横須賀 誠

The brown-eared bulbul (Hysipetes amaurotis) is commonly found in Japan where it is regarded as a harmful bird that causes damage to agricultural products. Few studies have investigated the sensory apparatus of this bird, and consequently little is known of the sensory modalities it uses. Here we analyzed the anatomical and histological properties of the nasal cavity and olfactory bulb (OB) of the bulbul in order to investigate the functional level of olfaction in this species. Although both anterior and maxillary conchae were observed in the bulbul nasal cavity, there was no structure equivalent to the posterior concha. The OB located on the ventral side of the anterior extremity of the cerebrum and the ratio of olfactory bulb size to that of the cerebral hemisphere were very small. Interestingly, the left and right OBs were completely fused at the midline of the cerebrum. Furthermore, certain types of lectins that bind to the olfactory nerve of vertebrates with a well-developed sense of smell also bound positively to the olfactory nerve and glomerular layers of the bulbul OB. These findings suggest that the brown-eared bulbul has an anatomically and functionally less well developed sense of smell compared to other avian species. Although the molecular and developmental mechanisms underlying the fusion of the OB remain unknown, we suggest that the fused OB may offer a unique model for studying the evolution and development of the central olfactory nervous system in vertebrates.

The nasal cavity and olfactory bulb (OB) of the Japanese jungle crow (Corvus macrorhynchos) were studied using computed tomography (CT) and histochemical staining. The nasal septum divided the nasal cavity in half. The anterior and maxillary conchae were present on both sides of the nasal cavity, but the posterior concha was indistinct. A small OB was present on the ventral surface of the periphery of the cerebrum. The OB-brain ratio--the ratio of the size of the OB to that of the cerebral hemisphere--was 6.13. The olfactory nerve bundles projected independently to the OB, which appeared fused on gross examination. Histochemical analysis confirmed the fusion of all OB layers. Using a neural tracer, we found that the olfactory nerve bundles independently projected to the olfactory nerve layer (ONL) and glomerular layer (GL) of the left and right halves of the fused OB. Only 4 of 21 lectins bound to the ONL and GL. Thus, compared with mammals and other birds, the jungle crow may have a poorly developed olfactory system and an inferior sense of olfaction. However, it has been contended recently that the olfactory abilities of birds cannot be judged from anatomical findings alone. Our results indicate that the olfactory system of the jungle crow is an interesting research model to evaluate the development and functions of vertebrate olfactory systems.

As members of Western societies age, sexual function of older (elderly) individuals becomes an important issue, particularly for men. Specifically, copulatory behavior increases circulatory load, which may be related to reports of cardiac sudden death following ejaculation. To further examine this relationship, we compared heart rate (HR) before and after ejaculation in 48-week-old (aged) and 10-week-old (young) male rats. As compared with resting HR, HR after ejaculation was increased by 54.2 +/- 3.5 and 41.7 +/- 2.7%, respectively, among aged and young male rats. These values were significantly higher than baseline levels (P<0.01). The increases in HR at each time point during copulation were significantly higher in aged male rats than in young male rats (P<0.05 or P<0.01). We also studied decreases in HR following ejaculation and found that aged male rats had a significantly higher HR at 1 and 2 min after ejaculation than young rats (P<0.01). These results suggest that the circulatory load on the aged rat heart is greater than that on a young rat heart during copulatory behavior, especially at ejaculation. In addition, the decrease in HR in aged rats after ejaculation was more gradual than in young male rats. These results suggest that there is a higher risk of sudden cardiac death during sexual behavior in older males.

Purpose: Sudden cardiac death after ejaculation has been reported in humans and highlights the important relationship between sexual behavior and the heart. The rat is an extremely useful animal model for investigating reproductive function in male mammals. In this study, we examined the relationship between autonomic nervous system activity and the circulatory system during sexual behavior in male rats. Methods: Male Wistar-Imamichi rats were exposed to female rats in estrous and heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and plasma noradrenaline (NA) and adrenaline (Ad) concentrations were measured by telemetry during copulation. Results: The resting HR was 365.5 ± 18.4 beats/min (mean ± SE), which increased to 531.2 ± 21.1 beats/min at ejaculation and decreased to 404.6 ± 30.7 beats/min 1 min after ejaculation. At rest, SBP and DBP were 123.8 ± 6.6 and 81.5 ± 4.1 mmHg, respectively, which increased to 154.5 ± 5.9 and 112.1 ± 7.3 mmHg at ejaculation. Baseline plasma Ad and NA concentrations were 151.6 ± 32.0 and 248.5 ± 22.5 pg/ml, respectively, and these increased to 393.8 ± 89.9 and 792.7 ± 154.0 pg/ml at ejaculation, after which they decreased to resting levels. The rate of increase in NA at ejaculation differed significantly from that of Ad. Conclusions: The load on the circulatory and autonomic nervous systems is controlled by a rapid decrease in HR and NA concentration immediately after ejaculation, such that the male rat is prepared for the next copulation.

