Hippocampal or cortical cultures from embryonic rat brain have been previously used as a model system to study mRNA and protein localization as these cells differentiate distinct axonal and dendritic processes and have demonstrated similar molecular distributions to that observed in vivo (Goslin and Banker 1991
). We have described the use of cerebrocortical cultures in studying the localization of β-actin mRNA to neuronal processes and growth cones (Bassell et al. 1998
). In this study, we used a chick forebrain neuronal culture system, which also differentiates axon and dendrite-like neurites (Sensenbrenner et al. 1978
; Chada et al. 1997
), as an alternative to rat cortical neurons. We observed that neurons within these cultures frequently have larger growth cones that have a flattened, lamellar morphology that is characteristic of the type that localize β-actin mRNA (Bassell et al. 1998
The general method of neuronal culture that we use has been described in detail (Goslin and Banker 1991
) and modified for use with chick forebrain neurons (Chada et al. 1997
). Cerebral hemispheres were dissected from 8-d-old chick embryos and trypsinized (0.15% in HBSS) at 37°C for 7 min, washed in HBSS, and placed in MEM with 10% FBS. Cells were mechanically dissociated by pipetting, washed three times in MEM, and plated at low density (6,000 cells/cm2
) on poly-l
-lysine (0.2 mg/ml, 16 h) and laminin (0.02 mg/ml, 12 min) coated coverslips or plates in MEM with 10% FBS for 2 h. Cells were inverted onto a monolayer of rat astrocytes in N2
-conditioned medium with serum (2% FBS) and cultured for 4 d at 37°C in 5% CO2
-conditioned medium contained glutamate-free MEM supplemented with transferrin (100 μg/ml), insulin (5 μg/ml), progesterone (20 nM), putrescine (100 μM), selenium dioxide (30 nM), glucose (6 mg/ml), sodium pyruvate (1 mM), and ovalbumin (0.1%). The cells were then fixed in paraformaldehyde (4% in PBS) at room temperature for 15 min and washed in PBS containing 5 mM MgCl2
Drug and Growth Factor Treatments
Neurons were cultured in N2-conditioned medium with 2% FBS for 4 d and then starved in minimal essential medium (MEM) without N2 supplements or serum for a period of 6 h at 37°C. Before fixation, cells were treated at various time points (10 min through 2 h) with drugs or growth factors as follows: FBS (8%), forskolin (5 μM; Calbiochem-Novabiochem), neurotrophin-3 (NT-3) (25 ng/ml; Austral Biologicals), db-cAMP (N6, O2-dibutyryl-adenosine 3′,5′-cyclic monophosphate) (25 μM; Calbiochem-Novabiochem), brain derived neurotrophic factor (BDNF) (25 ng/ml; Sigma), nerve growth factor (25 ng/ml; GIBCO-BRL). In some experiments, the cells were treated with NT-3 in a series of time points, i.e., 1, 2, 5, and 10 min, after starvation in MEM to observe the changes of β-actin mRNA and protein localization in growth cones.
K252a (200 nM; BioMol), an inhibitor for a broad range of serine and threonine protein kinases including Trk receptor tyrosine kinases (Knusel and Hefti 1992
) and Rp-cAMP (50 μM; Calbiochem-Novabiochem), a nonhydrolyzable analogue competitor of cAMP (Bothelo et al. 1988
), were both used to inhibit the NT-3 induction of β-actin mRNA localization. After 5 h of starvation in MEM, K252a or Rp-cAMP was added for 1 h followed by treatment with NT-3 (in the continued presence of the inhibitor). The cells were fixed in 4% paraformaldehyde in PBS after treatment with drugs or growth factors.
To disrupt microfilaments, cells were treated with 5 μg/ml of cytochalasin-D (Sigma Chemical Co.) in culture media at 37°C for 30 min before NT-3 stimulation. To depolymerize microtubules, cells were treated with colchicine (10 μg/ml, Sigma Chemical Co.) in culture media for 30 min before NT-3 stimulation. In each case, the cells were first starved in MEM for 5.5 h before addition of cytochalasin-D or colchicine. Stock solutions of cytochalasin-D and colchicine were made up in DMSO and ethanol, respectively, and the concentration of these solvents was diluted below 0.1% in the culture media, so as not to be toxic to neurons. Neurons were fixed in paraformaldehyde (4% in PBS with 5 mM MgCl2) after drug treatment and NT-3 stimulation.
