Neonatal immune stimulation results in sex-specific changes in ultrasonic vocalizations but does not affect seizure susceptibility in neonatal mice.

Neuroinflammation during the neonatal period has been linked to disorders such as autism and epilepsy. In this study, we investigated the early life behavioral consequences of a single injection of lipopolysaccharide (LPS) at postnatal day 10 (PD10) in mice. To assess deficits in communication, we performed the isolation-induced ultrasonic vocalizations (USVs) test at PD12. To determine if early life immune stimulus could alter seizure susceptibility, latency to flurothyl-induced generalized seizures was measured at 4 hours (hrs), 2 days, or 5 days after LPS injections. LPS had a sex-dependent effect on USV number. LPS-treated male mice presented significantly fewer USVs than LPS-treated female mice. However, the number of calls did not significantly differ between control and LPS for either sex. In male mice, we found that downward, short, and composite calls were significantly more prevalent in the LPS treatment group, while upward, chevron, and complex calls were less prevalent than in controls (p < 0.05). Female mice that received LPS presented a significantly higher proportion of short, frequency steps, two-syllable, and composite calls in their repertoire when compared with female control mice (p < 0.05). Seizure latency was not altered by early-life inflammation at any of the time points measured. Our findings suggest that early-life immune stimulation at PD10 disrupts vocal development but does not alter the susceptibility to flurothyl-induced seizures during the neonatal period. Additionally, the effect of inflammation in the disruption of vocalization is sex-dependent.

Neuronal deletion of phosphatase and tensin homolog in mice results in spatial dysregulation of adult hippocampal neurogenesis.

Adult neurogenesis is a persistent phenomenon in mammals that occurs in select brain structures in both healthy and diseased brains. The tumor suppressor gene, phosphatase and tensin homolog deleted on chromosome 10 (Pten) has previously been found to restrict the proliferation of neural stem/progenitor cells (NSPCs) in vivo. In this study, we aimed to provide a comprehensive picture of how conditional deletion of Pten may regulate the genesis of adult NSPCs in the dentate gyrus of the hippocampus and the subventricular zone bordering the lateral ventricles. Using conventional markers and stereology, we quantified multiple stages of neurogenesis, including proliferating cells, immature neurons (neuroblasts), and apoptotic cells in several regions of the dentate gyrus, including the subgranular zone (SGZ), outer granule cell layer (oGCL), molecular layer, and hilus at 4 and 10 weeks of age. Our data demonstrate that conditional deletion of Pten in mice produces successive increases in dentate gyrus proliferating cells and immature neuroblasts, which confirms the known negative roles Pten has on cell proliferation and maturation. Specifically, we observe a significant increase in Ki67+ proliferating cells in the neurogenic SGZ at 4 weeks of age, but not 10 weeks of age. We also observe a delayed increase in neuroblasts at 10 weeks of age. However, our study expands on previous work by providing temporal, subregional, and neurogenesis-stage resolution. Specifically, we found that Pten deletion initially increases cell proliferation in the neurogenic SGZ, but this increase spreads to non-neurogenic dentate gyrus areas, including the hilus, oGCL, and molecular layer, as mice age. We also observed region-specific increases in apoptotic cells in the dentate gyrus hilar region that paralleled the regional increases in Ki67+ cells. Our work is accordant with the literature showing that Pten serves as a negative regulator of dentate gyrus neurogenesis but adds temporal and spatial components to the existing knowledge.

Neocortical and cerebellar malformations affect flurothyl-induced seizures in female C57BL/6J mice.

Brain malformations cause cognitive disability and seizures in both human and animal models. Highly laminated structures such as the neocortex and cerebellum are vulnerable to malformation, affecting lamination and neuronal connectivity as well as causing heterotopia. The objective of the present study was to determine if sporadic neocortical and/or cerebellar malformations in C57BL/6J mice are correlated with reduced seizure threshold. The inhaled chemi-convulsant flurothyl was used to induce generalized, tonic-clonic seizures in male and female C57BL/6J mice, and the time to seizure onset was recorded as a functional correlate of brain excitability changes. Following seizures, mice were euthanized, and brains were extracted for histology. Cryosections of the neocortex and cerebellar vermis were stained and examined for the presence of molecular layer heterotopia as previously described in C57BL/6J mice. Over 60% of mice had neocortical and/or cerebellar heterotopia. No sex differences were observed in the prevalence of malformations. Significantly reduced seizure onset time was observed dependent on sex and the type of malformation present. These results raise important questions regarding the presence of malformations in C57BL/6J mice used in the study of brain development, epilepsy, and many other diseases of the nervous system.

