scholarly journals Functional and Pharmacological Evaluation of a Novel SCN2A Variant Linked to Early-onset Epilepsy

2020 ◽  
Author(s):  
Scott K. Adney ◽  
John J. Millichap ◽  
Jean-Marc DeKeyser ◽  
Tatiana Abramova ◽  
Christopher H. Thompson ◽  
...  
Keyword(s):  
Drug Response ◽  
Time Course ◽  
Voltage Dependence ◽  
De Novo ◽  
Patch Clamp Recording ◽  
Variants Of Unknown Significance ◽  
Whole Cell Patch Clamp ◽  
Genetic Features ◽  
Resurgent Current

ABSTRACTObjectiveWe identified a novel de novo SCN2A variant (M1879T) associated with infantile-onset epilepsy that responded dramatically to sodium channel blocker antiepileptic drugs. We analyzed the functional and pharmacological consequences of this variant to establish pathogenicity, and to correlate genotype with phenotype and clinical drug response.MethodsThe clinical and genetic features of an infant boy with epilepsy are presented. We investigated the effect of the variant using heterologously expressed recombinant human NaV1.2 channels. We performed whole-cell patch clamp recording to determine the functional consequences and response to carbamazepine.ResultsThe M1879T variant caused disturbances in channel inactivation including substantially depolarized voltage-dependence of inactivation, slower time course of inactivation, and enhanced resurgent current that collectively represent a gain-of-function. Carbamazepine partially normalized the voltage-dependence of inactivation and produced use-dependent block of the variant channel at high pulsing frequencies. Carbamazepine also suppresses resurgent current conducted by M1879T channels, but this effect was explained primarily by reducing the peak transient current. Molecular modeling suggests that the M1879T variant disrupts contacts with nearby residues in the C-terminal domain of the channel.InterpretationOur study demonstrates the value of conducting functional analyses of SCN2A variants of unknown significance to establish pathogenicity and genotype-phenotype correlations. We also show concordance of in vitro pharmacology using heterologous cells with the drug response observed clinically in a case of SCN2A-associated epilepsy.

scholarly journals Morphine-induced synaptic plasticity in the VTA is reversed by HDAC inhibition

2016 ◽  
Vol 116 (3) ◽  
pp. 1093-1103 ◽  
Author(s):  
Michael E. Authement ◽  
Ludovic D. Langlois ◽  
Haifa Kassis ◽  
Shawn Gouty ◽  
Matthieu Dacher ◽  
...  
Keyword(s):  
Behavioral Plasticity ◽  
Drugs Of Abuse ◽  
Patch Clamp Recording ◽  
Whole Cell Patch Clamp ◽  
Synaptic Changes ◽  
Induced Changes ◽  
And Behavior ◽  
H3 Acetylation

Dopamine (DA) dysfunction originating from the ventral tegmental area (VTA) occurs as a result of synaptic abnormalities following consumption of drugs of abuse and underlies behavioral plasticity associated with drug abuse. Drugs of abuse can cause changes in gene expression through epigenetic mechanisms in the brain that underlie some of the lasting neuroplasticity and behavior associated with addiction. Here we investigated the function of histone acetylation and histone deacetylase (HDAC)2 in the VTA in recovery of morphine-induced synaptic modifications following a single in vivo exposure to morphine. Using a combination of immunohistochemistry, Western blot, and whole cell patch-clamp recording in rat midbrain slices, we show that morphine increased HDAC2 activity in VTA DA neurons and reduced histone H3 acetylation at lysine 9 (Ac-H3K9) in the VTA 24 h after the injection. Morphine-induced synaptic changes at glutamatergic synapses involved endocannabinoid signaling to reduce GABAergic synaptic strength onto VTA DA neurons. Both plasticities were recovered by in vitro incubation of midbrain slices with a class I-specific HDAC inhibitor (HDACi), CI-994, through an increase in acetylation of histone H3K9. Interestingly, HDACi incubation also increased levels of Ac-H3K9 and triggered GABAergic and glutamatergic plasticities in DA neurons of saline-treated rats. Our results suggest that acute morphine-induced changes in VTA DA activity and synaptic transmission engage HDAC2 activity locally in the VTA to maintain synaptic modifications through histone hypoacetylation.

