Sections were independently examined by two observers (H.C.E. and T.F.). A neuron was considered to be a large spindle-shaped
neuron if it was located in layer 5b among typical pyramidal neurons, had a unique basal dendrite that was as thick as its apical dendrite, had an elongated perikaryon larger than or as large as Y-27632 chemical structure local pyramidal neurons, was symmetrical about its vertical and longitudinal axes, and had a nucleus and nucleolus located in the middle of the perikaryon (Nimchinsky et al., 1999). Spindle-shaped neurons that were much smaller than the local pyramidal neurons or had very thin basal or apical dendrite were excluded. These criteria prevented elongated pyramidal (or lanceolated) neurons, inverted pyramidal
neurons, see more and small vertical fusiform interneurons in layer 6 from being counted as large spindle-shaped neurons. A fork cell was defined by a unique basal dendrite and a “forked” or “bifid” apical dendrite (Ngowyang, 1932). The total number of VENs was estimated at 63× with the optical fractionator (West et al., 1991) using a set of 225 × 168 μm2 optical dissectors with a 225 × 168 μm2 scan grid size for an exhaustive counting of the VENs (Butti et al., 2009 and Stimpson et al., 2011) (Figure S2A). The targeted number of counted VENs was set at 300. Using this strategy, we obtained averages of 202.8 ± 85.9 (mean ± SD) and 177.2 ± 35.2 VENs in the rhesus and cynomolgus macaques, respectively. The coefficients of error (CE) were all below the threshold CE of 0.1 (Schmitz and Hof, 2005), except for one hemisphere with a tolerated CE of 0.11 (Table S1). The thickness of the optical dissector was set to 80% of the section thickness; the top and bottom guard zones were each set to 10%. The thickness of the section was measured for each counting frame and averaged for the calculation of the total number estimate by using the Fractionator method (West et al., 1991). The total number of pyramidal neurons was estimated within the same region of interest (ROI) and in the same sections.
The dimensions of the sampling grid were set to 260 × 165 μm2 and the counting frame to 50 × 50 μm2 to count at least 300 pyramidal Temsirolimus purchase neurons per hemisphere. The counts were made in a sequence of counting frames selected by using a systematic random sampling. Perikaryal volumes were estimated by using the optical vertical planar principle (Stark et al., 2007) in 10 VENs, 20 local pyramidal neurons, and 10 layer 6 fusiform neurons in 3 rhesus, 3 cynomolgus, and 4 human AICs (left or right) (Figure S2B). The measured VEN and fusiform cells were selected randomly. The measured pyramidal neurons were always randomly selected within the direct vicinity of the measured VEN. Comparisons of the number or volume of cells across species or sides of the brain were made by using one-way or repeated-measure ANOVA and post hoc t test.