Heterozygous loss of epilepsy gene KCNQ2 alters social,repetitive and exploratory behaviors

KCNQ/K_v7 channels conduct voltage‐dependent outward potassium currents that potently decrease neuronal excitability. Heterozygous inherited mutations in their principle subunits K_v7.2/KCNQ2 and K_v7.3/KCNQ3 cause benign familial neonatal epilepsy whereas patients with de novo heterozygous K_v7.2 mutations are associated with early‐onset epileptic encephalopathy and neurodevelopmental disorders characterized by intellectual disability, developmental delay and autism. However, the role of K_v7.2‐containing K_v7 channels in behaviors especially autism‐associated behaviors has not been described. Because pathogenic K_v7.2 mutations in patients are typically heterozygous loss‐of‐function mutations, we investigated the contributions of K_v7.2 to exploratory, social, repetitive and compulsive‐like behaviors by behavioral phenotyping of both male and female KCNQ2^+/? mice that were heterozygous null for the KCNQ2 gene. Compared with their wild‐type littermates, male and female KCNQ2^+/? mice displayed increased locomotor activity in their home cage during the light phase but not the dark phase and showed no difference in motor coordination, suggesting hyperactivity during the inactive light phase. In the dark phase, KCNQ2^+/? group showed enhanced exploratory behaviors, and repetitive grooming but decreased sociability with sex differences in the degree of these behaviors. While male KCNQ2^+/? mice displayed enhanced compulsive‐like behavior and social dominance, female KCNQ2^+/? mice did not. In addition to elevated seizure susceptibility, our findings together indicate that heterozygous loss of K_v7.2 induces behavioral abnormalities including autism‐associated behaviors such as reduced sociability and enhanced repetitive behaviors. Therefore, our study is the first to provide a tangible link between loss‐of‐function K_v7.2 mutations and the behavioral comorbidities of K_v7.2‐associated epilepsy.     

Activity‐regulated cytoskeleton‐associated protein (Arc/Arg3.1) regulates anxiety‐ and novelty‐related behaviors

The activity‐regulated cytoskeleton‐associated protein (Arc, also known as Arg3.1) regulates glutamatergic synapse plasticity and has been linked to neuropsychiatric illness; however, its role in behaviors associated with mood and anxiety disorders remains unclear. We find that stress upregulates Arc expression in the adult mouse nucleus accumbens (NAc)—a brain region implicated in mood and anxiety behaviors. Global Arc knockout mice have altered AMPAR‐subunit surface levels in the adult NAc, and the Arc‐deficient mice show reductions in anxiety‐like behavior, deficits in social novelty preference, and antidepressive‐like behavior. Viral‐mediated expression of Arc in the adult NAc of male, global Arc KO mice restores normal levels of anxiety‐like behavior in the elevated plus maze (EPM). Consistent with this finding, viral‐mediated reduction of Arc in the adult NAc reduces anxiety‐like behavior in male, but not female, mice in the EPM. NAc‐specific reduction of Arc also produced significant deficits in both object and social novelty preference tasks. Together our findings indicate that Arc is essential for regulating normal mood‐ and anxiety‐related behaviors and novelty discrimination, and that Arc's function within the adult NAc contributes to these behavioral effects.     

Atp1a3‐deficient heterozygous mice show lower rank in the hierarchy and altered social behavior

Atp1a3 is the Na‐pump alpha3 subunit gene expressed mainly in neurons of the brain. Atp1a3‐deficient heterozygous mice (Atp1a3^+/?) show altered neurotransmission and deficits of motor function after stress loading. To understand the function of Atp1a3 in a social hierarchy, we evaluated social behaviors (social interaction, aggression, social approach and social dominance) of Atp1a3^+/? and compared the rank and hierarchy structure between Atp1a3^+/? and wild‐type mice within a housing cage using the round‐robin tube test and barbering observations. Formation of a hierarchy decreases social conflict and promote social stability within the group. The hierarchical rank is a reflection of social dominance within a cage, which is heritable and can be regulated by specific genes in mice. Here we report: (1) The degree of social interaction but not aggression was lower in Atp1a3^+/? than wild‐type mice, and Atp1a3^+/? approached Atp1a3^+/? mice more frequently than wild type. (2) The frequency of barbering was lower in the Atp1a3^+/? group than in the wild‐type group, while no difference was observed in the mixed‐genotype housing condition. (3) Hierarchy formation was not different between Atp1a3^+/? and wild type. (4) Atp1a3^+/? showed a lower rank in the mixed‐genotype housing condition than that in the wild type, indicating that Atp1a3 regulates social dominance. In sum, Atp1a3^+/? showed unique social behavior characteristics of lower social interaction and preference to approach the same genotype mice and a lower ranking in the hierarchy.     

