Journal: The Journal of Neuroscience

Article Title: Regulation of Calcineurin by Growth Cone Calcium Waves Controls Neurite Extension

doi: 10.1523/JNEUROSCI.20-01-00315.2000

Figure Lengend Snippet: Ca2+ wave frequency and Ca2+-dependent expression of GABA are not affected by inhibition or activation of CN. A, Spontaneous Ca2+ waves are shown. Intracellular Ca2+ was monitored using the Ca2+indicator dye Fluo 3, and images were acquired at 10 sec intervals in the presence of 1 nm DM. Arrows indicate events scored as waves, the asterisk denotes a Ca2+ spike, and the dashed lineindicates the event threshold. B, Inhibition of CN does not significantly affect the frequency of Ca2+ waves: control, 7 ± 3/hr; CsA (10 nm), 6 ± 3/hr; DM (Delt, 1 nm), 7 ± 3/hr. Neurons expressing CA-xCN, indicated by the fluorescence of fura-2-dextran, generate spontaneous Ca2+ waves at normal frequencies in growth cones: control, 7 ± 3/hr; CA-xCN, 7 ± 2/hr. Neurons were imaged for 30 min intervals. Data are mean ± SEM (n≥ 15). C, Neurons treated with either 10 nmCsA or 1 nm DM were assayed for expression of GABA with a rabbit anti-GABA polyclonal antibody. CN-inhibited neurons express the same extent of GABA immunoreactivity as controls. Reciprocal experiments with neurons expressing CA-xCN, confirmed by myc immunoreactivity, demonstrate no effect on GABA expression. Data are mean ± SEM (n ≥ 100).

Article Snippet: GABA immunoreactivity was detected in an identical manner to myc, using a rabbit anti-GABA polyclonal primary antibody (Incstar, Stillwater, MN) at 1:5000 and a tetramethylrhodamine isothiocyanate-biotinylated goat anti-rabbit secondary antibody (Jackson ImmunoResearch) at 1:500.

Techniques: Expressing, Inhibition, Activation Assay, Control, Fluorescence