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![GABA released from astrocytes is synthesized from putrescine. (A-B) Concentration of ornithine, putrescine and GABA in hippocampal slice tissues and in the bathing medium following one hour incubation normal ACSF (control) or in [Mg2+] = 1 μM ACSF in the absence (A) and presence (B) of 100 μM SNAP-5114 as determined by LC-MS. Asterisks represent significant change (P < 0.05). (C) Holding currents in [Mg2+] = 1 μM buffer in the presence of the monoamino oxidase inhibitor, deprenyl (10 μM) and the diamino oxidase inhibitor, aminoguanidine (100 μM). Left, baseline currents plotted at 1 s intervals during control condition (black), SNAP-5114 application (red) and washout (blue); right, box-chart representation of GABAergic baselines during control condition, SNAP-5114 application and washout. Box edges represent 25th, 50th and 75th percentile, open squares represent means, circles connected by lines represent paired individual baseline values. Average holding current in control: 74.8 ± 12.8 (N = 8 cells/6 animals). (D) Intracellular [Na+] in the soma of SR101 identified astrocytes measured by SBFI fluorescence during 100 μM SNAP-5114 applications with (black line) or without (red line) preceding activation of the Glu transporters by 200 μM t-PDC in [Mg2+] = 10 μM ACSF in the presence of deprenyl (10 μM) and aminoguanidine (100 μM) (average of N = 13 cells/2 animals).](profile/Arpad-Dobolyi/publication/221965027/figure/fig5/AS:202583793770515@1425311148321/GABA-released-from-astrocytes-is-synthesized-from-putrescine-A-B-Concentration-of.png)
GABA released from astrocytes is synthesized from putrescine. (A-B) Concentration of ornithine, putrescine and GABA in hippocampal slice tissues and in the bathing medium following one hour incubation normal ACSF (control) or in [Mg2+] = 1 μM ACSF in the absence (A) and presence (B) of 100 μM SNAP-5114 as determined by LC-MS. Asterisks represent significant change (P < 0.05). (C) Holding currents in [Mg2+] = 1 μM buffer in the presence of the monoamino oxidase inhibitor, deprenyl (10 μM) and the diamino oxidase inhibitor, aminoguanidine (100 μM). Left, baseline currents plotted at 1 s intervals during control condition (black), SNAP-5114 application (red) and washout (blue); right, box-chart representation of GABAergic baselines during control condition, SNAP-5114 application and washout. Box edges represent 25th, 50th and 75th percentile, open squares represent means, circles connected by lines represent paired individual baseline values. Average holding current in control: 74.8 ± 12.8 (N = 8 cells/6 animals). (D) Intracellular [Na+] in the soma of SR101 identified astrocytes measured by SBFI fluorescence during 100 μM SNAP-5114 applications with (black line) or without (red line) preceding activation of the Glu transporters by 200 μM t-PDC in [Mg2+] = 10 μM ACSF in the presence of deprenyl (10 μM) and aminoguanidine (100 μM) (average of N = 13 cells/2 animals).
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![Changes in glial [Na+] in response to EAAT activation and GAT-2/3...](profile/Arpad-Dobolyi/publication/221965027/figure/fig4/AS:202583793770507@1425311148248/Changes-in-glial-Na-in-response-to-EAAT-activation-and-GAT-2-3-blockade-A-Confocal_Q320.jpg)
Glutamate and γ-aminobutyric acid (GABA) transporters play important roles in balancing excitatory and inhibitory signals in the brain. Increasing evidence suggest that they may act concertedly to regulate extracellular levels of the neurotransmitters.
Here we present evidence that glutamate uptake-induced release of GABA from astrocytes has a dire...