Maternal care of heterozygous dopamine receptor D4 knockout mice: Differential susceptibility to early‐life rearing conditions

The differential susceptibility hypothesis proposes that individuals who are more susceptible to the negative effects of adverse rearing conditions may also benefit more from enriched environments. Evidence derived from human experiments suggests the lower efficacy dopamine receptor D4 (DRD4) 7‐repeat as a main factor in exhibiting these for better and for worse characteristics. However, human studies lack the genetic and environmental control offered by animal experiments, complicating assessment of causal relations. To study differential susceptibility in an animal model, we exposed Drd4^+/? mice and control litter mates to a limited nesting/bedding (LN), standard nesting (SN) or communal nesting (CN) rearing environment from postnatal day (P) 2‐14. Puberty onset was examined from P24 to P36 and adult females were assessed on maternal care towards their own offspring. In both males and females, LN reared mice showed a delay in puberty onset that was partly mediated by a reduction in body weight at weaning, irrespective of Drd4 genotype. During adulthood, LN reared females exhibited characteristics of poor maternal care, whereas dams reared in CN environments showed lower rates of unpredictability towards their own offspring. Differential susceptibility was observed only for licking/grooming levels of female offspring towards their litter; LN reared Drd4^+/? mice exhibited the lowest and CN reared Drd4^+/? mice the highest levels of licking/grooming. These results indicate that both genetic and early‐environmental factors play an important role in shaping maternal care of the offspring for better and for worse.     

Relaxin‐3 receptor (Rxfp3) gene deletion reduces operant sucrose‐ but not alcohol‐responding in mice

The pervasive use of refined sugars in highly accessible, palatable foods and persistent exposure to reinforcing food‐associated cues has contributed to overconsumption of sugar‐rich diets and the current obesity epidemic in Western society. We have shown previously that brain relaxin‐3 mRNA levels positively correlate with sucrose and alcohol intake, and that central antagonism of relaxin‐3 receptors (RXFP3) attenuates alcohol self‐administration and alcohol‐seeking in rats, but food‐seeking behaviour and palatable food consumption in mice. To further examine the relationship between motivated appetitive behaviours and relaxin‐3/RXFP3 signalling, we investigated the effect of Rxfp3 gene deletion in C57BL/6J mice on sucrose and alcohol self‐administration and cue‐induced reinstatement (RNST) of sucrose‐ and alcohol‐seeking. Acquisition and maintenance of sucrose and alcohol self‐administration was assessed in male wild‐type (WT) and Rxfp3 knockout (KO) (C57BL/6J^{RXFP3TM1/DGen}) littermate mice using fixed ratio (FR) schedules of reinforcement. Mice were subsequently challenged with a progressive ratio (PR) test to measure motivation and, following extinction training, re‐exposed to reward‐associated cues to evaluate RNST of active lever‐responding. Wild‐type and Rxfp3 KO mice displayed similar acquisition of FR1 sucrose self‐administration, but Rxfp3 KO mice responded less when the instrumental requirement was increased to FR3. These mice also showed a lower breakpoint for sucrose and attenuated cue‐induced RNST of sucrose‐seeking. Notably, no marked genotype differences in alcohol‐responding were observed. In mice, endogenous relaxin‐3/RXFP3 signalling promotes self‐administration of sucrose under high response requirements and cue‐induced RNST of sucrose‐seeking, but does not apparently regulate motivation to consume alcohol or alcohol‐seeking behaviour.     

