Results

Developmental Dynamics of GABA Uptake and Its Dose‑Dependent Inhibition by Acute EtOH in the Embryonic Chicken Retina

Although the retina is a well-established model for studying neurotransmission and neurodevelopmental processes, few studies have explored its neurochemical dynamics in the context of EtOH exposure, particularly regarding GABA transport mechanisms. The embryonic chicken retina offers a unique opportunity to investigate these questions due to its well-characterized developmental timeline and accessibility. However, the temporal regulation of high-affinity GABA uptake, as well as its modulation by acute EtOH exposure, remains poorly understood in this system. To establish a baseline for GABA transporter function during key neurodevelopmental windows, two distinct embryonic stages were used: embryonic day 11 (E11), corresponding to the onset of synaptogenesis and early neural circuit formation, and embryonic day 16 (E16), a stage of retinal development with active synaptic refinement [28]. [3H]-GABA uptake assays were conducted in retinal explants collected at E11 and E16 (Fig. 1). GABA uptake was then evaluated using [3H]-GABA assays, and values were normalized to protein concentration (fmol/mg). At E11, we observed measurable GABA uptake (82.7 ± 12.63 fmol/mg), indicating that GABA transporters are functionally active at the beginning of synaptic development (Fig. 1). By E16, uptake values increased significantly to 416.6 ± 75.25 fmol/mg, suggesting an upregulation of transporter activity as the retina matures and synaptic connectivity becomes more complex (p = 0.0029 vs. E16) (Fig. 1).

Fig. 1: GABA uptake at different developmental stages in embryonic chicken retinas. Retinal tissues were collected at embryonic day 11 (E11) and 16 (E16) and incubated with [3H]-GABA to assess uptake activity. GABA uptake is present at E11, indicating early functional expression of GABA transporters. A significant increase in uptake was observed at E16, reflecting enhanced transporter activity during synaptic maturation. Data are expressed as mean ± SEM (n = 8–11). Statistical analysis was performed using an unpaired Welch’s t-test (i.e., not assuming equal variances). Different colors for individual dots correspond to different experiment batches. **p < 0.01

Once GABA uptake is functional at embryonic day 11 (E11), we asked whether EtOH could modulate this process. E11 retinal explants were incubated for 30 min in HBSS containing increasing concentrations of EtOH: 0.1%, 0.3% and 0.5% (v/v). Untreated retinas incubated in HBSS alone served as the control group. Welch’s ANOVA test indicated a significant effect for EtOH treatment in E11 (W4, 14.26 = 7.093; p = 0.0023). As shown in Fig. 2A for E11, only the lowest concentration (0.1%) showed a suggestive decrease of GABA uptake (p = 0.0946 vs. CTRL), whereas higher doses did not differ from control (p > 0.05). At E16, EtOH treatment also did not significantly affect GABA uptake at 0.1% (p = 0.1607 vs. CTRL) (Fig. 2B). Together, these results suggest a non-linear dose effect. Importantly, the consistent tendency observed at E11 supports the selection of this developmental stage and the 0.1% EtOH concentration for subsequent experiments.

Fig. 2: Dose-dependent effect of acute EtOH exposure on GABA uptake in E11 and E16 chicken retinas. A Retinal explants from E11 embryos were treated for 30 min with vehicle (CTRL) or increasing concentrations of ethanol (EtOH) (EtOH: 0.1%, 0.3% and 0.5%; v/v), and GABA uptake was measured using [3H]-GABA (n = 7–13). Acute treatment with 0.1% EtOH produced a suggestive reduction in GABA uptake compared with CTRL (p = 0.0946), whereas 0.3% and 0.5% EtOH did not differ from CTRL B A similar assay was conducted using explants from E16 embryos. Acute EtOH treatment did not significantly affect GABA uptake at this developmental stage. Results were normalized to protein content (fmol/mg). Data are presented as mean ± SEM. Sample sizes varied between groups (n = 3–11). Statistical analysis was performed using Welch’s one-way ANOVA, followed by Dunnett’s T3 post hoc test to compare each treatment group to CTRL. This approach was chosen to account for unequal variances and unbalanced sample sizes. *p < 0.05

