Transverse hippocampal slices (400 μm) were made using a standard mechanical tissue chopper and allowed to recover in oxygenated (95% O2/5% CO2) artificial cerebrospinal fluid (ACSF) at 32 °C for 1 h. cLTP was induced using 200 nM N-methyl-d -aspartate (NMDA; Cayman Chemical, Ann Arbor, MI, USA) in 0 Mg2+ ACSF for 10 min followed by 0.1 μM rolipram + 50 μM forskolin (Cayman Chemical, Ann Arbor, MI, USA) in 0 Mg2+ ACSF for 15 min [29 (link),30 (link)]. ACSF lacking Mg2+ was used as NMDA receptors are normally blocked by such ions.
After 1 h of recovery, slices undergoing the cLTP induction protocol were incubated in ACSF with 25 μM β-lactone for 30 min or the following specific kinase inhibitors for 1 h; 20 μM U0126 (Cayman Chemical, Ann Arbor, MI, USA), 5 μM KT5720 (Cayman Chemical, Ann Arbor, MI, USA), or 5 μM KT5823 (Cayman Chemical, Ann Arbor, MI, USA); or the neddylation inhibitor MLN4924 (2 μM) (Cayman Chemical, Ann Arbor, MI, USA) followed by cLTP induction. Slices were then collected at different timepoints and processed for immunohistochemistry.
After 1 h of recovery, slices undergoing the cLTP induction protocol were incubated in ACSF with 25 μM β-lactone for 30 min or the following specific kinase inhibitors for 1 h; 20 μM U0126 (Cayman Chemical, Ann Arbor, MI, USA), 5 μM KT5720 (Cayman Chemical, Ann Arbor, MI, USA), or 5 μM KT5823 (Cayman Chemical, Ann Arbor, MI, USA); or the neddylation inhibitor MLN4924 (2 μM) (Cayman Chemical, Ann Arbor, MI, USA) followed by cLTP induction. Slices were then collected at different timepoints and processed for immunohistochemistry.