In mice, genetic differences between inbred strains have been shown for several parameters of sle... more In mice, genetic differences between inbred strains have been shown for several parameters of sleep and circadian activity rhythms. Our previous studies have demonstrated that CS mice have three remarkable characteristics in the circadian rhythm of locomotor activity: (1) high activity both during the day and night, (2) unstable freerunning period and (3) spontaneous rhythm splitting. In order to characterize sleep properties of CS mice, we compared circadian sleep patterns of CS with those of C57BL/6J and C3H/He mice which have normal circadian activity rhythms. Although C57BL/6J and C3H/He mice exhibited clear daily sleep-wake rhythms in the amount of each sleep parameter (Awake, SWS, PS), CS mice did not show clear rhythms in these parameters. The differences were particularly conspicuous in PS; no apparent day-night differences in the amount of PS, PS counts and PS interval (the interval between successive PS episodes) in CS mice. In addition, the ratio of PS to total sleep time was significantly larger in CS mice than other strains. Of these parameters, the most considerable was PS latency which was extremely short and direct transition from Awake to PS without appearance of SWS frequently occurred in these mice. These results indicate that CS mice may be useful for the understanding of sleep mechanisms and its dysfunction.
The avian perivitelline layer (PL), a vestment homologous to the zona pellucida (ZP) of mammalian... more The avian perivitelline layer (PL), a vestment homologous to the zona pellucida (ZP) of mammalian oocytes, is composed of at least three glycoproteins. Our previous studies have demonstrated that the matrix's components, ZP3 and ZPD, are synthesized in ovarian granulosa cells. Another component, ZP1, is synthesized in the liver and is transported to the ovary by blood circulation. In this study, we report the isolation of cDNA encoding quail ZP2 and its expression in the female bird. By RNase protection assay and in situ hybridization, we demonstrate that ZP2 transcripts are restricted to the oocytes of small white follicles (SWF). The expression level of ZP2 decreased dramatically during follicular development, and the highest expression was observed in the SWF. Western blot and immunohistochemical analyses using the specific antibody against ZP2 indicate that the 80 kDa protein is the authentic ZP2, and the immunoreactive ZP2 protein is also present in the oocytes. Moreover, ultrastructural analysis demonstrated that the immunoreactive ZP2 localizes to the zona radiata, the perivitelline space, and the oocyte cytoplasm in the SWF. By means of western blot analysis and immunofluorescence microscopy, we detected a possible interaction of the recombinant ZP2 with ZP3 and that this interaction might lead to the formation of amorphous structure on the cell surface. These results demonstrate for the first time that the avian ZP gene is expressed in the oocyte, and that the ZP2 protein in the oocyte might play a role for the PL formation in the immature follicles of the ovary.
The circadian clock is a fundamental property of living organisms and is involved in seasonal (ph... more The circadian clock is a fundamental property of living organisms and is involved in seasonal (photoperiodic) time measurement. Among vertebrates, birds have multiple circadian pacemakers in the eye, the pineal gland, and the suprachiasmatic nucleus (SCN), and have highly sophisticated photoperiodic mechanisms. However, because the removal of these circadian pacemakers fails to abolish the photoperiodic response, the existence of another "photoperiodic clock" has been suggested. Recent studies have revealed that the mediobasal hypothalamus (MBH) and the adjacent pars tuberalis (PT) of the pituitary gland constitute key components of the photoperiodic signal transduction machinery. In the present study, we generated a polyclonal antibody against the chicken circadian clock protein BMAL1 to examine BMAL1 distribution in the Japanese quail brain by using immunohistochemistry. BMAL1-like immunoreactivity (lir) was confirmed in the pineal gland and the medial SCN, which are critical circadian pacemakers. We also observed strong immunoreactivity in the MBH, including the ependymal cells (ECs), the infundibular nucleus (IN), the median eminence (ME), and the adjacent PT. Furthermore, semiquantitative analysis suggested that BMAL1-lir shows daily fluctuation in these regions. It is possible that circadian clocks in the photoperiodic signal transduction machinery such as the PT and the EC may be involved in the regulation of photoperiodism.
CS mice show unique properties of circadian rhythms: unstable free-running periods and distinct b... more CS mice show unique properties of circadian rhythms: unstable free-running periods and distinct bimodal rhythms (similar to rhythm splitting, but hereafter referred to as bimodal rhythms) under constant darkness. In the present study, we compared clock-related gene expression (mPer1, mBmal1 and Dbp) in the SCN and peripheral tissues (liver, adrenal gland and heart) between CS and C57BL/6J mice. In spite of normal robust oscillation in the SCN of both mice, behavioral rhythms and peripheral rhythms of clock-related genes were significantly different between these mice. However, when daytime restricted feeding was given, no essential differences between the two strains were observed. These results indicate that unusual circadian behaviors and peripheral gene expression in CS mice do not depend on the SCN but rather mechanisms outside of the SCN.
