Retinal Cell Suspensions Research Articles

Nucleofection of whole murine retinas

The mouse retina constitutes an important research model for studies aiming to unravel the cellular and molecular mechanisms underlying ocular diseases. The accessibility of this tissue and its feasibility to directly obtain neurons from it has increased the number of studies culturing mouse retina, mainly retinal cell suspensions. However, to address many questions concerning retinal diseases and protein function, the organotypic structure must be maintained, so it becomes important to devise methods to transfect and culture whole retinas without disturbing their cellular structure. Moreover, the postmitotic stage of retinal neurons makes them reluctant to commonly used transfection techniques. For this purpose some published methods employ in vivo virus-based transfection techniques or biolistics, methods that present some constraints. Here we report for the first time a method to transfect P15-P20 whole murine retinas via nucleofection, where nucleic acids are directly delivered to the cell nuclei, allowing in vitro transfection of postmitotic cells. A detailed protocol for successful retina extraction, organotypic culture, nucleofection, histological procedures and imaging is described. In our hands the A-33 nucleofector program shows the highest transfection efficiency. Whole flat-mount retinas and cryosections from transfected retinas were imaged by epifluorescence and confocal microscopy, showing that not only cells located in the outermost retinal layers, but also those in inner retinal layers are transfected. In conclusion, we present a novel method to successfully transfect postnatal whole murine retina via nucleofection, showing that retina can be successfully nucleofected after some optimization steps.

Microglia derived IL-6 suppresses neurosphere generation from adult human retinal cell suspensions

Following retinal degeneration or inflammation that disrupts tissue architecture, there is limited evidence of tissue regeneration, despite evidence of cells with progenitor properties in the adult human retina at all ages. With the prospect of tissue/cell transplantation, redressing homeostasis whilst overcoming glial barrier or gliosis remains key to successful graft versus host integration and functional recovery. Activated human retinal microglia (MG) secrete cytokines, including IL-6, which may suppress neurogenesis or cellular (photoreceptor) replacement.To investigate this hypothesis, adult human retinal explants were cultured in cytokine-conditioned media (TNFα, TGFβ, LPS/IFNγ) to activate microglia in situ. Following culture of retinal explants for 4 days, supernatant conditioned by resulting migrated microglia was collected after a further 3 days and fed to retinal cell suspensions (RCS). Neurosphere (NS) generation and survival analysis was performed after 7 and 14 days in culture, with or without addition of conditioned media and with or without concomitant IL-6 neutralisation. Neurosphere phenotype was analysed by immunohistochemistry and cell morphology.Migratory MG from retinal explants were activated (iNOS-positive) and expressed CD45, CD11b, and CD11c. LPS/IFNγ-activated MG conditioned media (MG-CM) contained significant levels of IL-6 (1265 ± 143) pg/ml, which inhibited neurosphere generation within RCS in the presence of optimal neurosphere generating N2-FGF2 culture medium. Neutralising IL-6 activity reinstated NS generation and the differentiation capacity was maintained in the spheres that formed. Even in the presence of high levels of IL-6, those few NS that did form demonstrated a capacity to differentiate. The data supports activated MG-derived IL-6 influence retinal cell turnover.

Evidence against Functional Interaction between Aquaporin-4 Water Channels and Kir4.1 Potassium Channels in Retinal Müller Cells

Indirect evidence suggests that the Müller/glial cell water channel aquaporin-4 (AQP4) modulates K(+) channel function of the closely associated Kir4.1 protein. We used patch clamp to compare Kir4.1 K(+) channel function in freshly isolated Müller cells from retinas of wild-type (+/+) and AQP4 knock-out (-/-) mice. Immunocytochemistry showed a comparable Kir4.1 protein expression pattern in Müller cells from +/+ and -/- retinas, with greatest expression at their end feet. Osmotic water permeability was >4-fold reduced in -/- than in +/+ Müller cells. Resting membrane potential did not differ significantly in +/+ versus -/- Müller cells (-64 +/- 1 versus -64 +/- 1 mV, S.E., n = 24). Whole-cell K(+) currents recorded with a micropipette inserted into the cell soma were Ba(2+)-sensitive and showed no significant differences in magnitude in +/+ versus -/- Müller cells (1.3 +/- 0.1 versus 1.2 +/- 0.1 nA at -160 mV) or in inwardly rectifying current-voltage relationships. Spatially resolved K(+) currents generated by pulsed K(+) injections along Müller cell bodies were also comparable in +/+ versus -/- Müller cells. Single-channel cell-attached patch clamp showed comparable unitary conductance, current-voltage data, and open probability in +/+ versus -/- Müller cells. Thus, contrary to the generally accepted view, our results provide direct evidence against functionally significant AQP4 modulation of Müller cell Kir4.1 K(+) channel function.

