Chronic social defeat stress induces the down-regulation of the Nedd4L-GLT-1 ubiquitination pathway in the prefrontal cortex of mice.

Stressful life events contribute to the onset of major depressive disorder (MDD). We recently demonstrated abnormalities in ubiquitination in the pathophysiology of MDD. However, the underlying molecular mechanisms remain unclear. We investigated the involvement of the ubiquitination system-mediated glutamatergic dysfunction in social impairment induced by chronic social defeat stress (CSDS). Adult C57BL/6J mice were exposed to aggressor ICR male mice for 10 consecutive days. Social impairment was induced by CSDS in the social interaction test 1 days after the last stress exposure. In terms of brain microdialysis, CSDS reduced depolarization-evoked glutamate release in the prefrontal cortex (PFC), which was reversed by a glutamate transporter 1 (GLT-1) inhibitor. Interestingly, the expression of ubiquitinated, but not total GLT-1, was decreased in the PFC of mice exposed to CSDS. The expression of neural precursor cells expressing developmentally downregulated gene 4-like (Nedd4L: E3 ligase for GLT-1), and ubiquitin-conjugating enzyme E2D2 (Ube2d2: E2 ubiquitin-conjugating enzyme for Nedd4L) was also reduced in CSDS mice. Furthermore, the downregulation of the Nedd4L-GLT-1 ubiquitination pathway decreased SIT ratio, but up-regulation increased it even in non-CSDS mice. Taken together, the decrease in GLT-1 ubiquitination may reduce the release of extracellular glutamate induced by high-potassium stimulation, which may lead to social impairment, while we could not find differences in GLT-1 ubiquitination between susceptible and resistant CSDS mice. In conclusion, GLT-1 ubiquitination could play a crucial role in the pathophysiology of MDD and is an attractive target for the development of novel antidepressants.

Rho kinase inhibitors ameliorate cognitive impairment in a male mouse model of methamphetamine-induced schizophrenia.

Schizophrenia (SCZ) is a severe psychiatric disorder characterized by positive symptoms, negative symptoms, and cognitive deficits. Current antipsychotic treatment in SCZ improves positive symptoms but has major side effects and little impact on negative symptoms and cognitive impairment. The pathoetiology of SCZ remains unclear, but is known to involve small GTPase signaling. Rho kinase, an effector of small GTPase Rho, is highly expressed in the brain and plays a major role in neurite elongation and neuronal architecture. This study used a touchscreen-based visual discrimination (VD) task to investigate the effects of Rho kinase inhibitors on cognitive impairment in a methamphetamine (METH)-treated male mouse model of SCZ. Systemic injection of the Rho kinase inhibitor fasudil dose-dependently ameliorated METH-induced VD impairment. Fasudil also significantly suppressed the increase in the number of c-Fos-positive cells in the infralimbic medial prefrontal cortex (infralimbic mPFC) and dorsomedial striatum (DMS) following METH treatment. Bilateral microinjections of Y-27632, another Rho kinase inhibitor, into the infralimbic mPFC or DMS significantly ameliorated METH-induced VD impairment. Two proteins downstream of Rho kinase, myosin phosphatase-targeting subunit 1 (MYPT1; Thr696) and myosin light chain kinase 2 (MLC2; Thr18/Ser19), exhibited increased phosphorylation in the infralimbic mPFC and DMS, respectively, after METH treatment, and fasudil inhibited these increases. Oral administration of haloperidol and fasudil ameliorated METH-induced VD impairment, while clozapine had little effect. Oral administration of haloperidol and clozapine suppressed METH-induced hyperactivity, but fasudil had no effect. These results suggest that METH activates Rho kinase in the infralimbic mPFC and DMS, which leads to cognitive impairment in male mice. Rho kinase inhibitors ameliorate METH-induced cognitive impairment, perhaps via the cortico-striatal circuit.

Shati/Nat8l deficiency disrupts adult neurogenesis and causes attentional impairment through dopaminergic neuronal dysfunction in the dentate gyrus.

