Suzanne O. Nolan

Sex-specific and genotype-specific differences in vocalization development in FMR1 knockout mice.

Fragile X syndrome is a neurodevelopmental disorder caused by a trinucleotide (CGG) hyperexpansion in the FMR1 gene, functionally silencing transcription of the fragile X mental retardation protein (FMRP). This disorder is characterized by impaired cognition, communication, and social behavior. The aim of this study was to investigate the development of ultrasonic vocalization (USV) behavior in a Fmr1-deficient mouse model. On postnatal days (PD) 9–14, separate cohorts of FVB/NJ pups were removed from their homecage and isolation-induced USVs were recorded. There were significant genotype-dependent and sex-dependent differences in USV behavior across the different testing days. Fmr1 knockout (KO) mice showed a significant reduction in vocalizations across all days. There was also a significant difference in vocalizations between male and female mice. We found a significant decrease in the total number of calls for KO males on PD9 and PD13 as well as an increase in the total number of calls for KO males on PD12. The KO males also showed a significant increase in the total call duration on PD12 and a reduction on PD13. The KO female showed a significant decrease in the total number of calls on PD9 and PD10. They also showed a significant decrease in the total call duration on PD9 and a marginal decrease in the total call duration on PD10. These results provide additional evidence for communication deficits in Fmr1 deficient mice and provide new insight suggesting sexually dimorphic vocalizations during the neonatal period.

Deletion of Fmr1 results in sex‐specific changes in behavior

In 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.

Spectral and temporal properties of calls reveal deficits in ultrasonic vocalizations of adult Fmr1 knockout mice

&NA; The Fmr1 knockout (KO) mouse has commonly been used to investigate communication impairments, one of the key diagnostic symptoms observed in Fragile X syndrome (FXS) and Autism spectrum disorder (ASD). Many studies have found alterations in ultrasonic vocalizations (USVs) in neonatal Fmr1 KO mice, however, there is limited research investigating whether these deficits continue into adulthood. In the present study, we examine differences in female urine‐induced ultrasonic vocalizations, scent marking behavior, odor discrimination, and open field activity in adult male Fmr1 KO and wildtype (WT) mice. Overall, we found extensive alterations between genotypes in both spectral and temporal properties of ultrasonic vocalizations. There was no difference in the average number of calls emitted by both genotypes, however, Fmr1 KO mice emitted calls of a higher frequency, decreased amplitude, and shorter duration than WT mice. Spectrographic analyses revealed statistically significant differences between genotypes in the types of calls emitted. Contrastingly, we found no differences in scent marking behavior, a form of social communication, or in odor discrimination and activity levels of the mice. The results corroborate previous studies emphasizing the importance of qualitative differences observed in vocalization behavior of Fmr1 KO mice, rather than quantitative measurements such as number of calls emitted. Overall, the study confirms the presence of abnormalities in vocalization behavior in adult Fmr1 KO mice that we believe are consistent with communication deficits seen in the syndrome. HighlightsNo difference in average quantity of calls produced between Fmr1 KO and WT mice.Fmr1 KO mice emit vocalizations at a higher frequency and decreased amplitude.Fmr1 KO vocalizations were of significantly shorter duration.Spectral analysis reveal Fmr1 KO mice to produce different types of calls.Results emphasize qualitative differences in Fmr1 KO mice vocalizations.

A comparison of the Avisoft (5.2) and Ultravox (2.0) recording systems: Implications for early-life communication and vocalization research

Alterations in early-life communicative behaviors are a common feature of neurodevelopmental conditions, such as autism and epilepsy. One method of investigating communication in murine models is through analyzing ultrasonic vocalizations. These vocalizations are commonly recorded with either the Avisoft or the Ultravox recording programs. However, since no study has compared whether the systems are equally sensitive, the findings in one program may not be reproducible in the other. To directly compare the two programs, we elicited vocalizations from male and female 129SvEvTac and C57BL/6 mouse pups via the maternal isolation paradigm, recording vocalizations simultaneously with both systems. We held the detection parameters identical for each system and found that there was only a medium correlation between Avisoft and Ultravox overall. Further analysis indicated that Avisoft detected more total vocalizations, as well as more vocalizations at the set frequencies of 50, 60, and 70 kHz than Ultravox, p <  .05. No statistically significant difference was present at 80 kHz. These findings demonstrate that different recording systems do not detect the same quantity of vocalizations as one another, even when detection parameters are congruent. Therefore, it may be useful to revisit previous negative results obtained with Ultravox and repeat the experiments using Avisoft. Ultimately, ultrasonic vocalizations are a valuable tool, capable of examining early-life phenotypes. However, a more thorough understanding of the relationships between recording systems is necessary to achieve a more comprehensive and reproducible assessment of vocalizing behaviors.

Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse

Background: Loss of FMR1 is associated with Fragile X syndrome, amongst the most prevalent inherited intellectual disability. Despite extensive research in this area, previous studies have failed to detect consistent evidence of cognitive impairments in the Morris water maze (MWM) task in the Fmr1 knockout (KO) mouse. However, few studies have examined cognitive flexibility in a reversal form of the MWM task, which may illuminate subtle learning deficits. Methods: Adult male Fmr1 wildtype (WT) and KO mice were bred and tested in the MWM reversal paradigm. The testing paradigm consisted of two blocks per day, with 4 trials per block to locate a hidden platform. After the last trials on the fourth day of testing, the animals were given a probe trial with the platform removed. The following week, the location of the platform was switched to the opposite quadrant and the animals received 2 more days of testing, with 4 blocks in total. Results: As expected, Fmr1 KO mice did not display a learning deficit during the acquisition phase, F genotype (1, 24) = 0.034, p = 0.854, and performed similarly on the probe trial, F genotype (1, 23) = 0.024, p = 0.877. However, during the reversal phase of learning, Fmr1 KO mice showed deficits in their ability to learn the new location of the platform, F genotype (1, 23) = 3.93, p = 0.059. Further independent samples t-testing revealed that KO animals displayed significantly higher latency to reach the hidden platform during the third trial, t(23) = -2.96, p < 0.01. Conclusions: While previous studies have not demonstrated deficits in spatial memory in the Fmr1 KO model, it is possible that the acquisition phase of the task is less sensitive to deficits in learning. Future studies using this model to evaluate therapeutic interventions should consider utilizing the MWM reversal paradigm.

