Neuronal integration in an abuttingretinas culture system

PURPOSE. Limited integration is consistently observed between subretinal transplants and host retinas. In the current study, an in vitro model system for studying connections forming between two abutting retinas was developed. METHODS. Neuroretinas were dissected from normal wild-type (WT) mice and green fluorescent protein (GFP) transgenic mice (obtained at postnatal days [P]0, P5, or P60), as well as from adult rd mice. Pieces from two different retinas (WT-WT, GFP-WT, GFP-rd) were placed side-by-side (contacting each other at the margins) or overlapping each other in organ cultures for 7 or 12 days. The abutting retinal pieces derived from animals of the same age (P5-P5; P60-P60) or of different ages (P0-P60; P5-P60). Retinal cells and fibers were visualized in wholemount preparations and in cross sections by immunocytochemistry using antibodies against neurofilament (NFϩ), neuronal nitric oxide synthase (NOSϩ), and protein kinase C (PKCϩ) and by GFP fluorescence (GFPϩ). RESULTS. In side-by-side pairs (WT-WT, GFP-WT), numerous horizontal cell fibers (NFϩ) and amacrine cell fibers (NOSϩ) crossed the interface between the two pieces, forming continuous plexiform layers. In overlapping pairs, NFϩ, NOSϩ, and PKCϩ fibers displayed parallel plexiform layers, and no crossover of fibers was observed in any of the pair combinations examined (WT-WT, GFP-WT, GFP-rd). Some integration was seen only in small areas where the structure of both retinal pieces was disrupted at the interface. CONCLUSIONS. The results demonstrate the ability of neurites to extend between abutting retinas and to make appropriate target choices when they are placed side-by-side. However, this ability is limited when they overlap each other, similar to that observed in subretinal transplantation. (Invest Ophthalmol Vis Sci. 2003;44:4936 -4946 ). Prompted by the problem of poor graft-host integration, we developed a modified culture system in which the outgrowth of fibers between two retinal pieces could be analyzed. The system consists of two abutting retinal pieces, placed overlapping each other, which is analogous to the in vivo situation of subretinal transplantation, or side by side. Using specific neuronal markers, we examined in wholemount preparations and in transverse sections, whether neuronal fibers can extend from one retinal piece into the abutting piece. Pairs were formed using retinal pieces derived from 5-day-old (P5) mice and cultured for 7 days, thus encompassing a time window (P5-P12) during which, in normal mouse development, substantial outgrowth of retinal cell processes occurs within the synaptic layers, and synaptic maturation is initiated. 11,12 MATERIALS AND METHODS Animals and Tissue Culture Preparation The experiments were conducted with the approval of the local animal experimentation and ethics committee. Animals were handled according to the guidelines on care and use of experimental animals set forth by the Government Committee on Animal Experimentation at the University of Lund and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The organ culture condition has been described in detail. 13 Retinas were dissected from normal mice (C57BL/6), from GFP mice (harboring a transgene consisting of enhanced GFP [EGFP] cDNA under the control of a chicken ␤-actin promoter and a cytomegalovirus enhancer), After the superior and nasal cornea were marked, the eyes were enucleated under sterile conditions and transferred to a dish containing serum-free medium (R16; Invitrogen-Gibco, Gaithersburg, MD). 15 Retinas were dissected from the retinal pigment epithelium (RPE) and from hyaloid vessels. Each retina was cut under fresh medium into four pieces along the superior-inferior and the nasal-temporal axes (Fig

Linking learning with governance in networks and clusters: key issues for analysis and policy

In this paper we analyse the relationship between governance and learning in clusters and networks. In particular, we see these two key elements as interdependent, suggesting that, under particular circumstances, such interdependence may drive clusters and networks towards a dynamic development trajectory. A pure ‘governance perspective’ makes the development of any locality dependent on the system of powers which exists within the locality or across the global value chain. In parallel, a pure ‘competence-based approach’ focuses mainly on the capabilities of actors to learn and undertake activities. In contrast, we open the prospects for an interdependent relation that may change the actual competences of actors as well as the governance settings and dynamics in networks and clusters. When supported by public policies, the learning process may have the potential to modify the governance environment. Simultaneously, the learning process is intrinsically influenced by economic power, which may seriously affect the development prospects of clusters and networks. This is why an intertwined consideration of both aspects is necessary to promote specific approaches to learning and to design appropriate policies. In this paper we offer two preliminary case studies to clarify some of these dynamics: the first taken from the computers cluster in Costa Rica and the second from an Italian bio-pharmaceutical firm and its production network. The first case study refers to the software cluster that was created from scratch in Costa Rica thanks to an enlightened government policy in coordination with new local enterprises and an important foreign direct investor; while the second reflects on the ability of an individual company to create a network of relationships with large transnational companies in order to acquire new competences without falling into a subordinate position with respect to its larger partners

Ubiquitous presence of gluconeogenic regulatory enzyme, fructose-1,6-bisphosphatase, within layers of rat retina

To shed some light on gluconeogenesis in mammalian retina, we have focused on fructose-1,6-bisphosphatase (FBPase), a regulatory enzyme of the process. The abundance of the enzyme within the layers of the rat retina suggests that, in mammals in contrast to amphibia, gluconeogenesis is not restricted to one specific cell of the retina. We propose that FBPase, in addition to its gluconeogenic role, participates in the protection of the retina against reactive oxygen species. Additionally, the nuclear localization of FBPase and of its binding partner, aldolase, in the retinal cells expressing the proliferation marker Ki-67 indicates that these two gluconeogenic enzymes are involved in non-enzymatic nuclear processes

