Organotypic Tissue Culture of Adult Rodent Retina Followed by Particle-Mediated Acute Gene Transfer In Vitro

BACKGROUND: Organotypic tissue culture of adult rodent retina with an acute gene transfer that enables the efficient introduction of variable transgenes would greatly facilitate studies into retinas of adult rodents as animal models. However, it has been a difficult challenge to culture adult rodent retina. The purpose of this present study was to develop organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We established an interphase organotypic tissue culture for adult rat retinas (>P35 of age) which was optimized from that used for adult rabbit retinas. We implemented three optimizations: a greater volume of Ames' medium (>26 mL) per retina, a higher speed (constant 55 rpm) of agitation by rotary shaker, and a greater concentration (10%) of horse serum in the medium. We also successfully applied this method to adult mouse retina (>P35 of age). The organotypic tissue culture allowed us to keep adult rodent retina morphologically and structurally intact for at least 4 days. However, mouse retinas showed less viability after 4-day culture. Electrophysiologically, ganglion cells in cultured rat retina were able to generate action potentials, but exhibited less reliable light responses. After transfection of EGFP plasmids by particle-mediated acute gene transfer, we observed EGFP-expressing retinal ganglion cells as early as 1 day of culture. We also introduced polarized-targeting fusion proteins such as PSD95-GFP and melanopsin-EYFP (hOPN4-EYFP) into rat retinal ganglion cells. These fusion proteins were successfully transferred into appropriate locations on individual retinal neurons. CONCLUSIONS/SIGNIFICANCE: This organotypic culture method is largely applicable to rat retinas, but it can be also applied to mouse retinas with a caveat regarding cell viability. This method is quite flexible for use in acute gene transfection in adult rodent retina, replacing molecular biological bioassays that used to be conducted in isolated cultured cells

A Positive Feedback Synapse from Retinal Horizontal Cells to Cone Photoreceptors

Cone photoreceptors and horizontal cells (HCs) have a reciprocal synapse that underlies lateral inhibition and establishes the antagonistic center-surround organization of the visual system. Cones transmit to HCs through an excitatory synapse and HCs feed back to cones through an inhibitory synapse. Here we report that HCs also transmit to cone terminals a positive feedback signal that elevates intracellular Ca2+ and accelerates neurotransmitter release. Positive and negative feedback are both initiated by AMPA receptors on HCs, but positive feedback appears to be mediated by a change in HC Ca2+, whereas negative feedback is mediated by a change in HC membrane potential. Local uncaging of AMPA receptor agonists suggests that positive feedback is spatially constrained to active HC-cone synapses, whereas the negative feedback signal spreads through HCs to affect release from surrounding cones. By locally offsetting the effects of negative feedback, positive feedback may amplify photoreceptor synaptic release without sacrificing HC-mediated contrast enhancement

Depolarization of isolated horizontal cells of fish acidifies their immediate surrounding by activating V-ATPase

In order to interpret the formation of receptive field surrounds in retinal neurons, a proton-mediated mechanism was proposed to mediate feedback from horizontal cells (HCs) to cone photoreceptors. To verify the idea that depolarized HCs release protons, we measured, by a fluorescence ratiometric technique, the pH of the immediate external surface (pHs) of HCs isolated from the carp or goldfish retina. When HCs stained by 5-hexadecanoylaminofluorescein, a pH-sensitive lipophilic dye, were depolarized by bath-application of kainate or high-K+ medium, pHs was lowered. The amount of pHs change was monotonically dependent on the degree of depolarization, as much as 0.21 ± 0.05 pH units by 100 mV depolarization (induced by 100 mm K+). Acidification was suppressed by 400 nm bafilomycin A1, a specific inhibitor of the vacuolar type H+ pump (V-ATPase), suggesting that depolarization released protons from HCs via the voltage-sensitive H+ pump. Immunocytochemical analysis, using an anti-V-ATPase antibody, revealed the existence of V-ATPase in dissociated HCs. These results support the hypothesis that the feedback from HCs to cones could be proton mediated

Gating, permselectivity and pH-dependent modulation of channels formed by connexin57, a major connexin of horizontal cells in the mouse retina

Mouse connexin57 (Cx57) is expressed most abundantly in horizontal cells of the retina, and forms gap junction (GJ) channels, which constitute a structural basis for electrical and metabolic intercellular communication, and unapposed hemichannels (UHCs) that are involved in an exchange of ions and metabolites between the cytoplasm and extracellular milieu. By combining fluorescence imaging and dual whole-cell voltage clamp methods, we showed that HeLa cells expressing Cx57 and C-terminally fused with enhanced green fluorescent protein (Cx57-EGFP) form junctional plaques (JPs) and that only cell pairs exhibiting at least one JP demonstrate cell-to-cell electrical coupling and transfer of negatively and positively charged dyes with molecular mass up to ∼400 Da. The permeability of the single Cx57 GJ channel to Alexa fluor-350 is ∼90-fold smaller than the permeability of Cx43, while its single channel conductance (57 pS) is only 2-fold smaller than Cx43 (110 pS). Gating of Cx57-EGFP/Cx45 heterotypic GJ channels reveal that Cx57 exhibit a negative gating polarity, i.e. channels tend to close at negativity on the cytoplasmic side of Cx57. Alkalization of pHi from 7.2 to 7.8 increased gap junctional conductance (gj) of ∼100-fold with pKa= 7.41. We show that this gj increase was caused by an increase of both the open channel probability and the number of functional channels. Function of Cx57 UHCs was evaluated based on the uptake of fluorescent dyes. We found that under control conditions, Cx57 UHCs are closed and open at [Ca2+]o=∼0.3 mm or below, demonstrating that a moderate reduction of [Ca2+]o can facilitate the opening of Cx57 UHCs. This was potentiated with intracellular alkalization. In summary, our data show that the open channel probability of Cx57 GJs can be modulated by pHi with very high efficiency in the physiologically relevant range and may explain pH-dependent regulation of cell–cell coupling in horizontal cell in the retina