Hippocampal Upregulation of Complement Component C3 in Response to Lipopolysaccharide Stimuli in a Model of Fragile-X Syndrome.

The complement system is part of the innate immune system and has been shown to be altered in autism spectrum disorder (ASD). Fragile-X syndrome (FXS) is the main genetic cause of ASD and studies suggest a dysregulation in the immune system in patients with the disorder. To assess if an animal model of FXS presents with altered complement signaling, we treated male Fmr1 knockout (KO) mice with lipopolysaccharide (LPS) and collected the hippocampus 24 h later. Assessment of the expression of the complement genes C1q, C3, and C4 identified the upregulation of C3 in both wild-type (WT) and knockout mice. Levels of C3 also increased in both genotypes. Analysis of the correlation between the expression of C3 and the cytokines IL-6, IL-1ß, and TNF-α identified a different relationship between the expression of the genes in Fmr1 KO when compared to WT mice. Our findings did not support our initial hypotheses that the lack of the FMR1 gene would alter complement system signaling, and that the induction of the complement system in response to LPS in Fmr1 KO mice differed from wild-type conspecifics.

Lipopolysaccharide-induced sickness behavior is not altered in male Fmr1-deficient mice.

OBJECTIVES: Fragile X syndrome is the main monogenetic cause of intellectual disability and autism. Alterations in the immune system are commonly found in these developmental disorders. We and others have demonstrated that Fmr1 mutant mice present an altered response to immune stimuli. However, whether this altered immune response can influence the Fmr1 mutant behavioral outcomes in response to inflammation has not been fully investigated. MATERIALS AND METHODS: In the current study, we examine the behavioral sickness response of male wildtype and knockout  mice to the innate immune stimulus lipopolysaccharide (LPS) (0.1 mg/kg) to determine if Fmr1 mutants have altered sickness behavior. We used an enzyme-linked immunosorbent assay (ELISA) to measure changes in the cytokine interleukin-6 (IL-6) to determine that inflammation was induced in the mice. Sickness behavior was assessed in a wheel-running paradigm, and a tail suspension test was used to assess the depressive-like phenotype that follows sickness behavior in response to LPS. RESULTS: The ELISA using blood serum confirmed a significant increase in IL-6 in mice that were treated with LPS. Treated Fmr1 mutants exhibited decreased distance traveled in the wheel running after LPS administration, similar to treated controls. Another cohort of animals treated with LPS were tested in the tail suspension test and exhibited no alterations in immobility time in response to LPS. CONCLUSION: Together, our data suggest that Fmr1 mutant mice do not have altered sickness behavior in response to a low dose of LPS.

Rapamycin improves social and stereotypic behavior abnormalities induced by pre-mitotic neuronal subset specific Pten deletion.

The mechanistic target of rapamycin (mTOR) pathway is a signaling system integral to neural growth and migration. In both patients and rodent models, mutations to the phosphatase and tensin homolog gene (PTEN) on chromosome 10 results in hyperactivation of the mTOR pathway, as well as seizures, intellectual disabilities and autistic behaviors. Rapamycin, an inhibitor of mTOR, can reverse the epileptic phenotype of neural subset specific Pten knockout (NS-Pten KO) mice, but its impact on behavior is not known. To determine the behavioral effects of rapamycin, male and female NS-Pten KO and wildtype (WT) mice were assigned as controls or administered 10 mg/kg of rapamycin for 2 weeks followed by behavioral testing. Rapamycin improved social behavior in both genotypes and stereotypic behaviors in NS-Pten KO mice. Rapamycin treatment resulted in a reduction of several measures of activity in the open field test in both genotypes. Rapamycin did not reverse the reduced anxiety behavior in KO mice. These data show the potential clinical use of mTOR inhibitors by showing its administration can reduce the production of autistic-like behaviors in NS-Pten KO mice.

Vitamin D supplementation positively affects activity but impairs stimulus response behavior in an age and sex specific manner in C57BL/6 mice.