Deficiency in the anti‐aging gene Klotho promotes aortic valve fibrosis through AMPKα‐mediated activation of RUNX2

Fibrotic aortic valve disease (FAVD) is an important cause of aortic stenosis, yet currently there is no effective treatment for FAVD due to its unknown etiology. The purpose of this study was to investigate whether deficiency in the anti‐aging Klotho gene (KL) promotes high‐fat‐diet‐induced FAVD and to explore the underlying molecular mechanism. Heterozygous Klotho‐deficient (KL^+/?) mice and WT littermates were fed with a high‐fat diet (HFD) or normal diet for 13 weeks, followed by treatment with the AMPKα activator (AICAR) for an additional 2 weeks. A HFD caused a greater increase in collagen levels in the aortic valves of KL^+/? mice than of WT mice, indicating that Klotho deficiency promotes HFD‐induced aortic valve fibrosis (AVF). AMPKα activity (pAMPKα) was decreased, while protein expression of collagen I and RUNX2 was increased in the aortic valves of KL^+/? mice fed with a HFD. Treatment with AICAR markedly attenuated HFD‐induced AVF in KL^+/? mice. AICAR not only abolished the downregulation of pAMPKα but also eliminated the upregulation of collagen I and RUNX2 in the aortic valves of KL^+/? mice fed with HFD. In cultured porcine aortic valve interstitial cells, Klotho‐deficient serum plus cholesterol increased RUNX2 and collagen I protein expression, which were attenuated by activation of AMPKα by AICAR. Interestingly, silencing of RUNX2 abolished the stimulatory effect of Klotho deficiency on cholesterol‐induced upregulation of matrix proteins, including collagen I and osteocalcin. In conclusion, Klotho gene deficiency promotes HFD‐induced fibrosis in aortic valves, likely through the AMPKα–RUNX2 pathway.     

Autism‐related behaviors in the cyclooxygenase‐2‐deficient mouse model

Prostaglandin E2 (PGE2) is an endogenous lipid molecule involved in normal brain development. Cyclooxygenase‐2 (COX2) is the main regulator of PGE2 synthesis. Emerging clinical and molecular research provides compelling evidence that abnormal COX2/PGE2 signaling is associated with autism spectrum disorder (ASD). We previously found that COX2 knockout mice had dysregulated expression of many ASD genes belonging to important biological pathways for neurodevelopment. The present study is the first to show the connection between irregular COX2/PGE2 signaling and autism‐related behaviors in male and female COX2‐deficient knockin, (COX)‐2^?, mice at young (4‐6 weeks) or adult (8‐11 weeks) ages. Autism‐related behaviors were prominent in male (COX)‐2^? mice for most behavioral tests. In the open field test, (COX)‐2^? mice traveled more than controls and adult male (COX)‐2^? mice spent less time in the center indicating elevated hyperactive and anxiety‐linked behaviors. (COX)‐2^? mice also buried more marbles, with males burying more than females, suggesting increased anxiety and repetitive behaviors. Young male (COX)‐2^? mice fell more frequently in the inverted screen test revealing motor deficits. The three‐chamber sociability test found that adult female (COX)‐2^? mice spent less time in the novel mouse chamber indicative of social abnormalities. In addition, male (COX)‐2^? mice showed altered expression of several autism‐linked genes: Wnt2, Glo1, Grm5 and Mmp9. Overall, our findings offer new insight into the involvement of disrupted COX2/PGE2 signaling in ASD pathology with age‐related differences and greater impact on males. We propose that (COX)‐2^? mice might serve as a novel model system to study specific types of autism.     

Hoxb8 is required for normal grooming behavior in mice.

Repertoires of grooming behaviors critical to survival are exhibited by most animal species, including humans. Genes that influence this complex behavior are unknown. We report that mice with disruptions of Hoxb8 show, with 100% penetrance, excessive grooming leading to hair removal and lesions. Additionally, these mice excessively groom normal cagemates. We have been unable to detect any skin or PNS abnormalities in Hoxb8 mutants. These observations suggest that the excessive, pathological grooming exhibited by these mice results from CNS abnormalities. Consistent with this interpretation, we demonstrate Hoxb8 expression in regions of the adult mouse CNS previously implicated in the control of grooming. The aberrant behavior observed in Hoxb8 mutants is not unlike that of humans suffering from the OC-spectrum disorder, trichotillomania. Interestingly, Hoxb8 is expressed in regions of the CNS known as the "OCD-circuit."     

