Long-lasting increases in GABAB receptor subunit levels in hippocampal dentate gyrus of mice with a single systemic injection of trimethyltin.

We have recently shown delayed increases in GABAB receptor (GABABR) subunit protein levels in the hippocampal dentate gyrus (DG), but not in the pyramidal CA1 and CA3 regions, at 15-30 days after the systemic single administration of trimethyltin (TMT) in mice. An attempt was thus made to determine whether the delayed increases return to the control levels found in naive mice afterward. In the DG on hippocampal slices obtained at 90 days after the administration, however, marked increases were still seen in protein levels of both GABABR1 and GABABR2 subunits without significant changes in calbindin and glial fibrillary acidic protein (GFAP) levels on immunoblotting analysis. Fluoro-Jade B staining clearly revealed the absence of degenerated neurons from the DG at 90 days after the administration. Although co-localization was invariably detected between GABABR2 subunit and GFAP in the DG at 30 days on immunohistochemical analysis, GABABR2-positive cells did not merge well with GFAP-positive cells in the DG at 90 days. These results suggest that both GABABR1 and GABABR2 subunits would be tardily and sustainably up-regulated by cells other than neurons and astrocytes in the murine DG at 90 days after a systemic single injection of TMT.

Pharmacokinetics of L-theanine and the effect on amino acid composition in mice administered with L-theanine.

L-theanine, an amino acid component of the tea leaves of Camellia sinensis, is sold in Japan as a supplement for good sleep. Although several studies in humans and mice have reported the effects of L-theanine on brain function, only a few reports have comprehensively clarified the disposition of theanine administered to mice and its effects on concentrations of other blood amino acids. In this study, we aimed to determine the changes in the blood levels of L-theanine administered to mice and amino acid composition of the serum. L-theanine were administered to four-week-old Std-ddY male mice orally or via tail vein injection. L-theanine and other amino acids in serum prepared from blood collected at different time points post-dose were labeled with phenylisothiocyanate and quantified. The serum concentration of orally administered L-theanine peaked 15 min after administration. The area under the curve for tail vein injection revealed the bioavailability of L- theanine to be approximately 70%. L-theanine administration did not affect any amino acid levels in the serum, but a significant increase in the peak area overlapping the Glycine (Gly) peak was observed 30 min after administration. L-theanine administered to mice was rapidly absorbed and eliminated, suggesting that taking L-theanine as a supplement is safe without affecting its own levels or serum levels of other amino acids. However, considering that Gly, similar to L-theanine, is used as a dietary supplement for its anxiolytic effects and to improve sleep, determining the effects of L-theanine administration on Gly is important and needs further research.

Post-translational modifications of the apelin receptor regulate its functional expression.

Post-translational modifications (PTMs) are protein modifications that occur after protein biosynthesis, playing a crucial role in regulating protein function. They are involved in the functional expression of G-protein-coupled receptors (GPCRs), as well as intracellular and secretory protein signaling. Here, we aimed to investigate the PTMs of the apelin receptor (APLNR), a GPCR and their potential influence on the receptor's function. In an in vitro experiment using HEK cells, we only observed glycosylation as a PTM of the APLNR and ineffective receptor signaling by the agonist, (Pyr1)-apelin-13. In contrast, when analyzing mouse spinal cord, we detected glycosylation and other PTMs, excluding isopeptidation. This suggests that additional PTMs are involved in the functional expression of the APLNR in vitro. In summary, these findings suggest that the APLNR in vivo requires multiple PTMs for functional expression. To comprehensively understand the pharmacological effects of the APLNR, it is essential to establish an in vitro system that adequately replicates the receptor's PTM profile. Nonetheless, it is crucial to overcome the challenge of heat-sensitive proteolysis in APLNR studies. By elucidating the regulation of PTMs, further research has the potential to advance the analysis and pharmacological studies of both the apelin/APLNR system and GPCR signal modulation.

Knockdown of Piccolo in the Nucleus Accumbens Suppresses Methamphetamine-Induced Hyperlocomotion and Conditioned Place Preference in Mice.

