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Females of various species exhibit mounting behavior toward other mature females, i.e., female-female (f-f) mounting. Cows exhibit f-f mounting, which is commonly considered a sexual behavior based on a few similarities with male sexual mounting. Here, we hypothesized that the degree of f-f mounting exhibited by cows in estrus would increase when they are sexually motivated by sexually relevant stimuli. To test this hypothesis, two groups of four cows each were prepared. First, estrus was induced in one of the cows in each group by injecting estradiol in the presence of a non-sexually active male steer as a stimulus animal (steer-stimulus condition), and all f-f mounts were recorded. Estrus induction was repeated until induced in all cows. We then changed the stimulus animal from the steer to a sexually active bull (bull-stimulus condition), and the estrus induction procedures were repeated as before. We confirmed that estrus was induced successfully in cows injected with estradiol and that they were sexually motivated by switching the stimulus animal from steer to bull, as they spent relatively more time with the stimulus animal in the bull-stimulus than in the steer-stimulus condition and when not in estrus. However, f-f mounting frequency in the bull-stimulus condition showed no significant difference to that in the steer-stimulus condition. We observed that six of the eight subjects exhibited f-f mounting when not in estrus, which accounted for 44.6 % of f-f mounting we observed (n = 668). These findings suggest that f-f mounting in cows is not associated with sexual motivation., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Stress, a risk factor for major depressive disorder and Alzheimer disease, leads to the release of high-mobility group box-1 (HMGB1) protein, which in turn causes neuroinflammation. The mechanism underlying stress-induced HMGB1 release is unknown, but stress-associated glucocorticoids could be involved. Primary cultured rat cortical microglia and neurons were treated with corticosterone, a stress-associated glucocorticoid, and HMGB1 release was measured by ELISA and western blotting to test this hypothesis. With corticosterone treatment, significant HMGB1 was released in microglia but not in neuronal cell cultures. HMGB1 mRNA expression and HMGB1 protein expression in microglia were not affected by corticosterone treatment. Thus, the source of extracellular HMGB1 released into the medium is likely to be existing nuclear HMGB1 rather than newly synthesized HMGB1. Corticosterone-induced HMGB1 release in microglia culture was significantly attenuated by blocking glucocorticoid receptors but not mineralocorticoid receptors. Dexamethasone, a selective glucocorticoid receptor agonist, and dexamethasone-bovine serum albumin (BSA), a membrane-impermeable glucocorticoid receptor agonist used to confirm the membrane receptor-mediated effects of glucocorticoids, increased the release of HMGB1. Immunocytochemistry showed that HMGB1 translocated from the nucleus to the cytoplasm following dexamethasone or dexamethasone-BSA treatment through glucocorticoid receptors. The present findings suggest that glucocorticoids stimulate microglial membrane glucocorticoid receptors and trigger cytoplasmic translocation and extracellular release of nuclear HMGB1. Thus, under stress conditions, glucocorticoids induce microglial HMGB1 release, leading to a neuroinflammatory state that could mediate neurological disorders., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Takahisa Shimoda is working in Laboratory for Pharmacology, Pharmaceuticals Research Center, Asahi Kasei Pharma Co. Ltd.. This study was conducted fully independent of the project in the Asahi Kasei Pharma Co., Ltd. The other authors declare no conflict of interest., (Copyright © 2024 International Brain Research Organization (IBRO). Published by Elsevier Inc. All rights reserved.)

The brown rat (Rattus norvegicus) is known to show three types of behavioral responses to novel objects. Whereas some rats are indifferent to novel objects, neophobic and neophilic rats show avoidance and approach behavior, respectively. Here, we compared the dopaminergic, serotonergic, and noradrenergic systems immunohistochemically among these rats. Trapped wild rats and laboratory rats were first individually exposed to the novel objects in their home cage. Wild rats were divided into neophobic and indifferent rats depending on their behavioral responses. Similarly, laboratory rats were divided into neophilic and indifferent rats. Consistent with the behavioral differences, in the paraventricular nucleus of the hypothalamus, Fos expression in corticotropin-releasing hormone-containing neurons was higher in the neophobic rats than in the indifferent rats. In the anterior basal amygdala, the neophobic rats showed higher Fos expression than the indifferent rats. In the posterior basal amygdala, the neophobic and neophilic rats showed lower and higher Fos expressions than the indifferent rats, respectively. When we compared the neuromodulatory systems, in the dorsal raphe, the number of serotonergic neurons and Fos expression in serotonergic neurons increased linearly from neophobic to indifferent to neophilic rats. In the ventral tegmental area, Fos expression in dopaminergic neurons was higher in the neophilic rats than in the indifferent rats. These results demonstrate that approach/avoidance behavior to novel objects is correlated with the serotonergic and dopaminergic systems in the brown rat. We propose that the serotonergic system suppresses avoidance behavior while the dopaminergic system enhances approach behavior to novel objects., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Social buffering is a phenomenon where stress responses are ameliorated by an affiliative conspecific. Our previous findings suggest that the posterior complex of the anterior olfactory nucleus (AOP) is well positioned to participate in the neural mechanisms underlying social buffering. However, the lack of anatomical information prevents us from further estimating the role of the AOP. Here, we obtained anatomical information regarding the AOP in male rats. In Experiment 1 (n = 5), among 4',6-diamidino-2-phenylindole-positive cells in the AOP, the proportion of glutamic acid decarboxylase 67 (GAD67)-positive cells was 13.8% ± 1.2%. In Experiment 2 (n = 5), among the cells that were labeled by a retrograde tracer injected into the basolateral complex of the amygdala (BLA), the proportion of GAD67-positive cells was 18.6% ± 0.8%. In Experiment 3 (n = 5), we demonstrated the existence of cells that were labeled by the retrograde tracer injected into the posterior part of the medial amygdala (MeP), mostly into the ventral part of the MeP. In addition, the proportion of GAD67-positive cells among the tracer-labeled cells was 21.7% ± 1.7%. In Experiment 4 (n = 3), the retrograde tracers were injected into the BLA and MeP, mostly into the ventral part of the MeP. The proportion of double-labeled cells among the tracer-labeled cells was 2.1% ± 1.2%. Taken together, these results suggest that the AOP is predominantly composed of glutamatergic neurons. In addition, the AOP sends mutually independent glutamatergic-predominant projections to the BLA and MeP., (Copyright © 2023 IBRO. Published by Elsevier Ltd. All rights reserved.)

In rats, a mixture of hexanal and 4-methylpentanal is a main component of the alarm pheromone. When detected by the main olfactory system (MOS) and the vomeronasal system, respectively, they activate the anterior part of the bed nucleus of the stria terminalis (BNSTa). Therefore, the information from the two olfactory systems is expected to be integrated before being transmitted to the BNSTa. To specify the integration site, we examined Fos expression in 16 brain regions in response to water (n = 10), hexanal (n = 9), 4-methylpentanal (n = 9), the mixture (n = 9), or the alarm pheromone (n = 9) in male rats. The posteroventral part of the medial amygdala showed increased Fos expression to hexanal and 4-methylpentanal. The expression was further increased by the mixture. Therefore, this region is suggested as the integration site. In addition, the BNSTa, paraventricular nucleus of the hypothalamus, and anteroventral, anterodorsal, and posterodorsal parts of the medial amygdala were suggested to be located downstream of the integrated site because only the mixture increased Fos expression. We suggest that the posterolateral part of the cortical amygdala is upstream of the integration site in the MOS because all stimuli increased Fos expression. The posterior part of the bed nucleus of the stria terminalis and posteromedial part of the cortical amygdala were suggested as being located upstream in the vomeronasal system because 4-methylpentanal and the mixture increased Fos expression. These results provide information about the neural pathway underlying the alarm pheromone effects., (Copyright © 2023 IBRO. Published by Elsevier Ltd. All rights reserved.)

