Patches of disorganization in the neocortex of children with autism

Abstract

Background: Autism involves early brain overgrowth and dysfunction, which is most strongly evident in the prefrontal cortex. As assessed on pathological analysis, an excess of neurons in the prefrontal cortex among children with autism signals a disturbance in prenatal development and may be concomitant with abnormal cell type and laminar development.

Methods: To systematically examine neocortical architecture during the early years after the onset of autism, we used RNA in situ hybridization with a panel of layer- and cell-type-specific molecular markers to phenotype cortical microstructure. We assayed markers for neurons and glia, along with genes that have been implicated in the risk of autism, in prefrontal, temporal, and occipital neocortical tissue from postmortem samples obtained from children with autism and unaffected children between the ages of 2 and 15 years.

Results: We observed focal patches of abnormal laminar cytoarchitecture and cortical disorganization of neurons, but not glia, in prefrontal and temporal cortical tissue from 10 of 11 children with autism and from 1 of 11 unaffected children. We observed heterogeneity between cases with respect to cell types that were most abnormal in the patches and the layers that were most affected by the pathological features. No cortical layer was uniformly spared, with the clearest signs of abnormal expression in layers 4 and 5. Three-dimensional reconstruction of layer markers confirmed the focal geometry and size of patches.

Conclusions: In this small, explorative study, we found focal disruption of cortical laminar architecture in the cortexes of a majority of young children with autism. Our data support a probable dysregulation of layer formation and layer-specific neuronal differentiation at prenatal developmental stages. (Funded by the Simons Foundation and others.).

Figure 1. Methods and Results of Marker-Based Phenotyping of Autism Case Samples and Control Samples

Panel A shows the locations of the dorsolateral pre-frontal cortex (from which case and control samples were obtained from eight boys [M] and three girls [F] each), posterior superior temporal cortex (from which case samples were obtained from two boys, and control samples from three boys), and occipital cortex (from which case and control samples were obtained from three boys each). Panel B shows the sectioning and labeling procedure, in which blocks of tissue measuring 1 cm³ were cut from frozen slabs and sectioned into 10 series of 30 sections per series with a section thickness of 20 μm. Each section was then labeled and imaged at 1 μm per pixel, with sections analyzed by means of in situ hybridization (ISH, in orange) or Nissl staining (in blue). The remaining sections were kept un-stained for future use. Panel C shows raw images labeled with the use of ISH with the nearest Nissl-labeled images for anatomical reference. Laminar expression patterns appear to be similar in unaffected adults (top row), which have been described previously, and in unaffected children in our study (bottom row). Annotations demarcate regions of layer-specific expression labeling. Panel D shows raw images of an identified patchlike region of aberrant laminar expression in a sample obtained from Patient 20, which was labeled with the use of ISH. Arrows show decreased or absent expression across neuronal markers in layers 2 or 3 through 5, with normal-appearing expression for marker CTGF in deep layer 6b. The nearest Nissl-labeled image also shows indications of polymicrogyria. Panel E shows a false-color image overlay generated from multiple markers, indicating a focal region of aberrant expression adjacent to normal-appearing cortex. Panel F shows a detailed view of the inset area in Panel E, indicating the transition region from normal-appearing cortex to a patch region.

Figure 2. Comprehensive View of Identified Expression Abnormalities

The left panel shows the genetic markers and their classification: laminar layer, cell type, and whether the data are from study I (in which dorsolateral prefrontal cortex from boys was assayed) or study II (in which the expression of a subset of genes was evaluated in both sexes and additional regions). The middle panel shows expression abnormalities as identified by an aggregate decision by two independent investigators after initial examination, according to the brain region. The intensity of the blue color indicates the affected level. The charts below and to the right of the investigators’ ratings show the proportion of markers that were examined that fell into one of two categories of severity (severe or either mild or severe), according to the cell type (below) and brain location (at right). More intense red color indicates greater severity. A single asterisk indicates Patient 6, a 9-year-old unaffected girl who was the only control in whom a patch was identified. Double asterisks indicate Patient 16, a 7-year-old boy with autism in whom investigators found no patch in the dorsolateral prefrontal cortex but a clear patch in the posterior superior temporal cortex. Triple asterisks indicate Patient 21, a 14-year-old boy with autism in whom no patch was identified.

Figure 3. Three-Dimensional Reconstruction of a Patch Region in Cortical Microstructure

Panel A shows a three-dimensional reconstruction of the four markers, as seen on in situ hybridization of samples obtained from Patient 20 (as shown in Fig. 1, Panels D, E, and F). Panel B shows a cross section of the reconstructed structure at the original slice plane for the same four markers. Panel C shows angled renderings of individual layer-specific markers, indicating a focal patch of substantially reduced or absent expression. Panel D shows an angled reconstruction indicating a patch region with an independent, conserved location across several markers. The dashed oval indicates the patch region.

Figure 4. Post Hoc Quantitative Reverse-Transcriptase–Polymerase-Chain-Reaction (RT-PCR) Assay and In Situ Hybridization Validating Patch Region

In a post hoc experiment, laser-capture microdissection was performed on a frozen tissue block of dorsolateral prefrontal cortex obtained from severely affected patients with autism. Panel A shows a Nissl-stained tissue section indicating the location of a patch (green box) and an adjacent region of interest (red box). Panel B shows the corresponding CALB1 image on in situ hybridization, with a region of decreased expression (green box) and adjacent cortex (red box). Panel C shows a magnified view of the patch region with decreased CALB1 expression. Panel D shows a magnified view of an adjacent region with normal CALB1 expression. The approximate location of cortical layers 2 through 6 are indicated. Panel E shows the difference in expression between the patch region and the adjacent region on RT-PCR assay, indicating the factor reductions in CALB1 expression in the patch region, as compared with the adjacent region. The I bar indicates the standard deviation.