Objective Inhibition of phosphodiesterase-4 (PDE4) increases phosphorylation of the cAMP response element binding protein (pCREB) and hippocampal neurogenesis, and produces antidepressant-like and cognitive-enhancing effects.This study is to establish causal links among these actions and explore the splice variants of PDE4 responsible for emotional-related effects.Methods Using tests sensitive to antidepressant and anxiolytic drugs, the emotional-related behavioral effects were determined.The hippocampal neurogenesis and pCREB expression were measured by fluorescence immunohistochemistry.Long-form PDE4Ds were silenced by the bilateral microinfusion of lentiviral vector containing PDE4D microRNAs (4DmiR) into the prefrontal cortex (PFC).Cognitive behaviors were measured by novel object recognition test (NOR) and Morris water maze test (MWM).The emetic potential was evaluated by the assessment of the anaesthetic reversal effect.The levels of cAMP and proteins were determined by ELISA and immunoblotting analyses, respectively.Resnlts (1) Chronic administration ofrolipram (0.31-1.25 mg/ kg, 16-23 days) produced antidepressant-and anxiolytic-like effects in mice.It also increased cAMP and pCREB levels in the hippocampus and prefrontal cortex, but increased Sox2, a marker for mitotic progenitor cells, only in the hippocampus.(2) Chronic rolipram treatment also increased hippocampal neurogenesis; Methylazoxymethanol (MAM), a toxin to proliferating cells, reversed rolipram-induced increases in BrdU-positive cells and pCREB in the hippocampus and partially blocked its behavioral effects.(3) Rolipram and MAM didnt alter the percentage of newborn neurons or pCREB in BrdU-positive cells.(4) As rolipram-treated wild-type mice, PDE4D-deficient mice also displayed increased hippocampal neurogenesis and pCREB, which were not altered by rolipram.(5) Two weeks after the bilateral PFC microinfusion, 4DmiR down-regulated PDE4D4/5 splice variants, but not PDE4A, PDE4B.(6) Rolipram and/or 4DmiR treatments decreased immobility in tail-suspension test and forced swimming test in mice.(7) Rolipram and/or 4DmiR enhanced the long-term memory in mice.(8) Rolipram decreased the duration of anaesthesia in the alpha2 adrenergic receptor-mediated anesthesia, but not 4DmiR treatment alone.(9) Rolipram and/or 4DmiR treatments increased cAMP, pCREB and BDNF in PFC.Conclusion (1) PDE4 inhibition by rolipram produces its antidepressant-and anxiolytic-like effects in a manner that at least partially depends on its neurogenic action in the hippocampus.(2) The cAMP/CREB signaling in hippocampal newborn neurons is critical for neurogenesis and contributes to the behavioral effects of rolipram.(3) The PDE4D subtype plays a critical role in the mediation of hippocampal neurogenesis and cAMP/CREB signaling.(4) Long-form PDE4Ds, at least D4 and D5, play critical roles in the mediation of antidepressant-like and cognitive-enhancing effects.