Methamphetamine (METH), the most widely distributed psychostimulant, aberrantly activates the reward system in the brain to induce addictive behaviors. The presynaptic protein "Piccolo", encoded by Pclo, was identified as a METH-responsive protein with enhanced expression in the nucleus accumbens (NAc) in mice. Although the physiological and pathological significance of Piccolo has been identified in dopaminergic signaling, its role in METH-induced behavioral abnormalities and the underlying mechanisms remain unclear. To clarify such functions, mice with Piccolo knockdown in the NAc (NAc-miPiccolo mice) by local injection of an adeno-associated virus vector carrying miRNA targeting Pclo were generated and investigated. NAc-miPiccolo mice exhibited suppressed hyperlocomotion, sensitization, and conditioned place preference behavior induced by systemic administration of METH. The excessive release of dopamine in the NAc was reduced in NAc-miPiccolo mice at baseline and in response to METH. These results suggest that Piccolo in the NAc is involved in METH-induced behavioral alterations and is a candidate therapeutic target for the treatment of drug addiction.

Shati/Nat8l Overexpression Improves Cognitive Decline by Upregulating Neuronal Trophic Factor in Alzheimer's Disease Model Mice.

Alzheimer's disease (AD) is a type of dementia characterized by the deposition of amyloid ß, a causative protein of AD, in the brain. Shati/Nat8l, identified as a psychiatric disease related molecule, is a responsive enzyme of N-acetylaspartate (NAA) synthesis. In the hippocampi of AD patients and model mice, the NAA content and Shati/Nat8l expression were reported to be reduced. Having recently clarified the involvement of Shati/Nat8l in cognitive function, we examined the recovery effect of the hippocampal overexpression of Shati/Nat8l in AD model mice (5XFAD). Shati/Nat8l overexpression suppressed cognitive dysfunction without affecting the Aß burden or number of NeuN-positive neurons. In addition, brain-derived neurotrophic factor mRNA was upregulated by Shati/Nat8l overexpression in 5XFAD mice. These results suggest that Shati/Nat8l overexpression prevents cognitive dysfunction in 5XFAD mice, indicating that Shati/Nat8l could be a therapeutic target for AD.

Delayed Expression of Both GABABR1 and GABABR2 Subunits in Murine Hippocampal Dentate Gyrus After a Single Systemic Injection of Trimethyltin.

Trimethyltin (TMT) has been used as a cytotoxin to neurons rather than glial cells in the mammalian hippocampus. The systemic administration of TMT led to declined fluorescence of ZnAF-2 DA staining as a marker of intact mossy fibers and increased fluorescence of Fluoro-Jade B staining as a marker of degenerated neurons during the initial 2 to 5 days after the administration with later ameliorations within 30 days in the hippocampal dentate gyrus (DG) and CA3 region in mice. On immunoblotting analysis, both GABABR1 and GABABR2 subunit levels increased during 15 to 30 days after TMT along with significant decreases in glutamatergic GluA1 and GluA2/3 receptor subunit levels during 2 to 7 days in the DG, but not in other hippocampal regions such as CA1 and CA3 regions. Immunohistochemical analysis revealed the constitutive and inducible expression of GABABR2 subunit in cells immunoreactive to an astrocytic marker as well as neuronal markers in the DG with the absence of neither GABABR1a nor GABABR1b subunit from cells positive to an astrocytic marker. These results suggest that both GABABR1 and GABABR2 subunits may be up-regulated in cells other than neurons and astroglia in the DG at a late stage of TMT intoxication in mice.

Apelin/Apelin Receptor System: Molecular Characteristics, Physiological Roles, and Prospects as a Target for Disease Prevention and Pharmacotherapy.

Among the various orphan G protein-coupled receptors, apelin receptor (APJ), originally identified in the human genome as an orphan G-protein-coupled receptor, was deorphanised in 1998 with the discovery of its endogenous ligand, apelin. Apelin and APJ mRNA are widely expressed in peripheral tissues and the central nervous system in mammals. In this review, we discuss the characteristics, pharmacology, physiology, and pathology of the apelin/APJ system in mammals. Several physiological roles of the apelin/APJ system have been reported, including in homeostasis, cardiovascular maintenance, angiogenesis, and neuroprotection. In cellular signaling, apelin has been shown to drive the PI3K/Akt, MAPK, and PKA signaling pathways, leading to cell proliferation and protection from excitotoxicity. Apelin is also found in breast milk; therefore, apelin is believed to contribute to the establishment of the infant immune system. Furthermore, activation of the apelin/APJ system is reported to restore muscular weakness associated with aging. Thus, the apelin/APJ system represents a novel target for the prevention of several important cardiovascular and neurodegenerative diseases and the maintenance of health during old age.

