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.

In memoriam: Frode Fonnum (1937-2023).

Frode Fonnum died unexpectedly on 26th April 2023, at 86 years of age. He was a tower of strength-a primeval force-in neuroscience, neurochemistry and toxicology. His highly cited publications, comprised salient evidence for GABA and glutamate as brain neurotransmitters. He served as an expert, and as an organizer, including of European research cooperation and as President of the International Society for Neurochemistry (ISN). Photo credit: Per Kristian Opstad.

The origins and early history of neurochemistry and its societies.

At the 2017 joint meeting of the International Society for Neurochemistry (ISN) and the European Society for Neurochemistry, 150 years of neurochemistry - the 50th anniversary of ISN, 40 years of European Society for Neurochemistry, and 60 years of the Journal of Neurochemistry (JNC) - was celebrated with a historical symposium that explored the foundations of neurochemical societies, key international figures in the discipline of neurochemistry, and the pre-eminent role of the JNC. The foundations of neurochemistry were laid in Europe, notably France and Germany, in the late 18th and early 19th centuries. Neurochemists in the United Kingdom made globally relevant contributions before and after the Second World War, and Swedish contributions were especially prominent in the 1950s and 1960s. As neurochemistry is a truly international branch of neuroscience, the important contributions of neurochemists in the Americas and the Asia-Pacific were also recognized, as were the seminal roles of the American, Asia-Pacific, and Japanese Societies of Neurochemistry. Although ISN was only formed in 1967, earlier international meetings in Europe and the Americas reflected the growing recognition of the importance of chemistry and biochemistry for understanding and responding to the pathophysiology of clinical conditions and diseases of the central and peripheral nervous systems. JNC was first published in 1956, but the ISN only assumed complete ownership of the journal under tempestuous circumstances in 1970. The ISN-JNC interface and the sterling work of the JNC Editors has meant that the income generated by the journal has allowed the ISN Council to implement diverse programs for supporting neurochemistry internationally, including sustaining regional neurochemical societies, and supporting neurochemists in the developing world and schools of neurochemistry.

Optimising the transient expression of GABA(A) receptors in adherent HEK293 cells.

Owing to their therapeutic relevance, considerable efforts are devoted to the structural characterisation of membrane proteins. Such studies are limited by the availability of high quality protein due to the difficulty of overexpression in recombinant mammalian systems. We sought to systematically optimise multiple aspects in the process of transiently transfecting HEK293 cells, to allow the rapid expression of membrane proteins, without the lengthy process of stable clone formation. We assessed the impact of medium formulation, cell line, and harvest time on the expression of GABAA receptors, as determined by [3H]muscimol binding in cell membranes. Furthermore, transfection with the use of calcium phosphate/polyethyleneimine multishell nanoparticles was optimised, and a dual vector system utilising viral enhancing elements was designed and implemented. These efforts resulted in a 40-fold improvement in GABAA α1ß3 receptor expression, providing final yields of 22 fmol/cm2. The findings from this work provide a guide to the optimisation of transient expression of proteins in mammalian cells and should assist in the structural characterisation of membrane proteins.

Long-lasting effects of early-life intervention in mice on adulthood behaviour, GABAA receptor subunit expression and synaptic clustering.

Variations in the early postnatal environment of rodents produce long-term changes in responses to stress that may underlie neuropsychiatric diseases such as anxiety, depression and schizophrenia. GABAA receptors undergo marked changes in their subunit composition during this period, involving a regionally-dependent replacement of α2 with α1 subunits, the so-called α-subunit switch. In this study we examined the effects of early-life environment on adulthood GABAA receptor α1 and α2 subunit expression and the synaptic clustering of GABAA receptors. Male and female mice were exposed to either 15min daily handling sessions (EH) or no intervention (NH) over postnatal day (PND) 1-14. Adulthood behavioural differences in anxiety were assessed on the elevated plus-maze. Immunoperoxidase histochemistry was used to examine the density of the α1 and α2 subunit proteins. Double-labelling immunofluorescence and confocal microscopy were used to study GABAA receptor synaptic clustering. NH animals showed increased anxiety-type behaviours in the elevated plus maze relative to EH mice. NH males showed a loss of α2 subunits from the thalamus and lower layers of the somatosensory cortex, whilst NH females showed a reduction of α2 but increase in α1 protein in lower layers of the primary somatosensory cortex only. The NH condition also reduced α1 subunit expression in dentate gyrus (DG) in both males and females. Regardless of sex, NH mice showed reduced colocalisation of GABAA receptor α2 subunits with the synaptic marker gephyrin relative to the control condition. These findings suggest that early-life environment has long-lasting effects on GABAA receptors, leading to long-term changes in adulthood behaviour, and are of relevance to neurodevelopmental explanations of stress-augmented neuropsychiatric disorders.

