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

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

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

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

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

(A) Accelerated natural cell death in the RGC layer of EC rats.

<p>RGC layer apoptotic cell number in EC and SC rats, analyzed at the indicated ages with the Tunel method (top) and with cresyl violet staining of whole-mount retinas (bottom). With both methods, two-ways ANOVA showed an effect of age (<i>p</i><0.001) and housing condition (<i>p</i><0.05) and a significant age×housing condition interaction (<i>p</i><0.001). Mann-Whitney rank sum test with Bonferroni correction revealed a difference between EC and SC at E18, E20 and P1 (<i>p</i><0.001) for the tunel method, and at E18 and P1 (<i>p</i> = 0.002) for cresyl violet staining. (B) RGC number was not different between SC and EC adult rats either as estimated by calculating the 50% of total cell number in the RGC layer (A), or by subtracting the number of displaced amacrine cells remaining in the RGC layer 30 days after ipsilateral optic nerve transection from the number of cells counted in contralateral retinas (B) (<i>p</i> = 0.77 and 0.28, t-test). (C) Micrographs of RGC layer of P1 whole mount retinas labeled with B4 isolectin. No qualitative difference was detected in the shape and intensity of microglial cells between SC and EC pups. Scale bar: 20 µm. Graph: microglial cell number in the RGC layer of SC and EC rats. Mann-Whitney rank sum test showed no difference between the two groups (<i>p</i> = 0.429). Bars indicate s.e.m.</p

Identification of specific cell types involved in the accelerated migration of retinal cells in EC fetuses.

<p>(A, top) Micrographs of EC and SC retinal sections immunostained for ISLET-1 (a marker for ganglion and cholinergic amacrine cells) at E15, acquired at 5× (left, scale bar: 100 µm) or 20× (right, scale bar: 50 µm). (A, bottom) Number of cells stained for ISLET-1 in the outer retinal layers of SC and EC rats. The number of ISLET-1-labeled cells was higher in EC than in SC embryos (t-test, <i>p</i><0.001). Bars indicate s.e.m. (B) Micrographs of EC and SC retinal sections immunostained for calbindin (a marker for horizontal cells) at E15, acquired at 20× (scale bar: 50 µm). The number of calbindin-labeled cells did not differ between EC and SC embryos (t-test, <i>p</i> = 0.253). (C) Micrographs of EC and SC retinal sections co-immunostained for DCX (red) and ISLET-1 (green) at E15, acquired at 60x. Scale bar: 10 µm.</p

Environmental enrichment promotes recovery of behavioural perceptual abilities in adult amblyopic monocular animals.

<p>a) Statistical analysis showed that the visual acuity (VA) of the previously deprived eye was not different from that of the other eye (fellow eye) in OND-EE rats (paired t-test, p = 0.51); a statistical difference was instead present between the VA of the previously deprived eye and that of the fellow eye in OND-SC animals (paired t-test, p<0.05). Insert shows a schematic representation of the visual water-box task, the apparatus used for behavioral assessment of visual acuity. b) Representative examples of behavioral VA estimates for both the previously deprived and the fellow eye in OND-SC (left) and OND-EE (right) animals. Visual acuity is obtained by extrapolation to 70% of correct choices on the sigmoidal function fitting the psychometric function in which the percentage of correct choices is plotted against zero spatial frequency. *, statistical significance; error bars represent s.e.m.</p

IGF-I is the mediator of maternal enrichment effects on retinal development in the fetus.

<p>Number of DCX (A) and of ISLET-1 (B) positive cells in the outer retinal layers of EC, anti-IGF-I EC, SC and IGF-I SC rats at E15. For both (A) and (B), one-way ANOVA showed an effect of the treatment (<i>p</i><0.05). A difference was found between EC and SC, between EC and EC anti-IGF-I and between SC and SC IGF-I groups (<i>p</i><0.05, Post-hoc Tukey test). Neither EC anti-IGF-I and SC groups nor EC and SC IGF-I groups were instead found to differ between each other. (C) Quantitative analysis of IGF-I immunofluorescence intensity in the RGC layer of EC, anti-IGF-I EC, SC and IGF-I SC rats at E18. (D) Pyknotic cell number of EC, anti-IGF-I EC, SC and IGF-I SC rats at E18. After treatment with anti-IGF-I, levels of IGF-I expression and number of pyknotic profiles in the RGC layer of EC fetuses were lowered to those of SC rats while, after chronic IGF-I protein infusion, levels of IGF-I expression and the number of pyknotic profiles in the RGC layer of SC fetuses were enhanced up to those of EC rats. For both (C) and (D), one-way ANOVA showed an effect of the housing treatment (<i>p</i><0.001). A difference was found between EC and SC, between EC and EC anti-IGF-I and between SC and SC IGF-I groups (<i>p</i><0.05, Post-hoc Tukey test). Neither EC anti-IGF-I and SC groups nor EC and SC IGF-I groups were instead found to differ between each other. The bars indicate s.e.m.</p