Several studies have begun to demonstrate the possible cognitive and physiological benefits of a fortified vitamin D diet. However, the behavioral effects of a high vitamin D fortified diet during adolescence has not been fully explored. In the present study, a 4-week vitamin D fortified diet (20,000 IU/Kg) compared to controls (1500 IU/Kg) was administered during the juvenile (4 weeks old) or early adult period (8 weeks old) in C57BL/6 J mice to investigate the effects of fortification on cognition, behavior, and their bone phenotype. After 4 weeks on the diet, vitamin D-treated and control groups underwent a 4-week battery of behavioral tests while remaining on their respective diets. We found that a fortified diet affected behavior in both an age- and sex-specific manner. When vitamin D was administered to juveniles, both sexes displayed impaired habituation to a loud tone. However, females also presented with impaired prepulse inhibition compared to female controls. In the adult treated group, the fortified diet increased only time spent in the open field and had no effect on anxiety-like behavior in the elevated plus maze. Juvenile mice treated with a high vitamin D fortified diet showed a decrease in the total volume compared to the control group in the proximal metaphysis and midshaft region of their femur. There were no differences in bone measurements for mice treated during adulthood. Overall, our results suggest that the juvenile period is a more sensitive time point to the startle response and bone effects of a diet supplemented with high vitamin D, while adults exhibited alterations in locomotive behavior.

Fragile X Messenger Ribonucleoprotein 1 (FMR1), a novel inhibitor of osteoblast/osteocyte differentiation, regulates bone formation, mass, and strength in young and aged male and female mice.

Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene mutations lead to fragile X syndrome, cognitive disorders, and, in some individuals, scoliosis and craniofacial abnormalities. Four-month-old (mo) male mice with deletion of the FMR1 gene exhibit a mild increase in cortical and cancellous femoral bone mass. However, consequences of absence of FMR1 in bone of young/aged male/female mice and the cellular basis of the skeletal phenotype remain unknown. We found that absence of FMR1 results in improved bone properties with higher bone mineral density in both sexes and in 2- and 9-mo mice. The cancellous bone mass is higher only in females, whereas, cortical bone mass is higher in 2- and 9-mo males, but higher in 2- and lower in 9-mo female FMR1-knockout mice. Furthermore, male bones show higher biomechanical properties at 2mo, and females at both ages. Absence of FMR1 increases osteoblast/mineralization/bone formation and osteocyte dendricity/gene expression in vivo/ex vivo/in vitro, without affecting osteoclasts in vivo/ex vivo. Thus, FMR1 is a novel osteoblast/osteocyte differentiation inhibitor, and its absence leads to age-, site- and sex-dependent higher bone mass/strength.

Assessment of tau phosphorylation and ß-amyloid pathology in human drug-resistant epilepsy.

OBJECTIVE: Epilepsy can be comorbid with cognitive impairments. Recent evidence suggests the possibility that cognitive decline in epilepsy may be associated with mechanisms typical of Alzheimer's disease (AD). Neuropathological hallmarks of AD have been found in brain biopsies surgically resected from patients with drug-resistant epilepsies. These include hyperphosphorylation of the tau protein (p-tau) that aggregates into neuropil threads (NT) or neurofibrillary tangles (NFT), as well as the presence of ß-amyloid (Aß) deposits. While recent studies agree on these AD neuropathological findings in epilepsy, some contrast in their correlation to cognitive decline. Thus, to further address this question we determined the abundance of p-tau and Aß proteins along with their association with cognitive function in 12 cases of refractory epilepsy. METHODS: Cortical biopsies surgically extracted from the temporal lobes of patients with refractory epilepsy were processed for immunohistology and enzyme-linked immunoassays to assess distribution and levels, respectively, of p-tau (Antibodies: Ser202/Thr205; Thr205; Thr181) and Aß proteins. In parallel, we measured the activation of mechanistic target of rapamycin (mTOR) via p-S6 (Antibodies: Ser240/244; Ser235/236). Pearson correlation coefficient analysis determined associations between these proteins and neurophysiological scores for full-scale intelligence quotient (FSIQ). RESULTS: We found a robust presence of p-tau (Ser202/Thr205)-related NT and NFT pathology, as well as Aß deposits, and p-S6 (Ser240/244; Ser235/236) in the epilepsy biopsies. We found no significant correlations between p-tau (Thr205; Thr181), Aß, or mTOR markers with FSIQ scores, although some correlation coefficients were modest to strong. SIGNIFICANCE: These findings strongly support the existence of hyperphosphorylated tau protein and Aß deposits in patients with human refractory epilepsy. However, their relation to cognitive decline is still unclear and requires further investigation.

