The insulin-like growth factor 1 receptor is essential for axonal regeneration in adult central nervous system neurons

Axonal regeneration is an essential condition to re-establish functional neuronal connections in the injured adult central nervous system (CNS), but efficient regrowth of severed axons has proven to be very difficult to achieve. Although significant progress has been made in identifying the intrinsic and extrinsic mechanisms involved, many aspects remain unresolved. Axonal development in embryonic CNS (hippocampus) requires the obligate activation of the insulin-like growth factor 1 receptor (IGF-1R). Based on known similarities between axonal growth in fetal compared to mature CNS, we decided to examine the expression of the IGF-1R, using an antibody to the βgc subunit or a polyclonal anti-peptide antibody directed to the IGF-R (C20), in an in vitro model of adult CNS axonal regeneration, namely retinal ganglion cells (RGC) derived from adult rat retinas. Expression of both βgc and the β subunit recognized by C20 antibody were low in freshly isolated adult RGC, but increased significantly after 4 days in vitro. As in embryonic axons, βgc was localised to distal regions and leading growth cones in RGC. IGF-1R-βgc co-localised with activated p85 involved in the phosphatidylinositol-3 kinase (PI3K) signaling pathway, upon stimulation with IGF-1. Blocking experiments using either an antibody which neutralises IGF-1R activation, shRNA designed against the IGF-1R sequence, or the PI3K pathway inhibitor LY294002, all significantly reduced axon regeneration from adult RGC in vitro (∼40% RGC possessed axons in controls vs 2-8% in the different blocking studies). Finally, co-transfection of RGC with shRNA to silence IGF-1R together with a vector containing a constitutively active form of downstream PI3K (p110), fully restored axonal outgrowth in vitro. Hence these data demonstrate that axonal regeneration in adult CNS neurons requires re-expression and activation of IGF-1R, and targeting this system may offer new therapeutic approaches to enhancing axonal regeneration following trauma.Fil: Dupraz, Sebastian, Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Córdoba. Centro de Investigaciones en Química Biológica de Cordoba (p); Argentina;Fil: Grassi, Diego Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Córdoba. Centro de Investigaciones en Química Biológica de Cordoba (p); Argentina;Fil: Karnas, Diana. Rhythms, Life and Death in the Retina. Centre National de la Recherche Scientifique (CNRS). Université de Strasbourg. Institut des Neurosciences Cellulaires et Intégratives; France;Fil: Nieto Guil, Alvaro Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Córdoba. Centro de Investigaciones en Química Biológica de Cordoba (p); Argentina;Fil: Hicks, David. Rhythms, Life and Death in the Retina. Centre National de la Recherche Scientifique (CNRS). Université de Strasbourg. Institut des Neurosciences Cellulaires et Intégratives; France;Fil: Quiroga, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Córdoba. Centro de Investigaciones en Química Biológica de Cordoba (p); Argentina

Intrinsic photosensitive retinal ganglion cells in the diurnal rodent, Arvicanthis ansorgei.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) represent a new class of photoreceptors which support a variety of non-image forming physiological functions, such as circadian photoentrainment, pupillary light reflex and masking responses to light. In view of the recently proposed role of retinal inputs for the regulation of diurnal and nocturnal behavior, we performed the first deep analysis of the ipRGC system in a diurnal rodent model, Arvicanthisansorgei, and compared the anatomical and physiological properties of ipRGCs with those of nocturnal mice. Based on somata location, stratification pattern and melanopsin expression, we identified two main ipRGC types in the retina of Arvicanthis: M1, constituting 74% of all ipRGCs and non-M1 (consisting mainly of the M2 type) constituting the following 25%. The displaced ipRGCs were rarely encountered. Phenotypical staining patterns of ganglion cell markers showed a preferential expression of Brn3 and neurofilaments in non-M1 ipRGCs. In general, the anatomical properties and molecular phenotyping of ipRGCs in Arvicanthis resemble ipRGCs of the mouse retina, however the percentage of M1 cells is considerably higher in the diurnal animal. Multi-electrode array recordings (MEA) identified in newborn retinas of Arvicanthis three response types of ipRGCs (type I, II and III) which are distinguished by their light sensitivity, response strength, latency and duration. Type I ipRGCs exhibited a high sensitivity to short light flashes and showed, contrary to mouse type I ipRGCs, robust light responses to 10 ms flashes. The morphological, molecular and physiological analysis reveals very few differences between mouse and Arvicanthis ipRGCs. These data imply that the influence of retinal inputs in defining the temporal niche could be related to a stronger cone input into ipRGCs in the cone-rich Arvicanthis retina, and to the higher sensitivity of type I ipRGCs and elevated proportion of M1 cells.journal articleresearch support, non-u.s. gov't20132013 08 09importedFunding: Research was carried out within the scope of the Associated European Laboratory “European Laboratory for Circadian Research”, LEA CNRS-UdS-MPG (LEA No. 367) funded by the Max Planck Society, München, and CNRS, Paris. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Intrinsically photosensitive retinal ganglion cells (ipRGCs) in nocturnal and diurnal rodents : a morphological, molecular and physiological comparison