Introduction: The safety profiles of COVID-19 vaccines are incompletely evaluated in Japan., Objectives: To examine the risk of serious adverse effects after COVID-19 mRNA vaccination (BNT162b2 and mRNA-1273) in cohort studies and self-controlled case series (SCCS)., Methods: Using an administrative claims database linked with the COVID-19 vaccination registry in a city in Japan between September 2020 and September 2021, we identified health insurance enrolees aged ≥ 18 years. We evaluated the risk of acute myocardial infarction, appendicitis, Bell's palsy, convulsions/seizures, disseminated intravascular coagulation, immune thrombocytopenia, pulmonary embolism, haemorrhagic or ischemic stroke, venous thromboembolism, and all-cause mortality, 21 days following any COVID-19 mRNA vaccination, compared with non-vaccination periods. For the cohort studies, we estimated incidence rate ratios (IRRs) by Poisson regression and rate differences (IRDs) by weighted least-squares regression, adjusting for sex, age, and Charlson comorbidity index. We applied a modified SCCS design to appropriately treat outcome-dependent exposures. For the modified SCCS, we estimated within-subject IRRs by weighted conditional Poisson regression. Subgroup analyses stratified by sex and age were also conducted., Results: We identified 184,491 enrolees [male: 87,218; mean (standard deviation) age: 64.2 (19.5) years] with 136,667 first and 127,322 s dose vaccinations. The risks of any outcomes did not increase in any analyses, except for the fact that the modified SCCS indicated an increased risk of pulmonary embolism after the first dose in women (within-subject IRR [95%CI]: 3.97 [1.18-13.32])., Conclusion: The findings suggested that the COVID-19 mRNA vaccine was generally safe, whilst a signal of pulmonary embolism following the first dose of the COVID-19 mRNA vaccine was observed., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: YT has received consultant fees from Pharmaceuticals and Medical Devices Agency and EPARK, Inc. YT has received lecture fees from SAS Institute Japan Ltd. YT has been conducting a collaborative study, which is not related to this article, with Pfizer Inc. SO is a member of the Department of Eat-loss Medicine, a cooperative program between the University of Tokyo and ITO EN Ltd and received grants from KAKENHI, and Health, Labour, and Welfare Policy Research Grants, not related to the submitted work. No other potential competing interests relevant to this study are reported., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: A kinematic coupling relationship exists between foot joints during gait. In individuals with hallux valgus, forefoot or hallux kinematics may be affected by adjacent or nonadjacent joint motion. Thus, this study aimed to investigate the foot joint coordination pattern and variability during gait in young females with hallux valgus., Methods: Twenty-five young females with hallux valgus and 25 healthy young females without hallux valgus were enrolled. Reflective markers were attached according to a multisegment foot model. Kinematic data were obtained using a three-dimensional motion analysis system. Joint angles between distal and proximal segments were calculated using analysis software. Foot joint coordination pattern and variability were assessed using a vector-coding technique., Findings: Individuals with hallux valgus had a larger rearfoot relative to shank eversion and forefoot relative to midfoot dorsiflexion during terminal stance and pre-swing compared with those without hallux valgus. There were no significant differences in coordination patterns, but the consistency of coordination between the rearfoot relative to shank motion in the frontal plane and forefoot relative to midfoot motion in the sagittal plane during terminal stance was greater in the hallux valgus group than in the control group., Interpretation: The soft tissue composing the first ray might suffer from more severe stress due to the large motion that occurred with low variability in individuals with hallux valgus. This finding may suggest that the altered kinematics and coordination variability in foot joints are related to hallux valgus biomechanical etiology., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Most broadcast spawner corals have a vitellogenic phase that lasts at least 6 months. It is established that estrogen regulates vitellogenin synthesis in vertebrates. Although some research have been conducted on the physiological role of sex steroids in corals, little is known about their involvement in oocyte development. This study aimed to detect steroid hormones - progesterone, testosterone, and estradiol-17β (E2) - in Acropora tenuis and study the relationships between vitellogenesis/vitellogenin synthesis and these steroids. This study also investigated the effect of E2 on vitellogenin synthesis in corals and identified steroidogenic enzymes in A. tenuis genome. Branches from tagged coral colonies were collected monthly from March to November. Histological observations showed that oocytes were vitellogenic from March to May (Stage IV and V), but not in June, and that gonads were occupied by immature oocytes in September (Stage I). Real-time qPCR revealed that vitellogenin (vg1 and vg2) transcript levels in coral branches were high in April and May, implying that corals actively underwent vitellogenesis during these months, and spawned before June. Liquid chromatography-mass spectrometry revealed that E2 could be detected in coral branches in March, April, and May, but not in June, whereas testosterone and progesterone did not fluctuate much in the same months. Immersing branches in E2-containing seawater failed to increase vitellogenin transcript levels. The results indicate that E2 is involved in oogenesis but does not positively regulate vitellogenin synthesis. Steroidogenic enzymes (except CYP19A) were identified in A. tenuis, suggesting that corals may endogenously synthesize progestogens and androgens from cholesterol., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Erypoegin K, an isoflavone isolated from the stem bark of Erythrina poeppigiana, has potent apoptosis-inducing effect on human leukemia HL-60 cells. Erypoegin K has a chiral carbon at the C-2'' position of its furan ring and naturally occurs as a racemic mixture of (S)- and (R)-isomers. In the present study, we semi-synthesized (RS)-erypoegin K from genistein and separated the optical isomers by HPLC using a chiral column to characterize its apoptosis-inducing activity. Apoptotic cell death was assessed by analyzing caspase-3 and caspase-9 activation, nuclear fragmentation, and genomic DNA ladder formation. (S)-erypoegin K showed exclusive anti-proliferative and apoptosis-inducing activity, with an IC 50 value of 90 nM, about 50% lower than that of its racemic mixture (175 nM). By contrast, no apoptosis-inducing activity was shown by the (R)-isomer. In addition, methylglyoxal accumulation in the culture medium was observed only in cells treated with (S)-erypoegin K. These results demonstrated that (S)-erypoegin K is a unique bioactive component that has potent apoptosis-inducing activity on HL-60 cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The performance of transmission Raman spectroscopy (TRS) for quantifying a cocrystal and its dissociation in solid dosage form was investigated. Some tablets containing 0%-20% (w/w) of a cocrystal of carbamazepine (CBZ)/succinic acid (SUC), 0%-4% of CBZ, 0%-4% of SUC, and 75%-99% of D-mannitol were prepared. The Raman spectra of these tablets were preprocessed using the standard normal variate (SNV) or multiplicative scatter correction (MSC) as well as the Savitzky Golay second derivative, and then, these were used to generate calibration models using partial least squares (PLS) regression. The performance of the model was superior when the MSC preprocessing spectra of the cocrystal between 200 and 1800 cm -1 were used for calibration. The determination coefficient of the PLS calibration curve for the CBZ/SUC cocrystal between 200 and 1800 cm -1 with MSC was 0.97, root mean square error of cross validation (RMSECV) was 1.16, and root mean square error of prediction (RMSEP) was 1.10. As in the case of the CBZ/SUC cocrystal, the performance of the model was superior when the MSC preprocessing spectra of CBZ and SUC between 200 and 1800 cm -1 were used for calibration. These data suggest that TRS is useful for quantifying a cocrystal and its dissociation compounds in solid dosage forms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Social buffering is a phenomenon in which the presence of an affiliative conspecific (associate) mitigates stress responses in a subject. We assessed the relationship between the stress level of subjects and the benefit of social buffering. In Experiment 1, subjects fear-conditioned using 0.15-mA, 0.45-mA, or 0.70-mA foot shocks were re-exposed to a conditioned stimulus (CS) either alone or with an associate on the day following fear conditioning. We found that behavioral responses were reduced by the presence of an associate. The intensity of this decrease was similar among all subjects. These results suggest that the intensity of social buffering was similar regardless of the stress level of the subject. The high stress subjects showed residual stress responses after receiving social buffering, indicating that the residual stress responses may have been resistant to social buffering. To further examine this, subjects fear-conditioned using 0.70-mA foot shocks were re-exposed to the CS either alone, with one associate, or with three associates in Experiment 2. We found that behavioral responses decreased as the number of associates increased. These results suggest that residual stress responses are further ameliorated when the number of associates increases. Therefore, the residual stress responses were also sensitive to buffering. Taken together, our data indicate that the benefits of social buffering are maintained regardless of the stress level of the subject rat and enhanced by more conspecifics., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mycobacteriosis and nocardiosis in cultured fish caused by infections with acid-fast bacteria, are responsible for large economic losses globally. In this study, we suggest a novel adjuvant using glycolipids that activates host immune systems. The immune response to glycolipids stimulation was investigated using ginbuna crucian carp. Ginbuna vaccinated with FKC (formalin-killed cells) + glycolipids isolated from Mycobacterium sp., upregulated inflammatory- and Th1-related cytokines, and a DTH (delayed-type hypersensitivity) response was confirmed only in ginbuna vaccinated with FKC + glycolipids. These observations suggest that glycolipids activated host innate and cell-mediated immunity. Subsequently, we evaluated the adjuvant effect of glycolipids against amberjack nocardiosis. In a challenge test, a higher survival rate was observed in amberjack vaccinated with FKC + glycolipids emulsified with conventional oil adjuvant than in fish vaccinated with FKC + oil adjuvant without glycolipids. Therefore, glycolipids potentially could be used as a practical, economical and safe adjuvant for aquaculture fish., (Copyright © 2018. Published by Elsevier Ltd.)