Assessment of the effects of sex, age, and rearing condition on ultrasonic vocalizations elicited by pups during the maternal potentiation paradigm in C57BL/6J mice.

Isolation-induced ultrasonic vocalizations (USVs) are important to elicit parental retrieval. This behavior is critical for the animal's survival and can be altered in models of developmental disorders. The potentiation of vocalizations in response to reunion with the dam, also called maternal potentiation, has been extensively studied in rats. However, the assessment of this paradigm in mice is scarce. In rats, the potentiation of vocalizations is dependent on rearing conditions. Since mice are the main species used for genetic models of diseases, we aimed to investigate how different factors such as age, sex, and rearing conditions can affect the potentiation of vocalizations in the maternal potentiation paradigm in mice. We carried out experiments using biparental (dam and sire) or uniparental rearing (dam). Pups were tested on postnatal days (PD) 9 or 12. Pups showed increased potentiation in both sexes at PD9 with uniparental rearing. Both rearing conditions and ages changed the repertoire from the first to the second isolation. Spectral parameters were affected by sex, rearing condition and reunion at PD9. At PD12, only duration was altered by reunion. We conclude that the performance of the pups in the maternal potentiation paradigm is dependent on age, sex, and rearing condition.

Neuronal subset-specific phosphatase and tensin homolog knockout mice exhibit age and brain region-associated alterations in microglia/macrophage activation.

Seizures induce brain region-dependent enhancements in microglia/macrophage activation. Neuronal subset-specific phosphatase and tensin homolog (PTEN) knockout (KO) mice display hyperactive mammalian target of rapamycin (mTOR) signaling in the hippocampus, cerebellum, and cortex followed by seizures that increase in severity with age. To determine if KO mice also exhibit alterations in the spatiotemporal activation pattern of microglia, we used flow cytometry to compare the percentage of major histocompatibility complex-II activated microglia/macrophages between KO and wildtype (WT) mice at 5, 10, and 15 weeks of age. At 5 weeks, microglia/macrophage activation was greater in the cortex, P < 0.001, cerebellum, P < 0.001, and hippocampus, P < 0.001, of KO compared to WT mice. At 10 weeks, activation was greatest in the cortex of KO mice, P < 0.001, in the cerebellum of WT mice, P < 0.001, but similar in the hippocampus, P > 0.05. By 15 weeks, activation in the hippocampus was more than 25 times greater in KO mice compared to WT mice, P < 0.001. We show that hyperactive mTOR signaling is associated with an altered spatiotemporal pattern of microglia/macrophage activation in the brain and induces an enhanced neuroimmune response in the hippocampus.

Multiple Early-Life Seizures Alters Neonatal Communicative Behavior in Fmr1 Knockout Mice.

Fragile X syndrome (FXS) is the leading monogenic cause of intellectual disability and a significant contributor to Autism Spectrum Disorder. Individuals with FXS are subject to developing numerous comorbidities, one of the most prevalent being seizures. In the present study, we investigated how seizures affected neonatal communicative behavior in the FXS mouse model. On postnatal day (PD) 7 through 11, we administered 3 flurothyl seizures per day to both Fmr1 knockout and wild-type C57BL/6J male mice. Ultrasonic vocalizations were recorded on PD12. Statistically significant alterations were found in both spectral and temporal measurements across seizure groups. We found that induction of seizures across PD7-11 resulted in an increased fundamental frequency (pitch) of ultrasonic vocalizations produced (p < 0.05), a longer duration of calls (p < 0.05), and a greater cumulative duration of calls (p < 0.05) in both genotypes. Induction of seizures across PD7-11 also resulted in a decreased latency to the first emitted vocalization (p < 0.05) and a decrease in mean power (loudness) for their vocalizations (p < 0.05). Early-life seizures also resulted in an increase in the number of downward and frequency step call types (p < 0.05). There was a significant increase in the number of chevron calls emitted from the Fmr1 knockout mice that received seizures compared to knockout control and wild-type seizure mice (p < 0.05). Overall, this study provides evidence that early-life seizures result in communication impairments and that superimposing seizures in Fmr1 knockout mice does produce an additional deficit in vocalization.

