Methylscopolamine, terbutaline, pilocarpine, and sodium pentobarbital were purchased from Sigma‐Aldrich. SC‐58125 and kainic acid were purchased from Tocris Bioscience. 16,16‐dimethyl prostaglandin E2 (dmPGE2) was purchased from Cayman Chemical. Diazepam was purchased from Henry Schein. Compound TG6‐10‐1 was synthesized accordingly,22 and the purity was confirmed by LC/MS and NMR in the Medicinal Chemistry Core at the University of Tennessee Health Science Center.
Kainic acid
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
Kainic acid is a chemical compound that acts as a potent agonist of the kainate receptor, a type of glutamate receptor found in the central nervous system. It is commonly used as a research tool in neuroscience studies to investigate excitatory neurotransmission and neuronal excitotoxicity.
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Lab products found in correlation
Pentobarbital sodium, by Merck Group (2 mentions)
N methyl d aspartic acid, by Bio-Techne (2 mentions)
Glutamate, by Merck Group (2 mentions)
Methylscopolamine, by Merck Group (1 mentions)
Terbutaline, by Merck Group (1 mentions)
Pilocarpine, by Merck Group (1 mentions)
Sc 58125, by Bio-Techne (1 mentions)
L glutamate, by Merck Group (1 mentions)
Ivermectin, by Merck Group (1 mentions)
Picrotoxinin, by Merck Group (1 mentions)
D glutamate, by Merck Group (1 mentions)
Quisqualic acid, by Bio-Techne (1 mentions)
Ibotenic acid, by Fujifilm (1 mentions)
S ketamine, by Merck Group (1 mentions)
Kainic acid, by Fujifilm (1 mentions)
Y 27632, by Fujifilm (1 mentions)
Paraquat, by Santa Cruz Biotechnology (1 mentions)
Menadione, by Merck Group (1 mentions)
N methyl d aspartic acid nmda, by Merck Group (1 mentions)
18 crown 6, by Merck Group (1 mentions)
32 protocols using kainic acid
1
Synthesis and Characterization of Compound TG6-10-1
2
Neurotransmitter Receptor Ligand Sources
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L-Glutamate, D-glutamate, ivermectin and picrotoxinin (PTX) were obtained from Sigma-Aldrich (UK). Fipronil was a gift from Dr. Lance Hammerland (Merial Ltd). Kainic acid (referred to as kainate throughout this paper), N-methyl-D-aspartic acid (NMDA), quisqualic acid (referred to as quisqualate throughout this paper), L-aspartatic acid (referred to as aspartate throughout this paper), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were obtained from Tocris (UK), whereas ibotenic acid (referred to as ibotenate throughout this paper) was obtained from Wako Pure Chemical Industries (Osaka, Japan). Okaramine B was isolated from fermentation products of P. simplicissimum according to the original paper (Hayashi et al., 1989 ).
3
Two-Electrode Voltage Clamp of Oocytes
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Ion currents elicited by agonist perfusion were recorded by the two-electrode voltage clamp (TEVC) method between 18–36 h post injection [41 (link)]. Microelectrodes were filled with 3 M KCl and resistance of the microelectrodes ranged from 0.5–3.0 MΩ. Piercing and recording took place in a chamber (volume ∼0.1 ml) continuously perfused (6 ml/min) with Ringer’s solution [115 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 5 mM Hepes (pH 7.4)] at RT (19–21°C). Oocytes were voltage clamped to –80 mV. Ion currents were recorded and stored with WinEDR ver 2.3.8 Strathclyde Electrophysiology Software (John Dempster, Glasgow, UK). Kainic acid was purchased from Tocris (Minneapolis, MN). All other reagents were from Sigma (St. Louis, MO). Working solutions were made by diluting stock solutions in Ringer’s solution. Two different membrane preparations from the same subjects were tested in MSM experiments. Ion current responses were measured at least in triplicate (three oocytes) in batches of oocytes from three different frogs. All samples were injected and tested in each experiment.
4
Kainic Acid-Induced Seizures in Mice
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Kainic acid (Tocris Bioscience) was dissolved in PBS and injected subcutaneously (20 mg/kg) in mice (Luo et al., 2006 (link)). Mice were then weighed and examined daily for clinical signs of seizures.