4-Coumaroyl-CoA ligase (4CL) is ubiquitous in the plant kingdom, and plays a central role in the biosynthesis of phenylpropanoids such as lignins, flavonoids, and coumarins. 4CL catalyzes the formation of the coenzyme A thioester of cinnamates such as 4-coumaric, caffeic, and ferulic acids, and the regulatory position of 4CL in the phenylpropanoid pathway renders the enzyme an attractive target that controls the composition of phenylpropanoids in plants. In this study, we designed and synthesized mechanism-based inhibitors for 4CL in order to develop useful tools for the investigation of physiological functions of 4CL and chemical agents that modulate plant growth with the ultimate goal to produce plant biomass that exhibits features that are beneficial to humans. The acylsulfamide backbone of the inhibitors in this study was adopted as a mimic of the acyladenylate intermediates in the catalytic reaction of 4CL. These acylsulfamide inhibitors and the important synthetic intermediates were fully characterized using two-dimensional NMR spectroscopy. Five 4CL proteins with distinct substrate specificity from four plant species, i.e., Arabidopsis thaliana, Glycine max (soybean), Populus trichocarpa (poplar), and Petunia hybrida (petunia), were used to evaluate the inhibitory activity, and the half-maximum inhibitory concentration (IC 50 ) of each acylsulfamide in the presence of 4-coumaric acid (100 µM) was determined as an index of inhibitory activity. The synthetic acylsulfamides used in this study inhibited the 4CLs with IC 50 values ranging from 0.10 to 722 µM, and the IC 50 values of the most potent inhibitors for each 4CL were 0.10-2.4 µM. The structure-activity relationship observed in this study revealed that both the presence and the structure of the acyl group of the synthetic inhibitors strongly affect the inhibitory activity, and indicates that 4CL recognizes the acylsulfamide inhibitors as acyladenylate mimics., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Little is known about mechanisms of resistance to parasitic diseases in marine finfish. Benedenia disease is caused by infection by the monogenean parasite Benedenia seriolae. Previous quantitative trait locus (QTL) analyses have identified a major QTL associated with resistance to Benedenia disease in linkage group Squ2 of the Japanese yellowtail/amberjack Seriola quinqueradiata. To uncover the bioregulatory mechanism of Benedenia disease resistance, complete Illumina sequencing of BAC clones carrying genomic DNA for the QTL region in linkage group Squ2 was performed to reveal a novel C-type lectin in this region. Expression of the mRNA of this C-type lectin was detected in skin tissue parasitized by B. seriolae. Scanning for single nucleotide polymorphisms (SNPs) uncovered a SNP in the C-type lectin/C-type lectin-like domain that was significantly associated with B. seriolae infection levels. These results strongly suggest that the novel C-type lectin gene controls resistance to Benedenia disease in Japanese yellowtails., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Social buffering is a phenomenon in which stress in an animal is ameliorated when the subject is accompanied by a conspecific animal(s) during exposure to distressing stimuli. Previous studies of social buffering of conditioned fear responses in rats have typically used a 3-s auditory conditioned stimulus (CS) as a stressor, observing stress responses during a specified experimental period. Because a 3-s CS is extremely short compared with a typical experimental period, freezing has thus been observed primarily in the absence of the CS. Therefore, it has been unclear whether social buffering ameliorates conditioned fear responses in the presence of the CS. To clarify this issue, the current study assessed the effects of social buffering on conditioned fear responses in the presence of a 20-s CS. We measured the percentage of time spent freezing during the 20-s period following the onset of the CS. When conditioned subjects were exposed to the 20-s CS alone, they exhibited a high percentage of freezing in the presence of the CS. The presence of another non-conditioned rat completely blocked this response. The same result was observed when freezing was observed primarily in the absence of the 3-s CS. In addition, we confirmed that the presence of an associate ameliorated conditioned fear responses induced by a 20-s CS or 3-s CS when the duration and frequency of fear responses was measured. These findings indicate that social buffering ameliorates conditioned fear responses in the presence of an auditory CS., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The kisspeptin receptor (GPR54) mediates neuroendocrine control of kisspeptin in the brain and acts as a gateway for a pulsatile release of hypothalamic gonadotropin-releasing hormone. This study aimed to clone two GPR54 genes (gpr54-1 and gpr54-2) from the brain of the sapphire devil Chrysiptera cyanea, a tropical damselfish, and to study their involvement in reproduction. The partial sequences of the sapphire devil gpr54-1 cDNA (1059bp) and gpr54-2 cDNA (1098bp) each had an open reading frame encoding a protein of 353 and 366 amino acids, respectively, both of which had structural features of a G-protein-coupled receptor. The expression of gpr54-1 mRNA was observed in the diencephalon and telencephalon, and gpr54-2 mRNA was found in the optic tectum of sapphire devil. When gpr54-1 and gpr54-2 mRNA levels were examined in the brain of sapphire devil by real-time quantitative polymerase chain reaction (qPCR), they were found to increase during late vitellogenesis and post-spawning. Treatment of fish with estradiol-17β (Ε2) resulted in an increase in gpr54-1 and gpr54-2 expression in the brain of sapphire devil. Thus, kisspeptin receptors likely mediate the activity of kisspeptin in the brain and are involved in controlling reproductive events in a tropical damselfish., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In social species, the phenomenon in which the presence of conspecific animals mitigates stress responses is called social buffering. We previously reported that social buffering in male rats ameliorated behavioral fear responses, as well as hypothalamic-pituitary-adrenal axis activation, elicited by an auditory conditioned stimulus (CS). However, after social buffering, it is not clear whether rats exhibit fear responses when they are re-exposed to the same CS in the absence of another rat. In the present study, we addressed this issue using an experimental model of extinction. High stress levels during extinction training impaired extinction, suggesting that extinction is enhanced when stress levels during extinction training are low. Therefore, we hypothesized that rats that had received social buffering during extinction training would not show fear responses to a CS, even in the absence of another rat, because social buffering had enhanced the extinction of conditioned fear responses. To test this, we subjected male fear-conditioned rats to extinction training either alone or with a non-conditioned male rat. The subjects were then individually re-exposed to the CS in a recall test. When the subjects individually underwent extinction training, no responses were suppressed in the recall test. Conversely, when the subjects received social buffering during extinction training, freezing and Fos expression in the paraventricular nucleus of the hypothalamus and lateral amygdala were suppressed. Additionally, the effects of social buffering were absent when the recall test was conducted in a different context from the extinction training. The present results suggest that social buffering enhances extinction of conditioned fear responses., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In social animals, recovery from the adverse effects of distressing stimuli is promoted by subsequent cohousing with a conspecific animal(s). This phenomenon has been termed housing-type social buffering. We previously found that social housing induced housing-type social buffering in fear-conditioned male rats. This buffering took the form of attenuated conditioned hyperthermia in response to an auditory conditioned stimulus (CS). Here, we assessed whether this social buffering is sustained even if the subject is housed alone after a period of social housing. When fear-conditioned subjects were housed alone during a 48-h period between conditioning and re-exposure to the auditory CS, they exhibited conditioned hyperthermia in response to the CS. However, conditioned hyperthermia was not observed when the 12-h period of social housing began 24 and 36h after conditioning during the 48-h period. This was not the case when the 12-h period of social housing began 0 and 12h after the conditioning. These results suggest that housing-type social buffering is sustained for 12h after the 12-h period of social housing. We next considered whether increasing the duration of social housing would extend the period of social buffering. We observed social buffering of conditioned hyperthermia 24 and 48, but not 96h after a 24-h period of social housing. These results suggest that social buffering was extended when the duration of social housing was increased. Taken together, our findings indicate that housing-type social buffering is sustained after a period of social housing., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Rats are known to emit a series of ultrasonic vocalizations, termed 22-kHz calls, when exposed to distressing stimuli. Pharmacological studies have indicated that anxiety mediates 22-kHz calls in distressed rats. We previously found that exposure to the rat alarm pheromone increases anxiety in rats. Therefore, we hypothesized that the alarm pheromone would increase 22-kHz calls in pheromone-exposed rats. Accordingly, we tested whether exposure to the alarm pheromone induced 22-kHz calls, as well as whether the alarm pheromone increased 22-kHz calls in response to an aversive conditioned stimulus (CS). Rats were first fear-conditioned to an auditory and contextual CS. On the following day, the rats were either exposed to the alarm pheromone or a control odor that was released from the neck region of odor-donor rats. Then, the rats were re-exposed to the aversive CS. The alarm pheromone neither induced 22-kHz calls nor increased 22-kHz calls in response to the aversive CS. In contrast, the control odor unexpectedly reduced the total number and duration of 22-kHz calls elicited by the aversive CS, as well as the duration of freezing. These results suggest that the alarm pheromone does not affect 22-kHz calls in rats. However, we may have found evidence for an appeasing olfactory signal, released from the neck region of odor-donor rats., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The sapphire devil, Chrysiptera cyanea, is a reef-associated damselfish and their ovarian development can be induced by a long photoperiod. In this study, we demonstrated the existence of a photoinducible phase for the photoperiodic ovarian development in the sapphire devil. Induction of ovarian development under night-interruption light schedules and Nanda-Hamner cycles revealed that the photoinducible phase appeared in a circadian manner between ZT12 and ZT13. To characterize the effect of photoperiod on clock gene expression in the brain of this species, we determined the expression levels of the sdPer1, sdPer2, sdCry1, and sdCry2 clock genes under constant light and dark conditions (LL and DD) and photoperiodic (short and long photoperiods). The expression of sdPer1 exhibited clear circadian oscillation under both LL and DD conditions, while sdPer2 and sdCry1 expression levels were lower under DD than under LL conditions and sdCry2 expression was lower under LL than under DD conditions. These results suggest a key role for sdPer1 in circadian clock cycling and that sdPer2, sdCry1, and sdCry2 are light-responsive clock genes in the sapphire devil. After 1 week under a long photoperiod, we observed photoperiod-related changes in sdPer1, sdPer2, and sdCry2 expression, but not in sdCry1 expression. These results suggest that the expression patterns of some clock genes exhibit seasonal variation according to seasonal changes in day length and that such seasonal alteration of clock gene expression may contribute to seasonal recognition by the sapphire devil., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Using fear-conditioning model, we have used a 3-s auditory conditioned stimulus (CS) as a stressor and observed fear and stress responses during a specific experimental period regardless of the presence or absence of the CS. Because the CS was extremely short compared with the experimental period, we observed responses primarily in the absence of the CS. In contrast, most studies in the literature have analyzed responses in the presence of the CS. Therefore, the characteristics of fear and stress responses in the absence of the CS remain to be clarified. To clarify this, we compared the characteristics of fear and stress responses elicited by a 3-s auditory CS with those observed during a 20-s auditory CS. The basolateral complex of the amygdala (BLA), but not the bed nucleus of the stria terminalis (BNST), participated in the fear response elicited by the 3-s CS, whereas both the BLA and BNST were involved in the response observed during the 20-s CS. Additional analyses revealed that the BNST participated in the fear response during the 20-s CS when the CS was paired with a 0.75-mA, but not with a 0.9-mA, foot shock, and to the contextual CS. In addition, the fear response elicited by the 3-s CS was more resistant to extinction than that during the 20-s CS. Finally, the 3-s CS produced more intense freezing and corticosterone secretion than the 20-s CS. On the basis of these characteristics, we conclude that the 3-s auditory CS is a more effective stressor than the 20-s auditory CS. Our findings also suggest that foot shock intensity is an additional determinant in the type of fear response induced by the CS., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Nonsteroidal progesterone receptor (PR) full antagonists are needed as tools for elucidating the physiological functions of PR and as candidates for treatment of various diseases. We designed and synthesized 1,3-diphenyladamantane derivatives, and investigated their PR-antagonistic activity in comparison with our recently developed boron cluster-based PR antagonists. Among the synthesized adamantane derivatives, compound 9a exhibited the most potent PR-antagonistic activity (IC50: 25nM) and showed high binding affinity for the PR ligand-binding domain, comparable with that of the boron cluster-based PR antagonists. These results suggest that disubstituted adamantane, like the boron cluster m-carborane, is a promising hydrophobic pharmacophore for further structural development of nonsteroidal PR antagonists., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: Aldehyde dehydrogenase 2 (ALDH2) degrades acetaldehyde produced by the metabolism of alcohol. The inactive ALDH2 phenotype is prevalent in East Asians, and an association between this ALDH2 polymorphism and osteoporosis has been reported. In our previous study, we found that alcohol consumption results in decreased trabecular bone volume in aldh2 knockout (aldh2(-/-)) mice compared with the volume in wild-type (aldh2(+/+)) mice. However, the effect of aldh2 gene on the skeletal phenotype in the absence of alcohol consumption remains unknown. The aim of this study was to clarify the effect of aldh2 disruption on femoral bone structure and dynamics in aldh2-disrupted mice in the absence of alcohol consumption., Materials and Methods: We examined aldh2(-/-) and aldh2(+/+) mice at the ages of 4, 8 and 12weeks. The femoral bone length and bone mineral density (BMD) were measured using peripheral quantitative computed tomography. The mechanical strength was assessed by the three-point bending test at 12weeks, and cortical bone histomorphometry at the femur diaphysis was performed at all three time points. Osteogenic activities in aldh2(-/-) and aldh2(+/+) mice were assessed by osteoblast culture from calvariae of the neonatal mice. Bilateral femoral and tibial bones containing no bone marrow cells of 8-week-old mice were used for analysis of mRNA expression. In addition, mRNA expression in aldh2(-/-) and aldh2(+/+) mice after tail suspension or climbing exercise for 7days from 8weeks was analyzed to clarify the response to mechanical loading., Results: At 12weeks, there were no significant differences in femoral bone length, trabecular BMD in the distal metaphyses of the femurs, or mechanical strength between aldh2(-/-) and aldh2(+/)(+) mice, whereas cortical BMD and cortical thickness were significantly increased and cross-sectional area and bone marrow area were significantly decreased in the femoral diaphysis of aldh2(-/-) mice relative to the corresponding values in aldh2(+/+) mice. At 8weeks, bone formation rate and mineral apposition rate on the periosteal and endocortical surfaces were significantly increased in aldh2(-/-) mice relative to the rates in aldh(+/+) mice. Calvarial osteoblast culture study revealed that the percentage of alkaline phosphatase stained cells was significantly higher in aldh2(-/-) mice compared to that in aldh(+/+) mice. Quantitative real-time RT-PCR revealed a significant increase in the expressions of bmp2, osterix, runx2, and col1a1 mRNA in aldh2(-/-) mice, along with an increase in the expression of wnt5a mRNA and the lrp5/sost mRNA ratio. The mRNA expressions of bmp2, osterix, runx2 and pthr in aldh2(-/-) mice were significantly decreased after climbing exercise compared to those in the control, although the mRNA expressions of bmp2, osterix, runx2 were not significantly decreased and pthr mRNA expression was increased in aldh(+/+) mice after climbing exercise., Conclusion: Our results show that disruption of aldh2 gene resulted in altered cortical bone structure and dynamics in mice. Cross-sectional area was decreased. Cortical BMD was increased owing to the promotion of cortical bone formation on the periosteal and endocortical surfaces of the femoral diaphysis. The possible mechanisms underlying altered cortical bone structure in aldh2(-/-) mice were gene-related higher osteogenic activity of osteoblasts and weakened osteogenice response to mechanical loading in growth period., (Copyright © 2013 Elsevier Inc. All rights reserved.)

