Female BTBR Research Articles

Mannan-Binding Lectin Is Associated with Inflammation and Kidney Damage in a Mouse Model of Type 2 Diabetes.

Autoreactivity of the complement system may escalate the development of diabetic nephropathy. We used the BTBR OB mouse model of type 2 diabetes to investigate the role of the complement factor mannan-binding lectin (MBL) in diabetic nephropathy. Female BTBR OB mice (n = 30) and BTBR non-diabetic WT mice (n = 30) were included. Plasma samples (weeks 12 and 21) and urine samples (week 19) were analyzed for MBL, C3, C3-fragments, SAA3, and markers for renal function. Renal tissue sections were analyzed for fibrosis, inflammation, and complement deposition. The renal cortex was analyzed for gene expression (complement, inflammation, and fibrosis), and isolated glomerular cells were investigated for MBL protein. Human vascular endothelial cells cultured under normo- and hyperglycemic conditions were analyzed by flow cytometry. We found that the OB mice had elevated plasma and urine concentrations of MBL-C (p < 0.0001 and p < 0.001, respectively) and higher plasma C3 levels (p < 0.001) compared to WT mice. Renal cryosections from OB mice showed increased MBL-C and C4 deposition in the glomeruli and increased macrophage infiltration (p = 0.002). Isolated glomeruli revealed significantly higher MBL protein levels (p < 0.001) compared to the OB and WT mice, and no renal MBL expression was detected. We report that chronic inflammation plays an important role in the development of DN through the binding of MBL to hyperglycemia-exposed renal cells.

Effects of different types of induced neonatal inflammation on development and behavior of C57BL/6 and BTBR mice

Neuroinflammation in the early postnatal period can disturb trajectories of the completion of normal brain development and can lead to mental illnesses, such as depression, anxiety disorders, and personality disorders later in life. In our study, we focused on evaluating short- and long-term effects of neonatal inflammation induced by lipopolysaccharide, poly(I:C), or their combination in female and male C57BL/6 and BTBR mice. We chose the BTBR strain as potentially more susceptible to neonatal inflammation because these mice have behavioral, neuroanatomical, and physiological features of autism spectrum disorders, an abnormal immune response, and several structural aberrations in the brain. Our results indicated that BTBR mice are more sensitive to the influence of the neonatal immune activation (NIA) on the formation of neonatal reflexes than C57BL/6 mice are. In these experiments, the injection of lipopolysaccharide had an effect on the formation of the cliff aversion reflex in female BTBR mice. Nonetheless, NIA had no delayed effects on either social behavior or anxiety-like behavior in juvenile and adolescent BTBR and C57BL/6 mice. Altogether, our data show that NIA has mimetic-, age-, and strain-dependent effects on the development of neonatal reflexes and on exploratory activity in BTBR and C57BL/6 mice.

Deletion, but Not Heterozygosity, of eNOS Raises Blood Pressure and Aggravates Nephropathy in BTBR ob/ob Mice

Introduction: ob/ob mice are a leptin-deficient type 2 diabetes mellitus model, which, on a BTBR background, mimics the glomerular pathophysiology of diabetic nephropathy (DN). Since leptin deficiency reduces blood pressure (BP) and endothelial nitric oxide synthase (eNOS) lowers BP and is kidney protective, we attempted to develop a more robust DN model by introducing eNOS deficiency in BTBR ob/ob mice. Methods: Six experimental groups included littermate male and female BTBR ob/ob or wild-type for ob (control) as well as wild-type (WT), heterozygote (HET), or knockout (KO) for eNOS. Systolic BP (by automated tail-cuff) and GFR (by FITC-sinistrin plasma kinetics) were determined in awake mice at 27–30 weeks of age, followed by molecular and histological kidney analyses. Results: Male and female ob/ob WT presented hyperglycemia and larger body and kidney weight, GFR, glomerular injury, and urine albumin to creatinine ratio (UACR) despite modestly lower BP versus control WT. These effects were associated with a higher tubular injury score and renal mRNA expression of NGAL only in males, whereas female ob/ob WT unexpectedly had lower KIM-1 and COL1A1 expression versus control WT, indicating sex differences. HET for eNOS did not consistently alter BP or renal outcome in control or ob/ob. In comparison, eNOS KO increased BP (15–25 mm Hg) and worsened renal markers of injury, inflammation and fibrosis, GFR, UACR, and survival rates, as observed in control and, more pronouncedly, in ob/ob mice and independent of sex. Conclusions: Deletion, but not heterozygosity, of eNOS raises blood pressure and aggravates nephropathy in BTBR ob/ob mice.

