scholarly journals Desensitization of NMDA receptors depends of their association with plasma membrane sodium-calcium exchangers in lipid nanoclasters

2018 ◽  
Author(s):  
Dmitry A. Sibarov ◽  
Ekaterina E. Poguzhelskaya ◽  
Sergei M. Antonov
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Cortical Neurons ◽  
External Solution ◽  
Membrane Lipid ◽  
Before And After ◽  
Rat Cortical Neurons ◽  
Amplitude Decrease

AbstractThe plasma membrane Na+/Ca2+-exchanger (NCX) has recently been shown to regulate Ca2+-dependent N-methyl-d-aspartate receptor (NMDAR) desensitization, suggesting tight interaction of NCXs and NMDARs in lipid nanoclaster or “rafts”. To evaluate possible role of this interaction we studied effects of Li+ on NMDA-elicited whole-cell currents and Ca2+ responses of rat cortical neurons in vitro before and after cholesterol extraction by methyl-β-cyclodextrin (MβCD). Substitution Li+ for Na+ in the external solution caused a concentration-dependent decrease of steady-state NMDAR currents from 440 ± 71 pA to 111 ± 29 pA in 140 mM Na+ and 140 mM Li+, respectively. Li+ inhibition of NMDAR currents disappeared in the absence of Ca2+ in the external solution (Ca2+-free), suggesting that Li+ enhanced Ca2+-dependent NMDAR desensitization. Whereas the cholesterol extraction with MβCD induced NMDAR current decrease to 136 ± 32 pA in 140 mM Na+ and 46 ± 15 pA in 140 mM Li+, the IC50 values for the Li+ inhibition were similar (about 44 mM Li+) before and after this procedure. In Ca2+-free Na+ solution steady-state NMDAR currents after the cholesterol extraction were 47 ± 6 % of control currents. Apparently this amplitude decrease was not Ca2+-dependent. In 1 mM Ca2+ Na+ solution the Ca2+-dependent NMDAR desensitization was greater when cholesterol was extracted. Obviously, this procedure promoted its development. In agreement, Li+ and KB-R7943, an inhibitor of NCX, both considerably reduced NMDAR-mediated Ca2+ responses. The cholesterol extraction itself caused a decrease of NMDAR-mediated Ca2+ responses and, in addition, abolished the effects of Li+ and KB-R7943. Taken together our data suggest that NCXs downregulate the Ca2+-dependent NMDAR desensitization. Most likely, this is determined by co-localization and tight functional interaction of NCX and NMDAR molecules in membrane lipid rafts. Their destruction is accompanied by an enhancement of NMDAR desensitization and a loss of NCX-selective agent effects on NMDARs.

scholarly journals Regulation of Intracellular pH in Single Rat Cortical Neurons in vitro: A Microspectrofluorometric Study

1993 ◽  
Vol 13 (5) ◽  
pp. 827-840 ◽  
Author(s):  
Yibing Ou-Yang ◽  
Pekka Mellergård ◽  
Bo K. Siesjö
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Cortical Neurons ◽  
Ph Sensitive ◽  
Disulfonic Acid ◽  
Extrusion Rate ◽  
Rat Cortical Neurons ◽  
Acid Extrusion

Intracellular pH (pHi) and the mechanisms of pHi regulation in cultured rat cortical neurons were studied with microspectrofluorometry and the pH-sensitive fluorophore 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady-state pHi was 7.00 ± 0.17 (mean ± SD) and 7.09 ± 0.14 in nominally HCO3− -free and HCO3−-containing solutions, respectively, and was dependent on extracellular Na+ and Cl−. Following an acid transient, induced by an NH1 prepulse or an increase in CO2 tension, pHi decreased and then rapidly returned to baseline, with an average net acid extrusion rate of 2.6 and 2.8 mmol/L/min, in nominally HCO3− -free and HCO3− -containing solutions, respectively. The recovery was completely blocked by removal of extracellular Na+ and was partially inhibited by amiloride or 5- N-methyl- N-isobutylamiloride. In most cells pHi recovery was completely blocked in the presence of harmaline. The recovery of pHi was not influenced by addition of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) or removal of Cl−. The rapid regulation of pHi seen following a transient alkalinization was not inhibited by amiloride or by removal of extracellular Na+, but was partially inhibited by DIDS and by removal of extracellular Cl−. The results are compatible with the presence of at least two different pHi-regulating mechanisms: an acid-extruding Na+/H+ antiporter, possibly consisting of different subtypes, and a passive Cl−/HCO3− exchanger, mediating loss of HCO3− from the cell.