Aim: Elucidation of the neural mechanism of maternal behaviors is a medically and biologically important research task. The rat is the laboratory animal most extensively analyzed for maternal behaviors. However, the neural mechanism that maintains the motivation of postpartum rats for maternal behaviors has not yet been elucidated. In this study, we aimed to identify brain regions involved in the maintenance of motivation for maternal behaviors by detecting brain regions that exhibit changes in nerve activity when the mother rat is separated from her pups. Methods: Lactating mother rats were separated from their pups on postpartum day 3 and kept away from the pups for a certain period of time, and brain regions that exhibited changes in nerve activity when the rats were separated from their pups and those that exhibited changes in nerve activity when the pups are returned were detected by immunohistochemistry using anti-c-Fos antibody, a marker for increased nerve activity. Results: Rats that were separated from their pups and with the pups returned later showed increases in the number of c-Fos immunoreactive (c-Fos-IR) cells in the medial preoptic area (MPA), the bed nucleus of the stria terminalis (BST), the caudal portion of posterior hypothalamic area (PH) and the supramamillary nucleus (SUM). In mother rats permanently separated from their pups, only the PH and SUM exhibited an increase in the number of c-Fos-IR cells. Conclusion: In rats, the SUM is involved in aversive memory and changes in the postpartum anxiety level. The observed increase in the number of c-Fos-IR cells in the SUM of mother rats separated from their pups suggests that the nerve activity change in the SUM, which is involved in aversive memory and anxiety, is involved in the maintenance of maternal behaviors.

The purpose of the present study was to examine the in vitro effects of low-dose cadmium (Cd) on developing cortical cells. The cortical cells removed from fetuses (embryonic day 15) were treated with 10nM of Cd for 24h. The effects of Cd on dendritic and synaptic development were immunocytochemically observed with anti-microtubule associated protein-2 (MAP2) and anti-synapsin I antibodies, respectively. Administration of Cd suppressed dendritic as well as synaptic development at 10nM. By two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometric (LC/MS/MS) analysis, we identified three proteins with different expression after Cd-treatment; dihydropyrimidinase-related protein 2 (DRP-2/CRMP-2), 14-3-3-epsillon and calmodulin (CaM). Though the number of identified proteins was small, these proteins are known to be involved in neuronal development. The present study demonstrated the morphological effects as well as affected proteins in Cd-treated cortical cells, providing tools and insights in elucidating mechanisms how low-dose Cd distorts brain development.

In this study, in order to clarify the kinetics of leptin, we focused on the ratio of leptin concentrations in cerebrospinal fluid and serum in aged male rats, and examined the weight of epididymal fat, and the passage rate of leptin through the blood-brain barrier. In the lighter animals, the epididymal fat weight was low, while leptin concentrations in the serum and cerebrospinal fluid were also low. Conversely, in the heavier animals, the weight of epididymal fat and leptin concentrations in the serum and cerebrospinal fluid were higher. With regard to the ratio of leptin in the cerebrospinal fluid and serum, the passage rate of leptin through the blood-brain barrier was lower in the heavier animals than in the lighter animals.

Efficient reproduction using natural mating and reproduction technology [in vitro fertilization (IVF) and embryo transfer (ET)] was investigated in IRS2 deficient mice with C57BL/6JJcl genetic background (Irs2(-/-) mice) as a typical type 2 diabetes model. From the results using various combinations of Irs2(-/-) and Irs2(-/+) mice, the combination of female Irs2(-/+) x male Irs2(-/-) was found to be more efficient than other combinations. In applications of reproduction technology using IVF and ET, the combination of female Irs2(-/+) x male Irs2(-/-) involves the possibility of Irs2(-/-) production by repeats using female Irs2(-/+) mice. However, reproductive continuity using this combination is difficult because of dependence on human technique and the cost of ET. Therefore, we concluded that Irs2(-/-) mice should be produced by embryo transfer using Irs2(-/-) mice from a colony consisting of female Irs2(-/+) x male Irs2(-/-).

Aim:  In the rat, intraperitoneal injection of p-chloroamphetamine (PCA), which releases central 5-hydroxytryptamine (5-HT) from serotonergic nerve terminals, induces ejaculation, even in the absence of an estrus female or female-related smell information. It is well known that the medial preoptic nucleus (MPN) and the medial nucleus amygdala (MEA) play a major role in the control of male sexual behavior in mammals. We examined whether or not neuronal activity of the MPN and/or the MEA was associated with PCA-induced ejaculation. Methods:  Using c-Fos immunohistochemistry, we demonstrated a difference in the neural activities of the MPN and the MEA for ejaculation during copulation with an estrus female and ejaculation by PCA injection. Results:  Increased numbers of c-Fos-immunoreactive (c-Fos-IR) cells were found in the MPN and the MEA in the brains of the mating animals, whereas in the brains of the animals undergoing PCA-induced ejaculation there was no increase in the number of c-Fos-IR cells in the MPN and a small increase in the MEA. Conclusion:  Based on these results, ejaculation induced by PCA is not associated with the MPN. Moreover, the MEA is not the main act for this ejaculation. (Reprod Med Biol 2008; 7: 37-43).