Comprehensive analysis of the gene expression profile of the male and female BTBR mice with diabetic nephropathy

Comprehensive insight into the gender-based gene expression-related omics data in a rodent model of diabetic nephropathy (DN) is scarce. In the present study, the gender-based genes regulating different pathways involved in the progression of DN were explored through an unbiased RNA sequence of kidneys from BTBR mice with DN. We identified 17,739 and 17,981 genes in male and female DN mice; 1121 and 655 genes were expressed differentially (DEGs, differentially expressed genes) in male and female DN mice; both genders displayed only 195 DEGs. In the male DN mice, the number of upregulated genes was nearly the same as that of the down-regulated genes. In contrast, the number of upregulated genes was lesser than that of the down-regulated genes in the female DN mice, manifesting a remarkable gender disparity during the progression of DN in this animal model. Gene Ontology (GO) and KEGG-enriched results showed that most of these DEGs were related to the critical biological processes, including metabolic pathways, natural oxidation, bile secretion, and PPAR signaling; all are highly associated with DN. Notably, the DEGs significantly enriched for steroid hormone biosynthesis pathway were identified in both genders; the number of DEGs increased was 22 in male DN mice and 14 in female DN mice. Specifically, the Ugt1a10, Akr1c12, and Akr1c14 were upregulated in both genders. Interestingly, the Hsd11b1 gene was upregulated in female DN mice but downregulated in male DN mice. These results suggest that a significant gender-based variance in the gene expression occurs during the progression of DN and may be playing a role in the advancement of DN in the BTBR mouse model.

TNF-α Pathway and Podocyte Dysfunction in Male and Female BTBR ob/ob Mice

TNF-α Pathway and Podocyte Dysfunction in Male and Female BTBR ob/ob Mice

Serotonergic circuit dysregulation underlying autism-related phenotypes in BTBR mouse model of autism

The inbred mouse strain, BTBR T+Itpr3tf/J (BTBR), possesses neuronal and circuit abnormalities that underlie atypical behavioral profiles resembling the major symptoms of human autism spectrum disorder (ASD). Forebrain serotonin (5-HT) transmission has been implicated in ASD-related behavioral alterations. In this study, we assessed 5-HT signals and the functional responsiveness in BTBR mice compared to standard C57BL/6J (B6) control mice to elucidate how 5-HT alterations contribute to behavioral abnormalities in BTBR mice. A lower number of 5-HT neurons in the median raphe, but not in the dorsal raphe, was observed in male and female BTBR mice. Acute systemic injection of buspirone, a 5-HT1A receptor agonist, induced c-Fos in several brain regions in both B6 and BTBR mice; however, blunted c-Fos induction in BTBR mice was documented in the cingulate cortex, basolateral amygdala (BLA), and ventral hippocampus (Hipp). Decreased c-Fos responses in these regions are associated with a lack of buspirone effects on anxiety-like behavior in BTBR mice. Analysis of mRNA expression following acute buspirone injection indicated that 5HTR1a gene downregulation (or upregulation) occurred in the BLA and Hipp of B6 mice, respectively, but not BTBR mice. The mRNA expression of factors associated with neurogenesis or the pro-inflammatory state was not consistently altered by acute buspirone injection. Therefore, 5-HT responsivity via 5-HT1A receptors in the BLA and Hipp are linked to anxiety-like behavior, in which circuits are disrupted in BTBR mice. Other distinct 5-HT circuits from the BLA and Hipp that regulate social behavior are restricted but preserved in BTBR mice.

Deficits in cerebellum-dependent learning and cerebellar morphology in male and female BTBR autism model mice.

Recently, there has been increased interest in the role of the cerebellum in autism spectrum disorders (ASD). To better understand the pathophysiological role of the cerebellum in ASD, it is necessary to have a variety of mouse models that have face validity for cerebellar disruption in humans. Here, we add to the literature on the cerebellum transgenic and induced mouse models of autism with the characterization of the cerebellum in the BTBR T+Itpr3tf/J (BTBR) inbred mouse strain, which has behavioral phenotypes that are suggestive of ASD in patients. When we examined both male and female BTBR mice in comparison to C57BL/6J (C57) controls, we noted that both sexes of BTBR mice showed motor coordination deficits characteristic of cerebellar dysfunction, but only the male mice showed differences in delay eyeblink conditioning, a cerebellum-dependent learning task that is also disrupted in ASD patients. Both male and female BTBR mice showed considerable expansion of and abnormal foliation in the cerebellum vermis--including significant expansion of specific lobules in the anterior cerebellum. In addition, we found a slight but significant decrease in Purkinje cell density in both male and female BTBR mice, irrespective of lobule. Furthermore, there was a marked reduction of Purkinje cell dendritic spines density in both male and female BTBR mice. These findings suggest that, for the most part, the BTBR mouse model successfully phenocopies many of the characteristics of the subpopulation of ASD patients that have a hypertrophic cerebellum. We discuss the significance of strain differences in the cerebellum as well as the importance of this first effort to identify both concordances and difference between male and female BTBR mice with regard to the cerebellum.

