Hycanthone Inhibits Inflammasome Activation and Neuroinflammation-Induced Depression-Like Behaviors in Mice

Despite the various medications used in clinics, the efforts to develop more effective treatments for depression continue to increase in the past decades mainly because of the treatment-resistant population, and the testing of several hypotheses- and target-based treatments. Undesirable side effects and unresponsiveness to current medications fuel the drive to solve this top global health problem. In this study, we focused on neuroinflammatory response-mediated depression which represents a cluster of depression etiology both in animal models and humans. Several meta-analyses reported that proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) were increased in major depressive disorder patients. Inflammatory mediators implicated in depression include type-I interferon and inflammasome pathways. To elucidate the molecular mechanisms of neuroinflammatory cascades underlying the pathophysiology of depression, we introduced hycanthone, an antischistosomal drug, to check whether it can counteract depressive-like behaviors in vivo and normalize the inflammation-induced changes in vitro. Lipopolysaccharide (LPS) treatment increased proinflammatory cytokine expression in the murine microglial cells as well as the stimulation of type I interferon-related pathways that are directly or indirectly regulated by Janus kinase-signal transducer and activator of transcription (JAK-STAT) activation. Hycanthone treatment attenuated those changes possibly by inhibiting the JAK-STAT pathway and inflammasome activation. Hycanthone also ameliorated depressive-like behaviors by LPS. Taken together, we suggest that the inhibitory action of hycanthone against the interferon pathway leading to attenuation of depressive-like behaviors can be a novel therapeutic mechanism for treating depression.

Autism-Like Behavioral Phenotypes in Mice Treated with Systemic N-Methyl-D-Aspartate

Autism spectrum disorder (ASD) having core characteristics of social interaction problems and repetitive behaviors and interests affects individuals at varying degrees and comorbidities, making it difficult to determine the precise etiology underlying the symptoms. Given its heterogeneity, ASD is difficult to treat and the development of therapeutics is slow due to the scarcity of animal models that are easy to produce and screen with. Based on the theory of excitation/inhibition imbalance in the brain with ASD which involves glutamatergic and/or GABAergic neurotransmission, a pharmacologic agent to modulate these receptors might be a good starting point for modeling. N-methyl-D-aspartic acid (NMDA) is an amino acid derivative acting as a specific agonist at the NMDA receptor and therefore imitates the action of the neurotransmitter glutamate on that receptor. In contrast to glutamate, NMDA selectively binds to and regulates the NMDA receptor, but not other glutamate receptors such as AMPA and kainite receptors. Given this role, we aimed to determine whether NMDA administration could result in autistic-like behavior in adolescent mice. Both male and female mice were treated with saline or NMDA (50 and 75 mg/kg) and were tested on various behavior experiments. Interestingly, acute NMDA-treated mice showed social deficits and repetitive behavior similar to ASD phenotypes. These results support the excitation/inhibition imbalance theory of ASD and that NMDA injection can be used as a pharmacologic model of ASD-like behaviors.

Behavioral Deficits in Adolescent Mice after Sub-Chronic Administration of NMDA during Early Stage of Postnatal Development

Neurodevelopmental disorders are complex conditions that pose difficulty in the modulation of proper motor, sensory and cognitive function due to dysregulated neuronal development. Previous studies have reported that an imbalance in the excitation/ inhibition (E/I) in the brain regulated by glutamatergic and/or GABAergic neurotransmission can cause neurodevelopmental and neuropsychiatric behavioral deficits such as autism spectrum disorder (ASD). NMDA acts as an agonist at the NMDA receptor and imitates the action of the glutamate on that receptor. NMDA however, unlike glutamate, only binds to and regulates the NMDA receptor subtypes and not the other glutamate receptors. This study seeks to determine whether NMDA administration in mice i.e., over-activation of the NMDA system would result in long-lasting behavioral deficits in the adolescent mice. Both gender mice were treated with NMDA or saline at early postnatal developmental period with significant synaptogenesis and synaptic maturation. On postnatal day 28, various behavioral experiments were conducted to assess and identify behavioral characteristics. NMDAtreated mice show social deficits, and repetitive behavior in both gender mice at adolescent periods. However, only the male mice but not female mice showed increased locomotor activity. This study implies that neonatal exposure to NMDA may illicit behavioral features similar to ASD. This study also confirms the validity of the E/I imbalance theory of ASD and that NMDA injection can be used as a pharmacologic model for ASD. Future studies may explore the mechanism behind the gender difference in locomotor activity as well as the human relevance and therapeutic significance of the present findings.