Rat retinal ganglion cells culture enriched with the magnetic cell sorter

The magnetic cell sorter (MACS) technique was applied to isolate retinal ganglion cells (RGCs) for culture. RGCs were labeled retrogradely with 1.1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). Subsequently retinal cell suspensions were incubated with biotinylated anti-rat Thy-1 antibody and MACS Streptavidin MicroBeads, and then applied onto the column in the magnetic fields. Cells attached on the column were flashed out without magnetism and plated on glass cover slips. RGCs were enriched to 31.0% of all cells with MACS from 0.55% before applying onto the magnetic column. Mean diameters of DiI-labeled cells were significantly larger than those of unlabeled cells. All cells with soma diameter over 11 μm were labeled. The number of viable RGCs were counted in the 10 fields of six cultures at a magnification of ×200; the mean numbers on the 2nd, 7th and 14th culture-day were 53±3, 24±2 and 21±3, respectively (mean±SEM, n=6). Thus, the MACS technique was confirmed to be useful for enrichment of RGCs and long-term study of cultured RGCs.

Localization of Janusin mRNA in the Central Nervous System of the Developing and Adult Mouse

Janusin (formerly termed J1-160/180) is an oligodendrocyte-derived extracellular matrix molecule which is restricted to the central nervous system and which is expressed late during development (Pesheva et al., J. Cell Biol., 1765-1778, 1989). To gain insights into the molecule's morphogenetic functions and to identify its cellular source in vivo, we have studied the localization of janusin messenger RNA in the optic nerve, retina and spinal cord and the expression of janusin protein in the spinal cord of developing and adult mice. Moreover, we have analysed optic nerve cell cultures and retinal cell suspensions in double-labelling experiments using a janusin-specific anti-sense complementary RNA probe and cell type-specific antibodies to identify the cell types containing janusin transcripts. In developing animals, oligodendrocytes were strongly labelled with the janusin anti-sense cRNA probe during the period of myelination. The number of labelled cells and intensity of the hybridization signal decreased significantly with increasing age. Interestingly, expression of janusin was not confined to oligodendrocytes. Some neuronal cell types and type-2 astrocytes present in optic nerve cell cultures also contained janusin transcripts. In contrast to oligodendrocytes, the number and labelling intensity of neurons containing janusin transcripts remained constant during postnatal development and into adulthood. Expression of janusin protein in the spinal cord was developmentally regulated, with a peak of expression in 2- or 3-week-old animals. The molecule was visible in the white and grey matter. In myelinated regions, it was associated with myelinated fibres and accumulated at nodes of Ranvier. These observations suggest that janusin may be of functional relevance for myelination.

Cryopreservation of postnatal rat retinal ganglion cells: Persistence of voltage- and ligand-gated ionic currents

