4 aminopyridine 4 ap

Cytosolic Ca²⁺ signal was determined at RT in cells loaded with 4.5 μM FURA-2·AM (Molecular probes Life technologies) (30 min) as previously described [50 (link)]. Cytosolic Ca²⁺ increases are represented as the normalized ratio of emitted fluorescence (510 nm) after excitation at 340 and 380 nm, relative to the ratio measured prior to cell stimulation (FURA-2 ratio 340/380). Cells were bathed in an isotonic solution (ISO) containing (in mM): 140 NaCl, 5 KCl, 1.2 CaCl₂, 0.5 MgCl₂, 5 glucose, 10 HEPES (300 mosmol/L, pH 7.4 with Tris). The following reactives were used during calcium experiments: 10 μM threo ifenprodil hemitartrate (If; a selective inhibitor of NMDA receptor having a GluN2B subunit; Tocris), 100 μM NMDA plus 100 μM Gly, 50 μM-(-) bicuculine methiodide (BIC; an antagonist of GABA A receptor; Tocris) plus 2.5 mM 4-aminopyridine (4-AP; a selective blocker of potassium channel Sigma).

Cultures were left to recover for a day after preparation. Drug application was started at DIV1 and was renewed with each change of medium (3 times per week) until the assessment time points (DIV3, 10 or 15). NMDAR inhibition was induced by DL-2-Amino-5-phosphonopentanoic acid (APV) (50 μM; Tocris), which has been shown to completely abolish NMDAR mediated responses in our preparation^{69 (link)}. Induction of oxidative stress was achieved by co-application of the γ-glutamylcysteine synthetase inhibitor L-Buthionine sulfoximine (BSO, 0.5, 1, 5, 10 μM, Sigma-Aldrich), and by the thioredoxin reductase blocker Auranofin (Au, 1 μM; Sigma). Enhancement of network activity was induced with 4-Aminopyridine (4AP, 100 μM; Tocris), a non-selective blocker of voltage-activated K⁺ channels.

ACSF contained the following (mM): 126 NaCl, 3 KCl, 25 NaHCO3, 1 NaHPO4, 2 MgSO4, 2 CaCl2, 14 dextrose with an osmolarity of 315-325mOsm. The internal solution for analysis of excitability and evoked synaptic responses was as follows (mM): 116 K-Glu, 4 KCl, 10 K-HEPES, 4 Mg-ATP, 0.3 Na-GTP, 10 Na-phosphocreatine, 0.4% biocytin (V_rev [Cl⁻¹] = −81 mV). The pH of the internal solution was adjusted to 7.35 with KOH and the osmolarity adjusted to 295mOsm with sucrose. The internal solution to assess spontaneous excitatory and inhibitory charge and events contained (mM): 20 KCl, 100 Cs-sulfate, 10 K-HEPES, 4 Mg-ATP, 0.3 Na-GTP, 10 Na-phosphocreatine, 3 QX-314 (Tocris Bioscience), 0.2% biocytin (Vrev [Cl−1] = −49.3 mV). Drugs. To assess the monosynaptic nature of BLA-EPSCs recordings were obtained in the presence of (in μM): 1 TTX (Tocris Bioscience), 100 4-aminopyridine (4-AP) (Tocris Bioscience). To determine possible changes in intrinsic excitability, the following blockers were bath applied (in uM): 20 DNQX (Tocris Bioscience), and 50 APV (Tocris Bioscience), 20 picrotoxin (Tocris Bioscience).

All cytokines were purchased from PeproTech (Rocky Hill, NJ, USA, (IL-1β: #211-11B, TNF-α: #315-01A, IL-4: #214-14, IL-10: #210-10). These peptides were dissolved in water at 10 μg/mL and stored in aliquots at −80 °C. Bath cytokine concentrations were used at 10 ng/mL or 30 ng/mL. Blockers used were: CsOH solution (Sigma-Aldrich, St. Louis, MO, USA, #232041), quinine (1 mM, K⁺ channel blocker, Sigma-Aldrich, #Q1125), 4-aminopyridine (4-AP, 1 mM, Kdr channel blocker, Tocris, Bristol, UK, #0940), BaCl₂ (200 µM, Kir channel blocker, Sigma-Aldrich, #217565), ML133 HCl (20 µM, Kir2.1 channel blocker, Tocris, #4549), Paxilline (50 µM, K_Ca1.1 blocker, Tocris, #2006), A740003 (100 µM, P2X7R antagonist, Tocris, #3701) and Suramin hexasodium salt (500 µM, P2X&Y receptor antagonist, Tocris, #1472).

The electrophysiology experiments were done after the iM-Neurons for 22 days. MFs-neuron cells were placed in a submerged chamber with artificial cerebrospinal fluid (ACSF) containing the following (in mM): 125 NaCl, 25 NaHCO₃, 1.25 NaH₂PO₄, 2.5 KCl, 25 glucose, 2 CaCl₂, and 1 MgCl₂. For the whole-cell patch-clamp recordings, recording electrodes (4–8 MΩ) were pulled from borosilicate glass tubing (outer diameter, 1.5 mm; inner diameter, 0.86 mm; Harvard Apparatus) filled with low Cl⁻ internal solution, containing the following (in mM): 136.8 K-gluconate, 7.2 KCl, 0.2 EGTA, 4 MgATP, 10 HEPES, 7 Na₂-phosphocreatine, 0.5 Na₃GTP (pH 7.3 with KOH). Individual cells were selected for the recordings based on a small round or ovoid cell body (diameters, 5–10 μm). The voltage-clamp recording was measured at a holding potential of −70 mV. The current-clamp was performed at −70 mV by using a holding current to adjust the membrane potential compatible. The tetrodotoxin (TTX, a voltage-gated Na+ -channel blocker, 1 μM, Sigma) and 4-aminopyridine (4-AP, a voltage-gated A-type K⁺-channel blocker, 1 mM, Tocris) were used in some experiments.