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1From the Departments of Ophthalmology and Visual Sciences and 2Physiology, University of Wisconsin Medical School, Madison, Wisconsin; and the 3Department of Cell Biology and Genetics, North Texas Health Science Center, Fort Worth, Texas.
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METHODS. A method was identified for differentiating RGC-5 cells using the nonspecific protein kinase inhibitor staurosporine. Cultures were treated with 100 nM to 3.16 µM staurosporine and assessed for a variety of differentiation markers.
RESULTS. Differentiated RGC-5 cells expressed numerous neuronal properties, including arrest of proliferation without inducing apoptosis, induction of a neuronal morphology, upregulation of neuronal markers, and establishment of outward rectifying channels. Differentiation was not dependent on a single kinase-dependent pathway, based on profiling multiple kinase phosphorylation targets and attempts to replicate differentiation with multiple specific kinase inhibitors.
CONCLUSIONS. This method for producing an RGC-like cell from a proliferating cell line facilitates the following previously impractical techniques: high-throughput screening for agents that are neuroprotective or affect ionic channels; straightforward transduction of gene expression in central neurons by nonviral transfection techniques, including production of stable transfectants; biochemical and other assays of pure RGC-like cells without purification on the basis of cell-surface antigens or anatomic location.
A transformed retinal ganglion cell line, RGC-5, was derived by transforming postnatal day 1 rat retinal cells with 
2 E1A virus. This cell line expresses neuronal markers characteristic of RGCs (e.g., Thy-1, Brn-3c, neuritin, and the N-methyl D-aspartate [NMDA]-R1 and 
-aminobutyric acid [GABA]-B receptors), but does not express the astrocyte marker glial fibrillary acidic protein (GFAP).8 Despite the presence of shared antigens, the RGC-5 line has features significantly different from RGCs, the most significant being that the former is mitotically active. In addition, RGC-5 cells are morphologically more similar to glial cells in culture than to primary RGCs and do not express the repertoire of ion channels characteristic of RGCs.9
We sought a method for terminally differentiating RGC-5 cells that would allow pharmacological, biochemical, and electrophysiological studies relevant to primary cultured RGCs. We found that treatment of proliferating RGC-5 cells with the broad-spectrum protein kinase inhibitor staurosporine (SS) for as little as 60 seconds resulted in nondividing cells with multiple branched neurites characteristic of a neuronal morphology, without inducing apoptosis. We also defined several features of these cells and explored the profile of protein kinase inhibition resulting in the differentiated phenotype.
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98% purity; catalog number 380-014) from Alexis Biochemicals (San Diego, CA). Cell culture reagents, unless noted, were obtained from BioWhittaker (Rockland, ME). Fluorescent secondary antibodies and fluorescent dyes were obtained from Molecular Probes (Eugene, OR).
Cell Culture
RGC-5 cells were cultured in Dulbecco’s modified Eagle’s medium (Mediatech, Inc., Herndon, CA) containing 1 g/L glucose with L-glutamine, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were incubated at 37°C in humidified 5% CO2.
Pharmacological agents were used to induce or inhibit various signaling pathways within RGC-5 cells. Cells were replated on 12-mm round coverslips in 24-well plates 24 hours before pharmacological treatment. Drugs were added for various lengths of time and then processed for immunocytochemistry.
