Milestone review: GABA, from chemistry, conformations, ionotropic receptors, modulators, epilepsy, flavonoids, and stress to neuro‐nutraceuticals

Arising out of a PhD project more than 50 years ago to synthesise analogues of the neurotransmitter GABA, a series of new chemical entities were found to have selective actions on ionotropic GABA receptors. Several of these neurochemicals are now commercially available. A new subtype of these receptors was discovered that could be a target for the treatment of myopia, the facilitation of learning and memory, and the improvement of post‐stroke motor recovery. The development of these new chemical entities over many years demonstrates the importance of neurochemicals with which to investigate selective aspects of GABA receptors and illustrates the significance of collaboration between chemists and biologists in neurochemistry. Vital were the improvements in synthetic organic chemistry and the use of functional human receptors expressed in oocytes. Current interest in ionotropic GABA receptors includes the clinical development of subtype‐specific agents and the role of gain‐of‐function receptor variants in epilepsy. Dietary flavonoids were found to cross the blood–brain barrier to influence brain function. Natural and synthetic flavonoids had a range of effects on GABA receptors, ranging from positive, silent, and negative allosteric modulators, to even second‐order modulation of first‐order modulators. Flavonoids have been called “a new family of benzodiazepines.” Like benzodiazepines, flavonoids reduce stress. Stress produces changes in GABA receptors in the brain that may be because of changes in endogenous modulators, such as neurosteroids and corticosteroids. GABA also occurs naturally in the diet leading to studies of the effects of oral GABA on brain function. This finding has resulted in studies of GABA and related neurochemicals as neuro‐nutraceuticals. GABA systems in the gut microbiome are essential to such studies. The actions of oral GABA and of GABA‐enriched beverages and foodstuffs are now an area of considerable scientific and commercial interest. GABA is a deceptively simple chemical that can take up many shapes, which may underlie its complex functions. The need for new chemical entities with selective actions for further studies highlights the need for continuing collaboration between chemists and biologists.


| INTRODUC TI ON
The deceptively simple molecule GABA acts as the major inhibitory synaptic transmitter in the mammalian central nervous system, as well as having many other functions (Hinton & Johnston, 2018).This milestone review highlights some of our key findings where the authors intersected in their studies on GABA, as in the Oxford English Dictionary definition of a milestone as "a significant stage" in our understanding of GABA neurochemistry.The review provides both a historical overview of our findings consistent with both authors having served as Historians to the International Society of Neurochemistry, plus current knowledge, and future directions.It is confined to the action of GABA on ionotropic receptors in the brain, their modulation by agents such as flavonoids, the effects of stress, and the role of exogenous GABA as a neuro-nutraceutical.
Ionotropic GABA receptors are thought to be involved in anaesthesia, anxiety, epilepsy, memory, neurodegenerative disorders, schizophrenia, sleep, stress, and cardiovascular and neuroendocrine function.Many agents act through these GABA receptors including barbiturates, benzodiazepines, ethanol, flavonoids, general anaesthetics, and neuroactive steroids (Hinton & Johnston, 2018).
GABA is a non-protein amino acid that is ubiquitous in most, if not all, life forms.First found in potatoes (Steward, 1949), it occurs in animals, bacteria, fungi, and plants and is chemically a simple molecule with complex functions.It has many roles in the mammalian central nervous system, distributed in distinctly different cellular pools reflecting its multiple functions (Waagepetersen et al., 1999).
Its ubiquitous presence in the brain and spinal cord means that almost all neurones either release GABA, express GABA receptors, or are innervated by neurones that do.Thus, most brain functions are likely to involve GABA as a neurotransmitter of major significance, being released by up to 40% of neurones to activate chloride channels resulting generally in inhibition of firing (Bowery & Smart, 2006).For some reviews of GABAergic systems, see "GABA, the major inhibitory neurotransmitter in the brain" (Hinton & Johnston, 2018), "The role of the GABAergic system in diseases of the central nervous system" (Zhang et al., 2021), "GABA A receptors as targets for anaesthetics and analgesics and promising candidates to help treat coronavirus infections: A mini-review" (Luo & Balle, 2022), and "The GABA system, a new target for medications against cognitive impairment-associated with neuroactive steroids" (Backstrom et al., 2023).
Arising out of a PhD project (Beart, 1972) in which both student (Philip Beart) and supervisor (Graham Johnston) had trained as organic chemists, we viewed GABA as a chemical entity to be modified and manipulated to explore its interactions with a variety of biological targets.These targets included central nervous system enzymes, receptors, and transporters.Research on GABA has a long history in Australia dating from 1958 with David Curtis and his colleagues in Canberra (Johnston, 2017).When we started on our GABA studies in 1969, the action of bicuculline on GABA and central inhibition had yet to be discovered (Curtis et al., 1970) and there were less than 120 references on GABA in the Science Core Collection of the Web of Science.Now, there are more than 66 000!Both authors worked with Les Iversen in the Department of Pharmacology, Cambridge University, in the 1970s, and broadened their scientific expertise (Snyder et al., 2020).Notably, Graham Johnston learnt about GABA transport (Iversen & Johnston, 1971) and brought the technique employing rat brain mini-slices back to Australia where together we screened a large number of GABA analogues from an extensive library in Canberra (Beart & Johnston, 1973).Philip Beart collaborated on various topics in Cambridge, including endeavours focused on the role of GABA in glia (Minchin & Beart, 1975;Schon & Kelly, 1975).He also learnt about GABA's involvement in human neuropathologies since at this time Iversen's team undertook clinical studies uncovering the depletion of brain GABA in Huntington's disease (Bird et al., 1973).This background proved extremely valuable for subsequent work at Harvard University (Boston, USA) where he studied the degeneration of GABAergic neurones in a genetic mouse model (Roffler-Tarlov et al., 1979).Thus, both authors became well aware of the multiple drug targets which could be employed to manipulate GABAergic transmission.

