Fig. 5
From: Regulation of density of functional presynaptic terminals by local energy supply
Elevating [Mg2+]i might increase the efficiency of axonal transport of Ca2+-sensitivity-related proteins. (a) Western blot detection showed no significant increase in the total protein levels of each Ca2+-sensitivity-related protein or SYP after elevating [Mg2+]o for 6 hr (n = 12 coverslips from 4 batches). (b) The immunoreactivity of Rab3a in a soma island from the culture coverslips before and after elevating [Mg2+]o for 4 hr. The pseudo-colored images were the magnifications of the local somatic or terminal regions marked in the large images. Stars s1-s4: examples of immunoreactive cell bodies. Dashed boxes d1-d6: representative terminal regions. Pseudo-color scale: fluorescent intensity; the upper one was for s1-s4 and the lower one was for d1-d6. (c) Significant decreases in Ca2+-sensitivity-related proteins in somatic area after elevating [Mg2+]o for 4 hr (analyzed from the same raw data as in Fig. 4d, 50–98 cell bodies). Dotted line represents levels of proteins at [Mg2+]o 0.8 normalized to 100 %. (d) Quantity of immunostained SYT1 at terminals (n = 6–8 coverslips for each data bar) or soma (40–55 cells from the same AOIs) changed in opposite directions after elevating [Mg2+]o from 0.6 to 0.75-1.2 mM (in gradient) for 4 hr. Dotted line represents initial SYT1 at Terminal and Soma, normalized to 0 % change. Pseudo-colored images represented the immunoreactivity in somatic area at different [Mg2+]o levels. Pseudo-color scale: fluorescent intensity. (e and f) Blocking axonal transport by 0.5 mM Colchicine (Colch) caused a decrease in proteins in terminals at low (e) and high [Mg2+]o conditions (f) (n = 8–10 coverslips for e and 10–15 coverslips for f). Dotted lines represent initial protein levels before addition of Colch, normalized to 100 %. (g and h) Blocking axonal transport by 0.5 mM Colch led to a decrease in N 5AP but no effect on [Mg2+]i compared to not adding Colch (blue dashed lines) (n = 5–6 coverslips for g and 5–8 coverslips for h). Dotted line represented the initial [Mg2+]i and N 5AP normalized to 100 %. (i) Schematic cartoon: intracellular Mg2+ might majorly affect axonal transport efficiency, but not affect protein synthesis or degradation within a few hours. The mean ± SEM of all coverslips was presented. Two-tailed Student t-test for comparing 1.2 4hr to Ctrl 0.8 (c) and comparing pre and post treatment values (d-h), * p < 0.05, ** p < 0.01, *** p < 0.001