Atsushi Tsujimura

Relaxin-3-Deficient Mice Showed Slight Alteration in Anxiety-Related Behavior

Relaxin-3 is a neuropeptide belonging to the relaxin/insulin superfamily. Studies using rodents have revealed that relaxin-3 is predominantly expressed in neurons in the nucleus incertus (NI) of the pons, the axons of which project to forebrain regions including the hypothalamus. There is evidence that relaxin-3 is involved in several functions, including food intake and stress responses. In the present study, we generated relaxin-3 gene knockout (KO) mice and examined them using a range of behavioral tests of sensory/motor functions and emotion-related behaviors. The results revealed that relaxin-3 KO mice exhibited normal growth and appearance, and were generally indistinguishable from wild genotype littermates. There was no difference in bodyweight among genotypes until at least 28 weeks after birth. In addition, there were no significant differences between wild-type and KO mice in locomotor activity, social interaction, hot plate test performance, fear conditioning, depression-like behavior, and Y-maze test performance. However, in the elevated plus maze test, KO mice exhibited a robust increase in the tendency to enter open arms, although they exhibited normal performance in a light/dark transition test and showed no difference from wild-type mice in the time spent in central area in the open field test. On the other hand, a significant increase in the acoustic startle response was observed in KO mice. These results indicate that relaxin-3 is slightly involved in the anxiety-related behavior.

Characterization of Nuclear Import of the Domain-Specific Androgen Receptor in Association with the Importin α/β and Ran-Guanosine 5′-Triphosphate Systems

Androgen induces androgen receptor (AR) nuclear import, which allows AR to act as a transcriptional factor and ultimately leads to biological activity. However, the mechanism of AR translocation to the nucleus is still unclear. In the present study, we assessed the nuclear import abilities of each domain of AR and their mechanisms related to Ran and importin alpha/beta using green fluorescent protein real-time imaging. The localization of AR to the nucleus in the absence and presence of ligands was dependent upon a complex interplay of the amino terminal transactivation domain (NTD), the DNA binding domain (DBD), and the ligand binding domain (LBD). NTD and DBD showed ligand-independent nuclear import ability, whereas LBD had ligand-dependent transport. In addition, AR deletion mutant lacking DBD was distributed in the cytoplasm regardless of ligand existence, suggesting that the remaining domains, NTD and LBD, are responsible for AR cytoplasmic localization. Cotransfection with a dominant negative form of Ran dramatically inhibited the nuclear import of all AR domains, and a dominant negative form of importin alpha prevented AR and DBD import. Importin beta-knockdown strongly blocked DBD import. These results indicate that there are two additional nuclear localization signals (NLSs) in the NTD and LBD, and there are distinct pathways used to attain domain-specific AR nuclear import: the NLS of DBD is Ran and importin alpha/beta-dependent, whereas the NLSs of NTD and LBD are Ran dependent but importin alpha/beta-independent. Our data suggest that the nuclear import of AR is regulated by the interplay between each domain of the AR.

Psychological stress, but not physical stress, causes increase in diazepam binding inhibitor (DBI) mRNA expression in mouse brains

Effects of conditioned emotional stimuli (CES), which induce psychological stress, on the expression of cerebral diazepam binding inhibitor (DBI) mRNA in mouse were examined using a communication box. Cerebral DBI mRNA expression significantly increased in a time-dependent manner after the application of CES. The maximal enhancement of DBI mRNA expression was observed 2 days after the application of CES, and this increase faded out over 7 days after the treatment. Flunitrazepam (1 mg/kg), an agonist for central benzodiazepine (BZD) receptors, completely abolished the CES-induced elevation of cerebral DBI contents and its mRNA expressions. These results indicate that cerebral DBI is enhanced by psychological stress, which is regulated by central BZD receptors.

