Assessment of Brain Serotonin Receptors in an Fmr1 Knockout Mouse Model of Fragile X Syndrome

Abstract

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and autism. It is caused by a mutation of FMR1 that prevents transcription of mRNA coding for the protein, FMRP. FMRP regulates the translation of nearly 1000 proteins, including many involved in cell signaling, neurotransmission, synaptic development and plasticity. There are currently no approved medications to treat core symptoms of FXS. Several FMRP targets are functionally linked with the central serotonin system, and certain serotonin receptors can modulate neuropsychological states that are disrupted in FXS, suggesting distinct serotonin receptors may be viable targets for FXS pharmacotherapy. However, studies investigating the expression and function of serotonin receptors and the serotonin transporter (SERT) in the brains of Fmr1 knockout mice—a preclinical, genetic model of FXS—have not been conducted. Using radioligand autoradiography and saturation binding assays, we are assessing the expression of key serotonin proteins—5‐HT1, 5‐HT2A, 5‐HT2C, and 5‐HT7 receptors as well as SERT—in brains of male and female wild‐type (WT) compared to Fmr1 knockout (KO) mice. We are also assessing GABAA receptors (as a positive control), as alterations in brain GABAA receptor expression have been reported in Fmr1 KO mice. For autoradiography experiments, brains from WT and Fmr1 KO mice were cryosectioned at 20 μm and thaw‐mounted on glass slides. Slides were immersed in assay buffer with radioligands at ~Kd concentrations to target receptors of interest: 5‐HT1 (2 nM [3H]5‐CT with 1 μM SB269970); 5‐HT2A (2 nM [3H]Ketanserin and 2 nM [3H]Ketanserin with 1 μM M100907 to distinguish 5‐HT2A labeling from off‐targets); 5‐HT2C, (3 nM [3H]Mesulergine); 5‐HT7 (2 nM [3H]5‐CT with 500 nM cyanopindolol); SERT (3 nM [3H]Citalopram); and GABAA (3 nM [3H]Flumazenil). Slides were dried and exposed to film for 8–12 weeks prior to developing. We did not observe obvious differences between WT and Fmr1 KO mice in expression, across several neural systems (e.g. dorsal hippocampus, cingulate cortex), of any receptor assessed by autoradiography (N=3–6 mice/genotype), however, this may be due to the low resolution of the technique. Saturation binding experiments using homogenized whole brain tissue are in progress. Preliminary results suggest an increase in GABAA receptor expression in Fmr1 KO mice, whereas preliminary results suggest no genotypic differences in SERT expression. In vivo tests of 5‐HT2A receptor function, using the DOI‐elicited head‐twitch response (HTR) assay, revealed no significant differences between WT and Fmr1 KO mice in the number of HTRs elicited by 1 mg/kg (±)‐DOI (N=9 WT, N=9 Fmr1 KO, P=0.10). Locomotion and rearing, however, were significantly higher in Fmr1 KO compared to WT mice treated with (±)‐DOI (P<0.02 for each). In vivo pharmacology tests of 5‐HT1, 5‐HT2C, and SERT function are in progress. Although clear conclusions cannot yet be made, our data currently suggest unaltered expression and function of key 5‐HT proteins in Fmr1 KO mice.Support or Funding InformationFRAXA Research Foundation, DOD W81XWH‐17‐1‐0329This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.