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\ud \ud The GAD system is widely present in several types of organisms and is known to play an important role in bacterial acid tolerance. There is only one account of this system playing a role in oxidative stress in bacteria and one in yeasts. Here we show for first time that it affects the oxidative stress resistance of a Gram-positive bacterium, (L. monocytogenes, tested in three strains; 10403S, EGD-e, and LO28). We found a statistically significant reduction in survival after H2O2 exposure in ΔgadD3 and ΔgadD2 of EGD-e and in ΔgadD1 of LO28. Furthermore, we observed a lag phase prolongation in ΔgadD3 of 10403S and EGD-e and a larger inhibition zone in disk diffusion assay for ΔgadD1 and ΔgadD3 of EGD-e upon H2O2 exposure. All GAD genes playing a role in oxidative stress resistance are part of GADi system while this occurs partly through catalase activity, while the most potent GADe system plays no role. The latter effects could occur through the GABA shunt, but we show here that mutants in succinate semialdehyde dehydrogenase do not show a phenotype suggesting that either effects are through the GABA transaminase or, this pathway is not involved. Our study highlights for first time the role of the GAD system in oxidative stress resistance of a Gram-positive bacterium, which could be used in Food Hurdle Technology to eliminate pathogens such as L. monocytogenes, while it gives an insight on the general mechanism.

GABA and glutamate, the major inhibitory and excitatory neurotransmitters in the brain, are partly taken up by surrounding astrocytes subsequent to interaction with synaptic receptors. We describe the transporters and enzymes involved in metabolism of these neurotransmitters in astrocytes. Moreover, the mechanisms and interactions with the neurons necessary to sustain GABA and glutamate homeostasis are explained. GABA transaminase is an important pharmacological target, which is briefly mentioned.

Ethnopharmacological relevance A previous pilot ethnobotanical and ethnopharmacological study with the Q‘echi׳ Maya identified the family Piperaceae, as an important taxonomic group traditionally used for the treatment of epileptic and culture-bound anxiety disorders and possessing activity in the GABA system. Following that lead, a botanical survey was conducted in Peru, where 47 species of Piperaceae were collected including 21 plants traditionally used for folk illnesses by the Yanesha of Peru, an indigenous Amazonian group. Materials and methods Two high throughput bioassays were used to quantify the in vitro activity of botanical extracts on the GABA system. Results Plant extracts demonstrated moderate to high affinity to the γ-aminobutyric acid benzodiazepine (GABA-BZD) receptor. In addition, extracts demonstrated low to moderate activity in the inhibition of the GABA-transaminase, with select plants exhibiting significant activity. Plants indicated by the Yanesha showed comparable activity to the other Piperaceae plants collected. Piper cremii was the most active plant in the GABA-BZD receptor assay, and Drymaria cordata (Caryophyllaceae) in the GABA-T assay. Conclusion The study provides evidence that there is a pharmacological basis behind the use of plants in the treatment of susto and mal aire in both Central and South America, and we propose that the possible mechanism of action includes an interaction with the GABA-T enzyme and/or the GABA A -BZD receptor.

GABA is the major inhibitory neurotransmitter in mammalian brain. GABA is synthesized from glutamate and degraded to succinate in a bypath of the tricarboxylic acid cycle known as the GABA shunt. In rodent neocortex GABAergic neurons comprise ∼15–30% of neurons and consume ∼18% of the glucose oxidized by neurons. GABA produced in neurons by glutamate decarboxylase (GAD) and released by exocytosis is cleared from the synapse by reuptake into the terminal and also by uptake and metabolism in the glia via specialized GABA transporter proteins. GABA degraded in the astroglia is replenished by glial precursors mainly as glutamine in the glutamate/GABA–glutamine cycle, although other precursors may be involved as well. Different pools, cytoplasmic and vesicular, of GABA exist in the cell, and their synthesis may involve separate isoforms of GAD.

