Enhancement of morphine-induced antinociception after electroconvulsive shock in mice.

Electroconvulsive therapy (ECT) has been applied for chronic pain for decades. The amounts of opioids to treat pain are sometimes reduced after a series of ECT. The effect of ECT on morphine-induced analgesia and its mechanism underlying the reduction of morphine requirement has yet to be clarified. Therefore, we administered electroconvulsive shocks (ECS) to mice and investigated the antinociceptive effect of morphine in a hot plate test. We examined the expression level of µ-opioid receptor in the thalami of mice 25 h after administration of ECS compared to the thalami of mice without ECS administration using western blotting. ECS disturbed the development of a decrease in the percentage of maximal possible effect (%MPE), which was observed 24 h after a morphine injection, when ECS was applied 25, 23, 21, and 12 h before the second administration of morphine. We also examined the effect of ECS on the dose-response curve of %MPE to morphine-antinociception. Twenty-five hours after ECS, the dose-response curve was shifted to the left, and the EC50 of morphine given to ECS-pretreated mice decreased by 30.1% compared to the mice that were not pretreated with ECS. We also found that the expression level of µ-opioid receptors was significantly increased after ECS administration. These results confirm previous clinical reports showing that ECT decreased the required dose of opioids in neuropathic pain patients and suggest the hypothesis that this effect of ECT works through the thalamus.

Distinct Roles of Opioid and Dopamine Systems in Lateral Hypothalamic Intracranial Self-Stimulation.

Background: Opioid and dopamine systems play crucial roles in reward. Similarities and differences in the neural mechanisms of reward that are mediated by these 2 systems have remained largely unknown. Thus, in the present study, we investigated the differences in reward function in both µ-opioid receptor knockout mice and dopamine transporter knockout mice, important molecules in the opioid and dopamine systems. Methods: Mice were implanted with electrodes into the right lateral hypothalamus (l hour). Mice were then trained to put their muzzle into the hole in the head-dipping chamber for intracranial electrical stimulation, and the influences of gene knockout were assessed. Results: Significant differences are observed between opioid and dopamine systems in reward function. µ-Opioid receptor knockout mice exhibited enhanced intracranial electrical stimulation, which induced dopamine release. They also exhibited greater motility under conditions of "despair" in both the tail suspension test and water wheel test. In contrast, dopamine transporter knockout mice maintained intracranial electrical stimulation responding even when more active efforts were required to obtain the reward. Conclusions: The absence of µ-opioid receptor or dopamine transporter did not lead to the absence of intracranial electrical stimulation responsiveness but rather differentially altered it. The present results in µ-opioid receptor knockout mice are consistent with the suppressive involvement of µ-opioid receptors in both positive incentive motivation associated with intracranial electrical stimulation and negative incentive motivation associated with depressive states. In contrast, the results in dopamine transporter knockout mice are consistent with the involvement of dopamine transporters in positive incentive motivation, especially its persistence. Differences in intracranial electrical stimulation in µ-opioid receptor and dopamine transporter knockout mice underscore the multidimensional nature of reward.

Exposure to GABAA Receptor Antagonist Picrotoxin in Pregnant Mice Causes Autism-Like Behaviors and Aberrant Gene Expression in Offspring.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is characterized by impairments in social interaction and restricted/repetitive behaviors. The neurotransmitter γ-aminobutyric acid (GABA) through GABAA receptor signaling in the immature brain plays a key role in the development of neuronal circuits. Excitatory/inhibitory imbalance in the mature brain has been investigated as a pathophysiological mechanism of ASD. However, whether and how disturbances of GABA signaling in embryos that are caused by GABAA receptor inhibitors cause ASD-like pathophysiology are poorly understood. The present study examined whether exposure to the GABAA receptor antagonist picrotoxin causes ASD-like pathophysiology in offspring by conducting behavioral tests from the juvenile period to adulthood and performing gene expression analyses in mature mouse brains. Here, we found that male mice that were prenatally exposed to picrotoxin exhibited a reduction of active interaction time in the social interaction test in both adolescence and adulthood. The gene expression analyses showed that picrotoxin-exposed male mice exhibited a significant increase in the gene expression of odorant receptors. Weighted gene co-expression network analysis showed a strong correlation between social interaction and enrichment of the "odorant binding" pathway gene module. Our findings suggest that exposure to a GABAA receptor inhibitor during the embryonic period induces ASD-like behavior, and impairments in odorant function may contribute to social deficits in offspring.