It is known that the size of the components of the sound production apparatus in mammals may affect the acoustic structure of vocalizations. Therefore, some acoustic variables such as voice frequency may change with age in association with body size and body weight increases. However, whether this relationship also applies to ultrasonic vocalizations emitted by laboratory rats has not been investigated. Thus, in the present study, we first recorded changes in three acoustic variables (mean frequency, duration, and bandwidth) of air puff-induced 22-kHz calls in male rats during their growth period and assessed the relationship between these changes and body weight gain (Experiment 1). Then we directly recorded several body size measures including components of the sound production apparatus in 6- and 12-week-old male rats and examined the correlation between these values and the acoustic variables of 22-kHz calls (Experiment 2). In Experiment 1, the mean frequency of 22-kHz calls in male rats during the growth period showed negative correlations with body weight gain, while the duration of 22-kHz calls showed positive correlations. In Experiment 2, only a close negative correlation between the mean frequency of 22-kHz calls and vocal tract length in male rats was found. These results suggest that the age-related decrease in the mean frequency of 22-kHz calls may be ascribed to anatomical elongation of the vocal tract length in association with the growth of male rats. These acoustic differences could inform the receivers about the age of the signaler., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Ovarian development of the sapphire devil, Chrysiptera cyanea, exhibits photoperiodism and is stimulated under long-day conditions. Previous studies suggest that red light is more effective than green and blue lights for inducing ovarian development. In addition, the extra-retinal photoreception involved in the ovarian development is suggested in this species. The present study aimed to clone the red-light-sensitive cone opsin (sdLWS) of this species, to demonstrate its expression in the brain, and to confirm the effectiveness of red light on the initiation of ovarian development. A phylogenetic analysis revealed that sdLWS belongs to the long-wavelength-sensitive opsin (LWS opsin) group, showing high identity (77-92%) with the LWS opsin of other fishes. RT-PCR showed that sdLWS is expressed in the eye, brain, and ovary. In situ hybridization indicated that sdLWS is expressed in the third ventricle periventricular area in the anterior hypothalamus. Exposing fish to long-day conditions of red light resulted in the appearance of vitellogenic oocytes in the ovary and an increase in the gonadosomatic index within 2weeks, suggesting that fish immediately undergo oocyte development under red light conditions. These results indicate that sdLWS is a possible candidate of deep brain photoreceptor molecule involved in photoperiodic ovarian development in the sapphire devil., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Type II iodothyronine deiodinase (D2) converts 3,5,3',5'-tetraiodothyronine to 3,5,3'-triiodothyronine and is involved in regulating thyroid hormone-dependent processes in various tissues. D2 mRNA expression in the mediobasal hypothalamus is affected by photoperiod, which influences reproductive processes in temperate birds and mammals. We examined whether D2 mRNA is expressed in the hypothalamus (located in the forebrain within the diencephalon area) and whether its abundance is affected by day length, temperature, or food availability in the tropical spinefoot, Siganus guttatus, which is endemic to tropical monsoon areas. The reverse transcription-polymerase chain reaction (RT-PCR) revealed that D2 mRNA is expressed in various brain regions. The abundance of hypothalamic D2 mRNA was higher at 12.00h than at 06.00h or 24.00h. Rearing fish under constant dark conditions resulted in a decrease in D2 mRNA abundance during the subjective night. A single injection of melatonin lowered D2 mRNA abundance within 3h. Collectively, it appears that hypothalamic D2 mRNA abundance is regulated by the circadian system and/or melatonin. No differences in D2 mRNA abundance were observed, when fish were reared at 20, 25, and 30°C. However, food deprivation stimulated D2 mRNA expression during the daytime. These results suggest that photoperiodic and nutritive conditions affect hypothalamic D2 mRNA expression in S. guttatus., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Ultrasound is used as degradation of hazardous organic compounds. In this study, indirect ultrasonic irradiation method was applied to the degradation process of phenol, the model hazardous organic compound, and the effects of irradiation distance on radical generation and ultrasonic power were investigated. The chemical effect estimated by KI oxidation dosimetry and ultrasonic power measured by calorimetry fluctuated for the irradiation distance, and there was a relationship between the period of the fluctuation of ultrasonic effect and the wavelength of ultrasound. The degradation of phenol was considered to progress in the zero-order kinetics, before the decomposition conversion was less than 25%. Therefore, the simple kinetic model on degradation of phenol was proposed, and there was a linear relation in the degradation rate constant of phenol and the ultrasonic power inside the reactor. In addition, the kinetic model proposed in this study was applied to the former study. There was a linear relation in the degradation rate constant of phenol and ultrasonic energy in the range of frequency of 20-30 kHz in spite of the difference of equipment and sample volume. On the other hand, the degradation rate constant in the range of frequency of 200-800 kHz was much larger than that of 20-30 kHz in the same ultrasonic energy, and this behaviour was agreed with the former investigation about the dependence of ultrasonic frequency on chemical effect., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

