3h sch23390

Methylphenidate hydrochloride (MPD; Ritalin®) was produced by Novartis AG (Basel, Switzerland). Cocaine hydrochloride, R(+)-SCH 23390 hydrochloride (SCH 23390) and Dopamine Hydrochloride were purchased from Sigma Aldrich Co. (St. Louis. MO, USA). [³H]-Dopamine (specific activity: 56.8 Ci/mmol) and [³H]-cAMP (specific activity: 28.1 Ci/mmol) were acquired from PerkinElmer (Massachusetts, USA). [³H]-SCH23390 (specific activity: 70.3 Ci/mmol) was acquired from Amersham (USA). All other chemicals were of the highest purity obtainable from regular commercial sources. Each experiment was performed at least 3 times.

Brain regional D1DR-binding densities were examined as described previously [23 (link),26 (link)]. Slide-mounted sections were incubated in Tris-buffer (50 mM Tris–HCl containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, and 1 mM MgCl₂; pH 7.7) containing 0.2 nM [3H]SCH 23390 (specific activity 66.0 Ci/mmol, Amersham (Amersham, UK)) for 90 min at room temperature. Sections for non-specific binding were incubated in Tris-buffer containing 0.2 nM [3H]SCH 23390 and 10−7 M cis-flupenthixol. After incubation, the slides were drained, washed twice for 5 min in buffer at +4 °C and briefly dipped twice into distilled water (+4 °C). Sections were dried at room temperature overnight and exposed to a tritium-sensitive film (3H Hyperfilm^®, Amersham) at −20 °C for 6 weeks, together with Amersham 3H Microscale Autoradiography Standards^®.