[Neuropsychopharmacological effects of methylphenidate].

Methylphenidate (MPH) is widely used for narcolepsy, a sleep disorder, and attention-deficit/hyperactivity disorder (AD/HD). Considering that MPH has stimulating and awakening actions, the mechanisms underlying the MPH effect on narcolepsy are easy to understand. On the other hand, the mechanisms underlying effects of MPH on AD/HD are largely unknown. While MPH induces hyperactivity in healthy humans, MPH reduces hyperactivity in AD/HD patients. The main target molecules of MPH are dopamine transporter and norepinephrine transporter. Interestingly, mice lacking dopamine transporter show AD/HD-like behaviors and MPH reactions like those in AD/HD patients. Analyses of mice lacking dopamine transporter may lead to a better understanding of the neuropsychological MPH effects.

Effects of rapamycin on social interaction deficits and gene expression in mice exposed to valproic acid in utero.

The mammalian target of rapamycin (mTOR) signaling pathway plays a crucial role in cell metabolism, growth, and proliferation. The overactivation of mTOR has been implicated in the pathogenesis of syndromic autism spectrum disorder (ASD), such as tuberous sclerosis complex (TSC). Treatment with the mTOR inhibitor rapamycin improved social interaction deficits in mouse models of TSC. Prenatal exposure to valproic acid (VPA) increases the incidence of ASD. Rodent pups that are exposed to VPA in utero have been used as an animal model of ASD. Activation of the mTOR signaling pathway was recently observed in rodents that were exposed to VPA in utero, and rapamycin ameliorated social interaction deficits. The present study investigated the effect of rapamycin on social interaction deficits in both adolescence and adulthood, and gene expressions in mice that were exposed to VPA in utero. We subcutaneously injected 600 mg/kg VPA in pregnant mice on gestational day 12.5 and used the pups as a model of ASD. The pups were intraperitoneally injected with rapamycin or an equal volume of vehicle once daily for 2 consecutive days. The social interaction test was conducted in the offspring after the last rapamycin administration at 5-6 weeks of ages (adolescence) or 10-11 weeks of age (adulthood). Whole brains were collected after the social interaction test in the adulthood, and microarray and Western blot analyses were performed. Mice that were exposed to VPA and treated with vehicle exhibited a decrease in social interaction compared with control mice that were treated with vehicle. Rapamycin treatment in VPA-exposed mice improved social deficits. Mice that were exposed to VPA and treated with vehicle exhibited the aberrant expression of genes in the mTOR signaling pathway, and rapamycin treatment recovered changes in the expression of some genes, including Fyb and A330094K24Rik. Rapamycin treatment suppressed S6 phosphorylation in VPA-exposed mice. Aberrant gene expression was associated with social interaction deficits in VPA-exposed mice. Rapamycin may be an effective treatment for non-syndromic ASD in adolescent and adult patients who present impairments in the mTOR signaling pathway.

Early manifestation of depressive-like behavior in transgenic mice that express dementia with Lewy body-linked mutant ß-synuclein.

AIM: We previously generated transgenic (Tg) mice that expressed P123H ß-synuclein (ßS), a dementia with Lewy body-linked mutant ßS. Notably, these mice recapitulated neurodegenerative features of Lewy body disease, reflected by motor dysfunction, greater protein aggregation, and memory impairment. Since recent studies suggested that non-motor symptoms, such as depression, might be manifested in the prodromal stage of Lewy body disease, the main objective of the present study was to investigate the early expression of behavior in P123H ßS Tg mice. METHODS: Nest building, locomotor activity, and depressive-like behavior were assessed using 6- to 10-month-old male and female P123H ßS Tg and wildtype mice. KEY RESULTS: P123H ßS Tg mice exhibited hyperlocomotor activity in a novel environment, a decrease in mobility time in the tail suspension test, and impairments in nest building. CONCLUSIONS: Importantly, these non-motor behaviors were manifested before the onset of motor dysfunction, suggesting that P123H ßS Tg mice could be a valid model for investigating the early phase of Lewy body disease.

