Yusuke Onaka

Lipopolysaccharide affects exploratory behaviors toward novel objects by impairing cognition and/or motivation in mice: Possible role of activation of the central amygdala.

Lipopolysaccharide (LPS) produces a series of systemic and psychiatric changes called sickness behavior. In the present study, we characterized the LPS-induced decrease in novel object exploratory behaviors in BALB/c mice. As already reported, LPS (0.3-5 μg/mouse) induced dose- and time-dependent decreases in locomotor activity, food intake, social interaction, and exploration for novel objects, and an increase in immobility in the forced-swim test. Although the decrease in locomotor activity was ameliorated by 10h postinjection, novel object exploratory behaviors remained decreased at 24h and were observed even with the lowest dose of LPS. In an object exploration test, LPS shortened object exploration time but did not affect moving time or the frequency of object exploration. Although pre-exposure to the same object markedly decreased the duration of exploration and LPS did not change this reduction, LPS significantly impaired the exploration of a novel object that replaced the familiar one. LPS did not affect anxiety-like behaviors in open-field and elevated plus-maze tests. An LPS-induced increase in the number of c-Fos-immunoreactive cells was observed in several brain regions within 6h of LPS administration, but the number of cells quickly returned to control levels, except in the central amygdala where the increase continued for 24h. These results suggest that LPS most prominently affects object exploratory behaviors by impairing cognition and/or motivation including continuous attention and curiosity toward objects, and that this may be associated with activation of brain nuclei such as the central amygdala.

Improvement by methylphenidate and atomoxetine of social interaction deficits and recognition memory impairment in a mouse model of valproic acid-induced autism

Rodents exposed prenatally to valproic acid (VPA) show autism‐related behavioral abnormalities. We recently found that prenatal VPA exposure causes a reduction of dopaminergic activity in the prefrontal cortex of male, but not female, mice. This suggests that reduced prefrontal dopaminergic activity is associated with behavioral abnormalities in VPA‐treated mice. In the present study, we examined whether the attention deficit/hyperactivity disorder drugs methylphenidate and atomoxetine (which increase dopamine release in the prefrontal cortex, but not striatum, in mice) could alleviate the behavioral abnormalities and changes in dendritic spine morphology induced by prenatal VPA exposure. We found that methylphenidate and atomoxetine increased prefrontal dopamine and noradrenaline release in VPA‐treated mice. Acute treatment with methylphenidate or atomoxetine did not alleviate the social interaction deficits or recognition memory impairment in VPA‐treated mice, while chronic treatment for 2 weeks did. Methylphenidate or atomoxetine for 2 weeks also improved the prenatal VPA‐induced decrease in dendritic spine density in the prefrontal cortex. The effects of these drugs on behaviors and dendritic spine morphology were antagonized by concomitant treatment with the dopamine‐D1 receptor antagonist SCH39166 or the dopamine‐D2 receptor antagonist raclopride, but not by the α2‐adrenoceptor antagonist idazoxan. These findings suggest that chronic treatment with methylphenidate or atomoxetine improves abnormal behaviors and diminishes the reduction in spine density in VPA‐treated mice via a prefrontal dopaminergic system‐dependent mechanism. Autism Res 2016, 9: 926–939.

The Female Encounter Test: A Novel Method for Evaluating Reward-Seeking Behavior or Motivation in Mice

Background: Reduced motivation is an important marker of psychiatric disorders, including depression. We describe the female encounter test, a novel method of evaluating reward-seeking behavior in mice. Methods: The test apparatus consists of three open chambers, formed with partitions that allow the animal to move freely from one chamber to another. A test male mouse is habituated in the apparatus, and subsequently a female and male mouse are introduced into a wire-mesh box in the left and right chamber, respectively. The time the test male mouse spends in the female or male area is measured for 10min. Results: All six strains of mice tested showed a significant preference for female encounters. The preference was observed in 7–30-week-old mice. The preference was blocked by castration of the resident male test mouse, and was not affected by the phase of the menstrual cycle of the female intruder. The preference was impaired in mouse models of depression, including social isolation-reared, corticosterone-treated, and lipopolysaccharide-treated mice. The impairment was alleviated by fluvoxamine in isolation-reared and lipopolysaccharide-treated mice, and it was improved by the metabotropic glutamate 2/3 receptor antagonist LY341495 in corticosterone-treated mice. Encounter with a female, but not male, mouse increased c-Fos expression in the nucleus accumbens shell of test male mice. Furthermore, both the preference and encounter-induced increases in c-Fos expression were blocked by dopamine D1 and D2 receptor antagonists. Conclusions: These findings indicate that motivation in adult male mice can be easily evaluated by quantitating female encounters.