Does the stranger mouse strain matter to female BTBR mice?

Autism spectrum disorder (ASD) is characterized by deficits in social communication and repetitive behaviors/restricted interests. One mouse model of ASD is the BTBR T+Itprtf/J (BTBR) mice which display low levels of social behavior in several tests. The social approach test is used to examine the preference for social interaction between a stranger mouse or a novel object. While female BTBR mice have been used in the social approach test, no one has examined the degree to which the strain of the stranger mouse will affect social behavior. The current experiment tested female BTBR subject mice in the social approach test with stranger mice from different strains including the BTBR, 129S1/SvImJ (129), and C57BL/6J (B6) mice, of which the B6 mice are most social. The results show that female BTBR mice overall spent significantly more time in the stranger mouse chamber. However, further analysis revealed that the subject mice spent significantly more time in the stranger mouse chamber when the stranger was from the B6 strain, but not the BTBR or 129 strains. The BTBR female mice also sniffed the B6 and 129 stranger mice more than the novel object. This suggests that BTBR females are more social with mice that display high levels of social behavior, but less so with less social mice.

Impairments in operant probabilistic reversal learning in BTBR T+tf/J male and female mice

Autism spectrum disorder (ASD) presents with two core symptoms, impairments in social communication and the presence of restricted, repetitive behaviors (RRBs). RRBs are commonly linked to a lack of behavioral flexibility, having a significant negative impact on daily functioning for ASD individuals and their caregivers. Commonly utilized tests of behavioral flexibility employ a traditional deterministic reward approach where choices are either correct or incorrect throughout testing. The incorporation of an 80 %/20 % probabilistic reversal learning paradigm allows for the examination of flexible behavior in the face of variable outcomes, a more ecologically relevant approach. In this task, one specific choice is reinforced on 80 % of trials and the opposite or incorrect choice is reinforced on 20% of trials. Upon successful discrimination learning, the reward contingencies are switched so that the correct choice is now reinforced 20% of trials and the incorrect choice reinforced 80 % of trials, making it the new optimal choice. This translational task has been previously validated in ASD individuals and animal models of ASD, including the BTBR T + tf/J strain. Our lab and others have demonstrated that male BTBR T + tf/J mice have higher expression of lower order RRBs and display deficits in spatial probabilistic reversal learning tasks using a T-maze apparatus. Instead, female BTBR mice do not express the same lower order RRBs and results are mixed on whether females demonstrate similar probabilistic reversal learning deficits in a T-maze. Therefore, the purpose of this study was to assess the validity of using operant chambers to examine BTBR mouse performance on an 80 %/20 % probabilistic reversal learning task and to also examine the sex-specific differences in reversal learning performance in both mouse strains. Results show that BTBR mice, irrespective of sex, were impaired on the reversal learning, requiring more days and trials to reach reversal criterion compared to C57BL/6J mice. These results parallel previous strain findings in the spatial dependent T-maze task in male mice. Further error analysis showed that the impaired behavioral flexibility was due to elevated regressive errors and lose-shift probabilities. BTBR mice have more difficulty maintaining new choice patterns compared to C57BL/6J mice, which supports findings utilizing a spatial T-maze task. Together, these findings further support the use of the BTBR mouse as preclinical models of ASD due to their validity as an ASD model.

Expression of Key Modulators of Serotonergic Neurotransmission after Chronic Metformin Treatment

