Primary striatal neuronal cultures were using postnatal day 1 mouse neonates from homozygous breeding of WT or βarr2-KO mice. Striatal neurons were prepared as described (68). Neurons were plated on a poly-l-lysine–coated 96-well plate for forskolin-stimulated cAMP accumulation assays or a poly-l-lysine–coated glass-bottom confocal dish for KOR internalization assays. Media were replaced with one-third of refresh neural basal complete media supplemented with 10 μM B-D-arabinofuranoside (Sigma #C1768) from day in vitro 4 (DIV4) every other day until assay was performed.

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