Minae Niwa

Adolescent Stress–Induced Epigenetic Control of Dopaminergic Neurons via Glucocorticoids

Defeat, Distress, and Glucocorticoids Understanding how individuals control emotions and cope with stressful events is a major clinical concern and of importance for the treatment of psychiatric illnesses (see the Perspective by McEwen). Barik et al. (p. 332) discovered that aggressive defeat stress in mice caused glucocortioid release and increased activity in the dopamine system. Deleting the glucocorticoid receptors in dopaminoceptive neurons completely prevented the social avoidance that usually follows aggressive defeat. How the combination of genetic factors and environmental stressors during adolescence determines adult behavior and how their disturbance results in neuropsychiatric disorders is poorly understood. Niwa et al. (p. 335) found that isolation stress during adolescence, which does not cause any long-lasting changes in wild-type mice, induced significant neurochemical and behavioral alterations in mutant mice expressing a dominant-negative variant of the disrupted in schizophrenia 1 gene under the control of the prion protein promoter. These deficits could be reversed by a glucocorticoid receptor antagonist. Genetically susceptible mice isolated during adolescence can subsequently present schizophrenia-like symptoms. [Also see Perspective by McEwen] Environmental stressors during childhood and adolescence influence postnatal brain maturation and human behavioral patterns in adulthood. Accordingly, excess stressors result in adult-onset neuropsychiatric disorders. We describe an underlying mechanism in which glucocorticoids link adolescent stressors to epigenetic controls in neurons. In a mouse model of this phenomenon, a mild isolation stress affects the mesocortical projection of dopaminergic neurons in which DNA hypermethylation of the tyrosine hydroxylase gene is elicited, but only when combined with a relevant genetic risk for neuropsychiatric disorders. These molecular changes are associated with several neurochemical and behavioral deficits that occur in this mouse model, all of which are blocked by a glucocorticoid receptor antagonist. The biology and phenotypes of the mouse models resemble those of psychotic depression, a common and debilitating psychiatric disease.

Combined effect of neonatal immune activation and mutant DISC1 on phenotypic changes in adulthood

Gene-environment interaction may play a role in the etiology of schizophrenia. Transgenic mice expressing dominant-negative DISC1 (DN-DISC1 mice) show some histological and behavioral endophenotypes relevant to schizophrenia. Viral infection during neurodevelopment provides a major environmental risk for schizophrenia. Neonatal injection of polyriboinosinic-polyribocytidylic acid (polyI:C), which mimics innate immune responses elicited by viral infection, leads to schizophrenia-like behavioral alteration in mice after puberty. To study how gene-environmental interaction during neurodevelopment results in phenotypic changes in adulthood, we treated DN-DISC1 mice or wild-type littermates with injection of polyI:C during the neonatal stage, according to the published method, respectively, and the behavioral and histological phenotypes were examined in adulthood. We demonstrated that neonatal polyI:C treatment in DN-DISC1 mice resulted in the deficits of short-term, object recognition, and hippocampus-dependent fear memories after puberty, although polyI:C treatment by itself had smaller influences on wild-type mice. Furthermore, polyI:C-treated DN-DISC1 mice exhibited signs of impairment of social recognition and interaction, and augmented susceptibility to MK-801-induced hyperactivity as compared with vehicle-treated wild-type mice. Of most importance, additive effects of polyI:C and DN-DISC1 were observed by a marked decrease in parvalbumin-positive interneurons in the medial prefrontal cortex. These results suggest that combined effect of neonatal polyI:C treatment and DN-DISC1 affects some behavioral and histological phenotypes in adulthood.

Reduction of methamphetamine-induced sensitization and reward in matrix metalloproteinase-2 and -9-deficient mice.

