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The Journal of Neuroscience, March 9, 2005, 25(10):2687-2701; doi:10.1523/JNEUROSCI.0951-04.2005

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Cellular/Molecular
Canonical Transient Receptor Potential 1 Plays a Role in Basic Fibroblast Growth Factor (bFGF)/FGF Receptor-1-Induced Ca²+ Entry and Embryonic Rat Neural Stem Cell Proliferation

Alessandra Fiorio Pla,¹ Dragan Maric,¹ So-Ching Brazer,³ Paolo Giacobini,^2,4 Xibao Liu,³ Yoong Hee Chang,¹ Indu S. Ambudkar,³ and Jeffery L. Barker¹

¹Laboratory of Neurophysiology and ²Cellular and Developmental Neurobiology Section, National Institute of Neurological Disorders and Stroke, and ³Secretory and Physiology Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, and ⁴Department of Human and Animal Biology, University of Torino, 10123 Torino, Italy

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Basic fibroblast growth factor (bFGF) and its major receptor FGF receptor-1 (FGFR-1) play an important role in the development of the cortex. The mechanisms underlying the mitogenic role of bFGF/FGFR-1 signaling have not been elucidated. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in proliferating cortical neuroepithelial cells are markedly dependent on Ca²⁺ entry (Maric et al., 2000a). The absence of voltage-dependent Ca²⁺ entry channels, which emerge later, indicates that other membrane mechanisms regulate [Ca²⁺]_i during proliferation. Canonical transient receptor potential (TRPC) family channels are candidates because they are voltage independent and are expressed during CNS development (Strübing et al., 2003).

Here, we investigated the involvement of TRPC1 in bFGF-mediated Ca²⁺ entry and proliferation of embryonic rat neural stem cells (NSCs). Both TRPC1 and FGFR-1 are expressed in the embryonic rat telencephalon and coimmunoprecipitate. Quantitative fluorescence-activated cell sorting analyses of phenotyped telencephalic dissociates show that 80% of NSCs are TRPC1⁺, proliferating, and express FGFR-1. Like NSCs profiled ex vivo, NSC-derived progeny proliferating in vitro coexpress TRPC1 and FGFR1. Antisense knock-down of TRPC1 significantly decreases bFGF-mediated proliferation of NSC progeny, reduces the Ca²⁺ entry component of the Ca_i²⁺ response to bFGF without affecting Ca²⁺ release from intracellular stores or 1-oleoyl-2-acetyl-sn-glycerol-induced Ca²⁺ entry, and significantly blocks an inward cation current evoked by bFGF in proliferating NSCs. Both Ca²⁺ influx evoked by bFGF and NSC proliferation are attenuated by Gd³⁺ and SKF96365 two antagonists of agonist-stimulated Ca²⁺ entry. Together, these results show that TRPC1 contributes to bFGF/FGFR-1-induced Ca²⁺ influx, which is involved in self-renewal of embryonic rat NSCs.

Key words: calcium [Ca]; cortex; development; proliferation; TRPC; neural stem cells

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ca²⁺ is a ubiquitous second messenger, and fluctuations in cellular Ca²⁺ are critical in embryonic development (for review, see Webb and Miller, 2003). The development of the CNS initially involves extensive proliferation of cells in the neuroepithelium as uncommitted precursors divide to self-renew and generate committed progenitors, which in turn divide and differentiate into neural phenotypes. Steady-state levels of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in proliferating neuroepithelial cells in the embryonic rat cortex are markedly dependent on Ca²⁺ entry via unknown mechanisms (Maric et al., 2000a). The complete absence of classical voltage-dependent Ca²⁺ entry channels, which are expressed later during development, indicates that other mechanisms regulate [Ca²⁺]_i during proliferation.

Recently, Ca²⁺ entry channel proteins belonging to the canonical transient receptor potential (TRPC) family, which are voltage-independent channels (for review, see Clapham, 2003), have been identified in the embryonic rat CNS (Strübing et al., 2003). All five TRPC proteins (TRPC1, 3, 4, 5, 6) detected were more highly expressed in the embryonic CNS compared with the adult, suggesting developmental roles for TRPC channels. Heterotetramers composed of TRPC1/TRPC4/TRPC5 and TRPC1/TRPC3/TRPC6 were detected, and a role for TRPC1 expression in the formation of heterotetramers has been proposed (Strübing et al., 2003). Thus, these TRPC channels are candidates for mediating Ca²⁺ entry during proliferation of neuroepithelial cells. Recent studies have shown that TRPC1 plays a role in Ca²⁺ influx and smooth muscle cell proliferation (Golovina et al., 2001; Sweeney et al., 2002a,b) and mediates Ca²⁺ influx activated by basic fibroblast growth factor (bFGF; FGF-2) in endothelial cells (Antoniotti et al., 2002).

bFGF also plays important roles in cortical neuroepithelial cell proliferation in vitro and in vivo (Vaccarino et al., 1995, 1999a,b; Dono et al., 1998; Ortega et al., 1998; Raballo et al., 2000; Korada et al., 2002). A major receptor for bFGF, FGF receptor-1 (FGFR-1) is expressed as early as embryonic day 8.5 (E8.5) to E9.5 in the rat telencephalon, with its expression being relatively confined to the proliferating neuroepithelium (Orr-Urtreger et al., 1991; Vaccarino et al., 1999a,b). In addition, FGFR-1 expression and bFGF-mediated Ca²⁺ signaling have been detected in the majority of neural stem cells (NSC) and their proliferating progeny (Maric et al., 2003). However, the mechanisms underlying bFGF/FGFR-1 signaling in determining the various fates of NSCs including self-renewal and the role of Ca²⁺ in this process have not been studied. Furthermore, continued proliferation of cortical neuroepithelial cells in vitro depends on the presence of bFGF as well as physiological levels of intracellular Ca²⁺ (Ma et al., 2000).

Here, we investigate the role of TRPC1 in NSC proliferation. The results show that TRPC1 and FGFR-1 are coexpressed in the neuroepithelium, coimmunoprecipitate from extracts of telencephalic membrane preparations, and colocalize in NSC-derived proliferating progeny. Antisense knock-down of TRPC1 transcripts and proteins decrease bFGF-mediated Ca²⁺ influx, inward current responses, and proliferation of NSC progeny. Together, these results show that (1) bFGF/FGFR-1-mediated Ca²⁺ influx plays a role in embryonic rat NSC proliferation and (2) TRPC1 channels contribute to bFGF/FGFR-1-mediated Ca²⁺ influx.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Labeling of tissue sections
This research was performed in compliance with the Animal Welfare Act and the U.S. 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.

FGFR1 or TRPC1 and PCNA plus Tuj1 immunostaining. Sprague Dawley rat embryos at 13 d of gestation (E13) were fixed in 4% paraformaldehyde (PF) for 4 h, cryoprotected in 30% sucrose for 3-5 d at 4°C, and frozen in dry ice. Sixteen-micrometer-thick sagittal sections were cut using a Jung Frigocut cryostat (model 2800E; Leica, Nussloch, Germany). The cells were fixed in 70% ethanol for 20 min and immunoreacted with a monoclonal mouse class IgM anti-FGFR-1 antibody (Chemicon, Temecula, CA) or a polyclonal rabbit anti-TRPC1 antibody (Wang et al., 1999) overnight at 4°C. These primary antibodies were visualized with goat anti-mouse IgM or goat anti-rabbit Alexa Fluor 546 antibodies (Molecular Probes, Eugene, OR). The cells were finally immunoreacted with a mouse monoclonal class IgG2a anti-proliferative cell nuclear antigen (PCNA; Chemicon) that labels proliferating cells and mouse monoclonal class IgG2b anti-tubulin III antibody (Tuj1; Sigma, St. Louis, MO), which labels differentiating neurons. These reactions were visualized using goat anti-mouse IgG2a-Alexa Fluor 488 and goat anti-mouse IgG2b-Alexa Fluor 350 antibodies (Molecular Probes), respectively.