Impact of external odor on self‐grooming of lesser flat‐headed bats,Tylonycteris pachypus

Grooming is a common behavior of some mammals. Previous studies have shown that grooming is a means by which animals clean themselves, remove ectoparasites, and lower their body temperature. It is also involved in olfactory communication. Bats belong to the order Chiroptera and, like most mammals, are the natural host of many ectoparasites. Bat grooming, including licking and scratching, is one of the ways to reduce the adverse effects caused by ectoparasites. Bat grooming may also be induced by exogenous odor. In this study, we used lesser flat‐headed bats (Tylonycteris pachypus) to test the hypothesis that exogenous odor affects the self‐grooming behavior of bats. Results showed that external odor from distantly related species caused lesser flat‐headed bats to spend more time in self‐grooming. Lesser flat‐headed bats that received odor from humans spent the longest time in self‐grooming, followed by those that received odor from a different species of bats (T. robustula). Lesser flat‐headed bats that received odor form the same species of bats, either from the same or a different colony, spent the least amount of time in self‐grooming. These results suggest that bats can recognize conspecific and heterospecific through body scent.     

Mice lacking the Parkinson's related GPR37/PAEL receptor show non‐motor behavioral phenotypes: age and gender effect

Non-motor symptoms in Parkinson's disease (PD) have been often described at different stages of the disease but they are poorly understood. We observed specific phenotypes related to these symptoms in mice lacking the PD‐associated GPR37/PAEL receptor. GPR37 is an orphan G‐protein‐coupled receptor highly expressed in the mammalian central nervous system. It is a substrate of parkin and it is involved in the pathogenesis of PD. GPR37 interacts with the dopamine transporter (DAT), modulating nigro‐striatal dopaminergic signaling and behavioral responses to amphetamine and cocaine. GPR37 knockout (KO) mice are resistant to MPTP and exhibit several motor behavioral abnormalities related to altered dopaminergic system function. To evaluate non-motor behavioral domains, adult and aged, male and female GPR37 KO mice and their wild‐type (WT) littermates were analyzed in a series of cross‐sectional studies. Aged GPR37 KO female mice showed mild improvements in olfactory function, while anxiety and depression‐like behaviors appeared to be significantly increased. A reduction of the startle response to acoustic stimuli was observed only in adult GPR37 KO mice of both genders. Furthermore, HPLC analysis of major neurotransmitter levels revealed gender differences in the striatum, hippocampus and olfactory bulb of mutant mice. The absence of GPR37 receptor could have a neuroprotective effect in an age and gender‐dependent manner, and the study of this receptor could be valuable in the search for novel therapeutic targets.     

Decreased aggression and increased repetitive behavior in Pten haploinsufficient mice

Aggression is an aspect of social behavior that can be elevated in some individuals with autism spectrum disorder (ASD) and a concern for peers and caregivers. Mutations in Phosphatase and tensin homolog (PTEN), one of several ASD risk factors encoding negative regulators of the PI3K–Akt–mTOR pathway, have been reported in individuals with ASD and comorbid macrocephaly. We previously showed that a mouse model of Pten germline haploinsufficiency (Pten^+/?) has selective deficits, primarily in social behavior, along with broad overgrowth of the brain. Here, we further examine the social behavior of Pten^+/? male mice in the resident–intruder test of aggression, using a comprehensive behavioral analysis to obtain an overall picture of the agonistic, non‐agonistic and non‐social behavior patterns of Pten^+/? mice during a free interaction with a novel conspecific. Pten^+/? male mice were involved in less aggression than their wild‐type littermates. Pten^+/? mice also performed less social investigation, including anogenital investigation and approaching and/or attending to the intruder, which is consistent with our previous finding of decreased sociability in the social approach test. In contrast to these decreases in social behaviors, Pten^+/? mice showed increased digging. In summary, we report decreased aggression and increased repetitive behavior in Pten^+/? mice, thus extending our characterization of this model of an ASD risk factor that features brain overgrowth and social deficits.     

