Gabriella Nyitrai

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.

Extracellular Level of GABA and Glu: In Vivo Microdialysis-HPLC Measurements

In spite of several studies showing specific physiological functions of changes in the extracellular level of the major excitatory and inhibitory transmitters, Glu and GABA within the brain ([Glu](EXT), [GABA](EXT)) the exact origin (neuronal vs. astroglial, synaptic vs. extrasynaptic) of Glu and GABA present in dialysate samples is still a matter of debate. For better understanding the significance of in vivo microdialysis data, here we discuss methodological details and problems in addition to regulation of [Glu](EXT) and [GABA](EXT). Changes in [Glu](EXT) and [GABA](EXT) under pathological conditions such as ischemia and epilepsy are also reviewed. Based on recent in vivo microdialysis data we argue that ambient [Glu](EXT) and [GABA](EXT)may have a functional role. It is suggested that specific changes in concentrations of Glu and GABA in dialysate samples together with their alterations independent of neuronal activity indicate the involvement of Glu and GABA in the information processing of the brain as essential signaling molecules of nonsynaptic transmission as well. Since various drugs are able to interfere with extrasynaptic signals in vivo, studying the extracellular cell-to-cell communication of brain cells represents a new aspect to improve drugs modulating Gluergic as well as GABAergic neurotransmission.

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.

Uridine activates fast transmembrane Ca2+ ion fluxes in rat brain homogenates

The excitatory actions of the pyrimidine nucleoside uridine, and the nucleotides UDP and UTP, as well as the purine nucleotide ATP, were studied by fluorescent labeling of Ca2+ and K+ ion fluxes on the time scale of 0.04 ms to 10s in resealed plasmalemma fragments and nerve endings from the rat cerebral cortex. Two phases of Ca2+ ion influx with onsets of a few milliseconds and a few hundred milliseconds, showing different concentration dependencies, agonist sequences and subcellular localizations were distinguishable. [3H]Uridine identified high (K(D) approximately 15 nM) and low affinity (K(D)approximately 1 microM) specific binding sites in purified synaptosomal membranes. Labeled uridine taken up by synaptosomes in a dipyridamole-sensitive process was released by depolarization (1 mM 4-aminopyridine). Taken together, these results may qualify uridine as a neurotransmitter.

Slow wave sleep is accompanied by release of certain amino acids in the thalamus of cats.

TO determine whether EEG synchronization in sleep has a metabolic equivalent, we investigated state-dependent changes in extracellular concentrations of amino acids. In vivo microdialysis studies were performed in the ventroposterolateral (VPL) nuclei of the thalamus of cats during natural slow wave sleep (SWS), waking (W) and carbachol-induced paradoxical sleep (PS) like episodes. About two-fold increases in aspartate, glutamate, asparagine, glycine, alanine and γ-aminobutyric acid (GABA) were observed in SWS compared with control samples collected in W, but serine increased to 487 ± 211%. In the PS-like state, glutamine increased and GABA decreased. These results suggest changes in intra-cellular processes reflected by amino acid release in the thalamus, specific to slow wave generation in EEG during natural sleep.

Differential effects of nipecotic acid and 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol on extracellular γ-aminobutyrate levels in rat thalamus