Some personal thoughts on the Australasian Society of Clinical and Experimental Pharmacologists in the 1980s.

There were a number of highly significant events regarding ASCEP in the 1980s: a tour of China by ASCEP pharmacologists; the Sydney IUPHAR Congress; and the initiation of the Australasian Visitor to the BPS scheme. ASCEP appointed a professional secretariat, established an investment portfolio, and initiated Special Interest Groups. The Society entered the 1990s welcoming toxicologists into the new ASCEPT.

GABA Australis, some reflections on the history of GABA receptor research in Australia.

Research on GABA receptors has a long history in Australia dating from 1958 with David Curtis and his colleagues in Canberra. This review traces many of the advances made in Australia guided by highly cited publications and some obscure ones. It covers the discovery of key chemicals with which to investigate GABA receptor function including bicuculline, muscimol, phaclofen, THIP and (+)-CAMP. Also described are findings relevant to the involvement of mutant GABA receptors in inherited epilepsy. The modulation of GABA receptors by a bewildering range of chemicals, especially by flavonoids and terpenoids, is discussed.

Unsaturated Analogues of the Neurotransmitter GABA: trans-4-Aminocrotonic, cis-4-Aminocrotonic and 4-Aminotetrolic Acids.

Analogues of the neurotransmitter GABA containing unsaturated bonds are restricted in the conformations they can attain. This review traces three such analogues from their synthesis to their use as neurochemicals. trans-4-Aminocrotonic acid was the first conformationally restricted analogue to be extensively studied. It acts like GABA across a range of macromolecules from receptors to transporters. It acts similarly to GABA on ionotropic receptors. cis-4-Aminocrotonic acid selectively activates bicuculline-insensitive GABAC receptors. 4-Aminotetrolic acid, containing a triple bond, activates bicuculline-sensitive GABAA receptors. These findings indicate that GABA activates GABAA receptors in extended conformations and GABAC receptors in folded conformations. These and related analogues are important for the molecular modelling of ionotropic GABA receptors and to the development of new agents acting selectively on these receptors.

Antidepressant, Anxiolytic and Antinociceptive Activities of Constituents from Rosmarinus Officinalis.

PURPOSE: Rosmarinus officinalis, traditionally known as rosemary, has been widely used in traditional medicines and has long been known as the herb of remembrance. However, few studies have investigated the effects of non-volatile components of rosemary on central nervous system function. METHODS: Fractionation of R. officinalis led to the isolation of salvigenin, rosmanol and cirsimaritin, which were investigated in mouse models of acute toxicity, antinociception (tail immersion and hot plate tests), depression (tail suspension and forced swim tests) and anxiety (elevated plus maze and light/dark box paradigms). RESULTS: Rosmanol, cirsimaritin and salvigenin were not found to exhibit any signs of acute toxicity (50-200 mg/kg), but elicited antinociceptive, antidepressant and anxiolytic activities. CONCLUSION: Rosmanol, cirsimaritin and salvigenin, all previously shown to have biphasic modulation of GABAA receptors, demonstrated CNS activity in mouse models of antinociception, antidepressant and anxiolysis. The anxiolytic activity of all three compounds was not ameliorated by flumazenil, but was inhibited by pentylenetetrazol, suggesting a mode of action via GABAA receptors at a site other than the high affinity benzodiazepine binding site. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Muscimol as an ionotropic GABA receptor agonist.