Reward-enhancing effect of methylphenidate is abolished in dopamine transporter knockout mice: A model of attention-deficit/hyperactivity disorder.

AIM: Attention-deficit/hyperactivity disorder is a heterogeneous neurobiological disorder that is characterized by inattention, impulsivity, and an increase in motor activity. Although methylphenidate has been used as a medication for decades, unknown is whether methylphenidate treatment can cause drug dependence in patients with attention-deficit/hyperactivity disorder. This study investigated the reward-enhancing effects of methylphenidate using intracranial self-stimulation in an animal model of attention-deficit/hyperactivity disorder, dopamine transporter knockout mice. METHODS: For the intracranial self-stimulation procedures, the mice were trained to nosepoke to receive direct electrical stimulation via an electrode that was implanted in the lateral hypothalamus. After the acquisition of nosepoke responding for intracranial self-stimulation, the effects of methylphenidate on intracranial self-stimulation were investigated. RESULTS: In the progressive-ratio procedure, dopamine transporter knockout mice exhibited an increase in intracranial self-stimulation compared with wild-type mice. Treatment with 5 and 10 mg/kg methylphenidate increased intracranial self-stimulation responding in wild-type mice. Methylphenidate at the same doses did not affect intracranial self-stimulation responding in dopamine transporter knockout mice. We then investigated the effects of high-dose methylphenidate (60 mg/kg) in a rate-frequency procedure. High-dose methylphenidate significantly decreased intracranial self-stimulation responding in both wild-type and dopamine transporter knockout mice. CONCLUSIONS: These results suggest that low-dose methylphenidate alters the reward system (ie, increases intracranial self-stimulation responding) in wild-type mice via dopamine transporter inhibition, whereas dopamine transporter knockout mice do not exhibit such alterations. High-dose methylphenidate appears to suppress intracranial self-stimulation responding not through dopamine transporter inhibition but rather through other mechanisms. These results support the possibility that methylphenidate treatment for attention-deficit/hyperactivity disorder does not increase the risk of drug dependence, in attention-deficit/hyperactivity disorder patients with dopamine transporter dysfunction.

Brain hyperserotonemia causes autism-relevant social deficits in mice.

Background: Hyperserotonemia in the brain is suspected to be an endophenotype of autism spectrum disorder (ASD). Reducing serotonin levels in the brain through modulation of serotonin transporter function may improve ASD symptoms. Methods: We analyzed behavior and gene expression to unveil the causal mechanism of ASD-relevant social deficits using serotonin transporter (Sert) knockout mice. Results: Social deficits were observed in both heterozygous knockout mice (HZ) and homozygous knockout mice (KO), but increases in general anxiety were only observed in KO mice. Two weeks of dietary restriction of the serotonin precursor tryptophan ameliorated both brain hyperserotonemia and ASD-relevant social deficits in Sert HZ and KO mice. The expression of rather distinct sets of genes was altered in Sert HZ and KO mice, and a substantial portion of these genes was also affected by tryptophan depletion. Tryptophan depletion in Sert HZ and KO mice was associated with alterations in the expression of genes involved in signal transduction pathways initiated by changes in extracellular serotonin or melatonin, a derivative of serotonin. Only expression of the AU015836 gene was altered in both Sert HZ and KO mice. AU015836 expression and ASD-relevant social deficits normalized after dietary tryptophan restriction. Conclusions: These findings reveal a Sert gene dose-dependent effect on brain hyperserotonemia and related behavioral sequelae in ASD and a possible therapeutic target to normalize brain hyperserotonemia and ASD-relevant social deficits.

Light/dark phase-dependent spontaneous activity is maintained in dopamine-deficient mice.