Behavioral characterization of mice overexpressing human dysbindin-1

BackgroundThe dysbindin-1 gene (DTNBP1: dystrobrevin binding protein 1) is a promising schizophrenia susceptibility gene, known to localize almost exclusively to neurons in the brain, and participates in the regulation of neurotransmitter release, membrane-surface receptor expression, and synaptic plasticity. Sandy mice, with spontaneous Dtnbp1 deletion, display behavioral abnormalities relevant to symptoms of schizophrenia. However, it remains unknown if dysbindin-1 gain-of-function is beneficial or detrimental.ResultsTo answer this question and gain further insight into the pathophysiology and therapeutic potential of dysbindin-1, we developed transgenic mice expressing human DTNBP1 (Dys1A-Tg) and analyzed their behavioral phenotypes. Dys1A-Tg mice were born viable in the expected Mendelian ratios, apparently normal and fertile. Primary screening of behavior and function showed a marginal change in limb grasping in Dys1A-Tg mice. In addition, Dys1A-Tg mice exhibited increased hyperlocomotion after methamphetamine injection. Transcriptomic analysis identified several up- and down-regulated genes, including the immediate-early genes Arc and Egr2, in the prefrontal cortex of Dys1A-Tg mice.ConclusionsThe present findings in Dys1A-Tg mice support the role of dysbindin-1 in psychiatric disorders. The fact that either overexpression (Dys1A-Tg) or underexpression (Sandy) of dysbindin-1 leads to behavioral alterations in mice highlights the functional importance of dysbindin-1 in vivo.

CRTH2, a prostaglandin D2 receptor, mediates depression-related behavior in mice.

Depression is a complex neuropsychiatric disorder with an unclear molecular etiology. Inflammatory cytokines and molecular intermediates (including prostaglandins) are suggested to be involved in depression; however, the roles of prostaglandins and their respective receptors are largely unknown in depression. Using genetic and pharmacological approaches, we show here that chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2), a second receptor for prostaglandin D2 (PGD2), mediates depression-related behavior in mice. CRTH2-deficient (CRTH2(-/-)) mice showed antidepressant-like activity in a chronic corticosterone treatment-induced depression. Consistent with this observation, the pharmacological inhibition of CRTH2 via the clinically available drug ramatroban also rescued abnormal social interaction and depression-related behavior in well-established models, including chronic corticosterone-, lipopolysaccharide-, and tumor-induced pathologically relevant depression models. Importantly, chronic stress via corticosterone treatment increased mRNA levels in PGD2-producing enzymes, such as cyclooxygenase-2 and lipocalin-type PGD2 synthase, in the brain. Furthermore, the activity of the hippocampal noradrenergic system but not the dopaminergic or serotonergic systems was increased in CRTH2(-/-) mice. Together with the observation that untreated CRTH2(-/-) mice showed antidepressant-like activity in the forced swim test, these results provide evidence that central CRTH2-mediated signaling is critically involved in depression-related behavior.

Central CRTH2, a Second Prostaglandin D2 Receptor, Mediates Emotional Impairment in the Lipopolysaccharide and Tumor-Induced Sickness Behavior Model

Chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2) is a second prostaglandin D2 receptor involved in mediating the allergic response; however, its central function is not yet known. Here, we demonstrate that central CRTH2 mediates emotional impairment. Lipopolysaccharide (LPS)-induced decreases in social interaction and novel exploratory behavior were observed in wild-type (CRTH2+/+) mice but not CRTH2-deficient (CRTH2−/−) mice, but both genotypes showed hypolocomotion and anorexia following LPS injection. Tumor (colon 26) inoculation, a more pathologically relevant model, induced decreases in social interaction and novel exploratory behavior in CRTH2+/+, but not CRTH2−/− mice. In addition, the CRTH2 antagonists including clinically available ramatroban reversed impaired social interaction and novel exploratory behavior after either LPS or tumor inoculation in CRTH2+/+ mice. Finally, LPS-induced c-Fos expression in the hypothalamic paraventricular nucleus (PVN) and central amygdala (CeA) was selectively abolished in CRTH2−/− mice. These results show that CRTH2 participates in LPS-induced emotional changes and activation in the PVN and CeA. Our study provides the first evidence that central CRTH2 regulates specific emotional behaviors, and that CRTH2 antagonism has potential as a therapeutic target for behavioral symptoms associated with tumors and infectious diseases.

