ArticlesPrenatal exposure of rats to valproic acid reproduces the cerebellar anomalies associated with autism
Introduction
Autism is a disorder characterized by impairments in communication and social interaction as well as restricted and repetitive behaviors and interests; it occurs at a rate of at least 1/1,000 births [7]. The etiology of the disorder is thought to have a strong genetic component [2], and at least three teratogens appear to be risk factors: thalidomide [19], valproic acid 8, 22, and ethanol [15]. Imaging studies have revealed two subsets among the autistic populations examined. While some patients have hyperplasia of the cerebellum, the majority of people with autism have cerebellar hypoplasia, with the greatest reduction in the posterior vermis 9, 10, 12. Histological examinations of autopsy material from many regions of the brain have produced few consistent results, but abnormalities of the cerebellum are always observed. There are decreases in the number of Purkinje cells in the vermis 3, 17 and hemispheres 3, 4, 17. In addition, atrophy of the cerebellar cortex with reduced numbers of Purkinje cells in the posterior lobe hemispheres have been noted [4].
The fact that teratogenic agents increase the risk of autism allows the possibility of the creation of animal models of the disorder. We have previously reported that in vivo exposure of rat embryos to valproic acid during the period of neural tube closure results in diminished motor neuron numbers in the nuclei of cranial nerves III, V, VI, and XII [18], paralleling the malfunctions of the targets of these neurons in thalidomide-induced autism [19] and anomalies of the cranial nerve motor nuclei observed in tissue from a person with autism [18]. Unpublished data also indicate that the brain stems of these rats are altered such that the region rostral to the facial nucleus is lengthened, and the region caudal to the facial nucleus is shortened, another feature observed in a human case [18].
Do animals exposed to valproate exhibit the changes in cerebellar anatomy characteristic of autism? In a pilot study using cell counts from matched sections, we found significant reductions in Purkinje cell numbers, accompanied by an apparent reduction in the cephalo-caudal and medial-lateral dimensions of the one-cell-thick layer [13]. The corresponding dimensions of the granule cell layer reflect those of the Purkinje cell layer, but the cell density and depth of the granule cell layer were unchanged in treated animals. The preliminary results prompted a more thorough evaluation of the Purkinje cells of valproate-exposed rats; these results are reported herein.
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Animals and valproic acid exposure
Long Evans rats (Charles River, Portage, ME) were mated overnight, with pregnancy determined by the presence of a vaginal plug on embryonic day (E) 1. Dams were randomly assigned to either the treatment group or the control group.
Valproic acid (Sigma) was purchased as the sodium salt and was dissolved in 0.9% saline for a concentration of 250 mg/ml, pH 7.3. The dosing solution was tested for valproic acid concentration by enzyme-multiplied immunoassay in the hospital's clinical laboratory. The
Results
Valproate-treated animals were healthy but exhibited a 14.4% reduction in body weight and an 11.2% reduction in brain weight when they were sacrificed when compared to controls. The treated animals had fewer Purkinje cells in both the cerebellar vermis and the hemispheres, although the difference in the hemispheres failed to reach statistical significance (Fig. 2a). On average, there were 5.4 × 105 neurons in the vermis of the treated animals, compared to 7.7 × 105 neurons in the controls [F(1,
Discussion
The cerebella of animals prenatally exposed to valproic acid were significantly smaller in volume than the control cerebella; in particular, the posterior vermis of the NaVP-exposed animals was more reduced in size than was the anterior vermis. The treated cerebella also contained significantly fewer Purkinje cells in the vermis, and there was a trend toward fewer cells in the hemisphere. Because of the necessity of using a layer other than the Purkinje cell layer as a reference volume for this
Acknowledgements
These studies were supported by NIH grants RO1HD34295 and PO1HD35466, and EPA grant R824758. S.M.P. was supported by a Summer Fellowship from the Strong Children's Research Center supervised by Susan L. Hyman, M.D.
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