Zd7288

For all experiments, external AFSF saline included 20 µM 6,7 dinitroquinoxaline-2,3-dione (DNQX), 25 µM D-22-amino-5 phosphonovaleric acid (D-APV), which were obtained from Alomone Labs, and 2 µM SR-95531 (gabazine; Abcam). In some experiments, 10 µM ZD7288, 10 µM XE991, 2 µM CGP-55845, or 0.5 µM TTX was included in the ACSF (Abcam). In addition, for some experiments, 2 mM NiCl₂ or 50 µM BaCl₂ (Sigma-Aldrich) was included in the ACSF. ZD7288 was only introduced through the bath transiently, for 3–4 min. This prevented a nonspecific depolarization that occurs with continuous bath application, yet provided a stable block for ∼30 min (Kim and Johnston, 2015 (link)).

8-bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) sodium salt, 8-bromo-guanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) sodium salt (Tocris, R+D Systems, Wiesbaden, Germany), Cytidine-3′,5′-cyclic monophosphate (cCMP), sodium salt and Uridine-3′,5′-cyclic monophosphate (cUMP), sodium salt (BIOLOG Life Science Institute, Bremen, Germany) were added to the recording pipette. Properties of I_h were determined 10–15 min after obtaining the whole-cell configuration. The soluble guanylyl cyclase inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; Sigma, Munich, Germany) was prepared as a stock solution in DMSO (10 mM) and diluted in ACSF to obtain the final concentration (10 μM). The concentration of DMSO was below 0.1%. Slices were kept for 10 min in ODQ before starting the recordings. In order to block adenylyl cyclase, the slices were preincubated with 200 μM SQ 22536 (Tocris) for 2 h. HCN channels were blocked by washing the slices for 10 min with 20 μM ZD7288 (Abcam, Cambridge, UK). Inward-rectifier K⁺ channels were blocked by Tertiapin-Q (Tocris).

All drugs were added to the aCFS medium and bath perfused at the following final concentrations: 10 μM 4-Ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidin chloride (ZD7288, Abcam; HCN channel blocker), 1 μM tetrodotoxin (TTx, Alomone Labs; voltage-dependent Na⁺ channel blocker), 10 μM 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX, Tocris; AMPA receptors antagonist), 30 μM (RS)-3-(2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid [(RS)-CPP, Tocris; NMDA receptors antagonist] and 10 μM bicuculline methiodide (Sigma-Aldrich; GABA_A receptors antagonist).