The electrophysiological experiments were carried out essentially as described18 (link),52 (link). Perforated patch-clamp recordings were performed from genetically marked AgRP and POMC neurons in coronal slices (270 µm) containing the ARC from adult POMC-Gi or AgRP-Gq;POMC-Gi male and female mice. Neurons were identified by their anatomical location in the ARC and by their ZsGreen or tdTomato fluorescence. Unless otherwise stated, the artificial cerebrospinal fluid contained 10−4 M picrotoxin (P1675, Sigma-Aldrich), 5 × 10−6 M CGP (CGP-54626 hydrochloride, BN0597, Biotrend), 5 × 10−5 M DL-AP5 (dl -2-amino-5-phosphonopentanoic acid; BN0086, Biotrend) and 10−5 M CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; C127, Sigma-Aldrich) to block GABAergic and glutamatergic synaptic input. Perforated patch-clamp experiments were conducted using protocols modified from previous studies. The used DMSO concentration (0.1–0.3%) had no noticeable effect on the investigated neurons. CNO was bath-applied at a flow rate of ~2.5 ml min−1 at a concentration of 3 µM for 5 min.
6 cyano 7 nitroquinoxaline 2 3 dione
Manufactured by Merck Group
Sourced in Switzerland, United Kingdom, Canada
6-cyano-7-nitroquinoxaline-2,3-dione is a synthetic compound that acts as an antagonist for the NMDA receptor. It has applications in experimental research settings.
Automatically generated - may contain errors
Lab products found in correlation
Strychnine, by Merck Group (2 mentions)
Bn0086, by Biotrend (1 mentions)
P1675, by Merck Group (1 mentions)
Dl 2 amino 5 phosphonovaleric acid ap5, by Merck Group (1 mentions)
Substance p sp, by Merck Group (1 mentions)
Bicuculline, by Merck Group (1 mentions)
Clampfit software, by Molecular Devices (1 mentions)
Pentylenetetrazole, by Merck Group (1 mentions)
Topiramate, by Merck Group (1 mentions)
Stiripentol, by Merck Group (1 mentions)
Dl 2 amino 5 phosphonopentanoic acid, by Merck Group (1 mentions)
Carbamazepine, by Merck Group (1 mentions)
Picrotoxin, by Merck Group (1 mentions)
Glutamate, by Merck Group (1 mentions)
N methyl d aspartate, by Merck Group (1 mentions)
Gamma aminobutyric acid, by Merck Group (1 mentions)
D 2 amino 5 phosphonopentanoic acid ap 5, by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Gabazine, by Merck Group (1 mentions)
Dl 2 amino 5 phosphonopentanoic acid ap5, by Merck Group (1 mentions)
6 protocols using 6 cyano 7 nitroquinoxaline 2 3 dione
1
Electrophysiological Recordings of ARC Neurons
2
Pharmacological Isolation of Pacemaker Neurons
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The pharmacological cocktail used to synaptically isolate pacemaker neurons contained 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 10 μM DL-2-amino-5-phosphonovaleric acid (AP5), 1 μM strychnine, and 10 μM bicuculline (all from Sigma).
Substance P (SP, Sigma) and the μ opioid receptor agonist D-Ala2-N-Me-Phe4-Glycol5-enkephalin (DAMGO, Sigma) were dissolved in aCSF and bath-applied at their final concentration of 0.5 μM and 0.25 μM, respectively. A single modulatory drug was applied per preparation in order to avoid cross effects. Preparations were exposed to a drug for a 15 min period and its effects on respiratory rhythm cycle frequency were analyzed at the end of this period using Clampfit software (Molecular Devices). The reversal of any observed effects was tested for each drug after a 30 min period wash-out during which fresh aCSF was superfused in the recording chamber.
Values are given as mean ± SEM. Student's t tests with a Mann-Whitney Rank Sum test or ANOVA tests were used to assess differences (taken to be significant at P < 0.05) between values in the presence of the drugs and controls.
Substance P (SP, Sigma) and the μ opioid receptor agonist D-Ala2-N-Me-Phe4-Glycol5-enkephalin (DAMGO, Sigma) were dissolved in aCSF and bath-applied at their final concentration of 0.5 μM and 0.25 μM, respectively. A single modulatory drug was applied per preparation in order to avoid cross effects. Preparations were exposed to a drug for a 15 min period and its effects on respiratory rhythm cycle frequency were analyzed at the end of this period using Clampfit software (Molecular Devices). The reversal of any observed effects was tested for each drug after a 30 min period wash-out during which fresh aCSF was superfused in the recording chamber.