Muscimol, a psychoactive isoxazole from Amanita muscaria and related mushrooms, has proved to be a remarkably selective agonist at ionotropic receptors for the inhibitory neurotransmitter GABA. This historic overview highlights the discovery and development of muscimol and related compounds as a GABA agonist by Danish and Australian neurochemists. Muscimol is widely used as a ligand to probe GABA receptors and was the lead compound in the development of a range of GABAergic agents including nipecotic acid, tiagabine, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, (Gaboxadol(®)) and 4-PIOL.

Modulation of ionotropic GABA receptors by 6-methoxyflavanone and 6-methoxyflavone.

We evaluated the effects of 6-methoxyflavanone and 6-methoxyflavone on wild-type α1/α2ß2γ2L GABAA and ρ1 GABAC receptors and on mutant ρ1I307S, ρ1W328 M, ρ1I307S/W328 M GABAC receptors expressed in Xenopus oocytes using two-electrode voltage clamp and radioligand binding. 6-Methoxyflavanone and 6-methoxyflavone act as a flumazenil-insensitive positive allosteric modulator of GABA responses at human recombinant α1ß2γ2L and α2ß2γ2L GABAA receptors. However, unlike 6-methoxyflavone, 6-methoxyflavanone was relatively inactive at α1ß2 GABAA receptors. 6-Methoxyflavanone inhibited [(3)H]-flunitrazepam binding to rat brain membranes. Both flavonoids were found to be inactive as modulators at ρ1, ρ1I307S and ρ1W328 M GABA receptors but acted as positive allosteric modulators of GABA at the benzodiazepine sensitive ρ1I307S/W328 M GABA receptors. This double mutant retains ρ1 properties of being insensitive to bicuculline and antagonised by TPMPA and THIP. Additionally, 6-methoxyflavanone was also a partial agonist at ρ1W328 M GABA receptors. The relative inactivity of 6-methoxyflavanone at α1ß2 GABAA receptors and it's partial agonist action at ρ1W328 M GABA receptors suggest that it exhibits a unique profile not matched by other flavonoids.

A single amino acid determines the toxicity of Ginkgo biloba extracts.

Ginkgo biloba extracts are currently used for a wide range of health-related conditions. Some of the medical benefits of these extracts are controversial, but their lack of toxicity in humans is not in doubt. These extracts are, however, highly toxic to insects. Their active components (ginkgolides and bilobalide) have structures similar to the convulsant picrotoxin, a GABA(A) receptor antagonist, so their lack of toxicity in mammals is puzzling. Here, we show that the different compositions of insect and vertebrate GABA receptor pores are responsible for the differing toxicities. Insect GABA receptors contain Ala at their 2' position in the pore. Substitution with Val, which is the equivalent residue in vertebrate GABA(A) receptor α-subunits, decreases ginkgolide potency by up to 10,000-fold. The reverse mutation in vertebrate GABA(A) α1 subunits increased the sensitivity of α1ß2 and α1ß2γ2 receptors to ginkgolides. Mutant cycle analysis demonstrates a strong interaction between the ginkgolides and the 2' residue, a result supported by in silico docking of compounds into a model of the pore. We conclude that the insecticidal activity of G. biloba extracts can be attributed to their effects at insect GABA receptors, and the presence of a Val at the 2' position in vertebrate GABA(A) receptors explains why these compounds are not similarly toxic to humans.

Potency of GABA at human recombinant GABA(A) receptors expressed in Xenopus oocytes: a mini review.

GABAA receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (ß) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure-function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABA(A) receptor combinations expressed in Xenopus oocytes using the two-electrode voltage clamp technique. The results show that the α-subunits and to a lesser extent, the ß-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the ß2- or ß3-subunit conferred higher sensitivity to GABA than receptors containing the ß1-subunit with the exception of the α2ß1γ2L and α6ß1γ2L subtypes. Of the δ-subunit containing GABA(A) receptors, α4/δ-containing GABA(A) receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4ß1δ and α4ß3δ receptors. At α4ß2δ, GABA had low micromolar activity.

