Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (E1122), 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 SpragueDawley rats (Taconic Farms, Germantown, NY) during the last half of gestation. The embryonic (E) age was determined by comparing the crownrump 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 (E1113) and cortical tissues (E1422), 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 x 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 x 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 ß 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 3045 min at 37°C. The cells were then washed to remove the unincorporated dye and resuspended in NPM at a final density of 2 x 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 210 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 3050 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.
Digitized fura-2 fluorescence emissions were converted into estimated [Ca2+]c using Fura-2 Penta K+ salt as a Ca2+ indicator (Molecular Probes) and the following equation:
![]() |
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (E1112), 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.
|
|
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.
|
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.
|
Voltage-dependent Ca2+ Channels Appear during Neurogenesis
Voltage-dependent Ca2+o entry was investigated by exposing the cells to altered media containing 1150 mM [K+]o, which, over the 5150 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 5145 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 15 mM K+o (Fig. 5B1
). Addition of an extra 5145 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 ~510 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.
|
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
-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
-agatoxin VIA, 100 µM Ni+ or 100 nM
-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,
-conotoxin GVIA,
-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
).
|
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+oCa2+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 50100 nM lower relative to BrdU+ precursors, but still showed a sustained Ca2+o entry component (Fig. 7B
). These cells also stereotypically exhibited increased Na+oCa2+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 (nestinBrdUTUJ1+) 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+oCa2+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
).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (100200 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+oCa2+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+oCa2+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.
![]() |
Notes |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Behar TN, Li YX, Tran HT, Ma W, Dunlap V, Scott C, Barker JL (1996) GABA stimulates chemotaxis and chemokinesis of embryonic cortical neurons via calcium-dependent mechanisms. J Neurosci 16:18081818.[Abstract]
Behar TN, Schaffner AE, Scott CA, O'Connell C, Barker JL (1998) Differential response of cortical plate and ventricular zone cells to GABA as a migration stimulus. J Neurosci 18:63786387.
Berridge MJ (1998) Neuronal calcium signaling. Neuron 21:1326 [review].[ISI][Medline]
Brent LH, Gong Q, Ross JM, Wieland SJ (1993) Mitogen-activated Ca++ channels in human B lymphocytes. J Cell Physiol 155:520529.[ISI][Medline]
Bulan EJ, Barker JL, Mienville JM (1994) Immature maxi-K channels exhibit heterogeneous properties in the embryonic rat telencephalon. Dev Neurosci 16:2533.[ISI][Medline]
Ciapa B, Pesando D, Wilding M, Whitaker M (1994) Cell-cycle calcium transients driven by cyclic changes in inositol trisphosphate levels. Nature 368:875878.[ISI][Medline]
Fasolato C, Innocenti B, Pozzan T (1994) Receptor-activated Ca2+ influx: how many mechanisms for how many channels? Trends Pharmacol Sci 15:7783 [review].[ISI][Medline]
Frischknecht F, Randall AD (1998) Voltage- and ligand-gated ion channels in floor plate neuroepithelia of the rat. Neuroscience 85:11351149.[ISI][Medline]
Garaschuk O, Yaari Y, Konnerth A (1997) Release and sequestration of calcium by ryanodine-sensitive stores in rat hippocampal neurones. J Physiol 502:1330.[Abstract]
Gomez TM, Snow DM, Letourneau PC (1995) Characterization of spontaneous calcium transients in nerve growth cones and their effect on growth cone migration. Neuron 14:12331246.[ISI][Medline]
Gratzner HG (1982) Monoclonal antibody to 5-bromo- and 5-iodo- deoxyuridine: a new reagent for detection of DNA replication. Science 218:474475.[ISI][Medline]
Hebel R, Stromberg MW (1986) Anatomy and embryology of the laboratory rat. Worthsee: BioMed Verlag.
Hockfield S, McKay RD (1985) Identification of major cell classes in the developing mammalian nervous system. J Neurosci 5:33103328.[Abstract]
Holliday J, Spitzer NC (1990) Spontaneous calcium influx and its roles in differentiation of spinal neurons in culture. Dev Biol 141:1323.[ISI][Medline]
Holliday J, Adams RJ, Sejnowski TJ, Spitzer NC (1991) Calcium-induced release of calcium regulates differentiation of cultured spinal neurons. Neuron 7:787796.[ISI][Medline]
Komuro H, Rakic P (1996) Intracellular Ca2+ fluctuations modulate the rate of neuronal migration. Neuron 17:275285.[ISI][Medline]
Koulakoff A, Bizzini B, Berwald-Netter Y (1983) Neuronal acquisition of tetanus toxin binding sites: relationship with the last mitotic cycle. Dev Biol 100:350357.[ISI][Medline]
Kubo Y (1989) Development of ion channels and neurofilaments during neuronal differentiation of mouse embryonal carcinoma cell lines. J Physiol (Lond) 409:497523.[Abstract]
Kuga T, Kobayashi S, Hirakawa Y, Kanaide H, Takeshita A (1996) Cell cycle-dependent expression of L- and T-type Ca2+ currents in rat aortic smooth muscle cells in primary culture. Circulat Res 79:1419.