Cannabis-like activity of Zornia latifolia Sm. detected in vitro on rat cortical neurons: major role of the flavone syzalterin

2020 ◽  
pp. 1-13
Author(s):  
Susanna Alloisio ◽  
Marco Clericuzio ◽  
Mario Nobile ◽  
Annalisa Salis ◽  
Gianluca Damonte ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Rat Cortical Neurons

scholarly journals Astrocytes Exhibit a Protective Role in Neuronal Firing Patterns under Chemically Induced Seizures in Neuron–Astrocyte Co-Cultures

2021 ◽  
Vol 22 (23) ◽  
pp. 12770
Author(s):  
Annika Ahtiainen ◽  
Barbara Genocchi ◽  
Jarno M. A. Tanskanen ◽  
Michael T. Barros ◽  
Jari A. K. Hyttinen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Microelectrode Arrays ◽  
Cell Types ◽  
Protective Role ◽  
Neuronal Firing ◽  
Neuronal Homeostasis ◽  
Chemically Induced ◽  
Rat Cortical Neurons

Astrocytes and neurons respond to each other by releasing transmitters, such as γ-aminobutyric acid (GABA) and glutamate, that modulate the synaptic transmission and electrochemical behavior of both cell types. Astrocytes also maintain neuronal homeostasis by clearing neurotransmitters from the extracellular space. These astrocytic actions are altered in diseases involving malfunction of neurons, e.g., in epilepsy, Alzheimer’s disease, and Parkinson’s disease. Convulsant drugs such as 4-aminopyridine (4-AP) and gabazine are commonly used to study epilepsy in vitro. In this study, we aim to assess the modulatory roles of astrocytes during epileptic-like conditions and in compensating drug-elicited hyperactivity. We plated rat cortical neurons and astrocytes with different ratios on microelectrode arrays, induced seizures with 4-AP and gabazine, and recorded the evoked neuronal activity. Our results indicated that astrocytes effectively counteracted the effect of 4-AP during stimulation. Gabazine, instead, induced neuronal hyperactivity and synchronicity in all cultures. Furthermore, our results showed that the response time to the drugs increased with an increasing number of astrocytes in the co-cultures. To the best of our knowledge, our study is the first that shows the critical modulatory role of astrocytes in 4-AP and gabazine-induced discharges and highlights the importance of considering different proportions of cells in the cultures.

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

scholarly journals 209FUNCTIONAL ASSESSMENT OF WHITE RHINOCEROS CERATHOTERIUM SIMUM EPIDIDYMAL SPERMATOZOA BEFORE AND AFTER CRYOPRESERVATION

2004 ◽  
Vol 16 (2) ◽  
pp. 226 ◽  
Author(s):  
F. Martinez-Pastor ◽  
F. Olivier ◽  
T. Spies ◽  
L. Anel ◽  
P. Bartels
Keyword(s):  
Plasma Membrane ◽  
Assisted Reproduction ◽  
Phase Contrast ◽  
Egg Yolk ◽  
Phase Contrast Microscopy ◽  
White Rhinoceros ◽  
Contrast Microscopy ◽  
Before And After ◽  
Epididymal Spermatozoa

