László Héja

Astrocytes convert network excitation to tonic inhibition of neurons

BackgroundGlutamate 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.ResultsHere we present evidence that glutamate uptake-induced release of GABA from astrocytes has a direct impact on the excitability of pyramidal neurons in the hippocampus. We demonstrate that GABA, synthesized from the polyamine putrescine, is released from astrocytes by the reverse action of glial GABA transporter (GAT) subtypes GAT-2 or GAT-3. GABA release can be prevented by blocking glutamate uptake with the non-transportable inhibitor DHK, confirming that it is the glutamate transporter activity that triggers the reversal of GABA transporters, conceivably by elevating the intracellular Na+ concentration in astrocytes. The released GABA significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner. Blockade of the Glu/GABA exchange mechanism increases the duration of seizure-like events in the low-[Mg2+] in vitro model of epilepsy. Under in vivo conditions the increased GABA release modulates the power of gamma range oscillation in the CA1 region, suggesting that the Glu/GABA exchange mechanism is also functioning in the intact hippocampus under physiological conditions.ConclusionsThe results suggest the existence of a novel molecular mechanism by which astrocytes transform glutamatergic excitation into GABAergic inhibition providing an adjustable, in situ negative feedback on the excitability of neurons.

Glutamate Uptake Triggers Transporter-Mediated GABA Release from Astrocytes

Background Glutamate (Glu) and γ-aminobutyric acid (GABA) transporters play important roles in regulating neuronal activity. Glu is removed from the extracellular space dominantly by glial transporters. In contrast, GABA is mainly taken up by neurons. However, the glial GABA transporter subtypes share their localization with the Glu transporters and their expression is confined to the same subpopulation of astrocytes, raising the possibility of cooperation between Glu and GABA transport processes. Methodology/Principal Findings Here we used diverse biological models both in vitro and in vivo to explore the interplay between these processes. We found that removal of Glu by astrocytic transporters triggers an elevation in the extracellular level of GABA. This coupling between excitatory and inhibitory signaling was found to be independent of Glu receptor-mediated depolarization, external presence of Ca2+ and glutamate decarboxylase activity. It was abolished in the presence of non-transportable blockers of glial Glu or GABA transporters, suggesting that the concerted action of these transporters underlies the process. Conclusions/Significance Our results suggest that activation of Glu transporters results in GABA release through reversal of glial GABA transporters. This transporter-mediated interplay represents a direct link between inhibitory and excitatory neurotransmission and may function as a negative feedback combating intense excitation in pathological conditions such as epilepsy or ischemia.

Synchronization of GABAergic Inputs to CA3 Pyramidal Cells Precedes Seizure-Like Event Onset in Juvenile Rat Hippocampal Slices

Here we address how dynamics of glutamatergic and GABAergic synaptic input to CA3 pyramidal cells contribute to spontaneous emergence and evolution of recurrent seizure-like events (SLEs) in juvenile (P10-13) rat hippocampal slices bathed in low-[Mg(2+)] artificial cerebrospinal fluid. In field potential recordings from the CA3 pyramidal layer, a short epoch of high-frequency oscillation (HFO; 400-800 Hz) was observed during the first 10 ms of SLE onset. GABAergic synaptic input currents to CA3 pyramidal cells were synchronized and coincided with HFO, whereas the glutamatergic input lagged by approximately 10 ms. If the intracellular [Cl(-)] remained unperturbed (cell-attached recordings) or was set high with whole cell electrode solution, CA3 pyramidal cell firing peaked with HFO and GABAergic input. By contrast, with low intracellular [Cl(-)], spikes of CA3 pyramidal cells lagged behind HFO and GABAergic input. This temporal arrangement of HFO, synaptic input sequence, synchrony of GABAergic currents, and pyramidal cell firing emerged gradually with preictal discharges until the SLE onset. Blockade of GABA(A) receptor-mediated currents by picrotoxin reduced the inter-SLE interval and the number of preictal discharges and did not block recurrent SLEs. Our data suggest that dynamic changes of the functional properties of GABAergic input contribute to ictogenesis and GABAergic and glutamatergic inputs are both excitatory at the instant of SLE onset. At the SLE onset GABAergic input contributes to synchronization and recruitment of pyramidal cells. We conjecture that this network state is reached by an activity-dependent shift in GABA reversal potential during the preictal phase.

Astrocyte sodium signaling and the regulation of neurotransmission.