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... found that putrescine and ornithine concentra- tions were significantly decreased in the cytosolic pool ( Figure 5A) in the reduced- [Mg 2+ ] environment. Ornithine concentration was also decreased in the extra- cellular space when exposed to the reduced-[Mg 2+ ] ACSF ( Figure 5A). ...Context 2
... found that putrescine and ornithine concentra- tions were significantly decreased in the cytosolic pool ( Figure 5A) in the reduced- [Mg 2+ ] environment. Ornithine concentration was also decreased in the extra- cellular space when exposed to the reduced-[Mg 2+ ] ACSF ( Figure 5A). These data demonstrate the increased metabolic conversion of ornithine and putres- cine. ...Context 3
... GABA concentration was also signifi- cantly decreased compared to the control condition. However, a remarkable increase was observed in the bath concentration of GABA ( Figure 5A) in accordance with the tonic inhibition measurements (Figure 2). The increase in the bath concentration of GABA and the decrease in cytosolic [GABA] were both completely blocked by 100 μM SNAP-5114 ( Figure 5B), demon- strating that GABA release was indeed mediated by GAT-2/3 transporters. ...Context 4
... a remarkable increase was observed in the bath concentration of GABA ( Figure 5A) in accordance with the tonic inhibition measurements (Figure 2). The increase in the bath concentration of GABA and the decrease in cytosolic [GABA] were both completely blocked by 100 μM SNAP-5114 ( Figure 5B), demon- strating that GABA release was indeed mediated by GAT-2/3 transporters. When SNAP-5114 was present in the reduced- [Mg 2+ ] buffer, both cytosolic and bath [putrescine] were increased ( Figure 5B), suggesting that maintenance of the putrescine-GABA conversion requires the continual removal of the synthesized GABA. ...Context 5
... increase in the bath concentration of GABA and the decrease in cytosolic [GABA] were both completely blocked by 100 μM SNAP-5114 ( Figure 5B), demon- strating that GABA release was indeed mediated by GAT-2/3 transporters. When SNAP-5114 was present in the reduced- [Mg 2+ ] buffer, both cytosolic and bath [putrescine] were increased ( Figure 5B), suggesting that maintenance of the putrescine-GABA conversion requires the continual removal of the synthesized GABA. In the absence of GABA efflux the ornithine- derived putrescine is released to the extracellular space by the depolarization-induced polyamine secretory path- way [54,55]. ...Context 6
... confirm the contribution of the putrescine-derived glial GABA to the tonic inhibition of neurons, we inves- tigated the tonic GABAergic currents under blockade of the putrescine-GABA metabolic pathway using the monoamino oxidase inhibitor deprenyl in combination with the diamine oxidase inhibitor aminoguanidine [44]. In the [Mg 2+ ] = 1 μM buffer that was used to investigate the Glu-induced GABAergic currents, the presence of 10 μM deprenyl and 100 μM aminoguanidine eliminated the GAT-2/3 mediated tonic current component ( Figure 5C). ...Context 7
... the involvement of the putrescine-GABA pathway as the source of releasable glial GABA was also confirmed by measuring the glial [Na + ] changes in response to the EAAT substrate t-PDC (200 μM) and the GAT blocker SNAP-5114 (100 μM) ( Figure 5D Figure 5D) was comparable to that measured in the presence of SNAP- 5114 when the putrescine-GABA pathway was intact ( Figure 4B), indicating that this [Na + ] increase can be achieved when the GAT-2/3 transporters are not releas- ing the Na + to the extracellular space. ...Context 8
... the involvement of the putrescine-GABA pathway as the source of releasable glial GABA was also confirmed by measuring the glial [Na + ] changes in response to the EAAT substrate t-PDC (200 μM) and the GAT blocker SNAP-5114 (100 μM) ( Figure 5D Figure 5D) was comparable to that measured in the presence of SNAP- 5114 when the putrescine-GABA pathway was intact ( Figure 4B), indicating that this [Na + ] increase can be achieved when the GAT-2/3 transporters are not releas- ing the Na + to the extracellular space. ...Context 9
... conclusion, among the various physiological changes in response to the low- [Mg 2+ ] environment, we consider the activity-induced enhancement of the putrescine-GABA synthetic pathway as the major deter- minant of the appearance of the Glu/GABA exchange mechanism, because its blockade eliminated the GAT-2/ 3 mediated component of both the tonic inhibition (Fig- ure 5B) and the glial [Na + ] increase ( Figure 5C). ...Context 10
... conclusion, among the various physiological changes in response to the low- [Mg 2+ ] environment, we consider the activity-induced enhancement of the putrescine-GABA synthetic pathway as the major deter- minant of the appearance of the Glu/GABA exchange mechanism, because its blockade eliminated the GAT-2/ 3 mediated component of both the tonic inhibition (Fig- ure 5B) and the glial [Na + ] increase ( Figure 5C). ...Citations
... 72 This GABA transporter may contribute to reducing neuronal hyperexcitability in treated mice through glutamate-induced GABA release. [73][74][75] Phosphorylation of N-methyl-D-aspartate (NMDA) modulatory subunits (NR1, NR2A, and NR2B) and AMPA receptors regulates neuronal excitability, among other neuronal pathways. In the hippocampus of mice treated with rAAV-hEPM2A, we observed significant dephosphorylation of NR2A and AMPAR subunits GLUA1 and GLUA2, along with a notable decrease in GLUA4 abundance. ...