Cultures of dissociated cerebella from 7-day-old mice were maintained in vitro for 1–13 days. GABA biosynthesis and degradation were studied during development in culture and pharmacological agents were used to identify the enzymes involved. The amount of GABA increased, whereas that of glutamate was unchanged during the first 5 days and both decreased thereafter. The presence of aminooxyacetic acid (AOAA, 10 μM) which inhibits transaminases and other pyridoxal phosphate dependent enzymes including GABA-transaminase (GABA-T), in the culture medium caused an increase in the intracellular amount of GABA and a decrease in glutamate. The GABA content was also increased following exposure to the specific GABA-T inhibitor γ-vinyl GABA. From day 6 in culture (day 4 when cultured in the presence of AOAA) GABA levels in the medium were increased compared to that in medium from 1-day-old cultures. Synthesis of GABA during the first 3 days was demonstrated by the finding that incubation with either [1-13C]glucose or [U-13C]glutamine led to formation of labeled GABA. Synthesis of GABA after 1 week in culture, when the enzymatic machinery is considered to be at a more differentiated level, was shown by labeling from [U-13C]glutamine added on day 7. Altogether the findings show continuous GABA synthesis and degradation throughout the culture period in the cerebellar neurons. At 10 μM AOAA, GABA synthesis from [U-13C]glutamine was not affected, indicating that transaminases are not involved in GABA synthesis and thus excluding the putrescine pathway. At a concentration of 5 mM AOAA GABA labeling was, however, abolished, showing that glutamate decarboxylase, which is inhibited at this level of AOAA, is responsible for GABA synthesis in the cerebellar cultures. In conclusion, the present study shows that GABA synthesis is taking place via GAD in a subpopulation of the cerebellar neurons, throughout the culture period., The Danish State Medical Research Council (22-03-0250; 22-04-0314), the Norwegian Epilepsy, Hørslev, Lundbeck and Novo Nordisk Foundations are thanked for financial support.

Publisher Summary This chapter provides an overview of glutamate-γ-aminobutyrate transaminase. γ-aminobutyrate (GABA) is a major inhibitory transmitter of the central nervous system of vertebrates. GABA arises via decarboxylation of L-glutamate and is subsequently metabolized via transamination to succinic semialdehyde. Succinic semialdehyde is then oxidized to succinate. The enzymes catalyzing reactions are called the GABA shunt (or bypath). The shunt allows four of the five carbons lost from the tricarboxylic acid cycle at the level of α-ketoglutarate to be reincorporated into the cycle at the level of succinate. In tissue homogenates where succinic semiaidehyde dehydrogenase is in excess, GABA transaminase may be estimated by using endogenous succinic semialdehyde dehydrogenase to convert formed succinic semialdehyde to succinate. The increase in absorbance at 340 nm because of NADH production can be used to estimate GABA transaminase activities.

Preliminary experiments were performed to establish the optimal microwave irradiation time required to protect the brain from post mortem metabolism of GABA. Following this study, the high-energy phosphates, which deplete rapidly during brain ischemia and/or anoxia, were measured along with GABA to further acertain the utilization of the proper irradiation time. The metabolic enzymes in the GABA system are known to be more rapidly destroyed by microwave treatment than those in the high-energy phosphate system. The regional distribution of GABA in discrete regions of the rat brain was then determined and compared for animals sacrificed by decapitation or microwave irradiation (5kW, 1 sec). As generally accepted, the GABA content found in most regions after decapitation was higher than that after microwave irradiation. However, the degree of post mortem GABA increase varied from region to region. For example, the GABA content in the substantia nigra, superior colliculus, inferior colliculus and hypothalamus increased by 86, 112, 119 and 37 percent, respectively, when compared to the control levels obtained with microwave irradiation. In other regions, such as the striatum and the thalamus, the post mortem GABA increase was not significant. The regional distribution of GAD activity paralleled that of the GABA concentration, whereas the regional distribution of GABA-T activity was not necessarily consistent with the GABA concentration and the GAD activity. The correlation between the regional post mortem GABA increase and the GAD and GABA-T activities in the present study, however, indicate that both enzymes may be involved in determining the degree of post mortem GABA increase in individual brain regions.