Targeting GABAC Receptors Improves Post-Stroke Motor Recovery.

Ischemic stroke remains a leading cause of disability worldwide, with limited treatment options available. This study investigates GABAC receptors as novel pharmacological targets for stroke recovery. The expression of ρ1 and ρ2 mRNA in mice were determined in peri-infarct tissue following photothrombotic motor cortex stroke. (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (R)-4-ACPBPA and (S)-4-ACPBPA were assessed using 2-elecotrode voltage electrophysiology in Xenopus laevis oocytes. Stroke mice were treated for 4 weeks with either vehicle, the α5-selective negative allosteric modulator, L655,708, or the ρ1/2 antagonists, (R)-4-ACPBPA and (S)-4-ACPBPA respectively from 3 days post-stroke. Infarct size and expression levels of GAT3 and reactive astrogliosis were determined using histochemistry and immunohistochemistry respectively, and motor function was assessed using both the grid-walking and cylinder tasks. After stroke, significant increases in ρ1 and ρ2 mRNAs were observed on day 3, with ρ2 showing a further increase on day 7. (R)- and (S)-4-ACPBPA are both potent antagonists at ρ2 and only weak inhibitors of α5ß2γ2 receptors. Treatment with either L655,708, (S)-4-ACPBPA (ρ1/2 antagonist; 5 mM only), or (R)-4-ACPBPA (ρ2 antagonist; 2.5 and 5 mM) from 3 days after stroke resulted in a significant improvement in motor recovery on the grid-walking task, with L655,708 and (R)-4-ACPBPA also showing an improvement in the cylinder task. Infarct size was unaffected, and only (R)-4-ACPBPA significantly increased peri-infarct GAT3 expression and decreased the level of reactive astrogliosis. Importantly, inhibiting GABAC receptors affords significant improvement in motor function after stroke. Targeting the ρ-subunit could provide a novel delayed treatment option for stroke recovery.

Stress and GABA receptors.

GABA(A) receptors 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, perhaps via effects on phosphorylation and/or receptor trafficking. Interestingly, some studies have also provided evidence for long-lasting changes in GABA(A) receptors as a result of exposure to stressors in early-life. The short- and long-term stress sensitivity of the GABAergic system implicates GABA(A) receptors in the non-genetic etiology of psychiatric illnesses such as depression and schizophrenia in which stress may be an important factor.

Neurochemicals for the investigation of GABA(C) receptors.

GABA(C) receptors are being investigated for their role in many aspects of nervous system function including memory, myopia, pain and sleep. There is evidence for functional GABA(C) receptors in many tissues such as retina, hippocampus, spinal cord, superior colliculus, pituitary and the gut. This review describes a variety of neurochemicals that have been shown to be useful in distinguishing GABA(C) receptors from other receptors for the major inhibitory neurotransmitter GABA. Some selective agonists (including (+)-CAMP and 5-methyl-IAA), competitive antagonists (such as TPMPA, (±)-cis-3-ACPBPA and aza-THIP), positive (allopregnanolone) and negative modulators (epipregnanolone, loreclezole) are described. Neurochemicals that may assist in distinguishing between homomeric ρ1 and ρ2 GABA(C) receptors (2-methyl-TACA and cyclothiazide) are also covered. Given their less widespread distribution, lower abundance and relative structural simplicity compared to GABA(A) and GABA(B) receptors, GABA(C) receptors are attractive drug targets.

Microwave-enhanced synthesis of 2,3,6-trisubstituted pyridazines: application to four-step synthesis of gabazine (SR-95531).

Microwave-enhanced, highly efficient protocols for the synthesis of synthetically and biologically important 2,3,6-trisubstituted pyridazine architectures have been developed by sequential amination/Suzuki coupling/alkylation reactions. This powerful strategy is an economical and highly chemoselective protocol for the synthesis of diversified pyridazines. The total synthesis of gabazine (SR-95531) has been achieved using a versatile strategy in four steps and 73% overall yield.

