Infra-Slow Oscillation (ISO) of the Pupil Size of Urethane-Anaesthetised Rats

Multiplicity of oscillatory phenomena in a range of infra-slow frequencies (<0.01 Hz) has been described in mammalian brains at different levels of organisation. The significance and manifestation in physiology and/or behaviour of many brain infra-slow oscillations (ISO) remain unknown. Examples of this phenomenon are two types of ISO observed in the brains of urethane-anaesthetised rats: infra-slow, rhythmic changes in the rate of action potential firing in a few nuclei of the subcortical visual system and a sleep-like cycle of activation/deactivation visible in the EEG signal. Because both of these rhythmic phenomena involve brain networks that can influence autonomic nervous system activity, we hypothesised that these two brain ISOs can be reflected by rhythmic changes of pupil size. Thus, in the present study, we used simultaneous pupillography and ECoG recording to verify the hypothesised existence of infra-slow oscillations in the pupil size of urethane-anaesthetised rats. The obtained results showed rhythmic changes in the size of the pupils and rhythmic eyeball movements in urethane-anaesthetised rats. The observed rhythms were characterised by two different dominant components in a range of infra-slow frequencies. First, the long component had a period of ≈29 minutes and was present in both the irises and the eyeball movements. Second, the short component had a period of ≈2 minutes and was observed only in the rhythmic constrictions and dilations of the pupils. Both ISOs were simultaneously present in both eyes, and they were synchronised between the left and right eye. The long ISO component was synchronised with the cyclic alternations of the brain state, as revealed by rhythmic changes in the pattern of the ECoG signal. Based on the obtained results, we propose a model of interference of ISO present in different brain systems involved in the control of pupil size.     

Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga

The divalent cation Sr²⁺ induced repetitive transient spikes of the cytosolic Ca²⁺ activity [Ca²⁺]_cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca²⁺]_cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr²⁺-induced [Ca²⁺]_cy oscillations. Sr²⁺ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca²⁺]_cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca²⁺-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr²⁺-induced repetitive [Ca²⁺]_cy spikes, whereas the compartmentalization of Sr²⁺ was not influenced. A repetitive Ca²⁺ release and Ca²⁺ re-uptake by the ER probably generated repetitive [Ca²⁺]_cy spikes in E. viridis in the presence of Sr²⁺. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr²⁺-induced Ca²⁺ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca²⁺ channel. The blockage of Sr²⁺-induced repetitive [Ca²⁺]_cy spikes by La³⁺ or Gd³⁺ indicated the necessity of a certain influx of divalent cations for sustained [Ca²⁺]_cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca²⁺]_cy oscillations in E. viridis.     

Apical Vesicles Bearing Inositol 1,4,5-trisphosphate Receptors in the Ca2+Initiation Site of Ductal Epithelium of Submandibular Gland

In polarized epithelial cells, agonists trigger Ca²⁺ waves and oscillations. These patterns may be caused by the compartmentalization of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ pools into specific regions. We have investigated the relationship between the distribution of IP₃ receptors (IP₃Rs) and the spatiotemporal pattern of Ca²⁺ signaling in the duct cells of the rat submandibular gland (SMG). Using immunofluorescence, although labeling was somewhat heterogeneous, the IP₃Rs were colocalized to the apical pole of the duct cells. Immunoelectron microscopy identified small apical vesicles bearing IP₃R2 in some types of duct cells. Real-time confocal imaging of intact ducts demonstrated that, after carbachol stimulation, an initial Ca²⁺ spike occurred in the apical region. Subsequently, repetitive Ca²⁺ spikes spread from the apical to the middle cytoplasm. These apical Ca²⁺ initiation sites were found only in some “pioneer cells,” rather than in all duct cells. We performed both Ca²⁺ imaging and immunofluorescence on the same ducts and detected the strongest immunosignals of IP₃R2 in the Ca²⁺ initiation sites of the pioneer cells. The subcellular localization and expression level of IP₃Rs correlated strongly with the spatiotemporal nature of the intracellular Ca²⁺ signal and distinct Ca²⁺ responses among the rat SMG duct cells.     