Immunocytochemistry
RGC-5 cells plated on coverslips were fixed with 4% paraformaldehyde (Fisher) in PBS (pH 7.2) for 20 minutes at room temperature, rapidly rinsed with Tris-buffered saline (TBS; 100 mM Tris [pH 7.6], 0.9% NaCl) three times for a few seconds each and two times for 5 minutes each, and blocked with 5% normal goat serum (BioWhittaker) in TBS for 30 minutes at room temperature. The NMDA R1 receptor was dictated by incubating with purified mouse monoclonal anti-NMDA-R1 antibody (BD Biosciences; San Jose, CA; clone 54.1) at 2.5 µg/mL overnight at room temperature, followed by Alexa Fluor 594 (Molecular Probes, Eugene, OR) goat anti-mouse IgG at 10 µg/mL in blocking buffer at room temperature for 90 minutes. Alexa Fluor 594 fluorescence was detected with a Texas red filter set (excitation 560 ± 20 nm, dichroic 595 nm long-pass, emission 630 ± 30 nm). Thy-1 was visualized after fixing and blocking by incubating with rabbit anti-Thy-1 polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA) at 20 µg/mL overnight at 4°C, followed by Alexa Fluor 488 goat anti-rabbit IgG at 4 µg/mL at room temperature for 60 minutes. Alexa Fluor 488 fluorescence was detected with a FITC filter set (excitation 470 ± 20 nm, dichroic 505 nm long-pass, emission 540 ± 20 nm). Nuclear condensation changes that are characteristic of apoptosis were assessed by adding Hoechst 33258 dye at 10 µg/mL for the final 30 minutes of pharmacological treatment before cell fixation and viewed with a 4',6'-diamino-2-phenylindole (DAPI) filter set (excitation 365 ± 12.5 nm, dichroic 395 nm, emission 420 long pass). Coverslips were then transferred and mounted on microscope slides (Gel/Mount; Biomeda Corp., Foster City, CA). Slides were viewed with an upright microscope (Axiophot; Carl Zeiss Meditec, Dublin CA) with Nomarski optics and epifluorescence, and images acquired (Axiovision software; Carl Zeiss Meditec) at 1300 x 1030 resolution.
Immunoblot Analysis
Rat RGC-5 cells were grown to approximately 70% confluence on 100-mm tissue culture plates (BD Biosciences, Bedford, MA) and either treated with SS to a final concentration of 316 nM for 3 days or harvested without treatment. Cells were lysed in PBS containing 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (Fisher Scientific), 0.10 mg/mL phenylmethylsulfonyl fluoride (Sigma-Aldrich), and a protease inhibitor (Complete Mini Protease Inhibitor Cocktail Tablet; 5 mg/mL; Roche Diagnostics, Mannheim, Germany) for 15 minutes at 4°C, scraped off the plate, and collected. Cell lysates from two tissue culture plates were pooled for each sample, and incubated on ice for 60 minutes. After centrifugation at 10,000g for 10 minutes at 4°C, the pellets were sonicated on ice for two 15-second bursts and centrifuged again at 10,000g for 10 minutes. The protein concentrations of the supernatants were determined by Bradford assay, and 1 mg of total protein from differentiated and undifferentiated cells was boiled in the presence of 4x lithium dodecyl sulfate (LDS) sample buffer (Invitrogen, Carlsbad, CA) plus 5% ß-mercaptoethanol, resolved on a Bis-Tris 4% to 12% polyacrylamide gel (NuPAGE; Invitrogen), and transferred overnight at 50 mA to nitrocellulose membrane in a transfer apparatus (Mini Protean II; Bio-Rad Laboratories, Hercules, CA).
After transfer, the membrane was blocked with 5% nonfat milk in TBS (pH 8.0) for 30 to 60 minutes and then probed with primary antibodies to RGC marker proteins in blocking buffer. Antibodies used included purified rabbit polyclonal anti-microtubule-associated protein 2 (1:2000; Chemicon, Temecula, CA), polyclonal rabbit anti-Thy-1 (1:100; Santa Cruz Biotechnology), and polyclonal rabbit anti-actin (1:1000; Sigma-Aldrich). Blots were rinsed three times with TBS containing 0.05% Tween-20 (Fisher Scientific), then washed 5 times for 10 minutes each at room temperature on an orbital shaker. Secondary antibodies used were purified horse radish peroxidase (HRP)-conjugated goat anti-rabbit IgG and purified HRP-conjugated goat anti-mouse IgG (1:5000; Jackson ImmunoResearch Laboratories, West Grove, PA) and were incubated for 1 hour at room temperature, followed by 3 rinses and five 10-minute washes with TBS containing Tween-20 at room temperature on an orbital shaker. Blots were treated with freshly prepared ECL solution containing 100 mM Tris-HCl [pH 8.5], 1.25 mM luminol, 225 µM p-coumaric acid (Sigma-Aldrich), and 1 mM H2O2 (Fisher Scientific) for 1 minute, and excess solution was allowed to drip off. The blots were then exposed to film (BioMax XAR; Eastman Kodak Company, Rochester, NY) and developed. The films were scanned at 1600 dpi and band density was determined by comparing total intensity in an area containing the band of interest to the intensity of an equal size area of background using NIH ImageJ software. Band density readings are presented with respect to the density of the band from the control, untreated cell condition.