| G ABA CHEMIS TRY
Seemingly simple in structure, GABA (4-aminobutanoic acid) is a highly flexible molecule capable of existing in as many as 9 lowenergy conformers (Blanco et al., 2010).There are a variety of strategies to produce GABA analogues that represent restricted conformations of GABA using ring structures, double or triple bonds, or heavy substituents (Allan & Johnston, 1983).Muscimol is such a conformationally restricted GABA analogue, containing both a ring structure and double bonds, acting selectively on GABA A receptors (Johnston et al., 1968).One of us (Graham Johnston) has reviewed unsaturated analogues of GABA (Johnston, 2016) in a special issue of Neurochemical Research honouring the other (Philip Beart; may underlie its complex functions.The need for new chemical entities with selective actions for further studies highlights the need for continuing collaboration between chemists and biologists.

K E Y W O R D S
flavonoids, GABA, microbiome, modulators, nutraceuticals, stress Lawrence & Gundlach, 2016).This was reciprocating a special issue of Neurochemical Research honouring a significant birthday of the other (Graham Johnston;Beart & Balcar, 2009).In addition to synthesising GABA analogues, Philip Beart synthesised some analogues of the GABA A receptor antagonist bicuculline, including bicuculline methochloride, which was much more water soluble than the parent alkaloid and aided in our understanding of how these alkaloids may act as antagonists (Johnston et al., 1972).

| 4-Aminotetrolic acid (4-aminobut-2-ynoic acid)
The first molecule we synthesised was 4-aminotetrolic acid (Figure 1) in a hazardous multi-step synthesis (Beart & Johnston, 1972).This was a new chemical entity, CAS number 34014-16-9.It represented GABA stripped of 4 hydrogen atoms by incorporating a carbon-carbon triple bond.This triple bond held the molecule representing a conformationally extended form of GABA as confirmed by X-ray crystal and molecular orbital studies (Jones & Pauling, 1976;Warner et al., 1975) as indicated in Figure 1.
4-Aminotetrolic acid proved to have an inhibitory action on spinal neurones, 20-50% as strong as GABA and muscimol on GABA A receptors, antagonised by bicuculline and insensitive to strychnine.
It was inactive on GABA B receptors, a competitive inhibitor of GABA uptake into brain slices and an inhibitor of GABA aminotransferase (Beart et al., 1971).We wrote at the time, "The successful interaction of 4-aminotetrolic acid with bicuculline-sensitive postsynaptic receptors indicated that GABA may act on these receptors in an extended rather than a folded conformation." Reduction of the triple bond in 4-amionotetrolic acid with tritium provided a commercial route to GABA labelled to high specific radioactivity (Ahern et al., 2003).There are 22 references listed in CAS SciFinder containing 4-aminotetrolic acid, including 2 patents and 27 suppliers.
2.2 | cis-4-Aminocrotonic acid ((2Z)-4-aminobut-2enoic acid) cis-4-Aminocrotonic acid was prepared by partial hydrogenation of 4-aminotetrolic acid (Johnston et al., 1975).This was also a new chemical entity, CAS number 55199-25-2.An improved synthesis was subsequently reported (Allan & Johnston, 1985).It is now available commercially from several sources.With its cis-double bond resulting in making it a GABA analogue in a partially folded conformation (Figure 1), cis-4-aminocrotonc acid was particularly important in leading to the discovery of a new subtype of ionotropic GABA receptor (Johnston, 1996).cis-4-Aminocrotonic acid had an inhibitory action on the firing of spinal neurones.This action was insensitive to the GABA A receptor antagonist bicuculline (Johnston, 2013) and the glycine receptor antagonist strychnine (Curtis et al., 1971).While the GABA B agonist baclofen was relatively ineffective as an inhibitor of the firing of Renshaw cells in the spinal cord, cis-4-aminocrotonic acid was equally potent on Renshaw cells and other spinal neurones.This evidence suggested that cis-4-aminocrotonic acid was not an agonist for bicuculline-sensitive GABA A or baclofen-sensitive GABA B receptors in spinal neurones, thus raising the possibility that cis-4-aminocrotonic acid might be interacting with a new subtype of GABA receptor.
The finding that cis-4-aminocrotonic acid had no effect on the binding of radioactive baclofen to rat cerebellar membranes led to the concept of GABA acting on "a class of binding site (GABA C ?) insensitive to (-)-baclofen and bicuculline" (Drew et al., 1984).cis-4-Aminocrotonic acid was described as a "disturber of the peace" (Krogsgaard-Larsen et al., 1997), upsetting the A/B nomenclature for GABA receptors -GABA A referring to bicuculline-sensitive, baclofen-insensitive receptors and GABA B referring to baclofensensitive, bicuculline-insensitive receptors.
Once cis-4-aminocrotonic acid became commercially available, many experiments were carried out that supported the idea that this chemical was doing "something different."There are 79 references listed in CAS SciFinder for cis-4-aminocrotonic acid, together with 23 patents and 11 suppliers.