... When the intracellular Na + concentration is changed in astrocytes, lactate is released from astrocytes to the neuron as the energy source. Furthermore, EAAT2-induced intracellular Na + elevation drives the release of γ-aminobutyric acid (GABA) through the reversal of GABA transporter GAT-3, thereby resulting in tonic inhibition of neurons [13]. This mechanism provides an adjustable, in situ negative feedback on the excitability of neurons in physiological conditions. . ...
In our recent report, we clarified the direct interaction between the excitatory amino acid transporter (EAAT) 1/2 and polyunsaturated fatty acids (PUFAs) by applying electrophysiological and molecular biological techniques to Xenopus oocytes. Xenopus oocytes have a long history of use in the scientific field, but they are still attractive experimental systems for neuropharmacological studies. We will therefore summarize the pharmacological significance, advantages (especially in the study of EAAT2), and experimental techniques that can be applied to Xenopus oocytes; our new findings concerning L-glutamate (L-Glu) transporters and PUFAs; and the significant outcomes of our data. The data obtained from electrophysiological and molecular biological studies of Xenopus oocytes have provided us with further important questions, such as whether or not some PUFAs can modulate EAATs as allosteric modulators and to what extent docosahexaenoic acid (DHA) affects neurotransmission and thereby affects brain functions. Xenopus oocytes have great advantages in the studies about the interactions between molecules and functional proteins, especially in the case when the expression levels of the proteins are small in cell culture systems without transfections. These are also proper to study the mechanisms underlying the interactions. Based on the data collected in Xenopus oocyte experiments, we can proceed to the next step, i.e., the physiological roles of the compounds and their significances. In the case of EAAT2, the effects on the neurotransmission should be examined by electrophysiological approach using acute brain slices. For new drug development, pharmacokinetics pharmacodynamics (PKPD) data and blood brain barrier (BBB) penetration data are also necessary. In order not to miss the promising candidate compounds at the primary stages of drug development, we should reconsider using Xenopus oocytes in the early phase of drug development.
... Hefty evidence in the last decades shows the physiological role of glial cells in synaptic transmission, especially astrocytes [52,53]. Astrocytes transform glutamatergic excitation into GABAergic inhibition [54] and contribute to establishing negative feedback [55,56]. During synaptic transmission, astrocytes absorb glutamate, which releases GABA. ...
Network dynamics are crucial for action and sensation. Changes in synaptic physiology lead to the reorganization of local microcircuits. Consequently, the functional state of the network impacts the output signal depending on the firing patterns of its units. Networks exhibit steady states in which neurons show various activities, producing many networks with diverse properties. Transitions between network states determine the output signal generated and its functional results. The temporal dynamics of excitation/inhibition allow a shift between states in an operational network. Therefore, a process capable of modulating the dynamics of excitation/inhibition may be functionally important. This process is known as disinhibition. In this review, we describe the effect of GABA levels and GABAB receptors on tonic inhibition, which causes changes (due to disinhibition) in network dynamics, leading to synchronous functional oscillations.
