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1. MitoBK Ca channel is functionally associated with its regulatory β1 subunit in cardiac mitochondria

   https://doi.org/10.1113/JP277769  

   Balderas, Enrique ; Torres, Natalia S. ; Rosa‐Garrido, Manuel ; Chaudhuri, Dipayan ; Toro, Ligia ; Stefani, Enrico ; Olcese, Riccardo ( July 2019 , The Journal of Physiology)

   Association of plasma membrane BK_Cachannels with BK‐β subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BK_Cachannel (mitoBK_Ca) by BK‐β subunits is not established.

   MitoBK_Ca‐α and the regulatory BK‐β1 subunit associate in mouse cardiac mitochondria.

   A large fraction of mitoBK_Cadisplay properties similar to that of plasma membrane BK_Cawhen associated with BK‐β1 (left‐shifted voltage dependence of activation,V_1/2 = −55 mV, 12 µmmatrix Ca²⁺).

   In BK‐β1 knockout mice, cardiac mitoBK_Cadisplayed a lowP_oand a depolarizedV_1/2of activation (+47 mV at 12 µmmatrix Ca²⁺)

   Co‐expression of BK_Cawith the BK‐β1 subunit in HeLa cells doubled the density of BK_Cain mitochondria.

   The present study supports the view that the cardiac mitoBK_Cachannel is functionally modulated by the BK‐β1 subunit; proper targeting and activation of mitoBK_Cashapes mitochondrial Ca²⁺handling.

   Association of the plasma membrane BK_Cachannel with auxiliary BK‐β1–4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBK_Ca) with regulatory subunits is unknown. We report that mitoBK_Cafunctionally associates with its regulatory subunit BK‐β1 in adult rodent cardiomyocytes. Cardiac mitoBK_Cais a calcium‐ and voltage‐activated channel that is sensitive to paxilline with a large conductance for K⁺of 300 pS. Additionally, mitoBK_Cadisplays a high open probability (P_o) and voltage half‐activation (V_1/2 = −55 mV,n = 7) resembling that of plasma membrane BK_Cawhen associated with its regulatory BK‐β1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BK_Ca‐α and its BK‐β1 subunit. Mitochondria from the BK‐β1 knockout (KO) mice showed sparse mitoBK_Cacurrents (five patches with mitoBK_Caactivity out of 28 total patches fromn = 5 different hearts), displaying a depolarizedV_1/2of activation (+47 mV in 12 µmmatrix Ca²⁺). The reduced activity of mitoBK_Cawas accompanied by a high expression of BK_Catranscript in the BK‐β1 KO, suggesting a lower abundance of mitoBK_Cachannels in this genotype. Accordingly, BK‐β1subunit increased the localization of BKDEC (i.e. the splice variant of BK_Cathat specifically targets mitochondria) into mitochondria by two‐fold. Importantly, both paxilline‐treated and BK‐β1 KO mitochondria displayed a more rapid Ca²⁺overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBK_Caassociates with its regulatory BK‐β1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBK_Cachannel that helps to maintain mitochondrial Ca²⁺homeostasis.

    

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2. Functional and pharmacological properties of triheteromeric GluN1/2B/2D NMDA receptors

   https://doi.org/10.1113/JP278168  

   Yi, Feng ; Bhattacharya, Subhrajit ; Thompson, Charles M. ; Traynelis, Stephen F. ; Hansen, Kasper B. ( November 2019 , The Journal of Physiology)

   Triheteromeric NMDA receptors contain two GluN1 and two distinct GluN2 subunits and mediate excitatory neurotransmission in the CNS.

   Triheteromeric GluN1/2B/2D receptors have functional properties intermediate to those of diheteromeric GluN1/2B and GluN1/2D receptors.

   GluN1/2B/2D receptors are more sensitive to channel blockade by ketamine and memantine compared to GluN1/2B receptors in the presence of physiological Mg²⁺.

   GluN2B‐selective antagonists produce robust inhibition of GluN1/2B/2D receptors, and the GluN2B‐selective positive allosteric modulator spermine enhances responses from GluN1/2B/2D but not GluN1/2A/2B receptors.

   These insights into the properties of triheteromeric GluN1/2B/2D receptors are necessary to appreciate their physiological roles in neural circuit function and the actions of therapeutic agents targeting NMDA receptors.