GAT‑1 Inhibition Decreases GABA Uptake with no Change in Transporter Expression

To evaluate whether the suggestive effect in GABA uptake observed following acute EtOH exposure involves GAT-1 activity, E11 retinal explants were incubated for 30 min in four groups: vehicle control (HBSS), 0.1% EtOH (EtOH), 100 µM NO-711 (a selective GAT-1 inhibitor), and the combination of 0.1% EtOH + NO-711 (Fig. 3). Treatment with NO-711 reduced [3H]-GABA uptake levels compared to control (CTRL: 228 ± 37; NO-711: 102 ± 22 fmol/mg, p < 0.05) (Fig. 3). Similarly, retinas exposed to 0.1% EtOH showed a reduced uptake (123 ± 20 fmol/mg, p < 0.05). The group receiving the combined treatment (EtOH + NO-711) was similar (78 ± 15 fmol/mg) with no additional reduction beyond the levels observed in the single-treatment groups (Fig. 3). These data confirm that both NO-711 and 0.1% EtOH decrease GABA uptake at E11. The absence of a more pronounced effect in the co-treated group indicates that the EtOH and GAT-1 selective inhibitor does not produce additive effects in GABA uptake pattern.

Fig. 3: Effect of GAT-1 inhibition and EtOH on GABA uptake in E11 chicken retinas. Retinal explants were treated with vehicle (CTRL), 0.1% ethanol (EtOH), 100 µM NO-711 (GAT-1 inhibitor), or 0.1% EtOH + NO-711 for 30 min. [3H]-GABA uptake was measured and normalized to protein content (fmol/mg). Both EtOH and NO-711 significantly reduced GABA uptake compared to CTRL (CTRL: 228 ± 37; EtOH: 123 ± 20; NO-711: 102 ± 20 fmol/mg, p < 0.05). Co-treatment with EtOH + NO-711 resulted in similar uptake levels (79 ± 15 fmol/mg) with no additive effect. Data are presented as mean ± SEM (n = 5–14). Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test; p < 0.05 vs. CTRL. *p < 0.05 and **p < 0.01

We next sought to determine whether EtOH modulates GABA uptake by altering GAT-1 protein expression levels. Thus, we performed Western blot analysis to assess the protein content of GAT-1 in control and EtOH-treated retinas (Fig. 4). Retinal tissues were collected from E11 embryos incubated for 30 min in either vehicle (HBSS) or 0.1% EtOH (EtOH), followed by protein extraction and immunoblotting for GAT-1. Densitometric analysis revealed no significant difference in GAT-1 protein levels between control and EtOH-treated groups (t = 0.054, df = 3.934; p = 0.9592) (Fig. 4). This data suggests that EtOH-induced decrease in GABA uptake in E11 retinas is independent of GAT-1 protein levels modulation.

Fig. 4: Western blot analysis of GAT-1 expression in E11 chicken retinas after acute EtOH exposure. Retinal explants were incubated with vehicle (CTRL) or 0.1% ethanol (EtOH) for 30 min. GAT-1 protein levels were measured by Western blot and normalized to β-actin. No significant difference was observed between groups (p = 0.9592; n = 3 per group). Data are expressed as mean ± SEM. Statistical analysis was performed using unpaired Welch’s t-test, which accounts for unequal variances. Given the small sample size, these results are interpreted in an exploratory, hypothesis-generating context

Reversible EtOH‑Induced Inhibition of GABA Uptake Occurs Without Cytotoxicity in E11 Retinas

After demonstrating that acute exposure to 0.1% EtOH significantly reduces GABA uptake in E11 retinas, and that this effect is not accompanied by changes in GAT-1 protein expression, we next investigated whether the observed functional inhibition is reversible. This step is important to determine whether EtOH transiently modulates transporter activity or induces longer-lasting disruptions in inhibitory neurotransmission during early retinal development.

To assess the reversibility of EtOH’s effect, retinal explants from E11 chick embryos were exposed to 0.1% EtOH in HBSS for 30 min (Fig. 5). Next, explants were either immediately processed for [3H]-GABA uptake (EtOH group) or transferred to EtOH-free HBSS for a 10-min washout period before uptake GABA assessment (Washout group). A control group (CTRL) was incubated only in HBSS. Welch’s ANOVA pointed to statistical significance among groups (W2, 14.10 = 16.13; p = 0.0001). As shown in Fig. 5, EtOH pointed to a suggestive decrease GABA uptake compared to the control (p = 0.088 vs. CTRL). Remarkably, explants subjected to the washout protocol showed uptake levels that were fully restored to baseline (p = 0.5286 vs. CTRL; p < 0.0001 vs. EtOH 0.1%), indicating that the inhibitory effect of EtOH on GABA uptake is reversible under the conditions tested.