Les horloges circadiennes, permettant l´anticipation des changements environnementaux cycliques, sont synchronisés par la lumière du jour via un signal lumineux à la rétine. Outre les cônes et les bâtonnets, la rétine contient des cellules ganglionnaires intrinsèquement photosensibles (ipRGCs), subdivisées en sous-types distincts exprimant le pigment mélanopsine et impliquées dans l´entrainement de l´horloge biologique à la lumière. Le système circadien est très similaire chez les animaux nocturnes et diurnes. L'objectif de cette thèse était d'étudier les propriétés morphologiques, moléculaires etphysiologiques des ipRGCs de rongeurs nocturnes (souris) et diurnes (Arvicanthis ansorgei). Ce travail révèle des morphologies comparables des différents types d´ipRGCs pour les deux espèces, mais la proportion du type M1 était plus élevée chez Arvicanthis. Des immunomarquages spécifiques des cellules ganglionnaires de la rétine ont révélé que les ipRGCs constituent une population hétérogène. Chez les deux espèces, l'expression de neurofilaments et de Brn3 différait selon le type d´ipRGC. Les propriétés physiologiques des ipRGCs étaient principalement similaires pour les deux espèces. Chez Arvicanthis, les ipRGCs de type I étaient plus sensibles à de courts éclairs lumineux. En conclusion , les ipRGCs des rongeurs nocturnes et diurnes partagent des caractéristiques communes. Cette étude est la première à décrire la sensibilité des ipRGCs a des éclairs de courte durée. De plus, ce travail étend les connaissances sur l'hétérogénéité moléculaire des différents types d´ipRGCs.Circadian clocks permit anticipation of cyclic environmental changes and are synchronized to solar day through photic input from the retina. Besides rods and cones, the retina contains intrinsically photosensitive retinal ganglion cells (ipRGCs), consisting of distinct sub-types. IpRGCs express the photopigment melanopsin and are implicated in photoentrainment of the biological clock. Light information shapes the animal’s temporal behavior, but the circadian systems of nocturnal and diurnal animals appear to be very similar. The aim of this thesis was to investigate the morphological, molecular and physiologicalproperties of ipRGCs in nocturnal (C57BL/6 mouse) compared to diurnal (Arvicanthis ansorgei) rodents. The morphological analysis revealed comparable characteristics of the different ipRGC types in both species; however the proportion of M1 cells was higher in Arvicanthis than in mouse. Immunostaining patterns using RGC markers revealed that ipRGCs are a heterogeneous population. In both species, Brn3 and neurofilaments expression were partly distinct between the ipRGC types.The physiological properties of ipRGC types were mostly similar between the two species, but type I ipRGCs were more sensitive to short light flashes in Arvicanthis than in mouse. In conclusion, the melanopsin system of nocturnal and diurnal rodents shares many common features. Importantly, this study is the first describing responses of ipRGCs to short light flashes and the observed molecular heterogeneity extends the characterization of individual ipRGC types

Intrinsically photosensitive retinal ganglion cells (ipRGCs) in nocturnal and diurnal rodents : a morphological, molecular and physiological comparison