5
Investigating Granule Cell Maturation
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Rats were injected with bromodeoxyuridine (BrdU; 200 mg/kg, i.p.; 10 mg/ml in saline with 0.007N NaOH) to identify young granule cells for maturation and survival analyses. Beginning 2 d after BrdU injection (Day 2), rats were injected daily with saline for 14 d to match the daily injections used for long-term treatment in Experiment 3. On Day 16, rats were given a single injection of either S-ketamine (10 mg/kg, i.p.; Sigma-Aldrich) or saline. Sixteen hours later (Day 17), they were injected with kainic acid (15 mg/kg, i.p.; Tocris Bioscience) to drive immediate-early gene (IEG) expression in synaptically integrated granule cells (Snyder et al., 2009a (link),b (link)). Sodium pentobarbital (50 mg/kg, i.p.; Ovation Pharmaceuticals) was given to stop seizures 30 min after the onset of stage 5 seizure activity. Rats were perfused 90 min after the onset of stage 5 seizure onset.
6
Kainic Acid-Induced Seizures in Mice
7
Excitotoxic Injury Model of Cochlear Tissue
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Damaged cochlear tissue were created as described elsewhere (Wang and Green, 2011 (link)). Following the original method, N-methyl-D -aspartic acid 0.5 mM (NMDA; Tocris Bioscience, Ellisville, MO, United States) 0.5 mM and kainic acid (Tocris Bioscience; NK treatment) were reacted in a culture solution for 2 h.
The following three groups were compared to investigate the effects of a ROCK inhibitor, Y-27632 (257-00511, Wako) in a model of excitotoxic injury of cochlear tissue: a control group; an NK group (NMDA 0.5 mM + kainic acid 0.5 mM for 2 h, followed by washing and culture in normal culture medium); and a ROCK inhibition group (washed after treatment with NK with addition of 10 μM Y-27632 in normal culture medium). Immunohistochemical evaluations were performed at 24 and 72 h after culture.
The following three groups were compared to investigate the effects of a ROCK inhibitor, Y-27632 (257-00511, Wako) in a model of excitotoxic injury of cochlear tissue: a control group; an NK group (NMDA 0.5 mM + kainic acid 0.5 mM for 2 h, followed by washing and culture in normal culture medium); and a ROCK inhibition group (washed after treatment with NK with addition of 10 μM Y-27632 in normal culture medium). Immunohistochemical evaluations were performed at 24 and 72 h after culture.
8
Oxidative Stress and Excitotoxicity Assays
9
Kainic Acid-Induced Seizure Model
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Intrahippocampal Kainic acid injections were performed as described previously21 (link). Briefly, mice were placed under isoflurane anesthesia and given local anesthetic, 0.5% bupivacaine, at the site of incision. Kainic acid (60 nl, 20 mmol l−1 in saline; Tocris Bioscience) was injected into the dorsal hippocampus (from the bregma: −2.0 mm anterior-posterior (AP), +1.25 mm medial-lateral (ML), −1.6 mm dorsal–ventral (DV)). The above protocol was modified for intra-amygdala Kainic acid injections in which 100 nl, 20 mmol l−1 Kainic acid in saline was injected into the right basolateral amygdala (from the bregma: −1.2 mm AP, +3.3 mm ML, −4.2 from dura DV). For both intrahippocampal Kainic acid and intra-amygdala Kainic acid, kainic-acid-induced status epilepticus after injection was allowed to self-terminate. For experiments conducted in the setting of acute seizures, animals were allowed to recover for 2 h (scFLARE experiments in Fig. 1c,d and Extended Data Fig. 3b ) or returned to the vivarium for at least 2 weeks to allow for the emergence of chronic spontaneous seizures (scFLARE experiments in Extended Data Fig. 2b , and all calcium imaging and closed-loop optogenetic experiments).
10
Kainic acid-induced seizure susceptibility in Kcnb1 mice
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Susceptibility to seizures induced by the chemoconvulsant KA (kainic acid, Cat #0222, Tocris Bioscience, Minneapolis, MN) was tested in WT, Kcnb1C/+ and Kcnb1C/C mice at P41–50. KA dissolved in saline was administered by intraperitoneal injection (25 mg/kg) and mice were video recorded for 2 hours. Videos were scored offline by reviewers blinded to genotype using a modified Racine scale (Racine, 1972 (link)) (1- behavioral arrest; 2- forelimb and/or Straub tail, facial automatisms; 3- automatisms, including repetitive scratching, circling, forelimb clonus without falling; 4- forelimb clonus with rearing and/or falling, barrel roll; 5- repetition of stage 4; 6- generalized tonic-clonic seizure, wild running and/or jumping; 7- death). Latencies to the first occurrence of each stage and the highest seizure stage reached within 5 minutes bins were determined from video records by reviewers blinded to genotype. Latency to death following KA injection was compared by log-rank Mantel-Cox time to event analysis. Severity within time bins was compared between mutant alleles and WT by two-way ANOVA with Tukey’s post hoc comparisons. No difference in sex was identified, therefore groups were collapsed across sex (n=12–19 mice per genotype).
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