Modulation of Ca2+ oscillation following ischemia and nicotinic acetylcholine receptors in primary cortical neurons by high-throughput analysis

Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug development. Previous studies have demonstrated that Ca 2+ influx via NMDA and nicotinic acetylcholine receptors (nAChRs) modulates Ca 2+ oscillations. Neverth...

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Published inScientific reports Vol. 14; no. 1; pp. 27667 - 18
Main Authors Sasaki, Tsutomu, Hisada, Sunao, Kanki, Hideaki, Nunomura, Kazuto, Lin, Bangzhong, Nishiyama, Kumiko, Kawano, Tomohito, Matsumura, Shigenobu, Mochizuki, Hideki
Format Journal Article
LanguageEnglish
Published London Nature Publishing Group UK 12.11.2024
Nature Publishing Group
Nature Portfolio
Subjects
631/154
631/154/1438
631/154/570
631/378/1689
631/378/340
631/378/87
Acetylcholine receptors (nicotinic)
Agonists
Allosteric properties
Ca2+ oscillations
Calcium antagonists
Calcium influx
Calcium signalling
Dizocilpine
Drug development
Drug screening
Glutamate receptors
Glutamic acid receptors (ionotropic)
High-throughput screening
Humanities and Social Sciences
Inhibitory postsynaptic potentials
Ischemia
multidisciplinary
N-Methyl-D-aspartic acid receptors
nAChR
Neuromodulation
Neuroprotection
Neuroprotective agents
PAM
Science
Science (multidisciplinary)
α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors
High-throughput screening
Ischemia
oscillations
PAM
nAChR
Ca
Online AccessGet full text

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Abstract Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug development. Previous studies have demonstrated that Ca 2+ influx via NMDA and nicotinic acetylcholine receptors (nAChRs) modulates Ca 2+ oscillations. Nevertheless, there has been no comprehensive investigation into the impact of ischemia or nAChR-positive allosteric modulators (PAM) drugs on Ca 2+ oscillations at a level that would facilitate high-throughput screening. We investigated the effects of ischemia and nAChR subtypes or nAChR PAM agonists on Ca 2+ oscillations in high-density 2D and 3D-sphere primary neuronal cultures using 384-well plates with FDSS-7000. Ischemia for 1 and 2 h resulted in an increase in the frequency of Ca 2+ oscillations and a decrease in their amplitude in a time-dependent manner. The NMDA and AMPA receptor inhibition significantly suppressed Ca 2+ oscillation. Inhibition of NR2A or NR2B had the opposite effect on Ca oscillations. The potentiation of ischemia-induced Ca 2+ oscillations was significantly inhibited by the NMDA receptor antagonist, MK-801, and the frequency of these oscillations was suppressed by the NR2B inhibitor, Ro-256981. In the 3D-neurosphere, the application of an α7nAChR agonist increased the frequency of Ca 2+ oscillations, whereas the activation of α4β2 had no effect. The combination of nicotine and PNU-120596 (type II PAM) affected the frequency and amplitude of Ca 2+ oscillations in a manner distinct from that of type I PAM. These systems may be useful not only for detecting epileptogenicity but also in the search for neuroprotective agents against cerebral ischemia.
AbstractList Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug development. Previous studies have demonstrated that Ca2+ influx via NMDA and nicotinic acetylcholine receptors (nAChRs) modulates Ca2+ oscillations. Nevertheless, there has been no comprehensive investigation into the impact of ischemia or nAChR-positive allosteric modulators (PAM) drugs on Ca2+ oscillations at a level that would facilitate high-throughput screening. We investigated the effects of ischemia and nAChR subtypes or nAChR PAM agonists on Ca2+ oscillations in high-density 2D and 3D-sphere primary neuronal cultures using 384-well plates with FDSS-7000. Ischemia for 1 and 2 h resulted in an increase in the frequency of Ca2+ oscillations and a decrease in their amplitude in a time-dependent manner. The NMDA and AMPA receptor inhibition significantly suppressed Ca2+ oscillation. Inhibition of NR2A or NR2B had the opposite effect on Ca oscillations. The potentiation of ischemia-induced Ca2+ oscillations was significantly inhibited by the NMDA receptor antagonist, MK-801, and the frequency of these oscillations was suppressed by the NR2B inhibitor, Ro-256981. In the 3D-neurosphere, the application of an α7nAChR agonist increased the frequency of Ca2+ oscillations, whereas the activation of α4β2 had no effect. The combination of nicotine and PNU-120596 (type II PAM) affected the frequency and amplitude of Ca2+ oscillations in a manner distinct from that of type I PAM. These systems may be useful not only for detecting epileptogenicity but also in the search for neuroprotective agents against cerebral ischemia.
Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug development. Previous studies have demonstrated that Ca 2+ influx via NMDA and nicotinic acetylcholine receptors (nAChRs) modulates Ca 2+ oscillations. Nevertheless, there has been no comprehensive investigation into the impact of ischemia or nAChR-positive allosteric modulators (PAM) drugs on Ca 2+ oscillations at a level that would facilitate high-throughput screening. We investigated the effects of ischemia and nAChR subtypes or nAChR PAM agonists on Ca 2+ oscillations in high-density 2D and 3D-sphere primary neuronal cultures using 384-well plates with FDSS-7000. Ischemia for 1 and 2 h resulted in an increase in the frequency of Ca 2+ oscillations and a decrease in their amplitude in a time-dependent manner. The NMDA and AMPA receptor inhibition significantly suppressed Ca 2+ oscillation. Inhibition of NR2A or NR2B had the opposite effect on Ca oscillations. The potentiation of ischemia-induced Ca 2+ oscillations was significantly inhibited by the NMDA receptor antagonist, MK-801, and the frequency of these oscillations was suppressed by the NR2B inhibitor, Ro-256981. In the 3D-neurosphere, the application of an α7nAChR agonist increased the frequency of Ca 2+ oscillations, whereas the activation of α4β2 had no effect. The combination of nicotine and PNU-120596 (type II PAM) affected the frequency and amplitude of Ca 2+ oscillations in a manner distinct from that of type I PAM. These systems may be useful not only for detecting epileptogenicity but also in the search for neuroprotective agents against cerebral ischemia.
Abstract Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug development. Previous studies have demonstrated that Ca2+ influx via NMDA and nicotinic acetylcholine receptors (nAChRs) modulates Ca2+ oscillations. Nevertheless, there has been no comprehensive investigation into the impact of ischemia or nAChR-positive allosteric modulators (PAM) drugs on Ca2+ oscillations at a level that would facilitate high-throughput screening. We investigated the effects of ischemia and nAChR subtypes or nAChR PAM agonists on Ca2+ oscillations in high-density 2D and 3D-sphere primary neuronal cultures using 384-well plates with FDSS-7000. Ischemia for 1 and 2 h resulted in an increase in the frequency of Ca2+ oscillations and a decrease in their amplitude in a time-dependent manner. The NMDA and AMPA receptor inhibition significantly suppressed Ca2+ oscillation. Inhibition of NR2A or NR2B had the opposite effect on Ca oscillations. The potentiation of ischemia-induced Ca2+ oscillations was significantly inhibited by the NMDA receptor antagonist, MK-801, and the frequency of these oscillations was suppressed by the NR2B inhibitor, Ro-256981. In the 3D-neurosphere, the application of an α7nAChR agonist increased the frequency of Ca2+ oscillations, whereas the activation of α4β2 had no effect. The combination of nicotine and PNU-120596 (type II PAM) affected the frequency and amplitude of Ca2+ oscillations in a manner distinct from that of type I PAM. These systems may be useful not only for detecting epileptogenicity but also in the search for neuroprotective agents against cerebral ischemia.
Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug development. Previous studies have demonstrated that Ca2+ influx via NMDA and nicotinic acetylcholine receptors (nAChRs) modulates Ca2+ oscillations. Nevertheless, there has been no comprehensive investigation into the impact of ischemia or nAChR-positive allosteric modulators (PAM) drugs on Ca2+ oscillations at a level that would facilitate high-throughput screening. We investigated the effects of ischemia and nAChR subtypes or nAChR PAM agonists on Ca2+ oscillations in high-density 2D and 3D-sphere primary neuronal cultures using 384-well plates with FDSS-7000. Ischemia for 1 and 2 h resulted in an increase in the frequency of Ca2+ oscillations and a decrease in their amplitude in a time-dependent manner. The NMDA and AMPA receptor inhibition significantly suppressed Ca2+ oscillation. Inhibition of NR2A or NR2B had the opposite effect on Ca oscillations. The potentiation of ischemia-induced Ca2+ oscillations was significantly inhibited by the NMDA receptor antagonist, MK-801, and the frequency of these oscillations was suppressed by the NR2B inhibitor, Ro-256981. In the 3D-neurosphere, the application of an α7nAChR agonist increased the frequency of Ca2+ oscillations, whereas the activation of α4β2 had no effect. The combination of nicotine and PNU-120596 (type II PAM) affected the frequency and amplitude of Ca2+ oscillations in a manner distinct from that of type I PAM. These systems may be useful not only for detecting epileptogenicity but also in the search for neuroprotective agents against cerebral ischemia.Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug development. Previous studies have demonstrated that Ca2+ influx via NMDA and nicotinic acetylcholine receptors (nAChRs) modulates Ca2+ oscillations. Nevertheless, there has been no comprehensive investigation into the impact of ischemia or nAChR-positive allosteric modulators (PAM) drugs on Ca2+ oscillations at a level that would facilitate high-throughput screening. We investigated the effects of ischemia and nAChR subtypes or nAChR PAM agonists on Ca2+ oscillations in high-density 2D and 3D-sphere primary neuronal cultures using 384-well plates with FDSS-7000. Ischemia for 1 and 2 h resulted in an increase in the frequency of Ca2+ oscillations and a decrease in their amplitude in a time-dependent manner. The NMDA and AMPA receptor inhibition significantly suppressed Ca2+ oscillation. Inhibition of NR2A or NR2B had the opposite effect on Ca oscillations. The potentiation of ischemia-induced Ca2+ oscillations was significantly inhibited by the NMDA receptor antagonist, MK-801, and the frequency of these oscillations was suppressed by the NR2B inhibitor, Ro-256981. In the 3D-neurosphere, the application of an α7nAChR agonist increased the frequency of Ca2+ oscillations, whereas the activation of α4β2 had no effect. The combination of nicotine and PNU-120596 (type II PAM) affected the frequency and amplitude of Ca2+ oscillations in a manner distinct from that of type I PAM. These systems may be useful not only for detecting epileptogenicity but also in the search for neuroprotective agents against cerebral ischemia.
ArticleNumber 27667
Author Kanki, Hideaki
Hisada, Sunao
Kawano, Tomohito
Matsumura, Shigenobu
Mochizuki, Hideki
Nunomura, Kazuto
Nishiyama, Kumiko
Lin, Bangzhong
Sasaki, Tsutomu
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  givenname: Sunao
  surname: Hisada
  fullname: Hisada, Sunao
  organization: Department of Neurology, Graduate School of Medicine, Osaka University
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  givenname: Hideaki
  surname: Kanki
  fullname: Kanki, Hideaki
  organization: Department of Neurology, Graduate School of Medicine, Osaka University
– sequence: 4
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  surname: Nunomura
  fullname: Nunomura, Kazuto
  organization: Center for Supporting Drug Discovery and Life Science Research, Graduate School of Pharmaceutical Science, Osaka University
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  fullname: Kawano, Tomohito
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  organization: Graduate School of Comprehensive Rehabilitation, Osaka Prefecture University
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  surname: Mochizuki
  fullname: Mochizuki, Hideki
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Keywords High-throughput screening
Ischemia
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Snippet Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug development....
Abstract Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug...
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SubjectTerms 631/154
631/154/1438
631/154/570
631/378/1689
631/378/340
631/378/87
Acetylcholine receptors (nicotinic)
Agonists
Allosteric properties
Ca2+ oscillations
Calcium antagonists
Calcium influx
Calcium signalling
Dizocilpine
Drug development
Drug screening
Glutamate receptors
Glutamic acid receptors (ionotropic)
High-throughput screening
Humanities and Social Sciences
Inhibitory postsynaptic potentials
Ischemia
multidisciplinary
N-Methyl-D-aspartic acid receptors
nAChR
Neuromodulation
Neuroprotection
Neuroprotective agents
PAM
Science
Science (multidisciplinary)
α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors
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Title Modulation of Ca2+ oscillation following ischemia and nicotinic acetylcholine receptors in primary cortical neurons by high-throughput analysis
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