Fluorescence microscopy. Phase-contrast and fluorescence signals of cells in culture and tissue sections were imaged using an Axiovert 200 inverted fluorescence microscope (Carl Zeiss, Thornwood, NY) equipped with an Orca-ER cooled digital camera (CCD; Hamamatsu, Hamamatsu City, Japan). The cells were illuminated with a 100 W mercury arc lamp (Carl Zeiss), and the resulting fluorescence emissions were collected through a Plan-Neo 25x/0.8 oil immersion phase 2 objective (Carl Zeiss) using filter sets optimized to detect Alexa Fluor 350, Alexa Fluor 546, Alexa Fluor 488, Alexa Fluor 647, and Alexa Fluor 750 (Omega Optical, Brattleboro, VT). Fluorescence emissions for each fluorochrome signal were captured separately as a 12-bit image, using the video sensor of the Orca-ER camera at a 512 x 480 pixel resolution, and analyzed using Adobe Photoshop software (Adobe Systems, San Jose, CA). In some experiments, fluorescence microscopy was used to quantify the fluorescence intensity of individual cells from 10 fields selected randomly from TRPC1 antisense-treated and random oligonucleotide (ODN)-treated cells, which served as a control. OpenLab software (Improvision, Lexington, MA) was used to acquire and analyze the images. For each set of experiments, identical settings of the OpenLab system were used. The cells were outlined manually, and the mean fluorescence intensity was obtained for the delineated regions. Data are presented as relative fluorescence intensity in arbitrary units. For confocal analyses, cells were imaged with an LSM 510 inverted fluorescence microscope (Carl Zeiss), and the resulting fluorescence emissions were collected through a 63x, 1.4 numerical aperture oil immersion objective (Carl Zeiss). Alexa Fluor 488 (Molecular Probes) fluorescence signals of individual cells were excited by an argon-ion laser at 488 nm, and Alexa Fluor 546 (Molecular Probes) fluorescence signals of individual cells were excited by an argonion laser at 543 nm.

Labeling of cells in suspension
Surface epitope labeling for cell identification. E13 telencephalic cells were identified using lineage-specific surface markers, as described previously (Maric et al., 2003). Briefly, the tissues were optimally dissociated into single-cell suspensions with papain (Maric et al., 1997, 1998a). Neuroglial and oligodendrocyte type 2 astrocyte oligoglial progenitors were immunoidentified using anti-A2B5 (Chemicon) and JONES (Sigma) antibodies and optimally visualized with an appropriate phycoerythrin (PE)-conjugated secondary antibody (Caltag, Burlingame, CA). Neuronal progenitors and differentiating neurons were labeled with cholera toxin B subunit (ChTx; Sigma) and tetanus toxin fragment C (TnTx; Roche Diagnostics, Indianapolis, IN) and visualized using appropriate PE/carbocyanine dye 5 (CY5)-conjugated secondary reagents (Caltag). Annexin V (Trevigen, Gaithersburg, MD), which was visualized with PE/Texas Red (TR)-conjugated secondary reagent, was used as an additional surface marker in conjunction with forward angle light scatter, a property related to cell size, to discriminate among apoptotic, necrotic, and nonapoptotic cells (data not shown) (Maric et al., 2003).

Colabeling of surface-phenotyped cells for expressions of FGFR-1 and PCNA or TRPC1. After surface immunophenotyping (see above), E13 telencephalic cells were then double-fixed first in 4% PF for 20 min at room temperature, followed by 70% ethanol for 20 min at room temperature, and washed in Dulbecco's PBS (Quality Biological, Gaithersburg, MD) supplemented with 1 mg/ml bovine serum albumin (BSA). The cells were immunoreacted with rabbit anti-FGFR-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse IgG2a anti-PCNA antibody (Chemicon) for 1 h at room temperature, and these reactions were then visualized with Alexa Fluor 350-conjugated goat anti-rabbit and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG2a antibodies (Caltag). In another set of experiments, after immunophenotyping and double-fixation (see above), the cells were immunoreacted with rabbit anti-TRPC1 antibody (Wang et al., 1999) and the reaction was visualized with FITC-conjugated donkey anti-rabbit IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA). This multistaining protocol revealed the distribution of FGFR-1 and TRPC1 among the proliferating (PCNA⁺) and nonproliferating (PCNA^-) cells in each phenotyped population.

Quantitative flow cytometric analysis. The fluorescent labeling reactions of cells in suspension were analyzed using a FACSVantage SE flow cytometer (Becton Dickinson, Mountain View, CA) equipped with three lasers, which provide excitation wavelengths tuned to 488 nm, 647 nm, and broad UV (351-364 nm). The FITC and PE, PE/TR, and PE/CY5 fluorescence signals of individual cells were excited by an argon-ion laser at 488 nm, and the resulting fluorescence emissions from each cell were collected using bandpass filters set at 530 ± 30, 575 ± 25, 613 ± 20, and 675 ± 20 nm, respectively. Alexa Fluor 350 was excited using a UV laser, and its emission was collected through 424 ± 22 nm bandpass filter. Cell Quest Acquisition and Analysis software (Becton Dickinson) was used to acquire and quantify the fluorescence signal intensities and to graph the data either as single-parameter histograms or bivariate dot density plots. In multiple labeling experiments, fluorescence emissions of individual fluorophores were corrected for spectral overlap using electronic compensation.

Protein extraction and Western blot analysis
Telencephalic tissue from E13 embryos was dissected and homogenized with a Dounce homogenizer, diluted in lysis buffer (100 mM Tris-HEPES, pH 8.0, and 1 mM MgCl₂) supplemented with protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN), and centrifuged at 30,000 x g for 1 h to obtain the heavy membrane fraction that was frozen and stored at -80°C until further use. Protein concentration was determined using protein assay solution (Bio-Rad, Hercules, CA). Conditions for SDS-PAGE and Western blotting were as described previously (Wang et al., 1999). Polyvinylidene difluoride membranes were blocked and incubated for 1 h with rabbit IgG anti-TRPC1 antibody (Wang et al., 1999) and rabbit IgG anti-FGFR-1 antibody (Santa Cruz Biotechnology). As a control, the membrane fraction was incubated with anti-TRPC1 in the presence of the corresponding antigen peptide (1 mg). The membrane was washed with Tris-buffered saline containing 0.1% Tween 20, incubated as required with HRP-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch), washed, treated with Super Signal West Pico chemiluminescent substrate (Pierce, Rockford, IL), and exposed to X-Omat films (Eastman Kodak, Rochester, NY).

Detergent solubilization of cell membranes and immunoprecipitation
Telencephalic tissue from E13 embryos was dissected and homogenized with a Dounce homogenizer, diluted in sucrose buffer (0.25 M sucrose and 10 mM Tris-HEPES, pH 7.4) supplemented with protease inhibitor mixture (Roche Molecular Biochemicals), and centrifuged at 100,000 x g for 1 h to obtain a microsome fraction. Solubilization and immunoprecipitation were performed with an octyl glucoside (OG) and potassium iodide (KI) solubilized preparation of the E13 telencephalic microsome fraction, as described previously (Lockwich et al., 2001). Briefly, 2 mg of the microsome fraction was washed by dilution into 5-6 ml of a buffer containing (in mM) 200 KCl, 50 K-4-morpholinepropanesulfonic acid (MOPS), pH 7.5, 2.5MgCl₂, and 1 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF), followed by centrifugation at 50,000 x g for 30 min. The washed membranes were solubilized with 1.5% OG (Calbiochem, La Jolla, CA) in 0.96 ml of a medium containing 50 mM K-MOPS, pH 7.5, 20% (v/v) glycerol, 0.5 M KI, 1.5 mM MgCl₂, 1 mM dithiothreitol, 0.5 mM AEBSF, 0.167 mM pepstatin A, 0.167 mM leupeptin, and 10 mg of lipids (Avanti Polar Lipids, Alabaster, AL) from a 50 mg/ml aqueous stock suspension containing 2% (v/v) -mercaptoethanol (Sigma). The lipid stock solution consisted of 60% Escherichia coli ether-washed bulk lipids, 17.5% phosphatidylcholine, 10% phosphatidylserine, and 12.5% cholesterol. The detergent-treated membranes were incubated on ice for 20 min and then centrifuged for 1 h at 145,000 x g. OG and KI solubilized fractions of the E13 telencephalic microsome fraction were incubated with anti-TRPC1 antibody (1:20 dilution) overnight, then pulled down with precleaned protein A-Sepharose CL-4B beads (50 mg/ml; Amersham Biosciences, Piscataway, NJ). The immunoprecipitated proteins were recovered by centrifugation for 1 min at 1000 x g. The beads were washed three times with buffer containing 500 mM NaCl, 10 mM Tris-HCl, pH 7.5, 10% (w/v) sucrose, 1 mM EDTA, 0.2 mM sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride, 0.5% (v/v) Nonidet P-40 (Sigma), 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A. The washed beads were then incubated with 200 µl of SDS-PAGE sample buffer for 5 min at 95°C. The immunoprecipitated proteins (and coimmunoprecipitated proteins) were detected by SDS-PAGE followed by Western blotting, as described above.