The transmembrane Na+ concentration gradient is an important source of energy required not only to enable the generation of action potentials in excitable cells, but also for various transmembrane transporters both in excitable and non‐excitable cells, like astrocytes. One of the vital functions of astrocytes in the central nervous system (CNS) is to regulate neurotransmitter concentrations in the extracellular space. Most neurotransmitters in the CNS are removed from the extracellular space by Na+‐dependent neurotransmitter transporters (NeuTs) expressed both in neurons and astrocytes. Neuronal NeuTs control mainly phasic synaptic transmission, i.e., synaptically induced transient postsynaptic potentials, while astrocytic NeuTs contribute to the termination of phasic neurotransmission and modulate the tonic tone, i.e., the long‐lasting activation of extrasynaptic receptors by neurotransmitter that has diffused out of the synaptic cleft. Consequently, local intracellular Na+ ([Na+]i) transients occurring in astrocytes, for example via the activation of ionotropic neurotransmitter receptors, can affect the driving force for neurotransmitter uptake, in turn modulating the spatio‐temporal profiles of neurotransmitter levels in the extracellular space. As some NeuTs are close to thermodynamic equilibrium under resting conditions, an increase in astrocytic [Na+]i can stimulate the direct release of neurotransmitter via NeuT reversal. In this review we discuss the role of astrocytic [Na+]i changes in the regulation of uptake/release of neurotransmitters. It is emphasized that an activation of one neurotransmitter system, including either its ionotropic receptor or Na+‐coupled co‐transporter, can strongly influence, or even reverse, other Na+‐dependent NeuTs, with potentially significant consequences for neuronal communication. GLIA 2016;64:1655–1666

Role for GABA and Glu Plasma Membrane Transporters in the Interplay of Inhibitory and Excitatory Neurotransmission

Neurotransmitter plasma membrane transporters do have much more to perform than simply terminating synaptic transmission and replenishing neurotransmitter pools. Findings in the past decade have evidenced their function in maintaining physiological synaptic excitability, and their actions in critical or pathological conditions, also. Conclusively these findings indicated a previously unrecognized role for neurotransmitter plasma membrane transporters in both, synaptic and nonsynaptic signaling. Major inhibitory and excitatory neurotransmitters within the brain, GABA and Glu, have long been considered to operate through independent systems (GABAergic or Gluergic), each of them characterized by its own localization, function and dedicated GABAergic or Gluergic cell phenotypes. Recent advances, however, have challenged this long-standing paradigm. Localization of GABA in Gluergic terminals and Glu in GABAergic cells were reported. Specific plasma membrane transporters for GABA and Glu are also co-localized in different brain areas. Although, their role in regulating each others signal is still far from being understood, emerging lines of evidence on interplaying GABAergic and Gluergic processes through plasma membrane transporters opens up a new avenue in the field of more specific therapeutic intervention.

Polyamidoamine dendrimer impairs mitochondrial oxidation in brain tissue

BackgroundThe potential nanocarrier polyamidoamine (PAMAM) generation 5 (G5-NH2) dendrimer has been shown to evoke lasting neuronal depolarization and cell death in a concentration-dependent manner. In this study we explored the early progression of G5-NH2 action in brain tissue on neuronal and astroglial cells.ResultsIn order to describe early mechanisms of G5-NH2 dendrimer action in brain tissue we assessed G5-NH2 trafficking, free intracellular Ca2+ and mitochondrial membrane potential (ΨMITO) changes in the rat hippocampal slice by microfluorimetry. With the help of fluorescent dye conjugated G5-NH2, we observed predominant appearance of the dendrimer in the plasma membrane of pyramidal neurons and glial cells within 30 min. Under this condition, G5-NH2 evoked robust intracellular Ca2+ enhancements and ΨMITO depolarization both in pyramidal neurons and astroglial cells. Intracellular Ca2+ enhancements clearly preceded ΨMITO depolarization in astroglial cells. Comparing activation dynamics, neurons and glia showed prevalence of lasting and transient ΨMITO depolarization, respectively. Transient as opposed to lasting ΨMITO changes to short-term G5-NH2 application suggested better survival of astroglia, as observed in the CA3 stratum radiatum area. We also showed that direct effect of G5-NH2 on astroglial ΨMITO was significantly enhanced by neuron-astroglia interaction, subsequent to G5-NH2 evoked neuronal activation.ConclusionThese findings indicate that the interaction of the PAMAM dendrimer with the plasma membrane leads to robust activation of neurons and astroglial cells, leading to mitochondrial depolarization. Distinguishable dynamics of mitochondrial depolarization in neurons and astroglia suggest that the enhanced mitochondrial depolarization followed by impaired oxidative metabolism of neurons may be the primary basis of neurotoxicity.

Non-adenosine nucleoside inosine, guanosine and uridine as promising antiepileptic drugs: a summary of current literature.