Dopamine is important for motor control and involved in the regulation of circadian rhythm. We previously found that dopamine-deficient (DD) mice became hyperactive in a novel environment 72 h after the last injection of L-3,4-dihydroxyphenylalanine (L-DOPA) when dopamine was almost completely depleted. DD mice did not initially exhibit hyperactivity in their home cages, but the animals exhibited hyperactivity several hours after the last L-DOPA injection. The regulation of motor activity in a novel environment and in home cages may be different. A previous study reported that DD mice became active again approximately 24 h after the last L-DOPA injection. One speculation was that light/dark phase-dependent spontaneous activity might be maintained despite dopamine deficiency. The present study investigated whether spontaneous home cage activity is maintained in DD mice 24-43 h and 72-91 h after the last L-DOPA injection. Spontaneous activity was almost completely suppressed during the light phase of the light/dark cycle in DD mice 24 and 72 h after the last L-DOPA injection. After the dark phase began, DD mice became active 24 and 72 h after the last L-DOPA injection. DD mice exhibited a similar amount of locomotor activity as wildtype mice 24 h after the last L-DOPA injection. Although DD mice presented a decrease in activity 72 h after the last L-DOPA injection, they maintained dark phase-stimulated locomotor activation. Despite low levels of dopamine in DD mice, they exhibited feeding behavior that was similar to wildtype mice. Although grooming and rearing behavior significantly decreased, DD mice retained their ability to perform these activities. Haloperidol treatment significantly suppressed all of these behaviors in wildtype mice but not in DD mice. These results indicate that DD mice maintain some aspects of light/dark phase-dependent spontaneous activity despite dopamine depletion, suggesting that compensatory dopamine-independent mechanisms might play a role in the DD mouse phenotype.

Loss of GluN2D subunit results in social recognition deficit, social stress, 5-HT2C receptor dysfunction, and anhedonia in mice.

The N-methyl-d-aspartate (NMDA) receptor channel is involved in various physiological functions, including learning and memory. The GluN2D subunit of the NMDA receptor has low expression in the mature brain, and its role is not fully understood. In the present study, the effects of GluN2D subunit deficiency on emotional and cognitive function were investigated in GluN2D knockout (KO) mice. We found a reduction of motility (i.e., a depressive-like state) in the tail suspension test and a reduction of sucrose preference (i.e., an anhedonic state) in GluN2D KO mice that were group-housed with littermates. Despite apparently normal olfactory function and social interaction, GluN2D KO mice exhibited a decrease in preference for social novelty, suggesting a deficit in social recognition or memory. Golgi-Cox staining revealed a reduction of the complexity of dendritic trees in the accessory olfactory bulb in GluN2D KO mice, suggesting a deficit in pheromone processing pathway activation, which modulates social recognition. The deficit in social recognition may result in social stress in GluN2D KO mice. Isolation housing is a procedure that has been shown to reduce stress in mice. Interestingly, 3-week isolation and treatment with agomelatine or the 5-hydroxytryptamine-2C (5-HT2C) receptor antagonist SB242084 reversed the anhedonic-like state in GluN2D KO mice. In contrast, treatment with the 5-HT2C receptor agonist CP809101 induced depressive- and anhedonic-like states in isolated GluN2D KO mice. These results suggest that social stress that is caused by a deficit in social recognition desensitizes 5-HT2c receptors, followed by an anhedonic- and depressive-like state, in GluN2D KO mice. The GluN2D subunit of the NMDA receptor appears to be important for the recognition of individuals and development of normal emotionality in mice. 5-HT2C receptor antagonism may be a therapeutic target for treating social stress-induced anhedonia. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

Fluoxetine as a potential pharmacotherapy for methamphetamine dependence: studies in mice.

The monoamine transporters are the main targets of psychostimulant drugs, including methamphetamine (METH) and cocaine. Interestingly, the rewarding effects of cocaine are retained in dopamine transporter (DAT) knockout (KO) mice, while serotonin transporter (SERT) and DAT double KO mice do not exhibit conditioned place preference (CPP) to cocaine. These data suggest that SERT inhibition decreases the rewarding effects of psychostimulants. To further test this hypothesis, in the present study, we investigated the effects of intraperitoneal (i.p.) injections of 20 mg/kg fluoxetine, a selective serotonin reuptake inhibitor (SSRI), on 2 mg/kg METH (i.p.) CPP and locomotor sensitization to 1 mg/kg METH (i.p.) in C57BL/6J mice. Fluoxetine treatment before both the conditioning and preference tests abolished METH CPP. A two-way analysis of variance (ANOVA) revealed that METH CPP tended to be lower in mice pretreated with fluoxetine before the preference test than in control mice pretreated with saline before the preference test. Furthermore, pretreatment with fluoxetine had inhibitory effects on METH-induced locomotor sensitization. These results suggest that fluoxetine, a widely used medication for depression, may be also a useful tool for treating METH dependence.