Despite metformin’s popularity as a treatment for metabolic and developmental disorders its mechanisms of action are still unknown. The goals of this project were to (1) assess the behavioral benefits of chronic metformin treatment alone for core autism symptoms, (2) assess if metformin acts on melanocortin 4 (MCR4) receptors in the brain to alter hormone levels in serum, and (3) to determine if metformin increases brain levels of peroxisome proliferator‐activated receptors α, δ, and γ (PPARs), which modulate insulin signaling throughout the body and increase in the liver with chronic metformin treatment. Since BTBR social behavior is enhanced by selective serotonin reuptake inhibitors (SSRIs) such as sertraline, and their chronic use reduces or otherwise alters receptor and transporter expression in the brain, we hypothesized that with chronic metformin treatment, expression of key regulators of serotonin neurotransmission such as the serotonin transporter (SERT) or organic cation transporters (OCTs) responsible for serotonin clearance would be reduced. Also, given prior evidence that chronic SSRI treatment increases serotonin levels in brain extracellular fluid, we hypothesized that expression of serotonin receptors such as 5‐HT2A among others would be reduced. This compensatory response occurs with chronic SSRI treatments. To explore these hypotheses BTBR T+ Itpr3tf/J (BTBR) and C57BL/6J (C57) mice were given chronic metformin treatment for six weeks, with behavior tests concluding in week 5, before euthanasia and blood and brain collection. With chronic metformin we found social interaction preference and social dominance were enhanced in male but not female BTBR mice. Blood serum was analyzed for hormonal changes, with C57 metformin treated mice seeing a reduction in corticosterone compared to controls. PPARδ was lower for metformin treated female C57 mice than control females, while PPARγ was found to be different between strain controls. Chronic metformin elevated blood glucose levels in female but not male mice. Male mice showed no changes in MCR 4 or SERT expression. Immunohistochemistry revealed an increase in OCT3 labeling in the brain of a BTBR male treated with metformin compared to a BTBR control male. The density of 5‐HT2A receptors was decreased in the anterior olfactory nucleus for BTBR mice following chronic metformin treatment. Overall, these findings indicate chronic metformin treatments that enhance social behaviors in male, but not female BTBR mice also have divergent effects on blood sugar among sexes. We did not find any evidence of chronic metformin treatment altering MCR4 or SERT but there was a decrease in 5‐HT2A expression. Effects on hormones were not present for BTBR mice but corticosterone was lower for C57 metformin treated males than controls, and metformin may increase OCT expression in BTBR males.

Gut-Microbiome Composition in Response to Phenylketonuria Depends on Dietary Phenylalanine in BTBR Pahenu2 Mice.

Phenylketonuria (PKU) is a metabolic disorder caused by a hepatic enzyme deficiency causing high blood and brain levels of the amino acid Phenylalanine (Phe), leading to severe cognitive and psychological deficits that can be prevented, but not completely, by dietary treatment. The behavioral outcome of PKU could be affected by the gut-microbiome-brain axis, as diet is one of the major drivers of the gut microbiome composition. Gut-microbiome alterations have been reported in treated patients with PKU, although the question remains whether this is due to PKU, the dietary treatment, or their interaction. We, therefore, examined the effects of dietary Phe restriction on gut-microbiome composition and relationships with behavioral outcome in mice. Male and female BTBR Pahenu2 mice received either a control diet (normal protein, “high” Phe), liberalized Phe-restricted (33% natural protein restriction), or severe Phe-restricted (75% natural protein restriction) diet with protein substitutes for 10 weeks (n = 14 per group). Their behavioral performance was examined in an open field test, novel and spatial object location tests, and a balance beam. Fecal samples were collected and sequenced for the bacterial 16S ribosomal RNA (rRNA) region. Results indicated that PKU on a high Phe diet reduced Shannon diversity significantly and altered the microbiome composition compared with wild-type animals. Phe-restriction prevented this loss in Shannon diversity but changed community composition even more than the high-Phe diet, depending on the severity of the restriction. Moreover, on a taxonomic level, we observed the highest number of differentially abundant genera in animals that received 75% Phe-restriction. Based on correlation analyses with differentially abundant taxa, the families Entereococacceae, Erysipelotrichaceae, Porphyromonadaceae, and the genus Alloprevotella showed interesting relationships with either plasma Phe levels and/or object memory. According to our results, these bacterial taxa could be good candidates to start examining the microbial metabolic potential and probiotic properties in the context of PKU. We conclude that PKU leads to an altered gut microbiome composition in mice, which is least severe on a liberalized Phe-restricted diet. This may suggest that the current Phe-restricted diet for PKU patients could be optimized by taking dietary effects on the microbiome into account.

