Evaluation of Serotonin 5‐HT 1A , 5‐HT 2A , and 5‐HT 2C Receptors and the Serotonin Transporter in an Fmr1 Knockout Mouse Model of Fragile X Syndrome

Abstract

Fragile X syndrome (FXS)—caused by FMR1 gene silencing—is the leading monogenic cause of intellectual disability and autism spectrum disorder (ASD). Diminished serotonin (5-hydroxytryptamine, 5-HT) synthesis has been reported in ASD. The 5-HT transporter (SERT) and receptors (5-HTRs) have been targeted to manage ASD and FXS symptoms. Serotonergic neurons project to glutamate and GABA neurons expressing 5-HT1ARs, 5-HT2ARs, and 5-HT2CRs that modulate cellular excitation-inhibition which is altered in ASD and FXS. Here, we assessed the expression and function of these 5-HTRs and SERT in adult (P60-180) Fmr1 knockout (KO) mice, a model of FXS, compared to wild type (WT) mice. We performed autoradiography on brain sections using [3H]5-CT for 5-HT1ARs, [3H]Ketanserin for 5-HT2ARs, [3H]Mesulergine for 5-HT2CRs, [3H]Citalopram for SERT, and appropriate non-specific and off-target blocking ligands. Brain regions of interest were chosen and analyzed using ImageJ. Mean pixel intensity from the images was normalized to the mean of background intensity, and non-specific binding was subtracted from total binding to obtain normalized specific binding. Saturation binding assays, using whole brain membrane homogenates, were performed for 5-HT1ARs with [3H]5-CT and SB-269970 to block 5-HT7Rs; WAY-100635 was used to isolate 5-HT1AR specific binding. Behavioral effects of (R)-8-OH-DPAT, (±)-DOI, and lorcaserin were used to probe in vivo functional activity of 5-HT1A, 5-HT2A, and 5-HT2CRs, respectively. Responses to (R)-8-OH-DPAT (1 and 2 mg/kg), including flat body posture, head weaving, straub tail, hind limb abduction, piloerection, tremor, rearing, grooming, and head twitch responses (HTRs), were recorded by two independent observers. Locomotor effects of lorcaserin (1, 3, and 10 mg/kg) were recorded using Noldus Ethovision XT software, and HTRs elicited by (±)-DOI (1 mg/kg) were recorded using a hand-held tally counter. Sex effects were assessed in all tests. Preliminary results suggest that compared to WT males, Fmr1 KO males (N=3 per group) have higher 5-HT1AR expression in lateral septum and frontal cortex, lower 5-HT2AR expression in isocortex layer V, and lower 5-HT2CR expression in anterior olfactory nucleus and nucleus accumbens. Increased 5-HT1AR expression was confirmed by saturation binding analyses (N=6 per group). Male mice of both genotypes had lower 5-HT2CR expression in choroid plexus and caudate putamen (N=3 per group). Behavioral effects of (R)-8-OH-DPAT were significantly more pronounced in both male and female (N=5-7 per group) Fmr1 KO mice, relative to WT mice. A minor decrease in DOI-elicited HTRs was observed in Fmr1 KO males compared to WT males (14 vs. 17, N=8-9 per group). No genotype differences in locomotor activity were observed after lorcaserin administration (N=6 per group). Ongoing studies are assessing SERT expression and in vivo function via autoradiography and the tail suspension test, respectively. This study provides the first observations of perturbations in key 5-HTRs in Fmr1 KO mice, suggesting dysfunctions in the central 5-HT system in FXS.