Specific Human Astrocyte Subtype Revealed by Affinity Purified GFAP+1 Antibody; Unpurified Serum Cross-Reacts with Neurofilament-L in Alzheimer

The human GFAP splice variants GFAPΔ164 and GFAPΔexon6 both result in a GFAP protein isoform with a unique out-of-frame carboxy-terminus that can be detected by the GFAP⁺¹ antibody. We previously reported that GFAP⁺¹ was expressed in astrocytes and in degenerating neurons in Alzheimer''s disease brains. In this study we aimed at further investigating the neuronal GFAP⁺¹ expression and we started by affinity purifying the GFAP⁺¹ antibody. The purified antibody resulted in a loss of neuronal GFAP⁺¹ signal, although other antibodies directed against the amino- and carboxy-terminus of GFAPα still revealed GFAP-immunopositive neurons, as described before. With an in-depth analysis of a western blot, followed by mass spectrometry we discovered that the previously detected neuronal GFAP⁺¹ expression was due to cross-reactivity of the antibody with neurofilament-L (NF-L). This was confirmed by double-label fluorescent immunohistochemistry and western blotting with the unpurified GFAP⁺¹ antibody and an antibody against NF-L. Our data imply that NF-L can accumulate in some tangle-like structures in Alzheimer brains. More importantly, the purified GFAP⁺¹ antibody clearly revealed a specific subtype of astrocytes in the adult human brain. These large astrocytes are present throughout the brain, e.g., along the subventricular zone, in the hippocampus, in the striatum and in the spinal cord of controls, Alzheimer, and Parkinson patients. The presence of a specific GFAP-isoform suggests a specialized function of these astrocytes.     

Glucose Metabolism,Islet Architecture,and Genetic Homogeneity in Imprinting of [Ca2+]i and Insulin Rhythms in Mouse Islets

We reported previously that islets isolated from individual, outbred Swiss-Webster mice displayed oscillations in intracellular calcium ([Ca²⁺]_i) that varied little between islets of a single mouse but considerably between mice, a phenomenon we termed “islet imprinting.” We have now confirmed and extended these findings in several respects. First, imprinting occurs in both inbred (C57BL/6J) as well as outbred mouse strains (Swiss-Webster; CD1). Second, imprinting was observed in NAD(P)H oscillations, indicating a metabolic component. Further, short-term exposure to a glucose-free solution, which transiently silenced [Ca²⁺]_i oscillations, reset the oscillatory patterns to a higher frequency. This suggests a key role for glucose metabolism in maintaining imprinting, as transiently suppressing the oscillations with diazoxide, a K_ATP-channel opener that blocks [Ca²⁺]_i influx downstream of glucose metabolism, did not change the imprinted patterns. Third, imprinting was not as readily observed at the level of single beta cells, as the [Ca²⁺]_i oscillations of single cells isolated from imprinted islets exhibited highly variable, and typically slower [Ca²⁺]_i oscillations. Lastly, to test whether the imprinted [Ca²⁺]_i patterns were of functional significance, a novel microchip platform was used to monitor insulin release from multiple islets in real time. Insulin release patterns correlated closely with [Ca²⁺]_i oscillations and showed significant mouse-to-mouse differences, indicating imprinting. These results indicate that islet imprinting is a general feature of islets and is likely to be of physiological significance. While islet imprinting did not depend on the genetic background of the mice, glucose metabolism and intact islet architecture may be important for the imprinting phenomenon.     

Positive and Negative Coupling of the Metabotropic Glutamate Receptors to a G Protein–activated K+ Channel,GIRK, in Xenopus Oocytes

