Stereotypical Physiological Properties Emerge During Early Neuronal and Glial Lineage Development in the Embryonic Rat Neocortex

Dragan Maric, Irina Maric, Yoong Hee Chang and Jeffery L. Barker

Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Surface immunolabeling was used together with membrane potential and/or Ca2+ indicator dyes to characterize physiological properties emerging among precursors, neuroglial progenitors and differentiating neurons during neurogenesis of embryonic rat neocortex. Cells were immunoidentified with tetanus toxin (TnTx), which binds to gangliosides expressed by neurons, and anti-A2B5, which reacts with gangliosides expressed by neuroglial progenitors. Microdissection of the neocortex into ventricular/subventricular zone (VZ/SVZ) and cortical plate/subplate (CP/SP) regions further resolved the TnTx/A2B5-immunoidentified cells into pre- and post-migratory subpopulations. Quantitative immunocytochemistry revealed mainly proliferative (BrdU+) and immature (nestin+) elements among TnTxA2B5 precursors and TnTxA2B5+ progenitors in the VZ/SVZ, and the appearance of neuron-specific antigens among post-mitotic TnTx+ subpopulations of the CP/SP. Flow cytometry of acutely prepared cells in suspension and dual-imaging of cells in culture revealed that ionotropic amino acid receptors and metabotropic acetylcholine receptors closely paralleled the emergence of voltage-dependent Na+ and Ca2+ channels and Na+–Ca2+ exchange activity among TnTx+ neuronal progenitors migrating from VZ/SVZ to CP/SP. During this period, TnTxA2B5 precursors and TnTxA2B5+ neuroglial progenitors from VZ/SVZ predominantly exhibited Ca2+ responses to ATP. Thus, stereotypical and contrasting physiologies emerge among embryonic cortical cells in vivo as they initially progress from proliferating precursors and progenitors along neuronal and glial cell lineages.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Excitable membrane properties, including ion channels and transmitter receptors coupled to second messenger signaling systems emerge during the embryonic period of vertebrate central nervous system (CNS) development, where they are thought to function in morphogenic roles before mediating fast forms of intercellular communication (Lauder, 1993Go; Weiss et al., 1998Go). In the embryonic rat cortex, electrical recordings of cells in slices prepared during neurogenesis have revealed widespread and changing distributions of both electrical and chemical forms of excitability (Lo Turco and Kriegstein, 1991Go; Bulan et al., 1994Go; Mienville et al., 1994Go; Lo Turco et al., 1995Go; Mienville and Barker, 1997Go; Owens and Kriegstein, 1998Go). Optical recordings of dissociated cells with a potentiometric indicator dye and flow cytometry have been used to demonstrate the developmental appearance of K+-dependent resting membrane potential mechanisms and voltage-dependent Na+ channels during cortical neurogenesis (Maric et al., 1998aGo). However, these complementary strategies are each compromised in revealing the physiological correlates and determinants of early cell lineage progressions into neuronal and glial phenotypes. Electrical recordings are limited by the relatively small number of cells that are usually studied, by inherent cell-to-cell and experiment-to-experiment variabilities and by the difficulties of patch-clamping small-diameter embryonic cells, which could alter cell physiology. In contrast, flow cytometric recordings with physiological indicator dyes involve random and rapid sampling from large numbers (>10 000) of intact cells, but their precursor/progenitor status has only been inferred indirectly from correlative immunocytochemistry.

Therefore, to resolve more precisely and completely the emergent physiological properties during neurogenesis in the embryonic cortex, we have incorporated an immunolabeling strategy to phenotype live cells in terms of their precursor/ progenitor and differentiating state. We targeted surface gangliosides, which become expressed during phylogenetic evolution of vertebrate nervous systems and are recapitulated during ontogeny of the mammalian CNS (Hilbig et al., 1981Go). The structurally related di- and tri-sialogangliosides GD3 and GT1b are especially abundant during the embryonic period (Hilbig et al., 1982Go; Yu et al., 1988Go). GT1b binds tetanus toxin (TnTx) (Rogers and Snyder, 1981Go; Halpern and Loftus, 1993Go; Shapiro et al., 1997Go), while GD3 can be labeled with the monoclonal antibody anti-A2B5 (Schwarz and Futerman, 1996Go). TnTx and A2B5 have been used previously to identify cells as neurons or neuroglial progenitors respectively (Raff et al., 1979Go, 1983Go; Koulakoff et al., 1983Go; Behar et al., 1988Go). In this study, dual TnTx/A2B5-imunolabeling was combined with potentiometric and Ca2+ indicator dyes to reveal the concerted emergence of specific forms of excitability that are differentially expressed among precursor, neuroglial progenitor and differentiating neuronal populations during neurogenesis in the embryonic rat neocortex. These included the expressions of functional ionotropic GABAA and glutamate/kainate receptors, muscarinic acetylcholine receptors coupled to cytosolic Ca2+ signaling, voltage-dependent Na+ and L-type Ca2+ channels, Na+–Ca2+ exchange activity and cytosolic Ca2+ responses to ATP. The results demonstrate highly contrasting patterns of physiological properties emerging in vivo as proliferating precursors and progenitors in the embryonic cortex become post-mitotic and begin to differentiate along neuronal and glial lineages. Some of these results have been published in abstract form (Maric et al., 1997aGo).


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Cell Preparation

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, 1986Go). 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.

Telencephalic (E11–13) and neocortical tissues (E14–22) were optimally dissociated with papain into single-cell suspensions with >95% viability (Maric et al., 1997bGo). Other commonly used methods for dissociating tissue into single-cell suspensions (trypsin, collagenase, mechanical) each resulted in a substantial loss of viable cells (Maric et al., 1998bGo). At E19, 350 µm thick coronal sections of the brain at the level of the midposterior neocortex, which corresponded to coronal plates 9–12 (Altman and Bayer, 1995Go), were microdissected along the incipient white matter into a cortical plate/subplate (CP/SP) region (including layer I) and a ventricular/subventricular (VZ/SVZ) zone (including lower intermediate zone) before dissociation. The cells were then washed and finally resuspended at a density of 2 x 106 cells/ml in a normal physiological medium (NPM) consisting of (in mM): 145 NaCl, 5 KC1, 1.8 CaCl2, 0.8 MgCl2, 10 glucose and 10 HEPES (pH 7.3, osmolarity 290 mOsm), which was supplemented with 1 mg/ml bovine serum albumin (BSA, Sigma, St Louis, MO).

Immunocytochemistry in Single-cell Suspensions

We used a mouse monoclonal class IgM anti-A2B5 antibody (Boehringer Mannheim Biochemicals, Indianapolis, IN) to label the surface disialoganglioside GD3 expressed by O-2A progenitor cells, type 2 astrocytes and certain types of neurons (Abney et al., 1983Go; Raff et al., 1983Go; Behar et al., 1988Go) and a mixture of TnTx fragment C (Boehringer Mannheim) and a mouse monoclonal class IgG2b anti-TnTx antibody (obtained from Dr William Habig, FDA, Bethesda, MD) to label the surface trisialoganglioside GT1b expressed by terminally post-mitotic neurons (Koulakoff et al., 1983Go). Acutely dissociated cells in suspension were double-immunoreacted with anti-A2B5 and TnTx/anti-TnTx for 30 min at room temperature (RT), then washed in NPM and the primary immunoreactions visualized by immunostaining with phycoerythrin (PE)-conjugated goat anti-mouse IgM and PE/CY5-conjugated goat anti-mouse IgG2b antibodies (Caltag Laboratories, Burlingame, CA) for an additional 30 min at RT. Other surface markers used to identify cells included mouse monoclonal class IgM anti-O4 and mouse monoclonal class IgG3 anti-galactocerebroside (GalC) antibodies (Boehringer Mannheim), specific for early and late stages of oligodendrocyte development (Raff et al., 1978Go; Schachner et al., 1981Go), which were visualized with PE-conjugated goat anti-mouse IgM and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG3 antibodies (Caltag) respectively.

Actively proliferating cells in A2B5/TnTx-immunolabeled populations were identified using the thymidine analogue 5-bromo 2-deoxyuridine (BrdU, Sigma), which becomes incorporated into DNA during the S-phase of the cell cycle (Gratzner, 1982Go). S-phase cells were labeled in vivo over an 8 h period by injecting E19 dams with four i.p. doses of BrdU (50 µg/g body wt), each administered every 2 h. After killing the dams, the embryonic neocortex was quickly microdissected into the VZ/SVZ zone and CP/SP region, dissociated into single-cell suspensions and the live cells double-immunolabelled with A2B5/TnTx, as described above. Subpopulations of cells were then physically sorted based on surfacelabeling characteristics into unlabeled precursor, A2B5+ neuroglial progenitor and TnTx+ neuronal subpopulations using the FACStar+ flow cytometer, as previously described (Maric et al., 1999Go). The BrdU+ cells in each subpopulation were detected immunocytochemically with a protocol (Maric et al., 1997bGo) that initially included fixation in 70% ethanol at –20oC, then permeabilization and DNA denaturation with 2 N HCl/0.5% Triton-X100 (Sigma), followed by visualization with FITC- conjugated mouse monoclonal class IgG1 anti-BrdU antibody (Becton Dickinson, Mountain View, CA).