Cell Morphology
Photomicrographs were taken at 400x total magnification with Nomarski optics, digitized as above, and stored as JPEG images. The pictures were then batch analyzed off-line, to assess the development of neurites. A total of 50 cells from each condition were analyzed. The 50 cells assessed for neurite outgrowth in each condition were selected to include all cells in each photomicrograph for which the neurite tree was visible, and continuing with a new photomicrograph until 50 cells were analyzed. Projections from the cell were classified as neurites if they were equal to or greater in length than the cell soma. Only branches arising from the soma were counted. The neurite counts were expressed as the mean ± SEM. Neurite length and branching characteristics were assessed using NeuronJ.10 Individual neurite length was determined by adding the length of all segments from the soma to the end of each branch.
Cell Proliferation
Cells were incubated with 100 µM bromodeoxyuridine (BrdU; Sigma-Aldrich) at 37°C for 2 hours. The medium was aspirated and the cells immediately fixed with ice-cold glycine-ethanol (150 mM glycine, 70% EtOH [pH 2.0]) for 30 minutes at –20°C. The wells were washed with TBS, incubated with blocking buffer (0.3% Triton X-100, 5% normal goat serum in TBS) for 30 minutes at room temperature, and incubated overnight at 4°C with monoclonal mouse anti-BrdU (Sigma-Aldrich; clone Bu 33) at 4 µg/mL in blocking buffer. After washing, the cells were incubated for 1 hour at room temperature with AlexaFluor 594 (Molecular Probes) goat anti-mouse IgG (2 µg/mL) in TBS. Proliferating cells were identified by superimposing BrdU fluorescence and Nomarski microscopy images of the same fields.
Cell Number
Cells were placed in 96-well plates, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added immediately to a final concentration of 500 µg/mL. The plates were incubated for 5 hours at 37°C. The medium was then replaced with 200 µL of DMSO, which was pipetted up and down to dissolve the formazan crystals, and the plate was incubated at 37°C for 5 minutes. Absorbance was measured at 550 nm on a microplate reader (ThermoMax; Molecular Devices, Sunnyvale, CA). All readings were normalized to a standard curve derived from known numbers of cells.
Electrophysiology
Whole-cell voltage-clamp recordings were made at room temperature using borosilicate glass pipettes (3–6 M
resistance) filled with (in mM): 140 KCl, 10 EGTA, 2 MgATP, 20 phosphocreatine, and 10 HEPES [pH 7.3], 315 mOsM. The extracellular solution contained (in mM) 145 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 4 glucose (pH 7.4), 325 mOsM. Series resistance was monitored during recordings (5.4 ± 0.65 M, mean ± SEM, n = 17 acceptable recordings). No series resistance compensation was used. Currents were low-pass filtered at 1 to 5 kHz with a four-pole Bessel filter and digitized at a rate no less than twice the filter frequency. Data were collected using an amplifier (200B; Axopatch) and digitizer (1320A Digidata, controlled by AxoGraph software; Axon Instruments Inc., Foster City, CA, running on a Macintosh G4; Apple Computer Inc., Cupertino, CA). Analysis and curve fitting was also performed on computer (AxoGraph; Axon Instruments, Inc.).
Voltage-gated currents were studied by applying 10-ms voltage steps from –100 to +90 mV in 10-mV increments, from a holding potential of –60 mV. This protocol was run two to six times on each cell, and the currents evoked at each potential were averaged. Passive leak and capacitive currents were removed by scaling the average current evoked at –70 mV, and subtracting the result from the average current obtained at each voltage. Current amplitudes were measured at steady state (i.e., near the end of the voltage step), and the slope conductance was computed as the difference between successive amplitudes, divided by the 10-mV step increment.
