Blue light exacerbates and red light counteracts negative insults to retinal ganglion cells in situ and R28 cells in vitro

1. We have begun to analyze neurotransmitter-activated conductances in retinal ganglion cells by measuring the response of single voltage-clamped adult goldfish ganglion cells to gamma-aminobutyric acid (GABA). Here we describe 1) our method of identifying ganglion cells in vitro after their dissociation from papain-treated retinas, and 2) the response of these cells to GABA in the tight-seal whole cell configuration of the patch-clamp method (cf. 41) after 1-4 days of primary cell culture. 2. Ganglion cell somata were backfilled in situ by injections of horseradish peroxidase (HRP) into the optic nerve. After dissociation of the retinas containing these cells, HRP reaction product was localized to cells that retained the size, shape, and an intracellular organelle characteristic of ganglion cells in situ. These features enabled us thereafter to identify ganglion cells in vitro without retrograde marker transport. 3. GABA (3-10 microM) elicited inward currents and substantial noise increases in almost all ganglion cells at negative holding potentials. Reversal potential measurements in salines containing different chloride concentrations indicated that GABA produces a chloride-selective conductance increase in ganglion cells. Bicuculline (10 microM) reversibly inhibited ganglion cell GABA responses. Baclofen (10 microM) alone elicited no responses in ganglion cells. 4. Noise analysis of GABA-activated whole cell currents yielded elementary conductance estimates of 16 pS, with a slow time constant of 30 ms plus a faster component of 1-2 ms. No significant voltage dependence of these values was observed between -20 and -80 mV. 5. We have thus devised a means of identifying ganglion cells dissociated from adult retinas, identified GABAA receptors (cf. 16) on these cells, and found that the responses mediated by these receptors resemble those found in other regions of central nervous system (CNS). These results are consistent with the notion that GABA may function as an inhibitory transmitter at synapses on ganglion cells.

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Comparative lipidomic analysis of mammalian retinal ganglion cells and Müller glia in situ and in vitro using High-Resolution Imaging Mass Spectrometry

Scientific Reports ◽

10.1038/s41598-020-77087-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Xandra Pereiro ◽

Roberto Fernández ◽

Gabriel Barreda-Gómez ◽

Noelia Ruzafa ◽

Arantxa Acera ◽

...

Keyword(s):

Retinal Ganglion Cells ◽

Ganglion Cells ◽

Imaging Mass Spectrometry ◽

Cell Types ◽

Retinal Ganglion ◽

Muller Glia ◽

Müller Glia ◽

Ocular Diseases

AbstractIn order to better understand retinal physiology, alterations to which underlie some ocular diseases, we set out to establish the lipid signature of two fundamental cell types in the retina, Müller Glia and Retinal Ganglion Cells (RGCs). Moreover, we compared the lipid signature of these cells in sections (in situ), as well as after culturing the cells and isolating their cell membranes (in vitro). The lipidome of Müller glia and RGCs was analyzed in porcine retinal sections using Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS). Isolated membranes, as well as whole cells from primary cell cultures of RGCs and Müller glia, were printed onto glass slides using a non-contact microarrayer (Nano Plotter), and a LTQ-Orbitrap XL analyzer was used to scan the samples in negative ion mode, thereafter identifying the RGCs and Müller cells immunohistochemically. The spectra acquired were aligned and normalized against the total ion current, and a statistical analysis was carried out to select the lipids specific to each cell type in the retinal sections and microarrays. The peaks of interest were identified by MS/MS analysis. A cluster analysis of the MS spectra obtained from the retinal sections identified regions containing RGCs and Müller glia, as confirmed by immunohistochemistry in the same sections. The relative density of certain lipids differed significantly (p-value ≤ 0.05) between the areas containing Müller glia and RGCs. Likewise, different densities of lipids were evident between the RGC and Müller glia cultures in vitro. Finally, a comparative analysis of the lipid profiles in the retinal sections and microarrays identified six peaks that corresponded to a collection of 10 lipids characteristic of retinal cells. These lipids were identified by MS/MS. The analyses performed on the RGC layer of the retina, on RGCs in culture and using cell membrane microarrays of RGCs indicate that the lipid composition of the retina detected in sections is preserved in primary cell cultures. Specific lipid species were found in RGCs and Müller glia, allowing both cell types to be identified by a lipid fingerprint. Further studies into these specific lipids and of their behavior in pathological conditions may well help identify novel therapeutic targets for ocular diseases.

