Receptor Density and Distribution of 5-HT2 Receptors in the Cingulate Cortex in Autism: A Multiple Concentration Saturation Binding Study in Children and Adults

Background: Selective serotonin reuptake inhibitors (SSRIs) are among the most commonly prescribed medications in autism, however, several studies show evidence of harm and variable outcomes of efficacy from SSRI use. Some of the variability may be a result of differential changes in serotonin (5-HT) receptor subtypes across individuals.

Objectives: In an effort to determine differences in 5-HT₂ receptor expression between autism and neurotypical individuals, a multiple concentration ligand binding assay was performed in two parts of the cingulate cortex. These areas are important in socioemotional behavior, reward anticipation and in the default network, among other functions, and have been shown to be impacted in autism.

Methods: Using a large cohort of postmortem brain tissue, a saturation binding assay was conducted on 20µm sections from the anterior cingulate cortex (ACC) and posterior cingulate cortex (PCC) (n=16-19 autism, n=18-19 controls) by incubation with ³H ketanserin (Perkin Elmer) at concentrations of 120, 90, 30, 12, 6, 3 and 1.5 nM before being loaded into X-ray cassettes with tritium standards and apposed to tritium-sensitive hyperfilm. Non-specific binding was determined with a competitive displacer (Ritanserin 100µM). After exposure, films were developed and digitized to quantify measurements of binding in femtomoles per milligram of tissue in both superficial and deep layers of each region. A Welch’s t-test was utilized for statistical analysis.

Results: 5HT₂ receptor density in superficial and deep layers of the ACC and PCC did not have statistically significant binding density differences between total autism and control cases. However, the variance in the PCC layers was significantly higher in autism cases (p=0.038 deep; p=0.0031 superficial) using an F test and 30nM ³H ketanserin as a representative concentration. The groups were then split into children (≤16) (n=8-9) and adults (>16) (n=9), which resulted in an increased binding density in the PCC of autism cases in children only (363.4 ±26.38 fmol/mg PCC deep; 400.0 ±10.93 fmol/mg PCC superficial) compared to controls (290.2 ±11.98 fmol/mg PCC deep; 308.8 ±10.93 fmol/mg PCC superficial) [p=0.028 PCC deep; p=0.0403 PCC superficial] while the ACC had similar mean and variance between groups. Adult autism cases had similar binding density compared to neurotypical individuals.

Conclusions: Although the overall comparisons of autism and neurotypical cases did not reveal a mean binding density difference across regions and layers, the variance of autism cases was significantly higher in the PCC and in children when compared to adults. This is particularly interesting given the literature on SSRI use in autism. A Cochrane review concluded that SSRI use has variable outcomes of efficacy, with adverse effects negligible in adults but significant in children. It is possible that the variance of 5HT₂ density seen in child autism cases contributes to the variable reactions to SSRI use. Comparison of 5HT₂ expression to other 5HT receptors in these cases will provide useful insight into how the 5HT system may be differentially impacted.