Analysis of Kinase Phosphorylation Targets
Total cell lysates were prepared as previously described.11 Cells were washed with ice-cold PBS, scraped, and centrifuged at 3200g for 5 minutes. The pellets were resuspended in lysis buffer (Kinexus Bioinformatics, Vancouver, BC, Canada) supplemented with 5 mM pepstatin A (Roche), and protease inhibitor cocktail (Mini Complete tablet; Roche). The suspensions were sonicated on ice for two 15-second bursts, followed by centrifugation at 100,000g for 30 minutes at 4°C. The protein concentrations of the supernatants were determined by Bradford assay,12 and 500 µg protein from undifferentiated and differentiated cells resolved on 13% single lane SDS polyacrylamide gels. These were then transferred to nitrocellulose membranes. Using a 20-lane multiblotter (Bio-Rad), the membranes were incubated with different mixtures of up to three antibodies per lane that react with a distinct subset of at least 37 known phosphorylation sites of cell signaling proteins of distinct molecular masses (protocol KPSS 2.1; Kinexus Bioinformatics). After further incubation with a mixture of relevant HRP-conjugated secondary antibodies (Santa Cruz Biotechnology), the blots were developed with enhanced chemiluminescence (ECL Plus; GE Healthcare, Piscataway, NJ) and the signals quantified on computer (Quantity One software; Bio-Rad). (Detailed information and protocols of the Kinetworks analysis can be found at the Kinexus Bioinformatics Corporation Web site; www.kinexus.ca.)
Statistical Analysis
Comparisons between two groups were by Student’s unpaired t-test. Comparisons between more than two groups were by ANOVA and the Dunnett post hoc test. Significant differences required P < 0.05.
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Neurite branching patterns of differentiated RGC-5 cells were examined and the number of branches counted. Neurite branching, average neurite length, and total neurite length were negligible in untreated cells. Differentiation induced by exposure to 316 nM SS led to minimally branched neurites, whereas higher concentrations induced more branching (Fig. 1E) . In addition to increased branching, a moderate (1 µM) concentration of SS promoted longer average (Fig. 1F) and total (Fig. 1G) neurite outgrowth. At high (3.16 µM) SS concentrations, the neurite branching was greater, but the average and total process length decreased somewhat. All comparisons between differentiated and undifferentiated RGC-5 cells of neurite number, branches, average neurite length, and total length were significant at P < 0.05, for each concentration of SS.
Effect of SS on RGC-5 Postmitotic Cell Viability
RGC-5 cells are mitotically active. To test whether SS induces terminal differentiation, we measured incorporation of the thymidine analogue BrdU during the S phase in SS-treated and control cells. We found that SS (1 µM for 24 hours) causes RGC-5 cells to become BrdU– (i.e., postmitotic; Fig. 2 ). BrdU incorporation was seen in 69.5% of untreated control cells, compared with 2.2% of SS-treated cells, consistent with a switch to a nonproliferating state.
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Expression of Retinal Ganglion Cell Markers in Differentiated RGC-5 Cells
RGC-5 cells express NMDAR1 and Thy-1, which are both seen in mature RGCs.8 We hypothesized that differentiated RGC-5 cells would retain their expression of Thy-1 and NMDAR1. NMDAR1 and Thy-1 labeling was seen in both undifferentiated and differentiated RGC-5 cells, with visibly more intense staining in the latter (Figs. 3A 3B) .
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Expression of Voltage-Gated Ion Channels after SS Treatment
To determine whether the differentiating effects of SS treatment also involves changes in the electrophysiological properties of RGC-5 cells, we examined membrane currents using whole-cell voltage-clamp recording. Untreated cells had an input resistance of 55 ± 10 M (mean ± SEM), an apparent capacitance of 28 ± 2 pF, and a resting potential of –34 ± 0.5 mV (n = 4). After subtracting passive components, a small residual outward current was present at depolarized potentials, yielding a maximum conductance of 3.1 ± 0.5 nS at +90 mV (Fig. 4A) . Three SS treatment protocols were tested (given as hours in SS with duration of the following incubation in medium alone in parentheses): 1 hour (24 hours), 1 hour (48 hours), and 22 hours (2 hours). SS induced some changes in passive properties, as judged by ANOVA and Dunnett’s post hoc test. After treatment, input resistances were 397 ± 171 M (1 hour [24 hours], n = 5, not significant, NS), 322 ± 89 M (1 hour [48 hours], n = 4, NS), and 843 ± 201 (22 hours [2 hours]; n = 3, P < 0.05), apparent capacitances were 31 ± 5 pF (1 hour [24 hours], NS), 44 ± 3 pF (1 hour [48 hours], P < 0.05), and 38 ± 3 (22 hours [2 hours]; NS), and resting potentials were –27 ± 9 mV (1 hour [24 hours], n = 5, NS), –9 ± 6 mV (1 hour [48 hours], NS), and –11 ± 3 (22 hours [2 hours]; NS). The larger capacitance in some cells is consistent with the elaboration of neuronlike processes. In addition, although not significant by ANOVA, SS induced increases in voltage-gated conductance in all groups of treated cells. After leak subtraction, outward currents had conductances at +90 mV of 14 ± 5 nS (1 hour [24 hours], NS), 7 ± 3 nS (1 hour [48 hours], NS), and 5 ± 3 nS (22 hours [2 hours], NS). Inspection of the spread in the data (Fig. 4B) reveals that some treated cells had small conductance similar to untreated cells, whereas others had much larger conductance. This bimodal distribution probably accounts for the inability to detect significant differences. We therefore further examined cells with conductances greater than the mean of the untreated group plus twice its SD. The probability of finding such values should be extremely low if there were truly no differences between groups. However, half of the treated cells had conductances in this range, suggesting that SS induces the expression of voltage-gated current in a large fraction of cells. In these cells, the current increased monotonically as a function of voltage (Fig. 4C) and could be fit with a Boltzmann function in the most robust treatment group (i.e., 1 hour [24 hours]), yielding a maximum conductance of 26 nS and an apparent half-activation voltage of +48 mV. Because treated cells displayed neuronlike processes, we cannot be assured of there being an adequate space clamp. Thus, the strongly positive activation range and shallow slope factor we observed may not accurately reflect the biophysical characteristics of the expressed channels. Nonetheless, it seems clear that SS altered the electrical properties of a subset of treated cells.