... Reduced GAT-2 levels have been associated with temporal lobe epilepsy 59 . This GABA transporter may contribute to reducing neuronal hyperexcitability in treated mice through glutamateinduced GABA release [60][61][62] . Phosphorylation of NMDA modulatory subunits (NR1, NR2A and NR2B), and AMPA receptors regulates neuronal excitability, among other neuronal pathways. ...
Lafora disease is a rare and fatal form of progressive myoclonic epilepsy typically occurring early in adolescence. Common symptoms include seizures, dementia, and a progressive neurological decline leading to death within 5-15 years from onset. The disease results from mutations transmitted with autosomal recessive inheritance in the EPM2A gene, encoding laforin, a dual-specificity phosphatase, or the EPM2B gene, encoding malin, an E3-ubiquitin ligase. Laforin has glucan phosphatase activity, is an adapter of enzymes involved in glycogen metabolism, is involved in endoplasmic reticulum-stress and protein clearance, and acts as a tumor suppressor protein. Laforin and malin work together in a complex to control glycogen synthesis and prevent the toxicity produced by misfolded proteins via the ubiquitin-proteasome system. Disruptions in either protein can lead to alterations in this complex, leading to the formation of Lafora bodies that contain abnormal, insoluble, and hyperphosphorylated forms of glycogen called polyglucosans.
We used the Epm2a -/- knock-out mouse model of Lafora disease to apply a gene replacement therapy by administering intracerebroventricular injections of a recombinant adeno-associated virus carrying the human EPM2A gene. We evaluated the effects of this treatment by means of neuropathological studies, behavioral tests, video-electroencephalography recording, and proteomic/phosphoproteomic analysis.
Gene therapy with recombinant adeno-associated virus containing the EPM2A gene ameliorated neurological and histopathological alterations, reduced epileptic activity and neuronal hyperexcitability, and decreased the formation of Lafora bodies. Differential quantitative proteomics and phosphoproteomics revealed beneficial changes in various molecular pathways altered in Lafora disease. Improvements were observed for up to nine months following a single intracerebroventricular injection. In conclusion, gene replacement therapy with human EPM2A gene in the Epm2a -/- knock-out mice shows promise as a potential treatment for Lafora disease.
... GABA released by astrocytes activates extrasynaptic GABA receptors producing a tonic inhibition thereby controlling neuronal excitability (Fig. 6). Thus, GABAergic pathways again place astroglial Na + at the centre of excitatory/inhibitory balance in neural networks [118,[123][124][125]. ...
... Of note, the magnitude of tonic inhibition provided by glial Glu/GABA exchange was more profound during seizure-like activity in rat entorhinal-hippocampal slices. It is not unlikely that targeting the molecular mechanism by which astrocytes transform glutamatergic excitation into tonic GABAergic inhibition mediated by high-affinity, slowly desensitizing, extrasynaptic GABA A receptors may have potential therapeutic implications in epilepsy [64]. ...
The pharmacological treatment of epilepsy is purely symptomatic. Despite many decades of intensive research, causal treatment of this common neurologic disorder is still unavailable. Nevertheless, it is expected that advances in modern neuroscience and molecular biology tools, as well as improved animal models may accelerate designing antiepileptogenic and epilepsy-modifying drugs. Epileptogenesis triggers a vast array of genomic, epigenomic and transcriptomic changes, which ultimately lead to morphological and functional transformation of specific neuronal circuits resulting in the occurrence of spontaneous convulsive or nonconvulsive seizures. Recent decades unraveled molecular processes and biochemical signaling pathways involved in the proepileptic transformation of brain circuits including oxidative stress, apoptosis, neuroinflammatory and neurotrophic factors. The “omics” data derived from both human and animal epileptic tissues, as well as electrophysiological, imaging and neurochemical analysis identified a plethora of possible molecular targets for drugs, which could interfere with various stages of epileptogenetic cascade, including inflammatory processes and neuroplastic changes. In this narrative review, we briefly present contemporary views on the neurobiological background of epileptogenesis and discuss the advantages and disadvantages of some more promising molecular targets for antiepileptogenic pharmacotherapy.