A flurothyl-induced seizure does not disrupt hippocampal memory reconsolidation in C57BL/6 J mice.

Memory deficits are a prevalent and pervasive comorbidity of epilepsy that significantly decrease an individual's quality of life. Numerous studies have investigated the effects of a seizure on the encoding process of memory; however, few studies have assessed the effect of a seizure on the reconsolidation process of memory. We investigated how a single seizure affects memory reconsolidation in C57BL/6 J adult mice using a predominately hippocampal-dependent paradigm. Mice were presented with a tone (conditioned stimulus), that was proceeded by a mild shock (unconditioned stimulus) occurring 20 s after the tone. Three days later, a flurothyl-induced seizure was administered 1-h before a memory reconsolidation trial. The learned association was assessed by presenting a conditioned stimulus in a new context 24 h or 1-week after memory reconsolidation. We found that there were no differences in memory present between seizure and control mice at the 24 h or 1-week timepoints. Wheel running was also assessed to ensure that the seizure did not alter locomotion and bias the measure in the memory task. No differences in locomotion between seizure and control mice were observed at any timepoint. Altogether, these findings suggest that hippocampal dependent memory reconsolidation is resistant to flurothyl-induced seizure disruption.

Dietary rescue of adult behavioral deficits in the Fmr1 knockout mouse.

The current study aimed to further address important questions regarding the therapeutic efficacy of omega-3 fatty acids for various behavioral and neuroimmune aspects of the Fmr1 phenotype. To address these questions, our experimental design utilized two different omega-3 fatty acid administration timepoints, compared to both standard laboratory chow controls ("Standard") and a diet controlling for the increase in fat content ("Control Fat"). In the first paradigm, post-weaning supplementation (after postnatal day 21) with the omega-3 fatty acid diet ("Omega-3") reversed deficits in startle threshold, but not deficits in prepulse inhibition, and the effect on startle threshold was not specific to the Omega-3 diet. However, post-weaning supplementation with both experimental diets also impaired acquisition of a fear response, recall of the fear memory and contextual fear conditioning compared to the Standard diet. The post-weaning Omega-3 diet reduced hippocampal expression of IL-6 and this reduction of IL-6 was significantly associated with diminished performance in the fear conditioning task. In the perinatal experimental paradigm, the Omega-3 diet attenuated hyperactivity and acquisition of a fear response. Additionally, perinatal exposure to the Control Fat diet (similar to a "Western" diet) further diminished nonsocial anxiety in the Fmr1 knockout. This study provides significant evidence that dietary fatty acids throughout the lifespan can significantly impact the behavioral and neuroimmune phenotype of the Fmr1 knockout model.

Evaluating the DeepSqueak and Mouse Song Analyzer vocalization analysis systems in C57BL/6J, FVB.129, and FVB neonates.

BACKGROUND: Communication is an essential behavior in mammals. Alterations in communication (neonatal crying) characterize numerous human neurodevelopmental conditions. Mice produce communicative vocalizations, known as ultrasonic vocalizations, (USVs) that can be recorded. The Mouse Song Analyzer is an automated USV analysis system while DeepSqueak is a semi-automated USV detection system. METHOD: We used data from, C57BL/6J, FVB.129, and FVB neonates to compare the reliability of DeepSqueak and the Mouse Song Analyzer across various acoustic variables. RESULTS: We found that both systems detected a similar quantity of USVs for FVB.129 and FVB mice. However, DeepSqueak detected more USVs for C57BL/6J mice. High correlations were found between systems for each strain. When assessing duration, Deepsqueak detected USVs of a longer duration then the Mouse Song Analyzer across all strains. A low correlation between systems for duration was found for FVB.129 mice, while high correlations were found for C57BL/6J and FVB mice. When assessing fundamental frequency, the Mouse Song Analyzer detected a higher frequency than DeepSqueak for FVB.129 mice, with no other differences present. High correlations between systems were found for C57BL/6J and FVB.129 mice, while a low correlation was found for FVB mice. We also assessed each system's sensitivity and found that Deepsqueak was able to detect softer USVs than the Mouse Song Analyzer. CONCLUSIONS: These findings demonstrate that the strain of mouse used significantly affects the reliability of USV analysis systems. However, our data also indicates that DeepSqueak is more reliable and accurate than the Mouse Song Analyzer due to its increased sensitivity.

