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4 protocols using potassium chloride (kcl)

1

Cultured Rat Hippocampal Neuron Activation

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Cultured hippocampal neurons were prepared from embryonic day 18 Sprague-Dawley rat brains. Dissociated neurons on poly-l-lysine coated (1 mg ml−1) coverslips were placed in neurobasal medium supplemented with B27 (Invitrogen), 0.5 mM l-glutamate and penicillin–streptomycin. For activation, cultured neurons at days in vitro (DIV) 18–21 were treated with NMDA (Sigma; 20 µM, 3 min), KCl (Sigma; 50 mM, 5 min) or l-glutamate (Sigma; 50 µM, 30 min). For induction of NMDA receptor-dependent LTD or mGluR-LTD, cultured neurons were treated with NMDA (20 µM, 3 min) and DHPG (Tocris; 50 μM, 30 min), respectively, and returned to normal conditioning medium. For the blockade of NMDA receptors, cultured neurons were pretreated with APV (Tocris; d-2-amino-5-phosphovalerate; 50 µM, 30 min), followed by stimulation with NMDA (20 µM, 3 min) in the presence of APV or with l-glutamate (50 µM, 1 min) or KCl (30 mM, 1 min) in the absence of APV. For chronic neuronal inhibition or activation, neurons were treated with tetrodotoxin (Tocris; TTX; 1 µM, 48 h) or with bicuculline (Tocris; Bic; 10 µM, 36 h). For blockade of metalloproteinase and γ-secretase activity, neurons were pretreated with GM6001 (Enzo Life Sciences; 2.5 and 25 µM, 30 min), DAPT (Sigma; 250 nM, 2 h; 2 µM, 3 h) and L-685,458 (Calbiochem; 1 µM, 30 min) before and during NMDA stimulation (20 µM, 3 min).
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2

Npas4Cre;Ai14 Neuron Culture Protocol

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Cortical primary neuron culture was prepared from Npas4Cre;Ai14 P1 brains according to previous publication.28 (link) Neurons were plated on glass slides within 24-well plates with 150k neurons per slide and one slide per well. Neurons were cultured in Neurobasal A medium (NBA, Invitrogen) supplemented with B27 (Invitrogen), GlutaMAX (Invitrogen), TTX (0.5 mM, Tocris), and APV (50 mM, Tocris), and were maintained in a humidified incubator with 5% CO2 at 37°C.
On day in vitro (DIV) 4, the medium was removed and reserved, and neurons were stimulated with NBA containing one of the following: KCl (50 mM), KCl+EGTA (5mM), forskolin (10 μM, Tocris), BDNF (50 ng/ml, Pepro Tech), NT3 (50 ng/ml, Pepro Tech), NT4 (50 ng/ml, Pepro Tech), CNTF (100 ng/ml, Pepro Tech), or VEGF (100 ng/ml, R&D Systems). After 1 hour of stimulation, the medium was replaced with reserved conditioned medium. On DIV6, neurons were fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich) in phosphate buffered saline (PBS) for subsequent imaging and quantification.
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3

Insulin, Kinase Inhibitors, and Vanadium Compound Preparation

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NovoRapid® insulin aspart 100 units/mL (Novo Nordisk, Bagsværd, Denmark), sotrastaurin (Item No. 16726, Cayman Chemical, Ann Arbor, MI, USA) and (R)-(+)-etomoxir sodium salt (Cat. No. 4539, Tocris Bioscience) were diluted before use in sterile filtered PBS (137 mM NaCl, 2.7 mM KCl (cat. P9541, Merck KGaA), 10 mM Na2HPO4 (cat. 12695147, ACROS Organics™), 1.8 mM KH2PO4 (cat. 205920025, ACROS Organics™), pH 7.4). BVT948 (Item No. 16615, Cayman Chemical) and wortmannin (Cat. No. 1232, Tocris Bioscience) were dissolved in dimethyl sulfoxide (DMSO) (cat. D8418, Merck KGaA). BMOV was synthesized using the method previously described by Caravan el al. [48 (link)]
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4

Vasodilator Compounds in Saline

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Losartan and methacholine (used for vivo studies) were dissolved in 0.9% saline. KCl, PD123319 (Tocris), Angiotensin II and novokinin (Abcam) were dissolved in water. Unless stated otherwise, all chemicals were of the highest grade available and were purchased from Sigma Chemicals (St. Louis, MO).
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