We investigated effects of 17beta-estradiol (E(2)) and endocrine disrupters, nonylphenol (NP) and bisphenol-A (BPA), focusing on the neuronal development in cultures of fetal rat hypothalamic cells. We applied different concentrations of E(2), NP or BPA to the cultured hypothalamic cells and observed their effects on dendritic and synaptic development by immunocytochemistry using anti-microtubule associated protein-2 (MAP2) and anti-synapsin I antibodies, respectively. Administration of E(2) for 7 days affected MAP2-positive area as well as synapsin I-positive area. NP and BPA also influenced neuronal developments. The significant increase both in MAP2- and synapsin I-positive areas was observed at 10 and/or 100 nM of them, while 1 microM of them reduced the positive areas. Synaptic densities calculated from synapsin I-positive area/MAP2-positive area were not constant among different doses of three chemicals, but increased at 10 and/or 100 nM and decreased at 1 microM. Furthermore, immunostaining of NP-treated cells with the antibody against glial fibrillary acidic protein (GFAP) revealed that glial development was similarly influenced by NP. Therefore, the present results demonstrated that not only E(2) but also the environmental estrogenic chemicals, NP and BPA, affect development of fetal rat hypothalamic cells in vitro.

Ontogenetic changes of USVs were investigated to establish an index of stress in infant rats. The USVs were obtained by exposing infant rats to cold stress and were analyzed by real-time spectrography. USV waveforms consisted of four types, R-I, II, III and IV. These USVs were closely monitored at the age of 3-7 days in both sexes. From day 14, the frequency and incidence of USVs gradually decreased and had disappeared completely at the age of 21 days. Therefore, USVs should be useful in estimating the stress of infant rats between the ages of 3-7 days.

Eighteen lactating Wistar-Imamichi rats habituated to drinking 1M sucrose-solution were divided into three groups. Appetitive and maternal behaviour was observed during 30 minutes. Group 1 had access to sucrose solution and their pups, Group 2 had access to distilled water and their pups, and Group 3 had access to sucrose solution but not pups. The maternal behaviour seen in Group 2 was effectively inhibited by the presence of the sucrose-solution, in Group 1 where, during the observation period, the rats exhibited appetitive behaviour exclusively. We conclude that maternal appetitive ingestive behaviour is undiminished by the presence of pups.

R. AxelとL. Buckによる匂い受容体遺伝子の解明は、その後、嗅球(主嗅球)における"匂い地図"という概念を生むことになり、嗅覚系の脳内情報処理に関する理解を飛躍的に進めることとなった。しかし、不揮発性分子による情報を受容し、"もう一つの嗅覚系"あるいは"副嗅覚系"と称されてきた鋤鼻系の情報処理機構に関する理解は未だに不十分のままである。これまで明らかにされた鋤鼻系に関する所見を嗅覚系と比べてみると、両者には明らかな構造的相違が認められる。例えば、同じ匂い受容体を発現した嗅細胞からの投射は、主嗅球において少数の大きな糸球体に収束するのに対し、同じ鋤鼻受容体を発現した鋤鼻細胞からの投射は、副嗅球において多数の小さな糸球体に終わっている。マウス副嗅球の糸球体層の構造を詳しく検討すると、主嗅球の糸球体周辺細胞が糸球体の周囲を規則正しく取り囲んでいるのに対し、副嗅球の糸球体周辺細胞は小さな糸球体の周辺に不規則に分布している。

To estimate the potential risk of perinatal exposure to estrogenic endocrine disrupters, pregnant, female mice received daily oral administration of diethylstilbestrol (DES; either 0.3 or 3 mug/kg body weight) dissolved in corn oil from gestation days 11 to 17 and from postnatal days 2 to 6. Multiple behaviors that are sexually dimorphic were examined, and the numbers of estrogen receptor-a and tyrosine hydroxylase-immunoreactive (ER-IR and TH-IR) cells in some brain loci related to these behaviors were investigated. Perinatal exposure to DES caused significantly enhanced open-field activity in both males and females and significantly poorer passive avoidance performance in males. In addition, a significant increase in the number of ER-IR cells in the ventromedial hypothalamic nucleus (VMH) was demonstrated for the first time. The DES-induced increases in the sexual and aggressive behaviors, although statistically nonsignificant, and the increase in the number of ER-IR cells did not agree with those obtained in previous studies using high-dose DES, which suggests that DES may have a different effect on these endpoints depending on the dose used. The relationship between the increase in ER-IR cells and behavioral changes should be further examined. (C) 2004 Elsevier Inc. All rights reserved.

