Fig. 4.

Treatment with an agrin-specific antibody prevented synaptic differentiation; recombinant agrin countered the effects of the antisense oligonucleotides in hippocampal neurons. A, Rat P3–P5 hippocampal neurons were plated in the presence of the m247 antibody. Cultures were kept for 10 d. Immunofluorescence staining for synapsin-I was performed on untreated (Untr; a cluster of 3 neurons is shown) and m247-treated (αAgAb) cultures. The number of synapsin-I-immunoreactive puncta was greatly reduced in the treated neurons. Treatment with an unrelated antibody did not produce any phenotype (data not shown). Scale bar, 50 μm. B, Quantification of the effects of the m247 antibody on synaptic differentiation is shown. The number of synapsin-I-immunoreactive puncta per neurite area was greatly reduced (≥80%) in treated cells (αAgAb) compared with untreated (Untr) and mock antibody-treated (C) cells. Values are from a representative experiment (n = 3); mean ± SEM values were calculated from 20 fields of view (p < 0.01). C, Neurons were incubated with antisense oligonucleotides and purified recombinant agrin isoforms. P3–P5 rat hippocampal neurons were treated for 10 d with the AS oligonucleotide (10 μm) with either the Ag4,0 or Ag4,8 isoforms (50 pm). Immunofluorescence staining with synapsin-I-specific antibodies is shown for untreated (Untr), AS-treated (AS), AS and Ag4,0-treated (AS/Ag0), and AS and Ag4,8-treated (AS/Ag8) neurons. Only the cultures treated with the Ag4,8 isoforms appeared similar to control untreated cells.D, Quantification of the effects of the antisense oligonucleotide and agrin isoform treatment is shown. The number of synapses per neurite area was determined for 10-d-untreated (Untr), AS-treated (AS), AS and Ag4,0-treated (AS/Ag0), and AS and Ag4,8-treated (AS/Ag8) neurons. Mean ± SEM values (n = 20 neurites in 5 randomly chosen fields of view per treatment; p < 0.05) are shown from a representative experiment (n = 4 independent experiments). Scale bar, 40 μm.