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

Appearance of fast astrocytic component in voltage-sensitive dye imaging of neural activity.

BACKGROUND: Voltage-sensitive dye (VSD) imaging and intrinsic optical signals (IOS) are widely used methods for monitoring spatiotemporal neural activity in extensive networks. In spite of that, identification of their major cellular and molecular components has not been concluded so far. RESULTS: We addressed these issues by imaging spatiotemporal spreading of IOS and VSD transients initiated by Schaffer collateral stimulation in rat hippocampal slices with temporal resolution comparable to standard field potential recordings using a 464-element photodiode array. By exploring the potential neuronal and astroglial molecular players in VSD and IOS generation, we identified multiple astrocytic mechanisms that significantly contribute to the VSD signal, in addition to the expected neuronal targets. Glutamate clearance through the astroglial glutamate transporter EAAT2 has been shown to be a significant player in VSD generation within a very short (<5 ms) time-scale, indicating that astrocytes do contribute to the development of spatiotemporal VSD transients previously thought to be essentially neuronal. In addition, non-specific anion channels, astroglial K(+) clearance through Kir4.1 channel and astroglial Na(+)/K(+) ATPase also contribute to IOS and VSD transients. CONCLUSION: VSD imaging cannot be considered as a spatially extended field potential measurement with predominantly neuronal origin, instead it also reflects a fast communication between neurons and astrocytes

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

Sodium-Assisted Formation of Binding and Traverse Conformations of the Substrate in a Neurotransmitter Sodium Symporter Model

Therapeutics designed to increase synaptic neurotransmitter levels by inhibiting neurotransmitter sodium sym- porters (NSSs) classify a strategic approach to treat brain disorders such as depression or epilepsy, however, the critical elementary steps that couple downhill flux of sodium to uphill transport of neurotransmitter are not disti nguished as yet. Here we present modelling of NSS member neuronal GAT1 with the substrate � -aminobutyric acid (GABA), the major inhibitory neurotransmitter. GABA binding is simulated with the occluded conformation of GAT1 homodimer in an ex- plicit lipid/water environment. Simulations performed in the 1-10 ns range of time elucidated persistent formation of half- extended minor and H-bridged major GABA conformations, referred to as binding and traverse conformations, respec- tively. The traverse GABA conformation was further stabilized by GAT1-bound Na + (1). We also observed Na + (1) trans- location to GAT1-bound Cl - as well as the appearance of water molecules at GABA and GAT1-bound Na + (2), conjectur- ing causality. Scaling dynamics suggest that the traverse GABA conformation may be valid for developing substrate in- hibitors with high efficacy. The potential for this finding is significant with impact not only in pharmacology but wherever understanding of the mechanism of neurotransmitter uptake is valuable

Carriage Rates and Characteristics of Enterobacteriaceae Producing Extended-Spectrum Beta-Lactamases in Healthy Individuals: comparison of Applicants for Long-Term Care and Individuals Screened for Employment Purposes

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Asymptomatic faecal carriage of ESBL producing Enterobacteriaceae in Hungarian healthy individuals and in long-term care applicants: a shift towards CTX-M producers in the community

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Connexons Coupling to Gap Junction Channel: Potential Role for Extracellular Protein Stabilization Centers

Connexin (Cx) proteins establish intercellular gap junction channels (Cx GJCs) through coupling of two apposed hexameric Cx hemichannels (Cx HCs, connexons). Pre- and post-GJ interfaces consist of extracellular EL1 and EL2 loops, each with three conserved cysteines. Previously, we reported that known peptide inhibitors, mimicking a variety of Cx43 sequences, appear non-selective when binding to homomeric Cx43 vs. Cx36 GJC homology model subtypes. In pursuit of finding potentially Cx subtype-specific inhibitors of connexon-connexon coupling, we aimed at to understand better how the GJ interface is formed. Here we report on the discovery of Cx GJC subtype-specific protein stabilization centers (SCs) featuring GJ interface architecture. First, the Cx43 GJC homology model, embedded in two opposed membrane bilayers, has been devised. Next, we endorsed the fluctuation dynamics of SCs of the interface domain of Cx43 GJC by applying standard molecular dynamics under open and closed cystine disulfide bond ((C)S-S(C)) preconditions. The simulations confirmed the major role of the unique trans-GJ SC pattern comprising conserved (55N, 56T) and non-conserved (57Q) residues of the apposed EL1 loops in the stabilization of the GJC complex. Importantly, clusters of SC patterns residing close to the GJ interface domain appear to orient the interface formation via the numerous SCs between EL1 and EL2. These include central (54C)S-S(198C) or (61C)S-S(192C) contacts with residues 53R, 54C, 55N, 197D, 199F or 64V, 191P, respectively. In addition, we revealed that GJC interface formation is favoured when the psi dihedral angle of the nearby 193P residue is stable around 180° and the interface SCs disappear when this angle moves to the 0° to −45° range. The potential of the association of non-conserved residues with SC motifs in connexon-connexon coupling makes the development of Cx subtype-specific inhibitors viable

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