Antibodies specific for cytoskeletal proteins expressed by precursor, progenitor and differentiating cells were used to further immunotype A2B5/TnTx-labeled populations using triple-staining protocols. These included rabbit polyclonal anti-nestin antibody (a gift from Dr Ron McKay, NIH, Bethesda, MD), which identifies an intermediate filament protein associated with neuroepithelium-derived precursor and progenitor cells (Hockfield and McKay, 1985Go), mouse monoclonal class IgG1 anti-microtubule associated protein-2 (MAP-2; Sigma) and class IgG2a anti-tubulin ß III (TuJ1; Berkeley Antibody Company, Richmond, CA) antibodies, which identify neuron-specific cytosketetal proteins (Huber and Matus, 1984Go; Tucker et al., 1988Go; Lee et al., 1990Go; Menezes and Luskin, 1994Go), and rabbit polyclonal anti-glial fibrillary acidic protein antibody (GFAP), which is specific for astrocytes (Chemicon International Inc., Temecula, CA). Cells labeled with anti-A2B5-PE and anti-TnTx- PE/CY5 were fixed in 4% paraformaldehyde for 30 min at RT, then washed three times in PBS/1% (w/v) BSA, immunoreacted with antinestin, anti-MAP-2, anti-TuJ1 or anti-GFAP antibodies for 1 h at RT and visualized with appropriate FITC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) or goat anti-mouse IgG1 or IgG2a (Caltag) secondary antibodies.

Surface, cytoplasmic and nuclear epitopes among TnTx/A2B5-labeled subpopulations in suspension were quantified in 100 000 cell samples using the FACSTAR+ flow cytometer (Becton Dickinson). The FITC, PE and PE/CY5 fluorescence signals were 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 collected using bandpass filters set at 530 ± 30, 575 ± 25 and 670 ± 20 nm respectively. Cell Quest Analysis software (Becton Dickinson) was used to quantify the fluorescence signal intensities among the immunolabeled subpopulations.

Immunocytochemistry in Short-term Culture

In another series of experiments, cells dissociated from the VZ/SVZ and CP/SP at E19 were cultured for up to 48 h to permit morphological differentiation and to reveal the immunophenotype of proliferating and differentiating cells. The cells were plated at a density of 2 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 maintained in Neurobasal Medium supplemented with B27 and G5 additives (Life Technologies Inc., Frederick, MD). Two hours before the termination of culture, the cells were pulse labeled with 10 µM BrdU, then immunolabeled for surface (TnTx, A2B5, O4, GalC), cytoplasmic (nestin, TuJ1, GFAP) and nuclear (BrdU) epitopes using subtypespecific, fluorochrome-conjugated primary or secondary antibodies and sequential photo-bleaching and restaining. In one 5-epitope staining protocol, the cells were first surface-immunoreacted with mouse monoclonal class IgG2b anti-TnTx and mouse monoclonal class IgM anti-A2B5 antibodies, then visualized by immunostaining with subtype-specific goat anti-mouse FITC- or PE-conjugated secondary antibodies (Caltag Laboratories), and subsequently fixed in 4% paraformaldehyde. The resulting immunoreactions on individual cells were examined using an Axiovert 135 inverted fluorescence microscope (Carl Zeiss, Thornwood, NY) equipped with a standard FITC/PE filter set (Omega Optical, Brattleboro, VT). The cells were photographed with a 35 mm camera, then photo-bleached using filtered light from a mercury arc lamp until no FITC or PE fluorescence could be detected. The same cells were then fixed in 70% ethanol, washed in PBS/1% (w/v) BSA, reimmunoreacted with rabbit polyclonal anti-nestin and mouse monoclonal class IgG2a anti-Tuj1 antibodies and restained with aminomethylcoumarin (AMCA)-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories) or tetramethyl rhodamine isothiocyanate (TRITC)- conjugated goat anti-mouse IgG2a secondary antibodies (Southern Biotechnology Associates, Inc., Birmingham, AL) respectively. As a final step, the cells were immunostained with a mouse monoclonal class IgG1 anti-BrdU-FITC antibody (Becton Dickinson), relocated on the alphanumeric grid and rephotographed using a standard FITC/AMCA/TRITC filter set (Omega Optical). In another 5-epitope staining protocol, separate plates of cultured cells were first surface-immunoreacted with mouse monoclonal class IgM anti-O4-PE and class IgG3 anti-GalC-FITC, followed by photobleaching and restaining with rabbit polyclonal anti-GFAP-AMCA, mouse monoclonal class IgG2a anti-TuJ1-TRITC and mouse monoclonal class IgG1 anti-BrdU-FITC, as described above. This sequential immunolabeling of 5-epitopes in the same field of cultured cells allowed direct identification of pre-mitotic/immature precursors or progenitors and post-mitotic/differentiating cells at specific stages of neuronal or glial lineage progression.

Flow Cytometric Recordings of Physiological Properties Expressed by Immunoidentified Cells in Single-cell Suspensions

E19 cells immunolabeled with TnTx-PE/CY5 and A2B5-PE were stained with either potentiometric or Ca2+ indicator dyes to record their membrane potential or cytosolic Ca2+ (Ca2+c) levels under resting baseline conditions in NPM and in response to various agonists. Strategies to record and calibrate indicator dye fluorescence emissions using flow cytometry have recently been described in studies of unlabeled cortical cells (Maric et al., 1998aGo,bGo, 1999Go). Briefly, to record membrane potential (MP), immunolabeled cells were resuspended at a density of 2 x 106 cells/ml and stained with 200 nM bis-(1,3-dibutyl barbituric acid) trimethine oxonol (Molecular Probes, Eugene, OR) for 10 min at RT to allow complete equilibration of the negatively charged dye with cell plasma membranes before recording MP values. Alternatively, the immunolabeled cells were loaded with 100 nM fluo-3/AM, a Ca2+ indicator dye (Molecular Probes), for 20 min at RT, then washed to remove unincorporated dye and resuspended in NPM for 30 min to permit de-esterification before recording Ca2+c levels. Both oxonol and fluo-3 were excited at 488 nm and their fluorescence emissions detected with a bandpass filter set at 525 ± 15 nm. In triple-staining experiments, the spectral overlap of fluo-3 or oxonol fluorescence emissions into the PE detection window and PE fluorescence signals into the PE/CY5 detection window were electronically compensated at the preamplifier stage.

Modal values in the oxonol and fluo-3 fluorescence signal distributions were calibrated in terms of MP or [Ca2+]c using previously established protocols (Maric et al., 1998aGo,bGo, 1999Go). Typically, resting MP and Ca2+c levels were randomly recorded from 100 000 TnTx/A2B5- immunoidentified cells at the rate of ~2000 cells per second. The modal (approximately mean in symmetrically distributed signals) MP or Ca2+c values expressed by different subpopulations were quantified by gating electronically on TnTx and/or A2B5 immunofluorescence signals. This combined surface-labeling-and-indicator-dye-recording strategy allowed us to profile physiological properties of virtually all the cells in suspension in a rapid (~1–2 min) and statistically complete manner. TnTx/A2B5 labeling itself did not trigger detectable changes in either baseline membrane potential or Ca2+c (not shown).

Dual-imaging of Physiological Properties Expressed by Immunoidentified Cells Recovered in Culture

Dual-indicator dye digital videomicroscopic imaging of Ca2+c and membrane potential was carried out on fields of 20–30 isolated cells cultured from the VZ/SVZ and CP/SP for 24 h in order to record simultaneously both properties on the same cell after recovery from dissociation. The cells were pulse-labeled with BrdU during the last 2 h of culture, then loaded with 2 µM fura-2/AM (Molecular Probes) for 1 h at 37°C, washed in NPM and stained for 10 min at RT with 500 nM oxonol. Since oxonol equilibrates dynamically according to cell membrane potential, the dye was included in all recording solutions, which were delivered to the 150 ml recording chamber using gravity-driven perfusion at ~2 ml/min.

Digital videomicroscopic imaging was conducted using the Attofluor RatioVision workstation (Atto Instruments, Rockville, MD) equipped with an Axiovert 135 inverted microscope (Carl Zeiss) and an ICCD camera (Atto Instruments). The indicator dyes were sequentially excited at 1 s intervals with a 100 W mercury arc lamp filtered at 488 ± 5 nm for oxonol and at 334 ± 5 and 380 ± 5 nm for fura-2. 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).

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: , where Kd is the fura-2/Ca2+ binding constant at RT (225 nM), R is the ratio of fura-2 fluorescence at 334 and 380 nm, Rmin and Rmax are values of R in Ca2+-free saline and in NPM with 1.8 mM Ca2+ respectively, and F0/F{infty} is the ratio of fura-2 fluorescence at 380 nm in Ca2+-free and 1.8 mM Ca2+ salines. The ratioed signals of fura-2-loaded cells were converted online into [Ca2+]c values using the Attofluor RatioVision acquisition software (Atto Instruments). At the end of each experiment, Ca2+c signals were exported as an ASCII text format and combined with the corresponding membrane potential values, which were calibrated (see below) and plotted in parallel to illustrate the relationship between the two properties on individual cells.

Oxonol fluorescence (FLOX) emissions were calibrated in terms of estimated membrane potential, as previously described (Maric et al., 1998aGo). Briefly, the protocol involved permeabilization of cells with 1 µM gramicidin (a monovalent cationophore) and exposure to varying [Na+]o (see Figs 7 and 8GoGo). The membrane potential was then derived using the logarithmic values of FLOX and a simple Nernst equation involving Na+ and K+ and assuming that the total intracellular concentration of permeant monovalent cations remained constant at ~150 mM during the brief period required for calibration (Maric et al., 1998aGo).