Values are given as mean ± SEM. Student's t tests with a Mann-Whitney Rank Sum test or ANOVA tests were used to assess differences (taken to be significant at P < 0.05) between values in the presence of the drugs and controls.
3
Anticonvulsant Compounds: Preparation and Use
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Carbamazepine (CBZ), topiramate (TPR), stiripentol (STP), pentylenetetrazole (PTZ), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphonopentanoic acid (APV) were purchased from Sigma-Aldrich, sodium valproate (VPA) from Sanofi-Aventis, clobazam (CLB) from Lipomed AG, Switzerland and fenfluramine (FA) from Peak International Products B.V. Compounds were dissolved in DMSO and diluted in embryo medium to achieve a final DMSO concentration of 1% w/v, which served as a vehicle control (VHC). In case of treatment for the hyperthermia-experiment, a final DMSO concentration of 0.1% w/v was used.
4
Neuroactive Compound Preparation Protocol
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Recombinant-murine interferon gamma (RnD Systems), tumor necrosis factor-alpha (Prospec), and interleukin 1 beta (Prospec) were aliquoted at 1000x stocks and stored at −20C. All neuroactive compounds including gamma-aminobutyric acid (GABA), N-methyl-D-aspartate (NMDA), 1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), glutamate, glycine, strychnine, picrotoxin, and D(−)−2-Amino-5-phosphonopentanoic acid (AP5) were purchased from Sigma. All factors were diluted to 5x working solutions in pre-warmed neuron feed media and further diluted 5 fold upon addition to neuronal cultures.
5
Optogenetic Stimulation of Nkx2-1 Interneurons
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Cell-type-selective optogenetic stimulation was performed via expression of the P2X2R in Nkx2-1 interneurons in Nkx2-1Cre;Z/EG;R26::P2X2R-EGFP neonates. Our standard ultraviolet laser LSPS set-up30 (link) was used to focally release DMNPE-caged ATP (100 μm; adenosine 5′-triphosphate, P3-(1-(4,5-dimethoxy-2-nitrophenyl)ethyl) ester, disodium salt; Life Technologies, UK) across the extent of the target grid. The emitted power of the DPSL-355/30 laser (Rapp Optoelectronic GmbH, Germany) was adjusted to evoke action potentials in Nkx2-1 interneuron only when focused at the laser target spot immediately above the recorded cell. To establish these setting, the laser was fired repeatedly across the laser target grid, and the laser power adjusted, while recording the interneuron in either loose cell-attached or whole-cell current-clamp configuration. Photostimulation was performed in high divalent ACSF, which included CNQX (30 μm; 6-cyano-7-nitroquinoxaline-2,3-dione; Sigma, UK) and AP-5 (30 μm; DL-2-Amino-5-phosphonopentanoic acid solid; Sigma UK) to block glutamatergic activity. To observe IPSCs in the postsynaptic recorded PYRs, we used a high-chloride intracellular electrode solution (EGABA∼0 mV)30 (link) and voltage-clamped recorded cells at a holding potential of −70 mV.
6
Isolating mGluR1a-Mediated Excitatory Currents
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To isolate mGluR1a-mediated excitatory postsynaptic currents (mGluR1a-EPSCs), antagonists of non-NMDA (6-cyano-7-nitroquinoxaline-2,3-dione; CNQX, 20 μM), NMDA (DL-2-amino-5-phosphonopentanoic acid; AP5, 50 μM), and GABA-A (gabazine, 5 μM) receptors (all from Sigma, Oakville, ON, Canada), as well as the glutamate transporter blocker DL-threo-b-benzyloxyaspartic acid (TBOA, 30 μM) (Tocris, Ellisville, MO,USA) were bath-applied. In some experiments, the mGluR1a receptor antagonist (S)(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385, 100 μM), the TRP channel antagonist 1–2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)proproxy]ethyl-1H-imidazole (SKF96365, 30 μM) (both drugs from Tocris), or the phospholipase C inhibitor 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122, 10 μM) (Calbiochem, Gibbstown, NJ, USA), were added to the external solution.
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