Metabotropic glutamate receptors (mGluRs) control intracellular signaling cascades through activation of G proteins. The inwardly rectifying K⁺ channel, GIRK, is activated by the βγ subunits of G_i proteins and is widely expressed in the brain. We investigated whether an interaction between mGluRs and GIRK is possible, using Xenopus oocytes expressing mGluRs and a cardiac/brain subunit of GIRK, GIRK1, with or without another brain subunit, GIRK2. mGluRs known to inhibit adenylyl cyclase (types 2, 3, 4, 6, and 7) activated the GIRK channel. The strongest response was observed with mGluR2; it was inhibited by pertussis toxin (PTX). This is consistent with the activation of GIRK by G_i/G_o-coupled receptors. In contrast, mGluR1a and mGluR5 receptors known to activate phospholipase C, presumably via G proteins of the G_q class, inhibited the channel''s activity. The inhibition was preceded by an initial weak activation, which was more prominent at higher levels of mGluR1a expression. The inhibition of GIRK activity by mGluR1a was suppressed by a broad-specificity protein kinase inhibitor, staurosporine, and by a specific protein kinase C (PKC) inhibitor, bis-indolylmaleimide, but not by PTX, Ca²⁺ chelation, or calphostin C. Thus, mGluR1a inhibits the GIRK channel primarily via a pathway involving activation of a PTX-insensitive G protein and, eventually, of a subtype of PKC, possibly PKC-μ. In contrast, the initial activation of GIRK1 caused by mGluR1a was suppressed by PTX but not by the protein kinase inhibitors. Thus, this activation probably results from a promiscuous coupling of mGluR1a to a G_i/G_o protein. The observed modulations may be involved in the mGluRs'' effects on neuronal excitability in the brain. Inhibition of GIRK by phospholipase C–activating mGluRs bears upon the problem of specificity of G protein (GIRK interaction) helping to explain why receptors coupled to G_q are inefficient in activating GIRK.     

Calcium Spiking Patterns and the Role of the Calcium/Calmodulin-Dependent Kinase CCaMK in Lateral Root Base Nodulation of Sesbania rostrata

Nodulation factor (NF) signal transduction in the legume-rhizobium symbiosis involves calcium oscillations that are instrumental in eliciting nodulation. To date, Ca²⁺ spiking has been studied exclusively in the intracellular bacterial invasion of growing root hairs in zone I. This mechanism is not the only one by which rhizobia gain entry into their hosts; the tropical legume Sesbania rostrata can be invaded intercellularly by rhizobia at cracks caused by lateral root emergence, and this process is associated with cell death for formation of infection pockets. We show that epidermal cells at lateral root bases respond to NFs with Ca²⁺ oscillations that are faster and more symmetrical than those observed during root hair invasion. Enhanced jasmonic acid or reduced ethylene levels slowed down the Ca²⁺ spiking frequency and stimulated intracellular root hair invasion by rhizobia, but prevented nodule formation. Hence, intracellular invasion in root hairs is linked with a very specific Ca²⁺ signature. In parallel experiments, we found that knockdown of the calcium/calmodulin-dependent protein kinase gene of S. rostrata abolished nodule development but not the formation of infection pockets by intercellular invasion at lateral root bases, suggesting that the colonization of the outer cortex is independent of Ca²⁺ spiking decoding.     

PKC-dependent ERK phosphorylation is essential for P2X7 receptor-mediated neuronal differentiation of neural progenitor cells

Purinergic receptors have been shown to be involved in neuronal development, but the functions of specific subtypes of P2 receptors during neuronal development remain elusive. In this study we investigate the distribution of P2X₇ receptors (P2X₇Rs) in the embryonic rat brain using in situ hybridization. At E15.5, P2X₇R mRNA was observed in the ventricular zone and subventricular zone, and colocalized with nestin, indicating that P2X₇R might be expressed in neural progenitor cells (NPCs). P2X₇R mRNA was also detected in the subgranular zone and dentate gyrus of the E18.5 and P4 brain. To investigate the roles of P2X₇R and elucidate its mechanism, we established NPC cultures from the E15.5 rat brain. Stimulation of P2X₇Rs induced Ca²⁺ influx, inhibited proliferation, altered cell cycle progression and enhanced the expression of neuronal markers, such as TUJ1 and MAP2. Similarly, knockdown of P2X₇R by shRNA nearly abolished the agonist-stimulated increases in intracellular Ca²⁺ concentration and the expression of TUJ1 and NeuN. Furthermore, stimulation of P2X₇R induced activation of ERK1/2, which was inhibited by the removal of extracellular Ca²⁺ and treatment with blockers for P2X₇R and PKC activity. Stimulation of P2X₇R also induced translocation of PKCα and PKCγ, but not of PKCβ, whereas knockdown of either PKCα or PKCγ inhibited ERK1/2 activation. Inhibition of PKC or p-ERK1/2 also caused a decrease in the number of TUJ1-positive cells and a concomitant increase in the number of GFAP-positive cells. Taken together, the activation of P2X₇R in NPCs induced neuronal differentiation through a PKC-ERK1/2 signaling pathway.     