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Figure 7. Dual-imaging demonstrates the ionic determinants of baseline physiological properties in a SP-TnTxhi neuron. CP/SP neurons were cultured for 24 h, then double-loaded with oxonol and fura-2 and imaged simultaneously every second with digital videomicroscopy at RT with the same recording saline used in flow cytometry. After imaging, the oxonol dye was rinsed out and the cells were surface labeled with TnTx and A2B5 immunoreagents in order to correlate their recorded properties with immunolabeling characteristics. Oxonol fluorescence (FLOX) traces and Ca2+c levels imaged in one neuron (of 25 studied) illustrate the representative stereotypical phenomenology of SP-TnTxhi cells. Numbers above the oxonol trace indicate [K+]o or [Na+]o in mM. (A) FLOX is directly related to [K+]o over 1–150 mM and elevating [K+]o (>=20 mM) triggers transient and sustained increases in Ca2+c levels, which completely recover in 5 mM [K+]o. Switching to Na+o-free saline elicits a transient and sustained increase in Ca2+c levels with little change in FLOX. Permeabilization with the cationophore gramicidin followed by stepwise increases in Na+o leads to stepwise increases in FLOX. (B) Log FLOX is directly related to membrane potential (assuming constant [Na+ + K+]i) and resting membrane potential (RMP) is estimated to be ~–82 mV. (C) Assuming [Na+ + K+]i {approx} 150 mM, then membrane potential can be directly related to K+o in an approximately Nernstian manner (~56 mV/10-fold change in K+o over 5–150 mM). (D) Photomicrograph of the cell recorded in (A) after dual-imaging and surface immunostaining with TnTx-FITC (green) and A2B5-PE (red), followed by fixation in paraformaldehyde, reveals a typical immunofluorescence reaction of the SP-TnTxhi neuron from the CP/SP. (E) Photomicrograph of the same cell which was first completely photobleached of its FITC and PE fluorescence, then refixed in ethanol, washed and sequentially restained with BrdU-FITC (green), nestin-AMCA (blue) and TuJ1-TRITC (red), as described in Materials and Methods. The results show that the SP-TnTxhi neuron recorded in (A) exhibits a BrdUNestinTuj1+ immunophenotype which is overwhelmingly predominant in the CP/SP (see Fig. 3Go). Scale bars in (D,E) represent 10 µm.

 


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Figure 8. Dual-imaging demonstrates the ionic determinants of baseline physiological properties in a SP-A2B5 progenitor. VZ/SVZ cells were cultured for 24 h, then 34 were imaged simultaneously as described in Figure 7Go. FLOX and Ca2+c levels illustrate the representative phenomenology for a SP-A2B5 progenitor, which was immunoidentified after imaging as described in Figure 7Go. (A) FLOX is directly related to [K+]o over 1–150 mM, but elevated K+ does not trigger changes in Ca2+c and switching to Na+o-free does not alter Ca2+c levels. (B) Log FLOX is directly related to membrane potential and the RMP is estimated to be –80mV. (C) Membrane potential is related to K+o in an approximately Nernstian manner (~58 mV/10-fold change in K+o over 5–150 mM). (D) Photomicrograph of the cell recorded in (A) after dual-imaging and surface immunostaining with TnTx-FITC (green) and A2B5-PE (red), followed by fixation in paraformaldehyde, reveals a typical immunofluorescence reaction of the SP-A2B5 progenitor from the VZ/SVZ. (E) Photomicrograph of the same cell which was photobleached of its FITC and PE fluorescence, then restained with BrdU-FITC (green), nestin-AMCA (blue) and TuJ1-TRITC (red), as described in Materials and Methods. The results show that the SP-A2B5 cell recorded in (A) exhibits a BrdU+Nestin+Tuj1 immunophenotype which is characteristic of SP-A2B5 progenitors from the VZ/SVZ (see Fig. 3Go). Scale bars in (D,E) represent 10 µm.

 
After imaging, the field of recorded cells was photographed with a 35 mm camera using phase-contrast optics in order to reveal their location with respect to the underlying alpha-numeric grid. The cells were then washed in NPM to remove the oxonol dye, then sequentially immunoidentified with anti-A2B5-PE, anti-TnTx-FITC, anti-O4-PE, antiGalC-FITC, anti-nestin-AMCA, anti-GFAP-AMCA, anti-Tuj1-TRITC and anti-BrdU-FITC using 5-epitope immunostaining protocols, as described above. In this way, the physiological properties of individual cells at membrane and cytoplasmic levels could be recorded simultaneously and then correlated with their precursor, progenitor or differentiation state.

Pharmacology

We used both flow cytometric and dual-imaging strategies to examine the emergence of specific physiological properties in entire populations and in single cells respectively. Pharmacological experiments were carried out by exposing the cells to asymptotic concentrations of acetylcholine (ACh) ± atropine (ATR) to reveal expression of muscarinic receptors, {gamma}-aminobutyric acid (GABA) ± bicuculline (BIC) to reveal GABAA/Cl channels, L-glutamate (GLUT) ± 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and (5R,10S)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK801) to discriminate between ionotropic glutamate receptors of the AMPA/kainate and NMDA subtypes and adenosine trisphosphate (ATP) ± suramin (SUR) to reveal expression of P2 purinoreceptors. Veratridine (VTD) and tetrodotoxin (TTX) were used to detect voltage-dependent Na+ channels and nitrendipine was employed to block L-type Ca2+ channels. CNQX and MK801 were obtained from RBI (Natick, MA). All other reagents were purchased from Sigma. All recordings were carried out at RT in NPM containing 1 mg/ml BSA, which adequately stabilized the baseline cell properties during the experimental period.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Progressively More Cells Label with TnTx and/or A2B5 During Cortical Neurogenesis

TnTx/A2B5 immunolabeling of surface gangliosides expressed by telencephalic and neocortical cells dissociated during neurogenesis and analyzed by flow cytometry revealed four primary subpopulations (Fig. 1Go): (i) cells labeled with TnTx, but not A2B5 (TnTx+A2B5, or single-positive for TnTx, SP-TnTx); (ii) cells labeled with A2B5, but not TnTx (TnTxA2B5+, or single-positive for A2B5, SP-A2B5); (iii) cells labeled with both TnTx and A2B5 (TnTx+A2B5+, or double-positive, DP); and (iv) cells not labeled with either TnTx or A2B5 (TnTxA2B5, or double-negative, DN). DN cells were most abundant at the onset of cortical neurogenesis (E12), when most cells were actively proliferating (Maric et al., 1997bGo). At this embryonic age, DN cells constituted ~70% of all cells in the telencephalic dissociates, while ~20% were SP-TnTx, ~10% were SP-A2B5 and ~2% were DP (Fig. 1A,DGo). As neurogenesis proceeded (E13–19), progressively more cells became labeled with either TnTx and/or A2B5 (Fig. 1B,CGo), so that by the end of neurogenesis (E19) >90% of them could be labeled (Fig. 1DGo). After E19, the proportion of DN cells increased, reaching ~30% by birth. This re-emergence of DN cells coincided with the advent of gliogenesis in the prenatal period of cortical development.



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Figure 1. Developmental changes in the TnTx/A2B5 surface epitope labeling patterns during cortical neurogenesis. Cells were dissociated into single-cell suspensions on different embryonic days during neurogenesis and double-labeled with anti-A2B5-PE and anti-TnTx-PE/CY5 before quantitative flow cytometry. (A–C) Two-dimensional, log–log dot density plots of fluorescence signals emitted by ~100 000 cells recorded at random are displayed in pseudo-color, ranging from low (violet) to medium (green) to high (yellow and red) relative abundance. Four main subpopulations can be identified at each age studied, which are outlined here by the crosshairs. These include cells which are (1) double-negative for TnTx and A2B5 (DN or TnTx/A2B5), the fluorescence of which is equivalent to autofluorescence of unstained cells (not shown); (2) single-positive for TnTx (SP-TnTx or TnTx+/A2B5); (3) single-positive for A2B5 (SP-A2B5 or TnTx/A2B5+); and (4) double-positive (DP or TnTx+/A2B5+). The percentages of cells composing each subpopulation in dissociates prepared at E12, E13 and E15 are displayed within each density plot. (D) Over E12–15, the proportion of TnTx- and/or A2B5-labeled cells increases from ~30% to ~75%, while that of DN cells decreases in a complementary fashion. At E17 and E19, >90% of the cells are labeled by either TnTx and/or A2B5. After E19, there is a progressive increase in the proportion of DN cells with a corresponding decrease in SP-TnTx, SP-A2B5 and DP cells. Percentages represent means ± SEM from at least three independent determinations at each age studied.

 
Quantitative flow cytometric analyses of the TnTx/A2B5- labeling patterns evolving during cortical neurogenesis further revealed distinctive developmental changes in both TnTx and A2B5 labeling intensities (Fig. 1ACGo). At E12, the distribution of SP-TnTx labeling intensities ranged up to 10-fold above the values of DN cells, which were equivalent to the autofluorescence signals of cells not exposed to the labeling protocol, while the distribution of SP-A2B5 cell intensities extended >200-fold. At this embryonic age, the DP cells expressed only low levels of both TnTx and A2B5 labeling. Over the next several days of cortical development, the number of cells expressing higher levels of A2B5 or TnTx labeling progressively increased and by E19 the majority of the cells were moderately to intensely stained (Fig. 2B,CGo). Interestingly, the continuous distribution in labeling intensities evident between DN cells and both SP-TnTx and SP-A2B5 cells throughout neurogenesis (Figs 1AC, 2C1GoGo) indicated that both labeled subpopulations likely originated directly from DN cells.



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Figure 2. TnTx/A2B5-labeled subpopulations originate from different anatomical locations at the end of cortical neurogenesis. (A) At E19, the coronal sections of mid-posterior neocortex were microdissected into the ventricular zone/subventricular zone (VZ/SVZ) and the cortical plate/subplate (CP/SP) region by sectioning along the intermediate zone (IZ) and then cutting perpendicularly to exclude the hippocampus (HC), rhinencephalon (RC), amygdala and the basal ganglia, as illustrated on the right side. (B) A three-dimensional contour plot representation of the complex TnTx/A2B5 labeling patterns in cells dissociated from the intact neocortex reveals distinct modes of signal distribution corresponding to six major subpopulations (DN, SP-A2B5, SP-TnTxlo, SP-TnTxhi, DP-TnTxlo and DP-TnTxhi), which are identified according to their labeling intensity or lack thereof (see Results). (C) The pseudo-color dot density plots of TnTx/A2B5 labeling in cells dissociated from intact neocortex (C1) and after its microdissection into the CP/SP (C2) and VZ/SVZ (C3) reveal largely complementary distributions in TnTx/A2B5 labeling patterns and identify the anatomical locations of the six supopulations. Boundaries (crosshairs) between labeled and unlabeled cells and between cells expressing low and high levels of TnTx labeling (TnTxlo and TnTxhi) have been drawn empirically to quantify the percentages of cells in each of the six subpopulations recovered in the dissociates of microdissected tissues. Percentages of each subpopulation represent means ± SEM from at least five independent determinations.