| I ONOTROPI C G ABA RECEP TOR S
GABA receptors may be divided into two categories, ionotropic and metabotropic (Chebib & Johnston, 2000).The former are ligandgated ion channels (LGICs), while the latter are G-protein-coupled receptors (GCPRs).GABA A and GABA C receptors are LGICs gating chloride channels.They are part of the nicotinicoid superfamily of LGICs encompassing several families of receptors including nicotinic acetylcholine receptors, 5-HT 3 receptors, strychnine-sensitive glycine receptors, and some invertebrate anionic glutamate receptors.
The molecular diversity of these ligand-gated ion channels represents important challenges for neurochemists in the design of subunit-specific therapeutic agents.The most studied and best F I G U R E 1 The two extreme conformations of GABA, where the amino and carboxyl moieties are furthest apart and closest, with matching conformations of 4-aminotetrolic acid (a partial agonist selective for GABA A receptors) and cis-4-aminocrotonic acid (a partial agonist selective for GABA C receptors).Adapted from (Beart, 1972).
understood of this class of receptors are the nicotinic acetylcholine receptors, while the GABA A receptors may be the most complex and the GABA C receptors might be the simplest (Le Novere & Changeux, 1999).
Members of the nicotinicoid superfamily are considered to be composed of 5 protein subunits likely to have been derived from a common ancestor (Le Novere & Changeux, 1999).Each subunit has a large extracellular N-terminal domain which incorporates part of the agonist-/antagonist-binding site, followed by three membranespanning domains (M1-3), an intracellular loop of variable length, and a fourth membrane-spanning domain (M4), and the C-terminal end being extracellular.Each subunit arranges itself such that the second membrane-spanning domain (M2) forms the wall of the channel pore and the overall charge of the domain determines whether the channel conducts anions or cations.Both GABA A and GABA C receptors are GABA-gated chloride ion channels usually causing inhibition of neuronal firing, with GABA A receptors being heteromeric, composed usually of 3 different subunits, and GABA C receptors being homomeric, composed usually exclusively of one type of subunit (Chebib & Johnston, 2000).
The optimised alignment of the human GABA A and GABA C receptors' amino acid sequences (Le Novere & Changeux, 1999) shows overall some 170 matching amino acids between these proteins equating to an optimised sequence identity of the order of 40% and as high as 75% in the transmembrane region.The differences in the physiology and pharmacology between the GABA A and GABA C receptor subfamilies are substantial (Naffaa et al., 2017).

| GABA C receptors (also known as GABAρ, GABA ρ , and GABA Aρ receptors)
Following the commercial release of cis-4-aminocrotonic acid, 3 papers were published in 1993 describing the action of cis-4aminocrotonic acid on bicuculline-and baclofen-insensitive receptors in the retina (Feigenspan et al., 1993;Kusama et al., 1993;Qian & Dowling, 1993).The receptors became known as GABA C and were found to be made up of ρ-subunits, cloned from the retina (Bormann & Feigenspan, 1995), although the naming of these ionotropic GABA receptors remains unsettled (Chebib & Johnston, 2000).There are 692 journal articles listed in CAS SciFinder that mention GABA C receptors, including 11 patents.cis-4-Aminocrotonic acid is a partial agonist at human recombinant GABA C receptors (Bormann & Feigenspan, 1995;Naffaa et al., 2017) and is a weak substrate for GABA transport (Chebib & Johnston, 1997).It has been used to help characterise GABA C receptors in the retina, hippocampus, superior colliculus, pituitary, pelvic ganglia, and gut (Johnston, 2016).
Substituting the cis-double bond in cis-4-aminocrotonic acid with a cyclopropane ring yields (+)-CAMP (CAS 74028-33-4, 1S,2R-2-(aminomethyl)cyclopropane carboxylic acid) a selective full agonist for GABA C receptors being inactive at GABA A receptors (Allan et al., 1980;Naffaa et al., 2017).It is now commercially available.TPMPA (Figure 2, (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid) was the first selective antagonist described for GABA C receptors (Murata et al., 1996).It was a new chemical entity, CAS number 182485-36-5, first prepared by Ragozzino and colleagues (Ragozzino et al., 1996), and is now available from many commercial suppliers.In pharmacological terms, GABA C receptors may be described as TPMPA-sensitive, bicuculline-insensitive ionotropic GABA receptors.SGS742 (Figure 2, CAS number 123690-78-8, (3-aminopropyl)(nbutyl)phosphinic acid), the first GABA B receptor antagonist in clinical trials for the potential treatment of mild cognitive impairment and Alzheimer's disease (Froestl et al., 2004), was shown to be also a GABA C receptor antagonist, increasing spatial memory in rodents and inhibiting myopia progression in chicks (Chebib et al., 2009).However, it is not clear whether the physiological effects of SGS742 are because of its GABA B or GABA C activity or a mixture of both actions.

