Development of a spontaneously active dorsal root ganglia assay using multiwell multielectrode arrays

Newberry, Kim M.; Wang, Shuya; Hoque, Nina; Kiss, László; Ahlijanian, Michael K.; Herrington, James; Graef, John D.

doi:10.1152/jn.01122.2015

Cited by 47 publications

(51 citation statements)

References 52 publications

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“…The only added current to the original HH scheme is the Nav1.7, because it is strictly needed to implement the biological mechanisms under consideration. A detailed quantitative modelling would require Ca 2+ currents and several types of Na + and K + currents that have been found in mammalian DRG cells (e.g., Rush et al, 2007; Scroggs and Fox, 1992; Choi and Waxman, 2011; Newberry et al, 2016; Mandge and Manchanda, 2018; Zemel et al, 2018), and is beyond the scope of this study. Although more refined kinetic models of the Nav1.7 sodium and other channels have been proposed (e.g., Sigg, 2014), we think that we are adopting here a level of simplification in the modelling process that is fitted for our purpose.…”

Section: Discussionmentioning

confidence: 99%

On the role of Nav1.7 sodium channels in chronic pain: an experimental and computational study

Capurro

Thornton

Cessac

et al. 2019

Preprint

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Chronic pain is a global healthcare problem with a huge societal impact. Its management remains generally unsatisfactory, with no single treatment clinically approved in most cases. In this study we use an in vitro model of erythromelalgia consisting of dorsal root ganglion neurons derived from human induced pluripotent stem cells obtained from a patient (carrying the mutation F1449V) and a control subject. We combine neurophysiology and computational modelling to focus on the Nav1.7 voltage gated sodium channel, which acts as an amplifier of the receptor potential in nociceptive neurons and plays a critical role in erythromelalgia due to gain of function mutations causing the channel to open with smaller depolarisations.Using extracellular recordings, we found that the scorpion toxin OD1 (a Nav1.7 channel opener) increases dorsal root ganglion cell excitability in cultures obtained from the control donor, evidenced by an increase in spontaneous discharges, firing rate and spike amplitude. In addition, we confirmed previous reports of voltage clamp experiments concerning an increase in spontaneous discharge in the patient cell cultures and the analgesic effects of the Nav1.7 blocker PF-05089771. Our findings are explained with a conductance-based model of the dorsal root ganglion neuron, exploring its behaviour for different values of half activation voltage and inactivation removal rate of the Nav1.7 current. Erythromelalgia was simulated through a decrease of the Nav1.7 half activation voltage, turning previously subthreshold stimuli to pain-inducing, and successfully counteracted with the channel blocker. The painful effects of OD1 were simulated through a quicker removal of Nav1.7 inactivation that reproduced the effects of the toxin not only on the spike frequency but also on its amplitude.New & NoteworthyWe investigate Nav1.7 channel gating mechanisms in human iPSC-derived dorsal root ganglion cell cultures using multielectrode array recordings. The scorpion toxin OD1 increases firing frequency and spike amplitudes whilst the analgesic PF-05089771 decreases or even abolishes spontaneous activity. The antagonistic effect of these compounds is explained with a computational model to reach deeper understanding of changes in channel kinetics in erythromelalgia, a chronic pain disorder.

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Section: Discussionmentioning

confidence: 99%

On the role of Nav1.7 sodium channels in chronic pain: an experimental and computational study

Capurro

Thornton

Cessac

et al. 2019

Preprint

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“…Capsaicin, the spicy principle of pepper, causes intense pain if injected [26]. It is a TRPV1 agonist [27]. The vanilloid receptor is involved in orofacial nociception, contributing to the detection of thermal stimuli and the integration with fiber type C and Aδ [26].…”

Section: Discussionmentioning

confidence: 99%

Orofacial antinociceptive effect of the ethanolic extract of Annona vepretorum Mart. (Annonaceae)

Silva

Macedo

Souza

et al. 2016

Zeitschrift Für Naturforschung C

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Annona vepretorum Mart. (Annonaceae) is a species popularly known in Brazil as "araticum" and "pinha da Caatinga". We have evaluated the antinociceptive effects of A. vepretorum in formalin-, capsaicin-, and glutamate-induced orofacial nociception in mice. Male Swiss mice were pretreated with either saline (p.o.), A. vepretorum ethanol extract (Av-EtOH 25, 50 and 100 mg/kg, p.o.), or morphine (10 mg/kg, i.p.), before formalin, capsaicin, or glutamate was injected into the right upper lip. Pre-treatment with Av-EtOH at all doses produced a reduction in face-rubbing behavior induced by formalin in both phases, and these pre-treatments also produced a significant antinociceptive effect in the capsaicin and glutamate tests. Pre-treatment with naloxone (1.5 mg/kg, i.p.) did not reverse the antinociceptive activity of the extract at the dose of 100 mg/kg in the first phase of this test. Our results suggest that Av-EtOH might be useful in the treatment of orofacial pain.

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“…Recording was performed every day before or after dCas9-Tet1/CGG sgRNA, FMR1 mRNA and FMR1 ASO treatments. Data were analyzed using a custom MATLAB program [30] and presented as mean ± SD, and are considered statistically significant when p < 0.05 by paired Student's T-Test or one-way ANOVA, where appropriate.…”

Section: Mea and Data Analysismentioning

confidence: 99%

Partial FMRP expression is sufficient to normalize neuronal hyperactivity in Fragile X neurons

Graef

et al. 2019

Preprint

Self Cite

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Highlights:• CRISPR gene editing to generate FMRP KO and CGG-deleted isogenic iPSCs• MEA as an approach to identify FMR1 dependent phenotype in NGN2 neurons derived from FXS and FMRP KO iPSCs• Cell mixing paradigm as mosaicism in a dish to rescue phenotype • Minimal level of FMRP determined by FMR1 mRNA and targeted demethylation of CGG repeats to correct the hyperactive phenotype in FXS neurons • ASO titration-validated partial expression of FMRP is sufficient to normalize increased neuronal activityAbstract Fragile X Syndrome (FXS) is the most common genetic form of intellectual disability caused by a CGG repeat expansion in the 5'-UTR of the Fragile X mental retardation gene FMR1, triggering epigenetic silencing and the subsequent absence of the protein, FMRP. Reactivation of FMR1 represents an attractive therapeutic strategy targeting the genetic root cause of FXS. However, largely missing in the FXS field is an understanding of how much FMR1 reactivation is required to rescue FMRP-dependent mutant phenotypes. Here, we utilize FXS patient derived excitatory neurons to model FXS in vitro and confirm that the absence of FMRP leads to neuronal hyperactivity. We further determined the levels of FMRP and the percentage of FMRP positive cells necessary to correct this phenotype utilizing a mixed and mosaic neuronal culture system and a combination of CRISPR, antisense and expression technologies to titrate FMRP in FXS and WT neurons. Our data demonstrate that restoration of greater than 5% of overall FMRP expression levels or greater than 20% FMRP expressing neurons in a mosaic pattern is sufficient to normalize a FMRP-dependent, hyperactive phenotype in FXS iPSC-derived neurons.

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Development of a spontaneously active dorsal root ganglia assay using multiwell multielectrode arrays

Cited by 47 publications

References 52 publications

On the role of Nav1.7 sodium channels in chronic pain: an experimental and computational study

On the role of Nav1.7 sodium channels in chronic pain: an experimental and computational study

Orofacial antinociceptive effect of the ethanolic extract of Annona vepretorum Mart. (Annonaceae)

Partial FMRP expression is sufficient to normalize neuronal hyperactivity in Fragile X neurons

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