Colocalization of somatostatin, neuropeptide Y, neuronal nitric oxide synthase and NADPH-diaphorase in striatal inerneurons in rats

The neuropeptides somatostatin (SS), neuropeptide Y (NPY), the enzyme neuronal nitric oxide synthase (nNOS) and enzymatic activity for NADPH diaphorase (NADPHd) are extensively colocalized in striatal interneurons, which has led to the widespread tendency to operationally treat all four substances as being completely colocalized within a single class of striatal interneurons. We have explored the validity of this assumption in rat striatum using multiple-labeling methods. Conventional epi-illumination fluorescence microscopy was used to examine tissue triple labeled for SS, NPY and nNOS, or double-labeled for SS and nNOS or for SS and NPY. In tissue double-labeled for SS and nNOS, confocal laser scanning microscopy (CLSM) images of SS and nNOS labeling were compared to subsequent NADPHd labeling. We found that SS, NPY and nNOS co-occurred extensively, but a moderately abundant population of neurons containing SS and nNOS but not NPY was also observed, as were small populations of SS only and nNOS only neurons. About 80% of SS + neurons contained NPY, and no NPY neurons were devoid of SS or nNOS. All neurons containing nNOS in rat striatum were found to contain NADPHd. Combining our various quantitative observations, we found that of those striatal neurons containing any combination of SS, NPY, nNOS and NADPHd in rats, about 73% contained all four, 16% contained SS, nNOS and NADPHd, 5% contained SS only, and 6% contained only nNOS and NADPHd. These results indicate that while there is a large population of striatal neurons in which SS, NPY, nNOS and NADPHd are colocalized in rats, there may be smaller populations of striatal neurons devoid of NPY in which SS or nNOS/NADPHd are found individually or together.