Minicolumnar pathology in autism Manuel F. Casanova, MD Daniel P. Buxhoeveden, PhD Andrew E. Switala and Emil Roy, PhD Abstract���Objective: To determine whether differences exist in the configuration of minicolumns between the brains of autistic and control patients. Background: Autism is a severe and pervasive developmental disturbance of childhood characterized by disturbances in both social interactions and communication, as well as stereotyped patterns of interests, activities, and behaviors. Postmortem neuropathologic studies remain inconclusive. Methods: The authors used a comput- erized imaging program to measure details of cell column morphologic features in area 9 of the prefrontal cortex and areas 21 and posterior 22 (Tpt) within the temporal lobe of nine brains of autistic patients and controls. Results: The authors found significant differences between brains of autistic patients and controls in the number of minicolumns, in the horizontal spacing that separates cell columns, and in their internal structure, that is, relative dispersion of cells. Specifically, cell columns in brains of autistic patients were more numerous, smaller, and less compact in their cellular configuration with reduced neuropil space in the periphery. Conclusions: In autism, there are minicolumnar abnormalities in the frontal and temporal lobes of the brain. NEUROLOGY 2002 58:428���432 Existing neuropathologic studies of autism provide for subtle if not mixed results. The subjective nature of the methodology and small case series may par- tially explain the disparate findings. Alternatively, attempts based on classic neuropathologic methods and its emphasis on cellular changes would neces- sarily prove unrewarding if the underlying distur- bance is one of circuitry. Given the importance of neocortical cellular organization and circuitry during development, we focused on the single radial column or cell minicolumn. The minicolumn is a basic func- tional unit of the brain that organizes neurons in cortical space.1-4 Thus, changes based on circuitry or spatial morphologic features may affect this funda- mental unit of cortical structure. Materials and methods. Material for this study in- cluded nine autistic and four control subjects from the Autism Research Foundation. Drs. M. Bauman and T. Kemper provided for diagnosis, tissue collection and pro- cessing. Specimens are readily available at Boston Medical Center. Five additional controls were taken from the Yakovlev���Haleem Collection, National Museum of Health and Medicine, Armed Forces Institute of Pathology, Wash- ington, D.C. Mean age was 12 years for autistic cases and 15 for controls. Diagnostic and postmortem information on the autistic cases has been reported elsewhere (table).5-14 The brains had been celloidin imbedded and cut into 35- m serial sections and Nissl stained. Adjacent slides were stained with the Loyez technique. We obtained digitized images of lamina III in each of three cortical areas: 9, posterior 22, and the middle tempo- ral gyrus (area 21). Some areas were unavailable for sev- eral of the case subjects and were considered as an unbalanced design. Minicolumns. In a minicolumn, a core line of neurons ascends vertically between layers VI and II. Most of the cells aggregate in a linear fashion a cell-poor area, usually denoted as the peripheral neuropil space, surrounds both sides. This area, which is noticeable in a cell body stain, is rich in unmyelinated axon fibers, dendritic arborizations, and synapses.15 The core area of a column and its immedi- ate surroundings appear to contain most of the neurons, apical dendrites, cortical efferents, and corticocortical fi- bers, as well as unmyelinated axons and synapses.16 My- elinated axon bundles presumably are cortical efferents originating in pyramidal cells in layers II and III. These bundles descend toward the white matter, lying within or adjacent to the cellular core of a column. Apical dendrites originating in layer V pyramidal cells ascend in bundles through or adjacent to the cell column core.17 The edges of the column core contain vertical fiber bundles of GABAer- gic interneurons. These bundles���especially marked in lamina III���are a source of lateral inhibition that is be- lieved to delineate individual minicolumns from their neighbors.2,3,18 Cortical areas. Area 21 is located primarily within the middle temporal gyrus on the lateral surface of the brain hemisphere. It lies bordered by areas 20 (ventral), 22 (dor- sal), 37 (caudal), and 38 (rostral). The surrounding areas are paralimbic19,20 and auditory parasensory.21 Area 21 is considered visual parasensory association cortex. Myeloar- chitectonic studies have shown a scarcity of myelinated fibers. At higher magnification, area 21 has a well-defined layer IIIb, which lies rectilinearly opposed to layer IV. Area 9 lies in the superior and middle frontal gyrus. Researchers have found19,22 that it is located in the middle third of the superior frontal gyrus covering both its dorso- lateral and dorsomedial surfaces. Microscopically, area 9 has a poorly defined lamina IV.23 Within Brodmann area 22, we focused on the tem- From Medical College of Georgia (Dr. Casanova), Augusta Department of Anthropology (Dr. Buxhoeveden), University of South Carolina, Columbia and Downtown Veterans Administration Medical Center (Drs. Casanova and Roy, and A. Switala), Augusta, GA. Supported by grants from the Theodore and Vada Stanley Foundation and the VA Merit Review Board. Received November 27, 2000. Accepted in final form October 11, 2001. Address correspondence and reprint requests to Dr. Manuel F. Casanova, Downtown VA Medical Center, 26 Psychiatry Service, 3B-121, Augusta, GA 30910 e-mail: casanova@np2.mcg.edu 428 Copyright �� 2002 by AAN Enterprises, Inc.