 
Anatomical Locations of the Immunoidentified Subpopulations in the Cortex

We focused most of our experiments on the neocortex at E19, since the great majority of cells (>90%; Figs 1D, 2C1GoGo) could be surface-labeled with TnTx and/or A2B5 and these populations could be placed in their proper anatomical context in vivo by a microdissection of the cortical tissue into the proliferating VZ/SVZ and the differentiating CP/SP regions (Fig. 2AGo). At this stage of cortical development, a three-dimensional flow cytometric representation of the TnTx/A2B5 labeling intensities of the neocortical dissociates revealed a complex labeling pattern which included six major subpopulations whose fluorescence signal distributions exhibited variably defined modes (Fig. 2BGo) and relative abundance (Fig. 2C1Go).

After microdissection, TnTx/A2B5 labeling of cells dissociated from the VZ/SVZ (Fig. 2C3Go) and CP/SP (Fig. 2C2Go) revealed almost complementary distributions in labeling patterns, which together reflected those observed in dissociates from the intact neocortex (Fig. 2C1Go). These labeling intensities were quantified in terms of arbitrary fluorescence units (a.f.u.), with each a.f.u. being equivalent to a unitary log increase in signal intensity. Autofluorescence values of DN cells generated approximately symmetrical frequency distributions with clear modes typically forming at ~2–3 a.f.u. (Figs 1AC, 2B,CGoGo). TnTx-labeled cells were arbitrarily subclassed into low-expressing (TnTxlo, <500 a.f.u.) and high-expressing (TnTxhi, >500 a.f.u.) subpopulations (Fig. 2B,C1Go), since the former predominated in the VZ/SVZ and the latter mostly populated the CP/SP. Based on this criterion, the DP cells we subclassed into DP-TnTxlo and DP-TnTxhi subpopulations. Since we found empirically that SP-A2B5 cells, the great majority of which resided in the VZ/SVZ at E19, exhibited physiological properties that were relatively independent of surface labeling intensity, we did not subclassify them in characterizing their membrane excitability and Ca2+ signaling mechanisms. However, we found that their proliferative and bipotential (glial or neuronal) capacity, determined from sort-purified SP-A2B5 cells in short-term culture, clearly varied with labeling intensity, so we subclassified them into SP-A2B5lo (A2B5 intensity <500 a.f.u.) and SP-A2B5hi (A2B5 intensity >500 a.f.u.) in summarizing the developmental relationships among the different subpopulations (see Fig. 10Go).



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Figure 10. Stereotypical morphologies and physiological properties are differentially expressed by precursor, neuroglial progenitor and differentiating neuronal subpopulations in the rat neocortex at the end of neurogenesis. Cells were cultured from the CP/SP and the VZ/SVZ regions for 48 h, exposed to BrdU for 2 h and then sequentially immunolabeled with TnTx-FITC (green), A2B5-PE (red), nestin-AMCA (blue), Tuj1-TRITC (red) and BrdU-FITC (green) to reveal their precursor/progenitor or differentiating status, as described in Materials and Methods. TnTx/A2B5-labeled cells were identified as either SP-TnTx (green) or DP (yellow) neurons or SP-A2B5 (red) neuroglial progenitors. Cells remaining unlabeled for TnTx or A2B5 were classified as double negative (DN). Representative cell morphologies and their labeling characteristics are shown together with a summary of their expressed patterns of physiological properties derived from quantitative FACS and imaging studies. Color-coded legends to the left of each subpopulation identify the immunostained epitopes targeted for illustration (blue, green or red). The white-colored legends complete the quantitative 5-epitope immunocytochemical analysis (with the percentage immunopositive cells shown in parentheses), but their subcellular distribution is not displayed. Clustered DN precursors in the VZ/SVZ typically lack elaborate processes and are either nestin+ (blue) and/or BrdU+ (green). The SP-A2B5 cells from the VZ/SVZ, most of which exhibit nestin+Tuj1 phenotype and simple mono- or bipolar morphology, have been subclassified according to their A2B5 labeling intensity using flow cytometric measurements into SP-A2B5lo and SP-A2B5hi subpopulations, with the latter exhibiting few BrdU+ elements, indicating less proliferating potential and implying greater commitment of these cells to gliogenesis. In contrast, multipolar cells with more extensive arbors characterize the pre-migratory SP-TnTxlo and DP-TnTxlo neuronal progenitors from the VZ/SVZ, most of which still express nestin, with an increasing number expressing Tuj1 and a decreasing number incorporating BrdU. The post-migratory SP-TnTxhi and DP-TnTxhi neurons in the CP/SP also express multipolar morphologies with extensive processes and complex arbors, but the great majority of them are BrdUnestinTuj1+. Solid lines with arrows indicate probable relationships and directions in neuron and glial cell lineage progression from proliferating precursors, which become either TnTx-labeled neuronal progenitors (SP-TnTxlo) or bipotential (SP-A2B5lo) neuroglial progenitors. Bipotential progenitors progress along glial lineages (SP-A2B5hi) or acquire TnTx- reactive epitopes at low levels of expression (DP-TnTxlo) and progress along neuronal lineages. The dotted line with arrow indicates a possible progression from DP-TnTxlo to SP-TnTxlo subpopulations, as the expression of A2B5 decreases. The physiological properties identified at precursor, progenitor and differentiating neuronal stages, depicted by the yellow legend to the right of each subpopulation, are indicated by the percentages of cells exhibiting functional voltage-dependent Na+ and L-type Ca2+ (Ca2+L) channels, ionotropic GABAA and glutamate/kainate (GLUTKA) receptors, muscarinic acetylcholine receptors (ACHM) coupled to cytoplasmic Ca2+ signaling pathways and cytoplasmic Ca2+ responses to ATP.

 
Double immunostaining of E19 neocortical dissociates with anti-O4-PE and anti-GalC-FITC antibodies revealed the presence of only ~2% of the former and <1% of the latter population. Therefore, the results show that the majority of E19 neocortical cells can be subtyped based on live-cell surface immunolabeling into SP-TnTxhi and DP-TnTxhi cells (Fig. 2C2Go), which reside almost exclusively in the CP/SP, and DN, SP-A2B5, SP-TnTxlo and DP-TnTxlo cells, which predominate in the VZ/SVZ, as seen in Figure 2C3Go. In contrast, the O4+ and GalC+ cells represented a very small minority of E19 neocortical cells and were not further studied with the strategies described here.

Intracellular Immunostaining of TnTx/A2B5-labeled Cells Identifies the Proliferating Precursor/Progenitor and Differentiating Pre- and Post-migratory Neuronal Subpopulations

Nuclear BrdU incorporation and cytoplasmic protein expressions characteristic of proliferating precursor and progenitor cells (nestin+) or differentiating neurons (TuJ1+, MAP2+) or astrocytes (GFAP+) were quantified in TnTx/A2B5-labeled subpopulations dissociated from VZ/SVZ and CP/SP regions using triple-staining immunocytochemistry and flow cytometry. BrdU+ precursors accounted for slightly more than half of the DN cells in the VZ/SVZ during an 8 h BrdU labeling period in vivo (Fig. 3AGo). The percentage of BrdU+ cells decreased concomitant with an increase in both TnTx (Fig. 3AGo) and A2B5 labeling intensities (not shown, but see Fig. 10Go). Few SP-TnTxhi and DP-TnTxhi cells in the CP/SP (<2%) were BrdU+ following an 8 h pulse in vivo, demonstrating that virtually all of these cells were >8 h post-mitotic. However, if the 8 h BrdU labeling period was carried out in vivo on E18 and then followed by a 15 h chase interval, ~25% of the SP-TnTxhi and 5% of the DP-TnTxhi cells were BrdU+ (not shown). The delayed appearance of BrdU+TnTx+ cells in the CP/SP reflects the radial migration of terminally post-mitotic neuronal progenitors from the VZ/SVZ to the CP/SP during the 15 h interval (Rakic, 1978Go; Behar et al., 1996Go, 1998Go).



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Figure 3. Nuclear and cytoplasmic epitope expressions identify the proliferative and differentiating elements among the TnTx/A2B5-immunolabeled cells. CP/SP and VZ/SVZ cells were first surface labeled with TnTx/A2B5, then fixed and immunoreacted for intracellular epitopes characteristic of either proliferating precursor and progenitor cells (BrdU+, nestin+) or cells differentiating along neuronal (TuJ1+, MAP2+) or astrocyte-glial (GFAP+) lineages. Epitope-immunopositive cells distributed among the six subpopulations were quantified in samples of ~100 000 cells with flow cytometry. Almost all of the BrdU+ and nestin+ cells are distributed among the four VZ/SVZ subpopulations, identifying the great majority of them as proliferating precursors and progenitors or cells in the earliest stages of differentiation. Many of the SP-TnTxlo cells in the VZ/SVZ and almost all of the DP-TnTxhi and SP-TnTxhi cells in the CP/SP are either TUJ1+ or MAP2+, confirming their neuronal phenotype. Few SP-A2B5 cells are either TUJ1+ or MAP2+, consistent with the notion that these cells are predominantly glial progenitors. Less than 1% of E19 neocortical dissociates were GFAP+ and all exhibited DN phenotype (not shown). Data represent means ± SEM from at least three independent determinations.