Evaluation of Poorly-Bioavailable Cocoa Flavanols and Their Gut Microbial Metabolites in Potentiating Anti-diabetic Activities Through BTBR.Cg-Lepob/ob/WiscJ Mice

Abstract Objectives Cocoa (Theobroma cacao) is a concentrated dietary source of flavanols that have beneficial activities against type-2 diabetes. These compounds have limited small intestinal absorption and are metabolized by the microbiota to bioavailable metabolites that may exert anti-diabetic effects locally and in peripheral tissues. Our objectives were to 1) determine the role of the gut microbiome in facilitating protective effects of cocoa flavanols, and 2) evaluate these effects in a novel mouse model of progressive type-2 diabetes. Methods A small pilot study (n = 3) of male and female BTBR mice (wild-type and homozygous for the Lepob mutation) received either control or cocoa extract-supplemented diet for 10 weeks. Half the animals were administered antibiotics orally to knock down the commensal gut microbiota. Glucose and insulin tolerance tests were conducted at weeks 1 and 5 and 2 and 6, respectively. Weight gain and food intake were monitored weekly. Biomarkers of gut integrity and inflammation were assessed by ELISA. Results Baseline fasting blood glucose (FBG) levels in five-week-old homozygous males and females were measured at 211–271 mg/dL and 112–234 mg/dL, respectively. After five weeks, FBG measured at 281–438 mg/dL and 177–562 mg/dL, respectively. Cocoa provided moderate, yet not significant, protection against weight gain in homozygous males when compared to homozygous males fed control diet. Cocoa provided no significant protection against hypoglycemia in homozygous male or female mice when compared to homozygous controls. In treatment comparisons with and without antibiotics, knocking out the commensal gut microbiota appeared to have minimal effect on weight gain and glycemic control in both males and females. Conclusions Cocoa did provide a moderate level of protection for homozygous males when directly comparing weight gain and FBG across sex. While the microbiome has displayed a promising role in the bioavailability of large flavanols, in this particular model, the impact was minimal. Overall, cocoa was ineffective against the mediation of advanced diabetes and further work must be conducted to understand if this conclusion is isolated to this model of progressive type-2 diabetes. Funding Sources This work was supported by the US Department of Agriculture by AFRI grant 2020–67,017-30,846.

Deletion of eNOS enhances kidney injury in BTBR ob/ob mice

Background ob/ob mice are a leptin deficient murine model of type 2 diabetes mellitus (T2DM), characterized by hyperphagia, obesity and hyperglycemia. ob/ob mice on a BTBR background have enhanced insulin resistance and mimic the glomerular pathophysiology of diabetic nephropathy (DN) in humans. However, leptin deficiency reduces blood pressure (BP). Since reduced activity of endothelial nitric oxide synthase (eNOS) enhances BP and the progression of kidney disease, we attempted to develop a more robust DN model by introducing eNOS deficiency in BTBR ob/ob mice. Methods BTBR mice heterozygous for both ob and eNOS were bred to generate 6 experimental groups being wild-type for Ob (Con) or ob/ob (Ob) as well as wild-type (WT), heterozygote (HET) or knockout (KO) for eNOS: Con/WT, Con/HET, Con/KO, Ob/WT, Ob/HET & Ob/KO. Systolic BP (automated tail-cuff) and GFR (FITC sinistrin plasma kinetics) were determined in awake mice in weeks 27-30 followed by kidney harvest for RTqPCR analysis of injury markers. Statistics: 2-way ANOVA for Ob and eNOS followed by pairwise comparisons if interaction was significant. 13-24 mice (males plus females)/group for WT and HET, n= 5-8 mice/group for KO. Results In WT, Ob increased blood glucose (BG), body weight (Bw), kidney weight (Kw), GFR, and urinary albumin to creatinine ratio (UACR), and reduced BP vs Con. These responses to Ob were not different in HET. KO increased BP and UACR and reduced Kw and GFR vs WT in both Con and Ob. In Ob, KO reduced BG and Bw vs WT. All effects were independent of gender. In male mice: In WT, Ob increased renal mRNA expression of Ngal, Ccl2, and Timp1, whereas Kim1, Tnfa and Col1a1 were not significantly different vs Con. These responses to Ob were similar in HET. KO increased renal mRNA expression of Ngal, Timp1, Col1a1 vs WT in both Con and Ob; and KO, in addition, also increased Kim1, Ccl2 and Tnfa vs WT in Ob. In female mice: In WT, Ob did not change renal mRNA expression of Ngal, Ccl2, Timp1, and Tnfa and the expression of Kim1 and Col1a1 were actually reduced vs Con. Similar responses to Ob were observed in HET. KO increased renal mRNA expression of Ngal, Ccl2, Timp1, and Col1a1 vs WT in both Con and Ob; and KO in addition increased Tnfa vs WT in Ob. Conclusions Male and female BTBR ob/ob mice at 27-30 weeks of age presented hyperglycemia and increased Bw associated with larger kidneys, glomerular hyperfiltration and a 100x higher UACR vs Con. These effects were associated with modest increases in renal mRNA expression of Ngal, Ccl2, and Timp1 only in male mice, whereas female Ob mice unexpectedly had lesser expression of Kim1 and Col1a1 vs Con. eNOS heterozygosity did not induce robust renal changes vs WT in Con or Ob. In contrast, eNOS KO, which increased BP by 15-25 mmHg, enhanced kidney injury as indicated by lesser Kw and GFR, associated with higher UACR and renal mRNA expression of markers of injury, inflammation and fibrosis, and lower survival rates; these effects of eNOS KO were detectable in Con and more pronounced in Ob.