Adenosine (Ado) and some non-adenosine (non-Ado) nucleosides including inosine (Ino), guanosine (Guo) and uridine (Urd) are modulatory molecules in the central nervous system (CNS), regulating different physiological and pathophysiological processes in the brain such as sleep and epilepsy. Indeed, different drugs effective on adenosinergic system (e.g., Ado metabolism inhibitors, agonists and antagonists of Ado receptors) are being used in drug development for the treatment of epileptic disorders. Although (i) endogenous Ino, Guo and Urd showed anticonvulsant/antiepileptic effects (e.g., in quinolinic acid - induced seizures and in different epilepsy models such as hippocampal kindling models), and (ii) there is a need to generate new and more effective antiepileptic drugs for the treatment of drug-resistant epilepsies, our knowledge about antiepileptic influence of non-Ado nucleosides is far from complete. Thus, in this review article, we give a short summary of anticonvulsant/antiepileptic effects and mechanisms evoked by Ino, Guo, and Urd. Finally, we discuss some non-Ado nucleoside derivatives and their structures, which may be candidates as potential antiepileptic agents.

Astrocytic target mechanisms in epilepsy.

Although glial proliferation of the epileptic loci is recognized for more than a century in certain focal epilepsies, the role of astrocytes in epileptic conditions is receiving significant attention only in recent years. The present review will highlight current knowledge about the various ways astrocytes control neuronal excitability and contribute to genesis, maintenance and suppression of seizures. Besides the widely recognized astrocytic tasks like glutamate clearance, the role of gliotransmission, glutamate, GABA and ATP release as well as gap junctional communication will also be discussed along with the contribution of blood-brain barrier dysfunction, inflammatory pathways and alterations in mircoRNA expression profile to epilepsy. The mechanisms described will help to understand the astrocytic mechanisms contributing to the antiepileptic effect of existing anti-epileptic drugs (AEDs) and current therapeutic strategies and also signifies the potential of specific astrocyte-based AED development.

Characterisation of an uridine-specific binding site in rat cerebrocortical homogenates

Parameters of [3H]uridine binding to synaptic membranes isolated from rat brain cortex (K(D)=71+/-4 nM, B(max)=1.37+/-0.13 pmol/mg protein) were obtained. Pyrimidine and purine analogues displayed different rank order of potency in displacement of specifically bound [3H]uridine (uridine>5-F-uridine>5-Br-uridine approximately adenosine>>5-ethyl-uridine approximately suramin>theophylline) and in the inhibition of [14C]uridine uptake (adenosine>uridine>5-Br-uridine approximately 5-F-uridine approximately 5-ethyl-uridine) into purified cerebrocortical synaptosomes. Furthermore, the effective ligand concentration for the inhibition of [14C]uridine uptake was about two order of magnitude higher than that for the displacement of specifically bound [3H]uridine. Adenosine evoked the transmembrane Na(+) ion influx, whereas uridine the transmembrane Ca(2+) ion influx much more effectively. Also, uridine was shown to increase free intracellular Ca(2+) ion levels in hippocampal slices by measuring Calcium-Green fluorescence. Uridine analogues were found to be ineffective in displacing radioligands that were bound to various glutamate and adenosine-recognition and modulatory-binding sites, however, increased [35S]GTPgammaS binding to membranes isolated from the rat cerebral cortex. These findings provide evidence for a rather specific, G-protein-coupled site of excitatory action for uridine in the brain.

Neuronal and astroglial correlates underlying spatiotemporal intrinsic optical signal in the rat hippocampal slice.

Widely used for mapping afferent activated brain areas in vivo, the label-free intrinsic optical signal (IOS) is mainly ascribed to blood volume changes subsequent to glial glutamate uptake. By contrast, IOS imaged in vitro is generally attributed to neuronal and glial cell swelling, however the relative contribution of different cell types and molecular players remained largely unknown. We characterized IOS to Schaffer collateral stimulation in the rat hippocampal slice using a 464-element photodiode-array device that enables IOS monitoring at 0.6 ms time-resolution in combination with simultaneous field potential recordings. We used brief half-maximal stimuli by applying a medium intensity 50 Volt-stimulus train within 50 ms (20 Hz). IOS was primarily observed in the str. pyramidale and proximal region of the str. radiatum of the hippocampus. It was eliminated by tetrodotoxin blockade of voltage-gated Na+ channels and was significantly enhanced by suppressing inhibitory signaling with gamma-aminobutyric acid(A) receptor antagonist picrotoxin. We found that IOS was predominantly initiated by postsynaptic Glu receptor activation and progressed by the activation of astroglial Glu transporters and Mg2+-independent astroglial N-methyl-D-aspartate receptors. Under control conditions, role for neuronal K+/Cl− cotransporter KCC2, but not for glial Na+/K+/Cl− cotransporter NKCC1 was observed. Slight enhancement and inhibition of IOS through non-specific Cl− and volume-regulated anion channels, respectively, were also depicted. High-frequency IOS imaging, evoked by brief afferent stimulation in brain slices provide a new paradigm for studying mechanisms underlying IOS genesis. Major players disclosed this way imply that spatiotemporal IOS reflects glutamatergic neuronal activation and astroglial response, as observed within the hippocampus. Our model may help to better interpret in vivo IOS and support diagnosis in the future.