Les horloges circadiennes, permettant l´anticipation des changements environnementaux cycliques, sont synchronisés par la lumière du jour via un signal lumineux à la rétine. Outre les cônes et les bâtonnets, la rétine contient des cellules ganglionnaires intrinsèquement photosensibles (ipRGCs), subdivisées en sous-types distincts exprimant le pigment mélanopsine et impliquées dans l´entrainement de l´horloge biologique à la lumière. Le système circadien est très similaire chez les animaux nocturnes et diurnes. L'objectif de cette thèse était d'étudier les propriétés morphologiques, moléculaires etphysiologiques des ipRGCs de rongeurs nocturnes (souris) et diurnes (Arvicanthis ansorgei). Ce travail révèle des morphologies comparables des différents types d´ipRGCs pour les deux espèces, mais la proportion du type M1 était plus élevée chez Arvicanthis. Des immunomarquages spécifiques des cellules ganglionnaires de la rétine ont révélé que les ipRGCs constituent une population hétérogène. Chez les deux espèces, l'expression de neurofilaments et de Brn3 différait selon le type d´ipRGC. Les propriétés physiologiques des ipRGCs étaient principalement similaires pour les deux espèces. Chez Arvicanthis, les ipRGCs de type I étaient plus sensibles à de courts éclairs lumineux. En conclusion , les ipRGCs des rongeurs nocturnes et diurnes partagent des caractéristiques communes. Cette étude est la première à décrire la sensibilité des ipRGCs a des éclairs de courte durée. De plus, ce travail étend les connaissances sur l'hétérogénéité moléculaire des différents types d´ipRGCs.Circadian clocks permit anticipation of cyclic environmental changes and are synchronized to solar day through photic input from the retina. Besides rods and cones, the retina contains intrinsically photosensitive retinal ganglion cells (ipRGCs), consisting of distinct sub-types. IpRGCs express the photopigment melanopsin and are implicated in photoentrainment of the biological clock. Light information shapes the animal’s temporal behavior, but the circadian systems of nocturnal and diurnal animals appear to be very similar. The aim of this thesis was to investigate the morphological, molecular and physiologicalproperties of ipRGCs in nocturnal (C57BL/6 mouse) compared to diurnal (Arvicanthis ansorgei) rodents. The morphological analysis revealed comparable characteristics of the different ipRGC types in both species; however the proportion of M1 cells was higher in Arvicanthis than in mouse. Immunostaining patterns using RGC markers revealed that ipRGCs are a heterogeneous population. In both species, Brn3 and neurofilaments expression were partly distinct between the ipRGC types.The physiological properties of ipRGC types were mostly similar between the two species, but type I ipRGCs were more sensitive to short light flashes in Arvicanthis than in mouse. In conclusion, the melanopsin system of nocturnal and diurnal rodents shares many common features. Importantly, this study is the first describing responses of ipRGCs to short light flashes and the observed molecular heterogeneity extends the characterization of individual ipRGC types

The psychology of the environment in children's health care setting : James Whitcomb Riley Hospital for Children - Cancer Unit

Hospitals are constantly evolving to keep pace with the latest medical technologies. Whether it is a refurbishment of an existing facility or the addition of a new unit, the design process usually focuses on the technological requirements rather than the human elements of such an undertaking. The Riley Hospital for Children in Indianapolis presents an architectural opportunity to incorporate psychology of the environment into the design and construction of a new Cancer Unit. By balancing the technological requirements with the physical and psychological needs of the pediatric bone marrow transplant and hematology/oncology patients, one can create a healing environment more conducive to a rapid recovery.Thesis (M. Arch.)Department of Architectur

Density of melanopsin-positive ipRGCs in the retina of <i>Arvicanthis</i> at different ages.