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Masking of a circadian behavior in larval zebrafish involves the thalamo-habenula pathway

10.1101/097790 ◽

2017 ◽

Author(s):

Qian Lin ◽

Suresh Jesuthasan

Keyword(s):

Retinal Ganglion Cells ◽

Blue Light ◽

Calcium Imaging ◽

Thalamic Nucleus ◽

Ganglion Cells ◽

Red Light ◽

Retinal Ganglion ◽

Two Photon ◽

Larval Zebrafish ◽

Circadian Behavior

AbstractLight has the ability to disrupt or mask behavior that is normally controlled by the circadian clock. In mammals, masking requires melanopsin-expressing retinal ganglion cells that detect blue light and project to the thalamus. It is not known whether masking is wavelength-dependent in other vertebrates, nor is it clear what higher circuits are involved. Here, we address these questions in zebrafish. We find that diel vertical migration, a circadian behavior in larval zebrafish, is effectively masked by blue, but not by red light. Two-photon calcium imaging reveals that a retino-recipient thalamic nucleus and a downstream structure, the habenula, are tuned to blue light. Lesioning the habenula inhibits light-evoked climbing. These data suggest that a thalamo-habenula pathway may be involved in the ability of blue light to mask circadian behavior.

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A Safe GDNF and GDNF/BDNF Controlled Delivery System Improves Migration in Human Retinal Pigment Epithelial Cells and Survival in Retinal Ganglion Cells: Potential Usefulness in Degenerative Retinal Pathologies

Pharmaceuticals ◽

10.3390/ph14010050 ◽

2021 ◽

Vol 14 (1) ◽

pp. 50

Author(s):

Alicia Arranz-Romera ◽

Maria Hernandez ◽

Patricia Checa-Casalengua ◽

Alfredo Garcia-Layana ◽

Irene T. Molina-Martinez ◽

...

Keyword(s):

Cell Viability ◽

Retinal Ganglion Cells ◽

Neurotrophic Factor ◽

Retinal Pigment Epithelial ◽

Ganglion Cells ◽

Retinal Pigment ◽

Terminal Deoxynucleotidyl Transferase ◽

Retinal Ganglion ◽

Pigment Epithelial

We assessed the sustained delivery effect of poly (lactic-co-glycolic) acid (PLGA)/vitamin E (VitE) microspheres (MSs) loaded with glial cell-derived neurotrophic factor (GDNF) alone (GDNF-MSs) or combined with brain-derived neurotrophic factor (BDNF; GDNF/BDNF-MSs) on migration of the human adult retinal pigment epithelial cell-line-19 (ARPE-19) cells, primate choroidal endothelial (RF/6A) cells, and the survival of isolated mouse retinal ganglion cells (RGCs). The morphology of the MSs, particle size, and encapsulation efficiencies of the active substances were evaluated. In vitro release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability, terminal deoxynucleotidyl transferase (TdT) deoxyuridine dUTP nick-end labelling (TUNEL) apoptosis, functional wound healing migration (ARPE-19; migration), and (RF/6A; angiogenesis) assays were conducted. The safety of MS intravitreal injection was assessed using hematoxylin and eosin, neuronal nuclei (NeuN) immunolabeling, and TUNEL assays, and RGC in vitro survival was analyzed. MSs delivered GDNF and co-delivered GDNF/BDNF in a sustained manner over 77 days. The BDNF/GDNF combination increased RPE cell migration, whereas no effect was observed on RF/6A. MSs did not alter cell viability, apoptosis was absent in vitro, and RGCs survived in vitro for seven weeks. In mice, retinal toxicity and apoptosis was absent in histologic sections. This delivery strategy could be useful as a potential co-therapy in retinal degenerations and glaucoma, in line with future personalized long-term intravitreal treatment as different amounts (doses) of microparticles can be administered according to patients’ needs.

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