Ting average baseline (R0) in the ratiometric measurements as described above for nonratiometric measurements. Even though expression levels of GCaMP2 varied from cell to cell, this didn’t have an effect on the frequency of calcium transients reported. Raw baseline fluorescence didn’t correlate with frequency (Spearman correlation coefficient r 0.09, p 0.69). We validated our calcium transient measurements with additional energy spectral density evaluation (Uhlen, 2004; Bortone and Polleux, 2009), which measures periodicity within a time series signal with out an arbitrary definition of a transient. This evaluation (our unpublished observations) confirmed larger periodicity as measured by typical relative power in calcium signals in contralateral vs. ipsilateral axons at a frequency of 15 per hour, the frequency of calcium transients evoked by Wnt5a in vitro (Li et al., 2009).Dissociated Cortical Neuron Cultures and Wnt5a ExperimentsCulture of dissociated cortical neurons and bath application experiments with Wnt5a have been performed as previously described (Li et al., 2009). Briefly, cortical neurons were dissociated from P0 hamster sensorimotor cortex and electroporated with EGFP-CaMKIIN plasmids with an Amaxa Nucleofector. These neurons were plated onto coverslips coated with 0.five mg mL poly-D-lysine (Sigma) and 20 lg mL laminin (Sigma/Invitrogen) at a density of 20007000 per cm2 and had been incubated in five CO2 and 9 O2 at 378C for two days. For long-term axon outgrowth assays, 400 ng mL Wnt5a in 0.5 BSA is PBS, or BSA alone, was then added towards the cultures. Cultures have been then incubated for 72 h just before fixation. Axon lengths were measured in neurons expressing EGFP-CaMKIIN or in untransfected neurons in the identical dish as a handle.Dunn Chamber Axon Guidance Assay and AnalysisFor Dunn chamber axon guidance assays, P0 hamster cortical neurons were grown on appropriately coated (see above) 22-mm2 No. 1.5 coverslips (Corning) at a low density (10 k cells/well in a six effectively plate (Falcon). Assembly of your Dunn chamber (Hawksley, UK) was modified from previDevelopmental NeurobiologyHutchins et al. noted, comparisons in between two groups were made with Student’s t test and comparisons amongst a number of groups were made using a one-way ANOVA with Dunnett’s posttest. Measurements are given in mean six SEM unless otherwise noted. Images have been modified with a low-pass filter in MetaMorph to reduce single-pixel noise. The images presented in figures were enhanced with brightness-contrast adjustments in Adobe (Mountain View, CA) Photoshop, and with Flatten Background and Sharpen adjustments in MetaMorph for slice pictures taken in the Nikon epifluorescence technique [Fig. 3(C)].ous research (Yam et al., 2009). Dunn chambers have been rinsed by serum-free medium once and then both inner and outer wells have been filled by serum-free medium. To secure coverslips with neurons around the chamber, silicon sealant (Dow Corning) was applied at 0.five cm from the border of outer properly but omitted at one side to type a slit later for draining and refilling the outer properly. A coverslip with neurons was inverted over the Dunn chamber leaving a narrow slit at the edge with out the sealant. Media in the outer well was aspirated after which medium with 400 ng mL Wnt5a was added towards the outer well. The narrow slit was sealed by fixing a tiny piece of parafilm (American bis-PEG2-endo-BCN supplier National Can) towards the chamber with sealant. Images had been acquired immediately soon after Dunn chamber assembly and two h later having a 20 three 0.5 numerical aperture (NA).