Somatosensorimotor and Odor Modification, Along with Serotonergic Processes Underlying the Social Deficits in BTBR T+ Itpr3tf/J and BALB/cJ Mouse Models of Autism

Autism is a complex spectrum of disorders characterized by core behavioral deficits in social communicative behavior, which are also required for comprehensive analysis of preclinical mouse models. As animal models of the core behavioral deficits in autism, two inbred mouse strains, BTBR T+ Itpr3tf/J (BTBR) and BALB/cJ (BALB), were compared with the standard social strain, C57BL/6J (B6), regarding a variety of behavioral factors underlying social communicative interactions, including olfactory and tactile sensory processes, social recognition abilities and behavioral expression strategies. Although both female BTBR and BALB mice can express social recognition and approach behavior depending on the stimuli they encounter, the available sensory modalities, along with modulation of the serotonergic system, differ between the two strains. BALB mice have deficits in using volatile olfactory cues and tactile information in a social context; they fail to exhibit a social approach to volatile cues and seek nonvolatile cues by exhibiting substantial sniff/contact behavior when allowed direct contact with social opponents. Systemic injection of the serotonin (5-HT1A) agonist buspirone has little effect on these social deficits, suggesting a congenitally degraded serotonergic system in BALB mice. In contrast, BTBR mice exhibit impaired body coordination and social motivation-modified olfactory signals, which are relevant to a reduced social approach. A systemic injection of the 5-HT1A agonist restored these social deficits in BTBR mice, indicating that a downregulated serotonergic system is involved in the social deficits exhibited by BTBR mice.

Differences in the expression of restricted repetitive behaviors in female and male BTBR T + tf/J mice

Autism spectrum disorder (ASD) is characterized by the expression of restricted repetitive behaviors (RRBs) and impairments in social recognition and communication. Epidemiological studies demonstrate males are three times more likely than females to be affected. Although this is the case, more recent studies suggest females may be underrepresented in these numbers due to standard clinical measures of RRBs and social behaviors. In addition, many studies examining mouse models of ASD exclude females due to the sex disparity in diagnoses. The present study examined how female and male BTBR T + Itpr3tf /J (BTBR) compare to control C57BL/6J mice on tests of RRBs (probabilistic reversal learning, repetitive grooming, spontaneous alternation, and marble burying) and social behaviors (three chambered social approach task). Utilizing a spatial reversal learning test with 80/20 probabilistic feedback, in which ASD individuals have exhibited deficits, we find that female BTBR mice do not show the same impairment found in male BTBR mice. Interestingly, control female C57BL/6J mice required more trials to reach criterion. Female BTBR mice expressed comparable rates of repetitive grooming, marble burying and spontaneous alternation compared to female C57BL/6J mice. Male BTBR mice expressed higher rates of grooming behavior and locomotor activity compared to male C57BL/6J mice, as found in previous studies. Similarly, male BTBR mice showed a reduction in both measures of social approach compared to controls. Both male and female BTBR mice showed a reduction in sniff time for the stranger mouse compared to controls. Together these findings demonstrate how female BTBR mice do not display the RRB profile expressed by male BTBR mice. Testing of repetitive behaviors in ASD needs to better reflect the sex differences in how RRBs manifest in females compared to their extensively researched male counterparts.

792-P: Effect of Oral Anserine Supplementation on Type 2 Diabetes and Diabetic Nephropathy in BTBR ob/ob Mice