Successful completion of daily activities relies on the ability to select the relevant features of the environment for memory and recall. Disruption to these processes can lead to various disorders, such as attention-deficit hyperactivity disorder (ADHD). Dopamine is a neurotransmitter implicated in the regulation of several processes, including attention. In addition to the higher-order brain function, dopamine is implicated in the regulation of adult neurogenesis. Previously, we generated mice lacking Shati, an N-acetyltransferase-8-like protein on a C57BL/6J genetic background (Shati/Nat8l-/- ). These mice showed a series of changes in the dopamine system and ADHD-like behavioral phenotypes. Therefore, we hypothesized that deficiency of Shati/Nat8l would affect neurogenesis and attentional behavior in mice. We found aberrant morphology of neurons and impaired neurogenesis in the dentate gyrus of Shati/Nat8l-/- mice. Additionally, research has suggested that impaired neurogenesis might be because of the reduction of dopamine in the hippocampus. Galantamine (GAL) attenuated the attentional impairment observed in the object-based attention test via increasing the dopamine release in the hippocampus of Shati/Nat8l-/- mice. The α7 nicotinic acetylcholine receptor antagonist, methyllycaconitine, and dopamine D1 receptor antagonist, SCH23390, blocked the ameliorating effect of GAL on attentional impairment in Shati/Nat8l-/- mice. These results suggest that the ameliorating effect of GAL on Shati/Nat8l-/- attentional impairment is associated with activation of D1 receptors following increased dopamine release in the hippocampus via α7 nicotinic acetylcholine receptor. In summary, Shati/Nat8l is important in both morphogenesis and neurogenesis in the dentate gyrus and attention, possible via modulation of dopaminergic transmission. Cover Image for this issue: https://doi.org/10.1111/jnc.15061.

Prefrontal cortex miR-874-3p prevents lipopolysaccharide-induced depression-like behavior through inhibition of indoleamine 2,3-dioxygenase 1 expression in mice.

Indoleamine 2,3-dioxygenase 1 (IDO1) is the first rate-limiting enzyme that metabolizes tryptophan to the kynurenine pathway. Its activity is highly inducible by pro-inflammatory cytokines and correlates with the severity of major depressive disorder (MDD). MicroRNAs (miRNAs) are involved in gene regulation and the development of neuropsychiatric disorders including MDD. However, the role of miRNAs in targeting IDO1 in the pathophysiology of MDD is still unknown. In this study, we investigated the role of novel miRNAs in the regulation of IDO1 activity and its effect on lipopolysaccharide (LPS)-induced depression-like behavior in mice. LPS up-regulated miR-874-3p concomitantly with increase in IDO1 expression in the prefrontal cortex (PFC), increase in immobility in the forced swimming test as depression-like behavior and decrease in locomotor activity as sickness behavior without motor dysfunction. The miR-874-3p increased in both neuron and microglia after LPS. Its mimic significantly suppressed LPS-induced IDO1 expression in the PFC. Infusion of IDO1 inhibitor (1-methyl-l-tryptophan) and miR-874-3p into PFC prevented an increase in immobility in the forced swimming test, but did not decrease in locomotor activity induced by LPS. These results suggest that miR-874-3p may play an important role in preventing the LPS-induced depression-like behavior through inhibition of IDO1 expression. This may also serve as a novel potential target molecule for the treatment of MDD.

Heat-sterilized Bifidobacterium breve prevents depression-like behavior and interleukin-1ß expression in mice exposed to chronic social defeat stress.

