Localization of the Cyclic Guanosine 3,5-Monophosphate- Hydrolyzing Phosphodiesterase Type 9 in Rat Brain by Nonradioactive In Situ Hybridization

In this chapter, we describe a protocol for the localization of the cyclic guanosine 3′,5′-monophosphate-specific phosphodiesterase type 9 (PDE9) mRNA in the adult rat brain that uses digoxigenin-labeled riboprobes in a nonradioactive in situ hybridization (ISH). The three different riboprobes used all showed similar PDE9 mRNA expression patterns, detecting PDE9 in cell bodies throughout the whole brain. By using immunocytochemical double labeling of the ISH sections with the neuronal marker NeuN or the glial cell marker glial fibrillary acidic protein, the cells expressing PDE9 mRNA were further characterized. Double-labeling experiments revealed that PDE9 was predominantly expressed in neurons.