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

Polyamidoamine dendrimer impairs mitochondrial oxidation in brain tissue

Background: The 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.Results: In 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.Conclusion: These 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. © 2013 Nyitrai et al.; licensee BioMed Central Ltd

Activation of Astroglial Calcium Signaling by Endogenous Metabolites Succinate and Gamma-Hydroxybutyrate in the Nucleus Accumbens

Accumulating evidence suggests that different energy metabolites play a role not only in neuronal but also in glial signaling. Recently, astroglial Ca2+ transients evoked by the major citric acid cycle metabolite succinate (SUC) and gamma-hydroxybutyrate (GHB) that enters the citric acid cycle via SUC have been described in the brain reward area, the nucleus accumbens (NAc). Cells responding to SUC by Ca2+ transient constitute a subset of ATP-responsive astrocytes that are activated in a neuron-independent way. In this study we show that GHB-evoked Ca2+ transients were also found to constitute a subset of ATP-responsive astrocytes in the NAc. Repetitive Ca2+ dynamics evoked by GHB suggested that Ca2+ was released from internal stores. Similarly to SUC, the GHB response was also characterized by an effective concentration of 50 μM. We observed that the number of ATP-responsive cells decreased with increasing concentration of either SUC or GHB. Moreover, the concentration dependence of the number of ATP-responsive cells were highly identical as a function of both [SUC] and [GHB], suggesting a mutual receptor for SUC and GHB, therefore implying the existence of a distinct GHB-recognizing astroglial SUC receptor in the brain. The SUC-evoked Ca2+ signal remained in mice lacking GABAB receptor type 1 subunit in the presence and absence of the N-Methyl-d-Aspartate (NMDA) receptor antagonist (2R)-amino-5-phosphonovaleric acid (APV), indicating action mechanisms independent of the GABAB or NMDA receptor subtypes. By molecular docking calculations we found that residues R99, H103, R252, and R281 of the binding crevice of the kidney SUC-responsive membrane receptor SUCNR1 (GPCR91) also predict interaction with GHB, further implying similar GHB and SUC action mechanisms. We conclude that the astroglial action of SUC and GHB may represent a link between brain energy states and Ca2+ signaling in astrocytic networks

Calcium signals in the nucleus accumbens: activation of astrocytes by ATP and succinate

BACKGROUND: Accumulating evidence suggests that glial signalling is activated by different brain functions. However, knowledge regarding molecular mechanisms of activation or their relation to neuronal activity is limited. The purpose of the present study is to identify the characteristics of ATP-evoked glial signalling in the brain reward area, the nucleus accumbens (NAc), and thereby to explore the action of citric acid cycle intermediate succinate (SUC). RESULTS: We described the burst-like propagation of Ca2+ transients evoked by ATP in acute NAc slices from rat brain. Co-localization of the ATP-evoked Ca2+ signalling with immunoreactivities of the astroglia-specific gap junction forming channel protein connexin43 (Cx43) and the glial fibrillary acidic protein (GFAP) indicated that the responsive cells were a subpopulation of Cx43 and GFAP immunoreactive astrocytes. The ATP-evoked Ca2+ transients were present under the blockade of neuronal activity, but were inhibited by Ca2+ store depletion and antagonism of the G protein coupled purinergic P2Y1 receptor subtype-specific antagonist MRS2179. Similarly, Ca2+ transients evoked by the P2Y1 receptor subtype-specific agonist 2-(Methylthio)adenosine 5'-diphosphate were also blocked by MRS2179. These characteristics implied that intercellular Ca2+ signalling originated from the release of Ca2+ from internal stores, triggered by the activation of P2Y1 receptors. Inhibition by the gap junction blockers carbenoxolone and flufenamic acid and by an antibody raised against the gating-associated segment of Cx43 suggested that intercellular Ca2+ signalling proceeded through gap junctions. We demonstrated for the first time that extracellular SUC also evoked Ca2+ transients (EC50 = 50-60 μM) in about 15% of the ATP-responsive NAc astrocytes. By contrast to glial cells, electrophysiologically identified NAc neurons surrounded by ATP-responsive astrocytes were not activated simultaneously. CONCLUSIONS: We concluded, therefore, that ATP- and SUC-sensitive Ca2+ transients appear to represent a signalling layer independent of NAc neurons. This previously unrecognised glial action of SUC, a major cellular energy metabolite, may play a role in linking metabolism to Ca2+ signalling in astrocytic networks under physiological and pathological conditions such as exercise and metabolic diseases