Major depressive disorder (MDD) is a common and serious psychiatric disease that involves brain inflammation. Bifidobacterium breve is commonly used as a probiotic and was shown to improve colitis and allergic diseases by suppressing the inflammatory response. Heat-sterilized B. breve has beneficial effects on inflammation. We hypothesize, therefore, that this probiotic might reduce depression symptoms. We tested this is a mouse model of social defeat stress. C57BL/6J mice exposed to chronic social defeat stress (CSDS) for five consecutive days developed a mild depression-like behavior characterized by a social interaction impairment. CSDS also altered the gut microbiota composition, such as increased abundance of Bacilli, Bacteroidia, Mollicutes, and Verrucomicrobiae classes and decreased Erysipelotrichi class. The prophylactic effect of heat-sterilized B. breve as a functional food ingredient was evaluated on the depression-like behavior in mice. The supplementation started two weeks before and lasted two weeks after the last exposure to CSDS. Two weeks after CSDS, the mice showed deficits in social interaction and increased levels of inflammatory cytokines, including interleukin-1ß (IL-1ß) in the prefrontal cortex (PFC) and hippocampus (HIP). Heat-sterilized B. breve supplementation significantly prevented social interaction impairment, suppressed IL-1ß increase in the PFC and HIP, and modulated the alteration of the gut microbiota composition induced by CSDS. These findings suggest that heat-sterilized B. breve prevents depression-like behavior and IL-1ß expression induced by CSDS through modulation of the gut microbiota composition in mice. Therefore, heat-sterilized B. breve used as an ingredient of functional food might prevent MDD.

Indoleamine 2,3-dioxygenase 2 deficiency associates with autism-like behavior via dopaminergic neuronal dysfunction.

Indoleamine 2,3-dioxygenase 2 (IDO2) is an enzyme of the tryptophan-kynurenine pathway that is constitutively expressed in the brain. To provide insight into the physiological role of IDO2 in the brain, behavioral and neurochemical analyses in IDO2 knockout (KO) mice were performed. IDO2 KO mice showed stereotyped behavior, restricted interest and social deficits, traits that are associated with behavioral endophenotypes of autism spectrum disorder (ASD). IDO2 was colocalized immunohistochemically with tyrosine-hydroxylase-positive cells in dopaminergic neurons. In the striatum and amygdala of IDO2 KO mice, decreased dopamine turnover was associated with increased α-synuclein level. Correspondingly, levels of downstream dopamine D1 receptor signaling molecules such as brain-derived neurotrophic factor and c-Fos positive proteins were decreased. Furthermore, decreased abundance of ramified-type microglia resulted in increased dendritic spine density in the striatum of IDO2 KO mice. Both chemogenetic activation of dopaminergic neurons and treatment with methylphenidate, a dopamine reuptake inhibitor, ameliorated the ASD-like behavior of IDO2 KO mice. Sequencing analysis of exon regions in IDO2 from 309 ASD samples identified a rare canonical splice site variant in one ASD case. These results suggest that the IDO2 gene is, at least in part, a factor closely related to the development of psychiatric disorders.

Ido2 Deficiency Exacerbates Motor Impairment and Reduces Aryl Hydrocarbon Receptor Activity through Decreased Kynurenine in a Chronic Demyelinating Mouse Model.

Demyelinating diseases including multiple sclerosis (MS) are chronic inflammatory diseases of the central nervous system. Indoleamine 2,3-dioxygenase 2 (Ido2) is a recently identified as catalytic enzyme involved in the rate-limiting step of the tryptophan-kynurenine pathway that influences susceptibility to inflammatory diseases. However, the pathological role of Ido2 in demyelination remains unclear. In this study, we investigated whether Ido2 deficiency influences the pathogenesis of proteolipid protein transgenic (Plp tg) mice, an animal model of chronic demyelination. Ido2 deficiency exacerbates impairments of motor function in the locomotor activity test, wire hanging test, and rotarod test. Ido2 deficiency caused severe demyelination associated with CD68-positive microglial activation in Plp tg mice. In the cerebellum of Plp tg mice, Ido2 deficiency significantly increased the expression of Tnfα. Ido2 deficiency reduced tryptophan metabolite kynurenine (KYN) levels and subsequent aryl hydrocarbon receptor (AhR) activity, which play an important role in anti-inflammatory response. These results suggest that Ido2 has an important role in preventing demyelination through AhR. Taken together, Ido2 could be a potential therapeutic target for demyelinating diseases.

High salt induces cognitive impairment via the interaction of the angiotensin II-AT1 and prostaglandin E2-EP1 systems.

