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Dynamic microtubule-dependent interactions position homotypic neurones in regular monolayered arrays during retinal development

¹ Istituto di Neuroscienze CNR, Laboratorio di Neurofisiologia, Via G. Moruzzi 1, 56100 Pisa, Italy
² Scuola Normale Superiore, Via G. Moruzzi 1, 56100 Pisa, Italy

View larger version (192K): [in a new window]   Fig. 1. Regular mosaics of cholinergic and horizontal cells appear when and where a continuous net of dendrites connects neighbouring array cells. (A) The ChAT-positive cells of the GCL are regularly spaced at P0, and a continuous net of dendrites links neighbouring elements. (B) An incomplete net of ChAT immunoreactive processes is observed at E21, and irregular cell spacing is observed close to the gaps in the net of processes (e.g. cell clusters indicated by arrows). (C) At P4, the horizontal cells are regularly spaced in the central retina, and linked by a continuous net of processes. (D) At the same age, irregular cell spacing and gaps in the net of horizontal cell processes are observed in the peripheral retina. Confocal images. Scale bars: in B, 10 µm for A,B; in D 10 µm for C,D.

View larger version (106K): [in a new window]   Fig. 2. MAP2ab expression is limited to the ChAT-positive cells after ganglion cells removal. (A,B) Cross-sections through the P1 retina show that the two arrays of ChAT immunoreactive cells (A) are the only retinal cells where MAP2ab immunoreactivity is found (B) after optic nerve section at birth to remove RGCs. To help visualize the co-localization of the two proteins, the section in B is flipped horizontally so that each point in A corresponds to its mirror image in B. The top cell line is the ChAT-positive array in the inner nuclear layer (INL); the bottom cell line is the ChAT-positive array in the GCL. (C,D) Oligo penetration as assessed 30 minutes after injection of FITC conjugated oligo. In four out of five cases, the oligos appear to penetrate the entire retinal thickness (C); in a fifth case, FITC labelling indicates a penetration limited to the most vitreal retinal region (D). The approximate depths of the two ChAT-positive arrays are indicated by arrowheads. (E,F) ChAT immunoreactivity is still detected (E), while MAP2ab immunoreactivity is significantly reduced (F) 24 hours after treatment with MAP2 antisense oligonucleotides (P1). Arrows in E indicate cells that are at different retinal depths from those of their neighbouring elements. (A,B,E,F) Confocal images (4 µm thick). (G, left) Immunoblots for MAP2ab 24 hours after treatment with MAP2 antisense. (G, right) MAP2ab level assessed via immunoblot shows a 50% reduction 24 hours after oligo treatment (at P1), and a complete recovery 6 days later (at P7). Each column is the average of five different samples. Each sample pools together three retinas. C, control; T, treated. (H) Levels of immunoreactivity for MAP2ab, MAP5 and MAP1A detected in the ChAT-positive cell processes of five control (C) retinas and five treated (T) retinas analysed in cross sections 24 hours after intraocular injection of oligos antisense to MAP2 (P1). (I,J) No significant difference in retina stratification is observed 24 hours after treatment between normal (I) and treated (J) cases. At this age, the retina consists of the GCL (bottom), separated by a narrow region devoid of cell nuclei (IPL) from the proliferative zone that contains many postmitotic cells (Braekevelt and Hollenberg, 1970). Scale bars: in F, 10 µm for A,B,E,F; in J, 20 mm for C,D,I,J.

View larger version (63K): [in a new window]   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.