Investigating DNA Methylation of SHATI/NAT8L Promoter Sites in Blood of Unmedicated Patients with Major Depressive Disorder.

Major depressive disorder (MDD) is one of the most common psychiatric diseases. However, early detection and diagnosis of MDD is difficult, largely because there is no known biomarker or objective diagnostic examination, and its diagnosis is instead based on a clinical interview. The aim of this study was to develop a novel diagnostic tool using DNA methylation as a blood biomarker. We sought to determine whether unmedicated patients with MDD showed significant differences in DNA methylation in the promoter region of the SHATI/N-acetyltransferase 8 like (SHATI/NAT8L) gene compared to healthy controls. Sixty participants with MDD were recruited from all over Japan. They were diagnosed and assessed by at least two trained psychiatrists according to DSM-5 criteria. DNA was extracted from peripheral blood. We then assessed DNA methylation of the SHATI/NAT8L promoter regions in patients with MDD by pyrosequencing. Methylation levels of the SHATI/NAT8L promoter region at CpG sites in peripheral blood from unmedicated patients were significantly higher than in healthy controls. In contrast, medicated patients with MDD showed significantly lower methylation levels in the same region compared to healthy controls. Since previous studies of DNA methylation in MDD only assessed medicated patients, the methylation status of the SHATI/NAT8L promoter region in unmedicated patients presented herein may prove useful for the diagnosis of MDD. To our knowledge, this is the first attempt to measure methylation of the SHATI/NAT8L gene in drug-naïve patients with psychiatric diseases. Based on our findings, methylation of SHATI/NAT8L DNA might be a diagnostic biomarker of MDD.

A Single Medical Marker for Diagnosis of Methamphetamine Addiction - DNA Methylation of SHATI/NAT8L Promoter Sites from Patient Blood.

BACKGROUND: Methamphetamine (METH) is one of the most widely distributed psychostimulants worldwide. Despite active counter measures taken by different countries, neither overall usage of METH nor the frequency of repeat users has reduced over the past decade. METH induces abuse and dependence as it acts on the central nervous system and temporarily stimulates the brain. The recidivism rate for abuse of stimulants in Japan is very high and therefore prevention of repeated usage is paramount. However, we lack information about the relationship between METH users and genomic changes in humans in Japan, which would provide important information to aid such efforts. OBJECTIVE: Shati/Nat8l is a METH-inducible molecule and its overexpression has protective effects on the brain upon METH usage. Here we investigated the effect of METH usage on DNA methylation rates at the promoter site of SHATI/NAT8L. We used DNA samples from human METH users, who are usually difficult to recruit in Japan. METHODS: We measured DNA methylation at SHATI/NAT8L promoter sites by pyrosequencing method using 193 samples of METH users and 60 samples of healthy subjects. In this method, DNA methylation is measured by utilizing the property that only non-methylated cytosine changes to urasil after bisulfite conversion. RESULTS: We found that the rate of DNA methylation at six CpG islands of SHATI/NAT8L promoter sites is significantly higher in METH users when compared to healthy subjects. CONCLUSION: These results suggest that the DNA methylation rate of SHATI/NAT8L promotor regions offers a new diagnostic method for METH usage.

Inhibitory effects of Shati/Nat8l overexpression in the medial prefrontal cortex on methamphetamine-induced conditioned place preference in mice.

