Fig. 2.

Agrin antisense oligonucleotides induced a change in phenotype in rat hippocampal neurons. A, Morphology of cultured neurons at 3 d (3d), 7 d (7d), and 14 d (14d) in culture. Only neurons treated with the agrin antisense oligonucleotide AS appeared clustered in multicellular aggregates with extended neurites in fascicles. All three antisense oligonucleotides tested produced the same phenotype. The concentration of S (S) and AS (AS) oligonucleotides was 7.5 μm. Scale bar, 100 μm. B, Concentration dependence of the AS oligonucleotides. The severity of the phenotype described inA increases with increasing concentration of AS oligonucleotides. Decreased cell numbers were observed in cultures treated with all three AS oligonucleotides tested at concentrations ≥25 μm. Scale bar, 60 μm. C, Determination of the concentration–response curve of the AS oligonucleotide for synaptic differentiation. Synapsin-I-immunoreactive puncta per neurite area were measured in hippocampal neurons treated for 10 d with the AS oligonucleotide (AS oligo). Mean ± SEM values (synapses onn = 20 neurites in 5 randomly chosen fields) of a representative experiment (n = 3 independent experiments) are shown. D, Quantification of the number of synapsin-I-immunoreactive puncta per neurite area in cultured hippocampal neurons treated for 10 d with 5 μm agrin sense (S), scrambled (SC), and three agrin antisense oligonucleotides (AS, AS2, and ASY). Mean ± SEM values of a representative experiment (n = 3 independent experiments) are shown (n = 20 neurites in 5 randomly chosen fields of view/treatment; p < 0.05). Control cultures (C).