Differential effects of donepezil on methamphetamine and cocaine dependencies.

Donepezil, a choline esterase inhibitor, has been widely used as a medicine for Alzheimer's disease. Recently, a study showed that donepezil inhibited addictive behaviors induced by cocaine, including cocaine-conditioned place preference (CPP) and locomotor sensitization to cocaine. In the present study, we investigated the effects of donepezil on methamphetamine (METH)-induced behavioral changes in mice. In counterbalanced CPP tests, the intraperitoneal (i.p.) administration of 3 mg/kg donepezil prior to 2 mg/kg METH i.p. failed to inhibit METH CPP, whereas pretreatment with 3 mg/kg donepezil abolished the CPP for cocaine (10 mg/kg, i.p.). Similarly, in locomotor sensitization experiments, i.p. administration of 1 mg/kg donepezil prior to 2 mg/kg METH i.p. failed to inhibit locomotor sensitivity to METH, whereas pretreatment with 1 mg/kg donepezil significantly inhibited locomotor sensitivity to cocaine (10 mg/kg, i.p.). These results suggest that donepezil may be a useful tool for treating cocaine dependence but not for treating METH dependence. The differences in the donepezil effects on addictive behaviors induced by METH and cocaine might be due to differences in the involvement of acetylcholine in the mechanisms of METH and cocaine dependencies.

Repeated methamphetamine administration alters expression of the NMDA receptor channel epsilon2 subunit and kinesins in the mouse brain.

Repeated amphetamine administration results in behavioral sensitization. Behavioral sensitization related to abuse and/or relapse may be associated with stable changes in gene expression. To explore the participating genes, we examined the changes in gene expression levels 24 h or 21 days (long-term withdrawal period) after chronic methamphetamine (METH) treatment for 2 weeks. The expression of several genes related to glutamatergic neural transmission was altered, although changes in the corresponding protein expression were not always consistent with the results for mRNA expression. Of interest, in the frontal cortex of mice treated with METH for 2 weeks, protein expression levels of KIF17 and the N-methyl-D-asparate (NMDA) receptor channel epsilon2 subunit (NRepsilon2) were concomitantly increased. The alteration in expression of these proteins, KIF17 and NRepsilon2, might be a part of the molecular basis of the behavioral sensitization to METH.

Increased body weight in mice lacking mu-opioid receptors.

Opioids have been suggested to affect feeding behaviour. To clarify the role of mu-opioid receptors in feeding, we measured several parameters relating to food intake in mu-opioid receptor knockout mice. Here, we show that the knockout mice had increased body weight in adulthood, although the intake amount of standard food was similar between the wild-type and knockout littermates. Serum markers for energy homeostasis were not significantly altered in the knockout mice. Hypothalamic neuropeptide Y mRNA, however, was higher in knockouts than in wild-type mice. Our results suggest that the up-regulated expression of neuropeptide Y mRNA might contribute to the increased weights of adult mu-opioid receptor knockout mice.

G protein-activated inwardly rectifying K+ channel inhibition and rescue of weaver mouse motor functions by antidepressants.

Antidepressants, including tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs), have been widely used for the treatment of not only depression but also other psychiatric disorders, although the molecular mechanisms of the drug effects have not yet been sufficiently revealed. Here, we investigated the in vivo effects of these antidepressants on G protein-activated inwardly rectifying K+ (GIRK) channels, which are important for regulating the excitability of various cells, by using weaver (wv) mice, which have mutant GIRK channels and show abnormal neuronal cell death and motor disturbances. First, we found that a widely used SSRI fluoxetine (also known as Prozac) effectively inhibited wv GIRK2 channels like wild-type GIRK channels, expressed in Xenopus oocytes. Next, we found that weaver motor disturbances were remarkably alleviated by chronic treatment with fluoxetine or desipramine. Furthermore, the chronic fluoxetine treatment substantially suppressed the abnormal neuronal cell death in the weaver mouse cerebellum and pontine nuclei. These results suggest that continuous inhibition of wv GIRK2 channels by a group of antidepressants caused substantial suppression of the neuronal cell death and resulted in improvement of motor abilities in weaver mice. These results provide evidence for in vivo GIRK channel inhibition by a group of antidepressants.