 
The great majority (~85–90%) of DN, SP-A2B5, SP-TnTxlo and DP-TnTxlo cells from the VZ/SVZ expressed nestin (Fig. 3BGo), a neuroepithelium/stem cell-derived cytoskeletal protein, which was consistent with their proliferative and precursor/progenitor stages. In contrast, <15% of DP-TnTxhi and SP-TnTxhi cells in the CP/SP still retained this protein (Fig. 3BGo), which was expressed at markedly lower levels of signal intensity (not shown). Cytoskeletal protein expressions characteristic of differentiating neurons (TuJ1, MAP2) largely paralleled changes in TnTx labeling intensity (Fig. 3C,DGo). About 25% of the DN cells from VZ/SVZ expressed TuJ1, indicating the appearance of a neuronspecific cytoskeletal protein before surface TnTx labeling. Few SP-A2B5 cells at all levels of labeling intensity were either TuJ1+ (~5%) or MAP2+ (~2%), which is consistent with their surface epitope expression correlating more closely with a glial (astrocyte–oligodendrocyte) rather than a neuronal progenitor status. Furthermore, those SP-A2B5 progenitors that expressed intracellular neuronal antigens all exhibited low levels of surface A2B5 labeling (a.f.u. <500, not shown), thereby identifying SP-A2B5lo cells as a bipotential (neuroglial) stage in cell lineage progressions (see below). There were more TuJ1+ than MAP2+ cells among the SP-TnTxlo (~80% versus ~45%) and DP-TnTxlo (~30% versus ~10%) subpopulations of the VZ/SVZ, while the great majority of DP-TnTxhi and SP-TnTxhi cells from the CP/SP co-expressed both cytoskeletal proteins.

In contrast, <1% of E19 neocortical cells were GFAP+ and all exhibited DN phenotype (not shown). In sum, the results demonstrate that the surface labeling with TnTx and A2B5 can be used to identify the great majority of cells at the end of neurogenesis of the embryonic neocortex, with labeling intensity correlating closely both with different stages of early neuronal or neuroglial lineage progression, before the appearance of oligodendrocytes or astrocytes, and with anatomical location.

Voltage-dependent Na+ Channels Progressively Emerge with Neuronal Differentiation

The resting membrane potential and Ca2+c levels of TnTx/A2B5- labeled subpopulations and their pharmacological responses to ligands were investigated using physiological indicator dyes and flow cytometry. In the figures that follow (Figs 4–6GoGoGo), we have illustrated the most contrasting differences in the physiological properties emerging among SP-A2B5 neuroglial progenitors from the VZ/SVZ and SP-TnTxhi neurons from the CP/SP, while summarizing the results associated with the other four populations in bar graphs. Under resting conditions, almost all of the cells in each subpopulation were well polarized at negative membrane potentials (–80 to –90 mV) and the majority exhibited relatively low Ca2+c levels (50–100 nM), as illustrated by the modal values of potentiometric and Ca2+-indicator fluorescence signal distributions (Fig. 4A1,B1Go). The resting membrane potentials of most cells in each subpopulation were determined by K+ ions in an approximately Nernstian relationship (not shown, but see Figs 7 and 8GoGo), consistent with published results obtained with unlabeled cortical cells in suspension or in short-term culture throughout neurogenesis (Maric et al., 1998aGo). These results indicate that, under resting conditions, cells in suspension and in short-term culture both exhibit similar membrane potentials, which are characteristic of a relatively quiescent state.



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Figure 4. Voltage-dependent Na+ channel expression closely parallels neuronal differentiation. Cells dissociated from the CP/SP and VZ/SVZ were first labeled with TnTx/A2B5 and then stained with oxonol or fluo-3. Distributions of potentiometric or Ca2+ indicator dye signals were acquired on ~100 000 cells under resting conditions and 2–5 min after addition of 100 µM veratridine. Resting (baseline) fluorescence signal distributions (dotted-line frequency histograms in A1 and B1) and peak changes in the signal distribution induced by veratridine (solid-line frequency histograms in A1 and B1) are shown for SP-A2B5 progenitors in the VZ/SVZ and SP-TnTxhi neurons in the CP/SP to illustrate the highly contrasting responses. Results from the other four populations are summarized in bar graphs (A2,B2). At rest, the potentiometric and Ca2+c signal distributions are approximately symmetrical so that modes approximate means and therefore provide useful estimates of average resting membrane potentials (~–80 mV for both subpopulations) and Ca2+c levels (~70 nM for SP-A2B5 cells and ~120 nM for SP-TnTxhi neurons). (A1) Veratridine depolarizes 20% of SP-A2B5 cells, with almost all of them peaking at negative potentials. In contrast, 96% of SP-TnTxhi neurons depolarize to veratridine, forming a well-defined mode at ~+20 mV. (A2) Progressively more cells are depolarized to positive potentials (>0 mV, filled portions of bar graphs) and correspondingly fewer are depolarized to negative potentials (<0 mV, unfilled portions of bar graphs) by veratridine, as TnTx/A2B5 labeling intensity changes, with the order of appearance of the former response and disappearance of the latter effect being DP-TnTxlo, SP-TnTxlo, DP-TnTxhi and SP-TnTxhi. (B1,B2) Veratridine induces Ca2+c elevations to micromolar levels in all subpopulations, with progressively more TnTx+ cells responding in the same order as found for cells depolarized by veratridine to positive potentials. Bar graph data in Figures 4–6GoGoGo represent means ± SEM from at least three independent determinations.

 


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Figure 5. Pharmacological responses to GABA and glutamate are largely confined to neuronal subpopulations. Membrane potential and Ca2+c responses to 10 µM GABA or 100 µM L-glutamate (GLUT) were quantified in TnTx/A2B5-labeled dissociates as described in Figure 4Go. (A1) Few SP-A2B5 progenitors (~12%) depolarize to GABA, while almost all SP-TnTxhi neurons (93%) depolarize, forming a well-defined mode at ~–40 mV. (A2) The percentage of cells depolarized by GABA increases systematically in the same order of TnTx labeling intensity as observed for veratridine-mediated depolarization to positive potentials. (A3) Few SP-A2B5 cells (~10%) increase Ca2+c levels after exposure to GABA, while >50% of the SP-TnTxhi neurons respond, with most peaking at ~400 nM Ca2+c. (A4) Cells responding to GABA with an elevation in Ca2+c are largely restricted to the SP-TnTxlo, DP-TnTxhi and SP-TnTxhi subpopulations. (B1) Few SP-A2B5 progenitors (<5%) depolarize to glutamate, while most SP-TnTxhi neurons (~67%) respond, with many depolarizing to ~0 mV. (B2) The percentage of cells depolarized by glutamate progressively increases among the TnTx+ subpopulations in the same order as recorded for cells depolarized by veratridine to positive potentials and by GABA to negative potentials. (B3) Few SP-A2B5 progenitors (~10%) increase Ca2+c in response to glutamate, while >50% of the SP-TnTxhi neuronal population responds, generating a well-defined mode at ~500 nM. (B4) The proportions of cells responding to glutamate with an elevation in Ca2+c among the TnTx+ subpopulations closely mirror those whose Ca2+c levels are increased by GABA.

 


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Figure 6. Ca2+c responses to acetylcholine and ATP vary with cortical cell phenotype. Ca2+c responses to 10 µM acetylcholine (ACh) and 1 mM ATP were quantified in TnTx/A2B5-labeled dissociates as described in Figure 4Go. (A1) Few SP-A2B5 progenitors (~11%) elevate their Ca2+c to ACh, while most SP-TnTxhi neurons (~76%) respond, forming a mode at 1 µM. (A2) TnTx+ subpopulations contain most of the ACh-responsive elements. (B1) Many unlabeled precursors and SP-A2B5 progenitors (~60–70%) respond to ATP by elevating their Ca2+c to ~1 µM, while only a fraction of SP-TnTxhi neurons (~12%) respond in a similar manner. (B2) The percentage of responding cells that elevate their Ca2+c in response to ATP varies inversely with the intensity of TnTx labeling.

 
Veratridine, which activates voltage-dependent Na+ channels at resting potentials, depolarized cells in all subpopulations and elevated Ca2+c (Fig. 4A,BGo). However, the majority of VZ/SVZ cells depolarized to negative rather than to positive potentials, while only a fraction of DP-TnTxlo (~20%) and SP-TnTxlo (~30%) subpopulations depolarized above 0 mV. In contrast, many DP-TnTxhi (~40%) and most SP-TnTxhi (~80%) neurons in CP/SP dissociates depolarized to positive potentials. Depolarizations to positive potentials were Na+o-dependent and blocked by TTX (not shown, but see Fig. 9AGo), while depolarizations to negative potentials were TTX-insensitive (not shown). These pharmacological properties recorded in subpopulations at E19 recapitulate the developmental changes in the pharmacology of depolarizing responses to veratridine obtained in unlabeled cells during cortical neurogenesis using flow cytometry and potentiometric dye recordings (Maric et al., 1998aGo). In fact, the veratridine- induced responses of the VZ/SVZ subpopulations quantified in this study closely resembled those observed for cells recovered at the beginning of neurogenesis when the tissue is primarily a proliferating neuroepithelium.