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We differentiated RGC-5 cells with SS in the presence of two apoptosis inhibitors with different mechanisms of action, minocycline (which inhibits cytochrome c release) and ZVAD-fmk (a broad-spectrum caspase inhibitor). After 30-minute pretreatment with 10 µM minocycline or 20 µM ZVAD-fmk, SS was added to RGC-5 cells at a final concentration of 1 µM and incubated for 24 hours. Cells treated with minocycline or ZVAD-fmk and subsequently exposed to SS underwent morphologic differentiation, similar to cells that were not pretreated with apoptosis inhibitors (Fig. 5) . Undifferentiated cells treated with minocycline or ZVAD-fmk alone did not differentiate.
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Reproduction of Differentiation with SS by More Kinase-Specific Inhibitors
SS is a broad-spectrum kinase inhibitor. To determine which specific kinase(s) could be responsible for differentiation, we attempted to differentiate RGC-5 cells with 12 different relatively specific kinase inhibitors, alone or in combination, at a wide range of concentrations. Inhibitors were chosen based on published data14 demonstrating that SS inhibited the respective kinase (Table 1) . None of the kinase inhibitors alone or in combination differentiated RGC-5 cells to the same degree as SS, although two produced observable changes in morphology. H-1152, which primarily inhibits Rho-kinase (Ki = 1.6 nM) and H-89, which primarily inhibits protein kinase A (Ki = 50 nM), induced mild elevation in neurite counts, but did not produce rounding of the cell soma (Fig. 7) . SS-treated (3.16 µM) cells expressed 3.04 ± 0.16 neurites, whereas H-1152-treated (1 µM) and H-89-treated (56.2 µM) RGC-5 cells expressed 1.06 ± 0.15 and 1.80 ± 0.16 neurites, respectively. Despite mildly increased neurite expression with some kinase inhibitors, none reproduced the morphologic appearance of RGCs (Fig. 8) .
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Compared with sham-treated cells, there was a 90% increase in the S722 target of focal adhesion kinase (FAK). Other phosphorylation targets with notable differences from control were c-KitY703 (43% increase), ERK1T202/Y204 (not present in control), ERK2T185/Y187 (59% decrease), CDK1T14/Y15 (64% increase), MEK1S298 (100% decrease), and PKRT451 (100% decrease; Fig. 9 ).