Cell sorting
We used E13 telencephalic tissues as a primary source of NSCs for fluorescence-activated cell sorting (FACS) and experimentation in vitro. The methods are, for the most part, based on a recently published strategy (Maric et al., 2003). After dissociation, the cells were then labeled with antibodies against surface markers identifying early neuronal and/or glial progenitors (detailed briefly above) (see Fig. 2 A1). Vital NSCs were then physically purified using a FACSVantage SE flow cytometer (Becton Dickinson) in conjunction with a quintuple epitopenegative selection sorting protocol (Maric et al., 2003). During the initial cell preparation, labeling of surface epitopes, and FACS, the cells were maintained in a normal physiological medium (NPM) supplemented with 1 mg/ml BSA. NPM consisted of the following (in mM): 145 NaCl, 5 KC1, 1.8 CaCl₂, 0.8 MgCl₂, 10 glucose, and 10 HEPES (all from Sigma), with pH and osmolarity adjusted to 7.3 and 290 mOsm, respectively.

View larger version (59K): [in this window] [in a new window]   Figure 2. TRPC1, FGFR-1, and PCNA distributions among phenotyped E13 telencephalic cells and coexpression of TRPC1 transcripts with other TRPCs in NSCs. Dissociates of the E13 rat telencephalon were phenotyped ex vivo using surface markers for neuronal progenitors (ChTx, TnTx), neuroglial progenitors (A2B5, JONES), and apoptotic cells (annexin V) (data not shown), fixed, and immunoreacted to reveal their proliferation (PCNA) status and their expression of either TRPC1 or FGFR-1 (data not shown). The complex distribution of labeling reactions was quantified by FACS and displayed as dot density plots in pseudocolor to reveal the conjoint distributions of fluorescence signals reflecting extracellular and intracellular epitopes and the percentages of cells expressing them. A1, Surface labeling with differentiating markers identifies six cell populations composing E13 telencephalic dissociates that include NSCs, which are devoid of the distinguishing surface markers, neuroglial progenitors (NGP), oligoglial progenitors (OGP), ENPs, and two populations of late neuronal progenitors (LNP, which are ChTx+TnTx+A2B5-JONES-, and quadruple-positive cells, which are ChTx+TnTx+A2B5+JONES+). The boxed regions represent electronic FACS gates used to identify and quantify individual populations. A2-A6, Frequency histograms demonstrate the relative numbers of TRPC1+ cells in the five subpopulations identified in A1. B, Bar plot summarizes the relative abundance of TRPC1+ (gray bars), FGFR-1+ (red bars), and PCNA+ (blue bars) cells in each of the five populations. The percentages of TRPC1+ and PCNA+ cells are greatest among NSCs and decline as cells differentiate along neuronal and neuro-/oligoglial lineages. FGFR-1+ cells are widely distributed throughout the populations. Data are means ± SEM of three independent experiments. C1-C3, Confocal images of three representative NSCs show cellular localizations of TRPC1 (C1) and FGFR-1 (C2) in freshly sorted cells acutely cultured for 1 h. C3, Merge of C1 and C2 shows the colocalization of TRPC1 (red) and FGFR-1 (green) as a yellow signal. Color-coded epitopes are identified in each panel. Scale bar, 10 µm. D, PCR analysis of different TRPC mRNAs in E13 telencephalic dissociates (E13 tel) and in the freshly sorted NSC population (NSC). Equal amounts of RNA were reversed transcribed to generate cDNA. The cDNA was subjected to PCR amplification using paired primers specific for the different TRPCs. The right lane shows the negative control for each paired primer set used (H2O). NSCs clearly express TRPC1-4 and TRPC6, whereas the telencephalic dissociates express these and TRPC5. TRPC7 is barely detectable in NSCs.

 
Cell culture
Sort-purified NSCs were plated at clonal cell densities (5 x 10³ cells/cm²) on poly-D-lysine (Sigma)- and bovine plasma fibronectin (Invitrogen, Frederick, MD)-coated coverslips, which were photo-etched with an alphanumeric grid (Bellco Glass, Vineland, NJ) and preglued to 35 mm tissue culture dishes (MatTek, Ashland, MA). Clones derived from isolated cells were followed over a 7 d period using an Axiovert 200 inverted microscope (Carl Zeiss). The cells were cultured under control conditions in Neurobasal medium (Invitrogen) supplemented with 1x working stock of B27 additives (diluted 50-fold from commercially available stock; Invitrogen) and 10 ng/ml human recombinant bFGF (Intergen, Purchase, NY). Experimental plates of cells were treated with TRPC1 antisense or missense ODNs, or with antagonists of voltage-independent Ca²⁺ channels (SKF96365 Gd³⁺; Sigma), or L-type voltage-dependent Ca²⁺ channels (nitrendipine; Sigma).

Labeling and quantitative analysis of cells in culture
FGFR1 and TRPC1. Cells in culture were double-fixed in PF and ethanol, as described above, and immunoreacted with a polyclonal rabbit IgG anti-TRPC1 antibody (Wang et al., 1999), a mouse monoclonal class IgM anti-FGFR-1 antibody (Chemicon), a mouse monoclonal class IgG1 anti-vimentin antibody (Chemicon), which labels immature precursors and progenitors, and a mouse monoclonal class IgG2a anti-PCNA antibody. The reactions were visualized with goat anti-rabbit IgG-Alexa Fluor 546, goat anti-mouse IgM-Alexa Fluor 488, and goat anti-mouse IgG1-Alexa Fluor 350 antibodies (Molecular Probes), respectively. PCNA immunoreaction was visualized with biotinylated goat anti-mouse IgG2a antibody (Caltag), followed by Alexa Fluor 750-conjugated streptavidin (Molecular Probes).

Multi-epitope staining for proliferation and differentiation. The cells were first surface-immunoreacted with a mouse monoclonal class IgM JONES antibody (Sigma), followed by goat-anti mouse IgM-Alexa Fluor 350 antibody (Molecular Probes), and then sequentially double-fixed in PF and ethanol. The same cells were subsequently probed for lineage-specific cytoskeletal markers, including a mouse monoclonal class IgG1 anti-vimentin antibody (Chemicon), a mouse monoclonal class IgG2b anti-Tuj1 antibody. These immunoreactions were visualized with a biotinylated goat anti-mouse IgG1 antibody (Caltag), followed by Alexa Fluor 750-conjugated streptavidin (Molecular Probes) and a goat anti-mouse IgG2b-Alexa Fluor 647 antibody (Molecular Probes), respectively. The cells were finally probed for nuclear markers, including a proliferation-selective mouse monoclonal class IgG2a anti-PCNA antibody (Chemicon) and a total DNA stain (Sytox Orange; Molecular Probes). PCNA immunoreaction was visualized with goat anti-mouse IgG2a-Alexa Fluor 488 antibody (Molecular Probes). Sytox Orange was used to count the total cell number and to discriminate between viable cells and those undergoing apoptotic (fragmented nuclei) or necrotic (pyknotic nuclei) cell death.

In optimizing the above protocols, we have performed all of the appropriate control experiments to confirm the specificity of each immunoreagent. Control immunoreactions using single-, double-, or triple-staining protocols revealed no significant cross-epitope immunoreactivity among primary or secondary antibodies.