The aim of this study was to determine the involvement of neurohypophysial hormones in the diurnal patterns of the threespot wrasse Halichoeres trimaculatus, which is common in coral reefs and exhibits daily behavioral periodicity. Prohormone cDNAs of the neurohypophysial peptides, arginine vasotocin (AVT) and isotocin (IT), were cloned by 3'- and 5'-rapid amplification of cDNA ends (RACE). The distribution and expression patterns of pro-AVT and -IT mRNAs in the brain were determined using reverse-transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR, respectively. The respective full-length cDNAs of pro-AVT and -IT were 945 and 755 bp in length, respectively. The deduced amino acid sequences for pro-AVT and pro-IT were 154 and 156 residues in length, respectively. Both pro-peptides contained a signal sequence followed by the respective hormones and neurophysin connected by a Gly-Lys-Arg bridge. Pro-AVT mRNA was detected only in the hypothalamus area, while pro-IT mRNA in the whole part of the brain. The relative abundance of pro-AVT and -IT mRNA varied according to time of day; it was significantly greater at 12:00 h than at 24:00 h. Following intraperitoneal administration of melatonin, pro-AVT mRNA abundance in the brain decreased, while pro-IT mRNA abundance remained unchanged. These results demonstrate that daily fluctuations of pro-AVT and pro-IT levels in the brain of threespot wrasse are differentially regulated., (Copyright © 2010 Elsevier Inc. All rights reserved.)

