Clinton E. Canal - US grants

? DESCRIPTION (provided by applicant): The National Institute on Drug Abuse recognizes that the abuse of synthetic cathinones (SC) is a serious public health issue. SC use can cause panic, psychosis, neurological complications, cardiac and renal toxicity, and fatal overdose. SC is ?-ketone analogs of amphetamines, and have similar subjective effects (e.g., stimulant and hallucinogenic effects) as their respective amphetamine counterparts. However, humans report conspicuous differences in psychological effects between amphetamines and SC, highlighting that SC is a unique drug class with unique pharmacological properties and neurobiological effects, much of which are presently unknown. With the recent DEA scheduling of many widely abused SC, e.g. mephedrone, methylenedioxypyrovalerone (MDPV), and methylone, second-generation SC have emerged, most of which contain a pyrrolidine moiety in place of the secondary amine of first-generation SC. MDPV was the first of the pyrrolidine SC to gain popularity among stimulant users, however, due to MDPV's scheduling with other first-generation SC, illicit chemists have synthesized and distributed many other pyrrolidine SC. Pyrrolidine SC appear to have an unusually high propensity to induce panic and psychosis, which suggests they may be acting through receptor systems in addition to monoamine transporters. However, systematic studies testing the fear-inducing effects of pyrrolidine SC are lacking, and systematic pharmacological assessments of pyrrolidine SC at receptors that could contribute to psychiatric events are warranted. Also, to date, no approved medications exist for acute SC overdose, but our preliminary data demonstrate a promising, new pharmacological intervention, 5-HT2A receptor antagonism, to mitigate psychostimulant-induced psychosis, hyperthermia, convulsions, and fatal overdose. Our proposed studies will compare new and widely available, second-generation pyrrolidine SC to mephedrone and will determine: 1) how the pyrrolidine substitution affects affinity and function at serotonin 5-HT2 and adrenergic ?1 receptor subtypes expressed in HEK293 cells; 2) whether the pyrrolidine substitution contributes to fear, anxiety, and psychosis in mice; and 3) the efficacy of 5-HT2 and ?1 selective ligands to block clinically critical components of acute SC overdose in mice. The experiments in Aims 1 and 2 will address the goal of this public funding announcement to provide information regarding the pharmacology and unique effects of SC. The results from Aim 3 will guide discovery of novel fast-acting treatments for acute SC overdose.

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.