Reverse transcription-PCR
Total RNA was isolated from sort-purified NSC progeny, which had been treated with either TRPC1 antisense or control ODNs (see below) for 7 d in culture, using RNA STAT-60 (Tel-Test, Friendswood, TX) following the manufacturer's protocol. Briefly, cells were homogenized (1 ml of RNA STAT-60/50-100 mg), chloroform was added (0.2 ml/ml homogenate), and the mixture was spun. To the aqueous layer, isopropanol was added (0.5 ml) to precipitate RNA. The RNA pellet was washed (75% ethanol), air dried, and resuspended (DEPC-treated water). One microgram of each sample was used for reverse transcription (RT)-PCR.

First-strand cDNAs were synthesized using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) following the manufacturer's instructions.

PCR was performed to detect the different TRPC channel transcripts and the 60S ribosomal subunit gene, L-19. PCR primer pairs are reported in Table 1. All designed primers were screened using BLAST (Basic Local Alignment Search Tool) to ensure specificity of binding. Primers were used at a concentration of 250 nM.

View this table: [in this window] [in a new window]   Table 1. ODN sequences of primers used for RT-PCR

 
For each reaction, 30.5 µl of nuclease free H₂O, 5 µl of 10x PCR GOLD buffer (Applied Biosystems, Foster City, CA), 4 µl of 25 mM MgCl₂,5 µl of deoxynucleoside triphosphate mix (2.5 mM), and 0.5 µl of Amplitaq Gold (Applied Biosystems) were mixed. Primers (2 µl of each) and template cDNA (2 µl) were added to the mixture. The PCR program was as follows: 10 min 94°C pre-run, 30 s at 94°C, 30 s at 55°C, 2 min at 72°C for 35 cycles, and 10 min 72°C post-run. No products were amplified in water.

Antisense targeting
Phosphorothionate-modified TRPC1 ODNs (5'-TGCTCCTTTGAAGTATATCCTTTA-3') were synthesized and purified by Biognostick (Göttingen, Germany). The control, provided by Biognostik, was a GC-matched randomized-sequence ODN (missense). Cells were grown on glass coverslips for 7 d in the presence of 2 or 5 µM antisense/TRPC1 or control ODNs and then examined for downregulation of TRPC1 transcript expression by RT-PCR, as described above. For analyses of different TRPC protein expressions, single-cell staining immunofluorescence was performed using polyclonal rabbit IgG anti-TRPC1 (Wang et al., 1999), chicken TRPC3 antisera (a kind gift from Dr. C. Montell, Departments of Biological Chemistry and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD), rabbit IgG anti-TRPC4 (Chemicon), and IgG anti-TRPC6 (Sigma) antibodies at a 1:100 dilution factor. The stainings were revealed with goat anti-rabbit IgG-Alexa Fluor 546 (Molecular Probes) and with anti-chicken FITC-conjugated (Jackson ImmunoResearch) antibodies. In other sets of experiments, NSC progeny grown for 7 d in culture in the presence of TRPC1 antisense or control ODN were used for Ca²⁺ imaging, electrophysiological recordings, or multi-epitope staining for proliferation and differentiation, as described above.

Calcium imaging
Changes in [Ca²⁺]_i levels in cultured cells were measured according to methods described previously (Maric et al., 2000b,c, 2003). Briefly, the cells were loaded with fura-2 AM and imaged at 2 s intervals using the Attofluor RatioVision workstation (Atto Bioscience, Rockville, MD). All recording solutions were warmed to 37°C and delivered to the 150 µl recording chamber using gravity-driven perfusion at a constant flow rate of 2 ml/min.

Electrophysiological recordings
For patch-clamp experiments, coverslips of NSCs cultured for 7 d in antisense/TRPC1 or missense were transferred to the recording chamber and kept in a Ringer's solution of the following composition (in mM): 145 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 10 HEPES, and 10 glucose, pH 7.4 (NaOH). The patch pipettes had resistances of 3-5 M after filling with the standard intracellular solution that contained the following (in mM): 145 Cs methane-sulfonate, 8 NaCl, 10 MgCl₂, 10 HEPES, and 10 EGTA, pH 7.2 (CsOH). External solutions were composed as follows (in mM): 145 NaCl, 5 CsCl, 1 MgCl₂, 10 CaCl₂, 10 HEPES, and 10 glucose, pH 7.4 (NaOH). A divalent cation-free (DVF) solution contained the following (in mM): 165 NaCl, 5 CsCl, 10 EDTA, 10 HEPES, and 10 glucose, pH 7.4 (NaOH). An N-methyl-D-glucamine (NMDG) solution contained the following (in mM): 170 NMDG, 5 CsCl, 1 MgCl₂, 10 HEPES, and 10 glucose, pH 7.4 (HCl). The osmolality for all of the solutions was adjusted with mannose to 300-315 mmol/kg using a Vapor Pressure Osmometer (Wescor, Logan, UT).

Patch-clamp experiments were performed in the tight-seal whole-cell configuration at room temperature (22-25°C) using an Axopatch 200B amplifier (Axon Instruments, Union City, CA). The cells were routinely held at 0 mV, and the development of a current in response to bFGF was assessed by measuring the current amplitudes at a potential of -80 mV, taken from high-resolution currents in response to voltage ramps ranging from -90 to 90 mV over a period of 1 s every 4 s and digitized at a rate of 1 kHz. This electrophysiological protocol has been applied previously to study Ca²⁺-permeable cation currents in other cell types (Liu et al., 2004). A liquid-junction potential, which was <8 mV, was not corrected, and capacitative currents and series resistance were determined and minimized. For analysis, the first ramp obtained under baseline conditions was used for leak subtraction of the subsequent current-voltage (I-V) plots.

Statistical analysis
One-way ANOVA was performed to analyze sets of data. Post hoc tests were used to determine statistically significant differences among the groups (Student's t test). Data were considered significantly different if p < 0.05. All data are expressed as means ± SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Differential distributions of FGFR-1 and TRPC1 in the developing telencephalon
Immunohistochemistry was performed to reveal the distributions of FGFR-1 and TRPC1 immunoreactivities in the embryonic telencephalon at the beginning of neurogenesis. FGFR-1 was primarily distributed in cells lining the lateral ventricle (LV), although some signal was also apparent in the neuroepithelium away from the ventricle (Fig. 1A). This is consistent with previous studies (Raballo et al., 2000). Immunostaining with anti-tubulin III antibody (Tuj1), which labels differentiating neurons, showed the distributions of neuronal progenitors and postmitotic neurons in the telencephalon (Fig. 1A). Some of the FGFR-1 immunoreactivity was expressed by cells progressing along the neuronal lineage. Tissue sections were also immunostained for the presence of PCNA to reveal the distribution of proliferating precursors and progenitors. FGFR-1 was detected in PCNA⁺ cells primarily at the interface with the LV (Fig. 1B). These results show that FGFR-1 is widely distributed in the proliferating neuroepithelium. Unlike the broad distribution of FGFR-1, TRPC1 was predominantly confined to the cells lining the LV (Fig. 1C) and was coexpressed with proliferating (PCNA⁺) elements (Fig. 1D). In addition, TRPC1 and FGFR-1 signals were found to be colocalized in individual cells interfacing with the LV (data not shown). Together, these results reveal that FGFR-1 and TRPC1 are coexpressed in proliferating neuroepithelial cells.

View larger version (106K): [in this window] [in a new window]   Figure 1. TRPC1 and FGFR-1 are expressed by proliferating neuroepithelial cells in the E13 rat telencephalon. A, B, After fixation, sagittal sections of the E13 brain were immunostained for FGFR-1 (red), Tuj1 (blue), and PCNA (green). FGFR-1 is present throughout the neuroepithelium and is coexpressed with proliferating (PCNA+) cells along the LV. C, D, E13 sections were fixed and stained for TRPC1 (red), Tuj1 (blue), and PCNA (green). TRPC1 expression is more restricted to cells near the LV and, like FGFR-1, is coexpressed in PCNA+ cells lining the LV surface. The insets are magnified regions of the boxed areas. Scale bar, 10 µm.