Long-term cerebral thromboembolic complications of transapical endocardial resynchronization therapy

Purpose: Cardiac resynchronization therapy (CRT) is an established therapeutic option in selected heart failure patients (pts). However, the transvenous left ventricular (LV) lead implantation remains ineffectual in a considerable number of pts. Transapical LV (TALV) lead implantation is an alternative minimally invasive, surgical, endocardial implantation technique. The aim of the present prospective study is to determine the long-term outcome, including the cerebral thromboembolic complications, of pts

The Composition of Hyperacute Serum and Platelet-Rich Plasma Is Markedly Different despite the Similar Production Method

Autologous blood derived products, such as platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) are widely applied in regenerative therapies, in contrast to the drawbacks in their application, mainly deriving from the preparation methods used. Eliminating the disadvantages of both PRP and PRF, hyperacute serum (HAS) opens a new path in autologous serum therapy showing similar or even improved regenerative potential at the same time. Despite the frequent experimental and clinical use of PRP and HAS, their protein composition has not been examined thoroughly yet. Thus, we investigated and compared the composition of HAS, serum, PRP and plasma products using citrate and EDTA by simple laboratory tests, and we compared the composition of HAS, serum, EDTA PRP and plasma by Proteome Profiler and ELISA assays. According to our results the natural ionic balance was upset in both EDTA and citrate PRP as well as in plasma. EDTA PRP contained significantly higher level of growth factors and cytokines, especially platelet derived angiogenic and inflammatory proteins, that can be explained by the significantly higher number of platelets in EDTA PRP. The composition analysis of blood derivatives revealed that although the preparation method of PRP and HAS were similar, the ionic and protein composition of HAS could be advantageous for cell function

Környezettudományi terepgyakorlat

A környezettudomány terepi módszereit bemutató jegyzet célja, hogy átfogó képet adjon a leendő környezettan tanár szakos hallgatóknak a terepen viszonylag egyszerűen elvégezhetőmérési módszerekről. A mű több éves kipróbálás és tapasztalat birtokában egy konkrét mintaterületen elvégzett számos környezeti vizsgálat módszerét mutatja be, ám ezek a módszerek könnyen alkalmazhatók más területek felmérésére, vizsgálatára is. Az igen komplex, mintegy 10 tudományterületet felsorakoztató terepi program során a hallgatók szinte minden környezeti diszciplina terepi módszereivel megismerkedhetnek és a mérésekelvégzése után a gyakorlatokhoz tartozó jegyzőkönyvvel, feladatlappal ellenőrizhetik tudásukat. Az egyes gyakorlati részek modulokként épülnek fel, – s bár egy adott területen kerültek kipróbálásra – általánosan másutt is használhatók, jól kombinálhatók például az érintett szakterületenként, de a tanárképzésben akár korcsoportonként is

The Effects of Hyperacute Serum on the Elements of the Human Subchondral Bone Marrow Niche

Mesenchymal stem cells (MSCs) are widely used in laboratory experiments as well as in human cell therapy. Their culture requires animal sera like fetal calf serum (FCS) as essential supplementation; however, animal sera pose a risk for clinical applications. Human blood derivatives, for example, platelet-rich plasma (PRP) releasates, are potential replacements of FCS; however, it is unclear which serum variant has the best effect on the given cell or tissue type. Additionally, blood derivatives are commonly used in musculoskeletal diseases like osteoarthritis (OA) or osteonecrosis as "proliferative agents" for the topical MSC pool. Hyperacute serum (HAS), a new serum derivative, has been designed to approximate the natural coagulation cascade with a single-step, additive-free preparation method. We investigated the effects of HAS on monolayer MSC cultures and in their natural niche, in 3D subchondral bone and marrow explants. Viability measurements, RT-qPCR evaluation for gene expression and flow cytometry for cell surface marker analysis were performed to compare the effects of FCS-, PRP-, or HAS-supplemented culture media. Monolayer MSCs showed significantly higher metabolic activity following 5 days' incubation in HAS, and osteoblast-specific mRNA expression was markedly increased, while cells also retained their MSC-specific cell surface markers. A similar effect was observed on bone and marrow explants, which was further confirmed with confocal microscopy analysis. Moreover, markedly higher bone marrow preservation was observed with histology in case of HAS supplementation compared to FCS. These findings indicate possible application of HAS in regenerative solutions of skeletal diseases like OA or osteonecrosis

Glutamate Uptake Triggers Transporter-Mediated GABA Release from Astrocytes

Background: Glutamate (Glu) and c-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