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Figure 9. Dual-imaging demonstrates contrasting patterns of transmitter receptors and ion channels expressed in a SP-TnTxhi neuron and a SP-A2B5 progenitor. CP/SP and VZ/SVZ cells were cultured for 24 h, then imaged in dual mode and immunoidentified after imaging (inserts), as described in Fig. 7Go. Membrane potential and Ca2+c responses to five agonists and co-applications of corresponding antagonists reveal qualitative differences in the expression of functional receptors and voltage-dependent Na+ channels in two cells representative of SP-TnTxhi and SP-A2B5 subpopulations. (A) In the SP-TnTxhi (TnTx+A2B5NestinBrdUTuj1+) CP/SP neuron, 10 µM ACh triggers a transient and sustained increase in Ca2+c without changing baseline membrane potential. Co-application of 100 µM atropine (ATR) blocks the sustained phase. ATP (1 mM) depolarizes the neuron by ~20 mV without changing Ca2+c and this effect is blocked by co-application of 100 µM suramin (SUR). GABA (10 µM) depolarizes the neuron to ~–50 mV and triggers a transient and sustained increase in Ca2+c. The sustained changes are effectively blocked by 100 µM bicuculline (BIC). Glutamate (GLUT, 100 µM) depolarizes the neuron to ~–10 mV and elevates Ca2+c in a sustained manner. Co-application of 100 µM CNQX substantially reverses these effects, while 10 µM MK-801 has little further effect. Although membrane potential recovers completely in normal physiological medium (NPM), Ca2+c remains elevated. Veratridine (VTD, 100 µM) depolarizes the neuron to ~+50 mV and elevates Ca2+c still further. Addition of 1 µM tetrodotoxin (TTX) blocks most of the depolarization, but has no effect on the sustained levels of elevated Ca2+c. (B) In contrast, ATP is the only agonist that exerts an effect on membrane potential and Ca2+c in the SP-A2B5 (TnTxA2B5+Nestin+BrdU+Tuj1) VZ/SVZ progenitor, depolarizing the cell by ~20 mV and transiently elevating Ca2+c by 200 nM. The depolarization is blocked by co-application of suramin, implying the involvement of putative P2 purinergic receptors. Scale bars in the inserts represent 10 µm.

 
In the VZ/SVZ, the percentages of cells exhibiting Ca2+c elevation in response to veratridine ranged from ~25% among DN to ~40% among SP-TnTxlo cells (Fig. 4B1,B2Go). The percentages of cells exhibiting Ca2+c responses to veratridine were similar to those depolarized by veratridine with the notable exception of SP-A2B5 progenitors. About twice as many SP-A2B5 cells responded to veratridine with an elevation in Ca2+c (~30–35%) as were depolarized (~15–20%), indicating that the former effect could occur independently of the latter. The highest percentages of cells with Ca2+c responses to veratridine were recorded among SP-TnTxhi (~85%) and DP-TnTxhi postmitotic neurons (~60%) recovered from CP/SP dissociates (Fig. 4B1,B2Go). However, these Ca2+c elevations were entirely TTX- sensitive (not shown) and therefore dependent on depolarization and activation of voltage-dependent Ca2+ channels, which are also expressed by these neurons (see Fig. 7AGo).

Highly Contrasting Physiologies Distinguish Differentiating Neurons from Precursor and Progenitor Subpopulations

Membrane potential and Ca2+c responses to four neurotransmitters (GABA, glutamate, ACh and ATP) were quantified to reveal the distribution of functional receptors emerging among TnTx/A2B5-immunoidentified subpopulations. Few DN precursors (~5–18%) and <10% of the SP-A2B5 progenitors responded to either GABA, glutamate or ACh, either by depolarizing or by elevating Ca2+c (Figs 5 and 6GoGo). However, many DN (~60%) and SP-A2B5 cells (~70%) responded to ATP with an elevation in Ca2+c to micromolar levels (Fig. 6BGo) and a modest (~10–20 mV) depolarization (not shown, but see Fig. 9BGo).

The emergence of TnTx labeling among VZ/SVZ cells, identifying them as neuronal progenitors, correlated with the largely parallel expressions of responses to GABA (Fig. 5AGo), glutamate (Fig. 5BGo) and ACh (Fig. 6AGo), together with the concomitant disappearance of Ca2+c responses to ATP (Fig. 6BGo). However, >70% of SP-TnTxlo and DP-TnTxlo neuronal progenitors from the VZ/SVZ exhibited modest depolarizations to ATP similar to those observed with DN and SP-A2B5 cells (not shown). More SP-TnTxlo progenitors were affected by GABA, glutamate or ACh (~40–50%) than DP-TnTxlo progenitors (<=25%), while fewer SP-TnTxlo progenitors (<10%) exhibited Ca2+c responses to ATP than DP-TnTxlo cells (~30%). Depolarizing responses were uniformly more widespread than Ca2+c signals when changes in both membrane potential and Ca2+c levels were detected. For example, ~25% of the DP-TnTxlo progenitors were depolarized by GABA, but almost none of these responded with a rise in Ca2+c, indicating that the two effects could occur independently. Furthermore, these progenitors did not respond at membrane or cytoplasmic levels to either glutamate or ACh, suggesting that depolarizing GABA receptors detectable with this strategy emerged in vivo before functional glutaminergic or cholinergic receptors. GABA depolarized ~50% of SP-TnTxlo progenitors and elevated Ca2+c in ~25%, while glutamate depolarized ~40% and elevated Ca2+c in ~25% and ACh elevated Ca2+c in ~45% of these cells without depolarizing them. Hence, functional receptor-coupled signals to these three transmitters emerged together in 40–50% of SP-TnTxlo neuronal progenitors.

Similar percentages of DP-TnTxhi neurons, which had migrated from the VZ/SVZ and started differentiating in the CP/SP, responded to GABA, glutamate and ACh (~40–60%; Figs 5, 6AGoGo). SP-TnTxhi neurons, which had likely migrated prior to DP-TnTxhi cells, were the most responsive subpopulation to each of these three transmitters. While GABA depolarized more of SP-TnTxhi neurons (~90%; Fig. 5AGo) than glutamate (~70%; Fig. 5BGo), both amino acids elevated Ca2+c in the same percentage of cells (~50%). ACh increased Ca2+c in ~75% of these cells (Fig. 6AGo), without changing their membrane potential (see Fig. 9AGo). In marked contrast, <10% of either TnTxhi neuronal subpopulation responded to ATP with a rise in Ca2+c (Fig. 6BGo), although >70% of them still exhibited modest (~10–20 mV) depolarizations (not shown, but see Fig. 9AGo). Thus, depolarizing responses to ATP were ubiquitous in neocortical dissociates and occurred independently of the Ca2+c responses triggered by this ligand, which were predominantly restricted to precursors and neuronal and glial progenitors in the VZ/SVZ.

In all subpopulations studied, the GABA-induced responses were predominantly blocked by preincubation with 100 mM bicuculline, while those of glutamate, ACh and ATP were antagonized with 10 mM CNQX, 10 mM atropine and 100 mM suramin respectively (data not shown, but see Fig. 9Go). Hence, while the expression of functional P2 purinoreceptors was widespread, the concerted expressions of GABAA, glutamate/ kainate and muscarinic ACh receptor-coupled signals were restricted to neuronal progenitors migrating from the VZ/SVZ into the CP/SP, and these closely paralleled the developmental expression of voltage-dependent Na+ channels, suggesting that this stereotypical pattern of physiological properties characterizes early stages of neuronal lineage progression and radial migration in the embryonic neocortex.

Dual-imaging of Immunoidentified Cells in Culture Demonstrates the Coexistence of Physiological Properties

In order to correlate emergent physiologies with cell lineage progressions at the single-cell level and to allow cells to recover from the trauma of dissociation, VZ/SVZ and CP/SP cells were cultured for 24 h, then double-stained with oxonol and fura-2 and fields of ~20–30 cells dual-imaged at room temperature using the same saline employed in flow cytometry. After imaging, the live cells were immunoidentified initially with TnTx and A2B5, then fixed and the same cells further immunoidentified with sequential immunoreactions for nestin, Tuj1 and BrdU, as described in Materials and Methods.

The resting membrane potentials of cultured cells, like those in suspension, were determined by K+ in an approximately Nernstian manner over ~5–150 mM K+o (Figs 7A,C, 8A,CGoGo). K+o levels of 20 mM or more triggered similar increases in Ca2+c in almost all CP/SP neurons (Fig. 7AGo), while none of the SP-A2B5 progenitors exhibited Ca2+c responses even though they also depolarized in an approximately Nernstian manner (Fig. 8AGo). All K+o-dependent Ca2+c responses were Ca2+o-dependent and markedly blocked by 10 µM nitrendipine, but little affected by antagonists of other voltage-dependent Ca2+ channels (P-, Q-, N- and T-types; not shown). These results demonstrate the involvement of L-type Ca2+ channels in the generation of K+-dependent Ca2+c responses. In all CP/SP neurons, removal of Na+o immediately led to a transient increase in Ca2+c to ~1 µM (Fig. 7AGo). This response to Na+o-free saline did not occur in SP-A2B5 progenitors (Fig. 8AGo). Thus, active Na+-Ca2+ exchange functions to regulate baseline Ca2+c levels in differentiating neurons, but not neuroglial progenitors. These results are comparable to those obtained in an independent study of unlabeled cells using flow cytometry, which shows that during neurogenesis steady-state Ca2+c levels are initially Na+o- independent and become progressively Na+o-dependent (D. Maric et al., in press).

