Objectives: The aim of the stereological and quantitative immunofluorescence-based study of raphe nuclei in autistic subjects 5 to 15 years of age and age matched control subjects was (a) to establish methods of brainstem preservation and unbiased raphe nuclei quantitative evaluation, and (b) to characterize the pattern of developmental abnormalities which may contribute to autistic phenotype.
Methods: Preliminary evaluation of brainstem samples revealed that during routine brainstem dissection with midsaggital cut in the midline and transverse cut on the level of the substantia nigra, raphe nuclei are partially or completely lost. From 9 autistic and 6 control subjects only 4 pairs 5 to 15 years of age were qualified for the study of raphe nuclei. Formalin-fixed brainstem was dehydrated and embedded in polyethylene glycol and cut into serial 50-μm-thick sections. They were stained to estimate cell volume, and immunostained and examined in fluorescence to estimate amount of tryptophan hydroxylase reflecting serotonin synthesis level.
Results: 3-D reconstruction demonstrates topography and size of raphe nuclei and explains why preservation of raphe nuclei located in the midline requires modification of brainstem sampling. Nucleator (Microbrightfield) applied to cresyl violet stained sections revealed 24% smaller neuronal soma volume in the dorsal raphe nuclei of autistic subjects than in control group. Application of immunofluorescence and ImageJ software (NIH) revealed significant increase in tryptophan hydroxylase immunofluorescence in spite of smaller size of raphe neurons.
Conclusions: These data indicate developmental impairment of neuron growth comparable to that observed in cortex and in subcortical structures (Bauman and Kemper 1996; Casanova et al. 2006). Enhanced tryptophan hydroxylase immunofluorescence in raphe neurons is consistent with enhanced immunoreactivity in serotonergic fibers in several brain regions of autistic subjects (Azmitia et al. 2011). Pathology detected in raphe neurons suggests that target brain areas are exposed to altered levels of serotonin that may modify function of cerebral cortex and subcortical structures and contribute to the autistic phenotype.