In situ hybridization histochemical and immunohistochemical evidence that striatal projection neurons co-containing substance P and enkephalin are overrepresented in the striosomal compartment of striatum in rats

Abstract

In a prior study, we showed that the few striatal projection neurons that contain both substance P (SP) and enkephalin (ENK) in rats may preferentially project to the substantia nigra pars compacta. Since striatal neurons that project to the pars compacta are thought to preferentially reside in the striosomal compartment, we investigated if striatal neurons that contain both SP and ENK are preferentially localized to the patch compartment. We used in situ hybridization histochemistry to double-label sections for SP and ENK to identify SP/ENK co-containing neurons, and immunolabeling of adjacent sections for the mu opiate receptor (MOR) to define the striosomal compartment. We found that 32.3% of neurons containing both SP and ENK were localized to the striosomal compartment, which itself only made up 12.8% of the striatum. Our results further showed that the density of neurons co-containing SP and ENK was three-fold higher in striosomes than in the matrix compartment. These results are consistent with the notion that SP/ENK colocalizing neurons preferentially project to pars compacta, and these and our prior results additionally raise the possibility that neurons of this type in the striatal matrix may also project to the pars compacta.

1

Schematic drawing of a transverse section through the striatum of a 4 month old rat that had been double-labeled by ISHH for SP (PPT) by autoradiographic ISHH and for ENK (PPE) by DIG-ISHH, overlain with the distribution of MOR+ striosomes as visualizrd by immunolabeling of the adjacent section. Perikarya labeled by ISHH for both SP and ENK are shown as dots, while the striosomes are shown as irregular enclosed contours. Abbreviations: AC – anterior commissure; CC – corpus callosum; V – ventricle.

2

Images of striatum in a 4 month old rat in adjacent coronal sections that had been either immunolabeled for MOR (A, B, D) or double-labeled for SP (PPT) and ENK (PPE), using ARG to visualize the SP and DIG to visualize the ENK (C, E, F). The MOR images show successively higher power views of the region of two double-labeled neurons (arrows) shown in C, E, F, with the box in A showing the field enlarged in B, and the box in B showing the field enlarged in D. C and D are at the same magnification and show the alignment of the two double-labeled neurons with the striosomal compartment. The images E and F show two focal planes of view, one focusing on the ARG silver-grain labeling (E), and one focusing on the DIG labeling (F). E and F are at the same magnification.