ZFPIP/Zfp462 is involved in P19 cell pluripotency and in their neuronal fate

The nuclear zinc finger protein ZFPIP/Zfp462 is an important factor involved in cell division during the early embryonic development of vertebrates. In pluripotent P19 cells, ZFPIP/Zfp462 takes part in cell proliferation, likely via its role in maintaining chromatin structure. To further define the function of ZFPIP/Zfp462 in the mechanisms of pluripotency and cell differentiation, we constructed a stable P19 cell line in which ZFPIP/Zfp462 knockdown is inducible.We report that ZFPIP/Zfp462 was vital for mitosis and self-renewal in pluripotent P19 cells. Its depletion induced substantial decreases in the expression of the pluripotency genes Nanog, Oct4 and Sox2 and was associated with the transient expression of specific neuronal differentiation markers. We also demonstrated that ZFPIP/Zfp462 expression appears to be unnecessary after neuronal differentiation is induced in P19 cells.Taken together, our results strongly suggest that ZFPIP/Zfp462 is a key chromatin factor involved in maintaining P19 pluripotency and in the early mechanisms of neural differentiation but that it is dispensable in differentiated P19 cells.     

Behavioral flexibility in a mouse model for obsessive‐compulsive disorder: Impaired Pavlovian reversal learning in SAPAP3 mutants

Obsessive‐compulsive disorder (OCD) is characterized by obsessive thinking, compulsive behavior and anxiety, and is often accompanied by cognitive deficits. The neuropathology of OCD involves dysregulation of cortical‐striatal circuits. Similar to OCD patients, SAPAP3 knockout mice 3 (SAPAP3^?/?) exhibit compulsive behavior (grooming), anxiety and dysregulated cortical‐striatal function. However, it is unknown whether SAPAP3^?/? display cognitive deficits and how these different behavioral traits relate to one another. SAPAP3^?/? and wild‐type (WT) littermates were trained in a Pavlovian conditioning task pairing visual cues with the delivery of sucrose solution. After mice learned to discriminate between a reward‐predicting conditioned stimulus (CS+) and a non‐reward stimulus (CS?), contingencies were reversed (CS+ became CS? and vice versa). Additionally, we assessed grooming, anxiety and general activity. SAPAP3^?/? acquired Pavlovian approach behavior similarly to WT, albeit less vigorously and with a different strategy. However, unlike WT, SAPAP3^?/? were unable to adapt their behavior after contingency reversal, exemplified by a lack of re‐establishing CS+ approach behavior (sign tracking). Surprisingly, such behavioral inflexibility, decreased vigor, compulsive grooming and anxiety were unrelated. This study shows that SAPAP3^?/? are capable of Pavlovian learning, but lack flexibility to adapt associated conditioned approach behavior. Thus, SAPAP3^?/? not only display compulsive‐like behavior and anxiety, but also cognitive deficits, confirming and extending the validity of SAPAP3^?/? as a suitable model for the study of OCD. The observation that compulsive‐like behavior, anxiety and behavioral inflexibility were unrelated suggests a non‐causal relationship between these traits and may be of clinical relevance for the treatment of OCD.     

Effects of brain‐derived neurotrophic factor on dopaminergic function and motor behavior during aging