BACKGROUND AND PURPOSE: High salt (HS) intake has been associated with hypertension and cognitive impairment. It is well known that the angiotensin II (Ang II)-AT1 receptor and prostaglandin E2 (PGE2)-EP1 receptor systems are involved in hypertension and neurotoxicity. However, the involvement of these systems in HS-mediated hypertension and emotional and cognitive impairments remains unclear. EXPERIMENTAL APPROACH: Mice were loaded with HS solution (2% NaCl drinking water) for 12 weeks, and blood pressure was monitored. Subsequently, effects of HS intake on emotional and cognitive function and tau phosphorylation in the prefrontal cortex (PFC) and hippocampus (HIP) were investigated. The involvement of Ang II-AT1 and PGE2-EP1 systems in HS-induced hypertension and neuronal and behavioural impairments was examined by treatment with losartan, an AT1 receptor blocker (ARB), or EP1 gene knockout. KEY RESULTS: We demonstrate that hypertension and impaired social behaviour and object recognition memory following HS intake may be associated with tau hyperphosphorylation, decreased phosphorylation of Ca2+ /calmodulin-dependent protein kinase II (CaMKII), and postsynaptic density protein 95 (PSD95) expression in the PFC and HIP of mice. These changes were blocked by pharmacological treatment with losartan or EP1 receptor gene knockout. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that the interaction of Ang II-AT1 receptor and PGE2-EP1 receptor systems could be novel therapeutic targets for hypertension-induced cognitive impairment.

Mice with exonic RELN deletion identified from a patient with schizophrenia have impaired visual discrimination learning and reversal learning in touchscreen operant tasks.

The Reelin gene (RELN) encodes a large extracellular protein, which has multiple roles in brain development and adult brain function. It activates a series of neuronal signal transduction pathways in the adult brain that function in synaptic plasticity, dendritic morphology, and cognitive function. To further investigate the roles of Reln in brain function, we generated a mouse line using the C57BL/6 J strain with the specific Reln deletion identified from a Japanese patient with schizophrenia (Reln-del mice). These mice exhibited abnormal sociality, but the pathophysiological significance of the Reln deletion for higher brain functions, such as learning and behavioral flexibility remains unclear. In this study, cognitive function in Reln-del mice was assessed using touchscreen-based visual discrimination (VD) and reversal learning (RL) tasks. Reln-del mice showed normal learning in the simple VD task, but the learning was delayed in the complex VD task as compared to their wild-type (WT) littermates. In the RL task, sessions were divided into early perseverative phase (sessions with <50% correct) and later learning phase (sessions with ≥50% correct). Reln-del mice showed normal perseveration but impaired relearning ability in both simple RL and complex RL task as compared to WT mice. These results suggest that Reln-del mice have impaired learning ability, but the behavioral flexibility is unaffected. Overall, the observed behavioral abnormalities in Reln-del mice suggest that this mouse model is a useful preclinical tool for investigating the neurobiological mechanism underlying cognitive impairments in schizophrenia and a therapeutic strategy.

Pharmacological blockade of dopamine D1- or D2-receptor in the prefrontal cortex induces attentional impairment in the object-based attention test through different neuronal circuits in mice.

Dopamine is a key neurotransmitter that regulates attention through dopamine D1 and D2-receptors in the prefrontal cortex (PFC). We previously developed an object-based attention test (OBAT) to evaluate attention in mice. Disruption of the dopaminergic neuronal system in the PFC induced attentional impairment in the OBAT. However, previous studies have not systematically examined which specific brain regions are associated with the blockade of PFC dopamine D1 and D2-receptors in the OBAT. In this study, we investigated the association of dopamine D1 and D2-receptors in the PFC with attention and neuronal activity in diverse brain regions. We found that both dopamine D1 and D2-receptor antagonists induced attentional impairment in the OBAT by bilateral microinjection into the PFC of mice, suggesting that both dopamine D1 and D2-receptors were associated with attention in the OBAT. Our analysis of the neuronal activity as indicated by c-Fos expression in 11 different brain regions showed that based on the antagonist types, there was selective activation of several brain regions. Overall, this study suggests that both dopamine D1 and D2-receptors play a role in attention through different neuronal circuits in the PFC of mice.