Fig. 5: Reversibility of EtOH-induced inhibition of GABA uptake following washout. E11 chicken retinal explants were divided into three experimental groups: control (CTRL), ethanol (EtOH; 30-min exposure to 0.1% EtOH), and washout (30-min exposure to 0.1% EtOH followed by a 10-min wash with Hank’s solution 4). GABA uptake in the EtOH group showed a suggestive reduction compared to the control group (p = 0.08). Following washout, uptake was significantly restored p < 0.0001 vs. EtOH 0.1%). Data are presented as mean ± SEM (n = 7–12). Statistical analysis was performed using one-way Welch’s ANOVA followed by Dunnett T3 post hoc test, appropriate for unbalanced groups with unequal variances. Different colors for individual dots correspond to different experiment batches. *p < 0.05 and **** p < 0.0001

To demonstrate that EtOH is not cytotoxic, we assessed cell viability through quantification of lactate dehydrogenase (LDH) release, a widely used marker of membrane integrity and cell lysis. Elevated levels of LDH in the extracellular medium are indicative of cellular damage or death. As shown in Fig. 6, LDH levels in the EtOH-treated group were statistically similar to those in the control group in both ages (E11: t = 0.1913, df = 9.153; p = 0.8525; E16: t = 1.019, df = 9.437; p = 0.3335), indicating that acute exposure to 0.1% EtOH did not increase membrane permeability or induce cell death under these conditions. Triton-X addition to retinas were used as positive control and reference levels for cytotoxicity.

Fig. 6: EtOH exposure does not reduce cell viability in E11 and E16 chicken retinas. Cell death was assessed by measuring lactate dehydrogenase (LDH) release in retinal explants at embryonic day 11 (E11, A) and day 16 (E16, B) following a 30-min incubation with vehicle (CTRL) or 0.1% ethanol (EtOH). A third group treated with 0.01% Triton X-100 was included as a positive control for maximal lysis. LDH release values were normalized to the Triton X group (100%) to express results as percentage of total lysis. Both CTRL and EtOH groups showed comparable LDH levels (p > 0.05), indicating no EtOH-induced increase in cell death. Triton X-100 treatment resulted in complete cell lysis, validating the assay. Statistical comparisons were performed between CTRL and EtOH groups only, using Welch’s t-test (n = 5–7 per group). The Triton group was excluded from inferential analysis and used solely as a normalization reference. Values are presented as mean ± SEM. Different colors for individual dots correspond to different experiment batches.

EtOH Does Not Modify GluN2B Phosphorylation but Promotes GABA Release in E11 Retinas

We asked if EtOH may influence intracellular signaling pathways that regulate transporter function. To do so, we examined the levels of phosphorylated GluN2B (pGluN2B) in retinal explants from E11 embryos following acute exposure to 0.1% EtOH for 30 min (Fig. 7). As shown, no significant difference in pGluN2B expression was observed between EtOH-treated retinas and controls (t = 0.7889, df = 2.328; p = 0.5025). These results suggest that, under the experimental conditions tested, acute exposure to low-dose EtOH does not significantly alter the phosphorylation state of the GluN2B subunit in the developing chicken retina.

Fig. 7: EtOH does not alter phosphorylated GluN2B expression in E11 chicken retinas. Western blot analysis of phosphorylated GluN2B (pGluN2B) was performed in retinal explants from E11 embryos treated for 30 min with vehicle (CTRL) or 0.1% ethanol (EtOH). Densitometric quantification normalized to actin revealed no significant difference between groups (n = 3 per group; p = 0.5025). Data are presented as mean ± SEM. Statistical analysis was performed using Welch’s t-test. Given the small sample size, results are interpreted as preliminary and hypothesis-generating

Next, we asked whether acute EtOH exposure also modulates neurotransmitter release. To address this, E11 retinal explants were pulse-labeled with [3H]-GABA and exposed for 5 min to 0.1% EtOH, a time window selected consistent with pulse-based release paradigms in the chicken retina in previous results [17, 18, 22] (Fig. 8). A general effect was found in Welch’s ANOVA test (W2, 5.18 = 9.592; p = 0.0181). As shown in Fig. 8, EtOH increased GABA release, more than doubling the amount released relative to vehicle controls (p = 0.0421 vs. CTRL). To determine whether this effect involves NMDA receptors containing the GluN2B subunit, explants were co-treated with Ifenprodil (10 µM), a GluN2B antagonist. Co-application of Ifenprodil did not restore release to baseline, and GABA efflux remained elevated (p = 0.0285 vs CTRL; p = 0.9921 vs. EtOH 0.1%). These findings demonstrate that the EtOH-induced enhancement of GABA release in E11 retinas is robust and does not depend on activation of GluN2B-type NMDA receptors.