| Current knowledge regarding GABA C receptors
An extensive study by Mary Chebib, Jane Hanrahan, and colleagues of cyclopentane and cyclopentene derivatives with butylphosphinic acid moieties resulted in the development of a model pharmacophore for GABA C receptor antagonism that can be used for in silico screening (Kumar et al., 2008).The agonist-and antagonist-binding sites for GABA C receptors have been characterised by extensive structure-activity and molecular modelling studies (Naffaa et al., 2017).The development of these new chemical entities over many years demonstrates the importance of neurochemicals with which to investigate selective aspects of GABA receptors and illustrates the importance of collaboration between chemists and biologists in neurochemistry.Significant were the improvements in synthetic organic chemistry and the use of functional human receptors expressed in oocytes (Miledi et al., 2002).Extensive molecular modelling has been carried out distinguishing ρ1 and ρ2 GABA C receptors suggesting several GABA analogues which can be used as partial agonists to discover selective modulatory agents for these receptors (Naffaa et al., 2022).
Deficits of GABA C receptors in the inner retina are associated with the peripheral visual field defects resulting from the use of vigabatrin in the treatment of paediatric epilepsy (Pathan et al., 2023).Also in the retina, sensory deprivation influences GABA C receptors involved in night vision (Wisner et al., 2023).
It was recently shown that histamine negatively modulates ρ1 GABA C receptors expressed in oocytes, whereas it positively modulates a range of GABA A receptors (González et al., 2023).
Interestingly, opposite effects involving these receptors have been noted previously; for example, opposing roles for these receptors have been described for short-term memory in young chicks where GABA A antagonism inhibited memory and GABA C antagonism reinforced memory (Gibbs & Johnston, 2005).

| Current knowledge regarding GABA A receptors
There is much current interest in the development of new therapeutic agents targeting specific subtypes of GABA A receptors and also the role of such subtypes in epilepsy.

| Neurosteroids
Realising the therapeutic potential of either endogenous neurosteroids or synthetic "neuroactive" steroids has proven challenging (Belelli et al., 2022).Recent approval by the Food and Drug Administration of the use of allopregnanolone (brexanolone) to treat post-partum depression has rekindled enthusiasm for exploring their potential as new medicines.The development of subtype-selective modulators of GABA A receptors for the treatment of neuropsychiatric disorders is progressing well, with more than 20 possible therapeutic agents in commercial development (Thompson, 2023).These agents mainly target receptors containing α2/3, α5, and δ subunits.Receptors containing δ subunits are particular targets for the treatment of psychiatric disorders (Belelli et al., 2022).
Cryo-electromicroscopy analysis and single-molecule photobleaching experiments have revealed an important role for endogenous neurosteroids in modulating native GABA A receptors defining a structural landscape from which subtype-specific drugs can be developed (Sun et al., 2023).Subunit selectivity is not necessary, however, for useful therapeutic agents.An orally active neuroactive steroid, zuranolone, that displays no striking subunit preferences, has completed phase III clinical trials in major depressive disorder and was approved by the Food and Drug Administration for treating post-partum depression in 2023 (Epperson et al., 2023).

| Epilepsy and GABA A receptor variants
"In recent years, the number of epilepsy associated GABA A receptor variants discovered has grown at a seemingly exponential rate" (Absalom et al., 2023) The authors build on their previous studies on GABA A receptor variants (Absalom et al., 2022;Ahring, Liao, Gardella, et al., 2022;Ahring, Liao, Lin, et al., 2022).Of particular interest are their findings on gain-of-function variants, that is those variants when expressed in oocytes which show an increased activity on the recombinant receptors.The authors describe this as a "conundrum" as it goes against the simple view of the brain balance between glutamate-mediated excitation and GABA-mediated inhibition going out-of-balance in epilepsy because of either increased excitation or decreased inhibition in particular brain circuits.
In their gain-of-function study on β3 GABA A receptor variants in epileptic encephalopathies, Absalom and colleagues found that the gain-of-function in GABA A receptor variants in a cohort of 27 patients presented with a younger age of seizure onset (median age 2.5 months) than 47 patients in the loss-of-function cohort (median age 10.5 months; Absalom et al., 2022).The so-called GABA switch or shift might come into play here.The direction of GABA currents through ionotropic GABA receptors reverses during brain development from depolarising to hyperpolarising.This reversal reflects ionotropic GABA receptors being ligand-gated ion channels whose effects depend on the direction of chloride ion flow through the channel.This takes place in humans in the first year after birth but may be delayed in certain neurological disorders and differs in different brain regions (Peerboom & Wierenga, 2021).Further investigations are needed to address this interesting "conundrum" as acknowledged in the authors' extensive Journal of Neurochemistry review (Absalom et al., 2023).