In Situ Hybridization and Immunofluorescence Analysis
Six amino group modified oligonucleotides (50 bases each) complementary to 3′ untranslated sequences (3′-UTR) of chick β-actin mRNA were synthesized on a DNA synthesizer (Latham et al. 1994
). Each oligonucleotide was modified at five positions within the sequence and chemically labeled using digoxigenin succinamide ester (Boehringer Mannheim). To ensure isoform specificity, probes were selected from unique regions within the 3′-UTR and were of identical length, guanine/cytosine (GC) content and hapten incorporation. Oligonucleotide probes complementary to β-galactosidase mRNA or Mu phage DNA were used as controls. In situ hybridization for β-actin mRNA was completed as previously described (Bassell et al. 1998
). Cells were equilibrated in 1× SSC with 40% formamide for 5 min. Each coverslip was incubated at 37°C overnight in hybridization reactions containing 20 ng of oligonucleotide probe, 1× SSC, 40% formamide, 10% dextran sulfate, 0.4% BSA, 20 mM ribonucleotide vanadyl complex, salmon testes DNA (10 mg/ml), E. coli
tRNA (10 mg/ml), and 10 mM sodium phosphate. Cells were washed twice with 4× SSC/40% formamide and then twice with 2× SSC/40% formamide, both at 37°C, and then with 2× SSC three times at room temperature.
The hybridized probes labeled with digoxigenin were detected using Cy3-conjugated monoclonal antibody (mAb) to digoxigenin and anti–mouse mAb-Cy3 (from Jackson ImmunoResearch Labs.). After blocking in TBS with BSA (2%) and FBS (2%) at 37°C for 1 h, the coverslips were incubated with Cy3-mAb to digoxigenin in TBS (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with 1% BSA at 37°C for 1 h. After washes in TBS with 1% BSA, cells were mounted with n-propyl gallate (anti-fading agent). β-actin protein was detected with a mouse monoclonal antibody (Sigma) and secondary antibodies were conjugated with Cy3 (Jackson ImmunoResearch Labs.).
Microscopy and Digital Imaging
Immunofluorescence signal was viewed using an Olympus-IX70 microscope equipped with a 60× Plan-Neofluar objective and Nomarski (DIC) optics. Cells were viewed using a 100 watt mercury arc lamp and light was filtered using HiQ bandpass filters (ChromaTech). The images were captured with a cooled CCD camera (Photometrics) using a 35-mm shutter and processed using IP Lab Spectrum (Scanalytics) running on a Macintosh G3. After identification of growth cones using DIC optics, a fluorescence image was immediately acquired. All exposure times with the CCD camera were kept constant (1 s for β-actin mRNA, 0.5 s for β-actin protein) and below grey scale saturation to permit a linear response to light intensity and quantitative analysis of differences in fluorescence intensities. The perimeter of each growth cone was traced using the DIC image and IP Lab software to identify a region of interest (ROI) and measure total fluorescence intensity. For quantitative image analysis of β-actin mRNA and protein localization using this method (see and ), 20 cells were imaged for each cell culture condition.
Figure 3 NT-3 stimulated localization of β-actin mRNA and protein analyzed using quantitative digital imaging microscopy. Neurons were fixed for in situ hybridization to β-actin mRNA (A) and immunofluorescence detection of β-actin protein (more ...)
Figure 4 Visualization of NT-3–stimulated β-actin mRNA localization in cells treated with cytoskeletal disrupting drugs. (A) β-actin mRNA localization in cytochalasin-D–treated cell. Hybridization signal was prominent in the cell (more ...)
For quantitative analysis using a visual scoring method, 100 cells per coverslip were analyzed for each cell culture condition. Experiments were done with duplicate coverslips for each variable and each experiment was repeated at least three times. The scoring method involved visualization of the presence or absence of β-actin mRNA granules in the axon-like growth cone from each cell. Cells were scored as localized if several granules were observed, and scored as nonlocalized if the signal was not distinguishable from background levels (hybridization with control probe). Localized cells would be expected to have a higher amount of fluorescent signal in growth cones compared with nonlocalized cells. Examples of localized and nonlocalized cells are shown in . This scoring method was used to show that NT-3 promotes an increase in localization (see ) and that the results were comparable to the quantitation of fluorescence intensity using the CCD camera (see ). The value for each bar within the histogram reports the mean and standard deviation between independent samples (see ). The Student's t test was used to compare the percentage of localized cells under a number of experimental conditions. This scoring method produced similar results to that of quantitation of fluorescence intensity within each growth cone using digital imaging analysis (see above). The advantage of the scoring method is that one can rapidly score hundreds of cells within a population and evaluate multiple variables.
Figure 1 β-actin mRNA and protein localization is influenced by neurotrophin-3 (NT-3). (A) β-actin mRNA was prominent in the cell body and localized within the axonal process and growth cone in the form of spatially distinct granules (arrow; (more ...)
