Figure 7. Netrin-1 increases protein synthesis in SNs and strength of SN-target MN synapses.

(A) A bifurcated sensory neuron was cultured with an L7 target MN and an L11 non-target MN for 3 days. Alexa647 was microinjected into SNs and served as a volume filling control for the analysis of immunocytochemistry (ICC). Cultures were incubated with 250 ng/ml recombinant Fc human netrin-1 for 24 hr. Representative high magnification images of areas marked by white squares in (A) are shown in (B), with merged DIC/Alexa488 in the left panels, and sensorin ICC in the right panels. Group data (C) reveal that the concentration of sensorin in netrin-1-treated SNs is the same in neurites contacting L7 target MNs and L11 non-target MNs. We compared sensorin (D) and phospho-eIF4E immunoreactivity (not shown) in SN-LFS target MN cultures in the presence and absence of 250 ng/ml Fc-netrin-1 (24 hr). Representative high magnification images of areas denoted by white squares in left panels of (D) stained with anti-sensorin antibodies are shown in right panels of (D). Group data (E) reveal that netrin-1 triggers an increase in sensorin and phospho-eIF4E immunoreactivity that is equivalent to the increase observed 24 hr after 5 spaced applications of 5HT (which produce long-term facilitation) and that is blocked by the protein synthesis inhibitor anisomycin (10 mM). The effect of netrin-1 on sensorin immunoreactivity in isolated SN processes and on eIF4E and phospho-eIF4e immunoreactivity is shown in Figure 7—figure supplement 1. Netrin-1 also increased synaptic strength: representative traces of EPSPs evoked in LFS MNs after stimulation of SN, at time 0 and 24 hr after incubation with vehicle (artificial seawater, ASW) or netrin-1 are shown in (F); histogram of group data is shown in (G). Histogram in (H) shows fold change in the number of varicosities between SN and LFS MN after 24 hr of incubation with vehicle (ASW) and netrin-1. Error bars represent SEM. **p < 0.001, Student's unpaired t-test for (G) and (H); ANOVA and Dunnett's multiple comparison test for (E). Scale bars in (A) and (D, left panel) =100 μm; in (B), (D, right panel)= 10 μm.

DOI: http://dx.doi.org/10.7554/eLife.04158.014

Figure 7—figure supplement 1. Netrin-1 increases local translation in soma-free SN processes in SN-MN cultures.

SN-MNs were cultured for 3 days and the SN soma (denoted by dotted circle in A) was mechanically removed with sharp glass electrode. 12–18 hr later, cultures were incubated with recombinant Fc human netrin-1 (250 ng/ml for 24 hr) and then processed for immunocytochemistry with sensorin antibodies (B). As shown in the group data in (C), recombinant Fc human netrin-1 increased translation in SN processes in cultures in which the soma was present (with soma) or removed (without soma). Quantification of the effect of netrin-1 (250 ng/ml for 24 hr) in intact SN-MN cultures (3 DIV) on eIF4e and phospho-eIF4e immunoreactivity, with sensorin immunoreactivity as a positive control, is shown in (D). Error bars represent SEM. **p < 0.01 unpaired t-test. Scale bars in (A) =100 μm; in (B) =10 μm.

DOI: http://dx.doi.org/10.7554/eLife.04158.015