Mice lacking the kf-1 gene exhibit increased anxiety- but not despair-like behavior

KF-1 was originally identified as a protein encoded by human gene with increased expression in the cerebral cortex of a patient with Alzheimers disease. In mouse brain, kf-1 mRNA is detected predominantly in the hippocampus and cerebellum, and kf-1 gene expression is elevated also in the frontal cortex of rats after chronic antidepressant treatments. KF-1 mediates E2-dependent ubiquitination and may modulate cellular protein levels as an E3 ubiquitin ligase, though its target proteins are not yet identified. To elucidate the role of kf-1 in the central nervous system, we generated kf-1 knockout mice by gene targeting, using Cre-lox recombination. The resulting kf-1−/− mice were normal and healthy in appearance. Behavioral analyses revealed that kf-1−/− mice showed significantly increased anxiety-like behavior compared with kf-1+/+ littermates in the light/dark transition and elevated plus maze tests; however, no significant differences were observed in exploratory locomotion using the open field test or in behavioral despair using the forced swim and tail suspension tests. These observations suggest that KF-1 suppresses selectively anxiety under physiological conditions probably through modulating protein levels of its unknown target(s). Interestingly, kf-1−/− mice exhibited significantly increased prepulse inhibition, which is usually reduced in human schizophrenic patients. Thus, the kf-1−/− mice provide a novel animal model for elucidating molecular mechanisms of psychiatric diseases such as anxiety/depression, and may be useful for screening novel anxiolytic/antidepressant compounds.

Functional involvement of benzodiazepine receptors in ethanol-induced increases of diazepam binding inhibitor (DBI) and its mRNA in the mouse brain

We have attempted to clarify the mechanisms for alcohol (EtOH)-induced elevation of diazepam binding inhibitor (DBI) mRNA and to investigate whether the increase in DBI mRNA is paralleled with that in DBI using EtOH-treated mice and primary cultured neurons. Both the DBI content and the expression of DBI mRNA were elevated in the cerebral cortex of EtOH-inhaled and -withdrawn mice. Simultaneous administration of flunitrazepam (FLN) and Ro15-1788 with EtOH vapor completely abolished the EtOH-induced elevation of DBI mRNA. In addition, the exposure of the neurons for 3 days significantly elevated the expression of DBI mRNA, which was completely inhibited by concomitant exposure of FLN, Ro15-4513 and Ro-15-1788 with EtOH, while muscimol and bicuculline showed no effects on the EtOH-induced increase of DBI mRNA expression. These results indicate that functional interaction between EtOH and benzodiazepine (BDZ) receptors is a critical role in the increased expression of DBI mRNA.

Differential Expression of Alpha-Synuclein in Hippocampal Neurons

α-Synuclein is the major pathological component of synucleinopathies including Parkinsons disease and dementia with Lewy bodies. Recent studies have demonstrated that α-synuclein also plays important roles in the release of synaptic vesicles and synaptic membrane recycling in healthy neurons. However, the precise relationship between the pathogenicity and physiological functions of α-synuclein remains to be elucidated. To address this issue, we investigated the subcellular localization of α-synuclein in normal and pathological conditions using primary mouse hippocampal neuronal cultures. While some neurons expressed high levels of α-synuclein in presynaptic boutons and cell bodies, other neurons either did not or only very weakly expressed the protein. These α-synuclein-negative cells were identified as inhibitory neurons by immunostaining with specific antibodies against glutamic acid decarboxylase (GAD), parvalbumin, and somatostatin. In contrast, α-synuclein-positive synapses were colocalized with the excitatory synapse marker vesicular glutamate transporter-1. This expression profile of α-synuclein was conserved in the hippocampus in vivo. In addition, we found that while presynaptic α-synuclein colocalizes with synapsin, a marker of presynaptic vesicles, it is not essential for activity-dependent membrane recycling induced by high potassium treatment. Exogenous supply of preformed fibrils generated by recombinant α-synuclein was shown to promote the formation of Lewy body (LB) -like intracellular aggregates involving endogenous α-synuclein. GAD-positive neurons did not form LB-like aggregates following treatment with preformed fibrils, however, exogenous expression of human α-synuclein allowed intracellular aggregate formation in these cells. These results suggest the presence of a different mechanism for regulation of the expression of α-synuclein between excitatory and inhibitory neurons. Furthermore, α-synuclein expression levels may determine the efficiency of intracellular aggregate formation in different neuronal subtypes.

