Neurobiological investigations of autism symptomatology and cerebellar pathology

The cerebellum is the most commonly reported brain region to demonstrate pathology in autism. Multiple reports have indicated a lower density of Purkinje cells (PCs) in the posterolateral cerebellar hemispheres. We performed a stereological technique to precisely quantify PCs in postmortem autism cases compared to age- and sex-matched controls. We found that although PC density was lower in all cerebellar regions studied, the most significant difference was in lobule VIIa of the posterior lobe. The PCs in this region project to deep cerebellar nuclei that reciprocally connect to all aspects of the established broader sensorimotor gating network. Sensorimotor gating abnormalities are commonly observed in individuals diagnosed with autism, and may contribute to problems with sensory processing and behavioral inhibition in these individuals. We studied a rat model of sensorimotor gating impairment, in which the histamine H1 receptor antagonist, pyrilamine, improved sensorimotor gating. Using autoradiography, we found that pyrilamine treatment altered H1 receptor and α7 nicotinic receptor binding in the anterior cingulate and insular cortex, respectively, an effect which correlated with improved sensorimotor gating. Histamine functions as both a neurotransmitter as well as a regulator of glial activity throughout the brain. Using western blots, we quantified H1 receptor levels in lobule VIIa from postmortem autism cases but found no difference compared to controls. We further quantified additional proteins to investigate theories of neuroimmune and neuroendocrine dysregulation in the cerebellum in autism. These included IBA-1, GFAP, IL-6, androgen receptor, estrogen receptor β, and aromatase. We found no evidence to support these theories: all protein levels tested were found to be similar in the autism and control groups. We suggest further studies to better understand cerebellar pathogenesis and regulation of sensorimotor gating in autism. The implications of sensorimotor gating impairment in autism are discussed in relation to the established symptomatology of this neurodevelopmental behavioral disorder

Overall PC Density Decreases with Age.

<p>Overall PC density obtained from the anterior, posterior, and flocculonodular lobes negatively correlates with age (R² = −0.39±0.14, p = 0.030). Some cases were missing data from an individual region (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081255#s2" target="_blank">Methods</a>) and were not included: one female and one male case from the autism and control groups were not included.</p

Models of PC Arrangement.

<p>These figures represent the importance of estimating PC density in 3-dimensional space rather than along a 1-dimensional line (PC/mm³ rather than PC/mm). 7a and 7b display two views of a 3-dimensional model of a cerebellar folium, in which PCs (black) are arranged in a monolayer apposed to the granule cell layer (blue) that surrounds a central white matter tract (white). 7b displays two slices, one perfectly parallel to a plane of PCs, and the other, more realistically, transverse to this plane. 7c and 7d are cartoons demonstrating the PC arrangement within the parallel and transverse slice, respectively. Notice in 7d the PC layer is thicker, the degree to which depends on the slice position. Also notice that the selection of which PCs lie perfectly along a line is ambiguous (a few examples have been circled in red). This ambiguity introduces human error when performing an estimate of PC/mm that is eliminated in 3-dimensional estimates of PC/mm³ as are utilized in this study.</p

Photomicrograph of Transverse Section.

<p>This photomicrograph is an illustrative example of the arrangement of PCs on a transverse section. The PCs (purple) go out of focus to the left as the PC layer curves through the depth of the tissue section. The granule cell layer (blue) is visible below the PC layer, the molecular layer surrounds the PC layer (seen here as sparsely stained space), and the white matter tract is out of view to the right. Nissl-stained section from the celloidin collection used in this study, reference bar = 100 µm.</p

Prepulse inhibition of the startle response and radioligand binding assays

<p>Dataset 1. PPI data.<br>The percent PPI data is provided for each prepulse decibel level tested. This raw behavioral data reflects the percentage of inhibition in prepulse-pulse trials compared with pulse-only trials for each prepulse intensity tested. The data is graphically presented in Figure 2.</p> <p>Dataset 2. Regional radioligand binding data.<br>The radioligand binding data presented in Figures 3 – 7 is provided in fmol/mg tissue equivalent. In a few instances, tissue damage during processing prevented reliable densitometry within a region. These data were omitted from the study (indicated by blank cells in the spreadsheet). Additionally, we were unable to block nonspecific binding in all tissue samples from rat O0 for either radioligand, possibly due to tissue damage that was isolated to this case, so this data were omitted as well. AC = anterior cingulate, IC = insular cortex.</p> <p> </p

Lobule X PC Density is Associated with Direct Gaze.

<p>Lobule X PC density in the autism group negatively correlated with ADI-R question 50, which assessed the social and communicative use of eye contact (R² = −0.75±0.04, p = 0.012). Higher scores on the ADI-R correspond with increased symptom severity. One case from the autism group was lacking sufficient ADI-R data to be included in this analysis.</p

Clinical Characteristics.

a<p>Fresh brain weight (in grams) was measured prior to tissue processing.</p>b<p>Postmortem interval.</p>c<p>Mental Retardation.</p>d<p>Left brain hemisphere was investigated. (All other cases were investigated from the right brain hemisphere).</p

Linear Mixed Models.^a

a<p>Cells in the table that have bold data presented designate the target results for each variable and interaction, whereas cells with italicized data designate results that are more adequately assessed by a different test presented in the table: Test 1 was designed to investigate the overall effect of gender and autism diagnosis on PC density. It incorporated cerebellar regional volume as a WLS weight to adjust the test's significance relative to the regional volume for each PC density measurement. Test 3 was designed to test the regional differences in PC density as an effect of gender and diagnosis. Tests 2 and 4 are designed to investigate the contribution of fresh brain weight (grams), as a correlate of tissue volume <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081255#pone.0081255-Witelson1" target="_blank">[39]</a>, to Tests 1 and 3, respectively.</p>b<p>One control male case was missing brain weight information.</p