Biological Resource Banks represent a potentially valuable tool for species conservation. It is, however, necessary to understand the species-specific cryopreservation process and its consequences for spermatozoa to aid in the development of assisted reproduction as a future conservation tool. The aim of this study was to assess the in vitro functionality of white rhinoceros Cerathoterium simum epididymal spermatozoa both before and after cryopreservation. Testes from a harvested white rhino bull were removed and transported at 5°C to the laboratory within 4h. The cauda epididymis was dissected out and flushed with 2mL of Tris-citrate egg yolk extender (fraction A, Biladyl, Minitüb, Germany). A 0.1mL aliquot was removed for analysis and the balance (9mL; 2mL fraction A+7mL sperm sample) mixed with an additional 27.2mL of Tris-citrate egg yolk with glycerol (fraction B, Bidadyl). The extended sample was allowed to cool to 4°C over a 6-h period before an additional 29.2mL of cooled fraction B were added (final sperm concentration=150×106mL−1). Sperm samples were loaded into 0.25-mL straws and frozen over LN2 vapor (4cm for 20min) for later assessment. Sperm straws were thawed by placing the straws in water at 37°C for 30s. Pre-freeze and post-thaw evaluations were carried out in the same manner. Media used included: HEPES for washing (20mM HEPES, 355mM sucrose, 10mM glucose, 2.5mM KOH) and HEPES saline (197mM NaCl, instead of sucrose). An aliquot was diluted with HEPES (washing) and centrifuged for 5min at 600×g; the pellet was resuspended in HEPES saline. Sperm motility (total motility %, TM;; and progressive motility %, PM) was assessed using phase contrast microscopy (×200; 37°C). Sperm plasma membrane status was assessed using the fluorescent dye, propidium iodide (50ngmL−1 in HEPES saline;; 10min, RT). Percentage of cells with plasma membranes intact (unstained;; PMI) was recorded. Mitochondrial status was assessed with the fluorescent dye, JC-1 (7.5μM in HEPES saline;; 30min, 37°C). The % of cells with an orange-stained midpiece was recorded (active mitochondria;; MIT). Resilience to hypoosmotic shock (HOS test) was assessed by diluting a sample in 100mOsm/kg HEPES saline (1:20; 15min, RT). An aliquot was stained with PI to assess plasma membrane status (HOSPMI), and the rest was fixed with formaldehyde, and % coiled tails (positive endosmosis;; HOST) was estimated using phase contrast microscopy (×400). Evaluations of PMI, MIT and HOSPMI were performed using fluorescence microscopy (×400, 450–490nm excitation filter). The results indicated that quality was good pre-freezing (TM: 60%; PMI: 86%; MIT: 100%), except for a PM value of 15%. After thawing, although there was a drop in TM (30%), there was no decrease in PM (20%). Our in vitro functional assessment indicated a loss of quality between the pre-freeze and post-thaw evaluations, but PMI and MIT maintained their pre-thaw levels (60% and 72%, respectively). The HOS test, which indicates plasma membrane integrity, decreased from the pre-freeze level (91%) to a post-thaw value of 70%. HOSTPMI was 72% pre-freeze, and decreased to 54% post-thaw. In conclusion, epididymal spermatozoa from the white rhino may retain its functionality after cryopreservation in a commerically available cryo-extender (Bidadyl). The use of assisted reproduction techniques could someday play a role in the management and conservation of the white rhinoceros and related species.

scholarly journals Role of Ca2+ in pyruvate dehydrogenase interconversion in brain mitochondria and synaptosomes

1985 ◽  
Vol 227 (1) ◽  
pp. 129-136 ◽  
Author(s):  
R G Hansford ◽  
F Castro
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Pyruvate Dehydrogenase ◽  
Maximal Activity ◽  
Brain Mitochondria ◽  
Ruthenium Red ◽  
Ion Concentration ◽  
Maximal Effect ◽  
State Content

