ObjectiveIn this study, we used a systemic Fmr1 knockout in order to investigate both genotype‐ and sex‐specific differences across multiple measures of sociability, repetitive behaviors, activity levels, anxiety, and fear‐related learning and memory.BackgroundFragile X syndrome is the most common monogenic cause of intellectual disability and autism. Few studies to date have examined sex differences in a mouse model of Fragile X syndrome, though clinical data support the idea of differences in both overall prevalence and phenotype between the sexes.MethodsUsing wild‐type and systemic homozygous Fmr1 knockout mice, we assessed a variety of behavioral paradigms in adult animals, including the open field test, elevated plus maze, nose‐poke assay, accelerating rotarod, social partition task, three‐chambered social task, and two different fear conditioning paradigms. Tests were ordered such that the most invasive tests were performed last in the sequence, and testing paradigms for similar behaviors were performed in separate cohorts to minimize testing effects.ResultsOur results indicate several sex‐specific changes in Fmr1 knockout mice, including male‐specific increases in activity levels, and female‐specific increases in repetitive behaviors on both the nose‐poke assay and motor coordination on the accelerating rotarod task. The results also indicated that Fmr1 deletion results in deficits in fear learning and memory across both sexes, and no changes in social behavior across two tasks.ConclusionThese findings highlight the importance of including female subjects in preclinical studies, as simply studying the impact of genetic mutations in males does not yield a complete picture of the phenotype. Further research should explore these marked phenotypic differences among the sexes. Moreover, given that treatment strategies are typically equivalent between the sexes, the results highlight a potential need for sex‐specific therapeutics.

Early-life seizures are known to cause long-term deficits in social behavior, learning, and memory, however little is known regarding their acute impact. Ultrasonic vocalization (USV) recordings have been developed as a tool for investigating early communicative deficits in mice. Previous investigation from our lab found that postnatal day (PD) 10 seizures cause male-specific suppression of 50-kHz USVs on PD12 in 129 SvEvTac mouse pups. The present study extends these findings by spectrographic characterization of USVs following neonatal seizures. On PD10, male C57BL/6 pups were administered intraperitoneal injections of kainic acid or physiological saline. On PD12, isolation-induced recordings were captured using a broad-spectrum ultrasonic microphone. Status epilepticus significantly suppressed USV quantity (p=0.001) and total duration (p<0.05). Seizure pups also utilized fewer complex calls than controls (p<0.05). There were no changes in call latency or inter-call intervals. Spectrographic analysis revealed increased peak amplitude for complex, downward, short, two-syllable, and upward calls, as well as reduced mean duration for short and two-syllable calls in seizure mice. This investigation provides the first known spectrographic characterization of USVs following early-life seizures. These findings also enhance evidence for USVs as an indicator of select communicative impairment.

The fragile X mental retardation protein (FMRP), an RNA-binding protein that mediates the transport, stability, and translation of hundreds of brain RNAs, is critically involved in regulating synaptic function. Loss of FMRP, as in fragile X syndrome (FXS), is a leading monogenic cause of autism and results in altered structural and functional synaptic plasticity, widely described in the hippocampus and cortex. Though FXS is associated with hyperactivity, impaired social interaction, and the development of repetitive or stereotyped behaviors, all of which are influenced by striatal activity, few studies have investigated the function of FMRP here. Utilizing a cortical-striatal co-culture model, we find that striatal medium spiny neurons (MSNs) lacking FMRP fail to make normal increases in PSD95 expression over a short time period and have significant deficits in dendritic spine density and colocalized synaptic puncta at the later measured time point compared to wildtype (WT) MSNs. Acute expression of wtFMRP plasmid in Fmr1 KO co-cultures results in contrasting outcomes for these measures on MSNs at the more mature time point, reducing spine density across multiple spine types but making no significant changes in colocalized puncta. FMRP's KH2 and RGG RNA-binding domains are required for normal elimination of PSD95, and interruption of these domains slightly favors elimination of immature spine types. Further, KH2 is required for normal levels of colocalized puncta. Our data are largely consistent with a basal role for FMRP and its RNA-binding domains in striatal synapse stabilization on developing MSNs, and in light of previous findings, suggest distinct regional and/or cell type-specific roles for FMRP in regulating synapse structure.

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