Carnosine, a naturally occurring dipeptide present in an omnivorous diet, has been shown to ameliorate the development of metabolic syndrome, type 2 diabetes and early- and advanced-stage diabetic nephropathy (Albrecht et al. 2017) in different rodent models. The physiological mechanisms of the protection by carnosine is presumably related to its anti-lipidemic, anti-hyperglycemic and anti-glycation actions. As anserine, its methylated analogue, is more bio-available in humans upon supplementation (Everaert et al. 2018) without affecting its functionality, it was now aimed to investigate the effect of oral supplementation with anserine on the development of severe diabetes and advanced diabetic nephropathy in BTBR ob/ob mice. Male and female BTBR ob/ob mice were either supplemented with 4mM carnosine or anserine in drinking water for 18 weeks and compared with non-supplemented BTBR ob/ob and wt/wt mice. Tissue carnosine and anserine levels were determined with HPLC and metabolic markers were determined in clinical lab of University Hospital UZ Ghent. Gastrocnemius carnosine and anserine levels were lower in male ob/ob vs. wt/wt mice, but were not affected by supplementation. Kidney carnosine and anserine levels were not different between ob/ob and wt/wt mice, but anserine levels were significantly higher in anserine-treated mice compared to non-treated ob/ob mice. The evolution of fasting blood glucose, fructosamine, triglycerides and cholesterol was not affected by the supplementation regimen. The area of the glomerular tuft and whole glomerulus was bigger in ob/ob vs. wt/wt mice, but not affected by supplementation. Based on the currently measured markers, it is suggested that chronic oral anserine supplementation is not able to attenuate the development of diabetes or diabetic nephropathy in the severe model of BTBR ob/ob mice. Further research will have to elucidate whether anserine can attenuate milder forms of metabolic syndrome or type 2 diabetes. Disclosure I. Everaert: None. M. Hanssens: None. H.J. Baelde: None. W. Derave: None. Funding Research Foundation - Flanders

Comorbid epilepsy in autism spectrum disorder: Implications of postnatal inflammation for brain excitability.

In different cohorts, 5%-30% of individuals with autism spectrum disorder (ASD) also have epilepsy. The high co-occurrence of these disorders suggests that a common mechanistic link may exist. The underlying pathophysiology of this comorbidity remains unknown. To investigate the mechanism(s) involved in the pathogenesis of ASD and epilepsy, we developed and validated a novel mouse model that concurrently exhibits hallmark features of both disorders. We utilized inbred BTBR T+ Itpr3tf/J (BTBR) mice that exhibit the core behavioral characteristics of ASD (ie, impaired sociability, altered vocalizations, and restricted interests). BTBR mice received a lipopolysaccharide (LPS) or sterile saline injection at postnatal day (P)7, P14, or P21. Cytokine expression was analyzed for interleukin (IL)-1β, IL-10, IL-6, and tumor necrosis factor α in brain tissue of P7 and adult BTBR mice. Adult BTBR mice were behaviorally analyzed for seizure susceptibility, sociability, communication deficits, and motor stereotypies, and monitored using chronic video-electroencephalography (EEG). Adult male and female BTBR mice treated at P7-P14 with LPS were more sensitive to pentylenetetrazol-induced seizures than saline-treated controls. ASD-like behaviors and hippocampal cytokine levels were unchanged between P7 LPS-treated BTBR mice and controls. EEG recordings from the dorsal hippocampus revealed a significant increase in number and frequency of seizures over the 4-week recording period (P60-P88) in BTBR mice postnatally treated with LPS at P7. These results indicate the presence of a comorbid epileptic phenotype in BTBR mice. These findings suggest that an early postnatal immune challenge can increase brain excitability in adult BTBR mice and reveal an underlying epilepsy phenotype. This novel animal model may enable the elucidation of specific molecular alterations that are associated with the concurrent presentation of ASD and epilepsy, which could facilitate the development of targeted therapies for individuals affected by this comorbidity.

Gender-specific differences in diabetic neuropathy in BTBR ob/ob mice

AimsTo identify a female mouse model of diabetic peripheral neuropathy (DPN), we characterized DPN in female BTBR ob/ob mice and compared their phenotype to non-diabetic and gender-matched controls. We also identified dysregulated genes and pathways in sciatic nerve (SCN) and dorsal root ganglia (DRG) of female BTBR ob/ob mice to determine potential DPN mechanisms. MethodsTerminal neuropathy phenotyping consisted of examining latency to heat stimuli, sciatic motor and sural sensory nerve conduction velocities (NCV), and intraepidermal nerve fiber (IENF) density. For gene expression profiling, DRG and SCN were dissected, RNA was isolated and processed using microarray technology and differentially expressed genes were identified. ResultsSimilar motor and sensory NCV deficits were observed in male and female BTBR ob/ob mice at study termination; however, IENF density was greater in female ob/ob mice than their male counterparts. Male and female ob/ob mice exhibited similar weight gain, hyperglycemia, and hyperinsulinemia compared to non-diabetic controls, although triglycerides were elevated more so in males than in females. Transcriptional profiling of nerve tissue from female mice identified dysregulation of pathways related to inflammation. ConclusionsSimilar to males, female BTBR ob/ob mice display robust DPN, and pathways related to inflammation are dysregulated in peripheral nerve.