Subcellular Min Oscillations as a Single-Cell Reporter of the Action of Polycations,Protamine, and Gentamicin on Escherichia coli

Background

In Escherichia coli, MinD-GFP fusion proteins show rapid pole to pole oscillations. The objective was to investigate the effects of extracellular cations on the subcellular oscillation of cytoplasmic MinD within Escherichia coli.

Methodology/Principal Findings

We exposed bacteria to the extracellular cations Ca⁺⁺, Mg⁺⁺, the cationic antimicrobial peptide (CAP) protamine, and the cationic aminoglycoside gentamicin. We found rapid and substantial increases in the average MinD oscillation periods in the presence of any of these polyvalent cations. For Ca⁺⁺ and Mg⁺⁺ the increases in period were transient, even with a constant extracellular concentration, while increases in period for protamine or gentamicin were apparently irreversible. We also found striking interdependence in the action of the small cations with protamine or gentamicin, distorted oscillations under the action of intermediate levels of gentamicin and Ca⁺⁺, and reversible freezing of the Min oscillation at high cationic concentrations.

Conclusions/Significance

Intracellular Min oscillations provide a fast single-cell reporter of bacterial response to extracellular polycations, which can be explained by the penetration of polycations into cells.     

Postsynaptic scaffold protein Homer 1a protects against traumatic brain injury via regulating group I metabotropic glutamate receptors

Traumatic brain injury (TBI) produces excessive glutamate, leading to excitotoxicity via the activation of glutamate receptors. Postsynaptic density scaffold proteins have crucial roles in mediating signal transduction from glutamate receptors to their downstream mediators. Therefore, studies on the mechanisms underlying regulation of excitotoxicity by scaffold proteins can uncover new treatments for TBI. Here, we demonstrated that the postsynaptic scaffold protein Homer 1a was neuroprotective against TBI in vitro and in vivo, and this neuroprotection was associated with its effects on group I metabotropic glutamate receptors (mGluRs). Upon further study, we found that Homer 1a mainly affected neuronal injury induced by mGluR1 activation after TBI and also influenced mGluR5 function when its activity was restored. The ability of Homer 1a to disrupt mGluR-ERK signaling contributed to its ability to regulate the functions of mGluR1 and mGluR5 after traumatic injury. Intracellular Ca²⁺ and PKC were two important factors involved in the mediation of mGluR-ERK signaling by Homer 1a. These results define Homer 1a as a novel endogenous neuroprotective agent against TBI.     

Estrogen Receptor β-Selective Agonists Stimulate Calcium Oscillations in Human and Mouse Embryonic Stem Cell-Derived Neurons

Estrogens are used extensively to treat hot flashes in menopausal women. Some of the beneficial effects of estrogens in hormone therapy on the brain might be due to nongenomic effects in neurons such as the rapid stimulation of calcium oscillations. Most studies have examined the nongenomic effects of estrogen receptors (ER) in primary neurons or brain slices from the rodent brain. However, these cells can not be maintained continuously in culture because neurons are post-mitotic. Neurons derived from embryonic stem cells could be a potential continuous, cell-based model to study nongenomic actions of estrogens in neurons if they are responsive to estrogens after differentiation. In this study ER-subtype specific estrogens were used to examine the role of ERα and ERβ on calcium oscillations in neurons derived from human (hES) and mouse embryonic stem cells. Unlike the undifferentiated hES cells the differentiated cells expressed neuronal markers, ERβ, but not ERα. The non-selective ER agonist 17β-estradiol (E₂) rapidly increased [Ca2+]i oscillations and synchronizations within a few minutes. No change in calcium oscillations was observed with the selective ERα agonist 4,4′,4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT). In contrast, the selective ERβ agonists, 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN), MF101, and 2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-1,3 benzoxazol-5-ol (ERB-041; WAY-202041) stimulated calcium oscillations similar to E₂. The ERβ agonists also increased calcium oscillations and phosphorylated PKC, AKT and ERK1/2 in neurons derived from mouse ES cells, which was inhibited by nifedipine demonstrating that ERβ activates L-type voltage gated calcium channels to regulate neuronal activity. Our results demonstrate that ERβ signaling regulates nongenomic pathways in neurons derived from ES cells, and suggest that these cells might be useful to study the nongenomic mechanisms of estrogenic compounds.     