In most organisms living in temperate zones, reproduction is under photoperiodic control. Althoug... more In most organisms living in temperate zones, reproduction is under photoperiodic control. Although photoperiodic time measurement has been studied in organisms ranging from plants to vertebrates, the underlying molecular mechanism is not well understood. The Japanese quail (Coturnix japonica) represents an excellent model to study this problem because of the rapid and dramatic photoperiodic response of its hypothalamic-pituitary-gonadal axis. Recent investigations of Japanese quail show that long-day-induced type 2 deiodinase (Dio2) expression in the mediobasal hypothalamus (MBH) plays an important role in the photoperiodic gonadal regulation by catalyzing the conversion of the prohormone thyroxine (T(4)) to bioactive 3,5,3'-triiodothyronine (T3). The T3 content in the MBH is approximately 10-fold higher under long than short days and conditions, and the intracerebroventricular infusion of T3 under short days and conditions mimics the photoperiodic gonadal response. While Dio2 generates active T3 from T4 by outer ring deiodination, type 3 deiodinase (Dio3) catalyzes the conversion of both T3 and T4 into inactive forms by inner ring deiodination. In contrast to Dio2 expression, Dio3 expression in the MBH is suppressed under the long-day condition. Photoperiodic changes in the expression of both genes during the photoinduction process occur before the changes in the level of luteinizing hormone (LH) secretion, suggesting that the reciprocal changes in Dio2 and Dio3 expression act as gene switches of the photoperiodic molecular cascade to trigger induction of LH secretion.
In temperate zones, animals use changes in day length as a calendar to time their breeding season... more In temperate zones, animals use changes in day length as a calendar to time their breeding season. However, the photoreceptive and neuroendocrine mechanisms of seasonal reproduction are considered to differ markedly between birds and mammals. This can be understood from the fact that the eye is the only photoreceptive organ, and melatonin mediates the photoperiodic information in mammals, whereas in birds, photoperiodic information is directly received by the deep brain photoreceptors and melatonin is not involved in seasonal reproduction. Recent molecular and functional genomics analysis uncovered the gene cascade regulating seasonal reproduction in birds and mammals. Long day-induced thyroid stimulating hormone in the pars tuberalis of the pituitary gland regulates thyroid hormone catabolism within the mediobasal hypothalamus. Further, this local thyroid hormone catabolism appears to regulate seasonal gonadotropin-releasing hormone secretion. These findings suggest that although the light input pathway is different between birds and mammals (i.e. light or melatonin), the core mechanisms are conserved in these vertebrates.
Administration of vitamin B12 (VB12) has been reported to normalize human sleep-wake rhythm disor... more Administration of vitamin B12 (VB12) has been reported to normalize human sleep-wake rhythm disorders such as non-24-h sleep-wake syndrome (HNS), delayed sleep phase syndrome (DSPS) or insomnia. However, the mechanisms of the action of VB 12 on the ...
Appropriate timing of various seasonal processes is crucial to the survival and reproductive succ... more Appropriate timing of various seasonal processes is crucial to the survival and reproductive success of animals inhabiting temperate regions. When seasonally breeding animals are subjected to annual changes in daylength, dramatic changes in neuroendocrine-gonadal ...
It is known that retinally degenerate C57BL/6J (rd/rd) mice have unattenuated circadian photosens... more It is known that retinally degenerate C57BL/6J (rd/rd) mice have unattenuated circadian photosensitivity. However, the authors have previously found that CBA/J (rd/rd) mice that carry the same rd mutation have attenuated circadian photosensitivity compared to normal CBA/N (+/+) mice. In the present study, a quantitative trait locus (QTL) analysis using C57BL/6J (rd/rd) and CBA/J (rd/rd) mice was conducted in order to identify the genes affecting circadian photosensitivity of the rd mice. As a result, several putative QTLs onthree separate chromosomes (8, 12, 17) were detected, which indicates that circadian photosensitivity in rd mice is altered by multiple genes. Identification of these genes may provide new insights into the understanding of regulation of circadian photoentrainment and sleep-wake disorders.