Publisher Summary This chapter discusses the distribution and circadian variations of gamma-aminobutyric acid (GABA) in hypothalamic areas regulating food intake. Occupany of GABA receptors located in the brain can be varied indirectly by blocking GABA catabolism with specific blockers of GABA transaminase, such as Ethanolamin-O-Sulfate (EOS), or directly with GABA receptor agonists, such as muscimol. Intracisternally injected EOS raises GABA content and decreases food intake. EOS is able to reverse over eating produced by lesions of medial hypothalamus, injections of 2-deoxy-d-glucose, and a palatable diet. Direct stimulation of GABA receptors by intraventricular administration of muscimol increases the eating in free feeding rats. Therefore, this finding seems in contrast to the effect observed with EOS in normal animals. Moreover, peripheral administration of muscimol reduces short term food intake. Because brain GABA neurones are organized in various neuronal systems with different functional specifications, it is possible that simultaneous changes in total activity of brain GABAergic neurones, as obtained by intraventricular injections of EOS, may obscure specific action of GABA in specific hypothalamic nuclei.

Publisher Summary This chapter describes regulation of glutamatergic and GABAergic neuronal activity by astroglial cells. In contrast to acetylcholine and the monoamines, the amino acid neurotransmitters are compounds which not only serve as transmitters but also to a considerable extent take part in the energy metabolism in the central nervous system. The astrocytes play an extremely active role in the control of the neurotransmission processes mediated by glutamate and γ-aminobutyric acid (GABA). This holds true both at the level of inactivation and at the level of metabolism. GABA is accumulated into a variety of neuronal preparations and into nerve endings in vitro. The uptake capacity into the mainly GABAergic cortical neurons is 4–5 times higher than the uptake into astrocytes cultured from the same brain region and the affinity of the neuronal GABA carrier seems to be higher than that of the astrocytic GABA carrier. In contrast, glutamatergic cerebellar granule cells do not accumulate GABA by a high affinity transport mechanism. The metabolism of GABA is basically identical in neurons and astrocytes, being catalyzed by GABA transaminase. The activity of the GABA transaminase seems to be of the same magnitude in the two cell types, which is in agreement with histochemical localizations of the enzyme in tissue sections based on the classical nitroblue tetrazolium staining procedure as well as staining employing specific antibody to purified GABA transaminase.

Publisher Summary High concentrations of y-amino butyric acid (GABA) in blood did not result in an elevation of brain GABA levels. A barrier exists which restricts the passage of GABA from blood to nervous tissue. But recent histochemical studies on the distribution of GABA-a-keto-glutarate transaminase in the mammalian central nervous system suggested that the barrier may be in part a reflection of high GABA-transaminase activity in the walls of cerebral blood vessels and in the cells lining the cerebrospinal fluid spaces. To explore this suggestion further, the paper has a study that was conducted on the distribution of GABA-transaminase in kidney and liver. GABA appears to pass freely from blood to kidney, since as much as fifty per cent of the total GABA injected can be recovered from urine. Similarly, the concentration of GABA in liver after injection parallels that of the blood. The paper consists of the histochemical procedures for the localization of GABA-transaminase. The method is based on the fact that, in the course of the metabolism of GABA to succinic acid, NADH is formed, which in turn reduces a tetrazolium salt (Nitro BT) to its formazan form. Formazan is insoluble and will precipitate out at sites in the tissue where the combined presences of GABA-a-keto glutarate transaminase and succinic semialdehyde dehydrogenase have caused the conversion of GABA to succinic acid. When sections of mammalian brain are incubated with a transaminase medium, strong formazan precipitation occurs at sites where GABA-transaminase and succinic semialdehyde dehydrogenase are present in combination. Although the presence of both enzymes is a prerequisite for the reaction, recent studies suggested that the transamination step is rate limiting with respect to the histochemical demonstration of GABA metabolism. As a conclusion, the paper states that the ependymal cell layer which lines the cerebrospinal fluid spaces is among the sites in the central nervous system which exhibit strong GABA-transaminase activity. The paper also has a brief on the specificity of the histochemical reaction for GABA. In order to determine the specificity of the histochemical reaction for GABA transaminase in kidney, hydrazinopropionic acid (1 5 mg/kg) was injected into a mouse and its kidney was sectioned two hours later. Such sections, when incubated in the transaminase medium, were identical in appearance to normal sections which had been incubated in a control medium lacking GABA.