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Fig. 3
Neuronal cell cultures derived from embryos injected with APPb-MO had decreased neurite length.
(A) Neuronal culture from control embryos and APPb mRNA (350 pg) over-expression embryos exhibited on average 3 primary branches with several secondary branches. Neuronal cultures from embryos injected with 8 ng of APPb-MO exhibited on average 1 primary branch with many secondary branches. 8 ng of APPb-MO is sufficient to inhibit neruite growth, so embryos were injected with 8 ng of the APPb MO instead of the 10 ng to limit the potential for toxicity. The neurons were cultured for 2 days before fixing. (B) Down-regulation of APPb in cultured neurons affected normal neurite growth. The neurons from the embryos injected with 8 ng of APPb-MO showed a decrease in neurite length compared to neurons from the control embryos (un-injected). The average neurite length of a control neuron was about 130 µm, compared to only about 100 µm in the morphant embryos. Statistical significance was observed between control neurons and APPb knockdown neurons (8 ng APPb MO) (p = 0.0007, p<0.05 in two-tailed paired t-test). (C) Quantifying the effects of APPb mRNA in cultured neurons. No significant difference was observed in neurite length between control and APPb mRNA over-expression cultured neurons. Control cultured neurons expressed a shorter neurite length compared to APPb mRNA over-expression neurons. No statistical significance was observed (p = 0.1915, p>0.05 in two tailed paired t-test). (D) There were fewer branches of neurites in the APPb knockdown neurons (8 ng of APPb MO) than in control neurons (WT); only branches with more than 5 µm were counted. The neurons were cultured for 2 days. (E) Down-regulation of APPb in cultured neurons affected the number of branch tips. Compared to control neurons, APPb-MO cultured neurons showed a decreased number of branch tips of the longest neurite. Statistical significance was observed between control and APPb-MO cultured neurons (p = 0.0074, p<0.05 in two tailed paired t-test). (F) Down-regulation of APPb in cultured neurons affected the morphology and projection of growth cones and filopodia. APPb knockdown neurons expressed abnormal growth cone morphology and a decreased number of filopodia (20 hour neuron cultures). (G) Reduced number of filopodia in APPb knockdown cultured neurons. Compared to control cultured neurons, APPb-MO (8 ng per embryo) cultured neurons expressed a reduced number of filopodia. Statistical significance was observed between control and APPb cultured neurons (p = 0.0091, p<0.05 in two tailed paired t-test).