Liu QY, Schaffner AE, Barker JL (1998) Kainate induces an intracellular Na+-activated current in cultured embryonic rat hippocampal neurones. J Physiol 510:721734.
LoTurco JJ, Kriegstein AR (1991) Clusters of coupled neuroblasts in embryonic neocortex. Science 252:563566.[ISI][Medline]
Magee JC, Avery RB, Christie BR, Johnston D (1996) Dihydropyridine- sensitive, voltage-gated Ca2+ channels contribute to the resting intracellular Ca2+ concentration of hippocampal CA1 pyramidal neurons. J Neurophysiol 76:34603470.
Maric D, Maric I, Ma W, Lahjouji F, Somogyi R, Wen X, Sieghart W, Fritschy J-M, Barker JL (1997) Anatomical gradients in proliferation and differentiation of embryonic rat CNS accessed by buoyant density fractionation: 3, ß3 and
2 GABAA receptor subunit co-expression by post-mitotic neocortical neurons correlates directly with cell buoyancy. Eur J Neurosci 9:507522.[ISI][Medline]
Maric D, Maric I, Smith SV, Serafini R, Hu Q, Barker JL (1998a) Potentiometric study of resting potential, contributing K+ channels and the onset of Na+ channel excitability in embryonic rat cortical cells. Eur J Neurosci 10:25322546.[ISI][Medline]
Maric D, Maric I, Barker, JL (1998b) Buoyant density gradient fractionation and flow cytometric analysis of embryonic rat cortical neurons and progenitor cells. Neurosci Methods 16:247259.
Maric D, Maric I, Barker, JL (1999) Flow cytometric strategies to study CNS development. In: Neuromethods (Boulton AA and Baker GB, eds), pp. 287318. NJ: Humana Press Inc.
Memberg SP, Hall AK (1994) Dividing neuron precursors express neuron-specific tubulin. J Neurosci 27:2643.
Menezes JR, Luskin MB (1994) Expression of neuron-specific tubulin defines a novel population in the proliferative layers of the developing telencephalon. J Neurosci 14:53995416.[Abstract]
Mienville JM, Lange GD, Barker JL (1994) Reciprocal expression of cell-cell coupling and voltage-dependent Na current during embryogenesis of rat telencephalon. Brain Res Dev Brain Res 77:8995.[ISI][Medline]
Moody SA, Quigg, MS, Frankfurter A (1989) Development of the peripheral trigeminal system in the chick revealed by an isotope-specific anti-beta-tubulin monoclonal antibody. J Comp Neurol 279:567580.[ISI][Medline]
Montell C, Rubin GM (1989) Molecular characterization of the Drosophila trp locus: a putative integral membrane protein required for phototransduction. Neuron 2:13131323.[ISI][Medline]
Munaron L, Antoniotti S, Distasi C, Lovisolo D (1997) Arachidonic acid mediates calcium influx induced by basic fibroblast growth factor in Balb-c 3T3 fibroblasts. Cell Calcium 22:179188 [review].[ISI][Medline]
Omann GM, Harter JM (1991) Pertussis toxin effects on chemoattractant- induced response heterogeneity in human PMNs utilizing Fluo-3 and flow cytometry. Cytometry 12:252259.[ISI][Medline]
Owen DG, Segal M, Barker JL (1986) Voltage-clamp analysis of a Ca2+- and voltage-dependent chloride conductance in cultured mouse spinal neurons. J Neurophysiol 55:11151135.
Owens DF, Kriegstein AR (1998) Patterns of intracellular calcium fluctuation in precursor cells of the neocortical ventricular zone. J Neurosci 18:53745388.
Philipp S, Hambrecht J, Braslavski L, Schroth G, Freichel M, Murakami M, Cavalie A, Flockerzi V (1998) A novel capacitative calcium entry channel expressed in excitable cells. EMBO J 17:42744282.
Phillips AM, Bull A, Kelly LE (1992) Identification of a Drosophila gene encoding a calmodulin-binding protein with homology to the trp phototransduction gene. Neuron 8:631642.[ISI][Medline]
Sakaki Y, Sugioka M, Fukuda Y, Yamashita M (1997) Capacitative Ca2+ influx in the neural retina of chick embryo. J Neurobiol 32:6268.[ISI][Medline]
Santella L (1998) The role of calcium in the cell cycle: facts and hypotheses. Biochem Biophys Res Commun 244:317324 [review].[ISI][Medline]
Spitzer NC (1995) Spontaneous activity: functions of calcium transients in neuronal differentiation. Perspect Dev Neurobiol 2:379386.[ISI][Medline]
Steinhardt R, Zucker R, Schatten G (1977) Intracellular calcium release at fertilization in the sea urchin egg. Dev Biol 58:185196.[ISI][Medline]
Wilding M (1996) Calcium and cell cycle control in early embryos. Zygote 4:16 [review].[ISI][Medline]
Xian H, Maric D, Maric I, Barker JL (1996) Tonically active divalent cation entry into embryonic rat cortical cells in vitro. Soc Neurosci Abstr 22:537.
Zhu X, Jiang M, Peyton M, Boulay G, Hurst R, Stefani E, Birnbaumer L (1996) trp, a novel mammalian gene family essential for agonist- activated capacitative Ca2+ entry. Cell 85:661671.[ISI][Medline]