Mice carrying a schizophrenia-associated mutation of the Arhgap10 gene are vulnerable to the effects of methamphetamine treatment on cognitive function: association with morphological abnormalities in striatal neurons.

We recently found a significant association between exonic copy-number variations in the Rho GTPase activating protein 10 (Arhgap10) gene and schizophrenia in Japanese patients. Special attention was paid to one patient carrying a missense variant (p.S490P) in exon 17, which overlapped with an exonic deletion in the other allele. Accordingly, we generated a mouse model (Arhgap10 S490P/NHEJ mice) carrying a missense variant and a coexisting frameshift mutation. We examined the spatiotemporal expression of Arhgap10 mRNA in the brain and found the highest expression levels in the cerebellum, striatum, and nucleus accumbens (NAc), followed by the frontal cortex in adolescent mice. The expression levels of phosphorylated myosin phosphatase-targeting subunit 1 and phosphorylated p21-activated kinases in the striatum and NAc were significantly increased in Arhgap10 S490P/NHEJ mice compared with wild-type littermates. Arhgap10 S490P/NHEJ mice exhibited a significant increase in neuronal complexity and spine density in the striatum and NAc. There was no difference in touchscreen-based visual discrimination learning between Arhgap10 S490P/NHEJ and wild-type mice, but a significant impairment of visual discrimination was evident in Arhgap10 S490P/NHEJ mice but not wild-type mice when they were treated with methamphetamine. The number of c-Fos-positive cells was significantly increased after methamphetamine treatment in the dorsomedial striatum and NAc core of Arhgap10 S490P/NHEJ mice. Taken together, these results suggested that schizophrenia-associated Arhgap10 gene mutations result in morphological abnormality of neurons in the striatum and NAc, which may be associated with vulnerability of cognition to methamphetamine treatment.

Overexpression of astroglial major histocompatibility complex class I in the medial prefrontal cortex impairs visual discrimination learning in mice.

BACKGROUND: Immune molecules, such as cytokines, complement, and major histocompatibility complex (MHC) proteins, in the central nervous system are often associated with neuropsychiatric disorders. Neuronal MHC class I (MHCI), such as H-2D, regulate neurite outgrowth, the establishment and function of cortical connections, and activity-dependent refinement in mice. We previously established mice expressing MHCI specifically in astrocytes of the media prefrontal cortex (mPFC) using the adeno-associated virus (AAV) vector under the control of the GfaABC1D promoter. Mice expressing the soluble form of H-2D (sH-2D) in the mPFC (sH-2D-expressing mice) showed abnormal behaviors, including social interaction deficits and cognitive dysfunctions. However, the pathophysiological significance of astroglial MHCI on higher brain functions, such as learning, memory, and behavioral flexibility, remains unclear. Therefore, cognitive function in mice expressing sH-2D in astrocytes of the mPFC was tested using the visual discrimination (VD) task. METHODS: sH-2D-expressing mice were subjected to the VD and reversal learning tasks, and morphological analysis. RESULTS: In the pretraining, sH-2D-expressing mice required significantly more trials to reach the learning criterion than control mice. The total number of sessions, trials, normal trials, and correction trials to reach the VD criterion were also significantly higher in sH-2D-expressing mice than in control mice. A morphological study showed that dendritic complexity and spine density were significantly reduced in the dorsal striatum of sH-2D-expressing mice. CONCLUSION: Collectively, the present results suggest that the overexpression of astroglial MHCI in the mPFC results in impaired VD learning, which may be accompanied by decreased dendritic complexity in the dorsal striatum and mPFC.

Prefrontal cortex, dorsomedial striatum, and dentate gyrus are necessary in the object-based attention test in mice.