Intracellular Calcium Spikes in Rat Suprachiasmatic Nucleus Neurons Induced by BAPTA-Based Calcium Dyes

Background

Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of “Ca²⁺ spikes” (i.e., [Ca²⁺]_c transients having a bandwidth of 10∼100 seconds) in SCN neurons, but it is unclear if these SCN Ca²⁺ spikes are related to the slow circadian rhythms.

Methodology/Principal Findings

We addressed this issue based on a Ca²⁺ indicator dye (fluo-4) and a protein Ca²⁺ sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca²⁺ spikes in 18% of rat SCN cells in acute brain slices, but the Ca²⁺ spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca²⁺ spike was barely observed (<3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca²⁺ spikes was increased to 13∼14%.

Conclusions/Significance

Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca²⁺ spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca²⁺ spiking activity is caused by the Ca²⁺ chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca²⁺]_c in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca²⁺ spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca²⁺ spikes in the function of SCN.     

Nonlinear Gap Junctions Enable Long-Distance Propagation of Pulsating Calcium Waves in Astrocyte Networks

A new paradigm has recently emerged in brain science whereby communications between glial cells and neuron-glia interactions should be considered together with neurons and their networks to understand higher brain functions. In particular, astrocytes, the main type of glial cells in the cortex, have been shown to communicate with neurons and with each other. They are thought to form a gap-junction-coupled syncytium supporting cell-cell communication via propagating Ca²⁺ waves. An identified mode of propagation is based on cytoplasm-to-cytoplasm transport of inositol trisphosphate (IP₃) through gap junctions that locally trigger Ca²⁺ pulses via IP₃-dependent Ca²⁺-induced Ca²⁺ release. It is, however, currently unknown whether this intracellular route is able to support the propagation of long-distance regenerative Ca²⁺ waves or is restricted to short-distance signaling. Furthermore, the influence of the intracellular signaling dynamics on intercellular propagation remains to be understood. In this work, we propose a model of the gap-junctional route for intercellular Ca²⁺ wave propagation in astrocytes. Our model yields two major predictions. First, we show that long-distance regenerative signaling requires nonlinear coupling in the gap junctions. Second, we show that even with nonlinear gap junctions, long-distance regenerative signaling is favored when the internal Ca²⁺ dynamics implements frequency modulation-encoding oscillations with pulsating dynamics, while amplitude modulation-encoding dynamics tends to restrict the propagation range. As a result, spatially heterogeneous molecular properties and/or weak couplings are shown to give rise to rich spatiotemporal dynamics that support complex propagation behaviors. These results shed new light on the mechanisms implicated in the propagation of Ca²⁺ waves across astrocytes and the precise conditions under which glial cells may participate in information processing in the brain.     

Assembly of a Ca2+-dependent BK channel signaling complex by binding to beta2 adrenergic receptor

Large-conductance voltage and Ca²⁺-activated potassium channels (BKCa) play a critical role in modulating contractile tone of smooth muscle, and neuronal processes. In most mammalian tissues, activation of β-adrenergic receptors and protein kinase A (PKA_c) increases BKCa channel activity, contributing to sympathetic nervous system/hormonal regulation of membrane excitability. Here we report the requirement of an association of the β2-adrenergic receptor (β2AR) with the pore forming α subunit of BKCa and an A-kinase-anchoring protein (AKAP79/150) for β2 agonist regulation. β2AR can simultaneously interact with both BKCa and L-type Ca²⁺ channels (Ca_v1.2) in vivo, which enables the assembly of a unique, highly localized signal transduction complex to mediate Ca²⁺- and phosphorylation-dependent modulation of BKCa current. Our findings reveal a novel function for G protein-coupled receptors as a scaffold to couple two families of ion channels into a physical and functional signaling complex to modulate β-adrenergic regulation of membrane excitability.     

