Potential role of astrocyte on gamma-aminobutyric acid tone regulation during developmental period

The early developmental period is a critical window during which brain cells mature and contribute to both brain development and later life functions. Gamma-aminobutyric acid (GABA), recognized as a major neurotransmitter, plays a crucial role in coordinating synapse formation, neuronal proliferation, and migration during this time. Notably, recent studies have distinguished two modes of GABA action in the brain: a fast, phasic mode mediated by synaptic GABA release from GABAergic neurons, and a slower, tonic mode resulting from GABA release by non-neuronal cells, particularly astrocytes, which primarily inhibits neuronal activity and synaptic transmission, regulating various cognitive functions (Koh et al., 2023). However, the specific role of tonic GABA effects during the developmental period remains underexplored (Figure 1A). Here, we aim to examine the potential role of astrocytes in regulating GABA tone during development, thereby stimulating further investigation into GABA tone during this critical period.Figure 1: Potential mechanisms of astrocytic regulation of GABA tone in the developing brain.(A) The role of GABA tone in the adult brain has been associated with various processes, including learning and memory, sensory processing, circadian rhythm, motor coordination, emotional regulation, and addiction. However, its role in the developing brain is yet to be determined. (B) Astrocytes regulate GABA tone through diverse molecular mechanisms, including GABA synthesis, release, and clearance. GABA can be synthesized via two primary pathways: (1) following the blue arrows, putrescine-derived GABA synthesis is divided into DAO-dependent and MAO-B-dependent mechanisms; (2) following the red arrows, glutamate-derived GABA synthesis occurs via GAD-dependent mechanisms. Synthesized GABA is stored in vesicles by vesicular GAT (vGAT) and released into the extracellular space either via exocytosis or through BEST1 or LRRC8A channels, contributing to extracellular GABA tone. The extracellular GABA tone is then regulated by uptake through GABA transporters (GATs). Once inside the cell, GABA is degraded by GABA-T and SSADH into succinic acid, which enters the tricarboxylic acid (TCA) cycle for metabolic clearance. (C–F) Developmental expression levels of genes associated with GABA synthesis, release, and clearance in visual cortex astrocytes at postnatal days (P) 7, 14, 28, and adulthood (P120), obtained from a publicly available astrocyte transcriptome database (Farhy-Tselnicker et al., 2021; GEO Accession number GSE161398). (C) Developmental expression levels of genes associated with putrescine-derived GABA synthesis. (D) Developmental expression levels of genes associated with glutamate-derived GABA synthesis. (E) Developmental expression levels of genes associated with GABA release. (F) Developmental expression levels of genes associated with GABA clearance. Figure 1A and B was created with BioRender.com. Figure 1C–F was generated using GraphPad Prism 10 based on data from the publicly available astrocyte transcriptomic database (Farhy-Tselnicker et al., 2021). Abat: 4-Aminobutyrate aminotransferase; ALDH1A1: aldehyde dehydrogenase 1 family member A1; Aldh5a1: aldehyde dehydrogenase 5 family member A1; Aoc1: amine oxidase copper-containing 1; BEST1: bestrophin 1; DAO: diamine oxidase; GABA: gamma-aminobutyric acid; GABA-T: gamma-aminobutyric acid transaminase; GAD: glutamate decarboxylase; GAT: gamma-aminobutyric acid transporter; LRRC8A: leucine-rich repeat-containing protein 8A; MAO-B: monoamine oxidase B; PAT: putrescine acetyltransferase; Sat1: spermidine/spermine N1-acetyltransferase 1; SIRT2: sirtuin 2; Slc32a1: solute carrier family 32 member 1; Slc6a1: solute carrier family 6 member 1; Slc6a3: solute carrier family 6 member 3; SSADH: succinic semialdehyde dehydrogenase.In the adult brain, astrocytes regulate GABA tone through three main processes: synthesis, release, and clearance (Figure 1B). GABA synthesis is pivotal for its overall regulation, involving two primary precursors: glutamate and putrescine. While GABA synthesis from glutamate occurs through glutamate decarboxylases GAD1 and GAD2 (also known as GAD67 and GAD65, respectively), astrocytic GABA synthesis predominantly relies on putrescine degradation. This pathway consists of two distinct routes: the DAO-dependent pathway, which involves two steps (oxidation by DAO and dehydrogenation by ALDH1A1), and the MAO-B-dependent pathway, which includes four steps as follows: acetylation by putrescine acetyltransferase (PAT), oxidation by MAO-B, dehydrogenation by ALDH1A1, and deacetylation by NAD-dependent deacetylase sirtuin 2 (SIRT2). Following synthesis, GABA is released from astrocytes either via vesicles or more commonly through membrane channels such as BEST1 and LRRC8A. Clearance of extracellular GABA is mediated by GABA transporters (GATs), which uptake GABA into astrocytes. Intracellularly, GABA is degraded by GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH), ultimately entering the tricarboxylic acid cycle as succinic acid. These coordinated steps maintain GABA tone in the brain, predominantly affecting extrasynaptic GABAA receptors, which mediate tonic inhibition critical