Late Passage Cultivation Induces Aged Astrocyte Phenotypes in Rat Primary Cultured Cells

Astrocytes play various important roles such as maintaining brain homeostasis, supporting neurons, and secreting inflammatory mediators to protect the brain cells. In aged subjects, astrocytes show diversely changed phenotypes and dysfunctions. But, the study of aged astrocytes or astrocytes from aged subjects is not yet sufficient to provide a comprehensive understanding of their important processes in the regulation of brain function. In this study, we induced an in vitro aged astrocyte model through late passage cultivation of rat primary cultured astrocytes. Astrocytes were cultured until passage 7 (P7) as late passage astrocytes and compared with passage 1 (P1) astrocytes as early passage astrocytes to confirm the differences in phenotypes and the effects of serial passage. In this study, we confirmed the morphological, molecular, and functional changes of late passage astrocytes showing aging phenotypes through SA-β-gal staining and measurement of nuclear size. We also observed a reduced expression of inflammatory mediators including IL-1β, IL-6, TNFα, iNOS, and COX2, as well as dysregulation of wound-healing, phagocytosis, and mitochondrial functions such as mitochondrial membrane potential and mitochondrial oxygen consumption rate. Cultureconditioned media obtained from P1 astrocytes promoted neurite outgrowth in immature primary cultures of rat cortices, which is significantly reduced when we treated the immature neurons with the culture media obtained from P7 astrocytes. These results suggest that late passage astrocytes show senescent astrocyte phenotypes with functional defects, which makes it a suitable model for the study of the role of astrocyte senescence on the modulation of normal and pathological brain aging.

Effects of Intraperitoneal N-methyl-D-aspartate (NMDA) Administration on Nociceptive/Repetitive Behaviors in Juvenile Mice

Dysregulation of excitatory neurotransmission has been implicated in the pathogenesis of neuropsychiatric disorders. Pharmacological inhibition of N-methyl-D-aspartate (NMDA) receptors is widely used to model neurobehavioral pathologies and underlying mechanisms. There is ample evidence that overstimulation of NMDA-dependent neurotransmission may induce neurobehavioral abnormalities, such as repetitive behaviors and hypersensitization to nociception and cognitive disruption, pharmacological modeling using NMDA has been limited due to the induction of neurotoxicity and blood brain barrier breakdown, especially in young animals. In this study, we examined the effects of intraperitoneal NMDA-administration on nociceptive and repetitive behaviors in ICR mice. Intraperitoneal injection of NMDA induced repetitive grooming and tail biting/licking behaviors in a dose- and age-dependent manner. Nociceptive and repetitive behaviors were more prominent in juvenile mice than adult mice. We did not observe extensive blood brain barrier breakdown or neuronal cell death after peritoneal injection of NMDA, indicating limited neurotoxic effects despite a significant increase in NMDA concentration in the cerebrospinal fluid. These findings suggest that the observed behavioral changes were not mediated by general NMDA toxicity. In the hot plate test, we found that the latency of paw licking and jumping decreased in the NMDA-exposed mice especially in the 75 mg/kg group, suggesting increased nociceptive sensitivity in NMDA-treated animals. Repetitive behaviors and increased pain sensitivity are often comorbid in psychiatric disorders (e.g., autism spectrum disorder). Therefore, the behavioral characteristics of intraperitoneal NMDA-administered mice described herein may be valuable for studying the mechanisms underlying relevant disorders and screening candidate therapeutic molecules.