Fluorescent Pseudomonad Pyoverdines Bind and Oxidize Ferrous Ion

Major pyoverdines from Pseudomonas fluorescens 2-79 (Pf-B), P. aeruginosa ATCC 15692 (Pa-C), and P. putida ATCC 12633 (Pp-C) were examined by absorption and fluorescence spectroscopic techniques to investigate the interaction between ferrous ion and the pyoverdine ligand. At physiological pH, ferrous ion quenched the fluorescence of all three pyoverdines much faster than ferric ion did. Also, increased absorbance at 460 nm was observed to be much faster for Fe²⁺-pyoverdine than for Fe³⁺-pyoverdine. At pH 7.4, about 90% of Fe³⁺ was bound by pyoverdine Pa-C after 24 h whereas Fe²⁺ was bound by the pyoverdine completely in only 5 min. The possibility that Fe²⁺ underwent rapid autoxidation before being bound by pyoverdine was considered unlikely, since the Fe²⁺ concentration in pyoverdine-free samples remained constant over a 3-min period at pH 7.4. Incubating excess Fe²⁺ with pyoverdine in the presence of 8-hydroxyquinoline, an Fe³⁺-specific chelating agent, resulted in the formation of a Fe³⁺-hydroxyquinoline complex, suggesting that the iron in the Fe²⁺-pyoverdine complex existed in the oxidized form. These results strongly suggested that pyoverdines bind and oxidize the ferrous ion.     

Attenuation of Reactive Gliosis Does Not Affect Infarct Volume in Neonatal Hypoxic-Ischemic Brain Injury in Mice

Background

Astroglial cells are activated following injury and up-regulate the expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Adult mice lacking the intermediate filament proteins GFAP and vimentin (GFAP^−/−Vim^−/−) show attenuated reactive gliosis, reduced glial scar formation and improved regeneration of neuronal synapses after neurotrauma. GFAP^−/−Vim^−/− mice exhibit larger brain infarcts after middle cerebral artery occlusion suggesting protective role of reactive gliosis after adult focal brain ischemia. However, the role of astrocyte activation and reactive gliosis in the injured developing brain is unknown.

Methodology/Principal Findings

We subjected GFAP^−/−Vim^−/− and wild-type mice to unilateral hypoxia-ischemia (HI) at postnatal day 9 (P9). Bromodeoxyuridine (BrdU; 25 mg/kg) was injected intraperitoneally twice daily from P9 to P12. On P12 and P31, the animals were perfused intracardially. Immunohistochemistry with MAP-2, BrdU, NeuN, and S100 antibodies was performed on coronal sections. We found no difference in the hemisphere or infarct volume between GFAP^−/−Vim^−/− and wild-type mice at P12 and P31, i.e. 3 and 22 days after HI. At P31, the number of NeuN⁺ neurons in the ischemic and contralateral hemisphere was comparable between GFAP^−/−Vim^−/− and wild-type mice. In wild-type mice, the number of S100⁺ astrocytes was lower in the ipsilateral compared to contralateral hemisphere (65.0±50.1 vs. 85.6±34.0, p<0.05). In the GFAP^−/−Vim^−/− mice, the number of S100⁺ astrocytes did not differ between the ischemic and contralateral hemisphere at P31. At P31, GFAP^−/−Vim^−/− mice showed an increase in NeuN⁺BrdU⁺ (surviving newly born) neurons in the ischemic cortex compared to wild-type mice (6.7±7.7; n = 29 versus 2.9±3.6; n = 28, respectively, p<0.05), but a comparable number of S100⁺BrdU⁺ (surviving newly born) astrocytes.

Conclusions/Significance

Our results suggest that attenuation of reactive gliosis in the developing brain does not affect the hemisphere or infarct volume after HI, but increases the number of surviving newborn neurons.     

The Adhesion-Associated sca Operon in Streptococcus gordonii Encodes an Inducible High-Affinity ABC Transporter for Mn2+ Uptake