Figure 2 Quantitative analysis of neurotrophin stimulated β-actin mRNA localization. Neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), or NGF was added to the MEM (25 ng/ml) for the indicated time. NT-3 and BDNF, but not NGF, were observed (more ...)
SignaTECT cAMP-Dependent Protein Kinase Assay (Promega) was used to monitor neurotrophin-3 stimulation of PKA activity in primary neuronal cultures (Cobb et al., 1992; Goueli et al. 1995
; Walsh and Van Patten 1994
). This assay uses a biotinylated Kemptide substrate, derived from pyruvate kinase (Piklis et al. 1980
), which is highly specific for cAMP-dependent PKA (Km = 5–10 μM). The biotinylated peptide substrate is phosphorylated by cellular PKA using γ-[32
P]ATP. The 32
P-labeled biotin substrate is then recovered from the reaction mix and captured by a streptavidin-linked matrix (SAMTM
Membrane). In brief, cultured neurons were treated with NT-3 as described, washed with HBSS, and then lysed in 0.3 ml cold extraction buffer (25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM mercaptoethanol, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 0.5 mM PMSF). Extracts were centrifuged for 5 min at 4°C at 14,000 g
and 5 μl of the supernatant was incubated with 20 μl of the reaction mixture containing biotinylated peptide substrate and γ-[32
P]ATP for 5 min at 30°C. The reaction was terminated and 10 μl was spotted onto a biotin capture membrane. After washing and drying of the membrane, radioactivity was measured using a liquid scintillation counter. This assay detects 0.012 casein units (2.5 Kemptide units) or less of purified cAMP-dependent PKA. A linear regression analysis, using calibrated amounts of casein units, produced an R2 value >0.95. Triplicate samples were used for each time point and the PKA activities and standard deviation is shown in (expressed relative to starved cells not exposed to NT-3).
Figure 8 NT-3 signaling of β-actin mRNA localization through cAMP. NT-3 stimulation of β-actin mRNA localization was reduced by kinase inhibitors. K252a (200 nM) or Rp-cAMP (50 μM) was added to the MEM for 1 h after a 5-h starvation. Forskolin (more ...)
Northern Blot Hybridization
Neurons (8 × 105 cells) were cultured in N2-conditioned medium supplemented with 2% fetal bovine serum on poly-l-lysine and laminin-coated plastic dishes for 4 d. Total cellular RNA was isolated using Tri Reagent following the product instructions (Molecular Research Center, Inc.). Cellular RNA was dissolved in DEPC-treated, distilled water. RNA (8 μg) was run in each lane in 0.8% formaldehyde-denatured agarose gel and transferred to Zeta-probe GT Genomic Tested Blotting Membrane (Bio-Rad Lab). The cDNA fragments (372 bp) complementary to the β-actin mRNA reading frame sequence were labeled with 32P-dCTP (Amersham Corp.) by using the Random Primers DNA Labeling System (GIBCO-BRL) and purified by Quick Spin Columns (Boehringer Mannheim). The RNA binding membrane was prehybridized in 5× SSPE supplemented with 5× Denhardt's, 0.2% SDS, 5% dextran sulfate, 300 μg/ml salmon testes DNA, and 50% formamide at 45°C for 4 h, and hybridized with 32P-labeled cDNA probes at 45°C overnight. After washes, the membrane was exposed to x-ray film (Kodak). Bands on the exposure film were scanned using a densitometer (Molecular Dynamics) and the optical densities were analyzed quantitatively using ImageQuant software.
Extraction with Rhodamine-labeled Actin
After 4 d of culturing in N2
-conditioned medium with 2% FBS and 6 h of starvation in MEM, the cells were incubated in MEM with NT-3 (25 ng/ml) for 1, 2, 5, and 10 min. Cells were extracted in buffer (20 mM Hepes, pH 7.4, 138 mM KCl, 4 mM MgCl2
, 3 mM EGTA, 0.1 mg/ml saponin, and 1 mM ATP) with 0.45 μM of rhodamine-labeled actin (rho-actin) at room temperature for 1 min and fixed for immunofluorescence microscopy (Chan et al. 1998
). Rho-actin was rabbit G-actin labeled with rhodamine (provided by J. Condeelis, Albert Einstein College of Medicine). Rho-actin was thawed on ice and diluted in buffer (1 mM Hepes, pH 7.4, 0.2 mM MgCl2
, and 0.2 mM ATP) to a final concentration of 12 μM. Rho-actin (75 μl) was added into 2 ml of extraction buffer just before application to the cells.