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There are other examples of differentiation of cell lines with protein kinase inhibition, although there are significant differences between the mechanism in RGC-5 cells and other cell types. For example, the human prostatic cancer cell line TSU-Pr1 is differentiated by SS, with most cells assuming a neuronal morphology after 3 days.16 The mechanism is inhibition of CDK2 and consequent arrest in G1.17 Our studies demonstrated that RGC-5 differentiation did not occur with kenpaullone or roscovitine, inhibitors of CDK2, even at 13 to 200 times the IC50.14 SS also potentiates differentiation of human promyelocytic leukemic HL-60 cells, but requires the presence of another differentiating agent (e.g., retinoic acid).18
SS is a differentiating agent in retinal cells and induces rhodopsin expression and decreases green cone opsin expression in chick embryonic retinal cells.19 This differs from the better characterized CNTF-mediated differentiation pathway, in that the latter increases green cone opsin expression.19 The mechanism by which SS differentiates chick photoreceptors has not yet been elucidated. SS induces neurite extension in the PC12 pheochromocytoma cell line at high concentrations,20 although at lower concentrations it inhibits neurite induction by nerve growth factor.21 The differentiating effect involves upregulation of epidermal growth factor (EGF) receptor expression, along with dephosphorylation of EGF receptors.22 In contrast, we did not find evidence of EGF receptor phosphorylation in either undifferentiated or differentiated RGC-5 cells. Another difference between SS differentiation of PC12 cells and RGC-5 cells is decreased ion channel expression in the former23 and upregulation of at least two different ion channels in the latter.
SS differentiates the neuroblastoma-derived SH-SY5Y cell line, causing neurite extension and expression of voltage-gated calcium channels.24 Unlike RGC-5 cells, many (but not all25 ) of the SH-SY5Y–differentiated phenotype can be reproduced with protein kinase C inhibition.26 Also, unlike RGC-5 cells, these cells undergo transient apoptosis after treatment.27
Thus, the mechanism by which SS induces RGC-5 cell differentiation is different from that underlying SS differentiation of other cell types. It is unlikely to be a result of apoptosisbecause SS, a known apoptosis inducer, does not activate the apoptotic cascade in RGC-5 cells at concentrations that were used to induce differentiation. This is an important distinction, because differentiation resulting in apoptosis would not be useful for studying RGC pathophysiology. Instead, it likely reflects the inhibition of an as yet unidentified kinase not normally known to be inhibited by SS.
Differentiating RGC-5 cells has significant advantages for the study of RGC pathophysiology and elucidating diseases of the optic nerve. The undifferentiated RGC-5 cell line, which has some RGC characteristics, is significantly different from RGCs with respect to proliferation, morphology, and electrophysiology. Differentiation of RGC-5 cells into a RGC phenotype could make the following methods possible: high-throughput screens of neuroprotective and other agents, nonviral gene transduction and production of stable lines, reduced volume of experimental animals needed for primary cultures, production of 
0 cells for studying mitochondrial (mt)DNA and making cybrids, large-scale biochemical and other assays without need for purification of RGCs, and possible extrapolation to understanding the differentiation of stem cells or retinal neuronal precursors.
In summary, we showed that SS induces RGC-5 cells to differentiate, express neurites, become postmitotic and nonapoptotic, and alter their kinase phosphorylation patterns. A subset of differentiated cells also display a much larger voltage-gated conductance than undifferentiated cells, with the outward rectification typical of most voltage-gated potassium channels and some voltage-gated chloride channels. The mechanism for differentiation is not the result of inhibition of kinases normally inhibited by SS, nor by induction of apoptosis, and presumably results from other effects of SS. Differentiated RGC-5 cells are potentially useful targets for studying neuronal pathophysiology and screening new therapies for nervous system and visual system disease.
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Submitted for publication March 16, 2005; revised May 12 and October 3, 2005; accepted December 1, 2005.
Disclosure: L.J. Frassetto, None; C.R. Schlieve, None; C.J. Lieven, None; A.A. Utter, None; M.V. Jones, None; N. Agarwal, (P); L.A. Levin, (P)
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Leonard A. Levin, Department of Ophthalmology and Visual Sciences, University of Wisconsin Medical School, 600 Highland Avenue, Madison, WI 53792.
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and 
). (C) Conductance versus voltage curves for the six cells in which SS treatment was effective. Filled symbols: from the 1-hour (24 hours) condition in (B). Solid line: a fit to their mean, using the equation G = Gmax/{1+ exp[(V1/2 – V)/slope]}, and yielding a maximum conductance (Gmax) of 26 nS, a half-maximum voltage (V1/2) of +48 mV, and a slope factor of 31 mV. The parameters from individual fits were (mean ± SEM): Gmax = 29 ± 5 nS, V1/2 = +55 ± 12 mV, and slope = 33 ± 6 mV. Open symbols: from the 1-hour (48 hours) and 22-hour (2 hours) treatment conditions. These cells did not approach their maximum conductance even at +90 mV. Thus, they could not be fit, but appeared to display a different voltage dependence than that in the 1-hour [24-hour] condition.