| FL AVONOIDS
In 2004, we wrote a commentary titled "Flavonoids: some of the wisdom of sage?" (Johnston & Beart, 2004).This was regarding an article describing the positive allosteric modulation of recombinant GABA A receptors by the flavonoid hispidulin that is found in sage (Kavvadias et al., 2004).They showed that hispidulin acts as a positive allosteric modulator across a range of GABA A receptor subtypes.The benzodiazepine antagonist flumazenil reduced, but did not block, the action of hispidulin on any of the GABA A receptor subtypes tested, indicating that a part of the positive modulatory action of hispidulin is mediated through flumazenil-insensitive sites on GABA A receptors.It was shown that hispidulin crosses the blood-brain barrier and this is related to its anticonvulsant action (Kavvadias et al., 2004).for GABA (Hinton et al., 2017).They can act as positive or negative allosteric modulators, enhancing or reducing the effect of GABA.
They can elicit a direct activation of the receptors.They can also act to modulate the action of other modulators.This ability to influence function via their actions on GABA receptors permits a range of effects of flavonoids, including relief of anxiety, anticonvulsant, analgesic, and sedative actions (Hinton & Johnston, 2021).
In this sense, the actions of many flavonoids overlap with that of benzodiazepines.
Benzodiazepines act on GABA A receptors via two distinct and separable mechanisms (Walters et al., 2000).Studies using recombinant α1β2γ2 GABA A receptors with low concentrations of GABA showed a biphasic potentiation by diazepam with distinct components in the nanomolar (high-affinity) and micromolar (low-affinity) ranges.Mutations in the second transmembrane domain of the subunits abolished the low-affinity component.The low-affinity component was independent of the γ2 subunit and was insensitive to the benzodiazepine antagonist flumazenil.Flumazenil-insensitive benzodiazepine effects have been found in a variety of GABA A receptor subtypes and may be important for designing of subtype-or binding site-specific agents (Lian et al., 2020).
Flumazenil-insensitive modulation of GABA A is not restricted to flavonoids and benzodiazepines.A wide range of plant-derived natural products act on GABA A receptors in flumazenil-sensitive and flumazenil-insensitive ways.These include alkaloids, terpenes, phenolic acids, lignans, and saponins, which act as anxiolytics (Fedotova et al., 2017).Indeed, a wide chemical range of GABA A allosteric modulators do not act on the classical flumazenil-sensitive benzodiazepine sites (Solomon et al., 2019).

| Flavan-3-ol esters
The most well-known flavan-3-ol ester is EGCG (Figure 3, CAS number 989-51-5, (−)-epigallocatechin gallate) that occurs widely in plants and is the major polyphenol in tea.It has dose-dependent stress-reducing, anxiolytic, sedative, and hypnotic properties in a number of animal models.EGCG enhances the positive allosteric modulation by diazepam on recombinant GABA A receptor; that is, it modulates the modulator (Campbell et al., 2004).This effect is observed at flavonoid concentrations that do not influence GABA action in the absence of diazepam.Such second-order modulation may result from alterations in the coupling of flumazenil-sensitive benzodiazepine allosteric sites with the orthosteric GABA sites on GABA A receptor.EGCG is also able to reverse the negative allosteric modulation by ethyl β-carboline (Vignes et al., 2006).This evidence suggests that EGCG may act as a second-order modulator with respect to the first-order modulation by both positive and negative F I G U R E 3 Structures of some naturally occurring and synthetic flavonoids that interact with GABA A receptors.Hispidulin is found in sage, and EGCG is an important constituent of tea.
modulators that act via benzodiazepine-binding sites on GABA A receptors (Hinton et al., 2017).
Second-order modulation (or metamodulation) has also been noted in other systems (Mesce, 2002;Ribeiro & Sebastiao, 2010) and may represent an obscure novel mechanism of drug action deserving further investigation, with the potential to lead to decreased therapeutic doses of the drug.It is not easy to investigate as it involves the dose-dependent interactions between three ligands, requiring the study of a number of dose combinations, and thus may be difficult to observe.Synergistic interactions have been described between other flavonoids on GABA receptor-related behaviours and at glycine receptors between strychnine and flavonoids (Hinton et al., 2017).
Simplifying the structure of EGCG, Ken Mewett and colleagues (Mewett et al., 2009) synthesised a large number of flavan-3-ol derivatives of which Fa131 and Fa173 were the most interesting.
Both are now commercially available.Fa131 (Figure 3. CAS number 40106-96-5) is a non-sedating anxiolytic and a selective flumazenilinsensitive positive allosteric modulator of α2-containing GABA A receptors (Fernandez et al., 2008).Fa131 is the first positive allosteric modulator to distinguish between α2-and α3-containing GABA A receptors, highlighting the potential of targeting flumazenilinsensitive allosteric sites in the search for new anxio-selective drugs (Fernandez et al., 2008).
Fa173 (Figure 3. CAS number 1414586-08-5), structurally related to Fa131 with a different stereochemistry and an additional methoxy group, blocks the modulatory action of Fa131 (Fernandez et al., 2012).It also blocks enhancement of the GABA response by the anaesthetic etomidate, the sedative anticonvulsant loreclezole, and selectively blocks the low-affinity effect of diazepam (100 μM) at α1β2γ2L and α1β2 GABA A receptors, but not the high-affinity effect of diazepam (100 nM).Fa173 was the first silent allosteric modulator of the low-affinity flumazenil-insensitive benzodiazepinebinding site (Fernandez et al., 2012).

