Discussion

Acute EtOH Affects GABA Uptake in a Dose-Dependent Manner

Several drugs of abuse have been investigated using the chicken retina model exploring its unique neuro-glial circuit, such as caffeine [43], nicotine [38], EtOH [22], opiates [44] and cannabinoids [45, 46], among others [47]. Here, we demonstrate that a single exposure to 0.1% EtOH at the onset of synaptogenesis is probably enough to modify GABA homeostasis. A 30-min exposure was used to assess acute effects, consistent with previous studies in the chicken retina employing similar incubation times [22, 38, 45].

The presence of functional GABA uptake at E11 is consistent with the early establishment of GABAergic circuitry during retinal development [36, 48]. Consistent with these trends, [3H]-GABA uptake assays revealed a functional transport system at E11 and a ~50% increase by E16, reflecting progressive maturation of GABA transport during synaptogenesis. Importantly, previous studies from our group demonstrated that [3H]-GABA uptake is completely abolished when extracellular Na⁺ ions are replaced by Tris and markedly reduced at 4 °C, providing strong evidence that the measured signal reflects a specific sodium-dependent, transporter-mediated process requiring metabolic activity rather than nonspecific radiotracer incorporation [17]. Moreover, a single dose of 0.1% EtOH exposure at E11 suggested a reduction in GABA uptake without alteration in tissue viability or total GAT-1 levels. These effects disappear after EtOH washout, highlighting a reversibility mechanism. Importantly, the recovery of GABA uptake following washout indicates that the effect depends on the continuous presence of EtOH and does not reflect long-lasting or irreversible alterations in transporter function. The rapid recovery of the system is consistent with a dynamic modulation of transporter activity, potentially involving phosphorylation-dependent processes. However, we acknowledge that this assay alone does not resolve the precise temporal dynamics of EtOH action. A detailed time-course study, including pretreatment intervals and co-application protocols, would be necessary to distinguish between acute interference and downstream regulatory effects.

Although GAT-1 activity is relevant in E11, other high-affinity GABA transporters may contribute to total uptake at both stages [49]. Furthermore, the significant reduction in [3H]-GABA uptake produced by NO-711 here supports the participation of a GAT-1-mediated component in this process. Such early functionality aligns with the broader developmental roles of GABA in regulating proliferation, neuronal survival, differentiation and synapse formation [50, 51]. Alternatively, additional GABA derived from putrescine via alternative metabolic routes also supports early inhibitory tone in mammals, a mechanism that may also operate in the chicken retina at E11 when endogenous synthesis is still increasing [52].

EtOH, as a lipophilic molecule, might cross both the blood–brain and the placental barrier, due to its solubility, causing a wide range of toxicity [53]. Moreover, as stated by the WHO, no level of alcohol consumption is safe. In addition, doses that seem minimal already have an impact on neurodevelopment [54]. The dose–response curve ranging from 0.1% to 0.5% suggests a decrease in GABA uptake at 0.1%, with no incremental inhibition at higher doses. Comparable low-dose sensitivity has been documented in cortical cultures, where sub-millimolar EtOH alters synaptic viability and neurotransmitter release [55]. The 0.1% EtOH used here corresponds to ~ 0.1 g dL⁻1 (21.71 mM), an equivalent blood-alcohol level achieved in humans after a moderate-to-high consumption (four standard drinks in two hours) and widely employed in FASD research to model episodic maternal drinking. GABA transporters, particularly GAT-1, constitute early molecular targets of EtOH [56]; their modulation may influence the establishment of neural networks during critical periods of circuit assembly.

Future studies should determine whether this inhibition involves direct transporter–EtOH interactions, EtOH-induced shifts in membrane potential, or additional signaling cascades beyond PKA/PKC pathways, thereby clarifying how transient maternal alcohol exposure can leave lasting effects on inhibitory circuitry.

Inhibition of GAT‑1 Activity by EtOH is not Linked to a Downregulation of GAT-1

GAT-1 function is known to be regulated by phosphorylation-dependent mechanisms that affect transporter activity and membrane trafficking. Our previous work showed that GAT-1 can be modulated by drugs such as caffeine and cocaine [17, 39, 57]. In this sense, our results indicate that the suggestive acute reduction in GABA uptake induced by EtOH is not accompanied by changes in the total expression of GAT-1 protein within the 30-min treatment window. Hence, the inhibition we observe is unlikely to originate from transcriptional or translational down-regulation of the transporter and instead points to fast functional modulation of GAT-1. Similar discrepancies between function and abundance have been reported in other brain regions after chronic EtOH exposure, where altered uptake kinetics were detected despite unchanged GAT-1 levels [58]. In E11 chicken retinae, a 0.1% EtOH pulse lowered basal [3H]-GABA uptake by 43.9%, indicating that EtOH directly impairs transporter function. Application of the selective GAT-1 inhibitor NO-711 reduced uptake even further ( 53.5% versus baseline), confirming its potency. Addition of EtOH with NO-711 produced a ~67% decrease of [3H]-GABA uptake relative to control values, with a non-additive interaction between EtOH and pharmacological blockade at this early developmental stage. Furthermore, the persistence of a certain level of uptake even with pharmacologically blocked GAT-1 is due to the presence and functionality of GAT-3, which performs a small portion of uptake and release in glial cells of the retinal tissue [29]. To our knowledge, this is the first study to examine low-dose (0.1% v/v) EtOH effects on GABA uptake in E11 retina with NO-711, highlighting the transporter’s vulnerability at the onset of synaptogenesis. Although GAT-1 is a major contributor, other high-affinity transporters probably participate, as developmental surveys have shown multiple GAT isoforms in the retina [49]. Tyrosine phosphorylation of GAT-1 is known to modulate its transport capacity [59]. The absence of a measurable change in GAT-1 protein suggests that EtOH acts through post-translational or signaling-dependent mechanisms.