Shati/Nat8l is a novel N-acetyltransferase identified in the brain of mice treated with methamphetamine (METH). Shati/Nat8l mRNA is expressed in various brain areas, including the prefrontal cortex (PFC), where the expression level is higher than that in other brain regions. Shati/Nat8l overexpression in the nucleus accumbens (NAc) attenuates the pharmacological response to METH via mGluR3. Meanwhile, dopamine (DA) and glutamate dysregulations have been reported in the medial prefrontal cortex (mPFC) and NAc after METH self-administration and during reinstatement. However, the mechanism, the reward system, and function of Shati/Nat8l in the mPFC is unclear. Here, we injected an adeno-associated virus (AAV) vector containing Shati/Nat8l into the mPFC of mice, to overexpress Shati/Nat8l in the mPFC (mPFC-Shati/Nat8l). Interestingly, the METH-induced conditioned place preference (CPP) was attenuated in the mPFC-Shati/Nat8l mice, but locomotor activity was not. Additionally, immunohistochemical results from mice that were injected with AAV-GFP showed fluorescence in the mPFC and other brain regions, mainly the NAc, indicating an mPFC-NAc top-down connection. Finally, in vivo microdialysis experiments revealed that Shati/Nat8l overexpression in the mPFC reduced extracellular DA levels and suppressed the METH-induced DA increase in the NAc. Moreover, decreased extracellular glutamate levels were observed in the NAc. These results indicate that Shati/Nat8l overexpression in the mPFC attenuates METH-induced CPP by decreasing extracellular DA in the NAc. In contrast, Shati/Nat8l-mPFC overexpression did not alter METH-induced hyperlocomotion. This study demonstrates that Shati/Nat8l in the mPFC attenuates METH reward-seeking behaviour but not the psychomotor activity of METH.

Glycine receptor α4 subunit facilitates the early embryonic development in mice.

Oviduct fluid is essential for the fertilization and subsequent preimplantation development. Glycine is abundant in oviduct fluid and is reported to be critical for preimplantation development of fertilized eggs in mammals. However, the mechanism by which glycine exerts its action on fertilized eggs is yet to be understood. Here we show that glycine regulates the preimplantation development of mouse fertilized eggs via glycine receptors. Among them, the alpha-4 subunit (Glra4) and the ß subunit are expressed in mouse fertilized eggs, and lacking Glra4 inhibits embryonic development to the blastocyst stage, decreases the number of cells in the blastocysts and the litter size. Thus, we identify a novel function of the glycine receptor, which is considered to act mainly as a neurotransmitter receptor, as a regulator of embryonic development and our data provide new insights into the interactions between oviduct milieu and mammalian fertilized egg.

Regulatory effects associated with changes in intracellular potassium level in susceptibility to mitochondrial depolarization and excitotoxicity.

Excitotoxicity has been believed to be one of the causes of neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. So far, much research has been done to suppress the neuronal excessive excitations, however, we still have not achieved full control, which may be due to the lack of some factors. As a matter of course, there is an urgent need to clarify all mechanisms that inhibit the onset and progression of neurodegenerative diseases. We found that potassium ion level regulation may be important in the sense that it suppresses mitochondrial depolarization rather than hyperpolarization of cell membrane potential. Minoxidil, an opener of ATP-activated potassium (KATP) channels decreased injury with middle cerebral artery occlusion in vivo experiment using TTC staining. In the primary cortical neurons, N-methyl-D-aspartate (NMDA)-induced mitochondrial depolarization was suppressed by minoxidil treatment. Minoxidil inhibited the increase in levels of cleaved caspase 3 and the release of cytochrome c into the cytosol, further reducing potassium ion levels. It was observed decreased potassium levels in neurons by the treatment of minoxidil. Those effects of minoxidil were blocked by glibenclamide. Therefore, it was suggested that minoxidil, via opening of KATP channels, reduced intracellular potassium ion level that contribute to mitochondrial depolarization, and suppressed subsequent NMDA-induced mitochondrial depolarization. Our findings suggest that the control of ion levels in neurons could dominate the onset and progression of neurodegenerative diseases.