<p><b>A</b>) Representative photomicrographs showing the change of the density of melanopsin-positive ipRGCs at different stages of the postnatal development. Scale bar: 100 µm. <b>B</b>) Plot showing a change of mean density of ipRGCs in the course of postnatal development. In the new-born retina (P0, postnatal day 0) almost 300 cells/mm² were observed. The values considerably declined during the first 2 postnatal weeks reaching a density of 20–40 cells/mm² in adult animals and stayed at nearly constant levels until the late adulthood. Values are presented as mean density (cells/mm²) ± SEM (1–6 retinae per age group).</p

Morphology of orthopic and displaced ipRGCs in the retina of <i>Arvicanthis.</i>

<p><b>A</b>–<b>H</b>) Retinal wholemounts of adult (9 months old) <i>Arvicanthis</i> immunostained for melanopsin (A, C, E, G; green in B, D, F, H) and ChAT (magenta in B, D, F, H), a cholinergic amacrine cell marker. Pictures were taken from focal planes on the GCL (A–B), IPL (C–D), IPL/INL border (E–F) and the INL (G–H). <b>I</b>–<b>K</b>) Side view of the cells. Orthotopic M1 cell (arrow in A and C) have cell bodies located in the GCL and stratify in the outer portion of the IPL (OFF sublamina), close to the INL (E–H and I). These cells show stronger melanopsin-immunoreaction than M2 cells (arrowhead in A), including cell body and dendrites. Displaced M1 (M1-d) cells show a comparable intensity of melanopsin staining to the orthotopic M1 cells, nevertheless their cell bodies are located in the INL (two headed arrow in E and G). M1-d cells stratify in the outer portion of the IPL, similar to their orthotopic counterparts (E–H and J–K). Figure 2 I shows only one M1 cell, Figure 2 J a M1 and a M1-d cell, and Figure 2 K only a single M1-d cell. ChAT, choline acetyl transferase; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer. Scale bar: 50 µm.</p

Responses of ipRGCs from <i>Arvicanthis</i> and mouse retina to stimuli of different duration.

<p><b>A</b>) Light-induced responses of ipRGCs in <i>Arvicanthis</i> (left) and in the mouse retina (right) to short flashes of constant irradiance (14.3 log N_Q * cm^-2 * s^-1). The most sensitive ipRGC type (type I) respond to very short light flashes of up to 10 ms in <i>Arvicanthis</i> and 50 ms in the mouse, respectively, whereas type II and type III ipRGCs were insensitive to flashes shorter than 1 s. <b>B</b>) Response characteristics of ipRGCs type I of <i>Arvicanthis</i> to light flashes of different duration. The left graph shows that the mean spike rate (filled circles) and peak firing rate (open circles) reached a maximum with stimuli lengths of 1 s. The response duration (middle graph) continuously increased with the stimulus duration, whereas response latencies (right graph; filled circles: latency to the first spike, open circles: latency to peak firing rate) rapidly declined with increasing stimulus duration. Data presented as mean values ± SEM.</p

Morphology of M1 and M2 ipRGCs in the retina of <i>Arvicanthis.</i>

<p><b>A</b>–<b>I</b>) Wholemounts of the retina from adult (9 months old) <i>Arvicanthis</i> immunostained for melanopsin (A, D, G; green in C, F, I) and ChAT (B, E, H; magenta in C, F, I), a cholinergic amacrine cell marker. Pictures were taken from focal planes on the GCL (A–C), IPL (D–F) and IPL/INL border (G–I). M1 cells show an intense melanopsin staining (arrow in A and C). Strongly melanopsin-positive dendrites of M1 cells pass the IPL (arrow in D and F) and stratify in the outer portion of the IPL (OFF sublamina) in proximity to the INL (in G and I the arrow shows the branching point of the main dendrite); M2 cells show a less intense melanopsin staining (arrowhead in A and C). Weakly melanopsin-positive M2 dendrites stratify in the inner portion of IPL (ON sublamina) near to the GCL (A–C). <b>J</b>–<b>L</b>) Side view and stratification level of M1 cell (soma indicated by an arrow). <b>M</b>–<b>O</b>) Side view and stratification level of the M2 cell (soma indicated by an arrowhead); note that in (M) and (O) also the intense OFF plexus of processes from melanopsin cells is visible. ChAT, choline acetyl transferase; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; Off, OFF sublamina of IPL; On, ON sublamina of IPL. Scale bar: 50 µm.</p