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) function to remodel the pericellular environment. Their activation and regulation are associated with synaptic physiology and pathology. Here, we investigated whether MMP‐2 and MMP‐9 are involved in the rewarding effects of and sensitization to methamphetamine (METH) in animals, in which the remodelling of neural circuits may play a crucial role. Repeated METH treatment induced behavioural sensitization, which was accompanied by an increase in MMP‐2 and MMP‐9 activity in the brain. In MMP‐2‐ and MMP‐9‐deficient mice [MMP‐2‐(–/–) and MMP‐9‐(–/–)], METH‐induced behavioural sensitization and conditioned place preference, a measure of the rewarding effect, as well as METH‐increased dopamine release in the nucleus accumbens (NAc) were attenuated compared with those in wild‐type mice. In contrast, infusion of purified human MMP‐2 into the NAc significantly potentiated the METH‐increased dopamine release. The [3H]dopamine uptake into striatal synaptosomes was reduced in wild‐type mice after repeated METH treatment, but METH‐induced changes in [3H]dopamine uptake were significantly attenuated in MMP‐2‐(–/–) and MMP‐9‐(–/–) mice. These results suggest that both MMP‐2 and MMP‐9 play a crucial role in METH‐induced behavioural sensitization and reward by regulating METH‐induced dopamine release and uptake in the NAc.

Usp46 is a quantitative trait gene regulating mouse immobile behavior in the tail suspension and forced swimming tests

The tail suspension test (TST) and forced swimming test (FST) are widely used for assessing antidepressant activity and depression-like behavior. We found that CS mice show negligible immobility in inescapable situations. Quantitative trait locus (QTL) mapping using CS and C57BL/6J mice revealed significant QTLs on chromosomes 4 (FST) and 5 (TST and FST). To identify the quantitative trait gene on chromosome 5, we narrowed the QTL interval to 0.5 Mb using several congenic and subcongenic strains. Ubiquitin-specific peptidase 46 (Usp46) with a lysine codon deletion was located in this region. This deletion affected nest building, muscimol-induced righting reflex and anti-immobility effects of imipramine. The muscimol-induced current in the hippocampal CA1 pyramidal neurons and hippocampal expression of the 67-kDa isoform of glutamic acid decarboxylase were significantly decreased in the Usp46 mutant mice compared to control mice. These phenotypes were rescued in transgenic mice with bacterial artificial chromosomes containing wild-type Usp46. Thus, Usp46 affects the immobility in the TST and FST, and it is implicated in the regulation of GABA action.

Enduring vulnerability to reinstatement of methamphetamine-seeking behavior in glial cell line-derived neurotrophic factor mutant mice

Genetic factors are considered to play an important role in drug dependence/addiction including the development of drug dependence and relapse. With the use of a model of drug self‐administration in mutant mice, several specific genes and proteins have been identified as potentially important in the development of drug dependence. In contrast, little is known about the role of specific genes in enduring vulnerability to relapse, a clinical hallmark of drug addiction. Using a mouse model of reinstatement, which models relapse of drug‐seeking behavior in addicts, we provide evidence that a partial reduction in the expression of the glial cell line‐derived neurotrophic factor (GDNF) potentiates methamphetamine (METH) self‐administration, enhances motivation to take METH, increases vulnerability to drug‐primed reinstatement, and prolongs cue‐induced reinstatement of extinguished METH‐seeking behavior. In contrast, there was no significant difference in novelty responses, METH‐stimulated hyperlocomotion and locomotor sensitization, food‐reinforced operant behavior and motivation, or reinstatement of food‐seeking behavior between GDNF heterozygous knockout mice and wild‐type littermates. These findings suggest that GDNF may be associated with enduring vulnerability to reinstatement of METH‐seeking behavior and a potential target in the development of therapies to control relapse.–Yan, Y., Yamada, K., Niwa, M., Nagai, T., Nitta, A., Nabeshima, T. Enduring vulnerability to reinstatement of methamphetamine‐seeking behavior in glial cell line‐derived neurotrophic factor mutant mice. FASEB J. 21, 1994–2004 (2007)

Vulnerability in early life to changes in the rearing environment plays a crucial role in the aetiopathology of psychiatric disorders.