N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

N-acetylaspartate (NAA) is synthesized by aspartate N-acetyltransferase (gene: Nat8l) from acetyl-coenzyme A and aspartate. In the brain, NAA is considered an important energy metabolite for lipid synthesis. However, the role of NAA in peripheral tissues remained elusive. Therefore, we characterized the metabolic phenotype of knockout (ko) and adipose tissue-specific (ako) Nat8l-ko mice as well as NAA-supplemented mice on various diets. We identified an important role of NAA availability in the brain during adolescence, as 75% of Nat8l-ko mice died on fat-free diet (FFD) after weaning but could be rescued by NAA supplementation. In adult life, NAA deficiency promotes a beneficial metabolic phenotype, as Nat8l-ko and Nat8l-ako mice showed reduced body weight, increased energy expenditure, and improved glucose tolerance on chow, high-fat, and FFDs. Furthermore, Nat8l-deficient adipocytes exhibited increased mitochondrial respiration, ATP synthesis, and an induction of browning. Conversely, NAA-treated wild-type mice showed reduced adipocyte respiration and lipolysis and increased de novo lipogenesis, culminating in reduced energy expenditure, glucose tolerance, and insulin sensitivity. Mechanistically, our data point to a possible role of NAA as modulator of pancreatic insulin secretion and suggest NAA as a critical energy metabolite for adipocyte and whole-body energy homeostasis.-Hofer, D. C., Zirkovits, G., Pelzmann, H. J., Huber, K., Pessentheiner, A. R., Xia, W., Uno, K., Miyazaki, T., Kon, K., Tsuneki, H., Pendl, T., Al Zoughbi, W., Madreiter-Sokolowski, C. T., Trausinger, G., Abdellatif, M., Schoiswohl, G., Schreiber, R., Eisenberg, T., Magnes, C., Sedej, S., Eckhardt, M., Sasahara, M., Sasaoka, T., Nitta, A., Hoefler, G., Graier, W. F., Kratky, D., Auwerx, J., Bogner-Strauss, J. G. N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

Vulnerability to depressive behavior induced by overexpression of striatal Shati/Nat8l via the serotonergic neuronal pathway in mice.

The number of patients with depressive disorders is increasing. However, the mechanism of depression onsets has not been completely revealed. We previously identified Shati/Nat8l, an N-acetyltransferase, in the brain using an animal model of psychosis. In this study, we revealed the involvement of Shati/Nat8l in the vulnerability to major depression. Shati/Nat8l mRNA was increased only in the striatum of mice, which were exposed to chronic social defeat stress. Shati/Nat8l-overexpressed mice showed impairment in social interaction and sucrose preference after the subthreshold social defeat (microdefeat) stress. These depression-like behaviors were restored by fluvoxamine and LY341495 injection prior to these tests. Furthermore, the intracerebral administration of only fluvoxamine, but not of LY341495, to the dorsal striatum and direct infusion of LY341495 to the dorsal raphe also rescued. Taken together, Shati/Nat8l in the striatum has an important role in the vulnerability to depression onsets by regulating the origin of serotonergic neuronal system via GABAergic projection neuron in the dorsal raphe from the dorsal striatum.

Regulatory system of mGluR group II in the nucleus accumbens for methamphetamine-induced dopamine increase by the medial prefrontal cortex.

AIM: We previously reported that methamphetamine (METH)-induced conditioned place preference was attenuated by Shati/Nat8l overexpression in the medial prefrontal cortex (mPFC). Shati/Nat8l overexpression in the mPFC expressed lower levels of both glutamate and dopamine (DA) in the nucleus accumbens (NAc) and attenuated METH-induced DA elevation. We suggested a mechanism in which a decline of glutamate levels in the NAc decreases extracellular DA levels. However, the hypothesis has not confirmed. METHODS: We conducted a recovery experiments by pre-microinjection of an mGluR group II antagonist, LY341495, into the NAc shell of mPFC-Shati/Nat8l-overexpressed mice followed by METH injection and DA levels measurement by in vivo microdialysis. RESULTS: Pretreatment with LY341495 was able to restore METH-induced DA increase. Furthermore, mice injected with an adeno-associated virus vector containing GFP (AAV-GFP vector) in the mPFC expressed a colocalization of GFP with DARPP-32 a medium spiny neuron (MSN) marker. Next, co-immunostaining of DARPP-32 and neuronal nitric oxide synthase (nNOS: expressed in a subtype of gamma-Aminobutyric acid (GABA interneurons) in ventral tegmental area (VTA) showed a colocalization of nNOS and DARPP-32. CONCLUSION: These results provided a proof that Shati/Nat8l attenuation of METH-induced DA increase is mediated by mGluR group II in the NAc. Moreover, immunohistochemical study showed a direct connection of mPFC projection neurons with NAc MSN and a connection of MSN projection neurons with a subtype of GABA interneurons in VTA.