Using the microdialysis technique and HPLC (high-performance liquid chromatography) determination of amino acids, the extracellular concentrations of gamma-aminobutyrate (GABA), glutamate, aspartate and a number of other amino acids were determined in rat thalamus during infusion through the microdialysis tubing of the GABA transport inhibitors 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) and nipecotic acid. Administration of 5.0 mM THPO led to a 200% increase in the extracellular GABA concentration. Simultaneous infusion of THPO and GABA (50 microM) increased the extracellular GABA concentration to 1200% of the basal level whereas GABA alone was found to increase the GABA level to 500%. If nipecotic acid (0.5 mM) was administered together with GABA (50 microM) the extracellular concentration of GABA was not increased further. While administration of GABA alone or GABA together with nipecotic acid had no effect on the extracellular levels of glutamate and aspartate it was found that GABA plus THPO increased the extracellular concentration of these amino acids. GABA administered alone or together with nipecotic acid or THPO led to relatively small but significant increases in the extracellular concentrations of the amino acids glycine, glutamine, serine and threonine. The results demonstrate that THPO, which preferentially inhibits glial GABA uptake and which is not a substrate for the GABA carriers, was more efficient increasing the extracellular concentration of GABA than nipocotic acid which is a substrate and an inhibitor of both neuronal and glial GABA uptake. This indicates that GABA uptake inhibitors that are not substrates for the carrier and which preferentially inhibit glial GABA uptake may constitute a group of drugs by which the efficacy of GABAergic neurotransmission may be enhanced.

γ-Hydroxybutyrate (GHB) induces GABAB receptor independent intracellular Ca2+ transients in astrocytes, but has no effect on GHB or GABAB receptors of medium spiny neurons in the nucleus accumbens

We report on cellular actions of the illicit recreational drug gamma-hydroxybutyrate (GHB) in the brain reward area nucleus accumbens. First, we compared the effects of GHB and the GABA(B) receptor agonist baclofen. Neither of them affected the membrane currents of medium spiny neurons in rat nucleus accumbens slices. GABAergic and glutamatergic synaptic potentials of medium spiny neurons, however, were reduced by baclofen but not GHB. These results indicate the lack of GHB as well as postsynaptic GABA(B) receptors, and the presence of GHB insensitive presynaptic GABA(B) receptors in medium spiny neurons. In astrocytes GHB induced intracellular Ca(2+) transients, preserved in slices from GABA(B) receptor type 1 subunit knockout mice. The effects of tetrodotoxin, zero added Ca(2+) with/without intracellular Ca(2+) store depletor cyclopiazonic acid or vacuolar H-ATPase inhibitor bafilomycin A1 indicate that GHB-evoked Ca(2+) transients depend on external Ca(2+) and intracellular Ca(2+) stores, but not on vesicular transmitter release. GHB-induced astrocytic Ca(2+) transients were not affected by the GHB receptor-specific antagonist NCS-382, suggesting the presence of a novel NCS-382-insensitive target for GHB in astrocytes. The activation of astrocytes by GHB implies their involvement in physiological actions of GHB. Our findings disclose a novel profile of GHB action in the nucleus accumbens. Here, unlike in other brain areas, GHB does not act on GABA(B) receptors, but activates an NCS-382 insensitive GHB-specific target in a subpopulation of astrocytes. The lack of either post- or presynaptic effects on medium spiny neurons in the nucleus accumbens distinguishes GHB from many drugs and natural rewards with addictive properties and might explain why GHB has only a weak reinforcing capacity.

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.

Assessing toxicity of polyamidoamine dendrimers by neuronal signaling functions

Abstract We report for the first time on neuronal signaling for the evaluation of interactions between native plasmamembrane and polyamidoamine (PAMAM) dendrimers. Generation 5 polycationic (G5-NH2), novel β-D-glucopyranose-conjugated G5-NH2 and generation 4.5 polyanionic (G4.5-COONa) polyamidoamine (PAMAM) dendrimers (1–0.0001 mg/ml) were applied in acute brain slices. Functional toxicity assessments–validated by fluorescence imaging of dead cells–were performed by employing electrophysiological indicators of plasma membrane breakdown and synaptic transmission relapse. Irreversible membrane depolarization and decrease of membrane resistance predicted substantial functional neurotoxicity of unmodified G5-NH2, but not of the G4.5-COONa PAMAM dendrimers. Model calculations suggested that freely moving protonated NH2 groups of terminal monomeric units of PAMAM dendrimers may be able directly destroy the membrane or inhibit important K+ channel function via contacting the positively charged NH2. In accordance, conjugation of surface amino groups by β-D-glucopyranose units reduced functional neurotoxicity that may hold great potential for biomedical applications.