Percentage of bistratified RGCs during development in non-EE and EE mice.

<p>Data in bold are the percentage of bistratified RGCs obtained from whole-mount retinas (wm). For each retina, the total number of RGCs analyzed and the percentage of bistratified RGCs are indicated; for each group, the mean percentage of bistratified RGCs and the total number of RGCs analyzed are also reported.</p

EE affects the maturational refinement of RGC dendrites.

<p>(A) Mean percentage of bistratified RGCs in non-EE (black) and EE mice (red) at P10 (non-EE: 65,8±3,5%, N = 4, 79/115 cells; EE: 44,2±3,7%, N = 5, 47/107 cells), P16 (non-EE: 53,8±3,2%, N = 4, 91/169 cells; EE: 36,7±5,7%, N = 5, 66/193 cells) and P30 (non-EE: 30,8±2,9%, N = 5, 54/169 cells; EE: 32,9±3%, N = 3, 44/138 cells). Two Way ANOVA shows a significant effect of age (p = 0,006) and environmental housing condition (p<0,001). <i>Post-hoc</i> Tukey's test reveals a significant difference between EE and non-EE at P10 and P16 (asterisk). The bars indicate SEM. EE accelerates the process of the segregation of RGC arborizations. (B) Mean percentage of bistratified RGCs in non-EE (hatched, black, N = 3, 51,5±0,9%, 48/93 cells) and EE (hatched, red, N = 3, 37,8±4,2%, 37/97 cells) P16 mice obtained from confocal reconstructed images in whole-mount retinas after digital rotation, as exemplified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000346#pone-0000346-g001" target="_blank">Fig. 1</a>. Data obtained in retinal vertical sections are replotted from A for direct comparison (solid bars). There is no difference between the results obtained with these two methods of dendritic stratification analysis (Two Way ANOVA, housing×method, housing p = 0,006, method p = 0,911; no significant interaction). The size of the RGC sample and the percentage of bistratified RGCs are reported, for each retina, in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000346#pone-0000346-t001" target="_blank">Table 1</a>.</p

RGC stratification during postnatal development in normal non-EE Thy-1 mGFPmice.

<p>(A) Schematic representation illustrating the passage from immature to adult state during development of RGC dendritic stratification (cholinergic amacrine cells in red, RGCs in green). (B) Percentages of monostratified and bistratified RGCs during development in normal non-EE mice between P10 and P30 are respectively 34,2±3,5% at P10 (N = 4), 46,2±3,2% at P16 (N = 4), 69,2±2,9% at P30 (N = 5) for monostratified cells, and 65,8±3,5% at P10, 53,8±3,2% at P16, 30,8±2,9% at P30 for bistratified cells. Vertical bars indicate SEM. There is a significant decline of bistratified RGCs with age (One Way ANOVA, p<0,001). The size of the RGC sample (total number of RGCs analyzed) and the percentage of bistratified RGCs are reported for each retina in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000346#pone-0000346-t001" target="_blank">Table 1</a>.</p

Dendritic stratification pattern of RGCs in Thy-1 mGFP mice.

<p>(A) Confocal image of an RGC from whole-mount retina of a mGFP mouse. GFP expression is enhanced with a specific immunostaining. Immunostaining with anti-GFP antibody shows that RGC somata, dendrites and axons are GFP-positive [scale bar = 50 µm]. (B) Schematic representation of the patterning of cholinergic amacrine cell projections (red), which identify the a and b sublaminae of the IPL. An ON- and an OFF-center RGC, with dendrites monostratified in the b or a sublaminae, respectively, and an ON-OFF bistratified RGC are drawn in green. GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer. (C, D, E): Top row: examples of RGC (green) confocal reconstructed maximum projection images taken from whole-mount retinas from P16 mGFP mice; bottom row: 90 degrees rotation images of the cells displayed above. Red label denotes the immunolabeling pattern of Choline Acetyltransferase (ChAT) positive amacrine cells. Confocal microscopy was used to produce stacked images of three-dimensional reconstructed GFP-expressing RGCs and of ChAT positive amacrine cells. ChAT positive cell bodies are respectively in the GCL and in the INL, while their projections form two bands clearly visible in the rotated images (white arrow heads) that run along the sublamina a and b of the IPL. Bistratified RGCs present a double-layered segregated arborization with respect to the two anti-ChAT labeled bands (C, bottom), while monostratified ganglion cells have their dendrites proximal to the cell body and restricted to the ChAT positive band within sublamina b (D, bottom) or distal to the cell body and restricted to the outermost ChAT positive band in sublamina a (E, bottom) [scale bars = 50 µm]. (F, G, H) Examples of RGC (green) confocal images taken from 25 µm vertical retinal sections from P30 mGFP mice. The red bands representing the projections of cholinergic amacrine cells immunolabeled with ChAT, which denote the sublaminae of the IPL, are pointed at with white arrow heads [scale bar = 50 µm].</p

EE counteracts DR effects promoting RGC dendritic maturation.

<p>(A) The average percentage of bistratified RGCs in normal non-EE (white), DR (black), and EE-DR mice (grey) at P30. The percentage of bistratified RGCs is 30,8±2,9% in non-EE mice (N = 4, data replotted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000346#pone-0000346-g002" target="_blank">Fig. 2</a>); DR blocks RGC dendritic stratification (bistratified cells 55,9±5,2% at P30, N = 5 mice, 65/121 cells), while this process takes place normally in EE-DR mice (P30 EE-DR mice: bistratified cells 32,2±1,4%, N = 4, 40/126 cells). One Way ANOVA shows a statistically significant difference between normal non-EE and DR, and between EE-DR and DR mice; EE-DR are not different from non-EE mice (One Way ANOVA, p<0,001; <i>post-hoc</i> Tukey's test). The bars indicate SEM. EE from birth prevents DR effects on the developmental remodelling of RGC dendrites. (B) Percentage of bistratified RGCs and sample size for each retina in DR and EE-DR mice. Data for non-EE mice are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000346#pone-0000346-t001" target="_blank">Table 1</a>.</p

BDNF mediates the effects of EE on RGC dendritic segregation.

<p>(A) Examples of an ON-OFF RGC (left), an ON RGC (center), an OFF RGC (right) in retinal vertical sections of P16 EE antisense treated mice (top row) or sense treated mice (bottom row) (scale bar: 50 µm). Conventions as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000346#pone-0000346-g001" target="_blank">Fig. 1</a>. (B) Average percentage of bistratified RGCs in P16 non-EE mice (white), untreated EE mice (control, black), EE mice treated with BDNF antisense (antisense, vertical line-pattern) and EE mice treated with BDNF sense (sense, horizontal line-pattern). In the retinas of EE mice injected with BDNF antisense oligos (N = 5) the percentage of bistratified RGCs is similar to that of normal non-EE mice of the same age (51,9±2,5%, 43/84 cells <i>versus</i> 53,8±3,2%, 91/169 cells), whereas the control treatment with sense oligos (N = 5) has no effect on the accelerated development produced by EE (32,2±3,5%, 29/88 cells <i>versus</i> 36,9±5,2%, 31/81 cells in EE mice treated with BDNF sense oligos and EE untreated mice, respectively). One Way ANOVA indicates a statistical difference (asterisks) between control EE and antisense treated EE, between sense and antisense treated EE mice and between non-EE and control or sense treated EE mice; no difference is found between untreated (control) and sense treated EE mice and between non-EE and antisense treated EE mice (p = 0,001; <i>post-hoc</i> Tukey's test). The bars indicate SEM. The blockade of BDNF expression blocks the effects of EE on RGC dendritic stratification.</p