We previously reported that in male rats, 24 h of social housing with an associate male rat immediately following fear conditioning attenuated conditioned hyperthermia in response to an auditory conditioned stimulus. In this study, we investigated the time window and threshold of social housing where "social buffering" mitigates conditioned hyperthermia. When 6 h of social housing immediately followed the conditioning procedure, attenuation of conditioned hyperthermia did not occur. However, an 18-h social housing period that started 6h after the conditioning procedure successfully blocked the subsequent hyperthermia, as was observed in our previous study. We also shortened the duration of social housing and found that a 12-h, but not a 6-h, period of social housing mitigated the subsequent hyperthermia. In addition, a 24-h social housing period that started 24 h after the conditioning procedure mitigated the hyperthermia. These results suggest that 12 h is sufficient for social buffering of conditioned hyperthermia., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Alcohol ingestion affects both neuropsychological and motor functions. We hypothesized that one of the key factors involved in such functions are neurotrophins and their receptors. We have therefore examined the effects of short-term ethanol exposure on the mRNA expression and protein levels of neurotrophin ligands and receptors in the cerebellum using real-time RT-PCR and Western blotting techniques. Male BALB/C mice were fed a liquid diet containing 5% (v/v) ethanol. The pair-fed control mice were fed an identical liquid diet except that sucrose was substituted isocalorically for ethanol. The cerebellum of mice exhibiting intoxication signs of stage 1 or 2 were used in the present study. We found that exposure to ethanol resulted in elevated levels of nerve growth factor (NGF) and TrkA mRNA expression but a decreased level of brain-derived neurotrophic factor (BDNF) mRNA expression. The expression of TrkB and p73 mRNA was unchanged. Changes in the level of these proteins were found to mirror these mRNA expression levels. We conclude that exposure to ethanol for a short period can cause a differential responsive in the various neurotrophin ligand/receptor systems. The functional consequences of these changes are unknown at present.

The human natural killer-1 (HNK-1) glyco-epitope possesses a unique structural feature, a sulfated glucuronic acid attached to lactosamine on the non-reducing termini of glycans. The expression of HNK-1 is temporally and spatially regulated by glucuronyltransferase (GlcAT-P) in the brain. Our previous report showed that mice lacking GlcAT-P almost completely lost HNK-1 expression in the brain and exhibited reduced long-term potentiation (LTP) at hippocampal CA1 synapses. GlcAT-P-deficient mice also showed impaired hippocampus-dependent spatial learning. Although HNK-1 plays an essential role in synaptic plasticity and memory formation, it remains unclear how HNK-1 regulates these functions. In this study, we showed that loss of the HNK-1 epitope resulted in an increase of filopodium-like immature spines and a decrease of mushroom-like mature spines in both the early postnatal mouse hippocampus and cultured hippocampal neurons. However, HNK-1 had no influence on spine density or filopodium formation. Immunofluorescence staining revealed that loss of HNK-1 altered the distribution of postsynaptic proteins such as alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA)-type glutamate receptor subunit GluR2 and PSD-95 from spine heads onto dendritic shafts without affecting synapse formation, resulting in an increase of shaft synapses in cultured GlcAT-P-deficient neurons. GluR2, a major HNK-1 carrier glycoprotein in postsynaptic density, has the ability to promote spine morphogenesis. Overexpression of GluR2 promoted spine growth in both wild-type and GlcAT-P-deficient neurons, but the increase in GlcAT-P-deficient neurons was lower than that in wild-type neurons. This is the first evidence that HNK-1 is a key factor for normal dendritic spine maturation and is involved in the distribution of postsynaptic proteins.

A novel series of indoline-based acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors with a methanesulfonamide group at the 5-position were synthesized and their lipophilicity and biological activities were evaluated. Hepatic ACAT inhibitory and anti-foam cell formation activity increased dependent on lipophilicity of derivatives with various alkyl chains at the 1-position. The logD(7.0)-biological activity curve of the derivatives with a methanesulfonamide group shifted leftward compared to that of Pactimibe derivatives with a carboxymethyl group, and derivatives with no substituent, suggesting that a methanesulfonamide group plays an important role in the interaction with ACAT protein. Among derivatives, N-(1-ethyl-5-methanesulfonylamino-4,6-dimethylindolin-7-yl)-2,2-dimethylpropanamide (1b) had about twofold lower logD(7.0) than Pactimibe, while it showed twofold higher hepatic ACAT inhibition than and the same anti-foam cell formation as Pactimibe, respectively. The C(max) of 1b (10mg/kg, po) was higher than that of Pactimibe in rats. The plasma protein binding ratio of 1b was lower than that of Pactimibe: 64.8% and 97.9%, respectively. Compound 1b showed greater inhibitory effects on hepatic cholesterol secretion in mice than Pactimibe. In conclusion, the introduction of a methanesulfonamide group is effective to design less lipophilic, more efficacious and more bioavailable indoline-based ACAT inhibitors than previous indoline-based inhibitors.

The volatility of an alarm pheromone in male rats. PHYSIOL BEHAV 00(0) 000-000, 2008. We previously reported that an alarm pheromone released from the perianal region of male rats is perceived by the vomeronasal organ and evokes stress-induced hyperthermia and defensive and risk assessment behavior. In addition, we recently reported that the alarm pheromone enhances the acoustic startle reflex (ASR). However, in contrast to our knowledge about such biological aspects of the pheromone, information concerning the physical character of the alarm pheromone is extremely limited. In this study, we investigated the volatility of the alarm pheromone using enhancement of the ASR as an index of the pheromone effect. The alarm pheromone enhanced the ASR when it was presented at a distance of 10 mm but not at 200 mm. In addition, the pheromone effect was observed even after the pheromone was trapped in the adsorbent (Tenax) and then extracted using purified water. These results suggest that the alarm pheromone is both volatile and water soluble.

Social isolation has been shown to modify behavioral patterns and neuroendocrine function in many species. In this study, we examined the influence of post-weaning social isolation on the behavioral development and fecal testosterone and corticosterone levels of male Mongolian gerbils. In Experiment 1, isolation-reared (IR) males spent more time engaged in social-sniffing. IR males also showed more aggressive and anxiety-related behaviors than group-reared males (GR; three animals per cage). In Experiment 2, a screen-divided-reared condition (SDR) was examined in addition to GR (two males per cage) and IR males. In the SDR group, two subjects were separated by a wire mesh screen allowing for sensory communication and limited physical contact but not direct social interactions. SDR males showed similar patterns in sniffing and anxiety-related behaviors to IR males; however, an increase in aggression was not observed. No differences in fecal testosterone and corticosterone levels were found among males in different rearing conditions. These results suggest that post-weaning social isolation affects behavioral development without affecting testosterone and corticosterone levels. The behavioral changes observed may therefore be regulated by different mechanisms; increases in social-sniffing and anxiety-related behaviors may be caused by the deprivation of direct social interactions, whereas the enhancement of aggressive behaviors may be caused by the lack of sensory stimulation and limited physical contact with other individuals.

We designed chiral 2-nitroimidazole derivatives containing a 2-aminomethylene-4-cyclopentene-1,3-dione moiety as antiangiogenic hypoxic cell radiosensitizers. Based on results of molecular orbital calculations, the 2-aminomethylene-4-cyclopentene-1,3-dione moiety is expected to show high electrophilicity comparable to that of the 2-methylene-4-cyclopentene-1,3-dione moiety included in TX-1123 and tyrphostin AG17. We evaluated the antiangiogenic and radiosensitizing effects of the new compounds, along with other biological properties including their activities as hypoxic cytotoxicities and protein tyrosine kinase (PTK) inhibitory activities. Among the compounds tested, 5 (TX-2036) proved to be the strongest antiangiogenic hypoxic cell radiosensitizer. All the other chiral 2-nitroimidazole derivatives having 2-aminomethylene-4-cyclopentene-1,3-dione moiety tested were also antiangiogenic hypoxic cell radiosensitizers. The PTK inhibitory activity of 5 (TX-2036) showed this to be a promising and potent EGFR kinase inhibitor, having an IC(50) value of lower than 2microM. This compound also was an Flt-1 kinase inhibitor having an IC(50) value of lower than 20microM. Our results show that these chiral 2-nitroimidazole derivatives that contain the 2-aminomethylene-4-cyclopentene-1,3-dione moiety as a potent antiangiogenic pharmacophoric descriptor are promising lead candidates for the development of antiangiogenic hypoxic cell radiosensitizers.