Methamphetamine modulation of gene expression in the brain: analysis using customized cDNA microarray system with the mouse homologues of KIAA genes.

Amphetamine abuse may be associated with adaptive changes in gene expression. In the present study, we used a newly developed cDNA array system comprising mouse KIAA (mKIAA) cDNA clones to examine changes in gene expression after chronic methamphetamine (MAP) treatment. Mice were daily treated with saline or MAP (2 mg/kg, ip) for 2 weeks. Approximately 800 mKIAA clones were blotted onto a nylon membrane and hybridized with 33P-labeled DNA derived from mRNAs from mouse whole brain. MAP-induced changes were found in several clones by using whole brain mRNA. Since gene expression of Per2, one of the period protein-related proteins, was the most affected by MAP treatment, its expression was further analyzed in pooled hippocampi from 20 mice that had been treated with saline or MAP (2 mg/kg, ip) for 2 weeks. The gene expression and protein expression of Per2 in the hippocampus were increased by MAP treatment. In the hippocampus, Per2 gene expression was under the regulation of circadian rhythm and increases in Per2 expression were due to the phase shift induced by chronic MAP treatment. These findings suggest that unique expression changes of period protein-related proteins in the hippocampus occur in MAP abuse.

[Control of craving for methamphetamine: development of scales for dependence and search for medicines for treatment].

Methamphetamine dependence presents a serious problem not only for patients but also for society. Medical treatment has mainly targeted psychotic symptoms such as hallucination and delusion, and ignored the symptoms of craving, which are the major cause of dependence. Therefore, the risk of lapse into methamphetamine reuse remains very high. Although development of both medicines and programs for treatment of craving is needed, progress has been hampered by the lack of appropriate scales for assessing the severity of dependence and craving. On the other hand, recent breakthroughs in genomic sciences and molecular medicine have made it possible to investigate the molecular mechanisms underlying craving in animals. This paper reviews studies on the development of scales for assessing the severity of methamphetamine dependence and craving, together with recent data on candidate medicines for craving treatment in animals. The reliability and validity of the revised Addiction Severity Index -Japanese version (ASI-J) was confirmed after its administration to 100 drug abuse patients. The Craving Index was also newly developed, and its validity for prediction of relapse was confirmed. In animal experiments, fluoxetine, a selective serotonin reuptake inhibitor, was recognized as a candidate medicine for treatment of methamphetamine dependence.

Involvement of the N-methyl-D-aspartate receptor GluN2D subunit in phencyclidine-induced motor impairment, gene expression, and increased Fos immunoreactivity.

BACKGROUND: Noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists evoke a behavioral and neurobiological syndrome in experimental animals. We previously reported that phencyclidine (PCP), an NMDA receptor antagonist, increased locomotor activity in wildtype (WT) mice but not GluN2D subunit knockout mice. Thus, the aim of the present study was to determine whether the GluN2D subunit is involved in PCP-induced motor impairment. RESULTS: PCP or UBP141 (a GluN2D antagonist) induced potent motor impairment in WT mice but not GluN2D KO mice. By contrast, CIQ, a GluN2C/2D potentiator, induced severe motor impairment in GluN2D KO mice but not WT mice, suggesting that the GluN2D subunit plays an essential role in the effects of PCP and UBP141, and an appropriate balance between GluN2C and GluN2D subunits might be needed for appropriate motor performance. The level of the GluN2D subunit in the mature mouse brain is very low and restricted. GluN2D subunits exist in brainstem structures, the globus pallidus, thalamus, and subthalamic nucleus. We found that the expression of the c-fos gene increased the most among PCP-dependent differentially expressed genes between WT and GluN2D KO mice, and the number of Fos-positive cells increased after PCP administration in the basal ganglia motor circuit in WT mice but not GluN2D KO mice. CONCLUSION: These results suggest that the GluN2D subunit within the motor circuitry is a key subunit for PCP-induced motor impairment, which requires an intricate balance between GluN2C- and GluN2D-mediated excitatory outputs.