ScaA lipoprotein in Streptococcus gordonii is a member of the LraI family of homologous polypeptides found among streptococci, pneumococci, and enterococci. It is the product of the third gene within the scaCBA operon encoding the components of an ATP-binding cassette (ABC) transporter system. Inactivation of scaC (ATP-binding protein) or scaA (substrate-binding protein) genes resulted in both impaired growth of cells and >70% inhibition of ⁵⁴Mn²⁺ uptake in media containing <0.5 μM Mn²⁺. In wild-type and scaC mutant cells, production of ScaA was induced at low concentrations of extracellular Mn²⁺ (<0.5 μM) and by the addition of ≥20 μM Zn²⁺. Sca permease-mediated uptake of ⁵⁴Mn²⁺ was inhibited by Zn²⁺ but not by Ca²⁺, Mg²⁺, Fe²⁺, or Cu²⁺. Reduced uptake of ⁵⁴Mn²⁺ by sca mutants and by wild-type cells in the presence of Zn²⁺ was abrogated by the uncoupler carbonylcyanide m-chlorophenylhydrazone, suggesting that Mn²⁺ uptake under these conditions was proton motive force dependent. The frequency of DNA-mediated transformation was reduced >20-fold in sca mutants. The addition of 0.1 mM Mn²⁺ to the transformation medium restored only partly the transformability of mutant cells, implying an alternate role for Sca proteins in the transformation process. Cells of sca mutants were unaffected in other binding properties tested and were unaffected in sensitivity to oxidants. The results show that Sca permease is a high-affinity mechanism for the acquisition of Mn²⁺ and is essential for growth of streptococci under Mn²⁺-limiting conditions.     

Scaffolding protein Homer1a protects against NMDA-induced neuronal injury

Excessive N-methyl-D-aspartate receptor (NMDAR) activation and the resulting activation of neuronal nitric oxide synthase (nNOS) cause neuronal injury. Homer1b/c facilitates NMDAR-PSD95-nNOS complex interactions, and Homer1a is a negative competitor of Homer1b/c. We report that Homer1a was both upregulated by and protected against NMDA-induced neuronal injury in vitro and in vivo. The neuroprotective activity of Homer1a was associated with NMDA-induced Ca²⁺ influx, oxidative stress and the resultant downstream signaling activation. Additionally, we found that Homer1a functionally regulated NMDAR channel properties in neurons, but did not regulate recombinant NR1/NR2B receptors in HEK293 cells. Furthermore, we found that Homer1a detached the physical links among NR2B, PSD95 and nNOS and reduced the membrane distribution of NMDAR. NMDA-induced neuronal injury was more severe in Homer1a homozygous knockout mice (KO, Homer1a^−/−) when compared with NMDA-induced neuronal injury in wild-type mice (WT, Homer1a^+/+). Additionally, Homer1a overexpression in the cortex of Homer1a^−/− mice alleviated NMDA-induced neuronal injury. These findings suggest that Homer1a may be a key neuroprotective endogenous molecule that protects against NMDA-induced neuronal injury by disassembling NR2B-PSD95-nNOS complexes and reducing the membrane distribution of NMDARs.Glutamate (Glu) acts on glutamate receptors, such as the N-methyl-D-aspartate receptor (NMDAR), and leads to neuronal hyper-excitability and death in a dose-dependent manner.¹ NMDAR activation induces Ca²⁺ influx and specifically activates neuronal nitric oxide synthase (nNOS) and downstream signaling pathways.^{2, 3, 4} Ca²⁺ influx is involved in glutamate-induced apoptosis caused by the activation of apoptosis-related signaling pathways, mitochondrial dysfunction and ROS induction.^{3, 4} Additionally, nNOS has been reported to contribute to NMDA-induced excitotoxicity.^{5, 6} Considering that direct NMDAR inhibition has not yet demonstrated favorable efficacy in most clinic trails and further considering the remarkable role of nNOS in NMDA-induced neuronal death,⁷ measures that can effectively protect neurons from NMDA-induced neuronal injury are urgently needed and represent a worthwhile research goal.Homer proteins belong to the postsynaptic density (PSD) family and consist of two major groups: the short-form Homer proteins (Homer1a and Ania3) and the long-form Homer proteins (Homer1b/c, Homer2 and Homer3).⁸ Homer1b/c has a conserved N-terminal Ena/VASP homology 1 domain and binds to group I metabotropic glutamate receptors (mGluRs), inositol triphosphate receptors and Shank family proteins.^{9, 10, 11, 12} Homer1b/c regulates surface receptor expression,^{13, 14} clustering,¹⁵ transient receptor potential family channels and mGluRs coupled to ion channels.^{10, 16, 17, 18, 19} Additionally, because of its C-terminal coiled-coil (CC) domains, Homer1b/c can self-multimerize, form multiprotein complexes and facilitate signal transduction to downstream pathways. Homer1a, which lacks the CC domain, is believed to compete with constitutive Homer1b/c and disrupt the association of multiple Homer1b/c complexes.Notably, Homer1b/c can interact with the Glu-induced Ca²⁺ influx pathway by binding to Shank, a NMDAR complex adaptor protein (NMDAR-PSD95-GKAP-Shank-Homer1b/c).^{12, 20} Furthermore, Homer1a also interacts with Shank, NMDA, nNOS and other Homer1b/c target proteins. Homer1a has a negative regulatory role by physically replacing certain target proteins, and is involved in the regulation of a variety of cellular and molecular functions in neurological diseases.^{21, 22, 23, 24, 25} Nevertheless, the mechanisms of action and associations between Homer1a and NMDA-induced neuronal injury have not yet been studied. Here, we aimed to investigate the possible neuroprotective effects of Homer1a and explore the mechanisms underlying Homer1a activity in NMDA-induced neuronal injury.     