poroparietal auditory area (Tpt), which covers the lateral aspects of the superior temporal gyrus (STG) posterior to the caudal parabelt cortex up to the bank of the superior temporal sulcus, as well as the most posterior portions of the superior surface of the supratemporal plane (the pla- num temporale).21,24-31 Because no sulcal demarcation ex- ists for Tpt, it must be discerned microscopically from its general location on the STG. Tpt is located in the posterior regions of the STG: it appears immediately behind and lateral to the parabelt region labeled as caudal auditory parabelt (CP).26-28 It also appears on the most posterior portion of the superior surface of the supratemporal plane. Qualitatively, adult Tpt is characterized by having all six cortical layers well formed, an undulating border between layers III and IV, an indistinct border between IV and V, and an ���organ-pipe��� organization (distinct curvilinear col- umns of neurons arising in an irregular fashion from the white matter) in layer VI.25,31 Procedures. Our method is a modification of an earlier version described elsewhere.32 A region of interest (ROI) must be isolated and transferred to the computer imaging system. The ROI is obtained from a microscopic field of view at a chosen magnification. Magnifications of 100 are chosen to resolve both individual perikarya and the com- plete laminar depth. Usually, the ROI consists of the en- tire field of view as seen in the photomicrograph however, the field is cropped as needed to exclude tissue distortions and superfluous features such as vessels. The first stage of the column detection routine divides a region of interest into overlapping horizontal strips. Cell concentration is defined for each strip by (x) i e 8(x xi)2/d2 the sum being taken over all cells in that strip. The char- acteristic scale d is the width of a box that, placed at random in the field, would enclose one cell on average. The relative maxima and minima of mark the locations of the centers of aggregation of cell columns and the space be- tween them. Notice that the full width of the Gaussian in the definition of is such that cells within less than 1���2d of each other contribute to the same relative maximum of and are not resolved into distinct columns. For the primary contribution to come from a region containing, on aver- age, one cell, the height of the horizontal strips is approxi- mately 2d. Minicolumns are defined by the method as vertical clusters of large neurons delimited on either side by cell-sparse areas. Imaginary lines through the sparse areas partition a field into polygonal regions (figure 1). We refer to such a polygon together with the totality of small and large neurons contained within it as a minicolumn segment, from which we obtain several descriptive statistics. For this study, we report on five measures: columnar width (CW), peripheral neuropil space (NS), interneuronal distance (MCS), compactness (RDR), and gray level index (GLI). CW describes the diameter of a single minicolumn, or equivalently, the center-to-center distance between ad- jacent minicolumns. NS is the width of the cell-poor space on both sides of the minicolumn, found by subtracting the width of the column core from the CW. The column core is defined as that part of the column that contains 90% of the cell bodies. Up to 10% of the cells in the column may lie in the region designated as peripheral neuropil space so that the measurement of NS is insensitive to ���outlier��� points. MCS is the mean distance between nearby neurons within a column. The compactness parameter RDR is the ratio of second moments of the distribution of cells (the ratio of the greater to the lesser) divided by the ratio of second mo- ments of the entire minicolumn, including its empty space. Because ours is not a cell counting method, we prefer to account for overall cell density by measuring the area frac- tion occupied by Nissl-stained segments, called the GLI.33 Before column detection, the GLI is computed from the original image by thresholding. The total area of Nissl- stained objects, that is, the number of pixels below the threshold, is divided by the image area to yield the GLI. Whereas this measurement does not provide us with cell numbers, it estimates the amount of space within the minicolumns occupied by cell somas. Results. A multivariate analysis of variance was per- formed with diagnosis, that is, normal or autistic, hemi- sphere, and cortical area as a fixed factor and CW, NS, MCS, RDR, and GLI as the dependent variables. Age was included as a covariate. Preliminary testing included the source of material (Yakovlev or Bauman-Kemper) as a fixed factor to test for possible differences between control brains from the two collections (none were found). The Table Summary data on autistic patients Brain Age MR Seizures ADI Cause of death BCH-AUT-84 10 Yes Yes Yes Peritonitis BCH-AUT-85 9 * Yes Yes * BCH-AUT-87 28 Yes No Yes Cardiac arrest (diabetic) BCH-AUT-87-2 22 Yes Yes * Drowned BCH-AUT-87-3 12 No No Yes Bone tumor metastasized to lung BCH-AUT-88 9 Yes * No * BCH-AUT-88-2 7 Yes No Yes Drowned BCH-AUT-89-3 5 Yes Yes Yes Found dead in bed BCH-AUT-91 6 Yes Yes Yes Asphyxiation (secondary to drowning) * Information missing from patients��� records. ADI autism diagnostic interview MR mental retardation. February (1 of 2) 2002 NEUROLOGY 58 429