Environmental Enrichment Effects on Development of Retinal Ganglion Cell Dendritic Stratification Require Retinal BDNF

A well-known developmental event of retinal maturation is the progressive segregation of retinal ganglion cell (RGC) dendrites into a and b sublaminae of the inner plexiform layer (IPL), a morphological rearrangement crucial for the emergence of the ON and OFF pathways. The factors regulating this process are not known, although electrical activity has been demonstrated to play a role. Here we report that Environmental Enrichment (EE) accelerates the developmental segregation of RGC dendrites and prevents the effects exerted on it by dark rearing (DR). Development of RGC stratification was analyzed in a line of transgenic mice expressing plasma-membrane marker green fluorescent protein (GFP) under the control of Thy-1 promoter; we visualized the a and b sublaminae of the IPL by using an antibody selectively directed against a specific marker of cholinergic neurons. EE precociously increases Brain Derived Neurotrophic Factor (BDNF) in the retina, in parallel with the precocious segregation of RGC dendrites; in addition, EE counteracts retinal BDNF reduction in DR retinas and promotes a normal segregation of RGC dendrites. Blocking retinal BDNF by means of antisense oligos blocks EE effects on the maturation of RGC dendritic stratification. Thus, EE affects the development of RGC dendritic segregation and retinal BDNF is required for this effect to take place, suggesting that BDNF could play an important role in the emergence of the ON and OFF pathways

Environmental Enrichment Promotes Plasticity and Visual Acuity Recovery in Adult Monocular Amblyopic Rats

Loss of visual acuity caused by abnormal visual experience during development (amblyopia) is an untreatable pathology in adults. In some occasions, amblyopic patients loose vision in their better eye owing to accidents or illnesses. While this condition is relevant both for its clinical importance and because it represents a case in which binocular interactions in the visual cortex are suppressed, it has scarcely been studied in animal models. We investigated whether exposure to environmental enrichment (EE) is effective in triggering recovery of vision in adult amblyopic rats rendered monocular by optic nerve dissection in their normal eye. By employing both electrophysiological and behavioral assessments, we found a full recovery of visual acuity in enriched rats compared to controls reared in standard conditions. Moreover, we report that EE modulates the expression of GAD67 and BDNF. The non invasive nature of EE renders this paradigm promising for amblyopia therapy in adult monocular people

Altered adipocyte differentiation and unbalanced autophagy in type 2 Familial Partial Lipodystrophy: an in vitro and in vivo study of adipose tissue browning

Type-2 Familial Partial Lipodystrophy is caused by LMNA mutations. Patients gradually lose subcutaneous fat from the limbs, while they accumulate adipose tissue in the face and neck. Several studies have demonstrated that autophagy is involved in the regulation of adipocyte differentiation and the maintenance of the balance between white and brown adipose tissue. We identified deregulation of autophagy in laminopathic preadipocytes before induction of differentiation. Moreover, in differentiating white adipocyte precursors, we observed impairment of large lipid droplet formation, altered regulation of adipose tissue genes, and expression of the brown adipose tissue marker UCP1. Conversely, in lipodystrophic brown adipocyte precursors induced to differentiate, we noticed activation of autophagy, formation of enlarged lipid droplets typical of white adipocytes, and dysregulation of brown adipose tissue genes. In agreement with these in vitro results indicating conversion of FPLD2 brown preadipocytes toward the white lineage, adipose tissue from FPLD2 patient neck, an area of brown adipogenesis, showed a white phenotype reminiscent of its brown origin. Moreover, in vivo morpho-functional evaluation of fat depots in the neck area of three FPLD2 patients by PET/CT analysis with cold stimulation showed the absence of brown adipose tissue activity. These findings highlight a new pathogenetic mechanism leading to improper fat distribution in lamin A-linked lipodystrophies and show that both impaired white adipocyte turnover and failure of adipose tissue browning contribute to disease.We thank FPLD2 patients for donating biological samples. We thank the Italian Network for Laminopathies and the European Consortium of Lipodystrophies (ECLip) for support and helpful discussion. We thank Aurelio Valmori for the technical support. The studies were supported by Rizzoli Orthopedic Institute “5 per mille” 2014 project to MC, AIProSaB project 2016 and Fondazione Del Monte di Bologna e Ravenna grant 2015–2016 “New pharmacological approaches in bone laminopathies based on the use of antibodies neutralizing TGF beta 2” to GL. GL is also supported by PRIN MIUR project 2015FBNB5Y.S

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

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