Small blocks (approximately 1 cm3) of Broca's area 44 were dissected from the posterior portion of the third frontal convolution (pars opercularis) of the left hemisphere, embedded in celloidin, and sectioned at 40 µm (Figure 1). Nissl-stained sections were examined to identify sections containing area 44 based on cytoarchitectonic characteristics of this area as described in the "Results" section. Five sections evenly spaced throughout the sections containing area 44 were chosen for measurement of cell density. For example, if after identifying the extent of area 44 in slides from the block, area 44 was located in 15 slides (cut at 40 µm spaced 400 µm apart), we sampled every third slide or every 1.2 mm. All slides were coded so that the investigators (L.D.S. and J.M.) were blind to diagnosis during the analysis. Slides of area 44 were analyzed under an ×100 oil objective on either a Zeiss Standard (L.D.S.) or Zeiss Axiophot(J.M.) (Carl Zeiss, Oberkochen, Germany) microscope with video output to a personal computer. The custom designed software Quantify (Oguz Algan, MD, PhD, Department of Neurobiology, Yale University School of Medicine) was used to define a 3-dimensional box (dimensions: 80 µm [x] × 60 µm[y] × 25 µm [z]) within the thickness of the section, allowing at least 5-µm guard zones at the top and bottom of the section, and to apply a direct, 3-dimensional counting method.27,28 Neurons and glia were counted separately in a linear probe of stacked counting boxes extending from the pial surface to the underlying white matter. Small neurons were distinguished from glia on the basis of cytologic characteristics, including presence of visible cytoplasm, a less distinct nuclear membrane, and a less heterogeneous distribution of chromatin material in the nucleus as illustrated previously.29 Laminar boundaries, which were most clearly visible at low power (6.3×), were marked on the linear probes by switching back and forth between 6.3× and 100× objectives during the analysis so that cell density for each of the 6 layers could be calculated. Total length of the linear probe provided a measurement of cortical thickness. Values from the 5 linear probes were averaged to obtain a mean value of neuronal density, glial density, and cortical thickness for each case. Because layers II and IV are very thin in area 44, an additional 6 to 12 linear probes were analyzed in which only neurons in layers II and IV were counted. This ensured that a minimum volume of 3 × 10−3 mm3 (range, 3.00-4.59 × 10−3 mm3) was sampled for these small granular layers, a volume comparable to the sampling volume in layers V and VI (4.05 × 10−3 mm3 and 4.93 × 10−3 mm3, respectively). Linear probes of layers II and IV were located on the same 5 sections but spaced a minimum of 500 µm from the original probes. Pyramidal and nonpyramidal density was assessed in a minimum of 2 linear probes in all cases and in the additional 6 to 12 linear probes per case for layers II and IV. Pyramidal neurons were identified by the triangular shape of the nucleus; all other neurons were classified as nonpyramidal.