Identification of the role of bone morphogenetic protein (BMP) and transforming growth factor-β (TGF-β) signaling in the trajectory of serotonergic differentiation in a rapid assay in mouse embryonic stem cells in vitro

The mechanism by which extracellular molecules control serotonergic cell fate remains elusive. Recently, we showed that noggin, which inactivates bone morphogenetic proteins (BMPs), induces serotonergic differentiation of mouse embryonic (ES) and induced pluripotent stem cells with coordinated gene expression along the serotonergic lineage. Here, we created a rapid assay for serotonergic induction by generating knock‐in ES cells expressing a naturally secreted Gaussia luciferase driven by the enhancer of Pet‐1/Fev, a landmark of serotonergic differentiation. Using these cells, we performed candidate‐based screening and identified BMP type I receptor kinase inhibitors LDN‐193189 and DMH1 as activators of luciferase. LDN‐193189 induced ES cells to express the genes encoding Pet‐1, tryptophan hydroxylase 2, and the serotonin transporter, and increased serotonin release without altering dopamine release. In contrast, TGF‐β receptor inhibitor SB‐431542 selectively inhibited serotonergic differentiation, without changing overall neuronal differentiation. LDN‐193189 inhibited expression of the BMP signaling target gene Id, and induced the TGF‐β target gene Lefty, whereas the opposite effect was observed with SB‐431542. This study thus provides a new tool to investigate serotonergic differentiation and suggests that inhibition of BMP type I receptors and concomitant activation of TGF‐β receptor signaling are implicated in serotonergic differentiation.

Anxiolytic-like effects of restraint during the dark cycle in adolescent mice

Stress during developmental stage may cause psychological morbidities, and then the studies on stress are important in adolescent rodents. Restraint is used as a common stressor in rodents and the effects of restraint during the light cycle have been studied, but those of restraint during the dark cycle have not. The present study examined the effects of restraint during the light and dark cycles on anxiety behaviors in adolescent mice. Restraint for 3h during either the light or dark cycle impaired memory function in the fear conditioning test, but did not affect locomotor activity. In the elevated plus-maze test, restraint during the dark cycle reduced anxiety-like behaviors in mice. Repeated exposure to a 3-h period dark cycle restraint for 2 weeks had a similar anxiolytic-like effect. In contrast, restraint for 3h during the light cycle produced anxiety behavior in adolescent, but not adult, mice. The light cycle stress increased plasma corticosterone levels, and elevated c-Fos expression in the prefrontal cortex, paraventricular hypothalamic nucleus, basolateral amygdala and dentate gyrus, and enhanced serotonin turnover in the hippocampus and striatum, while the dark cycle stress did not. There was no difference in the stress-mediated reduction in pentobarbital-induced sleeping time between dark and light cycle restraint. These findings suggest that the anxiolytic effect of dark cycle restraint is mediated by corticosterone, serotonin or γ-aminobutyric acid-independent mechanisms, although the anxiogenic effect of light cycle restraint is associated with changes in plasma corticosterone levels and serotonin turnover in specific brain regions.

Deletion of JMJD2B in neurons leads to defective spine maturation, hyperactive behavior and memory deficits in mouse

JMJD2B is a histone demethylase enzyme that regulates gene expression through demethylation of H3K9me3. Although mutations of JMJD2B have been suggested to be responsible for neurodevelopmental disorders, the function of JMJD2B in the central nervous system (CNS) remains to be elucidated. Here we show that JMJD2B has a critical role in the development of the CNS. We observed JMJD2B expression, which was especially strong in the hippocampus, throughout the CNS from embryonic periods through adulthood. We generated neuron-specific JMJD2B-deficient mice using the cre-loxP system. We found an increase in total spine number, but a decrease in mature spines, in the CA1 region of the hippocampus. JMJD2B-deficient mice exhibited hyperactive behavior, sustained hyperactivity in a novel environment, deficits in working memory and spontaneous epileptic-like seizures. Together these observations indicate that JMJD2B mutant mice display symptoms reminiscent of neurodevelopmental disorders. Our findings provide evidence for the involvement of histone demethylation in the formation of functional neural networks during development.

Atomoxetine reverses locomotor hyperactivity, impaired novel object recognition, and prepulse inhibition impairment in mice lacking pituitary adenylate cyclase-activating polypeptide

Attention-deficit/hyperactivity disorder (ADHD) is a complex neurobehavioral disorder that is characterized by attention difficulties, impulsivity, and hyperactivity. A non-stimulant drug, atomoxetine (ATX), which is a selective noradrenaline reuptake inhibitor, is widely used for ADHD because it exhibits fewer adverse effects compared to conventional psychostimulants. However, little is known about the therapeutic mechanisms of ATX. ATX treatment significantly alleviated hyperactivity of pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient (PACAP(-/-)) mice with C57BL/6J and 129S6/SvEvTac hybrid background. ATX also improved impaired novel object recognition memory and prepulse inhibition in PACAP(-/-) mice with CD1 background. The ATX-induced increases in extracellular noradrenaline and dopamine levels were significantly higher in the prefrontal cortex of PACAP(-/-) mice compared to wild-type mice with C57BL/6J and 129S6/SvEvTac hybrid background. These results suggest that ATX treatment-induced increases in central monoamine metabolism may be involved in the rescue of ADHD-related abnormalities in PACAP(-/-) mice. Our current study suggests that PACAP(-/-) mice are an ideal rodent model with predictive validity for the study of ADHD etiology and drug development. Additionally, the potential effects of differences in genetic background of PACAP(-/-) mice on behaviors are discussed.