The steady-state content of active (dephospho) pyruvate dehydrogenase (PDHA) of suspensions of coupled rat brain mitochondria oxidizing succinate was found to be markedly increased with increasing free Ca2+ ion concentration of the medium, with a half-maximal effect at 10(-6.43) M Ca2+. Other ions were present in these studies at concentrations appropriate for the cytosol. Depolarization of the plasma membrane of synaptosomes caused an increase in the steady-state content of PDHA, with veratridine giving a larger increase than depolarization by 33 mM-KCl. Values were 68 +/- 1% (n = 13) and 81 +/- 1% (n = 19) of maximal activity, for control incubations and incubations in the presence of 30 microM-veratridine, respectively. Measurements of cytosolic free Ca2+ concentrations ([Ca2+]cyt.) in these suspensions of synaptosomes, with the use of the fluorescent Ca2+-indicator Quin-2, indicated an increase on depolarization, with the change due to 30 microM-veratridine being larger in extent than that due to 33 mM-KCl. Values were 217 +/- 21 nM (n = 15), 544 +/- 48 nM (n = 15) and 783 +/- 75 nM (n = 14) for control, KCl-depolarized and veratridine-depolarized synaptosomes respectively. Experiments in which synaptosomes were treated with Ruthenium Red, an inhibitor of mitochondrial Ca2+ uptake, gave much lower resting contents of PDHA (42 +/- 2% of maximal), but failed to prevent totally an increase on depolarization. Addition of an excess of EGTA to the synaptosomal suspension just before the addition of veratridine resulted in a partial diminution in the response of PDHA content. Parallel studies with Quin-2 indicated no increase in [Ca2+]cyt. on addition of veratridine, under these conditions. Thus an increase in [Ca2+]cyt. forms only a part of the mechanism whereby pyruvate dehydrogenase interconversion responds to depolarization. A decrease in the ATP/ADP ratio may also be important, as inferred from the results of experiments with ouabain, which inhibits the Na+ + K+-dependent ATPase.

Sensory regulation of quadrupedal locomotion: a top-down or bottom-up control system?

2012 ◽  
Vol 108 (3) ◽  
pp. 709-711 ◽  
Author(s):  
Yann Thibaudier ◽  
Marie-France Hurteau
Keyword(s):  
Control System ◽  
Central Pattern Generators ◽  
Top Down ◽  
Bottom Up ◽  
Sensory Inputs ◽  
Sensory Regulation ◽  
Before And After ◽  
Pattern Generators

Propriospinal pathways are thought to be critical for quadrupedal coordination by coupling cervical and lumbar central pattern generators (CPGs). However, the mechanisms involved in relaying information between girdles remain largely unexplored. Using an in vitro spinal cord preparation in neonatal rats, Juvin and colleagues ( Juvin et al. 2012 ) have recently shown sensory inputs from the hindlimbs have greater influence on forelimb CPGs than forelimb sensory inputs on hindlimb CPGs, in other words, a bottom-up control system. However, results from decerebrate cats suggest a top-down control system. It may be that both bottom-up and top-down control systems exist and that the dominance of one over the other is task or context dependent. As such, the role of sensory inputs in controlling quadrupedal coordination before and after injury requires further investigation.

scholarly journals Purinergic Modulation of Interleukin-1β Release from Microglial Cells Stimulated with Bacterial Endotoxin

1997 ◽  
Vol 185 (3) ◽  
pp. 579-582 ◽  
Author(s):  
Davide Ferrari ◽  
Paola Chiozzi ◽  
Simonetta Falzoni ◽  
Stefania Hanau ◽  
Francesco Di  Virgilio
Keyword(s):  
Plasma Membrane ◽  
P2x7 Receptor ◽  
Purinergic Receptor ◽  
Present Report ◽  
Physiological Role ◽  
Bacterial Endotoxin ◽  
Microglial Cells

Microglial cells express a peculiar plasma membrane receptor for extracellular ATP, named P2Z/P2X7 purinergic receptor, that triggers massive transmembrane ion fluxes and a reversible permeabilization of the plasma membrane to hydrophylic molecules of up to 900 dalton molecule weight and eventual cell death (Di Virgilio, F. 1995. Immunol. Today. 16:524–528). The physiological role of this newly cloned (Surprenant, A., F. Rassendren, E. Kawashima, R.A. North and G. Buell. 1996. Science (Wash. DC). 272:735–737) cytolytic receptor is unknown. In vitro and in vivo activation of the macrophage and microglial cell P2Z/P2X7 receptor by exogenous ATP causes a large and rapid release of mature IL-1β. In the present report we investigated the role of microglial P2Z/P2X7 receptor in IL-1β release triggered by LPS. Our data suggest that LPS-dependent IL-1β release involves activation of this purinergic receptor as it is inhibited by the selective P2Z/P2X7 blocker oxidized ATP and modulated by ATP-hydrolyzing enzymes such as apyrase or hexokinase. Furthermore, microglial cells release ATP when stimulated with LPS. LPS-dependent release of ATP is also observed in monocyte-derived human macrophages. It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1β secretion.

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