The molecular mechanism underlying photoperiodism is not well understood in any organism. Long-da... more The molecular mechanism underlying photoperiodism is not well understood in any organism. Long-day-induced conversion of prohormone T(4) to bioactive T(3) within the mediobasal hypothalamus (MBH) is critical for the photoperiodic regulation of reproduction. However, because thyroidectomy does not completely block the photoperiodic response in some species, the existence of a thyroid hormone-independent regulatory mechanism appears certain. To identify this novel mechanism, differential subtractive hybridization analysis was performed using MBH of quail kept under short-day and long-day conditions. This analysis identified a gene encoding TGFalpha. Expression of TGFalpha mRNA was induced in the median eminence by the stimulus of long days, and this induction was observed at dusk on the first long day. This rapid induction of TGFalpha mRNA was similar to induction of the thyroid hormone-activating enzyme gene [Dio2 (type 2 iodothyronine deiodinase)], which is the earliest event yet determined in the photo-induction process. Expression analysis of epidermal growth factor receptors revealed strong expression of erbB4 and weak expression of erbB1 and erbB2 in the median eminence. Intracerebroventricular infusion of physiological dose of TGFalpha induced LH secretion and testicular growth under short-day conditions. Finally, we demonstrate that T(3) implantation and TGFalpha infusion into the MBH, either of which causes testicular growth, do not affect the expression of TGFalpha and Dio2, respectively. Thus, long-day-induced activation of the TGFalpha signaling pathway appears to mediate a thyroid hormone-independent pathway for the photoperiodic regulation of reproduction.
L-Glutamate is a major neurotransmitter at the excitatory synapses in the vertebrate brain. It is... more L-Glutamate is a major neurotransmitter at the excitatory synapses in the vertebrate brain. It is also the excitatory neurotransmitter at neuromuscular junctions in insects, however its functions in their brains remain to be established. We identified and characterized two different subtypes (AmGluRA and AmGluRB) of metabotropic glutamate receptors (mGluRs) from an eusocial insect, honeybee. Both AmGluRA and AmGluRB form homodimers independently on disulfide bonds, and bind [3H]glutamate with K(D) values of 156.7 and 80.7 nM, respectively. AmGluRB is specifically expressed in the brain, while AmGluRA is expressed in the brain and other body parts, suggesting that AmGluRA is also present at the neuromuscular junctions. Both mGluRs are expressed in the mushroom bodies and the brain regions of honeybees, where motor neurons are clustered. Their expression in the brain apparently overlaps, suggesting that they may interact with each other to modulate the glutamatergic neurotransmission.
Using the magnitude of phase shift of circadian locomotor rhythms induced by a single pulse of wh... more Using the magnitude of phase shift of circadian locomotor rhythms induced by a single pulse of white fluorescent light, we compared the sensitivity of the circadian system to light in retinally degenerate mice and in normal mice. In the first experiment, phase response curves (PRCs) for 10-lux white light were generated in CBA/J mice with retinal degeneration (rd/rd) and CBA/N mice with normal retinas (+/+). Although large phase delays early in the subjective night and small phase advances in the late subjective night were observed in CBA/N mice, CBA/J mice showed only small phase delays early in the subjective night. In the second experiment, we found that the magnitude of phase shifts at circadian time (CT) 16 for delays and CT 24 for advances in CBA/J mice became larger with increasing light intensity, and that CBA/J mice could show the same amount of phase shift as CBA/N mice when higher intensities were used. These findings indicate that the differences in the shapes of PRCs are not due to differences in the nature of the oscillating system, but to differences in circadian photosensitivity between these strains. Because the genetic background for the rd loci was not completely identical in the CBA/N and CBA/J mice, it was possible that genes other than the rd gene might have caused different photosensitivity in these mice. Therefore, in the last experiment, we studied the circadian photosensitivity in F1 hybrids between CBA/N and CBA/J mice and in the backcross progeny with different genotypes (+/rd and rd/rd) obtained from the crossing between F1 hybrids between CBA/N and CBA/J mice and in the backcross progeny with different genotypes (+/rd and rd/rd) obtained from the crossing between F1 and CBA/J mice. In these mice with heterogeneous genetic backgrounds as well, mice with retinal degeneration were always less sensitive to light, suggesting that reduced circadian photosensitivity is caused by retinal degeneration. These results are discussed in relation to recent findings in retinally degenerate C57BL mice, which have been found to have normal circadian sensitivity to light.
