Fk506

Picrotoxin (Sigma #P1675), bicuculline (Sigma #505875), tetrodotoxin (Cayman #14964), BI2536 (Selleckchem #S1109), Calyculin A (Abcam #ab141784), DL-AP5 (Tocris #0105), CNQX (Tocris #0190), FK506 (Tocris #3631), digitonin (Sigma #D141), nimodipine (Sigma #482200), conotoxin (Sigma #343781-M), agatoxin (Abcam #ab120210). All combined treatment paradigms throughout the manuscript were carried out in the presence of PTX (unless otherwise stated). As controls, pharmacological inhibitor treatments were also carried out in the absence of PTX in the same experiment.

Drugs were dissolved in HBSS, which was used as the experimental vehicle, and applied directly to the media containing NGF. Drugs concentrations are based on published literature: choline (Acros Organics, Geel, Belgium) (10 mM, 3 mM, and 1 mM) [22 (link)]; α-bungarotoxin (Thermofisher) (BGTX) (50 nM) [32 (link)]; calpeptin (Sigma Aldrich, St. Louis MO, USA)(26 μM) [33 ], ryanodine (Santa Cruz) (30 μM) [21 (link)]; Xestospongin C. (Tocris Biosciences, Bristol, UK.) (Xest C.) (1 μM) [20 (link)]; FK506 (Tocris Biosciences) (40 μM) [34 (link)]; Substance P (Tocris Biosciences) (Sub P) (1 μM) [20 (link)]; PNU 282987 (Tocris Biosciences) (10 μM) [35 (link)].

Transcription and translation inhibitors were applied from the beginning of treatments at time = 0 and were maintained at the same concentration through all stages of the experiment. Experimental designs contained both inhibitor-added and inhibitor-free conditions for each biological replicate. FP (Sigma; catalog no.: F3055), CHC (Sigma; catalog no.: C7698), and FK506 (Tocris; catalog no.: 3631) treatments (1 μM) were applied from time = 0 at the primary stage for “FK primary” condition, and from time = 1 h 30 min at the start of the secondary stage for the “FK secondary” condition. A control condition was run alongside these treatments, and DMSO was added at each stage of treatment to keep total DMSO additions equivalent across all three conditions. Therefore, all three conditions contained 1 μl DMSO per ml from time = 0 through 1 h 30 min at the start of the 2° stage, and 3 μl DMSO per ml from there on, as the FK secondary condition received FK in 1 μl DMSO and all conditions received 1 μl 5 μM Bic or DMSO as the 2° treatment.

DMEM:F12 was obtained from Corning, and fetal bovine serum, horse serum, antibiotics, TNFα, human EGF and human bFGF was obtained from GIBCO. Nifedipine, methoxyverapamil hydrochloride (D-600), lanthanum chloride, Bapta-AM, caffeine, dantrolene, Stattic, Synta66, EGTA, BSA, cycloheximide and collagenase were obtained from Sigma-Merck; papain was obtained from Worthington Biochemicals. Dexamethasone, 2-APB, 5′-N-Ethylcarboxamidoadenosine (NECA), ATPγS, NNC55-0396, 78c (CD38 inhibitor), xestospongin C, FK506 and ryanodine were purchased from Tocris. Insulin and 8BrcADPr were obtained from Santa Cruz. Fura-2 AM was obtained from Invitrogen. All the lipophilic drugs were dissolved in dimethyl sulfoxide (ethanol in the case of xestospongin C). The final concentration of the solvent was ≤0.1%.

cLTD was induced as previously described (Oh et al., 2006 (link)). Cultures were first incubated in ACSF for 30 min at room temperature: 125 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 33 mM D-glucose, and 25 mM HEPES (pH 7.3), followed by stimulation with 50 μM NMDA in ACSF (no MgCl₂) for 5 min. After NMDA treatment, neurons were replaced in regular ACSF and then subjected to the corresponding procedure at indicated time points. Cultures were then washed with ice-cold PBS once and lysed in cold 1% NP-40 homogenization buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 1 mM Na₂VO₄, and 1× Sigma protease inhibitor and phosphatase inhibitor cocktails). When indicated, cultures were pre-treated or post-treated with BAPTA-AM (20 μM), FK506 (10 μM), MG132 (10 μM), or MK801 (10 μM; all from Tocris) and were present until cells were lysed. cLTP was induced as previously described (Otmakhov et al., 2004 (link)) by application of forskolin (50 μM) and rolipram (0.1 μM; both from Tocris), 60 min before or after cLTD. Cells were lysed 60 min after cLTP or cLTD treatment. Lysates were centrifuged at 12,000 × g for 10 min at 4°C and protein in the supernatant was quantified by Bradford method assay kit (Bio-Rad Laboratories). The intensity of the cLTD protocol may vary depending on the cultures.