Objectives: The aim of this study was to define the structure of the PiC with precision in post-mortem brains from 8 ASD and 10 control donors for qualitative neuropathological assessment and quantitative investigation of glial, non-pyramidal and pyramidal cell densities in layers II and III of this brain area.
Methods: Subjects were selected from the Harvard, Maryland and Oxford Thomas Willis brain banks with appropriate ethical approval. Samples were age-matched. 25 um-thick sections were obtained from paraffin-embedded blocks and 5 sections per case were cut and stained with Cresyl Violet (0.1%) to visualise Nissl-substance for neuropathological assessment. A section sampling fraction of 0.125 was used and unbiased cell density measurements were performed.
Results: We found that, to obtain the characteristic Nissl-stained S-shape of the three-layered structure, the PiC needs to be sampled from the anterior side of the first coronal slice that shows the beginning of the temporal lobe at the level of the endorhinal sulcus. Detailed qualitative anatomical assessment revealed no overall microscopic neuropathology in the structure of the PiC in ASD brains. Dorsally, layer III of the PiC is anatomically bound by the claustrum, laterally by the limen insula and ventrally by the endopiriform nucleus of the anterior amygdala. Preliminary data from the stereological investigation provided estimates of the different cell sub-populations including pyramidal, non-pyramidal and glial cells in layers II and III of this structure. In layer II, the average cell densities were: pyramidal neurons- 171 (x103) cells/mm3, non-pyramidal neurons- 24 (x103) cells/mm3, glial cells- 62 (x103) cells/mm3. In layer III, pyramidal neurons averaged- 19.1 (x103) cells/mm3, non-pyramidal neurons- 3.1 (x103) cells/mm3, glial cells- 36 (x103) cells/mm3.
Conclusions: The PiC is unusual due to its three-layered structure and constitutes a novel area of investigation in ASD. We report no visible neuropathological anomaly of the microscopic organisation of the PiC by qualitative assessment. Further stereological measurements will reveal if the balance between the different sub-populations is altered and whether this might relate to the olfactory impairments seen in ASD individuals.