scholarly journals Size-dependent cell-to-cell movement of defective interfering RNAs of Cymbidium ringspot virus

2002 ◽  
Vol 83 (6) ◽  
pp. 1505-1510 ◽  
Author(s):  
György Szittya ◽  
Dániel Silhavy ◽  
Tamás Dalmay ◽  
József Burgyán
Keyword(s):  
Transgenic Plants ◽  
Nicotiana Benthamiana ◽  
Time Course ◽  
De Novo ◽  
Cell Movement ◽  
Ringspot Virus ◽  
Size Dependent ◽  
Dependent Cell ◽  
Rna Accumulation

Co-inoculation of Nicotiana benthamiana plants with in vitro transcripts of both genomic and short defective interfering (DI) RNAs of Cymbidium ringspot virus results in an accumulation of de novo generated DI RNA dimers. Time-course analysis of DI RNA accumulation in the inoculated leaves showed early accumulation of DI RNA dimers followed by increased levels of DI RNA monomers. In contrast, DI RNA dimers were barely detectable in systems where cell-to-cell movement does not take place (protoplasts) or is less important (monomeric DI RNA-expressing transgenic plants). Our results also demonstrated that the size of DI RNAs is important in the colonization of inoculated leaves, suggesting that DI RNA dimers are quickly selected for cell-to-cell movement if short DI RNA monomers are used for infection.

In Vitro Properties of Neurons in the Rat Pretectal Nucleus of the Optic Tract

2007 ◽  
Vol 97 (5) ◽  
pp. 3574-3584 ◽  
Author(s):  
N. Prochnow ◽  
P. Lee ◽  
W. C. Hall ◽  
M. Schmidt
Keyword(s):  
Fluorescent Dyes ◽  
Optic Tract ◽  
Cell Populations ◽  
Patch Clamp Recording ◽  
Visual Activity ◽  
Optokinetic Reflex ◽  
Whole Cell Patch Clamp ◽  
Pretectal Nucleus ◽  
Efferent Projections

The nucleus of the optic tract (NOT) has been implicated in the initiation of the optokinetic reflex (OKR) and in the modulation of visual activity during saccades. The present experiments demonstrate that these two functions are served by separate cell populations that can be distinguished by differences in both their cellular physiology and their efferent projections. We compared the response properties of NOT cells in rats using target-directed whole cell patch-clamp recording in vitro. To identify the cells at the time of the recording experiments, they were prelabeled by retrograde axonal transport of WGA-apo-HRP-gold (15 nm), which was injected into their primary projection targets, either the ipsilateral superior colliculus (iSC), or the contralateral NOT (cNOT), or the ipsilateral inferior olive (iIO). Retrograde labeling after injections in single animals of either WGA-apo-HRP-gold with different particle sizes (10 and 20 nm) or two different fluorescent dyes distinguished two NOT cell populations. One projects to both the iSC and cNOT. These cells are spontaneously active in vitro and respond to intracellular depolarizations with temporally regular tonic firing. The other population projects to the iIO and consists of cells that show no spontaneous activity, respond phasically to intracellular depolarization, and show irregular firing patterns. We propose that the spontaneously active pathway to iSC and cNOT is involved in modulating the level of visual activity during saccades and that the phasically active pathway to iIO provides a short-latency relay from the retina to premotor mechanisms involved in reducing retinal slip.

scholarly journals Synthesis and secretion of alkaline phosphatase in vitro from first-trimester and term human placentas