To estimate the potential risk of perinatal exposure to estrogenic endocrine disrupters, pregnant, female mice received daily oral administration of diethylstilbestrol (DES; either 0.3 or 3 mug/kg body weight) dissolved in corn oil from gestation days 11 to 17 and from postnatal days 2 to 6. Multiple behaviors that are sexually dimorphic were examined, and the numbers of estrogen receptor-a and tyrosine hydroxylase-immunoreactive (ER-IR and TH-IR) cells in some brain loci related to these behaviors were investigated. Perinatal exposure to DES caused significantly enhanced open-field activity in both males and females and significantly poorer passive avoidance performance in males. In addition, a significant increase in the number of ER-IR cells in the ventromedial hypothalamic nucleus (VMH) was demonstrated for the first time. The DES-induced increases in the sexual and aggressive behaviors, although statistically nonsignificant, and the increase in the number of ER-IR cells did not agree with those obtained in previous studies using high-dose DES, which suggests that DES may have a different effect on these endpoints depending on the dose used. The relationship between the increase in ER-IR cells and behavioral changes should be further examined. (C) 2004 Elsevier Inc. All rights reserved.

In the present study, we tested whether the pineal organ of ayu (Plecoglossus altivelis), an osmerid teleost close relative of salmonids, harbours a circadian oscillator regulating rhythmic melatonin release using flow-through culture. The pineal organ maintained under light/dark cycles released melatonin in a rhythmic fashion with high levels during the dark phase. A circadian rhythm of melatonin release persisted in constant darkness for at least four cycles. Characteristics of the circadian rhythm (free-running period, phase and amplitude) exhibited small variations among cultures when the data was normalized, indicating that this system is sufficient for the analysis of the circadian rhythm both at qualitative and quantitative levels. Six-hour extension of the light phase from the normal onset time of the dark phase or exposure to constant light for 36 or 48 h before transfer to constant darkness significantly inhibited melatonin release. Phase shifts in the circadian rhythm of melatonin release were also observed. Thus, the ayu pineal organ contains all the three essential components of the circadian system (a circadian clock, the photoreceptor responsible for photic entrainment of the clock, and melatonin generating system as an output pathway). This system should provide a useful model for analysing the physiological and molecular basis of the vertebrate circadian system. in addition, further comparative studies using salmonics and related species including ayu will provide some insight into the evolution of the roles of the pineal organ in the vertebrate circadian system.

In the present study, we tested whether the pineal organ of ayu (Plecoglossus altivelis), an osmerid teleost close relative of salmonids, harbours a circadian oscillator regulating rhythmic melatonin release using flow-through culture. The pineal organ maintained under light/dark cycles released melatonin in a rhythmic fashion with high levels during the dark phase. A circadian rhythm of melatonin release persisted in constant darkness for at least four cycles. Characteristics of the circadian rhythm (free-running period, phase and amplitude) exhibited small variations among cultures when the data was normalized, indicating that this system is sufficient for the analysis of the circadian rhythm both at qualitative and quantitative levels. Six-hour extension of the light phase from the normal onset time of the dark phase or exposure to constant light for 36 or 48 h before transfer to constant darkness significantly inhibited melatonin release. Phase shifts in the circadian rhythm of melatonin release were also observed. Thus, the ayu pineal organ contains all the three essential components of the circadian system (a circadian clock, the photoreceptor responsible for photic entrainment of the clock, and melatonin generating system as an output pathway). This system should provide a useful model for analysing the physiological and molecular basis of the vertebrate circadian system. in addition, further comparative studies using salmonics and related species including ayu will provide some insight into the evolution of the roles of the pineal organ in the vertebrate circadian system.

The effects of light on the circadian rhythm in melatonin release from the pineal organ of a teleost, ayu (Plecoglossus altivelis) were investigated in flow-through culture. Under the reversed light-dark (LD) cycle, the melatonin rhythm phase shifted as compared with those under the normal LD cycle. This phase shift persisted even under constant darkness (DD). Single 6-h light pulses starting at six different circadian phases under DD acutely suppressed melatonin release. Phase-dependent phase shifts in the circadian rhythm of melatonin release were also observed. The phase response curve to light pulses in the ayu pineal organ is typical of that found in many circadian systems. Thus, the ayu pineal organ should provide a useful model for analyzing the physiological and molecular basis of the entrainment mechanism of vertebrate circadian system. (C) 2003 Elsevier B.V. All rights reserved.