| Current knowledge regarding flavonoids and GABA
Flavonoids have distinct pharmacological profiles from endogenous or synthetic benzodiazepines.As many flavonoids occur in the diet, they may be found in the brain where they are able to modulate the function of the major inhibitory neurotransmitter, GABA (Hinton & Johnston, 2021).
There is continuing extensive interest in the effects of flavonoids isolated from natural products on GABA systems, for example anxiolytic flavonoid compounds isolated from Tibetan herbs (Liu et al., 2021).The effects of quercetin as an anxiolytic acting through interaction with GABA receptors has been studied in vivo and in silico (Islam et al., 2022).The anxiolytic effects of plant-derived flavonoids may involve other neurotransmitters than GABA (Wang et al., 2023).

| G ABA AND S TRE SS
There is a substantial literature on GABA and stress (Hinton & Johnston, 2018).Agents that are known to influence stress have been shown to act directly on GABA receptors including drugs such as benzodiazepines used in the treatment of anxiety.Generally speaking, increased activity of GABA results in the acute relief of anxiety and stress (Skilbeck et al., 2010).
We organised an official satellite meeting of the International Society for Neurochemistry (ISN), called Amino Acids 91, held in July 1991 in the Hunter Valley, one of Australia's premier wine-growing regions just north of Sydney-the location of the 1991 ISN Congress (where Graham Johnston was Chairman of the Local Organising Committee).There were 77 registered participants for the satellite from 14 countries.The meeting was generously supported by 8 pharmaceutical companies.The International Organizing Committee was composed of famous neuroscientists, including Norman Bowery, Povl Krogsgaard-Larsen, Sam Enna, Alessandro Guidotti, Kinya Kuriyama, and Richard Olsen, all of whom made notable contributions to our understanding of GABAergic synaptic function.
At the Amino Acids 91, Graham Johnston and Mualla Akinci presented studies on stress and GABA-binding sites (Johnston & Akinci, 1991).This work arose out of studies by John Skerritt for his PhD describing substantial increases in GABA binding in the brains of female mice following a 3-min swim stress at 32 ο C (Skerritt et al., 1981).The acute swim stress of mice produced increases in the density of high-and low-affinity GABA-binding sites in the brain, together with increased analgesia as measured by an increase in tailflick latency.Apparent tolerance developed in repeated swimming with analgesia, and GABA binding returned towards control levels.
The time course of analgesia and increases in GABA binding following a single swim are also similar.Intriguingly, acute swim stress did not alter diazepam binding.GABA systems may be important in analgesia and in responses to environmental stress.Relief of stress is a feature of yoga (Beart et al., 2020), known to increase levels of GABA in the brain (Streeter et al., 2010), possibly via the vagus nerve and the gut-brain axis (Breit et al., 2018), though many systems may be involved (Padmavathi et al., 2023).
The swim stress changes were not observed in male mice and were less apparent in a well-washed membrane preparation commonly used in GABA-binding assays, consistent with the loss of endogenous modulators of GABA binding in the latter preparation.
These changes may be related to stress-induced alterations in part in the modulation by endogenous steroids, as the acute swim stress produced a larger increase in plasma corticosterone levels in female mice than in male mice (Akinci & Johnston, 1993).Studies on gonadectomised mice and hormone replacement indicated the involvement of corticosteroids and neurosteroids in these effects of stress on GABA receptors in the cerebral cortex (Akinci & Johnston, 1997).
Corticosteroids are known to have potent actions on GABA responses in the guinea pig isolated ileum (Ong et al., 1987).
Further studies on GABA A receptors in the brain using autoradiography showed that they are sensitive to subtle changes in the environment in both early-life and adulthood.These neurochemical responses to stress in adulthood are sex dependent.Acute stress induces rapid changes in GABA A receptors in experimental animals, with the direction of the changes varying according to the sex of the animals and the stress paradigm studied.These rapid alterations are of particular interest as they provide an example of fast neurotransmitter system plasticity that may be mediated by stress-induced increases in neurosteroids and corticosteroids, perhaps via effects on phosphorylation and/or receptor trafficking (Skilbeck et al., 2010).
Current interest in GABA systems in stress involves the gut-brain axis, and the importance of the vagus nerve is discussed in the following section (Hou et al., 2020).