   Triheteromeric NMDA‐type glutamate receptors that contain two GluN1 and two different GluN2 subunits contribute to excitatory neurotransmission in the adult CNS. In the present study, we report properties of the triheteromeric GluN1/2B/2D NMDA receptor subtype that is expressed in distinct neuronal populations throughout the CNS. We show that neither GluN2B, nor GluN2D dominate the functional properties of GluN1/2B/2D receptors because agonist potencies, open probability and the glutamate deactivation time course of GluN1/2B/2D receptors are intermediate to those of diheteromeric GluN1/2B and GluN1/2D receptors. Furthermore, channel blockade of GluN1/2B/2D by extracellular Mg²⁺is intermediate compared to GluN1/2B and GluN1/2D, although GluN1/2B/2D is more sensitive to blockade by ketamine and memantine compared to GluN1/2B in the presence of physiological Mg²⁺. Subunit‐selective allosteric modulators have distinct activity at GluN1/2B/2D receptors, including GluN2B‐selective antagonists, ifenprodil, EVT‐101 and CP‐101‐606, which inhibit with similar potencies but with different efficacies at GluN1/2B/2D (∼65% inhibition) compared to GluN1/2B (∼95% inhibition). Furthermore, the GluN2B‐selective positive allosteric modulator spermine enhances responses from GluN1/2B/2D but not GluN1/2A/2B receptors. We show that these key features of allosteric modulation of recombinant GluN1/2B/2D receptors are also observed for NMDA receptors in hippocampal interneurons but not CA1 pyramidal cells, which is consistent with the expression of GluN1/2B/2D receptors in interneurons and GluN1/2A/2B receptors in pyramidal cells. Altogether, we uncover previously unknown functional and pharmacological properties of triheteromeric GluN1/2B/2D receptors that can facilitate advances in our understanding of their physiological roles in neural circuit function and therapeutic drug actions.

    

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3. A mouse model of seizures in anti– N ‐methyl‐ d ‐aspartate receptor encephalitis

   https://doi.org/10.1111/epi.14662  

   Taraschenko, Olga ; Fox, Howard S. ; Pittock, Sean J. ; Zekeridou, Anastasia ; Gafurova, Maftuna ; Eldridge, Ember ; Liu, Jinxu ; Dravid, Shashank M. ; Dingledine, Raymond ( February 2019 , Epilepsia)

   Seizures develop in 80% of patients with anti–N‐methyl‐d‐aspartate receptor (NMDAR) encephalitis, and these represent a major cause of morbidity and mortality. Anti‐NMDAR antibodies have been linked to memory loss in encephalitis; however, their role in seizures has not been established. We determined whether anti‐NMDAR antibodies from autoimmune encephalitis patients are pathogenic for seizures.

   We performed continuous intracerebroventricular infusion of cerebrospinal fluid (CSF) or purified immunoglobulin (IgG) from the CSF of patients with anti‐NMDAR encephalitis or polyclonal rabbit anti‐NMDAR IgG, in male C57BL/6 mice. Seizure status during a 2‐week treatment was assessed with video‐electroencephalography. We assessed memory, anxiety‐related behavior, and motor function at the end of treatment and assessed the extent of neuronal damage and gliosis in the CA1 region of hippocampus. We also performed whole‐cell patch recordings from the CA1 pyramidal neurons in hippocampal slices of mice with seizures.

   Prolonged exposure to rabbit anti‐NMDAR IgG, patient CSF, or human IgG purified from the CSF of patients with encephalitis induced seizures in 33 of 36 mice. The median number of seizures recorded in 2 weeks was 13, 39, and 35 per mouse in these groups, respectively. We observed only 18 brief nonconvulsive seizures in 11 of 29 control mice (median seizure count of 0) infused with vehicle (n = 4), normal CSF obtained from patients with noninflammatory central nervous system (CNS) conditions (n = 12), polyclonal rabbit IgG (n = 7), albumin (n = 3), and normal human IgG (n = 3). We did not observe memory deficits, anxiety‐related behavior, or motor impairment measured at 2 weeks in animals treated with CSF from affected patients or rabbit IgG. Furthermore, there was no evidence of hippocampal cell loss or astrocyte proliferation in the same mice.

   Our findings indicate that autoantibodies can induce seizures in anti‐NMDAR encephalitis and offer a model for testing novel therapies for refractory autoimmune seizures.

    

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4. Advanced age protects resistance arteries of mouse skeletal muscle from oxidative stress through attenuating apoptosis induced by hydrogen peroxide

   https://doi.org/10.1113/JP278255  

   Norton, Charles E. ; Sinkler, Shenghua Y. ; Jacobsen, Nicole L. ; Segal, Steven S. ( July 2019 , The Journal of Physiology)

   Vascular oxidative stress increases with advancing age.

   We hypothesized that resistance vessels develop resilience to oxidative stress to protect functional integrity and tested this hypothesis by exposing isolated pressurized superior epigastric arteries (SEAs) of old and young mice to H₂O₂.