... ALLO targets the GABA A Rs family [118], but especially strongly potentiates δGABA A Rs (δGABA A Rs) [19][20][21]. These receptors are located extrasynaptically and produce a tonic form of inhibition [20], which plays an important role in controlling neuronal excitability [119] and gamma generation [116,120]. Furthermore, the elevated concentration of ALLO during the luteal phase affects GABA A Rs expression pattern: it potentiates the expression of δGABA A Rs and therefore further augments the effect of ALLO on tonic inhibitory neurotransmission [30,121]. ...
Premenstrual dysphoric disorder (PMDD) is a psychiatric condition characterized by extreme mood shifts during the luteal phase of the menstrual cycle (MC) due to abnormal sensitivity to neurosteroids and unbalanced neural excitation/inhibition (E/I) ratio. We hypothesized that in women with PMDD in the luteal phase, these factors would alter the frequency of magnetoencephalographic visual gamma oscillations, affect modulation of their power by excitatory drive, and decrease perceptual spatial suppression. Women with PMDD and control women were examined twice–during the follicular and luteal phases of their MC. We recorded visual gamma response (GR) while modulating the excitatory drive by increasing the drift rate of the high-contrast grating (static, ‘slow’, ‘medium’, and ‘fast’). Contrary to our expectations, GR frequency was not affected in women with PMDD in either phase of the MC. GR power suppression, which is normally associated with a switch from the ‘optimal’ for GR slow drift rate to the medium drift rate, was reduced in women with PMDD and was the only GR parameter that distinguished them from control participants specifically in the luteal phase and predicted severity of their premenstrual symptoms. Over and above the atypical luteal GR suppression, in both phases of the MC women with PMDD had abnormally strong GR facilitation caused by a switch from the ‘suboptimal’ static to the ‘optimal’ slow drift rate. Perceptual spatial suppression did not differ between the groups but decreased from the follicular to the luteal phase only in PMDD women. The atypical modulation of GR power suggests that neuronal excitability in the visual cortex is constitutively elevated in PMDD and that this E/I imbalance is further exacerbated during the luteal phase. However, the unaltered GR frequency does not support the hypothesis of inhibitory neuron dysfunction in PMDD.
... Interestingly, GAT1 is reported to be expressed both in neurons and astrocytes [71], while GAT3 is predominantly located to astroglial processes [72], suggesting that GAT-3, in particular, contributes to the tonic GABA release from astrocytes in the neocortex. In the hippocampus, however, astrocytes are capable of releasing GABA only after a strong elevation of [Na + ] i , as a result of facilitating the EAAT-mediated glutamate uptake, occurring, for example, during epileptic-like activity [21,73]. ...
... However, under physiological conditions, the astrocytic [GABA] i appears to be relatively low, and the GAT3 operate in the uptake mode, reducing extracellular [GABA] o . This suggests that in the hippocampus astrocytic, [GABA] i is similar to that in the cerebral cortex, i.e., about 1 mM [73], and [GABA] o amounts to 0.8 µM [41,73]; an elevation of [Na + ] i to about 25-30 mM is required to result in GAT reversal ( Figure 2D). Because EAATs cotransport three Na + ions with one glutamate molecule, EAAT-mediated glutamate uptake strongly affects [Na + ] i [76]. ...
... However, under physiological conditions, the astrocytic [GABA] i appears to be relatively low, and the GAT3 operate in the uptake mode, reducing extracellular [GABA] o . This suggests that in the hippocampus astrocytic, [GABA] i is similar to that in the cerebral cortex, i.e., about 1 mM [73], and [GABA] o amounts to 0.8 µM [41,73]; an elevation of [Na + ] i to about 25-30 mM is required to result in GAT reversal ( Figure 2D). Because EAATs cotransport three Na + ions with one glutamate molecule, EAAT-mediated glutamate uptake strongly affects [Na + ] i [76]. ...
