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

Diversity of retinal ganglion cells identified by transient GFP transfection in organotypic tissue culture of adult marmoset monkey retina.

The mammalian retina has more diversity of neurons than scientists had once believed in order to establish complicated vision processing. In the monkey retina, morphological diversity of retinal ganglion cells (RGCs) besides dominant midget and parasol cells has been suggested. However, characteristic subtypes of RGCs in other species such as bistratified direction-selective ganglion cells (DSGC) have not yet been identified. Increasing interest has been shown in the common marmoset (Callithrix jacchus) monkey as a "super-model" of neuroscientific research. Here, we established organotypic tissue culture of the adult marmoset monkey retina with particle-mediated gene transfer of GFP to survey the morphological diversity of RGCs. We successfully incubated adult marmoset monkey retinas for 2 to 4 days ex vivo for transient expression of GFP. We morphologically examined 121 RGCs out of more than 3240 GFP-transfected cells in 5 retinas. Among them, we identified monostratified or broadly stratified ganglion cells (midget, parasol, sparse, recursive, thorny, and broad thorny ganglion cells), and bistratified ganglion cells (recursive, large, and small bistratified ganglion cells [blue-ON/yellow-OFF-like]). By this survey, we also found a candidate for bistratified DSGC whose dendrites were well cofasciculated with ChAT-positive starburst dendrites, costratified with ON and OFF ChAT bands, and had honeycomb-shaped dendritic arbors morphologically similar to those in rabbits. Our genetic engineering method provides a new approach to future investigation for morphological and functional diversity of RGCs in the monkey retina

A list of 121 <i>GFP</i>-transfected marmoset retinal ganglion cells.

<p>A list of 121 <i>GFP</i>-transfected marmoset retinal ganglion cells.</p

A candidate for bistratified direction-selective retinal ganglion cells in the marmoset monkey retina.

<p>(A) and (B) A <i>GFP-F</i>-transfected candidate for bistratified direction-selective retinal ganglion cells, namely Cell #1 (A), and Cell #2 (B) (dendritic field size: 402 μm in diameter and 355 μm in diameter, respectively). Confocal images, tracings and stratifications are shown. Scale bars, 50 μm.</p

Morphological diversity of <i>GFP-F</i>-transfected monostratified or broadly stratified retinal ganglion cells.

<p>(A) An ON midget cell and a broad thorny cell (dendritic field size: 49.7 μm in diameter and 179 μm in diameter, respectively). (B) An OFF parasol ganglion cell (143 μm in diameter). (C) A OFF recursive monostratified ganglion cell (288 μm in diameter). (D) A sparse ganglion cell (695 μm in diameter). (E) An ON thony ganglion cell (256 μm in diameter). Left panels: projections of a confocal image. Middle: Tracings of dendritic structure. Right: Stratification of these ganglion cells compared with ON and OFF ChAT bands in the IPL. Scale bars, 50 μm.</p

Cofasciculation and costratification of dendrites of the candidate for DSGC with ChAT-positive starburst dendrites.

<p>(A) A tracing of Cell #2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054667#pone-0054667-g005" target="_blank">Figure 5</a> with two boxes that represent the analyzed areas in (B) and (C). (B) GFP-F-positive dendrites (green) were cofasciculated with ChAT-positive dendrites (red) to form a honeycomb-shaped meshwork. OFF layer is shown here. Bottom panel: Only cofasciculated pixels are shown (analyzed by ImageJ). (C) GFP-F-positive dendrites (green) were clearly costratified with ON and OFF starburst ChAT bands (red) in IPL. (D) and (E) Highly magnified images of Cell #1 (D) and Cell #3 (E) are shown. GFP-F-positive dendrites (green) were clearly cofasciculated with ChAT-positive dendrites (red) to form a typical honeycomb-shaped meshwork. Scale bars, 50 μm.</p

Morphological diversity of <i>GFP-F</i>-transfected bistratified retinal ganglion cells.

<p>(A) A recursive bistratified ganglion cell (dendritic field size: 391 μm in diameter). (B) A large bistratified ganglion cell (273 μm in diameter). (C) A small bistratified ganglion cell (Blue-ON/Yellow-OFF-like) (234 μm in diameter). Left panels: projections of a confocal image. Middle: Tracings of dendritic structure with ON (red trace) and OFF layers (black trace). Right: Stratification of these ganglion cells compared with ON and OFF ChAT bands in the IPL. (D) and (E) Highly magnified images of the OFF layer of the recursive bistratified ganglion cell (D; boxed area in A) and OFF layer of the small bistratified ganglion cell (E; boxed area in C) are shown. GFP-F-positive dendrites (green) were not obviously cofasciculated with ChAT-positive dendrites (red). Overlapped pixels are shown in white. The cofasciculation indexes (calculated on the OFF dendrites in D and E, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054667#s2" target="_blank">Materials and Methods</a>) were calculated to be 0.55 and 1.08, respectively. Scale bars, 50 μm.</p

Incubation of adult marmoset monkey retina with transient gene transfection.

<p>(A) Organotypic culture of adult marmoset retina after 42-hour incubation. Left, a photograph of the interphase chamber of our organotypic culture system. Right, a schematic diagram of the interphase chamber. Deep dishes were used with custom-made stands to support the tissue culture insert with a flat-mounted retina. The retina was in contact with the medium over the filter on the photoreceptor side, and the ganglion cell side faced the atmosphere. Dishes were agitated by a rotary shaker in the CO₂ incubator during the incubation. (B) Patch-clamp recordings of a cultured retinal ganglion cell after 72-hour incubation. Left: Voltage-clamp recordings. Holding voltage  = −71 mV. Voltages were clamped from −91 mV to +19 mV, in 10-mV steps, for 100 msec (bar). Right: Current-clamp experiment. Injected currents were from −20 pA to 50 pA, in 10-pA steps, for 100 msec (bar). Resting membrane potential was −59 mV. (C) A piece of marmoset retina after 3-day culture was stained with YO-PRO-1. YO-PRO-1-positive cells were clustered on the retina piece. The clusters formed an area with bright green fluorescence so that we defined a clustered area as a YO-PRO-1-positive area (black areas in left panel). Scale bars, 1 mm. (D) Quantification of YO-PRO-1-positive areas on cultured marmoset retinas. In the cultured retinas, 18.6±9.8% (mean ± standard deviation, n = 4 retina pieces) of the whole retinal area was YO-PRO-1-positive, while 22.0±6.0% (n = 4) of the retinal area was also YO-PRO-1-positive even in the acutely isolated retina (no significant difference). (E) A piece of <i>GFP-F</i>-transfected retina after 42-hour incubation. In this piece of retina, there were more than 452 cells expressing GFP-F. Scale bar, 1 mm. (F) Another piece of <i>GFP-F</i>-transfected retina after 42-hour incubation. Several retinal ganglion cells with different morphologies expressed GFP-F. Scale bar, 100 μm.</p

Eccentricity and stratification of ChAT-positive starburst amacrine cells.

<p>(A) ON and OFF ChAT-positive SAC in the central retina, mid-peripheral retina and far-peripheral retina. Scale bars, 50 μm. (B) Retinal eccentricity of ON SAC. From this graph, we defined the central retina (up to 1.5 mm), the mid-peripheral retina (>1.5 to 4 mm), and the far-peripheral retina (>4 mm). Scale bars, 50 μm. (C) Typical distinct stratifications of dendrites of ON and OFF ChAT-positive SAC in the IPL. These two bands are used as a landmark for checking stratification in the IPL. Scale bars, 50 μm.</p