Adverse events early in life, including maternal separation and social isolation, profoundly affect brain development and adult behaviour and may contribute to the occurrence of psychiatric disorders such as schizophrenia and mood disorders in genetically predisposed individuals. The molecular mechanisms underlying these environmentally induced developmental adaptations are unclear and best evaluated in animal paradigms with translational salience. In this study, we examined the effects in mice of maternal separation and/or social isolation for 6 h/d between postnatal days 15 and 21 on performance during adulthood in the open-field, social interaction, elevated plus-maze, forced swimming, Y-maze, novel object recognition, conditioned fear-learning, prepulse inhibition, and locomotor activity tests, to investigate whether this animal model could show the phenotypes for schizophrenia and mood disorders. The stress of maternal separation and isolation led to adult behavioural deficits, activation of the hypothalamic-pituitary-adrenal axis, and decreases in the levels of norepinephrine and dopamine in the frontal cortex and metabolites of dopamine and serotonin in the amygdala, showing the involvement of endocrine and neuronal risk in behavioural deficits. The results suggest that the frontal cortex and amygdala undergo structural remodelling induced by the stress of maternal separation and isolation, which alters behavioural and physiological responses in adulthood, including anxiety, memory and other cognitive processes. Further, social isolation enhanced the behavioural dysfunctions induced by maternal separation. These findings indicate that maternal separation and social isolation early in life can lead to long-lasting abnormal behaviour and pathophysiological impairments including schizophrenia and mood disorders.

A Novel Molecule “Shati” Is Involved in Methamphetamine-Induced Hyperlocomotion, Sensitization, and Conditioned Place Preference

Drug addiction places an enormous burden on society through its repercussions on crime rate and healthcare. Repeated exposure to drugs of abuse causes cellular adaptations in specific neuronal populations that ultimately can lead to a state of addiction. In the present study, we have identified a novel molecule “shati” from the nucleus accumbens (NAc) of mice treated with methamphetamine (METH) using the PCR-select complementary DNA subtraction method. Moreover, we investigated whether shati is involved in METH-induced hyperlocomotion, sensitization, and conditioned place preference (CPP). METH induced expression of shati mRNA dose dependently via dopamine (DA) receptors. We prepared antibodies against shati and, using them, found shati to be expressed in neuronal cells of the mouse brain. Treatment with the shati antisense oligonucleotide (shati-AS), which significantly inhibited the expression of shati mRNA, enhanced the acute METH response, METH-induced behavioral sensitization, and CPP. Blockage of shati mRNA by shati-AS potentiated the METH-induced increase of DA overflow in the NAc and the METH-induced decrease in synaptosomal and vesicular DA uptake in the midbrain. These results suggest that a novel molecule shati is involved in the development of METH-induced hyperlocomotion, sensitization, and CPP. The functional roles of shati in METH-regulated behavioral alternations are likely to be mediated by its inhibitory effects on the METH-induced increase of DA overflow in the NAc and the METH-induced decrease in DA uptake in the midbrain.

Role of matrix metalloproteinase and tissue inhibitor of MMP in methamphetamine-induced behavioral sensitization and reward: implications for dopamine receptor down-regulation and dopamine release.

Matrix metalloproteinases (MMPs) and its inhibitors (TIMPs) function to remodel the pericellular environment. We have demonstrated that methamphetamine (METH)‐induced behavioral sensitization and reward were markedly attenuated in MMP‐2‐ and MMP‐9 deficient [MMP‐2‐(−/−) and MMP‐9‐(−/−)] mice compared with those in wild‐type mice, suggesting that METH‐induced expression of MMP‐2 and MMP‐9 in the brain plays a role in the development of METH‐induced sensitization and reward. In the present study, we investigated the changes in TIMP‐2 expression in the brain after repeated METH treatment. Furthermore, we studied a role of MMP/TIMP system in METH‐induced behavioral changes and dopamine neurotransmission. Repeated METH treatment induced behavioral sensitization, which was accompanied by an increase in TIMP‐2 expression. Antisense TIMP‐2 oligonucleotide (TIMP‐AS) treatment enhanced the sensitization, which was associated with the potentiation of METH‐induced dopamine release in the nucleus accumbens (NAc). On the other hand, MMP‐2/‐9 inhibitors blocked the METH‐induced behavioral sensitization and conditioned place preference, a measure of the rewarding effect, and reduced the METH‐increased dopamine release in the NAc. Dopamine receptor agonist‐stimulated [35S]GTPγS binding was reduced in the frontal cortex of sensitized rats. TIMP‐AS treatment potentiated, while MMP‐2/‐9 inhibitor attenuated, the reduction of dopamine D2 receptor agonist‐stimulated [35S]GTPγS binding. Repeated METH treatment also reduced dopamine D2 receptor agonist‐stimulated [35S]GTPγS binding in wild‐type mice, but such changes were significantly attenuated in MMP‐2‐(−/−) and MMP‐9‐(−/−) mice. These results suggest that the MMP/TIMP system is involved in METH‐induced behavioral sensitization and reward, by regulating dopamine release and receptor signaling.