1981 ◽  
Vol 194 (3) ◽  
pp. 857-866 ◽  
Author(s):  
H Galski ◽  
S E Fridovich ◽  
D Weinstein ◽  
N De Groot ◽  
S Segal ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Time Course ◽  
De Novo ◽  
Placental Tissue ◽  
First Trimester ◽  
Alkaline Phosphatases ◽  
Heat Stable ◽  
Alkaline Phosphatase Isoenzymes ◽  
Specific Activities

The synthesis and secretion of alkaline phosphatases in vitro by human placental tissue incubated in organ culture were studied. First-trimester placenta synthesizes and secretes two different alkaline phosphatase isoenzymes (heat-labile and heat-stable), whereas in term placenta nearly all the alkaline phosphatase synthesized and secreted is heat-stable. The specific activities of alkaline phosphatases in first-trimester and term placental tissue remain constant throughout the time course of incubation. In the media, specific activities increase with time. Hence, alkaline phosphatase synthesis seems to be the driving force for its own secretion. The rates of synthesis de novo and of alkaline phosphatases were measured. The specific radioactivities of the secreted alkaline phosphatases were higher than the corresponding specific radioactivities in the tissue throughout the entire incubation period. The intracellular distribution of the alkaline phosphatase isoenzymes was compared.

Modulatory effects of 5-hydroxytryptamine on voltage-activated currents in cultured antennal lobe neurones of the sphinx moth Manduca sexta.

1995 ◽  
Vol 198 (3) ◽  
pp. 613-627 ◽  
Author(s):  
A R Mercer ◽  
J H Hayashi ◽  
J G Hildebrand
Keyword(s):  
Manduca Sexta ◽  
Adult Development ◽  
Antennal Lobe ◽  
Ionic Currents ◽  
Delayed Rectifier ◽  
Patch Clamp Recording ◽  
Whole Cell Patch Clamp ◽  
K Current ◽  
Reversible Reduction

The modulatory effects of 5-hydroxytryptamine (5-HT or serotonin) on voltage-gated currents in central olfactory neurones of the moth Manduca sexta have been examined in vitro using whole-cell patch-clamp recording techniques. Central olfactory neurones were dissociated from the antennal lobes of animals at stage 5 of the 18 stages of metamorphic adult development. The modulatory actions of 5-HT on voltage-activated ionic currents were examined in a subset of morphologically identifiable antennal lobe neurones maintained for 2 weeks in primary cell culture. 5-HT caused reversible reduction of both a rapidly activating A-type K+ current and a relatively slowly activating K+ current resembling a delayed rectifier-type conductance. 5-HT also reduced the magnitude of voltage-activated Ca2+ influx in these cells. The functional significance of 5-HT-modulation of central neurones is discussed.

An important functional role of persistent Na+ current in carotid body hypoxia transduction

2006 ◽  
Vol 101 (4) ◽  
pp. 1076-1084 ◽  
Author(s):  
Edward Vincent S. Faustino ◽  
David F. Donnelly
Keyword(s):  
Carotid Body ◽  
Acute Hypoxia ◽  
Whole Body ◽  
Conduction Time ◽  
Afferent Neurons ◽  
Patch Clamp Recording ◽  
Peripheral Chemoreceptor ◽  
Whole Cell Patch Clamp ◽  
Old Rats

