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 CaV_2.1/2.2 (P/Q- and N-type), CaV_2.3 (R-type), CaV_1.2/1.3 (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 Fig. 5 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.

Our experiments thus far have established that NMDARs are present at axons and growth cones of DIV3-6 primary hippocampal neurons. Next, we investigated whether these NMDARs are functional. Low density primary hippocampal neurons were cultured such that individual cells were isolated, and not in contact with other neurons or glia. At DIV3-6, the neurons were examined by whole-cell voltage clamp technique (−70 mV holding potential). NMDA (200 µM) was then locally applied to the axonal growth cone by pressure application (4–6 psi) for 100 ms. Recordings were performed in a HEPES-based, magnesium-free extracellular solution containing 0.2 mM CaCl₂. Our cesium-based internal recording solution contained 5 mM EGTA to avoid calcium-mediated excitotoxicity. To restrict the NMDA application to the growth cone, only neurons that were oriented with the cell body positioned upstream of the perfusion flow, and at least 100 µm from the growth cone, were chosen for analysis (Fig. 4A). The application pipets had resistances ranging from 4–6 MΩ, and were positioned 10–20 µm from the growth cone. This application system allows us to specifically target the NMDA application to the growth cones while avoiding the dendrites and cell body, though it is likely that regions of the distal axon were also within the area of the application (Fig. 4E). For each cell examined, NMDA was applied onto the cell body as a control. The pipet was then repositioned, and NMDA was applied to the growth cone for three trials (once per minute). Representative traces from a single cell (application at the cell body, growth cone, and growth cone in the presence of APV) are shown in Fig. 4B. Perfusion of extracellular solution containing the NMDAR antagonist DL-APV (100 µM) eliminated the currents induced by NMDA application to the growth cone, indicating that this response is mediated by NMDARs, and not due to a mechanical artifact. In addition, these experiments were performed in the presence of 1 µM tetrodotoxin (TTX) to improve voltage clamp and prevent spontaneous action potentials or subthreshold depolarization. Application of NMDA to the growth cone elicited a highly reproducible current with an average peak amplitude of 31 ± 3.4 pA compared to 265 ± 40.9 pA at the cell body (n=10, Fig. 4C). Based on a single-channel NMDA receptor conductance of 50 pS (Stern et al., 1992), currents evoked by application of NMDA to the growth cone may represent the opening of only ~10 channels. Therefore to quantify the effects of APV we integrated the traces to measure charge. NMDA application to the growth cone was reduced from 14.4 ± 5.1 fC under control conditions to 0.038 ± 0.5 fC in the presence of APV (Fig. 4D). The APV-sensitive currents evoked by local application of NMDA demonstrate that NMDARs on axonal growth cones are functional.

NMDARs are calcium-permeable ion channels. Therefore, to obtain further evidence for the localization of functional NMDARs at growth cones, we used fluorescence microscopy to image calcium transients in growth cones of DIV3-6 primary hippocampal neurons. We began by performing a qualitative analysis of NMDA-mediated calcium influx at growth cones using the high-affinity cell-permeable fluorescent calcium indicator, Fluo-4 AM. Neurons were loaded with Fluo-4 AM, and NMDA was applied to the growth cone by pressure application. Highly dynamic calcium transients, visualized using fluorescent microscopy, appeared at various regions of the growth cone and propagated down the axon shaft (Fig. 5B). For each cell examined, the addition of 100 µM DL-APV to the extracellular solution prevented these calcium transients (Fig. 5C). This response was NMDAR-specific, as the subsequent removal of the DL-APV-containing ACSF led to a recovery of the calcium transients (Fig. 5D). It is unlikely that propagation of the transients was due to diffusion, as calcium ions diffuse quite slowly in the cytoplasm with a coefficient of around 10 µm²s⁻¹ (al-Baldawi and Abercrombie, 1995; Murthy et al., 2000). However, release of calcium from internal stores mediated by IP₃ and ryanodine receptors can produce localized bursts of high levels of cytoplasmic calcium (Zheng and Poo, 2007). To address the possibility that NMDAR-mediated depolarization and activation of voltage-sensitive calcium channels (VSCCs) or calcium induced calcium release (CICR) from intracellular stores may contribute to these transients, we isolated the NMDA component through the use of pharmacological compounds. We loaded DIV3-6 neurons with the calcium indicator, Fluo-5F AM (a lower affinity indicator than Fluo-4 with improved dynamic range) and the cells were then treated by bath application, with a combination of blockers. An example of the application system is shown in Fig. 6A. To prevent potential contributions from VSCCs to the calcium transients, a combination of blockers including 0.1 µM ω-conotoxin MVIIC, 0.03 µM SNX-482, 20 µM nimodipine, and 10 µM mibefredil, was added to the extracellular solution. These compounds block VSCCs CaV_2.1/2.2 (P/Q- and N-type), CaV_2.3 (R-type), CaV_1.2/1.3 (L-type), and CaV₃ (T-type) classes of VSCCs, respectively. TTX (1 µM) was also added to the solution to block voltage-sensitive sodium channels and spontaneous action potentials. To block the replenishment of intracellular calcium stores, 20 µM cyclopliazonic acid (CPA) was added to the extracellular solution. In the presence of these blockers, NMDA application to growth cones still caused local calcium transients at growth cones with an average peak increase in fluorescence intensity (ΔF/F, n=15) of 62.7 ± 20.1% (Fig. 6B). As expected, bath application of APV blocked the transients (n=15) (Fig. 6B). To examine the spatial distribution of the calcium transients, the acquired images were analyzed and average ΔF/F images were generated using Metamorph. The fluorescent calcium transient extended throughout the entire growth cone structure, though there were often areas of increased intensity occurring at discrete locations on the growth cone (Fig. 6C). These areas in which calcium reached higher levels may represent regions where NMDARs are present at greater density. These results demonstrate that NMDARs at axonal growth cones are functional, and mediate localized influx of calcium.