Brain‐derived neurotrophic factor (BDNF) is critical in synaptic plasticity and in the survival and function of midbrain dopamine neurons. In this study, we assessed the effects of a partial genetic deletion of BDNF on motor function and dopamine (DA) neurotransmitter measures by comparing Bdnf^+/? with wildtype mice (WT) at different ages. Bdnf^+/? and WT mice had similar body weights until 12 months of age; however, at 21 months, Bdnf^+/? mice were significantly heavier than WT mice. Horizontal and vertical motor activity was reduced for Bdnf^+/? compared to WT mice, but was not influenced by age. Performance on an accelerating rotarod declined with age for both genotypes and was exacerbated for Bdnf^+/? mice. Body weight did not correlate with any of the three behavioral measures studied. Dopamine neurotransmitter markers indicated no genotypic difference in striatal tyrosine hydroxylase, DA transporter (DAT) or vesicular monoamine transporter 2 (VMAT2) immunoreactivity at any age. However, DA transport via DAT (starting at 12 months) and VMAT2 (starting at 3 months) as well as KCl‐stimulated DA release were reduced in Bdnf^+/? mice and declined with age suggesting an increasingly important role for BDNF in the release and uptake of DA with the aging process. These findings suggest that a BDNF expression deficit becomes more critical to dopaminergic dynamics and related behavioral activities with increasing age.     

Both spontaneous Ins2+/− and streptozotocin‐induced type I diabetes cause bone loss in young mice

The adolescent skeleton undergoes accelerated growth determining overall bone density, length, and quality. Diseases such as type 1 diabetes (T1D), most often diagnosed in adolescents, can alter bone processes and promote bone loss. Studies examining type 1 diabetic (T1D) bone pathologies typically utilize adult mice and rely on pharmacologic models such as streptozotocin (STZ)‐induced diabetic rodents. To test the effect of T1D on adolescent bone growth/density we used a novel juvenile genetic model (Ins2^+/? mice) that spontaneously develop T1D at approximately 5 weeks of age and compared our findings with STZ‐induced T1D mice. Compared to controls, both Ins2^+/? and STZ‐induced T1D mice displayed blood glucose levels greater than 300 mg/dl and reduced body, fat and muscle mass as well as femur trabecular bone density. STZ mice exhibited greater bone loss compared to Ins2^+/? mice despite having lower blood glucose levels. Cortical bone was affected in STZ but not Ins2^+/? mice. Osteocalcin serum protein and bone RNA levels decreased in both models. Consistent with studies in adult mice, STZ adolescent mice displayed increased marrow adiposity, however this was not observed in the Ins2^+/? mice. Reduced femur length, decreased growth plate thickness and decreased collagen II expression in both model simplies impaired cartilage formation. In summary, both pharmacologic and spontaneous adolescent T1D mice demonstrated a bone synthesis and growth defect. STZ appears to cause a more severe phenotype. Thus, the Ins2^+/? mouse could serve as a useful model to study adolescent T1D bone loss with fewer complications. J. Cell. Physiol. 228: 689–695, 2013. © 2012 Wiley Periodicals, Inc.     

Antidepressant‐like effects and basal immobility depend on age and serotonin transporter genotype

Monoamine uptake inhibitors are common treatments for depression; however, the therapeutic efficacy of these drugs varies widely. Two factors that are commonly linked to clinical outcome are age and serotonin transporter (SERT) genotype. Mouse models provide powerful tools to study consequences of age and genotype on antidepressant‐like efficacy; however, to date, systematic studies of this nature are lacking. Here, we used the tail suspension test (TST), a preclinical assay for antidepressant efficacy, to gain insight into age and SERT genotype dependency of immobility time in the TST under control conditions (saline injection) and in response to the tricyclic antidepressant, desipramine (DMI). Immobility after saline injection in juvenile, adolescent, adult, mature adult and middle‐aged mice (postnatal days 21, 28, 90, 210 and 300, respectively) significantly increased with age; however, the rate of increase was slower for SERT null (?/?) mice than for wild‐type (+/+) or heterozygote (+/?) mice. Desipramine reduced immobility across ages and SERT genotypes. Middle‐aged, but not adult, SERT^?/? mice were significantly more sensitive to DMI than age‐matched SERT^+/+ or SERT^+/? mice. Desipramine was less potent in middle‐aged SERT^+/+ and SERT^+/? mice than in adult SERT^+/+ or SERT^+/? mice. Regardless of age, DMI's maximal effects were greater in SERT^?/? mice than in SERT^+/+ or SERT^+/? mice. These results show that immobility time in the TST varies as a function of age and SERT genotype, underscoring the utility of the TST as a potential model to examine age‐ and SERT genotype‐dependent influences on antidepressant response.