Systemic hypoxia in mammals is sensed and transduced by the carotid body into increased action potential (AP) frequency on the sinus nerve, resulting in increased ventilation. The mechanism of hypoxia transduction is not resolved, but previous work suggested that fast Na+ channels play an important role in determining the rate and timing of APs (Donnelly, DF, Panisello JM, and Boggs D. J Physiol. 511: 301–311, 1998). We speculated that Na+ channel activity between APs, termed persistent Na+ current ( INaP), is responsible for AP generation that and riluzole and phenytoin, which inhibit this current, would impair organ function. Using whole cell patch clamp recording of intact petrosal neurons with projections to the carotid body, we demonstrated that INaP is present in chemoreceptor afferent neurons and is inhibited by riluzole. Furthermore, discharge frequencies of single-unit, chemoreceptor activity, in vitro, during normoxia (Po2 150 Torr) and during acute hypoxia (Po2 90 Torr) were significantly reduced by riluzole concentrations at or above 5 μM, and by phenytoin at 100 μM, without significant affect on nerve conduction time, AP magnitude (inferred from extracellular field), and AP duration. The effect of both drugs appeared solely postsynaptic because hypoxia-induced catecholamine release in the carotid body was not altered by either drug. The respiratory response of unanesthetized, unrestrained 2-wk-old rats to acute hypoxia (12% inspired O2 fraction), which was measured with whole body plethysmography, was significantly reduced after treatment with riluzole (2 mg/kg ip) and phenytoin (20 mg/kg ip). We conclude that INaP is present in chemoreceptor afferent neurons and serves an important role in peripheral chemoreceptor function and, hence, in the ventilatory response to hypoxia.

scholarly journals Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback.

1995 ◽  
Vol 105 (2) ◽  
pp. 209-226 ◽  
Author(s):  
A Zweifach ◽  
R S Lewis
Keyword(s):  
Time Course ◽  
Measurement Techniques ◽  
Fast Inactivation ◽  
Patch Clamp Recording ◽  
Crac Channels ◽  
Whole Cell Patch Clamp ◽  
Close Proximity ◽  
Rapid Inactivation ◽  
Voltage Dependent ◽  
Crac Channel

Rapid inactivation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole-cell patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca2+, the Ca2+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 mV. Several lines of evidence suggest that the fast inactivation process is Ca2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca2+ entry through open channels. Second, inactivation is substantially reduced when Ba2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 mM), a rapid Ca2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 mM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i. These results demonstrate that rapid inactivation of ICRAC results from the action of Ca2+ in close proximity to the intracellular mouths of individual channels, and that Ca2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca2+ diffusion in the presence of a mobile buffer predicts multiple Ca2+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.

Early anoxia-induced vesicular glutamate release results from mobilization of calcium from intracellular stores

1993 ◽  
Vol 70 (1) ◽  
pp. 1-7 ◽  
Author(s):  
A. N. Katchman ◽  
N. Hershkowitz
Keyword(s):  
Calcium Influx ◽  
Glutamate Release ◽  
Hippocampal Slices ◽  
Calcium Currents ◽  
Patch Clamp Recording ◽  
Ca1 Neurons ◽  
Whole Cell Patch Clamp ◽  
Mepsc Frequency ◽  
Synaptic Changes

1. The cause of the increased frequency of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) resulting from anoxia was investigated in CA1 neurons of the in vitro rat hippocampal slice. These neurons were examined by whole-cell patch-clamp recording, and hypoxia was induced by switching the perfusion of the slice from oxygenated artificial cerebral spinal fluid (ACSF) to ACSF saturated with 95% N2-5% O2. Except where noted, experiments were carried out in ACSF containing 1 microM tetrodotoxin (TTX). 2. Although anoxia resulted in a significant increase in the frequency of mEPSCs, the amplitude, rise time, and half-decay time of the mEPSCs were unchanged. This increase in frequency indicates that there is a change in presynaptic neurotransmitter release mechanisms, probably an increase in calcium concentration, soon after the onset of anoxia. The unchanged kinetics and amplitude of the mEPSCs indicate that anoxic-induced synaptic changes are not a result of changes in the postsynaptic glutamate receptor. 3. When hippocampal slices were exposed to anoxic conditions in ACSF with calcium excluded, an increase in mEPSC frequency equal to that in normal ACSF was observed. When 0.2 mM of CdCl2 was added to the zero-calcium ACSF, anoxia still resulted in increases in mEPSC frequency equal to those of normal ACSF. It is therefore concluded that the anoxia-induced increase in mEPSC frequency does not result from an increase in a transmembrane calcium influx. The zero-calcium plus 0.2 mM CdCl2 ACSF solution completely abolished orthodromically elicited synaptic potential (in the absence of TTX), indicating that calcium currents that mediate normal orthodromic transmitter release were completely abolished in the latter experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Characterization of norovirus 3Dpol RNA-dependent RNA polymerase activity and initiation of RNA synthesis