Modulation of the Local SR Ca2+ Release by Intracellular Mg2+ in Cardiac Myocytes

In cardiac muscle, Ca²⁺-induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) defines the amplitude and time course of the Ca²⁺ transient. The global elevation of the intracellular Ca²⁺ concentration arises from the spatial and temporal summation of elementary Ca²⁺ release events, Ca²⁺ sparks. Ca²⁺ sparks represent the concerted opening of a group of ryanodine receptors (RYRs), which are under the control of several modulatory proteins and diffusible cytoplasmic factors (e.g., Ca²⁺, Mg²⁺, and ATP). Here, we examined by which mechanism the free intracellular Mg²⁺ ([Mg²⁺]_free) affects various Ca²⁺ spark parameters in permeabilized mouse ventricular myocytes, such as spark frequency, duration, rise time, and full width, at half magnitude and half maximal duration. Varying the levels of free ATP and Mg²⁺ in specifically designed solutions allowed us to separate the inhibition of RYRs by Mg²⁺ from the possible activation by ATP and Mg²⁺-ATP via the adenine binding site of the channel. Changes in [Mg²⁺]_free generally led to biphasic alterations of the Ca²⁺ spark frequency. For example, lowering [Mg²⁺]_free resulted in an abrupt increase of spark frequency, which slowly recovered toward the initial level, presumably as a result of SR Ca²⁺ depletion. Fitting the Ca²⁺ spark inhibition by [Mg²⁺]_free with a Hill equation revealed a K_i of 0.1 mM. In conclusion, our results support the notion that local Ca²⁺ release and Ca²⁺ sparks are modulated by Mg²⁺ in the intracellular environment. This seems to occur predominantly by hindering Ca²⁺-dependent activation of the RYRs through competitive Mg²⁺ occupancy of the high-affinity activation site of the channels. These findings help to characterize CICR in cardiac muscle under normal and pathological conditions, where the levels of Mg²⁺ and ATP can change.     

Ligand-independent exhaustion of killer immunoglobulin-like receptor-positive CD8+ T cells in human immunodeficiency virus type 1 infection

Virus-specific CD8⁺ T cells play a central role in the control of viral infections, including human immunodeficiency virus type 1 (HIV-1) infection. However, despite the presence of strong and broad HIV-specific CD8⁺ T-cell responses in chronic HIV-1 infection, these cells progressively lose critical effector functions and fail to clear the infection. Mounting evidence suggests that the upregulation of several inhibitory regulatory receptors on the surface of CD8⁺ T cells during HIV-1 infection may contribute directly to the impairment of T-cell function. Here, we investigated the role of killer immunoglobulin receptors (KIR), which are expressed on NK cells and on CD8⁺ T cells, in regulating CD8⁺ T-cell function in HIV-1 infection. KIR expression was progressively upregulated on CD8⁺ T cells during HIV-1 infection and correlated with the level of viral replication. Expression of KIR was associated with a profound inhibition of cytokine secretion, degranulation, proliferation, and activation by CD8⁺ T cells following stimulation with T-cell receptor (TCR)-dependent stimuli. In contrast, KIR⁺ CD8⁺ T cells responded potently to TCR-independent stimulation, demonstrating that these cells are functionally competent. KIR-associated suppression of CD8⁺ T-cell function was independent of ligand engagement, suggesting that these regulatory receptors may constitutively repress TCR activation. This ligand-independent repression of TCR activation of KIR⁺ CD8⁺ T cells may represent a significant barrier to therapeutic interventions aimed at improving the quality of the HIV-specific CD8⁺ T-cell response in infected individuals.