Recently, we reported that an alarm pheromone released from the perianal region of male rats aggravated stress-induced hyperthermia and increased defensive and risk assessment behaviors in recipient male rats. Based on these results, we hypothesized that the primary effect of the alarm pheromone is to increase anxiety; however, there is still no clear evidence for this pheromone effect. Therefore, we examined this issue by assessing the effect of the alarm pheromone on the acoustic startle reflex (ASR), which is a useful index for studying negative emotions such as anxiety in rats. The alarm pheromone enhanced the ASR for 105-dB auditory stimuli, but not for those of 90 and 120 dB, when these three intensities of sound were used randomly. The same results were obtained when one of these three intensities was used repeatedly. In addition, pretreatment with diazepam (i.p.) at doses of 0.7 and 2.0 mg/kg suppressed the ASR of the pheromone recipients, whereas the lower dose (0.2 mg/kg) slightly attenuated the pheromone effect and the control injection (vehicle) had no effect. These results indicate that the alarm pheromone enhances the ASR by increasing anxiety in recipient rats, suggesting that the primary effect of the alarm pheromone is to increase the anxiety level.

Social affiliation-avoidance behaviors are essential indices of sociality. We examined changes in social affiliation-avoidance behaviors in an open-field apparatus while simultaneously measuring wheel-running activity. Recent studies suggest that mice increase wheel-running activity in stressful situations; thus, we hypothesized that wheel-running activity would reflect a state of social stress and avoidance. Mean duration of wheel-running increased significantly when mice were confronted with unfamiliar mice compared to cage mates. There were negative correlations between the amount of wheel-running and social affiliation indices. We also examined the effect of social defeat on wheel-running activity. Mice that had experienced social defeat significantly increased their wheel-running when an aggressor mouse was present. This social defeat-induced wheel-running activity was ameliorated by the administration of diazepam. Our results indicate that wheel-running activity is relevant to social affiliation-avoidance behaviors and may be a reliable index of anxiety induced by social stress.

The long-term cardiac effects of amiodarone resemble many aspects of hypothyroidism. The anti-arrhythmic potential of amiodarone may therefore be the result of a drug-induced, local hypothyroid-like condition. To investigate this controversial issue, we compared gene expression profiles in the hearts of rats treated with amiodarone with those of rats with hypothyroidism. Wistar male rats were assigned to 3 groups (n=6-8): Control, systemic hypothyroidism (hypothyroidism) and amiodarone treatment (amiodarone, 150 mg/kg/day, p.o., 4 weeks). Electrocardiogram (ECG) recordings, gene profiling by DNA microarray and Northern blotting were carried out. Amiodarone, like hypothyroidism, caused significant prolongation of RR and QT intervals in ECGs. Microarray analysis of 8435 genes in the left ventricular myocardium revealed a significant similarity in expression profiles between hypothyroidism and amiodarone (R=0.63, p<0.00001). The gene expression profiles of hypothyroidism and amiodarone showed closer correlation when top 100 up-regulated and 100 down-regulated genes in hypothyroidism (total 200 genes) were analyzed (R=0.78, p<0.00001). Northern blots of left ventricular myocardium showed a parallel decrease in mRNAs for myosin heavy chain (MHC)-alpha and a parallel increase for myosin heavy chain (MHC)-beta in hypothyroidism and amiodarone. In the liver and pituitary, in contrast, Northern blots showed quite different changes in the transcripts of the representative T3-responsive genes in the hypothyroidism and amiodarone. In conclusion, long-term treatment with amiodarone causes cardioselective hypothyroid-like alterations in gene expression profiles. The potent anti-arrhythmic activity of amiodarone may be attributable, in part at least, to this unique transcriptional remodeling.

Vitamin D insufficiency is a reemerging and common health problem for skeletal system. Pharmacological application of glucocorticoid inhibits intestinal calcium absorption and stimulates tubular calcium excretion, thus induces severely negative calcium balance. We report a patient presenting symptomatic hypocalcemia following high dose glucocorticoid administration. After a pulse-therapy with methylprednisolone, hypocalcemia with muscle cramp developed in association with hypercalciuria and secondary hyperparathyroidism in the absence of hypomagnesemia. Circulating level of 1,25-dihydroxyvitamin D was in a reference range, while that of 25-hydroxyvitamin D was insufficient. Treatment with alfacalcidol of 1 mug/day promptly improved serum calcium level within a couple of weeks. Vitamin D insufficiency could be a serious problem in patients with high dose glucocorticoid therapy.

Postnatal stimuli affect many aspects of physiological and behavioral development. In mice, earlier weaning augments anxiety, putatively as a result of removing mother-pup interactions during the weaning period. Here, we examined the ameliorating effects of social and environmental enrichment on anxiety related to early weaning. Mice weaned at postpartum day 14 were fostered by virgin females, who displayed some nursing behavior during the 1-week fostering period. In elevated plus-maze tests, 10-week-old pups reared with a foster mother spent more time in the open arms than early-weaned mice, and entered into the open arms at a rate between that of normally- and early-weaned mice. Subsequently, the mice from each rearing group were transferred into either standard housing or housing enriched with toys that were changed periodically. Elevated plus-maze tests were conducted again when the mice were 18 and 26 weeks old. The enriched environment increased the duration of time spent in the open arms, but the magnitude of the effect varied with the rearing condition. Furthermore, mice that lived in the enriched environment showed lower activity than those kept in standard housing. These results suggest that fostering after early weaning attenuates increases in anxiety levels, and maternal care during this period may be important in the development of an offspring's emotionality. Environmental stimuli in adulthood may act to blunt the effects deprivation in early life.

The golden rabbitfish, Siganus guttatus, is a reef fish exhibiting a restricted lunar-related rhythm in behavior and reproduction. Here, to understand the circadian rhythm of this lunar-synchronized spawner, a melatonin receptor subtype-Mel(1c)-was cloned. The full-length Mel(1c) melatonin receptor cDNA comprised 1747 bp with a single open reading frame (1062 bp) that encodes a 353-amino acid protein, which included 7 presumed transmembrane domains. Real-time PCR revealed high Mel(1c) mRNA expression in the retina and brain but not in the peripheral tissues. When the fish were reared under light/dark (LD 12:12) conditions, Mel(1c) mRNA in the retina and brain was expressed with daily variations and increased during nighttime. Similar variations were noted under constant conditions, suggesting that Mel(1c) mRNA expression is regulated by the circadian clock system. Daily variations of Mel(1c) mRNA expression with a peak at zeitgeber time (ZT) 12 were observed in the cultured pineal gland under LD 12:12. Exposure of the cultured pineal gland to light at ZT17 resulted in a decrease in Mel(1c) mRNA expression. When light was obstructed at ZT5, the opposite effect was obtained. These results suggest that light exerts certain effects on Mel(1c) mRNA expression directly or indirectly through melatonin actions.