 
Distributions of TRPC1, FGFR-1, and PCNA expressions among phenotyped telencephalic cells
We used multi-epitope immunophenotyping of telencephalic dissociates in conjunction with FACS to quantify the distributions of TRPC1 and FGFR-1 among proliferative (PCNA⁺) NSCs and committed neuronal and neuro-/oligoglial progenitors. NSCs were identified by their lack of four surface ganglioside markers (ChTx^-TnTx^-A2B5^-JONES^-), which emerge as NSCs differentiate into progenitor phenotypes, whereas different progenitors were identified by their specific patterns of ganglioside expression (Fig. 2A1) (Maric et al., 2003). Each subpopulation was further divided into vital (nonapoptotic) and apoptotic cells by their expression of annexin V binding to phosphatidylserine residues at the cell surface (data not shown). The results show that the great majority (80%) of NSCs expressed TRPC1 (Fig. 2A2), which progressively decreased in abundance during both neuronal and neuro-/oligolgial lineage progressions (Fig. 2A3-A6). Oligolgial progenitors were virtually devoid of TRPC1⁺ cells, whereas a minority of "late" or well advanced neuronal progenitors expressed TRPC1 (Fig. 2A3-A6,B). Most TRPC1⁺ NSCs coexpressed PCNA (Fig. 2B), consistent with the coexpression of TRPC1 in PCNA⁺ cells in vivo (Fig. 1D). Both TRPC1 and PCNA expression proportionately decreased as NSCs progressed along the neuronal lineage. About one-half of the newly committed neuronal progenitors were TRPC1⁺ PCNA⁺, whereas 15-20% of the more differentiated progenitors were double positive (Fig. 2B). Both TRPC1⁺ and PCNA⁺ cells also decreased during neuro-/oligoglial lineage progression (Fig. 2B). The coexpression of TRPC1 and PCNA among NSCs and neuroglial and neuronal progenitor cells demonstrate that the channel is specifically present in cells that are actively proliferating rather than in cells that are preterminally or terminally postmitotic.

Immunophenotyped populations were also probed for FGFR-1 expression in the context of PCNA staining. FGFR-1 and PCNA signals were highly coexpressed in NSCs (data not shown) (results plotted in Fig. 2B). Thus, NSCs actively proliferating in vivo express both TRPC1 and FGFR-1. However, whereas TRPC1⁺PCNA⁺ cells decreased during cell lineage progression, FGFR-1 immunoreactivity could be detected in the great majority of differentiating cells. This reveals that FGFR-1 persists in preterminally and terminally postmitotic cells.

Confocal microscopy was used to resolve the localization of TRPC1 and FGFR1 in single cells. NSCs, sorted as described above, were plated and allowed to attach for 1 h. Cells were then fixed in 4% PF and stained for FGFR-1 and TRPC1. No detergent was used to permeabilize the cells. The data show that TRPC1 and FGFR-1 were expressed in the plasma membrane region, as well as intracellularly (Fig. 2C1,C2). A similar intracellular localization for TRPC1 has been described previously in human salivary gland (HSG) cells (Brazer et al., 2003). Notably, in another study, a considerable amount of TRPC1 appeared to be localized intracellularly in hippocampal neurons, and the presence of TRPC1 in the surface membrane fraction was detected by biotinylation (Bezzerides et al., 2004). In NSCs, colocalization of TRPC1 and FGFR-1 can be detected as a yellow fluorescence signal in the cytoplasmic and plasma membrane regions of single cells (Fig. 2C3 shows overlays of the green and red fluorescence signals).

TRPC1 transcripts are expressed by NSCs
We used PCR to detect the expression of TRPC1 mRNA as well as those of six other TRPCs in dissociates of the telencephalon and in freshly sorted NSCs (Fig. 2D). TRPC1-6 transcripts were all present in dissociates of the E13 telencephalon, although TRPC4 mRNA was barely detectable. In contrast, TRPC7 was not detectable in dissociates of the E13 telencephalon, whereas a very faint band was present in the freshly sorted NSC population. TRPC1-4 and TRPC6 mRNAs were all present in the NSC population. Thus, TRPC4 seemed to be more enriched in the NSC fraction compared with that detected in the E13 telencephalic dissociate. Interestingly, TRPC5 transcripts were present in the E13 telencephalic dissociate but not in the NSC population. The TRPC5 band was instead found in the FACS-sorted early neuronal progenitor (ENP) population (data not shown). Thus, TRPC1 mRNA is well expressed in NSCs along with four other TRPCs.

TRPC1 and FGFR-1 coimmunoprecipitate in telencephalic membranes
Predominant coexpression of TRPC1 and FGFR-1 among proliferating NSCs led us to investigate possible interactions between them. A band of 92 kDa corresponding to TRPC1 protein was detected in Western blots of telencephalic plasma membranes (Fig. 3A, lanes 1-3). HSG cells, which are known to express TRPC1, were used as a positive control (Fig. 3A, lane 4). The lower band (52 kDa) that was detected in the crude membrane fraction of HSG cells has been described previously (Wang et al., 1999; Liu et al., 2000). Interestingly, this band was not detected in crude membranes prepared from the E13 rat telencephalon. FGFR-1 was also detected in the same membrane fraction as an 120 kDa band, which represents the nonglycosylated form of the receptor, in accordance with the literature (Reilly et al., 2000; Kilkenny et al., 2003). Figure 3C shows the specificity of the anti-TRPC1 antibody. Detection of TRPC1 (Fig. 3C, lane 1) was completely blocked by incubation of anti-TRPC1 with the antigenic peptide (Fig. 3C, lane 2). To determine whether TRPC1 and FGFR-1 associate in vivo, we examined whether FGFR-1 coimmunoprecipitated with TRPC1 by using the TRPC1 antibody (Fig. 3D). Both proteins were detected in the total lysate and the solubilized fraction of the crude microsomal fraction (Fig. 3D, lanes 1, 2). FGFR-1 was highly enriched in the immunoprecipitate as well as TRPC1 (Fig. 3D, lane 3). Taken together, these data demonstrate that both TRPC1 and FGFR-1 are present in telencephalic membranes and can be immunoprecipitated as a complex, indicating that they are tightly associated with each other.

View larger version (71K): [in this window] [in a new window]   Figure 3. TRPC1 and FGFR-1 coimmunoprecipitate in membranes prepared from the E13 rat telencephalon. A, B, Membrane fractions obtained from the E13 rat telencephalon were probed on Western blots with anti-TRPC1-specific (A) and anti-FGFR-1-specific (B) antibodies. A, Lanes 1-3 were loaded with different amounts of proteins (lane 1, 75 µg; lane 2, 50 µg; lane 3, 25 µg). HSG membrane fractions were used as a positive control (lane 4). Bands at the expected molecular weight (92 kDa) are evident in all lanes. C, Reactivity to anti-TRPC1 was detected in the microsomal fraction obtained from the E13 telencephalon (arrowhead; lane 1). Reactivity was blocked by preincubation of antibody with peptide (lane 2). D, Immunoprecipitation of TRPC1 and FGFR-1 in the microsomal fraction. Detections of TRPC1 and FGFR-1 in the total lysate (lane 1), the solubilized fraction (lane 2), and in immunoprecipitates (lane 3) using the anti-TRPC1 antibody (Western blots using either anti-FGFR-1 or anti-TRPC1 antibodies) are shown. TRPC1 and FGFR-1 proteins are indicated by arrowheads.

