Primary hippocampal cultures
Embryonic day 18 (E18) primary hippocampal cultures were prepared as previously described (Wang et al., 2008
). Briefly, hippocampi from E18 rats of either sex were dissected, cells were dissociated and plated onto coverslips at high density (50,000 cells per ml, or 50K) for transfection, or low density (0.5–5K) for electrophysiology and calcium imaging experiments. Growth cones were examined between DIV3-DIV6. Beyond this age range, growth cones become increasingly difficult to find even in low-density cultures because isolated neurons form autapses. To minimize variability and contribution of somatic responses for electrophysiology and calcium imaging experiments, strict morphological and geometric criteria were utilized in the selection of neurons to analyze. Only isolated neurons (with no visible contacts with adjacent cells) that had morphologically distinct axonal growth cones positioned downstream of the perfusion flow, relative to the cell body, were chosen for examination.
Transfections and Immunocytochemistry
Transfections and immunocytochemistry were performed as previously described (Wang et al., 2008
). Briefly, neurons were fixed with 4% paraformaldehyde (PFA), washed with phosphate buffered saline (PBS) and permeabilized with 0.25% Triton-X-100/PBS. Neurons were then blocked with 10% normal goat serum (NGS)/PBS/0.1% Triton X-100 for one hour, and then incubated with primary antibodies at room temperature in 3% NGS/PBS/0.1% Triton X-100 for one hour. Neurons were washed and incubated with AlexaFluor 488 or 555 secondary antibodies (Invitrogen, Carlsbad, CA) for 30 minutes, and washed and mounted on slides using Prolong Antifade Gold (Invitrogen). For surface staining experiments, live neurons were incubated with primary antibodies for 30 minutes at 4 °C, fixed with 4% PFA for 20 minutes, blocked with 10% NGS/PBS, and incubated with secondary antibodies for 30 minutes. Neurons were transfected with cDNA constructs at various ages in culture (DIV4-14) using the calcium phosphate method. Plasmids encoding GFP-NR2A, GFP-NR2B, and YFP-NR1-1 were generously provided by Dr. Stefano Vicini. Surface or permeabilized immunocytochemistry was performed 48 hours after transfection, and visualized using a Nikon E1000M microscope equipped with a CCD camera using a Plan Apo 60× (1.4 NA) or Plan Fluor 40× (1.3 NA) oil-immersion objective, or a Zeiss LSM 710 laser scanning confocal microscope with a 63× (1.4 NA) oil immersion Plan Apo objective. GFP polyclonal antibodies were purchased from Millipore (Billerica, MA) and used at 1:2000 dilution for surface and permeabilized immunocytochemistry. The NR1 rabbit monoclonal antibody was from Millipore, based on a 30 amino acid sequence in the NR1 C-term tail (amino acids 909–938: LQNQKDTVLPRRAIEREEGQLQLCSRHRES) and used at a 1:1:000 dilution for permeabilized immunocytochemistry. This sequence corresponds to four of the eight known NR1 splice variants (NR1-1a, NR1-1b, NR1-2a, NR1-2b).
The extracellular recording solution contained 1.25 mM NaH2PO4, 150 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 10 mM glucose, 10 µM D-serine, and 0.2 mM CaCl2 (pH 7.3). Somatic whole-cell voltage-clamp recordings were performed with borosilicate glass electrodes (6–8 MΩ) filled with a cesium-based internal solution containing 130 mM CsMeSO4, 10 mM HEPES, 5 mM EGTA, 1 mM MgCl2, 10 mM TEA-Cl, 2 mM Mg-ATP, 0.3 mM Na-GTP, 10 mM phosphocreatine (tris), and 2 mM QX-314. Whole-cell voltage clamp recordings were obtained from primary hippocampal neurons using a MultiClamp 700B (Molecular Devices, Sunnyvale, CA) patch clamp amplifier. Application of NMDA (200 µM, in extracellular solution) was performed using a Picospritzer III (Parker Hannifen, Cleveland, OH) pressure application system. Compounds were applied at 4–6 psi for 100 ms. Cells were maintained at −70 mV for all conditions, and recordings were conducted at room temperature and analyzed using Igor Pro (Wavemetrics, Portland, OR) software.