View larger version (24K): [in a new window]   Fig. 4. Antisense oligos to MAPs disrupt the orderly spacing of the ChAT-positive cells and scatter these cells at different retinal depths. (A) Delaunay segments link array cells (dots) with their surrounding neighbours. (B) Examples of the altered distributions of Delaunay segments (DS) associated to the GCL ChAT-positive array 24 hours after treatment with MAP2 phosphorothiated antisense oligos (black circles, 25 µM PPRM21; black squares, 25 µM PPRM38), or with MAP2 antisense with phosphorothioate links only at the 3' and 5' ends (continuous line indicates 50 µM ppRM21). The DS associated with normal and vehicle injected cases are shown as grey lines. Each curve is the average DS distribution of a single retina: as shown, the extent of the oligo effect varies significantly between treated retinas, but it always includes DS lengths outside the normal range. Each treated DS plot is significantly different from the normal DS histogram (KS test; P<0.0001). The difference between the treated and the normal cases was confirmed by the bootstrap method, which allows the comparison of datasets, taking into consideration their internal variability (see Materials and Methods). (C) Examples showing that the DS distribution of the GCL ChAT-positive array did not vary significantly with: (1) the phosphorothioate sense sequences complementary to the antisense oligos used (black circles, 25 µM RM21 sense; black squares, 25 µM RM11 sense); (2) blocking antibodies to ßFGF (white circles, 10 µg/ml), used to simulate potential nonspecific effects of PP oligos. Normal DS distributions are grey lines. Each treated DS plot was not significantly different from the normal DS histogram (KS test P<0.01). This result was confirmed by bootstrap analysis (see Materials and Methods). (D) Examples of the alteration of the DS distribution after treatment with a phosphorothioate antisense to tau (PPRT11: upward facing triangles, 12 µM; downward facing triangles, 25 µM). Normal DS are the grey curves. Each treated DS plot is significantly different from the normal DS histogram (KS test; P<0.0001). The difference between the treated and the normal cases was confirmed by the bootstrap method, which allows the comparison of datasets, taking into consideration their internal variability (see Materials and Methods). (E) Schematic representation of array cells at different retinal depths. Dots represent cells, the line an arbitrary reference depth. (F-H) Examples of the scatter of the GCL ChAT-positive cells at different retinal depths after treatment with MAP2 antisense (F), control treatments (G) and tau antisense (H). Each curve represents data from a single retina (see above). Symbols are as in B-D. The treated cases are illustrated upside-down to facilitate comparison with the normal controls (grey curve). The statistical significance of the effects was assessed by means of the bootstrap method and the K-S test (P<0.001). Between 5 and 20% of each retina was sampled for this analysis.

View larger version (94K): [in a new window]   Fig. 5. When array disruption occurs, processes that link array cells are still observed and contain microtubules throughout. (A) Double labelling for ChAT (green) and ß-tubulin (red) of a region of the GCL cholinergic array after treatment with MAP2 antisense. (A) Stack of confocal sections. (B-D) Confocal section through A (0.5 µm) , showing the double labelling (B) and ChAT (C) and ß-tubulin (D) immunoreactivity. Although the array cells are irregularly spaced, the ChAT-positive processes linking array cells contain microtubule throughout, as revealed by ß-tubulin immunoreactivity (e.g. the processes linking cells 2-3 or 3-5).

View larger version (78K): [in a new window]   Fig. 6. Disruption of the horizontal cell array 24 hours after treatment with oligo antisense to tau at P6. (A,B) The regular horizontal cell array (A, P6) is turned into an irregular distribution of cells (B) 24 hours after intraocular injections of 25 µM PP oligo RT11 antisense to tau (P7). Scale bar: 20 µm. (C,D) Array disorganization is quantified by the irregular distribution of Delaunay segments (C) and depth scatter (D) (symbols represent examples of treated cases, the grey curves represent normal cases). The treated cases in D are illustrated upside-down to facilitate comparison with the normal controls (grey curve). Bootstrap analysis confirms the statistically significant difference between the treated and the normal datasets.

View larger version (94K): [in a new window]   Fig. 7. Disruption of the GCL ChAT-positive array within hours of exposure to pharmacological agents affecting MTs. (A) The low doses of drugs used do not significantly alter the retina stratification 4 hours after treatment (normal is on the left; demecolchid treated is on the right). Notice the accumulation of mitotic cells at the top of the demecolchid treated retina (right). (B,C) The regular organization of the GCL ChAT-positive array is altered 2 hours after administration of demecholcid (B) or paclitaxel (C). Scale bars: in A, 20 µm in A; in C, 10 µm in B,C. (D, top) The distribution of Delaunay segments associated with the GCL ChAT-positive array is altered 2 hours after administration of 10 µM demecolchide, and recovers by 24 hours (black symbols are examples of treated cases; grey lines correspond to normal plots). (D, bottom) Examples of the effects of 5 µM paclitaxel 4 and 24 hours after administration (black symbols indicate treated cases, grey line indicates normal cases). The treated cases are statistically different from normal 2 and 4 hours after treatment, but not 24 hours after treatment (KS test, P<0.01 and bootstrap comparison of the datasets).