Established methods for cryopreservation of living cells were modified for freeze-storage of postnatal retinal ganglion cells from rat. Retinal cell suspensions containing fluorescently labeled ganglion cells were frozen after addition of 8% dimethyl sulfoxide and stored at −80°C for up to 66 days. Viability of identified retinal ganglion cells was assessed by their ability to take up and cleave fluorescein diacetate to fluorescein. No significant difference was found in the number of living retinal ganglion cells when cells obtained from the same dissociation were counted before and after freezing ( 6.65 ± 2.37 × 10 4 vs 7.05 ± 3.67 × 10 4 retinal ganglion cells per ml, respectively; mean ±S.D., n = 4. In culture following cryopreservation, the cells appeared morphologically normal, and developed neurites and growth cones similar to their freshly dissociated counterparts. Since very little is known about the electrophysiology and membrane properties of neurones after cryopreservation, we used the whole-cell configuration of the patch-clamp technique to study voltage- and ligand-gated conductances in cryopreserved retinal ganglion cells. The cryopreserved retinal ganglion cells studied under current-clamp maintained resting potentials of -60.9 ± 6.6mV(n = 10) and upon depolarization fired action potentials. During voltage-clamp in the whole-cell mode, depolarizing voltage steps activated Na +-(I Na),Ca 2+-(I Ca) , and K + -currents in all cells tested (n = 122). I Na could be reversibly blocked by 1 μM tetrodotoxin added to the external solution. I Ca was blocked by external 250 μM Cd 2+ or 3 mM Co 2+. In some cells, I Ca consisted of both a transient and prolonged component. The outward K +-current consisted of Ca 2+-dependent and -independent components. The Ca 2+-intensitive portion of the K + outward current was separated into four distinct components based upon pharmacological sensitivity and biophysical properties. In many cells, a rapidly inactivating current similar to the A-type K +-current (I A) observed in freshly cultured retinal ganglion cells was isolated by its greater sensitivity to 4-aminopyridine (5 mM) than to tetraethylammonium (20 mM). A tetraethylammonium-sensitive current with a more prolonged time course reminiscent of I K , the delayed rectifier, was also found. When the 4-aminopyridine- and tetraethylammonium-insensitive portions of the outward current were further analysed with voltage protocols, and additional slowly decaying potassium current became apparent. The inhibitory aminom acids, GABA (20 μM) and glycine (100 μM), activated chloride-selective currents that were selectively blocked by bicuculline methiodide (10 μM) and strychnine (5 μM), respectively. The excitatory amino acids glutamate (100 μM), N-methyl- d-aspartate (200 μM), and kinate (125 μM) activated currents that were seletively blocked by 2-amino-5-phosphonovalerate (100 μM) and 6-cyano-7-nitroquinoxaline-2,3-dione (10 μM), respectively. Acetylcholine-(20 μM) induced currents were blocked by hexamethonium (20 μM), but not atropine (10 μM). The integrity of these ligand receptors and ionic channels in cryopreserved neurons is of critical importance for their normal function. In addition to the traditional role of ion channels in mediating fast cellular information signalling, these channels are also important in neuronal outgrowth and survival. Most importantly, cryopreservation can help reduce the number of animals used for research purposes; by collecting large numbers of cells from a single animal, multiple, temporally-separated electrophysiological experiments can be performed. Also, the capability of storing cells may facilitate the application of both molecular biology and electrophysiology techniques to defined subsets of CNS neurons and the exchange of specific batches of cells between laboratories.

Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning

Two different monoclonal antibodies to the Thy-1 antigen, T11D7 and 2G12, were used to purify and characterize retinal ganglion cells from postnatal rat retina. Although Thy-1 has been reported to be a specific marker for ganglion cells in retina, retinal cell suspensions contained several other types of Thy-1-positive cells as well. Nevertheless, a simple two-step “panning” procedure allowed isolation of ganglion cells to nearly 100% purity. We found that postnatal ganglion cells differed in antigenic, morphological, and intrinsic electrophysiological characteristics, and that these properties were correlated with one another. Minor variations of this panning protocol should allow rapid, high yield purification to homogeneity of many other neuronal and glial cell types.

Intraretinal transplantation of fluorescently labeled retinal cell suspensions

Dissociated cell suspensions of neonatal neural retina, labeled with the fluorescent dyes Fast blue or Fluoro-gold, were transplanted into the retina of normal adult rats or of rats affected by late stage phototoxic retinopathy. Light microscopy showed good survival, differentiation, and integration of the transplants, as well as permanence of the label up to 100 days. The results indicate that the transplantation of dissociated, fluorescently labeled retinal cells has a number of advantages over the transplantion of solid fragments of retinal tissue, previously performed by ourseleves and others. The following are some of the most immediate procedural advantages: the number of transplanted cells can be assessed, the transplanted cells are in a more intimate contact with the host tissue and therefore integrate better with the host, and the fluorescent tags permit precise determination of the survival and distribution of the transplanted cells.

Drug-induced changes of growth kinetics in brain tissue primary cultures

The responses of cultured chick embryo retinal neurons to several extracellular matrix molecules are described. Retinal cell suspensions in serum-free medium containing the “N1” supplement (J. E. Bottenstein, S. D. Skaper, S. Varon, and J. Sato, 1980, Exp. Cell Res.125, 183–190) were seeded on tissue culture plastic surfaces pretreated with polyornithine (PORN) and with one of the factors to be tested. Substantial cell survival could be observed after 72 hr in vitro on PORN pretreated with serum or laminin, whereas most cells appeared to be degenerating on untreated PORN, PORN-fibronectin, and PORN-chondronectin. Cell attachment, although quantitatively similar for all these substrata, was temperature-dependent on serum and laminin but not on fibronectin or untreated PORN. In a short-term bioassay, neurite development was abundant on laminin, scarce on serum and fibronectin, and absent on PORN. No positive correlation between cell spreading and neurite production could be seen: cell spreading was more extensive on PORN and fibronectin than on laminin or serum, while on laminin-treated dishes, spreading was similar for neurite-bearing and non-neurite-bearing cells. Laminin effects on retinal neurons were clearly substratum dependent. When bound to tissue culture plastic, laminin showed a dose-dependent inhibitory effect on cell attachment and did not stimulate neurite development. PORN-bound laminin, on the other hand, did not affect cell attachment but caused marked stimulation of neurite development, suggesting that laminin conformation and/or the spatial distribution of active sites play an important role in the neurite-promoting function of this extracellular matrix molecule. Investigation of the embryonic retina with ELISA and immunocytochemical methods showed that laminin is present in this organ during development. Therefore, in vivo and in vitro observations are consistent with the possibility that laminin might influence neuronal development in the retina.