Disturbances of attention are a common behavioral feature associated with neuropsychiatric disorders with largely unknown underlying causes. We previously developed an object-based attention test (OBAT) as a simple and practical method for evaluating attention in mice. Since its establishment, the test has become a popular method for assessing attention and related underlying mechanisms in various mouse models. However, the underlying neuronal network involved in this test has yet to be studied. The purpose of this study was to identify the principal brain regions activated in the OBAT. Accordingly, C57BL/6J mice were subjected to the OBAT and thereafter prepared for immunohistochemical quantification of c-Fos, an immediate early gene that is frequently used as a marker of neuronal activity, in 13 different brain regions. The number of c-Fos-positive cells was significantly higher in the prefrontal cortex (PFC), dorsomedial striatum (DMS), and dentate gyrus (DG) in the test group as compared to the control group. The neuronal activation of these brain regions during the OBAT indicates that these brain regions are necessary for the regulation of attention in this test. This was supported by excitotoxic lesioning of these brain regions, leading to impaired attention without causing locomotor dysfunction. This study is one of the first attempts to analyze the brain regions that regulate attention in the OBAT. These findings provide an initial insight into the role of these brain regions and ideas for studying the underlying neural and molecular mechanisms.

Comprehensive analysis of a novel mouse model of the 22q11.2 deletion syndrome: a model with the most common 3.0-Mb deletion at the human 22q11.2 locus.

The 22q11.2 deletion syndrome (22q11.2DS) is associated with an increased risk for psychiatric disorders. Although most of the 22q11.2DS patients have a 3.0-Mb deletion, existing mouse models only mimic a minor mutation of 22q11.2DS, a 1.5-Mb deletion. The role of the genes existing outside the 1.5-Mb deletion in psychiatric symptoms of 22q11.2DS is unclear. In this study, we generated a mouse model that reproduced the 3.0-Mb deletion of the 22q11.2DS (Del(3.0 Mb)/ +) using the CRISPR/Cas9 system. Ethological and physiological phenotypes of adult male mutants were comprehensively evaluated by visual-evoked potentials, circadian behavioral rhythm, and a series of behavioral tests, such as measurement of locomotor activity, prepulse inhibition, fear-conditioning memory, and visual discrimination learning. As a result, Del(3.0 Mb)/ + mice showed reduction of auditory prepulse inhibition and attenuated cue-dependent fear memory, which is consistent with the phenotypes of existing 22q11.2DS models. In addition, Del(3.0 Mb)/ + mice displayed an impaired early visual processing that is commonly seen in patients with schizophrenia. Meanwhile, unlike the existing models, Del(3.0 Mb)/ + mice exhibited hypoactivity over several behavioral tests, possibly reflecting the fatigability of 22q11.2DS patients. Lastly, Del(3.0 Mb)/ + mice displayed a faster adaptation to experimental jet lag as compared with wild-type mice. Our results support the validity of Del(3.0 Mb)/ + mice as a schizophrenia animal model and suggest that our mouse model is a useful resource to understand pathogenic mechanisms of schizophrenia and other psychiatric disorders associated with 22q11.2DS.

Cell type-specific activation of mitogen-activated protein kinase in D1 receptor-expressing neurons of the nucleus accumbens potentiates stimulus-reward learning in mice.

Medium spiny neurons (MSN) in the nucleus accumbens (NAc) are a fundamental component of various aspects of motivated behavior. Although mitogen-activated protein kinase (MAPK) signaling plays a crucial role in several types of learning, the cell type-specific role of MAPK pathway in stimulus-reward learning and motivation remains unclear. We herein investigated the role of MAPK in accumbal MSNs in reward-associated learning and memory. During the acquisition of Pavlovian conditioning, the number of phosphorylated MAPK1/3-positive cells was increased significantly and exclusively in the NAc core by 7-days of extensive training. MAPK signaling in the respective D1R- and D2R-MSNs was manipulated by transfecting an adeno-associated virus (AAV) plasmid into the NAc of Drd1a-Cre and Drd2-Cre transgenic mice. Potentiation of MAPK signaling shifted the learning curve of Pavlovian conditioning to the left only in Drd1a-Cre mice, whereas such manipulation in D2R-MSNs had negligible effects. In contrast, MAPK manipulation in D2R-MSNs of the NAc core significantly increased motivation for food rewards as found in Drd1a-Cre mice. These results suggest that MAPK signaling in the D1R-MSNs of NAc core plays an important role in stimulus-reward learning, while MAPK signaling in both D1R- and D2R-MSNs is involved in motivation for natural rewards.