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

The increase in LDH release is often correlated with the severity of EtOH exposure and is a reliable indicator of retinal damage [60]. During retinal development in the chick, a wave of programmed cell death unfolds between E10 and E14, peaking at E11, particularly within the inner nuclear layer [9, 61]. Since E11 is a period of intense cell death, one question that remains to be answered is whether cell death is being potentiated by EtOH. Previous work has demonstrated that an acute exposure to 1% EtOH in human retinal organoids led to a significant increase in apoptosis and cell cycle block, reflecting cell death and significant alterations in neuronal differentiation [62]. As LDH release was not further increased upon acute 0.1% EtOH challenge, this supports the interpretation that the observed reduction in GABA uptake reflects a transient functional modulation of GABA transporters rather than cell damage. A comparable phenomenon was reported by [63], who used the same LDH assay in murine retinal explants and showed that activation of the lactate-sensitive receptor HCA1R with 3.5 mM 3,5-DHBA preserved neuronal viability without elevating LDH. We reinforce that membrane integrity is maintained and that EtOH-induced interference (0.1%) with the GABAergic system in E11 chicken retina constitutes a physiological adjustment, not a cytotoxic event. It is important to note that, as the experiment was performed ex vivo, a basal level of LDH release is expected due to the intrinsic stress of tissue dissection and handling. Nonetheless, the absence of increased LDH release in the EtOH group supports the conclusion that the functional modulation of GABA uptake observed previously occurs in the absence of extensive cell damage. These findings strengthen the interpretation that the apparent effect in GABA uptake induced by acute EtOH exposure reflects a physiological regulatory mechanism rather than a pathological response associated with cell death. Following that, we hypothesized that, functionally, if we remove EtOH from the challenge after a previous exposure, we would be able to have the activity return, reinforcing that we are not experiencing death. The ability of developmental systems to recover from EtOH exposure may be linked to the timing and dosage of exposure. Studies in mice have shown that the timing of EtOH exposure significantly influences the severity and persistence of behavioral deficits, suggesting that early intervention or removal of EtOH can mitigate long-term effects [64]. In C. elegans, developmental delays were observed during and immediately after EtOH exposure (10%), but the effects were dynamic and not uniform, indicating that some recovery is possible after the removal of EtOH in these terms [65]. Similarly, research using zebrafish models has shown that short-term exposure to EtOH (low doses at 24-h post-fertilization and up to 2 h of exposure) might lead to developmental disruptions, but these effects may not be permanent [66]. For instance, zebrafish embryos exposed to EtOH exhibited behavioral anomalies, yet these were not always sustained into adulthood, suggesting a potential for recovery [67]. While the potential for recovery from EtOH-induced disruptions is promising, it is important to consider that the reversibility may vary across different species and developmental stages. These findings contribute novel functional evidence that short-term exposure to EtOH during critical developmental windows may produce transient, rather than sustained, disruption of GABA transporter function. To our knowledge, this is the first report to evaluate the reversibility of EtOH-induced inhibition of GABA uptake in the embryonic chicken retina, particularly at low EtOH concentrations.

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

NMDA receptor signaling—particularly involving the GluN2B subunit—has been identified as a potential mediator of EtOH’s effects in the developing nervous system [68]. Previous studies from our group have shown that pharmacological agents such as caffeine might modulate GluN2B-dependent signaling in the embryonic retina, which is blocked by Ifenprodil [18]. EtOH exposure might also modulate NMDA receptor function, potentially through phosphorylation of GluN2B subunits, leading to altered glutamatergic neurotransmission [69]. Although GluN2B is a known molecular target of EtOH in several brain regions and developmental models [8], our findings indicate that this specific signaling pathway is probably not involved during short-term exposure at this stage of retinal development (E11). Our results demonstrated that EtOH exposure significantly increased GABA release compared to control. Specifically, the amount of [3H]-GABA released during the 5-min pulse more than doubled in the EtOH-treated group relative to the vehicle-treated group (Fig. 8). To test whether this effect involved GluN2B-containing NMDA receptors, we included Ifenprodil, an allosteric inhibitor of GluN1-GluN2B subunits, during the EtOH pulse. Co-application of Ifenprodil with EtOH did not significantly reduce GABA release compared to EtOH alone.