Etoposide Induces Mitochondrial Dysfunction and Cellular Senescence in Primary Cultured Rat Astrocytes

Brain aging is an inevitable process characterized by structural and functional changes and is a major risk factor for neurodegenerative diseases. Most brain aging studies are focused on neurons and less on astrocytes which are the most abundant cells in the brain known to be in charge of various functions including the maintenance of brain physical formation, ion homeostasis, and secretion of various extracellular matrix proteins. Altered mitochondrial dynamics, defective mitophagy or mitochondrial damages are causative factors of mitochondrial dysfunction, which is linked to age-related disorders. Etoposide is an anti-cancer reagent which can induce DNA stress and cellular senescence of cancer cell lines. In this study, we investigated whether etoposide induces senescence and functional alterations in cultured rat astrocytes. Senescence-associated β-galactosidase (SA-β-gal) activity was used as a cellular senescence marker. The results indicated that etoposide-treated astrocytes showed cellular senescence phenotypes including increased SA-β-gal-positive cells number, increased nuclear size and increased senescence-associated secretory phenotypes (SASP) such as IL-6. We also observed a decreased expression of cell cycle markers, including Phospho-Histone H3/Histone H3 and CDK2, and dysregulation of cellular functions based on wound-healing, neuronal protection, and phagocytosis assays. Finally, mitochondrial dysfunction was noted through the determination of mitochondrial membrane potential using tetramethylrhodamine methyl ester (TMRM) and the measurement of mitochondrial oxygen consumption rate (OCR). These data suggest that etoposide can induce cellular senescence and mitochondrial dysfunction in astrocytes which may have implications in brain aging and neurodegenerative conditions.

Comparative Behavioral Correlation of High and Low-Performing Mice in the Forced Swim Test

Behavioral analysis in mice provided important contributions in helping understand and treat numerous neurobehavioral and neuropsychiatric disorders. The behavioral performance of animals and humans is widely different among individuals but the neurobehavioral mechanism of the innate difference is seldom investigated. Many neurologic conditions share comorbid symptoms that may have common pathophysiology and therapeutic strategy. The forced swim test (FST) has been commonly used to evaluate the “antidepressant” properties of drugs yet the individual difference analysis of this test was left scantly investigated along with the possible connection among other behavioral domains. This study conducted an FST-screening in outbred CD-1 male mice and segregated them into three groups: high performers (HP) or the active swimmers, middle performers (MP), and low performers (LP) or floaters. After which, a series of behavioral experiments were performed to measure their behavioral responses in the open field, elevated plus maze, Y maze, three-chamber social assay, novel object recognition, delay discounting task, and cliff avoidance reaction. The behavioral tests battery revealed that the three groups displayed seemingly correlated differences in locomotor activity and novel object recognition but not in other behaviors. This study suggests that the HP group in FST has higher locomotor activity and novelty-seeking tendencies compared to the other groups. These results may have important implications in creating behavior database in animal models that could be used for predicting interconnections of various behavioral domains, which eventually helps to understand the neurobiological mechanism controlling the behaviors in individual subjects.

Social Interaction Test in Home Cage as a Novel and Ethological Measure of Social Behavior in Mice

Sociability is the disposition to interact with one another. Rodents have a rich repertoire of social behaviors and demonstrate strong sociability. Various methods have been established to measure the sociability of rodents in simple and direct ways, which includes reciprocal social interaction, juvenile social play, and three-chamber social tests. There are possible confounding factors while performing some of these tasks, such as aggression, avoidance of interaction by the stimulus mouse, exposure to a new environment, and lengthy procedures. The present study devised a method to complement these shortcomings and measure sociability as a group in the home cage setting, which prevents group-housed mice from isolation or exposure to a new environment. The home cage social test can allow high-throughput screening of social behaviors in a short amount of time. We developed two types of home cage setup: a home cage social target interaction test that measures sociability by putting the wire cage in the center area of the cage and a home cage two-choice sociability and social preference test that measures both sociability or social preference by putting cage racks at opposite sides of the cage. Interestingly, our results showed that the two types of home cage setup that we used in this study can extract abnormal social behaviors in various animal models, similar to the three-chamber assay. Thus, this study establishes a new and effective method to measure sociability or social preference that could be a complementary assay to evaluate the social behavior of mice in various setup conditions.