Ethanol physical dependence is accompanied by up-regulated expression of L-type high voltage-gated calcium channel α1 subunits in mouse brain

We investigated how functional changes in high voltage-gated calcium channels (HVCCs) occurred in the cerebral cortex of mouse with ethanol physical dependence. The continuous treatment of mice with ethanol vapor for 8 days significantly increased the expressions of alpha1C, alpha1D, and alpha2/delta1 subunits of L-type HVCCs in association with decreased level of beta4 subunit of L-type HVCCs, although alpha1A and alpha1B subunits of P/Q- and N-type HVCCs, respectively, showed no alterations. [(3)H]Diltiazem binding to the particulate fractions increased with increased Bmax value and no changes of Kd value. These results indicate that ethanol physical dependence accompanies the up-regulation of L- type HVCCs, which is due to increased expression of alpha1C, alpha1D, and alpha2/delta1 subunits.

Ethanol stimulates diazepam binding inhibitor (DBI) mRNA expression in primary cultured neurons.

Changes in expression of diazepam binding inhibitor (DBI) mRNA in cerebral cortical neurons following long-term ethanol (EtOH) exposure were examined. A significant increase in DBI mRNA expression was observed by the exposure of neurons to 50 mM EtOH for up to 5 days and to EtOH (1-100 mM) for 3 days. These EtOH-induced increases in DBI mRNA expression were further elevated after the additional cultivation of neurons under EtOH-free condition. beta-Actin mRNA expression was not altered by similar EtOH treatments. These results indicate that EtOH possesses the activity to increase the expression of DBI mRNA in cerebral cortical neurons.

Nicotine increases diazepam binding inhibitor (DBI) mRNA in primary cultured neurons

The effect of nicotine on the expression of diazepam binding inhibitor (DBI) mRNA in primary cultured cerebral cortical neurons was examined using Northern blot analysis. Nicotine exposure (0.001-10 microM) for 24 h increased the DBI mRNA level in a dose-dependent manner, whereas the beta-actin mRNA level showed no change. This effect of nicotine was faded out over 48 h of its exposure. Hexamethonium (100 microM) completely abolished the nicotine-induced increase in DBI mRNA expression. These results indicate that nicotine increases the expression of DBI mRNA in cerebral cortical neurons via the activation of nicotinic acetylcholine receptor.

Up‐regulation of L‐type high voltage‐gated calcium channel subunits by sustained exposure to 1,4‐ and 1,5‐benzodiazepines in cerebrocortical neurons

The aim of this study is to examine how sustained exposure to two 1,4‐benzodiazepines (BZDs) with different action period, diazepam and brotizolam, and a 1,5‐BZD, clobazam, affects L‐type high voltage‐gated calcium channel (HVCC) functions and its mechanisms using primary cultures of mouse cerebral cortical neurons. The sustained exposure to these three BZDs increased [45Ca2+] influx, which was due to the enhanced [45Ca2+] entry through L‐type HVCCs but not through of Cav2.1 and Cav2.2. Increase in [3H]diltiazem binding after the exposure to these three BZDs was due to the increase in the binding sites of [3H]diltiazem. Western blot analysis showed increase of Cav1.2 and Cav1.3 in association with the increased expression of α2/δ1 subunit. Similar changes in [3H]diltiazem binding and L‐type HVCC subunit expression were found in the cerebral cortex from mouse with BZD physical dependence. These results indicate that BZDs examined here have the potential to increase L‐type HVCC functions mediated via the enhanced expression of not only Cav1.2 and Cav1.3 but also α2/δ1 subunit after their sustained exposure, which may participate in the development of physical dependence by these BZDs.