Fig. 3. Intraocular injections of MAP2 antisense reversibly disrupt the ChAT-positive arrays. (A-C) The regular intercellular spacing of the ChAT-positive arrays observed before treatment (A, P0) is disrupted 24 hours after treatment with oligo antisense to MAP2 (B, P1). At P7, when MAP2 has recovered (see Fig. 2G), the ChAT-positive cells are reorganized into regular arrays (C). Scale bars: 20 µm. (D) An example of the density oscillation of the cholinergic neurones across the retina 24 hours after treatment with MAP2 antisense oligonucleotide at P1. As a comparison, the grey band represents the interval between the maximal and minimal density values observed in either cholinergic arrays of a normal littermate retina. Notice that cell density in either ChAT-positive arrays does not normally vary with eccentricity at this age, as it has been shown to occur until P12 (Galli-Resta and Novelli, 2000). Density is measured in clusters of adjacent 125x125 µm² sampling fields taken at four different retinal eccentricities (eccentricity is the distance from the centre of the retina; 0%=centre). Filled symbols refer to the GCL ChAT-positive cells of the treated retina, open symbols to the treated INL ChAT-positive cells. (E) No difference in the density variation of ChAT-positive cells is observed between treated and normal littermates on P7, indicating complete recovery. Symbols as in D. The significant growth of the retina between P1 and P7 (McCall et al., 1987) explains the decrease in the average density of ChAT-positive cells observed between P1 (E) and P7 (D). (F) Treatment with MAP2 antisense affects the INL and GCL ChAT-positive arrays independently of one another, as shown by the ratio between the cell density in these two arrays at different retinal locations. The ratio between the density of INL ChAT-positive cells and the density of the GCL ChAT-positive cells is plotted as a function of eccentricity for the same treated retina shown in D. As a comparison, the grey band comprises the values obtained for this ratio in the normal retina shown in D. Although the INL/GCL density ratio variance is limited for the normal retinas, it varies considerably from field to field in the treated retinas, showing that there is no correlation between the effects on density observed in the GCL and in the INL ChAT-positive array of the treated retinas. (G) No significant difference in the number of cholinergic cells was observed between treated (T) and normal control (C) retinas at the time of array disassembly (P1) or after recovery (P7). Open triangles, INL ChAT-positive cells; black triangles, GCL ChAT-positive cells. The cholinergic cells were labelled with two different markers (ChAT and Islet 1), which produced indistinguishable results.

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