Materials and Methods

Subjects

Fertilized White Leghorn eggs (Gallus gallus domesticus) were obtained from a local hatchery and staged as previously described [37]. Embryonic retinas (E11 and E16) were dissected and separated from the other ocular tissues in calcium-magnesium free solution (CMF), at 37 °C. Subsequently, retinas were placed in 35 mm petri dishes containing 1 ml of Hanks’ 4 Balanced Salt Solution (HBSS; NaCl 128 mM; KCl 4 mM; MgCl2 1 mM; –CaCl2 3 mM; HEPES 20 mM; glucose 4 mM; pH 7.4), for neurochemical assays.

Drugs and Reagents

EtOH P.A. (obtained from Isofar), H-89 dihydrochloride hydrate (H-89, 10 µM; #B1427), NO-711 hydrochloride (NO-711, 100 µM; N142), Gö 6983 (2-[1-(3-dimethylaminopropyl)− 5-methoxyindol-3-yl]− 3-(1H-indol-3-yl) maleimide, 100 nM; G1918), and Ifenprodil (10 µM; I-2892) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Radiolabeled GABA ([3H]-GABA) specific activity 35 Ci/mmol was purchased from PerkinElmer (Massachusetts, USA). LDH Cytotoxicity Assay Kit was obtained from Cayman Chemical (Ann Arbor, MI, USA). All other reagents were of analytical grade, obtained from high-grade sources.

Tissue Preparation

Retinas from Fertilized White Leghorn eggs were isolated. To increase the contact surface, the tissue amount was equally sliced in sections of approximately 200 µm already in DMEM/F12 or Tris–HCl solution (pH 7.4) before the addition of [3H]-GABA (at 37 °C) or the addition of lysis buffer for western immunoblotting (20 mM Tris–HCl pH 8.0, 137 mM NaCl, 10% glycerol supplemented with protease inhibitors (10% v/v; Sigma) and phosphatase (Roche).

EtOH and Drug Treatment

EtOH was prepared by serial dilution in HBSS solution, starting from a 1% (v/v) EtOH concentration and reaching a final concentration of 0.1% (v/v), corresponding to approximately 21.71 mM (see Table 1). In most assays, EtOH was administered for 30 min prior to the addition of [3H]-GABA. An exception was made for the release assay, in which EtOH was perfused for 5 min, either alone or in combination with other drugs.

Drug | Function | Concentration
Ethanol | Positive allosteric modulator of GABAA receptors | 17 mM
NO-711 | GABA transporter-1 (GAT-1) inhibitor | 50 µM
Gö 6983 | Protein kinase C (PKC) inhibitor | 100 nM
H89 | Protein kinase A (PKA) inhibitor | 100 nM
Ifenprodil | GluN2B-selective N-methyl-d-aspartate (NMDA) receptor antagonist | 10 µM

[3H]-GABA Uptake Assay

[3H]-GABA uptake was performed as described before [38]. After pre-exposure to NO-711 (100 μM), Gö 6983 (100 nM), H89 (10 µM), or HBSS alone (control group), each retina was incubated for 1 h in 1 mL of HBSS pH 7.4 at 37 °C containing 0.5 μCi of [3H]-GABA (35 Ci/mmol = 35.106 μCi) and 20 μM of non-radioactive GABA as a carrier without changing the initial medium. The GABA concentration of 20 μM and the incubation time (1 h) were selected to ensure the steady state of the uptake curve, while 0.5 μCi [3H]-GABA (35 Ci/mmol) was only used as a radioactive tracer to evaluate [3H]-GABA transport activity [18, 39]. After 60 min incubation with [3H]-GABA, the solution was removed, and the tissue was washout three times with 3 mL of cold HBSS. This process was sufficient to washout the radioactivity not taken up by the cells. Then, 1 mL of Milli-Q water (Milli pore) was added to disrupt cell membranes, followed by a freeze–thaw cycle. The intracellularly accumulated radioactivity was determined using a liquid scintillation counter. Uptake values were calculated as fmol of [3H]-GABA and normalized by protein concentration, estimated with Lowry protein assay using BSA as standard, as described previously [38].

[3H]-GABA Release Assay

Briefly, [3H]-GABA release was performed as described before [38]. Following the [3H]-GABA uptake, the medium was removed, and the tissue was washed three times with 1 mL of warm HBSS at 37 °C to washout the radioactivity not taken up by the cells. Afterward, the retina was superfused with 0.5 mL of HBSS alone (basal), EtOH 0.1% (vol/vol) and/or Ifenprodil (10 µM) at 37 °C for 5 min. At the end of the superfusion period, the superfusate was collected and its radioactivity was quantified by liquid scintillation. To determine the proportion of released radioactivity relative to total cellular [3H]-GABA content, the corresponding retinal tissues were subsequently processed according to the uptake protocol. Release values were normalized as a percentage of total [3H]-GABA previously incorporated by each retina [40].