GABA-enriched teas as neuro-nutraceuticals.

Teas enriched in GABA are consumed for their beneficial effects on blood pressure, stress and anxiety. These effects may involve actions of GABA on the central and peripheral nervous systems. The anaerobic procedures for the production of GABA-enriched teas increase GABA levels by 10-20 times. They also significantly alter the levels of other constituents that may interact with the actions of GABA. These include epigallocatechin gallate, caffeine and theanine. The possible interactions of these active constituents make the understanding of the effects of GABA-enriched teas complex. More data is needed to establish where and how GABA is acting following consumption of GABA-enriched teas. While there is considerable evidence that such GABA is acting on GABA receptors in the periphery, there is rather less evidence that is acting directly in the brain. Certainly, there is more to the action of GABA-enriched teas than GABA itself.

Novel, potent, and selective GABAC antagonists inhibit myopia development and facilitate learning and memory.

This study reports pharmacological and physiological effects of cis- and trans-(3-aminocyclopentanyl)butylphosphinic acid (cis- and trans-3-ACPBPA). These compounds are conformationally restricted analogs of the orally active GABA(B/C) receptor antagonist (3-aminopropyl)-n-butylphosphinic acid (CGP36742 or SGS742). cis-[IC(50)(rho1) = 5.06 microM and IC(50)(rho2) = 11.08 microM; n = 4] and trans-3-ACPMPA [IC(50)(rho1) = 72.58 microM and IC(50)(rho2) = 189.7 microM; n = 4] seem competitive at GABA(C) receptors expressed in Xenopus laevis oocytes, having no effect as agonists (1 mM) but exerting weak antagonist (1 mM) effects on human GABA(A) and GABA(B) receptors. cis-3-ACPBPA was more potent and selective than the trans-compound, being more than 100 times more potent at GABA(C) than GABA(A) or GABA(B) receptors. cis-3-ACPBPA was further evaluated on dissociated rat retinal bipolar cells and dose-dependently inhibited the native GABA(C) receptor (IC(50) = 47 +/- 4.5 microM; n = 6). When applied to the eye as intravitreal injections, cis- and trans-3-ACPBPA prevented experimental myopia development and inhibited the associated vitreous chamber elongation, in a dose-dependent manner in the chick model. Doses only 10 times greater than required to inhibit recombinant GABA(C) receptors caused the antimyopia effects. Using intraperitoneal administration, cis- (30 mg/kg) and trans-3-ACPBPA (100 mg/kg) enhanced learning and memory in male Wistar rats; compared with vehicle there was a significant reduction in time for rats to find the platform in the Morris water maze task (p < 0.05; n = 10). As the physiological effects of cis- and trans-3-ACPBPA are similar to those reported for CGP36742, the memory and refractive effects of CGP36742 may be due in part to its GABA(C) activity.

GABA analogues derived from 4-aminocyclopent-1-enecarboxylic acid.

The incorporation of extra binding groups onto known ligands is a powerful tool for the development of more potent and selective agents at target sites such as the GABA receptors. In the present work we have attempted to build on the activity of the know potent GABA(A) agonist 4-ACP-3-CA and its cis and trans saturated analogues CACP and TACP. We have investigated reactions to add thiol substituents to the alpha,beta-unsaturated system of 4-ACP-3-CA. The reaction was successful with a limited number of thiols but gave products of mixed stereochemistry. The resultant thioether amino acids were screened for activity at human recombinant alpha(1)beta(2) gamma(2L) GABA(A) receptors. The most interesting derivative was the benzylthioether which acted as an antagonist with an IC(50) of 42 microM for the inhibition of a GABA EC(50) dose (50 microM). This study has shown that GABA analogues derived by thiol addition to 4-aminocyclopent-1-enecarboxylic acid display interesting antagonist activity at the alpha(1)beta(2)gamma(2L) GABA(A) receptor.