Overexpression of transmembrane protein 168 in the mouse nucleus accumbens induces anxiety and sensorimotor gating deficit.

Transmembrane protein 168 (TMEM168) comprises 697 amino acid residues, including some putative transmembrane domains. It is reported that TMEM168 controls methamphetamine (METH) dependence in the nucleus accumbens (NAc) of mice. Moreover, a strong link between METH dependence-induced adaptive changes in the brain and mood disorders has been evaluated. In the present study, we investigated the effects of accumbal TMEM168 in a battery of behavioral paradigms. The adeno-associated virus (AAV) Tmem168 vector was injected into the NAc of C57BL/6J mice (NAc-TMEM mice). Subsequently, the accumbal TMEM168 mRNA was increased approximately by seven-fold when compared with the NAc-Mock mice (controls). The NAc-TMEM mice reported no change in the locomotor activity, cognitive ability, social interaction, and depression-like behaviors; however, TMEM168 overexpression enhanced anxiety in the elevated-plus maze and light/dark box test. The increased anxiety was reversed by pretreatment with the antianxiety drug diazepam (0.3 mg/kg i.p.). Moreover, the NAc-TMEM mice exhibited decreased prepulse inhibition (PPI) in the startle response test, and the induced schizophrenia-like behavior was reversed by pretreatment with the antipsychotic drug risperidone (0.01 mg/kg i.p.). Furthermore, accumbal TMEM168 overexpression decreased the basal levels of extracellular GABA in the NAc and the high K+ (100 mM)-stimulated GABA elevation; however, the total contents of GABA in the NAc remained unaffected. These results suggest that the TMEM168-regulated GABAergic neuronal system in the NAc might become a novel target while studying the etiology of anxiety and sensorimotor gating deficits.

Behavioral impairment in SHATI/NAT8L knockout mice via dysfunction of myelination development.

We have identified SHATI/NAT8L in the brain of mice treated with methamphetamine. Recently, it has been reported that SHATI is N-acetyltransferase 8-like protein (NAT8L) that produces N-acetylaspatate (NAA) from aspartate and acetyl-CoA. We have generated SHATI/NAT8L knockout (Shati -/-) mouse which demonstrates behavioral deficits that are not rescued by single NAA supplementation, although the reason for which is still not clarified. It is possible that the developmental impairment results from deletion of SHATI/NAT8L in the mouse brain, because NAA is involved in myelination through lipid synthesis in oligodendrocytes. However, it remains unclear whether SHATI/NAT8L is involved in brain development. In this study, we found that the expression of Shati/Nat8l mRNA was increased with brain development in mice, while there was a reduction in the myelin basic protein (MBP) level in the prefrontal cortex of juvenile, but not adult, Shati -/- mice. Next, we found that deletion of SHATI/NAT8L induces several behavioral deficits in mice, and that glyceryltriacetate (GTA) treatment ameliorates the behavioral impairments and normalizes the reduced protein level of MBP in juvenile Shati -/- mice. These findings suggest that SHATI/NAT8L is involved in myelination in the juvenile mouse brain via supplementation of acetate derived from NAA. Thus, reduction of SHATI/NAT8L induces developmental neuronal dysfunction.

Striatal N-Acetylaspartate Synthetase Shati/Nat8l Regulates Depression-Like Behaviors via mGluR3-Mediated Serotonergic Suppression in Mice.

