorsal root ganglion neurons (Owen et al., 1986), as well as in secretory epithelia (Begenisich and Melvin, 1998).
Ca2+i Homeostatic Mechanisms Emerging during Precursor Differentiation into Neurons
Ca2+o entry not only modulates cellular potential indirectly via K+- and Cl–-dependent mechanisms, it also supplies Ca2+ for intracellular storage and regulated release. E12 BrdU– cells and E18 neurons, but not E12 BrdU+ cells, exhibited similar store capacities, which could be depleted by thapsigargin, indicating Ca2+ ATPase activity in the endoplasmic reticulum of these cells. However, E12 cells were not sensitive to caffeine and ryanodine, while E18 cells were, suggesting a differentiation of Ca2+ release channels, which regulate the Ca2+i stores, during embryonic cortical neurogenesis. Preliminary experiments have also revealed the expression of putative IP3 receptor-mediated Ca2+i release in neurons, but not precursors triggered by activation of muscarinic receptors (D. Maric, unpublished observations). Thus, differentiation of precursors into neurons includes expression of both types of Ca2+ release channels to generate store-derived Ca2+c signals. Similar findings were reported by Holliday et al. (Holliday et al., 1991), who demonstrated the presence of Ca2+ ATPase associated with the endoplasmic reticulum in both young and mature cultured spinal cord neurons, while observing the expression of caffeine-sensitive stores only in the more mature neurons.
The ~50% reduction in baseline Ca2+c levels observed in differentiating neurons was accompanied by a decrease in the absolute level of Ca2+o entry and the emergence of Na+o–Ca2+c exchange activity. Both of these changes could lower Ca2+c levels. In addition, voltage-dependent Ca2+o entry mechanisms, which were noticeably absent in precursors, emerged in differentiating neurons. Practically all of the Ca2+o entry evoked in E18 neurons by 50 mM K+o was blocked by nitrendipine, which blocks Ca2+ channels with an L-type structure. The expression of these Ca2+ channels, which provide an important pathway for Ca2+o entry regulated by voltage and/or K+, has also been shown to increase with neuronal maturation (Kubo, 1989). Finally, there was no evidence for voltage-dependent Ca2+i release in either the precursors or differentiating cortical neurons during embryonic development.
Address correspondence to Dragan Maric, Laboratory of Neuro- physiology, NINDS, NIH, Bldg 36, Room 2C02, Bethesda, MD 20892, USA. Email: email@example.com.