After 24 h in culture, the majority of TnTx+ neurons (~90%) responded to more than one transmitter. Over 40% of neuronal progenitors cultured from the VZ/SVZ and >70% of neurons from the CP/SP responded to ACh, GABA and glutamate (Fig. 9AGo), while the remaining cells progressing along a neuronal lineage(s) predominantly responded to GABA and glutamate, but not to ACh. The ACh-induced response was completely blocked by addition of atropine before (not shown) or during the sustained phase of Ca2+c elevation (Fig. 9AGo), indicating the involvement of muscarinic rather than nicotinic cholinergic receptors in the response. ATP modestly depolarized most cells by ~10–20 mV (Fig. 9A,BGo) and this was antagonized by suramin, implicating P2 purinergic receptors. A self-limiting Ca2+c response (to ~300 nM), which relaxed completely during ATP application and could be blocked by preincubation with suramin, was also triggered together with the depolarization in SP-A2B5 progenitors (Fig. 9BGo), but not in CP/SP neurons (Fig. 9AGo). GABA evoked a sustained depolarizing response of many neurons in both VZ/SVZ and CP/SP cultures to ~–40 mV, which was accompanied by a transient and sustained elevation in Ca2+c (Fig. 9AGo, VZ/SVZ neuron responses not shown), while SP-A2B5 progenitors were unaffected (Fig. 9BGo). The sustained effects of GABA on potential and Ca2+c levels were antagonized by bicuculline, indicating involvement of GABAA receptor/Cl channels and, indirectly, voltage-dependent Ca2+ channels in the Ca2+c responses. Glutamate depolarized many neuronal progenitors in the VZ/SVZ and neurons in the CP/SP near 0 mV and elevated their Ca2+c levels (Fig. 9AGo), but did not affect SP-A2B5 progenitors (Fig. 9BGo). These pharmacological effects were largely antagonized by CNQX, with little further block by MK-801, indicating the primary involvement of ionotropic receptors of the kainate/AMPA type. Veratridine depolarized >90% of CP/SP neurons to positive potentials (~+50 mV, Fig. 9AGo), while not affecting many SP-A2B5 progenitors (Fig. 9BGo). Veratridine-responsive cells also increased their Ca2+c levels. The depolarizing effects were significantly reversed by co-application of TTX, demonstrating the involvement of voltage-dependent Na+ channels in the phenomenology. Addition of TTX before veratridine eliminated both depolarizing effects and Ca2+c responses in neuronal progenitors from VZ/SVZ and neurons from CP/SP (not shown). This suggests that the latter were due to activation of voltage-dependent Ca2+ channels.

Collectively, the dual-imaging results of physiological properties expressed by individual immunoidentified cells after 24 h recovery in culture reinforce the flow cytometric findings on acutely prepared, immunolabeled populations in suspension. Furthermore, they demonstrate that depolarizing P2 purinoreceptors are co-expressed with GABAA, glutamate/kainate and muscarinic ACh receptors, voltage-dependent Na+ and L-type Ca2+ channels and Na+-Ca2+ exchange activity, as neuronal progenitors migrate and begin to differentiate in the CP/SP. In contrast, putative neuroglial (SP-A2B5) progenitors in the VZ/SVZ primarily exhibit P2 purinergic receptors coupled to both depolarizing and Ca2+c responses.

How Are the TnTx/A2B5-immunoidentified Subpopulations Related to Each Other?

In order to reveal the lineage relationships among the six subpopulations studied at the end of neurogenesis, we doublelabeled cells with TnTx and A2B5 at the beginning of neurogenesis (E13) and then sorted subpopulations for short-term culture. SP-TnTx , SP-A2B5 and DN cells were sorted to >98% purity, then cultured and photographed before and after a second round of TnTx/A2B5 labeling. After 2 days in culture, the majority (60%) of DN precursors, which accounted for ~40% of the original dissociate (Fig. 1DGo), consistently remained unlabeled, while the rest became either SP-TnTx (23%) or SP-A2B5 (12%), with few (<5%) becoming DP. SP-TnTx neuronal progenitors retained their original surface label, which had become internalized, and, following repeat immunostaining, surface-labeled only with TnTx and not A2B5, indicating that these cells progress along a neuronal lineage(s) without passing through a DP intermediate stage. In contrast, sorted SP-A2B5 cells, which had also internalized their original surface signal, became surface-labeled either with A2B5 (84%), thereby remaining SP-A2B5, or with both A2B5 and TnTx (16%), thus becoming DP. Therefore, these results in vitro indicate that (i) both SP-TnTx and SP-A2B5 cells can originate directly from the DN precursor subpopulation; (ii) SP-TnTx progenitors progressed directly along a neuronal lineage(s) without passing through a DP stage; and (iii) bipotential SP-A2B5 cells either remained SP-A2B5 and differentiated along a glial lineage(s), or became DP and differentiated into putative neuronal phenotypes.

These relationships evolving in vitro complement those apparent in the dot density displays and multimodal frequency distributions (Figs 1 and 2GoGo) derived from flow cytometric analyses of E19 cells, which reflect the relationships emerging in vivo at the end of neurogenesis. Continuities in the distributions of labeling intensities between DN precursors at E19, which only account for ~10% of the total cortical dissociate, and emergent SP-TnTx and SP-A2B5 progenitor subpopulations are consistent with the origins of the labeled cells from unlabeled precursors, mirroring the results obtained in vitro. Further-more, the continuities evident between SP-A2B5 progenitors expressing low-to-moderate labeling and both SP-A2B5 cells with higher levels of staining and DP-TnTxlo neuronal progenitors are consistent with the bipotential characteristics of sorted SP-A2B5 cells discovered in vitro. Continuities in dot density displays indicating probable relationships were also evident among neuronal progenitors in the VZ/SVZ (SP-TnTxlo and DP-TnTxlo), and between neuronal progenitors migrating from the VZ/SVZ to the CP/SP (DP-TnTxlo and DP-TnTxhi, and SP-TnTxlo and SP-TnTxhi). Since, after sorting, SP-TnTxlo neuronal progenitors did not become DP, it is likely that DP-TnTxlo progenitors either become SP-TnTxlo while in the VZ/SVZ or migrate to the CP/SP region, thus becoming DP-TnTxhi. These relationships have been schematized in Figure 10Go, together with a summary of the principal immunophenotypes and their primary physiological properties identified in this study. Changes in epitope expression/co-expression reflect different stages in neuronal and glial lineage progressions evolving among the precursors and progenitors in the VZ/SVZ and after neuronal migration from VZ/SVZ to the CP/SP region.


    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Novel and complementary experimental strategies allowed us to characterize the cellular distribution of physiological properties emerging among precursor, progenitor and differentiating neuronal populations during embryonic rat neocortical development in a relatively comprehensive manner. Diverse patterns of membrane mechanisms evolved as precursors progressed along neuronal and glial lineages and closely paralleled changes in surface epitope expression/co-expression. Dual-imaging of membrane potential and Ca2+c levels in cells cultured for 1–2 days, then epitope-identified post hoc, complemented and extended the FACS findings in dissociates. Together, the results reveal a concerted expression of ionotropic GABAA and glutamate/kainate receptors, muscarinic ACh receptors, voltage-dependent Na+ and L-type Ca2+ channels and Na+–Ca2+ exchange activity among neuronal progenitors migrating from the VZ/SVZ to the CP/SP. In contrast, proliferating precursors and A2B5+ neuroglial progenitors in the VZ/SVZ primarily exhibited P2 purinoreceptors.

Flow Cytometry Complements Dual-imaging of Embryonic Cortical Cell Physiology

Flow cytometry allowed us to quantify individual membrane or cytoplasmic properties expressed among practically all cells within 2–4 h after dissociation, while dual-imaging of both properties revealed the pattern of their co-distribution in adherent cells after recovery and process formation in culture. Immunolabeling of cells before flow cytometry and after dual-imaging allowed precise correlations between the developmental stage and the expressed pattern of physiological properties both at population and single-cell levels. Furthermore, dual-imaging of cells recovered in culture, which had generated or regenerated processes that could have been sheared off during dissociation, allowed us to characterize properties on cell bodies that had differentiated processes.

There was excellent agreement between patterns of physiological properties quantified on cortical cell populations in suspension and those characterized on single cells in culture, demonstrating that the contrasting patterns of emergent properties during lineage progression did not result from the trauma of dissociation, or the absence of sheared-off processes or the recording technique of flow cytometry. The latter strategy is both exceptionally rapid and completely random, thus providing a quick and complete account of near-average values expressed among thousands of individual cells, which are only recorded once. The close correspondence using complementary experimental strategies strongly suggests that the properties emerge in vivo and do not change during the initial 24 h in vitro, as processes are generated or regenerated. Since immunolabeling was carried out after dual-imaging, immunolabeling per se did not generate the contrasting physiological patterns. In addition, dual-imaging revealed patterns of physiological properties emerging at both membrane and cytoplasmic levels. These results verified and extended those obtained with flow cytometry, where only one parameter at a time could be studied. Altogether, the results provide compelling evidence for the contrasting expression patterns of physiological properties emerging in vivo as cortical precursors progress to become progenitors and their differentiating progeny.

TnTx/A2B5 Surface-labeling Patterns Discriminate between Precursors, Neuroglial Progenitors and Differentiating Neurons

The probable and possible relationships among TnTx/A2B5labeled subpopulations outlined in Figure 10Go were derived from the results obtained in vitro with cells sorted at the beginning of neurogenesis (E13), then differentiated in short-term culture, together with results derived both from FACS analyses of cortical dissociates and from imaging/immunocytochemistry of cultured cells at the end of neurogenesis. We conclude that during neurogenesis proliferating embryonic cortical precursors (BrdU+nestin+TnTxA2B5TUJ1 cells) differentiate directly into either neuronal progenitors (SP-TnTxlonestin+TnTx+TUJ1+) or bipotential progenitors (SP-A2B5lonestin+TnTxTUJ1). SPTnTxlo neuronal progenitors migrating from the VZ/SVZ to the CP/SP develop higher levels of TnTx labeling, reflecting higher levels of trisialoganglioside expression, as they become part of the CP/SP, while the SP-A2B5lo bipotential progenitors in the VZ/SVZ either develop higher levels of surface-detectable A2B5 labeling indicative of increased disialoganglioside expression and become SP-A2B5hi glial (oligodendrocyte-type 2 astrocyte) progenitors or begin to express TnTx labeling, thus becoming DP-TnTxlo neuronal progenitors. DP-TnTxlo progenitors appear to be bipotential in that they either migrate to the CP/SP, increase TnTx labeling intensity and become DP-TnTxhi, or they decrease the low levels of surface-detectable A2B5 expression and become SP-TnTxlo before migrating to the CP/SP. The loss of surface-detectable A2B5 expression may reflect the addition of another sialic acid moiety to the A2B5-sensitive disialoganglioside GD3, thereby generating the structurally related TnTx-sensitive trisialoganglioside, GT1b (Hilbig et al., 1981Go).