 
TRPC1 and FGFR-1 colocalize in proliferating NSC-derived progeny in vitro
We used immunophenotyping and flow cytometry to isolate NSCs by negative selection (Maric et al., 2003) to study the role of TRPC1 channels in NSC proliferation and self-renewal without differentiation, which can be sustained in defined serum-free medium containing bFGF. After 7 d in culture, NSC-derived progeny remained morphologically immature, resembling the initial NSC founders. Multi-epitope immunostaining revealed that the majority of the progeny were actively proliferating (91% PCNA⁺), and virtually all were immature precursors (99% vimentin⁺) (Fig. 4A1) devoid of differentiating epitopes (data not shown), confirming previous results (Maric et al., 2003). The great majority (84%) of proliferating progeny coexpressed TRPC1 and FGFR-1 (Figs. 4A2-A4, 5C), whereas few were either single-positive TRPC1⁺ (9%) or FGFR-1⁺ (6%) and only 1% were TRPC1^-FGFR-1^-. Most PCNA⁺ cells were TRPC1⁺ (79%) or FGFR-1⁺ (81%) (Fig. 4C). These widespread coexpressions resulted in the majority of immature NSC-derived progeny (77%) being TRPC1⁺ FGFR-1⁺ PCNA⁺ (Fig. 4C). Therefore, TRPC1 and FGFR-1 were coexpressed in most NSC-derived progeny proliferating in vitro in the presence of bFGF.

View larger version (48K): [in this window] [in a new window]   Figure 4. NSC-derived progeny coexpress TRPC1 and FGFR-1, which colocalize in the plasma membrane. NSCs were expanded for 7 d under self-renewing conditions (Neurobasal/B27 supplemented with 10 ng/ml bFGF), then their progeny were fixed and stained for TRPC1 and FGFR-1 in conjunction with PCNA and vimentin to reveal their proliferative (PCNA+) and undifferentiated (vimentin+) state. A1, The merged image of PCNA and vimentin immunostaining demonstrates the proliferative and undifferentiated state of the NSC-derived progeny. A2, A3, Epifluorescence images of a representative field show that the great majority of the NSC-derived progeny express TRPC1 (A2) and FGFR-1 (A3). A4, The merged image of both fluorescence signals reveals extensive colocalization of TRPC1 and FGFR-1. B1, B2, Confocal images show cellular localizations of TRPC1 (B1) and FGFR-1 (B2). B3, Enlargement of the boxed field in B2 reveals widespread membrane colocalization of TRPC1 and FGFR-1 immunoreactions. C, Bar plot summarizes the quantitative analysis of 10 different fields. TRPC1 and FGFR-1 are coexpressed with PCNA and with each other in the majority of the NSC-derived progeny (75-80%). Color-coded epitopes are identified in each panel.

 

View larger version (37K): [in this window] [in a new window]   Figure 5. TRPC1 antisense treatment of NSC-derived progeny decreases TRPC1 transcripts and protein expression. Phase contrast (A1) and epifluorescence (A2) images of a representative field of NSC-derived progeny show that FITC-labeled ODNs are taken up by virtually all progeny after 7 d in culture. B, Analysis of TRPC1 mRNA levels reveals clear effects of NSCs treated with control (Cntrl) and TRPC1 antisense (AS) ODNs (5 µM). Equal amounts of RNA were reversed transcribed to generate cDNA. The cDNA was subjected to TRPC1-specific PCR amplification (500 bp product) using paired primers. cDNA from all samples was also subjected to L-19-specific PCR amplification (195 bp product). The left lane shows a ladder of molecular weight markers (M). Note the dramatic reduction of TRPC1 mRNA in NSC-derived progeny treated for 7 d with antisense ODNs compared with treatment with control ODNs. C1-C4, Representative images of antisense/TRPC1-treated cells (AS) and missense-treated cells (CNTRL) stained for TRPC1 or TRPC6. The data show a clear difference in TRPC1 expression between CNTRL and AS, whereas no difference is evident in TRPC6 expression. D, Fluorescence intensities of TRPC1, TRPC3, TRPC4, and TRPC6 protein expressions in NSC-derived progeny shows a significant (**p<0.005) reduction in progeny treated with 5 µM antisense TRPC1 (AS) compared with treatment with equimolar control ODNs (C) for TRPC1 but not for the other TRPCs studied. Scale bars, 10 µm

 
Confocal microscopy was used to resolve the localization of TRPC1 and FGFR1 in single cells. TRPC1 and FGFR-1 signals were localized in the plasma membrane and subplasma membrane regions, as well as in the cytoplasm of cells (Fig. 4B1,B2). Subcellular colocalization of TRPC1 and FGFR-1 in individual cells was reflected by the presence of intricate yellow fluorescence signals corresponding to the merge of the individual green and red immunoreactivities (Fig. 4B3).

Collectively, the data indicate that, similar to NSCs profiled ex vivo, TRPC1 and FGFR-1 are coexpressed in the majority of the actively proliferating NSC-derived progeny, where they are colocalized in both cytoplasmic and membrane regions.

Antisense/TRPC1 treatment attenuates TRPC1 expression but not other TRPCs in proliferating NSC-derived progeny
NSCs were plated at clonal density and incubated with either antisense/TRPC1 ODN or missense ODNs over a 7 d period. FITC-labeled ODNs were taken up by the majority of proliferating progeny as reflected by the green fluorescence signals associated with many of the cells (Fig. 5A2). RT-PCR showed that treatment with antisense ODN markedly reduced the presence of TRPC1 mRNA, whereas treatment with missense did not (Fig. 5B). Treatment with the antisense/TRPC1 ODN did not affect the transcript abundance of a housekeeping gene (L-19), demonstrating that the lack of TRPC1 transcripts was not attributable to improper amplification (Fig. 5B). Quantitation of TRPC1 immunofluorescence signal intensities in fields of NSC-derived progeny revealed that antisense/TRPC1 significantly reduced the intensity of the fluorescence signals compared with missense treatment (Fig. 5C1,C2,D). To test whether antisense/TRPC1 treatment was specific for TRPC1 protein expression, we analyzed the effect of antisense/TRPC1 ODN on other TRPC expressions. As an example, Figure 5, C3 and C4, shows that TRPC6 expression was not affected by antisense/TRPC1 treatment compared with missense-treated cells. Quantitation of immunofluorescence signal intensities revealed that TRPC3, TRPC4, or TRPC6 was not affected by antisense/TRPC1 treatment (Fig. 5D). In addition, RT-PCR data show that TRPC2 levels did not change in antisense/TRPC1-treated cells compared with missense-treated cells (data not shown).

These results establish that antisense/TRPC1 effectively knocks down TRPC1 transcripts as well as proteins without affecting the expressions of other TRPCs expressed by NSC-derived progeny.

Antisense/TRPC1 treatment attenuates NSC proliferation
To test whether antisense treatment affected proliferation, NSCs were plated at clonal density and allowed to expand under selfrenewing conditions in defined medium with bFGF for 7 d. Two concentrations of antisense/TRPC1 ODNs were used (2 and 5 µM). Both concentrations significantly reduced the total number of cells per clone compared with missense treatments and decreased the percentage of proliferating (PCNA⁺) cells per clone (Fig. 6A,B). There was not a significant difference (p > 0.05%) in the percentage of total cell death (because of either pyknosis or apoptosis) between the antisense/TRPC1-treated cells (4.9 ± 1.1 and 5.2 ± 1.5%, respectively, at 2 and 5 µM) and missense-treated cells (3.1 ± 0.7 and 2.5 ± 0.6%, respectively, at 2 and 5 µM), indicating that cell death did not account for the decrease in clone size. The results indicate that knock-down of TRPC1 channel proteins attenuates NSC proliferation without causing significant cell death. Furthermore, the knock-down of TRPC1 proteins did not induce NSCs to differentiate.

View larger version (19K): [in this window] [in a new window]   Figure 6. NSC proliferation is inhibited by antisense/TRPC1 and antagonists of voltage-independent Ca2+ entry. NSCs were plated at clonal density and cultured for 7 d under different conditions. After multi-epitope staining with lineage-specific markers, 10 clones from three independent experiments were quantified to reveal the total cell number per clone (A, C, D) and the percentage of proliferative cells per clone (B, E, F). A, B, NSC progeny were grown for 7 d in the presence of antisense/TRPC1 and control ODNs to test for a role of TRPC1 in bFGF-mediated NSC proliferation. Both 2 and 5 µM antisense TRPC1 (AS) induce a significant reduction in the number of cells per clone as well as in the percentage of proliferative cells compared with treatment with control ODNs (CNTRL). C-F, NSC progeny were grown for 7 d under either self-renewing conditions (CNTRL) or in the presence of organic (SKF96365 1 µM) or inorganic (Gd3+; 1 µM) antagonists of voltage-independent Ca2+ channels or an organic blocker of L-type Ca2+ channels [nitrendipine (NTDP); 10 µM]. Both SKF69365and Gd3+ significantly reduce clone size and the percentage of proliferative cells in clones. Nitrendipine is ineffective (*p < 0.01; **p < 0.005).