| G ABA A S A NEURO -N UTR ACEUTI C AL
More than 400 GABA-enriched food and beverage products, such as chocolate, milk, rice, soya, tea, and yoghurt, are already on the Japanese market (Waltz, 2022).The market is overflowing with GABA, produced chemically and enzymatically, for use as a nutritional supplement in the main to lower high blood pressure, reduce anxiety, or promote sleep.
One of us (Philip Beart) has a special interest in neuronutraceuticals having co-edited 3 special issues on this topic (Williams et al., 2015(Williams et al., , 2016(Williams et al., , 2021)).The effects of EGCG on GABA A receptors and its widespread consumption in tea led to the study of GABA-enhanced tea (Hinton & Johnston, 2020).The GABA content in tea was enhanced to produce a therapeutically useful beverage for consumption.Processing Camellia sinensis leaves under anoxic conditions using nitrogen rather than oxidative conditions leads to a significant increase in GABA content of up to 20 times (Dai et al., 2020) along with changes in other constituents (Hinton et al., 2022).There are numerous GABA-enriched teas produced in this way from green, oolong, and black teas, the most famous of which is Gabaron tea (Tsushida et al., 1987).Gabaron and related GABA-enriched teas were found to reduce the blood pressure of spontaneously hypertensive rats and influence sleep, stress, anxiety, and depression, thus generating much interest (Hinton & Johnston, 2020).Tina Hinton and her colleagues found that consumption of a cup of GABA-enhanced oolong tea reduced stress in a university student cohort in a controlled study (Hinton et al., 2019).Chemical analysis of GABA-enhanced tea did indeed find an eightfold enrichment of GABA and a 10-fold decrease in EGCG along with a 1.5-fold increase in caffeine (Hinton et al., 2022).Thus, there are changes in multiple constituents in this GABA-enriched oolong tea that may contribute to the biological effects observed in students consuming these teas (Figure 4).
Gene-edited tomatoes have come onto the market with a 20-fold increase in GABA content and have hypotensive effects upon consumption (Ezura, 2022;Minako et al., 2023;Nagamine et al., 2022). of GABA-enriched products.The relative proportions of GABA and glutamate may be of significance given that glutamate imparts the umami taste to foodstuffs and the enriched GABA may be achieved at the expense of glutamate (Pencheva et al., 2023).

| GABA, blood-brain barrier, microbiome, and gut-brain axis
There is extensive evidence suggesting that oral GABA supplementation reaches the brain in concentrations able to exert biological effects in humans and animals affecting mood and activities of central nervous system (Boonstra et al., 2015).For example, in a recent study, a randomised, double-blind, placebo-controlled trial, 75 mg of oral GABA was shown to promote sleep in patients with insomnia (Yoon et al., 2022).Nonetheless, the ability of oral GABA to directly influence brain function is contentious and may depend on the state of the individual as in the case of people with insomnia and people who are stressed or have hypertension.
There are, however, other ways in which oral GABA might act besides acting directly on the brain (Hinton et al., 2022).Extensive effects of GABA on the enteric nervous system have been observed that may explain the actions of oral GABA (Diez-Gutierrez et al., 2020).Exogenous GABA may influence GABA action in the brain indirectly via actions on neurosteroids and other modulators (Belelli et al., 2021).In addition, GABA may be acting as an energy source via the GABA shunt that bypasses the normal TCA cycle to increase ATP synthesis as GABA has multiple roles as metabolite, neurotransmitter, neurotrophin, and beyond (Waagepetersen et al., 1999).
GABA is extensively distributed within the enteric nervous system and is an important metabolite of the gut microbiome (Diez-Gutierrez et al., 2020).There are ample GABA-producing bacteria in the human gut microbiome (Strandwitz et al., 2019).Its natural occurrence in the gut microbiome, as well as enrichment in foods and beverages, therefore potentially enables its contribution to functions and disorders mediated by the gut-brain axis, including depression and anxiety, and inflammatory and cardiovascular disorders (Breit et al., 2018;Carabotti et al., 2015).Chronic treatment of mice with GABA-producing Lactobacilli, for example, reportedly reduced anxiety behaviours in the elevated plus maze and fear conditioning tasks and reduced markers of depression in the forced swim test (Bravo et al., 2011).These mice also showed altered GABA A receptor subunit expression in key regions of the brain involved in regulating mood and anxiety, consistent with the behavioural changes observed.The same changes were not observed in vagotomised mice, suggesting the important role of vagal afferents in communicating GABAergic activity from the gut to the brain (Bravo et al., 2011;Breit et al., 2018).
Dietary GABA activates vagal afferents in collaboration with meals to regulate brain function including feeding behaviour (Nakamura et al., 2022).There may be gut-brain bidirectional communications via the vagus in connection with GABAergic systems (Hou et al., 2020).A therapeutic anti-depressant potential has been proposed for microbial GABA produced in the gut by Lactobacillus strains (Tette et al., 2022).Interestingly, GABA A and GABA B receptor agonists were shown many years ago to activate vagal efferent transmission in the rat (Yamasaki et al., 1991).
Given the extensive distribution of GABA systems in the periphery, further investigation of the action of oral GABA on peripheral tissues and the gut-brain axis is warranted (Hinton & Johnston, 2020).
Perhaps studies could be carried out with agents that do not cross the blood-brain barrier.There are some apparent anomalies in our understanding of GABA neurochemistry; for example, while most GABA A receptor antagonists, such as bicuculline (Johnston, 2013), are potent convulsants, a number are known that are inactive as convulsants, for example terpenoids isolated from Salvia triloba L (Abdelhalim et al., 2014).Such agents may prove useful in probing the peripheral sites of action of oral GABA.