2006 ◽  
Vol 87 (9) ◽  
pp. 2621-2630 ◽  
Author(s):  
Jacques Rohayem ◽  
Katrin Jäger ◽  
Ivonne Robel ◽  
Ulrike Scheffler ◽  
Achim Temme ◽  
...  
Keyword(s):  
Time Course ◽  
Rna Synthesis ◽  
De Novo ◽  
Polymerase Activity ◽  
Opposite Polarity ◽  
Transferase Activity ◽  
Structural Protein ◽  
Double Stranded Rna ◽  
Terminal Transferase

Norovirus (NV) 3Dpol is a non-structural protein predicted to play an essential role in the replication of the NV genome. In this study, the characteristics of NV 3Dpol activity and initiation of RNA synthesis have been examined in vitro. Recombinant NV 3Dpol, as well as a 3Dpol active-site mutant were expressed in Escherichia coli and purified. NV 3Dpol was able to synthesize RNA in vitro and displayed flexibility with respect to the use of Mg2+ or Mn2+ as a cofactor. NV 3Dpol yielded two different products when incubated with synthetic RNA in vitro: (i) a double-stranded RNA consisting of two single strands of opposite polarity or (ii) the single-stranded RNA template labelled at its 3′ terminus by terminal transferase activity. Initiation of RNA synthesis occurred de novo rather than by back-priming, as evidenced by the fact that the two strands of the double-stranded RNA product could be separated, and by dissociation in time-course analysis of terminal transferase and RNA synthesis activities. In addition, RNA synthesis was not affected by blocking of the 3′ terminus of the RNA template by a chain terminator, sustaining de novo initiation of RNA synthesis. NV 3Dpol displays in vitro properties characteristic of RNA-dependent RNA polymerases, allowing the implementation of this in vitro enzymic assay for the development and validation of antiviral drugs against NV, a so far non-cultivated virus and an important human pathogen.

scholarly journals Reduced GABAergic transmission in the ventrobasal thalamus contributes to thermal hyperalgesia in chronic inflammatory pain

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Chan Zhang ◽  
Rong-Xiang Chen ◽  
Yu Zhang ◽  
Jie Wang ◽  
Feng-Yu Liu ◽  
...  
Keyword(s):  
Inflammatory Pain ◽  
Thermal Hyperalgesia ◽  
Thalamic Reticular Nucleus ◽  
Reticular Nucleus ◽  
Patch Clamp Recording ◽  
Gabaergic Transmission ◽  
Chronic Inflammatory Pain ◽  
Whole Cell Patch Clamp ◽  
Ventrobasal Thalamus

Abstract The ventrobasal (VB) thalamus is innervated by GABAergic afferents from the thalamic reticular nucleus (TRN) and participates in nociception. But how the TRN-VB pathway regulates pain is not fully understood. In the present study, we reported decreased extracellular GABA levels in the VB of rats with CFA-induced chronic inflammatory pain, measured by microdialysis with HPLC analysis. In vitro whole-cell patch-clamp recording showed decreased amplitudes of tonic currents, increased frequencies of mIPSCs, and increased paired-pulse ratios in thalamic slices from chronic inflammatory rats (7 days). Microinjection of the GABAAR agonist muscimol and optogenetic activation of the TRN-VB pathway relieved thermal hyperalgesia in chronic inflammatory pain. By contrast, microinjecting the extrasynaptic GABAAR agonist THIP or selective knockout of synaptic GABAAR γ2 subunits aggravated thermal hyperalgesia in the chronic stage of inflammatory pain. Our findings indicate that reduced GABAergic transmission in the VB contributes to thermal hyperalgesia in chronic inflammatory pain, which could be a synaptic target for pharmacotherapy.

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