Background: Several clinical studies have suggested that N-acetylaspartate and N-acetylaspartylglutamate levels in the human brain are associated with various psychiatric disorders, including major depressive disorder. We have previously identified Shati/Nat8l, an N-acetyltransferase, in the brain using an animal model of psychosis. Shati/Nat8l synthesizes N-acetylaspartate from L-aspartate and acetyl-coenzyme A. Further, N-acetylaspartate is converted into N-acetylaspartylglutamate, a neurotransmitter for metabotropic glutamate receptor 3. Methods: Because Shati/Nat8l mRNA levels were increased in the dorsal striatum of mice following the exposure to forced swimming stress, Shati/Nat8l was overexpressed in mice by the microinjection of adeno-associated virus vectors containing Shati/Nat8l gene into the dorsal striatum (dS-Shati/Nat8l mice). The dS-Shati/Nat8l mice were further assessed using behavioral and neurochemical tests. Results: The dS-Shati/Nat8l mice exhibited behavioral despair in the forced swimming and tail suspension tests and social withdrawal in the 3-chamber social interaction test. These depression-like behaviors were attenuated by the administration of a metabotropic glutamate receptor 2/3 antagonist and a selective serotonin reuptake inhibitor. Furthermore, the metabolism of N-acetylaspartate to N-acetylaspartylglutamate was decreased in the dorsal striatum of the dS-Shati/Nat8l mice. This finding corresponded with the increased expression of glutamate carboxypeptidase II, an enzyme that metabolizes N-acetylaspartylglutamate present in the extracellular space. Extracellular serotonin levels were lower in the dorsal striatum of the dS-Shati/Nat8l and normal mice that were repeatedly administered a selective glutamate carboxypeptidase II inhibitor. Conclusions: Our findings indicate that the striatal expression of N-acetylaspartate synthetase Shati/Nat8l plays a role in major depressive disorder via the metabotropic glutamate receptor 3-mediated functional control of the serotonergic neuronal system.

Involvement of the accumbal osteopontin-interacting transmembrane protein 168 in methamphetamine-induced place preference and hyperlocomotion in mice.

Chronic exposure to methamphetamine causes adaptive changes in brain, which underlie dependence symptoms. We have found that the transmembrane protein 168 (TMEM168) is overexpressed in the nucleus accumbens of mice upon repeated methamphetamine administration. Here, we firstly demonstrate the inhibitory effect of TMEM168 on methamphetamine-induced behavioral changes in mice, and attempt to elucidate the mechanism of this inhibition. We overexpressed TMEM168 in the nucleus accumbens of mice by using an adeno-associated virus vector (NAc-TMEM mice). Methamphetamine-induced hyperlocomotion and conditioned place preference were attenuated in NAc-TMEM mice. Additionally, methamphetamine-induced extracellular dopamine elevation was suppressed in the nucleus accumbens of NAc-TMEM mice. Next, we identified extracellular matrix protein osteopontin as an interacting partner of TMEM168, by conducting immunoprecipitation in cultured COS-7 cells. TMEM168 overexpression in COS-7 cells induced the enhancement of extracellular and intracellular osteopontin. Similarly, osteopontin enhancement was also observed in the nucleus accumbens of NAc-TMEM mice, in in vivo studies. Furthermore, the infusion of osteopontin proteins into the nucleus accumbens of mice was found to inhibit methamphetamine-induced hyperlocomotion and conditioned place preference. Our studies suggest that the TMEM168-regulated osteopontin system is a novel target pathway for the therapy of methamphetamine dependence, via regulating the dopaminergic function in the nucleus accumbens.

Cleavage Speed and Blastomere Number in DBA/2J Compared with C57BL/6J Mouse Embryos.

DBA/2J mice are among the oldest and most important inbred strains still used in many research fields. However, this strain has reproductive problems, which may consume considerable time and effort during experiments requiring a large population. Because the quality of DBA/2J embryos has not yet been described in detail, we compared DBA/2J mice with the reproductively efficient C57BL/6J strain. Compared with C57BL/6J embryos, DBA/2J embryos had a slower cleavage speed (mean ± 1 SD; first cleavage: C57BL/6J, 16.87 ± 1.32 ; DBA/2J, 19.64 ± 0.96 h; P < 0.01; second cleavage: C57BL/6J, 41.12 ± 2.02 h; DBA/2J, 46.20 ± 2.68 h, P < 0.01) and lower cell counts at the morula and blastocyst stages (morula stage: C57BL/6J, 15 ± 3 cells per embryo; DBA/2J, 9 ± 5 cells per embryo; P < 0.05; blastocyst stage: C57BL/6J, 52 ± 6 cells per embryo; DBA/2J, 35 ± 14 cells per embryo; P < 0.05). In addition, the results of reciprocal in vitro fertilization and male-female reciprocal crosses revealed that these phenotypes were not affected by the sperm genome and were recessively inherited. These findings likely will facilitate the production of DBA/2J mice and genetically modified mice with their background. Our results also suggest that, due to their slow cleavage speed, DBA/2J mice can serve as a new model for human infertility.