Seizures occur in ~12% of individuals with autism spectrum disorder (ASD) and in ~25% of individuals with fragile X syndrome (FXS)?mutations in FMR1 that cause FXS are also the most common, known genetic risk factor for ASD. Seizure susceptibility in ASD and FXS is higher in children than in adults, inferring a critical neurodevelopmental window. Juvenile Fmr1 knock-out mice, useful for the study of FXS and ASD, exhibit robust sensory-evoked, audiogenic seizures, a phenotype that models sensory hypersensitivity and seizures in individuals with FXS. New studies show that individuals with FXS and Fmr1 knock-out mice also have perturbations in electroencephalogram (EEG) activity at rest, e.g., increased gamma power in the cortex, which we reproduced and report here as preliminary data. Thus, etiologically-valid, Fmr1 knock-out mice exhibit two phenotypes with translational relevance to individuals with FXS. Numerous studies have reported alterations in the serotonin (5-hydroxytryptamine, 5-HT) system in FXS and ASD, yet knowledge regarding the impact of specific 5-HT receptors (5-HTRs) on FXS phenotypes are conspicuously lacking. We now report data showing that audiogenic seizures in juvenile Fmr1 knock-out mice are entirely prevented by a novel 5-HTR modulator with 5-HT1AR and 5-HT7R partial agonist activity. Herein, we propose definitive in vivo behavioral pharmacology and EEG experiments together with ex vivo receptor pharmacology experiments to mechanistically probe 5-HT1ARs and 5-HT7Rs as targets that can prevent audiogenic seizures (Aim 1) and correct EEG abnormalities (Aim 2) in juvenile Fmr1 knock-out mice. We hypothesize in Aim 1 that selective 5-HT1AR activation or selective 5-HT7R inactivation will attenuate audiogenic seizures and combined 5-HT1AR activation/5-HT7R inactivation will prevent audiogenic seizures in juvenile Fmr1 knock-out mice. We extend the test of this hypothesis in Aim 2, testing this pharmacodynamic effect to correct EEG phenotypes, e.g. to correct abnormally high gamma band power in the auditory cortex and somatosensory cortex of juvenile Fmr1 knock-out mice. 5-HT1ARs and 5-HT7Rs are densely expressed in the hippocampus, a neural system with a low seizure threshold that is altered in Fmr1 knock-out mice and in individuals with FXS.
In Aim 3, selective radioligands will be used for saturation binding experiments to evaluate 5-HT1AR and 5-HT7R expression in the hippocampus, and [35S]GTP?S assays will be conducted to determine the function of 5-HT1ARs and 5-HT7Rs in the hippocampus of juvenile Fmr1 knock-out mice compared to wild-type mice. 5-HT1ARs and 5-HT7Rs couple to regulate adenylate cyclase activity in opposing ways, i.e., 5-HT1ARs stimulate G?i, whereas 5-HT7Rs stimulate G?s signaling. Since cAMP is known to be altered in FXS and ASD, outcomes from this project will provide knowledge to build a critical infrastructure regarding the putative impact of 5-HTR regulation of cAMP on translationally-valid FXS and ASD phenotypes. Results will also provide important information regarding whether 5-HT1ARs and/or 5-HT7Rs are viable pharmacotherapeutic targets for FXS or ASD.
Fragile X syndrome (FXS), caused by inactivation of the FMR1 gene, is the most common inherited form of intellectual disability and autism spectrum disorder (ASD), and up to 31% of children with FXS also have epilepsy. Closely modeling individuals with FXS, Fmr1 knock-out mice exhibit increased susceptibility to seizures and have abnormally elevated gamma band power in electroencephalogram recordings from several cortical areas. Synthesizing robust preliminary findings with results from the literature, this project adds rigor by building and testing the hypothesis that selectively activating serotonin 5-HT1A and selectively inactivating 5-HT7 receptors during an early developmental sensitive period prevents audiogenic seizures and corrects cortical EEG alterations in juvenile Fmr1 knockout mice.