AMPAKINE enhancement of social interaction in the BTBR mouse model of autism

Autism is a neurodevelopmental disorder in which the first diagnostic symptom is unusual reciprocal social interactions. Approximately half of the children diagnosed with an autism spectrum disorder also have intellectual impairments. General cognitive abilities may be fundamental to many aspects of social cognition. Cognitive enhancers could conceivably be of significant benefit to children and adults with autism. AMPAKINE compounds are a novel class of pharmacological agents that act as positive modulators of AMPA receptors to enhance excitatory glutamatergic neurotransmission. This class of compounds was reported to improve learning and memory in several rodent and non-human primate tasks, and to normalize respiratory abnormalities in a mouse model of Rett syndrome. Here we evaluate the actions of AMPA compounds in adult male and female BTBR mice, a well characterized mouse model of autism. Acute treatment with CX1837 and CX1739 reversed the deficit in sociability in BTBR mice on the most sensitive parameter, time spent sniffing a novel mouse as compared to time spent sniffing a novel object. The less sensitive parameter, time in the chamber containing the novel mouse versus time in the chamber containing the novel object, was not rescued by CX1837 or CX1739 treatment. Preliminary data with CX546, in which β-cyclodextrin was the vehicle, revealed behavioral effects of the acute intraperitoneal and oral administration of vehicle alone. To circumvent the artifacts introduced by the vehicle administration, we employed a novel treatment regimen using pellets of peanut butter for drug delivery. Absence of vehicle treatment effects when CX1837 and CX1739 were given in the peanut butter pellets, to multiple cohorts of BTBR and B6 control mice, confirmed that the pharmacologically-induced improvements in sociability in BTBR were not confounded by the administration procedures. The highest dose of CX1837 improved the cognitive deficit in novel object recognition in BTBR. No drug effects were detected on the high levels of repetitive self-grooming in BTBR. In open field tests, CX1837 and CX1739 did not induce hyperactivity or sedation in either strain. It is interesting to speculate that the ability of CX1837 and CX1739 to restore aspects of sociability in BTBR mice could utilize synaptic mechanisms regulating social cognition, suggesting a potential pharmacological target for interventions to treat symptoms of autism.This article is part of a Special Issue entitled ‘Cognitive Enhancers’.

Abstract 18296: A Murine Model of Diet-Induced Insulin Resistance and Cardiomyopathy

Background: Diabetes is associated with a 2- to 3-fold ↑ in risk for congestive heart failure (CHF), due primarily to cardiac hypertrophy and fibrosis. We recently have shown that leptin-deficient, female BTBR mice (BTBR ob/ob ) develop cardiomyopathy, with a 24-25% ↑ in heart weight and a 5-fold ↑ in fibrosis. We investigated whether the cardiomyopathy phenotype might be replicated with diet-induced obesity in wild-type, BTBR mice. Methods: At 4 weeks of age, male and female BTBR mice (n=7-8/group) were placed on one of three diets: 1) standard rodent chow (“Chow”), 2) a “diabetogenic” diet, with 58% of calories from fat and 14% of calories from sucrose (“DD”), or 3) the “diabetogenic” diet with 0.15% cholesterol added (“DDC”). Oral glucose tolerance tests were performed at 8 and 16 weeks on diets. Mice were sacrificed after 16 weeks on diets for determination of heart weights and fibrosis. Results: By 8 weeks on DD or DDC, both female and male BTBR mice had normal fasting glucose, but glucose tolerance (as judged by area under the glucose excursion curve) was significantly impaired vs. Chow-fed controls. Prior to sacrifice at 16 weeks, as compared to Chow, mice on DD or DDC had similar blood pressures and fasting glucose levels. However, necropsy heart weights were 17-18% higher (P&lt;0.01 vs. Chow for both DD and DDC for both genders). In addition, Masson's trichrome stain demonstrated significant increases in fibrosis vs. Chow for BTBR mice fed either DD (P=0.015) or DDC (P=0.029). Conclusions: Thus, in both genders, 16 weeks feeding of high-fat, high-sucrose diets to wild-type BTBR mice results in a 2-fold ↑ in fibrosis, and achieves &gt;60% of the cardiac hypertrophy seen in BTBR ob/ob females by age 16 weeks. These findings suggest that diabetogenic diet feeding in BTBR mice may represent an efficient model for studying the pathogenic mechanisms underlying cardiomyopathy in insulin resistance and diabetes.