Responses of cultured neural retinal cells to substratum-bound laminin and other extracellular matrix molecules

The responses of cultured chick embryo retinal neurons to several extracellular matrix molecules are described. Retinal cell suspensions in serum-free medium containing the “N1” supplement ( J. E. Bottenstein, S. D. Skaper, S. Varon, and J. Sato, 1980, Exp. Cell Res. 125, 183–190 ) were seeded on tissue culture plastic surfaces pretreated with polyornithine (PORN) and with one of the factors to be tested. Substantial cell survival could be observed after 72 hr in vitro on PORN pretreated with serum or laminin, whereas most cells appeared to be degenerating on untreated PORN, PORN-fibronectin, and PORN-chondronectin. Cell attachment, although quantitatively similar for all these substrata, was temperature-dependent on serum and laminin but not on fibronectin or untreated PORN. In a short-term bioassay, neurite development was abundant on laminin, scarce on serum and fibronectin, and absent on PORN. No positive correlation between cell spreading and neurite production could be seen: cell spreading was more extensive on PORN and fibronectin than on laminin or serum, while on laminin-treated dishes, spreading was similar for neurite-bearing and non-neurite-bearing cells. Laminin effects on retinal neurons were clearly substratum dependent. When bound to tissue culture plastic, laminin showed a dose-dependent inhibitory effect on cell attachment and did not stimulate neurite development. PORN-bound laminin, on the other hand, did not affect cell attachment but caused marked stimulation of neurite development, suggesting that laminin conformation and/or the spatial distribution of active sites play an important role in the neurite-promoting function of this extracellular matrix molecule. Investigation of the embryonic retina with ELISA and immunocytochemical methods showed that laminin is present in this organ during development. Therefore, in vivo and in vitro observations are consistent with the possibility that laminin might influence neuronal development in the retina.

GABA uptake and release in purified neuronal and nonneuronal cultures from chick embryo retina

Uptake and release of γ-aminobutyric acid (GABA) have been studied using glia-free, purified neuronal cultures from 8-day chick embryo retina. At 3 days in vitro 65% of the neurons showed high-affinity GABA uptake. These neurons appeared heavily labeled after incubation in 5 × 10 −8 M [ 3H]GABA, but no labeling was detected when the incubation was carried out at 4°C, or in the absence of Na +ions. Diaminobutyric acid (DABA) also blocked completely the neuronal uptake of GABA, while β-alanine was ineffective at similar concentrations. At 6 days in vitro Na +-and temperature-dependent GABA uptake was present in 50% of the neurons. In addition, in 80% of those neurons the uptake was insensitive to DABA or β-alanine, whereas in the remaining 20% it was blocked by DABA but not by α-alanine. Important developmental changes were also found in the capacity of the neurons to release GABA into the medium. Spontaneous GABA release (i.e. that taking place in regular medium, containing 5 mM K +) was higher at 3 than at 6 days in vitro. However, increasing the K + concentration to 56 mM had minimal effects at 3 days in vitro, but induced a 2 to 3-fold increase in GABA release at 6 days in vitro. This K +-induced release appeared to be Ca 2+-dependent, since it was substantially reduced in the presence of 10 mM Co 2+. Cultures containing a confluent monolayer of nonneuronal flat cells were generated by seeding retinal cell suspensions on poorly adhesive substrate. Retina nonneuronal cells showed, during the first 10 days in vitro, a high-affinity mechanism for GABA uptake which was Na +- and temperature-dependent, and was reduced by 85% by DABA but was practically unaffected by β-alanine. This uptake mechanism seemed to be lost towards the end of the second week in vitro, and could not be detected after 21 days culture.

