Introduction

Ethanol (EtOH) abuse is a critical global health challenge, promoting several deaths annually and accounting for 5.3% of all global mortality [1]. An elevated alcohol abuse is found in Brazil and other American countries, reflecting a growing public health burden [2]. The abuse of EtOH among pregnant women can result in Fetal Alcohol Spectrum Disorders (FASD)—a range of irreversible conditions that include structural, cognitive, and behavioral abnormalities [3]. EtOH is able to cross the placental barrier, reaching fetal concentrations equivalent to those in maternal circulation [4, 5]. During early development, the central nervous system (CNS) is vulnerable to EtOH-induced insults disrupting neuronal differentiation, synaptogenesis, and neurotransmission [6–8].

One of the most affected neurotransmitter systems by EtOH is the GABAergic system, which is a key regulator of excitatory-inhibitory balance in the CNS [9, 10]. EtOH modulates the GABAergic system at multiple levels. It acts as a positive allosteric modulator of GABA-A receptors [8], affects GABA synthesis and receptor subunit expression [9, 10], and may alter GABA transporter (GAT) activity, GAT-1, a neuronal high-affinity GABA transporter, is responsible for the majority of GABA reuptake in the CNS and its regulation is critical for maintaining synaptic and extra synaptic GABA levels [11, 12]. Specifically in the chicken retina, over 90% of GABA uptake is GAT-1-dependent [13–16]. Pharmacological studies have established that GAT-1 activity can be selectively inhibited by compounds such as NO-711 (also referred to as NNC-711), a selective blocker of GABA uptake [17, 18]. In the chicken retina, NO-711-sensitive GABA uptake has been associated predominantly with neuronal GAT-1 activity: although both GAT-1 and GAT-3 are expressed in Müller glial cells, NO-711 does not significantly affect [3H]-GABA uptake in purified Müller glia cultures, where uptake is mainly mediated by GAT-3. On the other hand, NO-711 inhibits approximately 90% of [3H]-GABA uptake in mixed neuron–glia cultures [19, 20].

Additionally to pharmacological regulation, GAT-1 activity is modulated by PKA and PKC pathways [21], which are themselves susceptible to EtOH-mediated changes [25–27]. In addition, chronic EtOH exposure has been shown to influence glutamatergic signaling, including NMDA receptor activity. In the developing chicken retina, it enhances glutamate-evoked GABA release through NMDA receptor-dependent mechanisms [22]. Consistent with this, chronic EtOH exposure also modifies the expression of NMDA receptor subunits, particularly GluN2B [23–25]. Prenatal EtOH exposure is able to increase GluN2B-containing NMDA receptor expression and increases sensitivity to a GluN2B-selective antagonist, as ifenprodil [26], indicating functional upregulation of this subunit. Therefore, GluN2B-containing receptors seems to contribute to chronic EtOH-induced modulation of neurotransmission during development.

GABA is detectable in chicken retina at embryonic day 6 (E6) [27]. GABA is predominantly expressed in amacrine and horizontal neurons, with Müller glial cells contributing to its homeostasis via GABA transporters expression [17, 28, 29]. Remarkably, GABA uptake and release depend on GATs activity, particularly GAT-1, with evidence of physiological transporter reversal under specific ionic conditions in the chicken retina [30, 31].

Several experimental models have contributed to advancing the understanding of the effects of prenatal alcohol exposure on neurodevelopment including rodents, zebrafish, and human cerebral organoids. In rodent models, prenatal EtOH acute exposure modifies GABAergic and glutamatergic signaling, and promotes neuronal apoptosis in regions such as the hippocampus and prefrontal cortex, and modifies the expression of GABA-A receptor subunits and transporters [32, 33]. Embryonic exposure to EtOH leads to impairments in several models, as zebrafish [40], human cerebral organoids [41], rodents [18, 42, 43]. In addition, chronic gestational EtOH showed a reduction of 30% of cortical GABAergic interneurons in exposed mice Smiley et al. (2015). In other regions, like the hippocampus, prenatal EtOH similarly dysregulated GABA signaling when adult guinea pigs were exposed in utero, exhibiting elevated hippocampal GABA-A subunit expression and impairments in spatial learning [34].

Despite the contributions of these systems, the chicken retina is a powerful and accessible model for studying early neurodevelopmental processes [35]. With a highly organized laminar architecture, well-characterized developmental timeline, and a rich repertoire of neurotransmitters, it offers a tractable platform for dissecting the molecular and cellular effects of EtOH during critical windows of synaptogenesis [22, 35]. Its simplicity does not compromise its representativeness: neurotransmitter dynamics in the retina mirror those observed in central brain region [36], making it possible to investigate both GABA uptake and release, transporter regulation, and kinase involvement under precise experimental control [35].

Although chronic EtOH-induced alterations in retinal GABAergic signaling have been described [22], whether acute EtOH exposure directly modulates GABA uptake and the intracellular mechanisms involved during critical windows of synaptogenesis is largely unexplored. Given that early disruptions in GABAergic signaling have been implicated in the neurodevelopmental consequences of prenatal alcohol exposure, investigating how EtOH modulates GABA transporter function during development is crucial for advancing our understanding of neurochemical alterations associated with these effects. This study examines the effect of acute EtOH exposure on GABA uptake in the developing chicken retina, focusing on embryonic days 11 (E11) and 16 (E16) stages, characterized by active synaptogenesis and circuit refinement. By analyzing transporter activity and the involvement of signaling pathways such as PKA and PKC, this work aims to elucidate mechanisms through which acute EtOH exposure may disrupt GABAergic homeostasis during early neurodevelopment, contributing to a broader understanding of how prenatal alcohol exposure affects the developing nervous system.