Stereological quantification

Quantitative analyses were performed by computer-assisted stereology using a Dell workstation receiving live video feed from an Optronics MicroFire color video camera (East Muskogee, OK) attached to a Nikon 80i light microscope, equipped with a Ludl MAC5000 motorized stage and a Heidenhain z-axis encoder. The optical dissector probe was used in conjunction with fractionator sampling using StereoInvestigator software (v. 10, MBF Bioscience, Williston, VT). Regions of interest were traced at 4× magnification (N.A. 0.75), and a square grid no larger than 1,000 μm² was superimposed in random orientation over the region of interest. Six sections per subject spanning the rostrocaudal extent of the head of the caudate nucleus, putamen, and nucleus accumbens regions (800 μm apart) were sampled for density of Ch+ and PV+ interneurons. Interneurons were counted at 20× magnification (N.A. 0.75) in a 350 μm² counting frame with a dissector height of 9 μm, with a coefficient of error (Gundersen m = 1) less than or equal to 0.1. Interneurons were counted only if they had a well-defined, darkly stained soma that came into view within the counting frame and initial segments of at least one visibly stained process. Incomplete neurons were excluded and comprised only a small proportion of stained neurons (typically less than 2 in approximately 20–25 sampling sites per section). Soma area for interneurons was additionally measured in one in every five interneurons counted using the Nucleator probe with a 4-point array in StereoInvestigator. Total density of interneurons was calculated as the estimated population divided by the planimetric volume of the region sampled. We additionally compared the density of Ch+ and PV+ interneurons relative to the total density of neurons for all regions of interest, as previously determined from Nissl-stained sections and quantified using previously published parameters (Hanson et al. 2018a, b) in the newly sampled subjects and territories. Additional stereological parameters, including average number of sampling sites and coefficient of error by region of interest, are listed in Tables 2 and ​and33.

Stereological sampling parameters for Ch+ interneurons

Stereological sampling parameters for PV+ interneurons