Astrocytes are the most abundant glial cells in the central nervous system (CNS) mediating a variety of homeostatic functions, such as spatial K+ buffering or neurotransmitter reuptake. In addition, astrocytes are capable of releasing several biologically active substances, including glutamate and GABA. Astrocyte-mediated GABA release has been a matter of debate because the expression level of the main GABA synthesizing enzyme glutamate decarboxylase is quite low in astrocytes, suggesting that low intracellular GABA concentration ([GABA]i) might be insufficient to support a non-vesicular GABA release. However, recent studies demonstrated that, at least in some regions of the CNS, [GABA]i in astrocytes might reach several millimoles both under physiological and especially pathophysiological conditions, thereby enabling GABA release from astrocytes via GABA-permeable anion channels and/or via GABA transporters operating in reverse mode. In this review, we summarize experimental data supporting both forms of GABA release from astrocytes in health and disease, paying special attention to possible feedback mechanisms that might govern the fine-tuning of astrocytic GABA release and, in turn, the tonic GABAA receptor-mediated inhibition in the CNS.
... In addition to the imbalance of neurotransmitter systems, changes in the activity and/or amount of potential-dependent ion channels, in particular, Na-, K-and Ca channels, play an important role in epileptogenesis, leading to the increased entry of Na + and Ca 2+ ions into the cell and K + ions exiting from the cell [35,[79][80][81]. These changes decrease the voltage of membrane potential and the threshold of neuronal excitation, leading to the generation of the action potential. ...
... These changes decrease the voltage of membrane potential and the threshold of neuronal excitation, leading to the generation of the action potential. Increased intracellular Ca 2+ concentration could also lead to the activation of glutamate release, decreased GABA levels, and activation of proinflammatory signaling cascades [81,82]. Together with neuronal excitation changes, an important role in epileptogenesis is now ascribed to glia. ...
Animal models of epilepsy are of great importance in epileptology. They are used to study the mechanisms of epileptogenesis, and search for new genes and regulatory pathways involved in the development of epilepsy as well as screening new antiepileptic drugs. Today, many methods of modeling epilepsy in animals are used, including electroconvulsive, pharmacological in intact animals, and genetic, with the predisposition for spontaneous or refractory epileptic seizures. Due to the simplicity of manipulation and universality, genetic models of audiogenic epilepsy in rodents stand out among this diversity. We tried to combine data on the genetics of audiogenic epilepsy in rodents, the relevance of various models of audiogenic epilepsy to certain epileptic syndromes in humans, and the advantages of using of rodent strains predisposed to audiogenic epilepsy in current epileptology.
... However, it has been found that a considerable amount of GABA can be produced and released from glial cells [129,130]. Endogenous putrescine has been considered the source of astrocytic GABA synthesis [131,132], which is suggested to be the dominant source of gliotransmitter GABA [133]. Thus, metabolism and transport of astrocytic polyamines may influence the levels of GABA and subsequently affect epileptiform activity. ...
... Under the action of these enzymes, increased GABA formed by putrescine boosts the Glu/GABA exchange that mediates the release of GABA through astroglial GABA transporters GAT-2/3 and exerts tonic inhibition under epileptic conditions [137]. As the exchange mechanism can be prevented by blocking MAO-B and DAO, putrescine is thought to play a key role in this process [132,137]. It has also been found that glial GABA is released via the bestrophin1 (Best1) channel in the cerebellum and striatum [129]. ...
Epilepsy is one of the most common neurological disorders and severely impacts the life quality of patients. Polyamines are ubiquitous, positively charged aliphatic amines that are present at a relatively high level and help regulate the maintenance of cell membrane excitability and neuronal physiological functions in the central nervous system. Studies have shown abnormalities in the synthesis and catabolism of polyamines in patients with epilepsy and in animal models of epilepsy. The polyamine system seems to involve in the pathophysiological processes of epilepsy via several mechanisms such as the regulation of ion permeability via interaction with ion channels, involvement in antioxidation as hydroperoxide scavengers, and the induction of cell damage via the production of toxic metabolites. In this review, we try to describe the possible associations between polyamines and epilepsy and speculate that the polyamine system is a potential target for the development of novel strategies for epilepsy treatment.