Sex-specific Behavioral Features of Rodent Models of Autism Spectrum Disorder

Sex is an important factor in understanding the clinical presentation, management, and developmental trajectory of children with neuropsychiatric disorders. While much is known about the clinical and neurobehavioral profiles of males with neuropsychiatric disorders, surprisingly little is known about females in this respect. Animal models may provide detailed mechanistic information about sex differences in autism spectrum disorder (ASD) in terms of manifestation, disease progression, and development of therapeutic options. This review aims to widen our understanding of the role of sex in autism spectrum disorder, by summarizing and comparing behavioral characteristics of animal models. Our current understanding of how differences emerge in boys and girls with neuropsychiatric disorders is limited: Information derived from animal studies will stimulate future research on the role of biological maturation rates, sex hormones, sex-selective protective (or aggravating) factors and psychosocial factors, which are essential to devise sex precision medicine and to improve diagnostic accuracy. Moreover, there is a strong need of novel strategies to elucidate the major mechanisms leading to sex-specific autism features, as well as novel models or methods to examine these sex differences.

Sex Differences in Autism-Like Behavioral Phenotypes and Postsynaptic Receptors Expression in the Prefrontal Cortex of TERT Transgenic Mice

Autism spectrum disorder (ASD) remains unexplained and untreated despite the high attention of research in recent years. Aside from its various characteristics is the baffling male preponderance over the female population. Using a validated animal model of ASD which is the telomerase reverse transcriptase overexpressing mice (TERT-tg), we conducted ASD-related behavioral assessments and protein expression experiments to mark the difference between male and females of this animal model. After statistically analyzing the results, we found significant effects of TERT overexpression in sociability, social novelty preference, anxiety, nest building, and electroseizure threshold in the males but not their female littermates. Along these differences are the male-specific increased expressions of postsynaptic proteins which are the NMDA and AMPA receptors in the prefrontal cortex. The vGluT1 presynaptic proteins, but not GAD, were upregulated in both sexes of TERT-tg mice, although it is more significantly pronounced in the male group. Here, we confirmed that the behavioral effect of TERT overexpression in mice was male-specific, suggesting that the aberration of this gene and its downstream pathways preferentially affect the functional development of the male brain, consistent with the male preponderance in ASD.

Sex Differences in Autism-Like Behavioral Phenotypes and Postsynaptic Receptors Expression in the Prefrontal Cortex of TERT Transgenic Mice

Autism spectrum disorder (ASD) remains unexplained and untreated despite the high attention of research in recent years. Aside from its various characteristics is the baffling male preponderance over the female population. Using a validated animal model of ASD which is the telomerase reverse transcriptase overexpressing mice (TERT-tg), we conducted ASD-related behavioral assessments and protein expression experiments to mark the difference between male and females of this animal model. After statistically analyzing the results, we found significant effects of TERT overexpression in sociability, social novelty preference, anxiety, nest building, and electroseizure threshold in the males but not their female littermates. Along these differences are the male-specific increased expressions of postsynaptic proteins which are the NMDA and AMPA receptors in the prefrontal cortex. The vGluT1 presynaptic proteins, but not GAD, were upregulated in both sexes of TERT-tg mice, although it is more significantly pronounced in the male group. Here, we confirmed that the behavioral effect of TERT overexpression in mice was male-specific, suggesting that the aberration of this gene and its downstream pathways preferentially affect the functional development of the male brain, consistent with the male preponderance in ASD.

Clinical and Neurobiological Relevance of Current Animal Models of Autism Spectrum Disorders

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social and communication impairments, as well as repetitive and restrictive behaviors. The phenotypic heterogeneity of ASD has made it overwhelmingly difficult to determine the exact etiology and pathophysiology underlying the core symptoms, which are often accompanied by comorbidities such as hyperactivity, seizures, and sensorimotor abnormalities. To our benefit, the advent of animal models has allowed us to assess and test diverse risk factors of ASD, both genetic and environmental, and measure their contribution to the manifestation of autistic symptoms. At a broader scale, rodent models have helped consolidate molecular pathways and unify the neurophysiological mechanisms underlying each one of the various etiologies. This approach will potentially enable the stratification of ASD into clinical, molecular, and neurophenotypic subgroups, further proving their translational utility. It is henceforth paramount to establish a common ground of mechanistic theories from complementing results in preclinical research. In this review, we cluster the ASD animal models into lesion and genetic models and further classify them based on the corresponding environmental, epigenetic and genetic factors. Finally, we summarize the symptoms and neuropathological highlights for each model and make critical comparisons that elucidate their clinical and neurobiological relevance.