Western Blot Assay

Western Blot was performed as described before [38]. Briefly, both retinas were extracted and homogenized together in a RIPA buffer containing a cocktail of protease inhibitors. The homogenate was used to assess the expression of GAT-1 protein and phosphorylated subunit GluN2B in control and EtOH-treated groups. Protein concentration was estimated [41] and the samples were diluted in a buffer composed of 10% glycerol (v/v), 1% ß-mercaptoethanol, 3% SDS and 62.5 mm Tris base, which were boiled for 5 min. Approximately 15 μg of protein from each sample was used in electrophoresis in 8% (GAT-1) and 10% (GluN2B) SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (ECL-Hybond) by a semi-dry transfer method. Membranes were washed with a Tween 20 Tris-buffered saline (TTBS) and blocked for 2 h with TTBS plus 1% BSA. After blocking, membranes were lightly washed with TTBS and incubated with primary anti-GAT-1 (1:500 in TTBS; Sigma-Aldrich; HPA013341) and anti-pGluN2B antibodies (1:2000 in TTBS; Sigma-Aldrich; M2442) overnight at 4 °C. On the following day, the primary antibodies were removed, and the membranes were washed three times with TTBS to remove unconjugated antibodies. Subsequently, incubation was carried out with anti-rabbit secondary antibodies conjugated to peroxidase (1:5000 in TTBS; Sigma-Aldrich; F1262) for 2 h at room temperature. After incubation, the membrane was again washed three times with TTBS (10 min each), and the probe was detected using an ECL kit (Amersham).

Membranes were re-probed with mouse anti-β-tubulin antibody (1:25,000 in TTBS, Cell Signaling; T5201) or mouse anti-β-actin antibody (1:2000 in TTBS; #CBL171, Millipore) for 1 h at room temperature, washed 3 times with TTBS, and incubated with a secondary anti-mouse antibody conjugated to peroxidase (1:5000 in TTBS; Santa Cruz Biotechnology; SC533657) for 45 min at room temperature, followed by three new TTBS washes. Immunostaining was detected with the ECL kit. The intensity of the bands was analyzed using ImageLab 5.2.1 software (Bio-Rad Laboratories Inc).

Lactate Dehydrogenase (LDH) Cytotoxicity Assay

Cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) release, which reflects plasma membrane disruption and loss of cellular integrity [42]. The assay was performed as previously described, with adaptations for the ex vivo chick retina preparation [42]. Briefly, the LDH Cytotoxicity Assay Kit (Cayman Chemical) was used according to the manufacturer's instructions. E11 chick retinas were incubated with 0.1% EtOH or vehicle for 30 min at 37 °C, while 10% Triton X-100 was used as a positive control for maximal LDH release. After treatment, the tissues were transferred to microcentrifuge tubes, vortexed and homogenized to promote tissue dissociation. Following a brief centrifugation, 10 μL of the supernatant from each sample was transferred to a 96-well plate containing HBSS and incubated with the LDH reaction solution, freshly prepared according to the manufacturer's instructions, for 30 min at 37 °C. Absorbance was measured at 490 nm using a microplate reader. Cytotoxicity was expressed as the percentage of maximal LDH release, calculated from spontaneous and maximum LDH release controls.

Statistical Analysis

All statistical analyses were conducted using GraphPad Prism 10 (GraphPad Software, LLC). Data are presented as mean ± standard error of the mean (SEM) (or as percentage of control where applicable), and results were considered significant at p < 0.05. Potential outliers were assessed using the ROUT (Robust Regression and Outlier Removal) method with Q = 1% in GraphPad Prism. Only values objectively identified by this criterion were excluded from the statistical analyses. For comparisons between two groups, unpaired two-tailed t-tests were used, with Welch’s correction applied whenever variances were unequal or group sizes were unbalanced. For comparisons involving three or more groups, one-way analysis of variance (ANOVA) was performed (using Welch’s ANOVA when heteroscedasticity warranted), and factorial experiments with a 2×2 design were analyzed by two-way ANOVA including the interaction term. Following Welch’s one-way ANOVA, Dunnett’s T3 post hoc test was used when the experimental design required comparisons of each treatment group with a single reference group, as this procedure appropriately accounts for unequal variances and unbalanced sample sizes. For two-way ANOVA, group differences were further examined using planned post hoc comparisons (simple effects contrasts) rather than exhaustive pairwise testing, with Šídák’s method employed to adjust for multiple comparisons, as it appropriately controls the family-wise error rate while maintaining greater statistical power for a limited number of predefined pairwise comparisons. Tukey's multiple comparisons test was used to evaluate all possible pairwise comparisons among the experimental groups while controlling the family-wise error rate. The sample size (n) refers to the number of retinas per experimental group, and retinas were randomly assigned to treatment groups.

Assumptions of normality and homogeneity of variances were verified primarily by visual inspection of residuals and Q–Q plots. Formal tests of normality (e.g., Shapiro–Wilk) were performed (Supplementary data 1) but not relied upon in isolation, given their limited sensitivity with small sample sizes. In comparisons involving small group sizes (n ≤ 4), formal statistical testing was still performed as specified above; however, these results are interpreted cautiously and are presented as exploratory and hypothesis-generating, given the limited precision and generalizability associated with small samples. No logarithmic or other data transformations were applied; all analyses were conducted on raw (untransformed) data.