A vitamin D enriched diet attenuates sex-specific behavioral deficits, increases the lifespan, but does not rescue bone abnormalities in a mouse model of cortical dysplasia.

Individuals who experience recurrent spontaneous seizures often show behavioral and physiological comorbidities. Those with epilepsy are at a high risk of bone fractures (independent of seizure-related falls) and show a higher rate of a diagnosis of Autism Spectrum Disorder. The neural subset-specific (NS) Pten knockout (KO) mouse has an epilepsy phenotype, has been characterized to show autistic-like deficits, and has an osteoporosis phenotype. The current study examined the effect of a vitamin D enriched diet (20,000 IU VD) in the NS-Pten KO and wildtype mice. Mice were placed onto a vitamin D enriched diet at 4 weeks of age and maintained on that diet throughout testing. Behavioral testing began at 6 weeks of age and included tests for general activity, anxiety, repetitive behaviors, social behaviors, and memory. Results indicated that a vitamin D diet attenuated hypoactivity levels in male KO mice (p < 0.05). In a social partition task, vitamin D increased sociability in male wildtype mice, (p < 0.05). Most significantly, vitamin D fortified diet increased percent survival in KO animals and decreased the level of microglia marker IBA-1 and mTOR (mammalian target of rapamycin) downstream targets pS6 and pAKT. A high vitamin D diet did not reverse bone deficits in male or female KO mice. Overall, these findings suggest that a vitamin D enriched diet had a significant impact on the behavioral phenotype of NS-Pten KO mice, suggesting that dietary manipulations could be a potential therapeutic option for autistic-like behavior.

A comparison of four commercially available RNA extraction kits for wastewater surveillance of SARS-CoV-2 in a college population.

Localized wastewater surveillance has allowed for public health officials to gain a broader understanding of SARS-CoV-2 viral prevalence in the community allowing public health officials time to prepare for impending outbreaks. Given variable levels of virus in the population through public health interventions, proper concentration and extraction of viral RNA is a key step in ensuring accurate detections. With many commercial RNA extraction kits and methodologies available, the performance of 4 different kits were evaluated for SARS-CoV-2 RNA detection in wastewater, specifically focusing on their applicability to lower population densities such as those at university campus dorms. Raw wastewater samples were collected at 4 sites on a college campus over a 24 hour period as a composite sample. Included in these sites was an isolation site that housed students that tested positive for Covid-19 via nasopharyngeal swabs. These samples were analyzed using the following kits: Qiagen All Prep PowerViral DNA/RNA kit, New England BioLabs Monarch RNA MiniPrep Kit, and Zymo Quick RNA-Viral Kit, and the Zymo Quick-RNA Fecal/Soil Microbe MicroPrep Kit. All four sites were processed according to the manufacturer's guidelines. Extractions were then quantified with RT-qPCR one-step reactions using an N2 primer and a linearized plasmid standard. While the Zymo Quick-RNA Fecal/Soil Microbe MicroPrep Kit (also known as the Zymo Environ Water RNA Kit) only recovered approximately 73% (±38%) SARS-CoV-2 RNA compared to the Zymo Quick-RNA Viral kit, it was the most time efficient kit to yield comparable results. This extraction kit had a cumulative processing time of approximately 5 h compared, while the other three kits had processing times between approximately 9 and 9.5 h. Based on the current research, the most effective kits for smaller population densities are pellet based and include a homogenization, inhibitor removal, and RNA preservation step.

Rapamycin, but not minocycline, significantly alters ultrasonic vocalization behavior in C57BL/6J pups in a flurothyl seizure model.

