To measure seizure propensity, both male and female mice were subjected to intraperitoneal (i.p.) injection of vehicle control (saline) or kainate (Abcam, 15 mg/kg) as described.54 The mice were at 6‐month old, and weighed 24 to 33 g when kainate injections were performed in the laboratory under the bright light. Mice were returned to their home cage and monitored for their behavioral seizures every 10 minutes using modified Racine, Pinal and Rovner scale,71 which consists of eight stages: (stage‐1) facial movements only, (stage‐2) facial movements and head nodding, (stage‐3) facial movements, head nodding and forelimb clonus, (stage‐4) facial movements, head nodding, forelimb clonus and rearing, (stage‐5) facial movements, head nodding, forelimb clonus, rearing, loss of balance and falling, (stage‐6) a stage‐5 terminating with multiple rearing and falling episodes, (stage‐7) a stage‐6 with a violent jumping and running fit, (stage‐8) a stage‐7 with periods of tonus and (stage‐9) death. At 2 hours post injection, the mice were euthanized and their brains were stored at −80°C until use.
Kainate
Manufactured by Abcam
Sourced in Germany
Kainate is a chemical compound that acts as an agonist of the kainate receptor, a type of ionotropic glutamate receptor found in the central nervous system. It is commonly used in research applications as a tool to study the physiological and pharmacological properties of this receptor.
Automatically generated - may contain errors
Lab products found in correlation
Gyki 53655, by Bio-Techne (1 mentions)
Iem 1460, by Bio-Techne (1 mentions)
Jnj 55511118, by Bio-Techne (1 mentions)
Naspm, by Alomone (1 mentions)
Cyclothiazide, by Abcam (1 mentions)
Glutamate, by Merck Group (1 mentions)
C fos, by Cell Signaling Technology (1 mentions)
Anti β tubulin, by Cell Signaling Technology (1 mentions)
C jun, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
4 protocols using kainate
1
Evaluating Seizure Propensity in Mice
2
Whole-cell Patch-clamp Recording of Freshly Isolated Cells
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Freshly isolated cells were obtained from slices after protease treatment as described (Matthias et al., 2003 (link)). K+ channel blockers (100 μM Ba2+, 100 μM quinine) as well as AMPA receptor agonists, blockers and modulators were applied by transferring the cells with a tube electrode to the different solutions (Seifert and Steinhäuser, 1995 (link)). Membrane currents were measured in the whole-cell configuration as described above. The resistance of the patch pipettes was 4 MΩ, the input resistance was determined as described above. The bath solution contained (in mM): 150 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose (pH 7.4). The pipette solution was the same as for slice recordings. For recordings in high Ca2+ solution, 150 mM NaCl was replaced by 50 mM CaCl2 and adjusted with N-methyl-D -glucamine (NMDG) to an osmolarity of 320 mOsm (pH 7.4). As for recording synaptic currents (cf. above), CsCl-based pipette solution was used. Recordings were obtained at room temperature. Salts and buffers were purchased from AppliChem (Darmstadt, Germany), kainate and CTZ were received from Abcam (Milton, United Kingdom), Naspm from Alomone Labs (Israel), and IEM-1460, JNJ 55511118 and GYKI53655 from Tocris (Bristol, United Kingdom).