The following pharmacological compounds were utilized, as described in the text. In control electrophysiology and calcium imaging experiments, NMDARs were blocked by adding 100 µM DL-APV (Tocris Bioscience, Ellisville, MO) to the extracellular solution, and introduced into the recording chamber by perfusion. Voltage-sensitive calcium channels (VSCCs) were blocked with 0.1 µM ω-conotoxin MVIIC (Peptides International, Louisville, KY), 0.03 µM SNX-482 (Peptides International), 20 µM nimodipine (Sigma-Aldrich, St. Louis, MO), and 10 µM mibefredil (Sigma-Aldrich). These compounds block VSCCs CaV2.1/2.2 (P/Q- and N-type), CaV2.3 (R-type), CaV1.2/1.3 (L-type), and CaV3 (T-type) classes of VSCCs, respectively. Replenishment of intracellular calcium stores was blocked using 10 µM cyclopiazonic acid (CPA) (Tocris Bioscience). All reagents were dissolved in distilled water except for nimodipine and CPA, which were dissolved in dimethyl sulfoxide (DMSO).
Our standard extracellular solution was prepared with 1 mM CaCl2 for these experiments. For AM-loading experiments, DIV3-6 primary hippocampal cultures were treated with the fluorescent calcium indicators Fluo-4 AM or Fluo-5F AM (Invitrogen). Fluo-4 AM or Fluo-5F AM (50 µg) was solubilized in DMSO/0.8% pluronic F-127 to create a 1 mM stock solution. The AM calcium indicator stock was applied to neurons in B27-supplemented neurobasal medium at a dilution of 1:100 and incubated at 37 °C for 3 minutes. Neurons were then washed with fresh culture media and incubated for 30 minutes at 37 °C. NMDA (200 µM) was applied to axonal growth cones, and calcium transients were imaged using a QImaging Rolera Mgi EM CCD camera in conjunction with QCapture Pro 6 (QImaging, Surrey, BC) and IgorPro software (Wavemetrics, Portland, OR). During control experiments performed to examine the spatial specificity of the local application system, we verified that NMDA application to the soma did not produce fluorescent calcium transients in the growth cone. As an additional measure to prevent diffusion of NMDA towards the cell body during experiments in which NMDA was locally applied to growth cones, neurons were selected with an orientation so that the flow of bath solution directed the NMDA solution away from the cell body. Images (100 ms exposures) were taken at 138 ms frame intervals, verified by output signals from the CCD camera. The exposure at the 0 ms time point in began at the onset of NMDA application. Calcium transients were quantified using Metmorph v7.0r3 (Molecular Devices). Regions of interest (ROIs) were selected to encompass the entire growth cone structure (P and C areas), and background ROIs were selected within close proximity to the growth cone. ΔF/F measurements were performed using the following formula (F-F0)/F0, where F was the background subtracted fluorescent value, and F0 was the average of the first five background subtracted frames (baseline levels). To examine the spatial distribution of calcium transients, ΔF/F images were generated using Metamorph. All images were background subtracted, and the F and F0 images were generated through averaging pixel values of frames 10–14 (frames immediately following NMDA application) and frames 1–5 (baseline), respectively. The ΔF image (F-F0) was generated by subtracting the pixel intensity values of the F and F0 with a constant value of +1, and the ΔF/F image was generated by the division function with a constant value of 100 in the numerator. Median filtering (3×3) was applied to the resulting ΔF/F image to eliminate background pixels.
Activation of NMDARs at axonal growth cones induces calcium influx
Immunogold electron microscopy
Postembedding immunogold labeling was performed as previously described (Petralia and Wenthold, 1999
; Petralia et al.). Briefly, two postnatal day 2 (P2) rats were perfused with 4% paraformaldehyde plus 0.5% glutaraldehyde, and sections were cryoprotected and frozen in a Leica EM CPC and further processed and embedded in Lowicryl HM-20 resin using a Leica AFS freeze-substitution instrument. Thin sections were incubated in 0.1% sodium borohydride plus 50 mM glycine/Tris-buffered saline plus 0.1% Triton X-100 (TBST), followed by 10% NGS in TBST, and primary antibody in 1% NGS/TBST overnight, and then immunogold labeling in 1% NGS in TBST plus 0.5% polyethylene glycol (20,000 MW). Finally, sections were stained with uranyl acetate and lead citrate. Corresponding controls, lacking the primary antibody, showed only rare gold labeling. Images were stored in their original formats and final images for figures were prepared in Adobe Photoshop. Brightness and contrast of images were minimally adjusted, evenly over the entire micrograph.