| FUTURE DIREC TIONS
The ubiquitous distribution of GABA throughout all life forms and its multitude of functions suggest that its chemical flexibility of being able to adopt many low-energy conformations is important.GABA may interact with many different recognition sites on enzymes, receptors, and transporters in different ways and thus be a common "key" to such sites, which may have had evolutionary benefits.It also provides varying targets for the development of agents that have relatively specific actions on these targets.The interest in the development of subtype-specific agents for ionotropic GABA receptors is but one example of this strategy.Such development also applies to GABA enzymes and transporters as human and veterinary medicines and to insecticides.We can expect significant cross-fertilisation in these areas.
Given its ubiquitous nature, GABA is likely to be involved, directly or indirectly, in most brain functions.Hence, it is not surprising to find it is involved in COVID-19 infection and treatment (Luo & Balle, 2022;Sfera et al., 2022) and disorders such as Alzheimer's disease (Carello-Collar et al., 2023).These authors state that "the GABAergic system is vulnerable to AD pathology and should be considered a potential target for developing pharmacological strategies and novel AD biomarkers."The problem is discovering specific agents and techniques to do this, which is a significant challenge.
Increasingly, drug development involves computer-aided design employing 3D models of receptors, enzymes, and transporters in order to discover agents with specific actions.AI will have an impact on this, especially in improving our understanding of protein folding.
The continuing studies on GABA A receptor function in various epilepsies, especially where there is a gain of function, will reveal a greater understanding of the glutamate/GABA excitation/inhibition balance in specific areas of the brain.
Antisense oligonucleotide therapy might be useful where gain of function in GABA receptors is the target (Absalom et al., 2023).
cis-3-ACPBPA (Figure2, CAS number 536756-57-7, rel-P-[(1R,3S)-3-aminocyclopentyl]-P-butylphosphinic acid) is under investigation in animal models of cognition, the inhibition of myopia development, and as a pharmacological tool for elucidating the role of GABA C receptors in vivo(Chebib et al., 2009).It is one of the F I G U R E 2 Structures of some key GABA C receptor antagonists.structurallydiverse GABA C receptor antagonists that act differentially on open and closed conformational states of the ρ1 receptor(Yamamoto et al., 2012).(R)-4-ACPBPA(Figure2, CAS number 872471-80-2, (R)-4amino-cyclopent-1-enyl butylphosphinic acid) is a selective GABA C ρ2 receptor antagonist that affords significant improvement in motor function after stroke in mice, suggesting that targeting these receptors might provide a novel delayed treatment option for stroke recovery(van Nieuwenhuijzen et al., 2021).
. This excellent review in the Journal of Neurochemistry by Nathan Absalom, Philip Ahring, Mary Chebib, Vivian Liao, Rikke Møller, and their colleagues in Australia and Denmark highlights recent progress in our understanding of the advances and complexities of the role of GABA A receptors in epilepsy.It reviews "the clinical manifestations of genetic GABA A receptor variants, the early-stage efforts undertaken by research laboratories to determine their functional consequences and the complex challenges faced in the field."This review also provides "recommendations for conducting future laboratory efforts to generate functional data that align with clinical guidelines to determine variant pathogenicity rapidly and accurately."

Flavonoids
are not made in mammals and only enter the mammalian brain from external sources.They are found in fruits and vegetables.Plant-derived beverages have diverse pharmacological actions in the central and peripheral nervous systems.Extracts from plants are used in herbal medicines as sedatives and tranquillisers.It is likely that the active ingredients in some of these extracts are flavonoids possessing activity for GABA A receptors.Marder, Medina, Paladini, and their co-workers in Argentina played a major role in drawing attention to the central actions and therapeutic potential of flavonoids (Medina et al., 1997).They called flavonoids "a new family of benzodiazepines."Flavonoids, both naturally occurring and synthetic, are known to have multiple effects on the activation of ionotropic receptors While the market may indicate that GABA is indeed a neuronutraceutical, other constituents, such as flavonoids and other polyphenols, may contribute to the increased beneficial effects of GABA-enriched products.The levels of all likely active constituents need to be considered when attempting to understand the effects F I G U R E 4 A representative collection of GABA-enhanced commercial products available in Japan assembled by Graham Johnston from publicly accessible advertisements on the internet.