for neuronal activity modulation and cognitive functions (for more details, see Koh et al., 2023). These previous works, however, primarily focused on adult brains, leaving the mechanisms during the developmental period largely elusive. We utilized a publicly available developmental database of astrocytic transcriptomes (Farhy-Tselnicker et al., 2021) alongside other reports to show the potential role of astrocytes in regulating GABA tone during the developmental period. This database, generated through bulk RNA sequencing, provides a developmental profile of the astrocyte transcriptome in the postnatal visual cortex of mice on postnatal days 7, 14, 28, and, postnatal day 120 (Figure 1C–F). During development, the expression of GABA-synthesizing enzymes and levels of GABA precursors undergo significant changes in the brain. For instance, MAO-B, a key enzyme for putrescine-derived GABA synthesis in astrocytes, shows low to moderate mRNA expression levels from postnatal day 0 (Jones and Raghanti, 2021), suggesting that MAO-B-dependent GABA synthesis occurs as early as the postnatal stage. PAT enzyme is expressed slightly higher than MAO-B on postnatal day 7 based on the database, reaching adulthood expression level by postnatal day 14 (Figure 1C). Since PAT is an initiator enzyme for the MAO-B-dependent pathway, a slightly higher expression of PAT is not surprising, considering its function for converting putrescine into N-acetylputrescine for MAO-B-dependent oxidation and polyamine regulation. ALDH1A1 enzyme is roughly at a similar level to MAO-B and is expressed in a time‐dependent manner from postnatal day 7 to 28, but showing a decrease by adult, postnatal day 120 (Figure 1C). Finally, SIRT2 shows the highest expression among MAO-B-dependent GABA synthesis enzymes. SIRT2 reaches the maximum peak by postnatal day 14 and shows a gradual decrease toward adulthood (Figure 1C). The increase in these enzymes during the developmental period may reflect astrocyte differentiation coupled with GABA synthesis, or it could arise from their multifaceted roles, as GABA synthesis is just one of the diverse functions attributed to ALDH1A1 and SIRT2. Conversely, DAO expression is negligible during embryonic and early postnatal stages. Albeit with limited evidence, DAO existence in the adult brain may indicate a distinct temporal window for DAO-dependent GABA synthesis during the developmental period (Figure 1C). ALDH1A1 carries a functional role in both MAO-B and DAO-dependent pathways. The minimal expression of DAO may indicate that the MAO-B-dependent GABA synthesis pathway is more predominant during development. Regarding GABA synthesis from glutamate in astrocytes, GAD enzymes, GAD1 and GAD2, are present during the embryonic period, with expression upregulated to adult levels within the first three postnatal weeks in both the brain and astrocytes (Figure 1D), suggesting that glutamate-derived GABA synthesis in astrocytes may play a functional role during early development. As for substrate availability, putrescine levels peak on postnatal day 0 and decline as the brain matures (Laitinen et al., 1982). Although it is unclear whether these levels correspond to extracellular or intracellular pools or specific brain regions, the high concentration of putrescine in the developing brain might serve as a critical GABA source, particularly given the significance of GAD-independent pathways for GABA synthesis during early postnatal stages (Sequerra et al., 2007). Similarly, glutamate levels are higher during early development and decline in adulthood (Hanson et al., 2019). However, whether this elevated glutamate is utilized for GABA synthesis in astrocytes during development remains uncertain. For GABA tone formation in the developing brain, GABA must be released from astrocytes following synthesis. Release mechanisms also exhibit developmental variations. BEST1 expression appears relatively low in both the brain and astrocytes during early development (Figure 1E). This may reflect experimental limitations in measuring transcript abundance, as analyses often rely on cell dissociation methods that can inadvertently lose transcripts localized to distal astrocytic processes (Oh and Lee, 2017). LRRC8A is expressed as early as the embryonic period and reaches adult levels by the second postnatal week (Figure 1E). Lastly, mRNA expression of vesicular GAT showed an initial expression level similar to the rest of the postnatal days 14, 28, and adult postnatal day 120 (Figure 1E). However, its existence may still suggest the potential use of vGAT for vesicular GABA release. Together, these synthesis and release mechanisms likely contribute to forming GABA tone in the developing brain, though further investigation is warranted. GABA tone can be downregulated by uptake via GATs (GAT1 and GAT3) and intracellular degradation of GABA via GABA-T and SSADH. While the transcriptomic database shows higher GAT1 expression, GAT3 is expressed relatively lower (Figure 1F). However, owing to the known expression level of GAT3 in different brain regions of both adult and developing brains from other reports, the negligible level might be due to the distinct localization pattern of expression and experimental limitations. Still, GAT1 shows a significant expression level and reaches the