Sensory chemo-signals conveying information on sex and familiarity are important to the manifestation of aggressive behaviors in male mice. In this study, we examined the role of familiarity conveyed by urine odor in the induction of aggressive behavior using a resident-intruder paradigm. First, an intact ICR male mouse (resident) was grouped with a castrated DBA mouse (cage-mate) and a female ICR mouse to allow the resident mouse to establish its territory. The resident male showed vigorous aggression, not only toward intact male DBA intruders, but also toward unfamiliar castrated DBA mice (UFC). In contrast, the aggression was markedly reduced toward its castrated DBA cage-mate. Next, to reveal how residents discriminate their cage-mates from unfamiliar intruders, we examined whether urine odor affected this familiarity-related aggression. When part of the body surface of a UFC was swabbed with the urine of a resident's cage-mate, the resident attacked the UFC much less often. These results suggest that the information about familiarity conveyed by urine odor plays an important role in controlling the territorial aggression of a resident male mouse toward castrated intruders.

We have previously demonstrated that gabapentin supraspinally activates the descending noradrenergic system to produce analgesic effects after peripheral nerve injury. To further establish the neurochemical basis for its supraspinally mediated analgesic action, concentrations of spinal noradrenaline, 4-hydroxy-3-methoxyphenylglycol (MHPG), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and dopamine were measured using high-performance liquid chromatography in a murine neuropathic pain model that was prepared by partial ligation of the sciatic nerve (the Seltzer model). Intracerebroventricularly (i.c.v.) administered gabapentin (100 and 300 microg) increased the spinal MHPG concentration and the MHPG/noradrenaline ratio and alleviated mechanical hypersensitivity, whereas the concentrations of noradrenaline, 5-HT, 5-HIAA and dopamine were unchanged. By contrast, i.c.v. gabapentin neither affected the spinal MHPG concentration and MHPG/noradrenaline ratio nor exhibited analgesic effects in animals subjected to a sham operation. In addition, spinal monoamine levels in ligated animals were not changed after intrathecal administration of gabapentin which however generated analgesic effects. Thus, the supraspinally mediated analgesic effects of gabapentin are correlated with an increase in spinal noradrenaline turnover.

Social hierarchy plays an important role in the lives of gregarious species. We investigated how social hierarchy between male Mongolian gerbils (Meriones unguiculatus) housed together before sexual maturity affected their scent-marking behavior, endocrine status and physical traits as adults. The social rank between paired males was assessed based on sexual activity in adulthood at 18 and 20 weeks of age. In most cases, the male that showed higher sexual activity at 18 weeks also exhibited greater sexual performance at 20 weeks, suggesting a precedence relationship between paired males regarding reproductive opportunity. Sexually active males scent-marked more often than their cage mate, particularly after a sexual experience. No significant differences in physical traits (e.g., the weight of the ventral gland, testes and adrenal glands) or endocrine status (e.g., fecal testosterone and corticosterone levels) were found between the two males. These findings suggest that social rank between familiar males formed during cohabitation, and rank was closely correlated with scent-marking activity, but not with endocrine status.

In many species, social-defeat stress induces behavioral changes in subordinate individuals. The present study examined how social-defeat stress affected scent-marking and approach behaviors in male Mongolian gerbils and whether socially defeated males selectively avoided dominant males. In Experiment 1, each of 11 gerbils was defeated by a dominant gerbil during a short social confrontation (10 min in total). For Experiment 2, the same animals were used two weeks after the end of Experiment 1. Each subject was again similarly defeated, but the two gerbils were then kept in the same cage, separated by a wire mesh for 24 h. Behavioral changes in socially defeated males were observed after social defeat; both experiments were replicated three times. After acute social defeat in Experiment 1, male gerbils did not change their scent-marking activity and frequently approached the dominant males. In contrast, males decreased scent-marking activity and approach behavior toward dominant males in Experiment 2. The amount of time defeated males approached unfamiliar males was also reduced, suggesting that social-defeat stress reduces scent-marking motivation and conspecific approach behavior rather than inducing social avoidance toward a specific individual. These findings showed that the severity of social-defeat stress is an important factor in subsequent behavioral changes in male Mongolian gerbils.

Previously, we reported that alarm pheromone released from the perianal region of male rats aggravated stress-induced hyperthermia and increased Fos expression in the vomeronasal pathway and stress-related nuclei in pheromone-recipient rats. However, the alarm property of this pheromone in terms of behavior modification is still unclear. We recently found that this alarm pheromone could be trapped in water. Based on this finding, we developed an experimental paradigm to assess the effect of alarm pheromone on recipient behavior. Male Wistar rats were acclimatized for 5 min to an open field, where two pieces of filter paper soaked with 750 microl of either pheromone-containing water or vehicle water were attached to the wall. Then, a small "hiding box" was placed in one corner of the field and the behavioral responses of the subject rat were recorded for 10 subsequent minutes. Exposure to alarm pheromone significantly increased defensive and risk assessment behaviors and decreased exploratory and grooming behaviors compared to the vehicle control group, indicating the alarm property of the pheromone. In addition, the comparison with previous results suggests that the alarm pheromone released from the perianal region of the male rat increases anxiety in recipients, rather than evoking a stereotyped autonomic response.

Orobanche minor is a non-photosynthetic root holoparasitic plant. Although it is known that photosynthesis-related genes are inactivated or have been eliminated from the plastid genomes of holoparasites, little is known about the alterations in their genes involved in the signaling networks by which light regulates photosynthesis. Cryptochromes (crys), which are blue-light receptors, appear to control both photosynthesis-related and non-photosynthetic responses to light in higher plants. Because we are interested in to what extent a cry-mediated light signaling network remains in the holoparasites, we cloned CRY homologous cDNA from O. minor (OmCRY1) and used real-time RT-PCR to compare its expression under natural daylight and darkness. We found that the OmCRY1 has a high degree of homology with CRY1 s from photosynthetic plants. Expression of the OmCRY1 gene was higher in plants grown in the dark than that in the plants grown under natural daylight. This is the first report of the gene expression of a blue-light receptor in non-photosynthetic plants.

(R)- and (S)-Epichlorohydrins were used to prepare the enantiomers of sterically diverse haloacetylcarbamoyl-2-nitroimidazoles that function as hypoxic cell radiosensitizers. The synthetic design allowed for introduction of a side chain of varying bulk that permitted an examination of the steric effects on enantio-discrimination in biological assay systems. The single stereocenter also connected the two pharmacophores--a 2-nitroimidazole moiety critical to hypoxic cell radiosensitization, and a haloacetylcarbamoyl group to function as an anti-angiogenesis pharmacophore. In the chick embryo chorioallantoic membrane (CAM) assay, the R-enantiomers possessing the bulky p-tert-butylphenyl group showed higher anti-angiogenic activity than the corresponding S-enantiomers, while there were no differences in the activity between the enantiomers containing the less bulky methyl and tert-butyl groups. Among the compounds we report, R-p-tert-butylphenyl-bromoacetylcarbamoyl-2-nitroimidazole, TX-1898, was found to be the most promising candidate for further development of as anti-angiogenic hypoxic cell radiosensitizer.

Morphological features and functional implications of projections of the parabrachial nucleus to the central nucleus of the amygdala were investigated in the rat. The anatomical study was based on injections of the tracers horseradish peroxidase and biotinylated dextran amine. An extremely dense concentration of labeled fibers was found in the lateral and lateral capsular subdivisions of the central nucleus of the amygdala, originating mainly from the external lateral and ventral lateral subnuclei of the parabrachial nucleus. The parabrachial fibers exhibited the morphological characteristic of forming dense pericellular terminal arborizations. The functional implications of this pathway in cardiovascular functions were verified using Fos protein induction in response to hypotension induced by continuous intravenous administration of hydralazine-hydrochloride. In this paradigm, Fos immunoreactivity was found to be confined to the lateral and lateral capsular subdivisions of the central nucleus of the amygdala. Double immunostaining methods were used to visualize, at the electron microscopic level, terminals labeled by biotinylated dextran amine and Fos cell labeling. With this approach, we were able to confirm that Fos-immunoreactive neurons in the central nucleus of the amygdala receive axosomatic terminals from the parabrachial nucleus. The present findings point out that parabrachial inputs to the central nucleus of the amygdala play a relevant role in regulating cardiovascular function.