 
Because TRPC1 channel proteins comprise voltage-independent Ca²⁺ channels, we compared the effects of an inorganic (Gd³⁺) and an organic (SKF96365 (Beech et al., 2003) antagonist of Ca²⁺ entry, which are commonly used to block voltage-independent Ca²⁺ channels, and an antagonist at L-type voltage-dependent Ca²⁺ entry (nitrendipine) on NSC proliferation. Both Gd³⁺ (1 µM) and SKF96365(1 µM) significantly reduced clone size and the percentage of proliferative cells (Fig. 6C-F). In contrast, nitrendipine did not affect either of these parameters (Fig. 6C,E). Furthermore, in the presence of Gd³⁺ or SKF96365 the percentages of total cell death (10.5 ± 2% for Gd³⁺ and 8.9 ± 1.5% for SKF96365 were not significantly different (p > 0.01) compared with controls (3.8 ± 1.9 and 5.8 ± 0.7%, respectively, for Gd³⁺ and SKF96365. These results indicate that the effects of the drugs were not simply attributable to toxic actions and that cell death did not account for the decrease in clone size. Thus, different blockers of receptor-operated Ca²⁺ entry channels mimic the effect of antisense treatment, whereas antagonism of a major voltage-dependent pathway does not. These results are consistent with a role for voltage-independent Ca²⁺ channels including TRPC1 in sustaining NSC proliferation.

Antisense/TRPC1 treatment attenuates bFGF-induced Ca_i²⁺ responses
NSC-derived progeny proliferating in bFGF respond to bFGF with elevations in [Ca²⁺]_i containing an initial peak and a later sustained phase (Maric et al., 2003). To test whether antisense/TRPC1 treatment affected regulation of [Ca²⁺]_i by bFGF, we performed imaging experiments in cells treated with missense or antisense/TRPC1 ODN for 7 d. Peak and sustained Ca_i²⁺ responses to bFGF were readily detected in proliferating NSC-derived progeny treated with missense (Fig. 7A1). In contrast, there was a modest decrease in the peak amplitude of the bFGF-induced Ca_i²⁺ signal and a dramatic decrease in the amplitude of the sustained phase after treatment with antisense/TRPC1 ODN (Fig. 7A2). Statistical analysis of the results showed that the reduction in peak amplitude was not significant (Fig. 7B1), whereas the decline in the sustained phase was significant (Fig. 7B2).

View larger version (24K): [in this window] [in a new window]   Figure 7. bFGF-mediated Ca2+ entry is inhibited by antisense/TRPC1 treatment. NSCs were cultured in the presence of antisense/TRPC1 and missense ODNs; after 7 d, the cells were loaded with fura-2 and then imaged to detect bFGF-induced Cai2+ signals. A, Two representative traces show bFGF-mediated Cai2+ signals in cells treated with missense ODN (CNTRL) (A1) and TRPC1 antisense (AS) (A2). B1, B2, The amplitudes of the initial transient peak and the sustained phase were quantified, and the results have been summarized in bar plots. There is no statistically significant difference in the amplitudes of the initial transient peak (B1), whereas there is a significant reduction in the sustained phase (*p < 0.005) after antisense/TRPC1 treatment compared with control ODN. C1-C3, bFGF (10 ng/ml) triggers a self-limiting Cai2+ transient in Ca2+-free medium, reflecting exhaustive Ca2+ release from intracellular stores, which is virtually identical in cells treated with either missense ODN (C1) or TRPC1 antisense (C2). C3, Quantitative summary of the results in bar plot format. D1, D2, A concentration of 25 µM OAG applied to cells treated with missense ODN (D1) or TRPC1 antisense (D2) evokes similar Cai2+ responses. D3, Bar plot of the quantified peak amplitudes of the OAG-induced Cai2+ signals show no significant difference between OAG-induced amplitudes evoked in cells treated with TRPC1 antisense compared with control ODNs. In A1, A2, C1, C2, D1, D2, the experimental logs are shown above the Ca2+ traces, and the dashed lines at the bottom indicate baselines. Double-ended arrows in A1 and A2 indicate the sustained phase amplitude. The numbers inside the bars indicate the total number of cells analyzed.

 
Additionally, we compared bFGF-induced Ca_i²⁺ signals in Ca²⁺-free medium to exclude Ca²⁺ entry. Transient Ca_i²⁺ responses to bFGF, reflecting Ca²⁺ release from intracellular stores, were virtually identical in cells treated with missense and antisense/TRPC1 ODNs (Fig. 7C1-C3). Thus, downregulation of TRPC1 mRNA and proteins did not affect bFGF/FGFR-1 signaling that triggers Ca²⁺ release. Rather, the external Ca²⁺-dependent contribution to the response (i.e., Ca²⁺ entry) is attenuated. To show that Ca²⁺ entry can also be triggered independently of bFGF, we exposed cells treated with missense or antisense/TRPC1 ODN to the membrane-permeant analog of diacylglycerol (DAG), 1-oleoyl-2-acetyl-sn-glycerol (OAG), which is thought to activate Ca²⁺ entry via TRPC channels not composed of TRPC1 subunits (for review, see Clapham, 2003). OAG evoked Ca_i²⁺ responses that were not significantly different in amplitude and time course in missense- and antisense/TRPC1-treated cells (Fig. 7D1-D3). Together, these results demonstrate that antisense/TRPC1 treatment affects the Ca²⁺ entry components of the Ca_i²⁺ response to bFGF without apparently affecting Ca²⁺ release. The lack of effect of antisense treatment on OAG-induced Ca_i²⁺ responses suggests that other voltage-independent Ca²⁺ channels putatively containing DAG-sensitive TRPC channel proteins are not affected.

The effect of antisense/TRPC1 treatment on the biphasic peak-and-plateau Ca_i²⁺ response to bFGF in the absence of any effects on bFGF-evoked Ca²⁺ signals in Ca_i²⁺-free medium, which involve Ca²⁺ release, led us to study agents known to block Ca²⁺ entry in other cell types. Both SKF96365(Fig. 8A2) and Gd³⁺ (Fig. 8A3) simplified the peak-and-plateau Ca_i²⁺ response to bFGF (Fig. 8A1) into one dominated by only a transient phase. This would be expected if both agents blocked the Ca²⁺ entry component but not the Ca²⁺ release component. The Ca_i²⁺ transients illustrated in Figure 8, A2 and A3, closely resemble those shown in Figure 7, C1 and C2, which demonstrate bFGF-induced Ca_i²⁺ transients in the absence of extracellular Ca²⁺. Not only was the sustained phase of the biphasic response to bFGF mostly eliminated by SKF96365and Gd³⁺, but the peak amplitudes of the Ca_i²⁺ responses were significantly reduced (Fig. 8A4). This shows that Ca²⁺ entry also contributes to the initial peak of the Ca_i²⁺ response to bFGF. A clear and partly reversible depression of the sustained phase was readily apparent with both Gd³⁺ (Fig. 8B1) and SKF96365(data not shown). Both of these effects were significant (Fig. 8B2). Finally, nitrendipine did not significantly affect the peak-and-plateau Ca_i²⁺ response to bFGF (Fig. 8C1,C2), demonstrating that voltage-dependent Ca²⁺ channels are not involved.