Regulation of tyrosine hydroxylase activity in retinal cell suspensions: Effects of potassium and 8-bromo cyclic AMP

Tyrosine hydroxylase (TH) contained in dopamine (DA) neurons of rat retina is activated in vivo as a consequence of photic stimulation. Experiments were conducted to test the effects of changes of membrane potential and of cyclic AMP-dependent protein phosphorylation on TH activity of these retinal neurons. Retinas were dissociated into suspensions of apparently viable cells to test the direct effects of pharmacological manipulations of TH activity in the absence of trans-synaptic influences. To test the effects of changes of membrane potential on TH activity we examined the effects of a depolarizing agent, potassium. Incubation of cell suspensions in Ringer's solution containing a depolarizing concentration of K + (52 mM) resulted in a significant increase of TH activity, suggesting that membrane depolarization may trigger a series of molecular events that leads to TH activation. Incubation of cell suspensions in the presence of 8-bromo cyclic AMP, a cyclic AMP analog that is known to activate cyclic AMP-dependent processes following extra-cellular application, resulted in a significant activation of TH that was comparable to that produced in vitro by cyclic AMP-dependent protein phosphorylation. These data support the hypothesis that membrane potential plays a role in the regulation of TH activity, and indicate that cyclic AMP-dependent phosphorylation can activate retinal TH in situ . The apparent viability of the retinal cells in suspension suggests that this preparation may be useful for studying synaptic regulatory mechanisms.

The role of GABA in the regulation of the dopamine/tyrosine hydroxylase-containing neurons of the rat retina

Dopamine (DA) and gamma-aminobutyric acid (GABA) are putative neurotransmitters in two separate populations of amacrine neurons in the mammalian retina. Pharmacological studies have been conducted to determine if GABA neurons regulate the neuronal activity of the neurons that secrete DA. Tyrosine hydroxylase (TH) activity, a biochemical indicator of changes in activity of DA/TH-containing neurons, was low in dark-adapted retinas and high in light-exposed retinas. Muscimol (a GABA receptor agonist) produced a dose-related, biphasic effect on the light-evoked activation of TH, when the drug was injected into the vitreous (intravitreal injection) of dark-adapted rats. At low doses, (35 and 60 pmol) muscimol enhanced the light-evoked activation of TH, but at higher doses (≥ 120 pmol) it inhibited the light-evoked increase in enzyme activity. Muscimol had no significant effect on the TH activity of dark-adapted retinas. GABA antagonists, bicuculline and picrotoxin, produced effects on TH activity that were dependent on both dose and light-exposure. At low doses (0.4–0.5 nmol), bicuculline and picrotoxin both inhibited the light-evoked activation of TH, but had no effect on TH activity of the dark-adapted retinas. At higher dose (2.0 nmol), both antagonists increased TH activity in the dark-adapted retina and attenuated the further activation of the enzyme by light. Rat retinas were dissociated into suspensions of viable cells in order to investigate the direct effects of muscimol and picrotoxin on the DA/TH-containing cells. The process of dissociating dark-adapted retinas resulted in an apparent activation of TH. Incubation of the cells with muscimol resulted in a decrease of TH activity in a concentration-dependent manner. Picrotoxin antagonized the inhibitory effect of muscimol, but had no effect when incubated alone. The biphasic effects of GABA agonists and antagonists in vivo suggest that a certain subpopulation of GABA neurons are involved in the activation of the DA/TH-containing neurons by photic stimulation, while another subpopulation of GABA neurons produce a tonic inhibition of the DA/TH-containing neurons in darkness. The experiments with retinal cell suspensions indicate that the tonic inhibition is probably mediated by synapses of GABA neurons directly onto the DA/TH-containing cells.

The relation between cortisol binding and glutamine synthetase induction in the neural retina of the chick embryo.

Cortisol induces high levels of glutamine synthetase (EC 6.3.2.1) activity in the embryonic neural retina of chick embryos. This response to the hormone is impaired when the tissue is dissociated to a single cell suspenion and is gradually lost when the cells are maintained in monolayer cultures. To elucidate the mechanism that controls this tissue-specific function, the interaction of [3H]cortisol with retinal cell suspensions and retinal monolayers was studied and compared to the interaction of the hormone with the responsive intact tissue in culture. The growth rate of neural retina cells, dissociated from the 10-day-old embryonic tissue, is higher than that of the intact tissue; there is a 2-fold increase when grown in suspension cultures and a 3- to 4-fold increase when grown in monolayers. Under these conditions, retinal cells in suspension retain the full binding capacity characteristic to the intact tissue in culture. The translocation of the hormone-receptor complexes to the nucleus is also uncha...