Flow cytometry of TnTx/A2B5 surface-labeling patterns in cells dissociated throughout the embryonic rat CNS revealed parallel changes in labeling patterns in each region similar to those quantified in the embryonic cortex (Maric et al., 1999Go). Thus, similar surface-labeling patterns emerge in both laminating and non-laminating regions of the developing CNS. Furthermore, CNS regions of other mammalian embryos (mouse, ferret, human) also exhibited similar labeling patterns (results to be published separately). Therefore, surface ganglioside epitope expression patterns can be used during the embryonic period to identify and isolate cells at precursor, progenitor and differentiating stages from different species for detailed cellular and molecular studies.

Contrasting Patterns of Physiological Properties Emerge during Early Cortical Neuron and Neuroglial Lineage Development

The vast majority of embryonic cortical cells at all stages of neurogenesis exhibited K+-dependent resting membrane potentials when studied with potentiometric dyes and flow cytometry (Maric et al., 1998aGo). The ion channel mechanisms underlying K+-dependent resting membrane potentials include both charybdotoxin- and Cs+-sensitive components, whose progressively widespread distribution closely parallels neurogenesis in the embryonic rat cortex (Bulan et al., 1994Go; Maric et al., 1998aGo). Potentiometry of immunoidentified cell populations at the end of cortical neurogenesis revealed that these two components, which reflect functional expression of Ca2+-dependent K+ channels (Bulan et al., 1994Go) and inwardly rectifying K+ channels (Maric et al., 1998aGo), were almost exclusively expressed among neuronal progenitors in the VZ/SVZ and neurons in the CP/SP, but not by unlabeled precursors or A2B5+ progenitors (D. and I. Maric, unpublished observations). These results among cells progressing along neuronal lineages at the end of neurogenesis reinforce the previously observed correlation apparent throughout neurogenesis. The ion channel mechanisms regulating the K+-dependent resting potentials of precursors and A2B5+ progenitors have yet to be elucidated.

Previous electrophysiological studies of individual embryonic cortical cells using patch-clamp recording techniques (LoTurco et al., 1995; Maric et al., 1998aGo) have led to resting potential measurements somewhat less negative (~15–30 mV) than those reported using imaging of calibrated potentiometric fluorescence signals emitted by cortical cells in suspension or in culture (Maric et al., 1998aGo; this study). The less negative potential values are apparent whether whole-cell (Bulan et al., 1994Go; Maric et al., 1998aGo) or perforated-patch configurations of the patch-clamp recording techniques are used (LoTurco et al., 1995), or whether the patched cells are isolated (Maric et al., 1998aGo) or electrically coupled in an acutely prepared slice of the cortex (Bulan et al., 1994Go; LoTurco et al., 1995). The majority of embryonic cortical cells are relatively small in diameter (<=10 µm). It has been well documented that patch-clamp recordings of such small diameter cells are subject to ‘small-cell’ effects, which may underestimate the resting potential to a variable extent depending on the relative proportions of membrane and pipette tip resistances (Barry and Lynch, 1991Go). Furthermore, patch-clamp recordings might also be compromised to an unknown degree by the mechanical force applied to the membrane during the formation of a tight seal. In this regard, embryonic cortical cells exhibit large-conductance Ca2+-dependent K+ channels, which are themselves mechanosensitive (Mienville et al., 1996Go). Thus, mechanical aspects of patching plasma membranes could affect the activity of these K+ channels, which make a critical contribution to the resting potentials of many cortical cells (Maric et al., 1998aGo). In addition, electrophysiological recordings are limited by the relatively small numbers of cells typically studied in a given experiment. Accumulation of sizeable samples requires a number of independent experiments, which could introduce further variability in the results. These clear differences in sampling techniques and in sample sizes may both contribute to the difference between values averaged from electrical and potentiometric measurements.

The expression of functional transmitter receptors and voltage-dependent ion channels varied dramatically among the subpopulations undergoing neuronal or glial lineage progression. Few TnTxA2B5 precursors or SP-A2B5 progenitors exhibited the pattern of physiological properties that was characteristic of differentiating neurons: tetrodotoxin-sensitive, voltage-dependent Na+ and nitrendipine-sensitive L-type Ca2+ channels, bicuculline-sensitive GABAA receptor/Cl channels and CNQX-sensitive glutamate/kainate receptor-coupled cation channels along with muscarinic cholinergic Ca2+c signaling, and Na+-Ca2+ exchange activity. Rather, these cells predominately exhibited functional purinergic receptors coupled to Ca2+c levels, which decreased dramatically in cells progressing along neuronal lineages.

The results using veratridine to survey the developmental expression of voltage-dependent Na+ channels on immunoidentified subpopulations at E19 generally reinforced those regarding changing veratridine pharmacology and ion dependency, which evolved in non-immunoidentified populations during early and late cortical neurogenesis from E11 to E22 (Maric et al., 1998aGo). During the early period of cortical neurogenesis (E11–13), veratridine-induced responses in the majority of cells were insensitive to TTX and the cells typically elevated Ca2+c levels and depolarized to negative membrane potentials in a Ca2+- and Cl-dependent manner. By contrast, during late neurogenesis (E17–20), progressively more cells became depolarized to positive potentials in a tetrodotoxin-sensitive, Na+-dependent manner after treatment with veratridine.

In this study, a detectable number of TnTxA2B5 precursors and SP-A2B5 progenitors from VZ/SVZ depolarized to veratridine, although the depolarizing effects were modest, with the cells becoming polarized at negative potentials (Fig. 4Go). Interestingly, there were more SP-A2B5 progenitors that responded to veratridine with an increase in Ca2+c than were depolarized. This indicates that veratridine stimulated elevation in Ca2+c levels without necessarily depolarizing the cells. Since SP-A2B5 progenitors did not exhibit Ca2+c increases when the cells were depolarized by elevated K+o, they did not express voltage-dependent Ca2+c channels at this stage. Thus, the effects of veratridine to elevate Ca2+c in these progenitors most likely do not involve either depolarization or activation of voltage- dependent Ca2+ channels. This may also be true in the other VZ/SVZ subpopulations, each of which exhibited depolarization to negative potentials and an elevation in Ca2+c level in response to veratridine. The mechanism of this veratridine effect has not been elucidated. However, it has recently been reported that Veratrum alkaloids (cyclopamine) inhibit sonic hedgehog signal transduction mechanisms (Incardona et al., 1998Go), which have been shown to play critical roles in precursor and progenitor cell biology in the CNS (Pringle et al., 1996Go; Hatten, 1999Go; Oppenheim et al., 1999Go; Orentas et al., 1999Go; Rowitch et al., 1999Go). Thus, it is possible that the Ca2+c-elevating effects of veratridine involve engagement of receptors for sonic hedgehog on precursors and progenitors. This remains to be determined.

Previous studies of astrocytes and oligodendrocytes have reported widespread expressions of functional GABAA, glutamate/kainate and muscarinic ACh receptors, and voltage- dependent Na+ and Ca2+ channels [reviewed recently by a number of authors (Gallo and Russell, 1995Go; Porter and McCarthy, 1997Go; Verkhratsky et al., 1998Go)], as well as Na+–Ca2+ exchanger activity (Takuma et al., 1996Go; Golovina et al., 1996Go). Our data show that at E19 most of these properties were either not yet expressed or present in only a fraction (<10%) of the A2B5+ glial progenitors. In contrast, functional purinergic receptors, which have also been reported on developing glial cells (King et al., 1996Go), were widely expressed at this stage. Thus, these results indicate that the expression of purinergic receptors precedes the aforementioned properties, which likely appear at later stages of glial lineage progression(s).

Possible Roles for Emergent Physiological Properties in Morphogenesis of the Cortex

The possible roles of physiological properties described in this study in cortical morphogenesis, which includes cell proliferation, apoptosis, migration and differentiation, remain to be elucidated. In this regard, a physiological role for endogenous GABA in modulating precursor cell proliferation in the VZ/SVZ has been suggested following results obtained in acutely prepared cortical slices (Lo Turco et al., 1995Go; Haydar et al., 1999Go). Acetylcholine acting via muscarinic receptors and phosphorylation of MAP kinase stimulates the proliferation of cortical precursors previously expanded with bFGF (Ma et al., 2000Go). In addition, GABA has been reported to be motogenic in the embryonic rat cortex, stimulating the directed migration of VZ/SVZ neurons (chemotaxis) and the random motility of CP/SP neurons (chemokinesis) via Ca2+c-dependent and pertussis toxin-sensitive mechanisms (Behar et al., 1996Go, 1998Go). The emergence of bicuculline-sensitive depolarization and Ca2+c elevation in response to GABA among neuronal progenitors migrating from the VZ/SVZ to CP/SP may be related to GABA's chemoattractant roles. Pharmacological activation of GABAA receptor/Cl channels arrests motility, while competitive block of their activation with bicuculline promotes motility induced by exogenous GABAmimetics in CP/SP neurons (Behar et al., 1998Go). Preliminary in vitro experiments have also revealed a critical role for GABA acting at GABAA receptor/Cl channels in the process of neurite outgrowth by differentiating CP/SP neurons (results to be published separately).

The emergence of neuronal- and glial-specific properties as precursors turned progenitors targets them for elucidation of their physiological roles. Precursor cells sorted at the beginning of neurogenesis and maintained in culture are currently being investigated in vitro to discover how the contrasting patterns of physiological properties characterized in this study among progenitors are involved in lineage progression. In this regard, we have found significant involvement of muscarinic receptors in precursor and progenitor cell proliferation (D. Maric, unpublished observations). In addition, sorted subpopulations are also being analyzed with molecular techniques (e.g. cDNA microarray plate technology) in order to resolve changes in specific gene expressions occurring in vivo as proliferating precursors progress to progenitor stages. Mapping arrays of genes expressed among developing subpopulations will provide a database for guiding further investigations into the cellular and molecular determinants of commitment and differentiation.


    Notes
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Address correspondence to Dragan Maric, Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.


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