3
Glutamate-Induced Currents in HEK Cells and Neurons
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For glutamate-induced whole-cell currents in HEK cells, cells were cultured and
transfected with GluA1, GluA1 plus GSG1L (ratio at 1:1) and GluA1/GSG1L (the
cDNA amount for GluA1 or GluA1/GSG1L is the same, 0.2 μg, and an
empty plasmid was used to make the total cDNA amount of 0.4 μg per
transfection). glutamate-induced whole-cell currents in HEK cells were recorded
at −70 mV by local application of 1 mM glutamate and
100 μM cyclothiazide, in presence of 10 μM NBQX in
the external solution. The tip of Mini-manfold was placed at
∼100 μM away from the recorded HEK cells. For the whole-cell
currents in neurons evoked by Kainate or glutamate, dual whole recording were
performed. The tip of the Mini-manfold was placed at ∼100 μM
away from the recorded neurons. Kainate (1 mM, Abcam) and glutamate
(1 mM, Sigma) solutions containing 100 μM cyclothiazide
(Abcam), 0.5 μM TTX, 100 μM D-APV and
100 μM picrotoxin were sequentially applied for 0.5 s with
an interval of 50 s. ACSF also contained 100 μM D-APV,
0.5 μm TTX, 100 μM picrotoxin and
10 μM NBQX (to facilitate the decay of the AMPAR-mediated
whole-cell currents). IKa/IGlu ratios were
calculated by dividing the peak current evoked by Kainate with peak current
evoked by glutamate. The data were presented as mean±s.e.m.
transfected with GluA1, GluA1 plus GSG1L (ratio at 1:1) and GluA1/GSG1L (the
cDNA amount for GluA1 or GluA1/GSG1L is the same, 0.2 μg, and an
empty plasmid was used to make the total cDNA amount of 0.4 μg per
transfection). glutamate-induced whole-cell currents in HEK cells were recorded
at −70 mV by local application of 1 mM glutamate and
100 μM cyclothiazide, in presence of 10 μM NBQX in
the external solution. The tip of Mini-manfold was placed at
∼100 μM away from the recorded HEK cells. For the whole-cell
currents in neurons evoked by Kainate or glutamate, dual whole recording were
performed. The tip of the Mini-manfold was placed at ∼100 μM
away from the recorded neurons. Kainate (1 mM, Abcam) and glutamate
(1 mM, Sigma) solutions containing 100 μM cyclothiazide
(Abcam), 0.5 μM TTX, 100 μM D-APV and
100 μM picrotoxin were sequentially applied for 0.5 s with
an interval of 50 s. ACSF also contained 100 μM D-APV,
0.5 μm TTX, 100 μM picrotoxin and
10 μM NBQX (to facilitate the decay of the AMPAR-mediated
whole-cell currents). IKa/IGlu ratios were
calculated by dividing the peak current evoked by Kainate with peak current
evoked by glutamate. The data were presented as mean±s.e.m.
4
Kainate-Induced Hippocampal Alterations
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To elicit status epilepticus, male rodents were subjected to intraperitoneal (i.p.) injection of vehicle control (autoclaved H2O or saline) or kainate (abcam) as described40 (link) at 9 mg/kg for Sprague-Dawley rats weighing 200–250 g (Fig. 8b,c ), 20 and 30 mg/kg for CD001 rats weighing 110–160 g (Fig. 8d,e ), 15 and 30 mg/kg for C57BL/6 J mice weighing 20 g (Supplementary Fig. S8 ). Rodents were returned to their home cage and video monitored. At 3 h and 8 h post injection, 2 hippocampi per rat was homogenized in modified RIPA buffer (1 mL) containing 1% SDS and protease inhibitors, sonicated briefly, and centrifuged at 14,000 rpm. Two hippocampi per mouse was homogenized as described65 (link) to isolate crude homogenate (S1), soluble (S2) and membrane (P2) protein fractions. Hippocampal lysates were immunoblotted with antibodies for GIRK1 and GIRK2 N termini (1:200, Supplementary Fig. S2 ), c-Fos, c-Jun, anti-β-tubulin, or GAPDH (1:500–1:1,000, all Cell Signaling). Several hippocampal lysates from vehicle-treated rats did not show any immunoblot bands for cleaved GIRK (Fig. 8b–e ). Therefore, background-subtracted immunoblot band intensity ratios of cleaved GIRK/loading controls (β-tubulin or GAPDH) from kainate-injected rats were taken as 100%. The ratio from vehicle-injected rats were then normalized to the ratio from kainate-injected rats to obtain % KA.
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