Fig. 8: The effect of EtOH on GABA release is not inhibited by Ifenprodil. Acute exposure to 0.1% ethanol (EtOH) for 5 min significantly increased [3H]-GABA release in E11 chicken retinal explants compared to control. Co-application of Ifenprodil (10 µM), a selective allosteric inhibitor of GluN1-GluN2B NMDA receptors, did not reverse GABA release to baseline levels. Data are expressed as mean ± SEM (n = 3–6). Statistical analysis was performed using Welch’s ANOVA followed by Dunnett’s T3 post hoc test. p = 0.0421 vs. CTRL (EtOH 0.1%); p = 0.0285 vs. CTRL (EtOH + Ifenprodil). Given the modest sample size and variation, results are interpreted as preliminary and hypothesis-generating. Different colors for individual dots correspond to different experiment batches. *p < 0.05

Inhibition of PKA and PKC Reverses EtOH-Induced Reduction in GABA Uptake

Since our data suggest that EtOH-decreased GABA uptake does not involve modulation of GAT levels, we asked whether PKA- and PKC-dependent signaling pathways contribute to this effect, as both kinases are central to control GAT activity. In this context, we tested whether pharmacological blockade of each enzyme could reverse EtOH-induced effects (Fig. 9 A, B). To do so, E11 retinal explants were treated for 30 min with 0.1% EtOH alone or in combination with either 10 mM H-89 (PKA inhibitor), or 100 nM Gö6983 (PKC inhibitor). Uptake of [3H]-GABA was then quantified and compared across experimental groups. For PKC analysis, two-way ANOVA test did not point to significance with EtOH treatment (F1,41 = 0.2999; p = 0.5869) but detected a significant statistical difference for treatment with G0 (F1, 41 = 17.62; p = 0.0001) and interaction between drugs (F1, 41 = 4.172; p = 0.0476). As shown in Fig. 9A, GABA uptake was reduced following 30-min treatment with 0.1% EtOH, however, this comparison did not reach statistical significance (p = 0.0511 vs. EtOH), confirming previous observations. Notably, Gö6983-treated retinas did not differ from control (p = 0.4523 vs. CTRL). However, when compared to EtOH, G0 significantly increased GABA uptake levels (p = 0.0002 vs. G0 + EtOH), indicating that its primary effect is to counteract EtOH’s inhibition rather than to elevate uptake beyond baseline. Similarly, for PKA analysis, the two-way ANOVA test did not indicate a statistical difference with EtOH treatment (F1,41 = 0.2054; p = 0.6528) but found a significant statistical difference for treatment with H89 (F1, 41 = 23.72; p < 0.0001), with interaction between drugs (F1, 41 = 5.314; p = 0.0263). While EtOH decreased GABA uptake (p = 0.0335 vs. CTRL), the PKA inhibitor H-89 did not affect GABA uptake by itself (p = 0.285 vs. CTRL) but increased GABA uptake relative to EtOH alone (p < 0.0001 vs. H89 + EtOH) (Fig. 9B**)**, suggesting that the observed increase reflects reversal of EtOH’s effect rather than enhancement above physiological levels.

Fig. 9: Inhibition of PKA and PKC reverses EtOH-induced reduction in GABA uptake in E11 chicken retinas. A GABA uptake was reduced following 30-min treatment with 0.1% ethanol (EtOH), however, this comparison did not reach statistical significance (p = 0.0511). Treatment with the PKC inhibitor Gö6983 (100 nM) increased uptake (p = 0.0002 vs. EtOH) while it did not induce any effect on control group (p = 0.4523 vs. CTRL). B Similarly, the PKA inhibitor H89 (10 µM) reversed the EtOH-induced reduction in GABA uptake (p < 0.0001 vs. EtOH), whereas H89 alone did not significantly affect GABA uptake (p = 0.2852 vs. CTRL). Data are presented as mean ± SEM (n = 5–20). Statistical analysis was performed using Welch’s one-way ANOVA followed by Šidák’s post hoc test. Different colors for individual dots correspond to different experiment batches. *p < 0.05, *** p < 0.001 and **** p < 0.0001

These results provide evidence that the inhibitory effect of EtOH on GABA uptake is mediated, at least in part, by signaling pathways dependent on PKA and PKC. Pharmacological inhibition of either kinase is sufficient to normalize transporter function, supporting the idea that EtOH acts through these pathways to suppress GABA uptake and that blocking them restores uptake to control levels without over-shooting baseline.