View larger version (22K): [in this window] [in a new window]   Figure 8. bFGF-mediated Ca2+ entry is blocked by antagonists of voltage-independent Ca2+ channels. NSC-derived progeny were imaged using methods outlined in the Figure 7 legend. Experimental conditions are logged above the corresponding Cai2+ traces (A1-A3, B1, C1), which are representative of the results (summarized in A4, B2, and C2). The numbers (N) at the ends of the traces indicate the total number of cells analyzed. The dashed lines depict the baselines of individual cells and in B1 and C1, the trajectory of the sustained phase of the Cai2+ response during prolonged exposure to bFGF. A1, bFGF (10 ng/ml) triggers Cai2+ signals composed of a transient and a sustained phase. A2, A3, A significant decrease in the initial peak amplitude of the Ca2+ signal and elimination of the sustained phase is observed in the presence of 1 µM SKF69365or 1 µM Gd3+ compared with the control. A4, Bar plot summarizes the significant decreases in peak amplitudes compared with control (CNTRL). B1, Prolonged exposure to 10 ng/ml bFGF sustains Cai2+ levels, which are blocked by 1 µM Gd3+ in a partially reversible manner. B2, Bar plot summarizes the effects of Gd3+ and SKF69365on the sustained phase (*p < 0.005). Double-ended arrows in B1 indicate the sustained phase amplitude before and during the applications of Gd3+. C1, C2, Nitrendipine (NTDP; 10 µM) has no significant effect on the bFGF-induced Cai2+ response. C2, Bar plot summarizes the results and shows no significant difference.

 
Based on these data, we suggest that a significant part of the biphasic Ca_i²⁺ response to bFGF involves Ca²⁺ entry mediated by voltage-independent Ca²⁺ channels. Our data showing that antisense/TRPC1 treatment attenuates this Ca²⁺ entry are consistent with this suggestion and indicate that TRPC1 channels mediate, at least in part, the Ca²⁺ influx induced by bFGF/FGFR-1 signaling.

Antisense/TRPC1 treatment depresses bFGF-induced current responses and conductance changes
The presence of a Ca²⁺ entry component throughout the biphasic Ca²⁺ response to bFGF and its sensitivity to both antisense and pharmacological treatments led us to use patch-clamp techniques to measure the currents stimulated by bFGF. Proliferating NSC-derived progeny were washed in bFGF-free saline solution and then clamped in the whole-cell mode to record cation currents in response to brief applications of bFGF. Cells were held at 0 mV and subjected to voltage ramps from -90 to +90 mV every 4 s. The traces shown in Fig. 9, A-C and E, represent bFGF-stimulated inward currents that have been reconstructed from the current amplitudes at -80 mV obtained from current responses to the intermittently imposed 1 s voltage ramps. The latter were used to construct I-V curves (Fig. 9D,F). We have illustrated bFGF-induced currents stimulated at -80 mV because this membrane potential is in the range previously reported for intact neuroepithelial cells studied with a calibrated potentiometric dye strategy and flow cytometry (Maric et al., 1998b) and because the current responses were readily detectable at this potential. bFGF triggered inwardly directed currents in all of the cells recorded, consistent with there being a quite widespread distribution of bFGF-induced Ca²⁺ responses among these cells. Superfusion of either Gd³⁺ (Fig. 9A) (n = 3 cells) or SKF96365(Fig. 9B) (n = 3 cells) completely inhibited the bFGF-induced inward currents, which returned to baseline levels in all cells tested. The results are consistent with bFGF activation of voltage-independent cation conductance. From previous studies of mechanisms regulating [Ca²⁺]_i in neuroepithelial cells at the beginning of neurogenesis, we found no evidence of voltage-dependent Ca²⁺ entry contributing to [Ca²⁺]_i at this stage of development (Maric et al., 2000a). In addition, nitrendipine did not affect the peak-and-sustained Ca_i²⁺ responses to bFGF (Fig. 8C1,C2). Perfusion of DVF medium during the bFGF-induced current response transiently increased the amplitude of the inwardly directed current (Fig. 9C), which then relaxed to baseline levels (n = 4 cells). The current relaxation in the cell illustrated began before exposure to the DVF solution, which accelerated the decline. The transient increase in current is consistent with the nonspecific cation selectivity of voltage-independent channels. I-V curves under control and perfusion with DVF conditions show that, in the latter, the reversal potential of the I-V curve shifts slightly in the negative direction but still remains positive (approximately +5mV). In addition, the slope of the I-V curve increased, reflecting a transient increase in the membrane conductance in DVF saline in response to bFGF. These results are consistent with the well known unblocking effect of DVF solutions on voltage-independent channels with a relatively broad selectivity for cations. In the absence of divalent cations, ambient monovalent cations (Na⁺ and K⁺) are conducted more easily, thus generating more inward current at negative potentials and leading to a reversal potential nearer to 0 mV. These results led us to test the effect of exposure to a saline in which all the extracellular Na⁺ and Ca²⁺ ions were replaced by NMDG. Switching from normal to NMDG saline rapidly eliminated the bFGF-induced current response (Fig. 9E) and associated conductance (Fig. 9F) in a completely reversible manner (n = 3 cells). Thus, bFGF activates inward currents at negative potentials, which reverse polarity at approximately +15 mV and are completely blocked by both Gd³⁺ and SKF96365 and by either exposure to a DVF solution or to an Na⁺- and Ca²⁺-free saline. These characteristics are consistent with the activation of bFGF of voltage-independent cation channels with relatively little selectivity over monovalent and divalent cations.

View larger version (17K): [in this window] [in a new window]   Figure 9. Pharmacological block of bFGF-induced currents and their sensitivity to DVF and Na+/Ca2+-free saline. NSC-derived progeny were expanded in medium with 10 ng/ml bFGF and then rinsed in bFGF-free saline before patch-clamp recordings were performed in the whole-cell mode. The cells were clamped at 0 mV, and 1 s voltage ramps over the -90 to +90 mV range were applied every 4 s. Current traces at -80 mV before and during perfusion of bFGF in different salines were reconstructed from the protocol. A, Perfusion of 10 ng/ml bFGF triggers an inwardly directed current that peaks in 60 s. Coapplication of 1 µM Gd3+ rapidly and completely reduces the current response, which does not recover after stopping the Gd3+ perfusion. B, Coapplication of 1 µM SKF96365(SKF) rapidly blocks the bFGF-induced current response, which does not recover during the recording period. C, bFGF induces an inward current (I), which transiently increases in amplitude (II) when DVF saline is perfused instead of normal saline containing Ca2+ and Mg2+ before relaxing to baseline. D, I-V plot of bFGF-induced currents in normal saline containing Ca2+ and Mg2+ (I) and in DVF (II) demonstrate relatively linear plots with similar reversal potentials (+15 mV in normal saline, +5 mV in DVF). The slope of the I-V plot increases in DVF, reflecting an increase in conductance. E, Perfusion of NMDG solution, which does not contain either Na+ or Ca2+, rapidly and completely blocks the bFGF-induced current response in a reversible manner. F, The I-V plot demonstrates the reversibility in the underlying bFGF-induced conductance. The traces shown are representative of three cells in Gd3+ and SKF96365experiments, four cells in the DVF experiments, and three cells in the NMDG experiments.

 
In missense-treated cells, the current activated by bFGF increased, reached a maximum in 40-60 s and remained sustained for 40-60 s after bFGF was washed out, then gradually declined (Fig. 10A1), similar to responses recorded under control conditions (Fig. 9). In antisense/TRPC1-treated cells, the current response was lower in amplitude (1083 ± 27 pA in six missense-treated cells, 358 ± 104 pA in eight antisense/TRPC1-treated cells) and declined quickly after removal of bFGF (Fig. 10A1). There was a significant decrease in the bFGF-activated current density measured at the peak of the current response in antisense/TRPC1-treated cells compared with control cells (Fig. 10A2). The individual voltage ramps obtained at the peak of the bFGF-evoked responses showed relatively linear I-V relationships with a reversal potential of approximately +15 mV (Fig. 10B), similar to those recorded in untreated cells (Fig. 9). In antisense/TRPC1-treated cells, neither the reversal potential nor the character of the I-V curve was altered (Fig. 10B). These results indicate that the decrease in the peak amplitude of the bFGF-evoked inward current was not attributable to a change in the driving force acting on the activated channels but rather to a decrease in their conductance.

View larger version (21K): [in this window] [in a new window]   Figure 10. Antisense/TRPC1 depresses bFGF-induced currents in NSC-derived progeny. NSC-derived progeny proliferating in medium with bFGF were rinsed with bFGF-free saline before recordings from the cells in whole-cell patch-clamp mode. A1<