adulthood level by postnatal day 14 (Figure 1F). GABA-T and SSADH are expressed during embryonic and early postnatal periods, peaking on postnatal day 14 before decreasing towards adulthood. The expression of GABA-T is considerably higher than SSADH (Figure 1F), while the expectation would be to see a similar degree of expression as both cooperatively work for GABA clearance. Together with GATs, notably high expression levels of these enzymes suggest that GABA clearance is actively functioning. Overall, these developmental changes strongly suggest that astrocytic mechanisms regulating GABA tone during development differ from those in the adult brain, potentially involving additional or alternative enzymes and transporters depending on the brain region. Interpreting these findings requires caution, as the heterogeneity of astrocytes across brain regions limits direct generalization to the entire brain. Unlike GABA’s inhibitory role in the adult brain, GABA exerts an excitatory influence during the developmental period (Ganguly et al., 2001; Cancedda et al., 2007; Ji et al., 2024), with astrocytes potentially regulating this excitatory tone through GABA synthesis, release, and clearance. By activating GABAA receptors, GABA causes membrane depolarization in immature neurons, leading to calcium influx via NMDA receptor activation, a process critical for neuronal proliferation, migration, and synaptogenesis during development (Peerboom and Wierenga, 2021). For instance, GABA can either enhance or suppress DNA synthesis depending on cellular context; blocking GABAA receptors with bicuculline increases DNA synthesis in GFAP-expressing cells in the subventricular zone, suggesting that ambient GABA inhibits proliferation in specific populations. GABA also acts as a chemoattractant, guiding neuronal migration in various regions, but serves as a stop signal in the cortical plate, with dysregulation potentially causing over-migration and cortical layer misplacement. These findings highlight the need for precise regulation of GABA tone (Peerboom and Wierenga, 2021), possibly mediated by astrocytes. During synaptogenesis, GABA provides the first excitatory drive for newborn neurons before the expression of glutamate receptors, emphasizing its foundational role in early neural circuits. In addition to its excitatory actions, GABA regulates neuronal excitability through shunting inhibition, and clamping membrane potential near the chloride reversal point to prevent excessive calcium influx and excitotoxicity (Peerboom and Wierenga, 2021). In the hippocampus, GABA also generates giant depolarizing potentials through tonic GABAA receptor-mediated currents, which are essential for synaptogenesis and early neural circuit development (Ji et al., 2024). Astrocytic regulation of GABA tone through synthesis pathways involving MAO-B, DAO, or GAD, release via channels such as BEST1 or LRRC8A, and clearance through GAT, GABA-T, and SSADH is thus crucial in maintaining the delicate balance required for proper neuronal development and preventing excitotoxicity. Importantly, the developmental switch of GABA from excitatory to inhibitory is driven by increased expression of the K+-Cl- cotransporter 2, which lowers intracellular chloride and shifts the GABAA receptor reversal potential (Ganguly et al., 2001). Notably, this switch can be induced by GABA itself, as the GABAA receptor plays a central role: chronic blockade of these receptors delays the transition, while enhanced activation accelerates it. This GABA-dependent process is independent of glutamatergic signaling or action potential activity, highlighting the unique role of GABA in neuronal maturation. Early depolarizing GABA activity is also critical for the morphological maturation of cortical neurons, although it does not significantly affect their radial migration (Cancedda et al., 2007). Disruptions in GABA synthesis, release, or clearance from astrocytes could lead to altered GABA tone, potentially contributing to neurodevelopmental disorders. For example, excitatory/inhibitory balance has been implicated in neurodevelopmental disorders such as autism spectrum disorders and developmental seizures (van van Hugte et al., 2023). Understanding astrocytic regulation of GABA tone during development could provide novel insights into the pathophysiology of these disorders and identify new targets for therapeutic intervention. Future studies should focus on elucidating the specific roles of astrocytic enzymes and transporters in regulating GABA tone during development and their impact on neuronal maturation and network formation. In conclusion, astrocytes play a potentially significant but underexplored role in regulating GABA tone during brain development. The dynamic changes in precursor availability and enzyme expression suggest that astrocytic GABA synthesis, release, and clearance mechanisms may differ from those in the adult brain. Elucidating these mechanisms is crucial for a comprehensive understanding of GABA’s role in developmental processes and may have important implications for therapeutic strategies targeting neurodevelopmental disorders. This work was supported by the Center for Cognition and Sociality, Institute for Basic Science (IBS) (IBS-R001-D2) (to WK). C-Editors: Zhao M, Sun Y, Qiu Y; T-Editor: Zhou H