The GABAergic system plays a crucial role in retinal development, and EtOH exposure can modulate GABA release through multiple mechanisms [70]. Activation of group III metabotropic glutamate receptors by EtOH decreases GABA immunoreactivity and alter glutamate release in the chicken retina, suggesting a complex interaction between glutamatergic and GABAergic systems [71]. Additionally, EtOH exposure can potentiate the depolarizing action of GABA in GABAergic interneurons, leading to aberrant migration of these cells in the embryonic cortex [72, 73]. Moreover, EtOH exposure has been shown to disrupt calcium signaling in the developing retina, which is critical for GABA release [74]. Several molecular targets may mediate the EtOH-induced increase in GABA release in the E11 chicken retina, since this effect was not blocked by Ifenprodil, whose pharmacological actions at 10 μM include near-maximal inhibition of GluN2B-containing NMDA receptors (IC₅₀ = 0.34 μM) [75]. The complete absence of an Ifenprodil effect suggests that GluN2B-containing NMDA receptors are not critically involved in this process. This negative result, while not definitive, did not warrant further investment in more selective GluN2B antagonists.

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

Protein kinase A (PKA) and C (PKC) are essential serine/threonine kinases involved in diverse cellular functions [76, 77]. These kinases exert contrasting effects on GABAergic signaling, vital for inhibitory neurotransmission in the developing retina. PKA and PKC influence the trafficking, surface expression, and functionality of GABAA receptors, which are critical for GABA uptake and synaptic inhibition [55, 78–80]. After observing a suggestive reduction in GABA uptake following acute EtOH exposure in the developing retina without altering GAT-1 expression or inducing cytotoxicity, and that this effect is reversible upon EtOH removal, we next sought to investigate the intracellular signaling pathways potentially involved in this modulation.

This uptake reduction is sensitive to pharmacological inhibition of GAT-1 activity and is abolished by pharmacological inhibition of PKA or PKC pathway, suggesting the involvement of kinase-dependent signaling in transporter regulation. Previous studies from our group have consistently [20] demonstrated the involvement of kinase-dependent signaling in the regulation of GABA transport in the chicken retina: Caffeine-induced reduction in GABA uptake in the chicken retina is mediated via PKA-dependent mechanisms [17], while caffeine also potentiates GAT-1-mediated D-aspartate-induced GABA release via adenosine A1 receptor inhibition and PKA activation [18]. Furthermore, pharmacological inhibition of PKC attenuates the nicotine-induced modulation of GABA uptake [38], reinforcing the role of intracellular kinase signaling in the drug-induced regulation of GABA transporter function in the chicken retina. Additionally, literature supports that EtOH can influence both PKA and PKC signaling pathways in neuronal systems [55, 81]. The activation of PKA by EtOH significantly enhances the surface expression of GABAA α1 subunits in cerebral cortical neurons, correlating with increased zolpidem potentiation of GABA responses, thus suggesting a facilitative role of PKA in GABAA receptor functionality [79]; concurrently, PKA activation also influences extra synaptic GABAA α4δ receptors, promoting tonic inhibition and potentially contributing to EtOH's neuroprotective properties under very specific conditions [80]. On the other hand, EtOH exposure activates PKC, specifically the PKCγ isoform, resulting in the internalization of GABAA α1 subunits, thereby diminishing their surface expression and impairing GABAergic inhibition, which may contribute to the hyperexcitability observed in the developing retina [82, 83]. The interplay of PKA and PKC within GABAergic signaling is not autonomous but rather interconnected with various signaling cascades; for instance, EtOH-initiated PKA activation is regulated by GABAB receptor activity, influencing intracellular cAMP levels and CREB phosphorylation [73, 84], while PKC activation through EtOH is affected by glycine receptor modulation, potentially leading to the inhibition of glycine-activated currents in retinal neurons [85]. To our knowledge, this is the first study to provide evidence supporting the involvement of kinase-dependent intracellular signaling in the modulation of GABA transport by acute EtOH in the developing chicken retina. Although additional mechanisms cannot be excluded, these findings identify PKA- and PKC-dependent signaling as a plausible component of the acute response and provide a basis for future mechanistic studies.

Study Limitations

While some experimental groups in this study had unbalanced or modest sample sizes, we believe this does not reduce the biological consistency or relevance of our findings. Across different assays, consistent patterns were observed in independently repeated experiments, supporting a coherent biological effect. In specific cases such as Western blot analyses, statistical testing was conducted even when group sizes were small (n = 3), and although no significant differences were found, these results were interpreted as preliminary indications of the absence of detectable changes in marker expression, rather than definitive conclusions. All experiments were performed under standardized conditions, and positive controls such as Triton X-100 were used to confirm assay sensitivity, while excluded from inferential statistics when not appropriate. While we acknowledge the limitations of some comparisons in terms of statistical power, we prioritized internal consistency, biological plausibility, and reproducibility across assays. Taken together, the data present a robust and biologically grounded model of EtOH acute effects on GABAergic transport during retinal development, contributing valuable insights and guiding future confirmatory studies.