   H₂O₂‐induced death was greater in smooth muscle cells (SMCs) than endothelial cells (ECs) and lower in SEAs from oldvs. young mice; the rise in vessel wall [Ca²⁺]_iinduced by H₂O₂was attenuated with ageing, as was the decline in noradrenergic vasoconstriction; genetic deletion of IL‐10 mimicked the effects of advanced age on cell survival.

   Inhibiting NO synthase or scavenging peroxynitrite reduced SMC death; endothelial denudation or inhibiting gap junctions increased SMC death; delocalization of cytochrome C activated caspases 9 and 3 to induce apoptosis.

   Vascular cells develop resilience to H₂O₂during ageing by preventing Ca²⁺overload and endothelial integrity promotes SMC survival.

   Advanced age is associated with elevated oxidative stress and can protect the endothelium from cell death induced by H₂O₂. Whether such protection occurs for intact vessels or differs between smooth muscle cell (SMC) and endothelial cell (EC) layers is unknown. We tested the hypothesis that ageing protects SMCs and ECs during acute exposure to H₂O₂(200 µm, 50 min). Mouse superior epigastric arteries (SEAs; diameter, ∼150 µm) were isolated and pressurized to 100 cmH₂O at 37˚C. For SEAs from young (4 months) mice, H₂O₂killed 57% of SMCs and 11% of ECs in malesvs. 8% and 2%, respectively, in females. Therefore, SEAs from males were studied to resolve the effect of ageing and experimental interventions. For old (24 months) mice, SMC death was reduced to 10% with diminished accumulation of [Ca²⁺]_iin the vessel wall during H₂O₂exposure. In young mice, genetic deletion of IL‐10 mimicked the protective effect of ageing on cell death and [Ca²⁺]_iaccumulation. Whereas endothelial denudation or gap junction inhibition (carbenoxolone; 100 µm) increased SMC death, inhibiting NO synthase (l‐NAME, 100 µm) or scavenging peroxynitrite (FeTPPS, 5 µm) reduced SMC death along with [Ca²⁺]_i. Despite NO toxicity via peroxynitrite formation, endothelial integrity protects SMCs. Caspase inhibition (Z‐VAD‐FMK, 50 µm) attenuated cell death with immunostaining for annexin V, cytochrome C, and caspases 3 and 9 pointing to induction of intrinsic apoptosis during H₂O₂exposure. We conclude that advanced age reduces Ca²⁺influx that triggers apoptosis, thereby promoting resilience of the vascular wall during oxidative stress.

    

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5. Activation of nicotinic acetylcholine receptors induces potentiation and synchronization within in vitro hippocampal networks

   https://doi.org/10.1111/jnc.14938  

   Djemil, Sarra ; Chen, Xin ; Zhang, Ziyue ; Lee, Jisoo ; Rauf, Mikael ; Pak, Daniel T. S. ; Dzakpasu, Rhonda ( December 2019 , Journal of Neurochemistry)

   Nicotinic acetylcholine receptors (nAChRs) are known to play a role in cognitive functions of the hippocampus, such as memory consolidation. Given that they conduct Ca²⁺and are capable of regulating the release of glutamate and γ‐aminobutyric acid (GABA) within the hippocampus, thereby shifting the excitatory‐inhibitory ratio, we hypothesized that the activation of nAChRs will result in the potentiation of hippocampal networks and alter synchronization. We used nicotine as a tool to investigate the impact of activation of nAChRs on neuronal network dynamics in primary embryonic rat hippocampal cultures prepared from timed‐pregnant Sprague‐Dawley rats. We perturbed cultured hippocampal networks with increasing concentrations of bath‐applied nicotine and performed network extracellular recordings of action potentials using a microelectrode array. We found that nicotine modulated network dynamics in a concentration‐dependent manner; it enhanced firing of action potentials as well as facilitated bursting activity. In addition, we used pharmacological agents to determine the contributions of discrete nAChR subtypes to the observed network dynamics. We found that β4‐containing nAChRs are necessary for the observed increases in spiking, bursting, and synchrony, while the activation of α7 nAChRs augments nicotine‐mediated network potentiation but is not necessary for its manifestation. We also observed that antagonists of N‐methyl‐D‐aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs) partially blocked the effects of nicotine. Furthermore, nicotine exposure promoted autophosphorylation of Ca²⁺/calmodulin‐dependent kinase II (CaMKII) and serine 831 phosphorylation of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPAR) subunit GluA1. These results suggest that nicotinic receptors induce potentiation and synchronization of hippocampal networks and glutamatergic synaptic transmission. Findings from this work highlight the impact of cholinergic signaling in generating network‐wide potentiation in the form of enhanced spiking and bursting dynamics that coincide with molecular correlates of memory such as increased phosphorylation of CaMKII and GluA1.

   This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. More information about the Open Practices badges can be found athttps://cos.io/our-services/open-science-badges/

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