A single seizure selectively impairs hippocampal-dependent memory and is associated with alterations in PI3K/Akt/mTOR and FMRP signaling

A single brief seizure before learning leads to spatial and contextual memory impairment in rodents without chronic epilepsy. These results suggest that memory can be impacted by seizure activity in the absence of epilepsy pathology. In this study, we investigated the types of memory affected by a seizure and the time course of impairment. We also examined alterations to mammalian target of rapamycin (mTOR) and fragile X mental retardation protein (FMRP) signaling, which modulate elements of the synapse and may underlie impairment.

Neuronal subset-specific deletion of Pten results in aberrant Wnt signaling and memory impairments

The canonical Wnt and PI3K/Akt/mTOR pathways both play critical roles in brain development early in life. There is extensive evidence of how each pathway is involved in neuronal and synaptic maturation, however, how these molecular networks interact requires further investigation. The present study examines the effect of neuronal subset-specific deletion of phosphatase and tensin homolog (Pten) in mice on Wnt signaling protein levels and associated cognitive impairments. PTEN functions as a negative regulator of the PI3K/Akt/mTOR pathway, and mutations in Pten can result in cognitive and behavioral impairments. We found that deletion of Pten resulted in elevated Dvl2, Wnt5a/b, and Naked2, along with decreased GSK3β hippocampal synaptosome protein expression compared to wild type mice. Aberrations in the canonical Wnt pathway were associated with learning and memory deficits in Pten knockout mice, specifically in novel object recognition and the Lashley maze. This study demonstrates that deletion of Pten not only significantly impacts PI3K/Akt/mTOR signaling, but affects proper functioning of the Wnt signaling pathway. Overall, these findings will help elucidate how the PI3K/Akt/mTOR pathway intersects with Wnt signaling to result in cognitive impairments, specifically in memory.

Adult Fmr1 knockout mice present with deficiencies in hippocampal interleukin-6 and tumor necrosis factor-α expression

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by a single genetic mutation in the FMR1 gene. Mutations in the FMR1 gene are the largest monogenic cause of autism spectrum disorder (ASD), and thus both disorders share many of the same cognitive and behavioral impairments. There is increasing evidence suggesting that dysregulated immune responses play a role in the pathophysiology of ASD; however, the association between FXS and altered immunity requires further investigation. This study examined whether Fmr1 knockout (KO) and wild-type mice on a FVB/NJ background strain had altered cytokine expression at baseline levels in the hippocampus. Results showed Fmr1 KO mice to have decreased proinflammatory cytokine hippocampal mRNA expression, specifically interleukin (IL)-6 and tumor necrosis factor-&agr;, compared with wild-type mice. However, no differences were detected in the expression levels of IL-1&bgr;, MCP-1, interferon-&ggr;, or IL-10. Despite the high comorbidity between FXS and ASD, these results suggest that the Fmr1 KO mouse does not mimic the increased proinflammatory cytokine expression commonly found in ASD mouse models and patients. Further investigation of the immune profile of the Fmr1 KO mouse is critical to understand whether this deficiency of cytokines in the hippocampus is indicative of a broader immunologic deficit associated with FXS.

Early-life status epilepticus acutely impacts select quantitative and qualitative features of neonatal vocalization behavior: Spectrographic and temporal characterizations in C57BL/6 mice

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.

Characterization of the behavioral phenotype and neuroinflammatory profile of the Fmr1 knockout mouse

1986 The effects of dietary supplementation with n-3 fatty acids on the behavioral and neuroinflammatory phenotype of the Fmr1 knockout mouse S.O. Nolan, S.L. Hodges, G.D. Smith, B. Escobar, T. Jefferson, A. Holley, J.N. Lugo Baylor University, One Bear Place, Department of Psychology and Neuroscience, Waco, Texas 76798, USA FragileX Syndrome (FXS) is a neurodevelopmental disorder caused by a genetic trinucleotide (CGG) overexpansionmutation in the FMR1 gene coding for fragile X mental retardation protein (FMRP). This disorder is characterized by marked intellectual disability, as well as other autistic-like behavioral phenotypes. Supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFAs) has been proposed as an alternative treatment for a variety of neurodevelopmental disorders, including Autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD). Previous results have examined the efficacy of this treatment paradigm in the FXS mouse model, though it is not known how the efficacy varies between background strains. In the present study, male FVB/129 Fmr1 knockout and Fmr1 wildtype littermates were assigned to one of three diet conditions following weaning on PD21: standard lab chow, EPA/DHA enriched chow, and a diet controlling for the increase in fat associated with the former. Upon reaching postnatal day 90, animals were tested in a several behavioral assays, which included elevated plus maze, social partition, nose poke assay, delay fear conditioning and prepulse inhibition. Results revealed that supplementation with omega-3 fatty acids attenuated alterations in prepulse inhibition behavior and hyperactivity. Results also revealed that the increased dietary fatty acid composition significantly impacted sociability and repetitive behavior. These behavioral changes were associated with reduction in hippocampal expression of IL-6 resulting from dietary supplementation omega-3 fatty acids. http://dx.doi.org/10.1016/j.bbi.2017.07.114