Epilepsy is one of the most common neurological disorders, with individuals having an increased susceptibility of seizures in the first few years of life, making children at risk of developing a multitude of cognitive and behavioral comorbidities throughout development. The present study examined the role of PI3K/Akt/mTOR pathway activity and neuroinflammatory signaling in the development of autistic-like behavior following seizures in the neonatal period. Male and female C57BL/6J mice were administered 3 flurothyl seizures on postnatal (PD) 10, followed by administration of minocycline, the mTOR inhibitor rapamycin, or a combined treatment of both therapeutics. On PD12, isolation-induced ultrasonic vocalizations (USVs) of mice were examined to determine the impact of seizures and treatment on communicative behaviors, a component of the autistic-like phenotype. Seizures on PD10 increased the quantity of USVs in female mice and reduced the amount of complex call types emitted in males compared to controls. Inhibition of mTOR with rapamycin significantly reduced the quantity and duration of USVs in both sexes. Changes in USVs were associated with increases in mTOR and astrocyte levels in male mice, however, three PD10 seizures did not result in enhanced proinflammatory cytokine expression in either sex. Beyond inhibition of mTOR activity by rapamycin, both therapeutics did not demonstrate beneficial effects. These findings emphasize the importance of differences that may exist across preclinical seizure models, as three flurothyl seizures did not induce as drastic of changes in mTOR activity or inflammation as observed in other rodent models.

Increased expression of Fragile X mental retardation protein in malformative lesions of patients with focal cortical dysplasia.

OBJECTIVE: Focal cortical dysplasia (FCD) accounts for nearly half of all cases of medically refractory epilepsy in the pediatric and adult patient populations. This neurological disorder stems from localized malformations in cortical brain tissue due to impaired neuronal proliferation, differentiation, and migration patterns. Recent studies in animal models have highlighted the potential role of the Fragile X mental retardation protein (FMRP) levels in FCD. The purpose of this study was to investigate the status of FMRP activation in cortical brain tissues surgically resected from patients with FCD. In parallel, this study also investigated protein levels within the PI3K/AKT/mTOR and canonical Wnt signaling pathways. METHODS: Pathologic tissue from malformative lesions of FCD patients with medically refractory epilepsy was compared to relatively normal control non-epileptic tissue from patients with intracranial neoplasms. A series of western blotting assays were performed to assess key proteins in the PI3K/AKT/mTOR, canonical Wnt signaling pathways, and FMRP. RESULTS: There was suppression of S235/236-phosphorylated S6, GSK3α, and GSK3ß protein levels in samples derived from FCD patients, compared to non-epileptic controls. FCD samples also had significantly greater levels of total and S499-phosphorylated FMRP. CONCLUSION: These findings support our hypothesis that malformative lesions associated with FCD are characterized by high levels of FMRP activation along with dysregulation of both PI3K/AKT/mTOR and canonical Wnt signaling. These novel clinical findings extend previous work in animal models, further suggesting a potential unforeseen role of GSK3α and GSK3ß in the pathophysiology of FCD and refractory epilepsy.

A comparison of the Avisoft (v.5.2) and MATLAB Mouse Song Analyzer (v.1.3) vocalization analysis systems in C57BL/6, Fmr1-FVB.129, NS-Pten-FVB, and 129 mice.

BACKGROUND: Communicative behaviors play a vital role in mammals and are highly relevant to human neurodevelopmental conditions. Mice produce communicative vocalizations that occur in the ultrasonic range, which are commonly analyzed within the Avisoft recording system. Fully automated programs such as the Mouse Song Analyzer in MATLAB, have been developed to analyze USVs in a shorter time period, however, no study has compared the accuracy of MATLAB to Avisoft. NEW METHOD: In order to determine MATLAB's accuracy, we used data from four different mouse strains and assessed whether the total number of USVs detected was similar between systems. RESULTS: We found that there was a high correlation between systems for the number of USVs emitted from C57BL/6 and NS-Pten mice however, Avisoft detected significantly more USVs than MATLAB for both strains. For Fmr1-FVB.129 and 129 mice, large correlations were observed between systems and no significant difference was present in the USVs detected. A partial correlation was run to control for the covariates: sex, age, strain, and treatment, and found that only strain substantially influences the relationship between the USVs detected in Avisoft and those detected in MATLAB. COMPARISON WITH EXISTING METHOD: These findings demonstrate that there is a high degree of agreement between Avisoft and the Mouse Song Analyzer however, Avisoft does detect significantly more USVs depending on the strain assessed. CONCLUSIONS: Therefore, there are relative advantages and disadvantages with both systems that vocalization researchers should be aware of when interpreting USV results, and when using either system.