Architecture of the mammalian pituitary cholinergic system with observations on a putative blood acetylcholine sensor

Acetylcholine ( ACh) play s an important role in pituitary gland function. Little is known, however, about the source and trajectory of pituitary ACh, the location of pituitary cholinergic receptors. and the pathways along which the release of pituitary ACh is controlled. Therefore choline acetyltransferase (ChAT) immunoreactive profiles have been investigated in the rat median eminence and pituitary. Furthermore, both muscarinic- (mAChRp-L) and nicotinic receptor proteinlike (nAChRp-L) immunoreactivity have been examined in the rat, rabbit, and cat pituitary. The results have demonstrated that the rat pituitary ChAT network is composed of neurons in the hypothalamic arcuate nucleus and a great number of terminals in the median eminence. In the pituitary, ChAT immunolabeled profiles were virtually absent. This suggests that much of the ACh acting on pituitary cells is released as a humoral factor from the median eminence. Al1 the examined animals expressed mAChRp-L immunostained endocrine cells in the intermediate lobe. Apart from this, marked species differences in AChRp-L immunolabeled profiles have been found. In addition, strong mAChRp-L immunoreactive rod to cone-shaped bodies were detected associated with blood vessels of the anterior and intermediate lobes in the rat and rabbit, but not in the cat. The immunolabeling was present in particles on the body plasma membrane. These characteristics suggest that the function of these structures might be to sense pituitary blood ACh levels. Consequently the name hlood acetylcholine reading hodies (BARBs) was adopted to indicate these stmctures. It is proposed that the BARBs may play a role in the feedback control of ACh release from the median eminence

Thyroid-beta2 and the retinoid RAR-alpha, RXR-gamma and ROR-beta2 receptor mRNAs; expression profiles in mouse retina, retinal explants and neocortex.

In neonatal retinal explants cultured long-term green cones are missing. Recently it was reported that thyroid hormone beta2 receptors (TR-beta2) are essential for these green cones to differentiate. Therefore transcript level of these receptors was investigated in our mouse retinal explants. However, thyroid receptors function as heterodimers with retinoid receptors (RR); so the fate of selected RRs was similarly analyzed using semi-quantitative RT-PCR. Loss of TR-beta2 and RR (RXR-gamma and ROR-beta2) mRNAs was observed after culturing the neonatal retina for 12 days. This indicates that these proteins are involved in determination of green cone identity. In addition, levels of the selected RR transcripts are differentially affected by short- or long-term culture. In the latter case an attached retinal pigment epithelium seems to play a protective role. Furthermore, divergent diurnal peaks of RR mRNAs are present in young as well as aged mouse retina and neocortex. This data might be relevant in the context of human ageing disorders

Neuronal integration in an abutting-retinas culture system

PURPOSE: Limited integration is consistently observed between subretinal transplants and host retinas. In the current study, an in vitro model system for studying connections forming between two abutting retinas was developed.METHODS: Neuroretinas were dissected from normal wild-type (WT) mice and green fluorescent protein (GFP) transgenic mice (obtained at postnatal days [P]0, P5, or P60), as well as from adult rd mice. Pieces from two different retinas (WT-WT, GFP-WT, GFP-rd) were placed side-by-side (contacting each other at the margins) or overlapping each other in organ cultures for 7 or 12 days. The abutting retinal pieces derived from animals of the same age (P5-P5; P60-P60) or of different ages (P0-P60; P5-P60). Retinal cells and fibers were visualized in wholemount preparations and in cross sections by immunocytochemistry using antibodies against neurofilament (NF+), neuronal nitric oxide synthase (NOS+), and protein kinase C (PKC+) and by GFP fluorescence (GFP+).RESULTS: In side-by-side pairs (WT-WT, GFP-WT), numerous horizontal cell fibers (NF+) and amacrine cell fibers (NOS+) crossed the interface between the two pieces, forming continuous plexiform layers. In overlapping pairs, NF+, NOS+, and PKC+ fibers displayed parallel plexiform layers, and no crossover of fibers was observed in any of the pair combinations examined (WT-WT, GFP-WT, GFP-rd). Some integration was seen only in small areas where the structure of both retinal pieces was disrupted at the interface.CONCLUSIONS: The results demonstrate the ability of neurites to extend between abutting retinas and to make appropriate target choices when they are placed side-by-side. However, this ability is limited when they overlap each other, similar to that observed in subretinal transplantation

Mouse retina explants after long-term culture in serum free medium

Abstract The neonatal mouse retina remains viable as an explant in serum-supplemented growth media for more than 4 weeks. Interpretation of drug effects on this tissue is compromised by the enigmatic composition of the serum. We sought to remove this ambiguity by culturing neonatal as well as late postnatal mouse retina in serum-free nutrient medium. In this study three important observations were made, (1) there is histotypic development of neonatal as well as preservation of late postnatal mouse retinal structure during long-term culture in serum-free medium, although the late postnatal tissue tends to show some loss of cells in the outer nuclear layer. (2) Protein expression in explant photoreceptor cells was similar to that in the litter-matched ones, except for green cone opsin and interphotoreceptor retinoid-binding protein, although mRNA of the latter is present at similar amounts as in age-matched in vivo controls. (3) Cells of the inner retina stained by antibodies to calcium-binding proteins display some novel sprouting of processes. The results show that the mouse retina can be cultured as an explant for more than 4 weeks in a serum-free medium. This represents an important step forward because, (1) the possibility of interference of drug effects by unknown serum factors has been eliminated; and (2) the spent culture medium can be analyzed to investigate biomolecules released by the retina in vitro