Silibinin attenuates cognitive deficits and decreases of dopamine and serotonin induced by repeated methamphetamine treatment

Cognitive deficits are a core feature of patients with methamphetamine (METH) abuse. It has been reported that repeated METH treatment impairs long-term recognition memory in the novel object recognition test (NORT) in mice. Recent studies indicate that silibinin, a flavonoid derived from the herb milk thistle, has potent neuroprotective effects in cell cultures and several animal models of neurological diseases. However, its effect on the cognitive deficit induced by METH remains unclear. In the present study, we attempt to clarify the effect of silibinin on impairments of recognition memory caused by METH in mice. Mice were co-administered silibinin with METH for 7 days and then cognitive function was assessed by NORT after 7-day withdrawal. Tissue levels of dopamine and serotonin as well as their metabolites in the prefrontal cortex and hippocampus were measured 1 day after NORT. Silibinin dose-dependently ameliorated the impairment of recognition memory caused by METH treatment in mice. Silibinin significantly attenuated the decreases in the dopamine content of the prefrontal cortex and serotonin content of the hippocampus caused by METH treatment. We also found a correlation between the recognition values and dopamine and serotonin contents of the prefrontal cortex and hippocampus. The effect of silibinin on cognitive impairment may be associated with an amelioration of decreases in dopamine and serotonin levels in the prefrontal cortex and hippocampus, respectively. These results suggest that silibinin may be useful as a pharmacological tool to investigate the mechanisms of METH-induced cognitive impairments.

Silibinin Attenuates Amyloid β25–35 Peptide-Induced Memory Impairments: Implication of Inducible Nitric-Oxide Synthase and Tumor Necrosis Factor-α in Mice

In Alzheimer’s disease (AD), the deposition of amyloid peptides is invariably associated with oxidative stress and inflammatory responses. Silibinin (silybin), a flavonoid derived from the herb milk thistle, has potent anti-inflammatory and antioxidant activities. However, it remains unclear whether silibinin improves amyloid β (Aβ) peptide-induced neurotoxicity. In this study, we examined the effect of silibinin on the fear-conditioning memory deficits, inflammatory response, and oxidative stress induced by the intracerebroventricular injection of Aβ peptide25–35 (Aβ25–35) in mice. Mice were treated with silibinin (2, 20, and 200 mg/kg p.o., once a day for 8 days) from the day of the Aβ25–35 injection (day 0). Memory function was evaluated in cued and contextual fear-conditioning tests (day 6). Nitrotyrosine levels in the hippocampus and amygdala were examined (day 8). The mRNA expression of inducible nitric-oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α) in the hippocampus and amygdala was measured 2 h after the Aβ25–35 injection. We found that silibinin significantly attenuated memory deficits caused by Aβ25–35 in the cued and contextual fear-conditioning test. Silibinin significantly inhibited the increase in nitrotyrosine levels in the hippocampus and amygdala induced by Aβ25–35. Nitrotyrosine levels in these regions were negatively correlated with memory performance. Moreover, real-time RT-PCR revealed that silibinin inhibited the overexpression of iNOS and TNF-α mRNA in the hippocampus and amygdala induced by Aβ25–35. These findings suggest that silibinin (i) attenuates memory impairment through amelioration of oxidative stress and inflammatory response induced by Aβ25–35 and (ii) may be a potential candidate for an AD medication.