Using the mPer1::luc real-time monitoring technique, the authors observed the bimodal patterns of... more Using the mPer1::luc real-time monitoring technique, the authors observed the bimodal patterns of mPer1 bioluminescence on each side of the SCN, in parallel with maintaining synchronization between the left and right sides of the SCN under an artificial light:dark:light:dark (LDLD) 7:5:7:5 condition. In situ hybridization analysis of mPer1 and mBmal1 mRNA distribution in the SCN showed that in 1 photophase (morning photophase; M) of LDLD, the mPer1 level in the ventrolateral-like (VL-like) subdivision of the SCN was higher than that in the dorsomedial-like (DM-like) subdivision, and this regional distribution pattern was reversed in another photophase (evening photophase; E). In contrast, the mBmal1 level was higher in the DM-like subdivision than in the VL-like subdivision in the M phase, and this distribution changed in the E phase. The prokineticin 2 (PK2) mRNA that encodes an SCN output molecule that is thought to transmit the circadian locomotor rhythms was reduced in both the DM-like and VL-like SCN and did not clearly correlate with the activity under the LDLD condition. The expression of mPer1 and mPer2 in the liver was clearly bimodal, whereas the expressions of other clock genes were not synchronized to the LDLD condition. These results may provide important insights into the mechanism underlying the splitting or bimodal rhythms that may in turn facilitate the understanding of the ability to measure the seasonal day length in mammals.
In mice, genetic differences between inbred strains have been shown for several parameters of sle... more In mice, genetic differences between inbred strains have been shown for several parameters of sleep and circadian activity rhythms. Our previous studies have demonstrated that CS mice have three remarkable characteristics in the circadian rhythm of locomotor activity: (1) high activity both during the day and night, (2) unstable freerunning period and (3) spontaneous rhythm splitting. In order to characterize sleep properties of CS mice, we compared circadian sleep patterns of CS with those of C57BL/6J and C3H/He mice which have normal circadian activity rhythms. Although C57BL/6J and C3H/He mice exhibited clear daily sleep-wake rhythms in the amount of each sleep parameter (Awake, SWS, PS), CS mice did not show clear rhythms in these parameters. The differences were particularly conspicuous in PS; no apparent day-night differences in the amount of PS, PS counts and PS interval (the interval between successive PS episodes) in CS mice. In addition, the ratio of PS to total sleep time was significantly larger in CS mice than other strains. Of these parameters, the most considerable was PS latency which was extremely short and direct transition from Awake to PS without appearance of SWS frequently occurred in these mice. These results indicate that CS mice may be useful for the understanding of sleep mechanisms and its dysfunction.
The avian perivitelline layer (PL), a vestment homologous to the zona pellucida (ZP) of mammalian... more The avian perivitelline layer (PL), a vestment homologous to the zona pellucida (ZP) of mammalian oocytes, is composed of at least three glycoproteins. Our previous studies have demonstrated that the matrix's components, ZP3 and ZPD, are synthesized in ovarian granulosa cells. Another component, ZP1, is synthesized in the liver and is transported to the ovary by blood circulation. In this study, we report the isolation of cDNA encoding quail ZP2 and its expression in the female bird. By RNase protection assay and in situ hybridization, we demonstrate that ZP2 transcripts are restricted to the oocytes of small white follicles (SWF). The expression level of ZP2 decreased dramatically during follicular development, and the highest expression was observed in the SWF. Western blot and immunohistochemical analyses using the specific antibody against ZP2 indicate that the 80 kDa protein is the authentic ZP2, and the immunoreactive ZP2 protein is also present in the oocytes. Moreover, ultrastructural analysis demonstrated that the immunoreactive ZP2 localizes to the zona radiata, the perivitelline space, and the oocyte cytoplasm in the SWF. By means of western blot analysis and immunofluorescence microscopy, we detected a possible interaction of the recombinant ZP2 with ZP3 and that this interaction might lead to the formation of amorphous structure on the cell surface. These results demonstrate for the first time that the avian ZP gene is expressed in the oocyte, and that the ZP2 protein in the oocyte might play a role for the PL formation in the immature follicles of the ovary.
The circadian clock is a fundamental property of living organisms and is involved in seasonal (ph... more The circadian clock is a fundamental property of living organisms and is involved in seasonal (photoperiodic) time measurement. Among vertebrates, birds have multiple circadian pacemakers in the eye, the pineal gland, and the suprachiasmatic nucleus (SCN), and have highly sophisticated photoperiodic mechanisms. However, because the removal of these circadian pacemakers fails to abolish the photoperiodic response, the existence of another "photoperiodic clock" has been suggested. Recent studies have revealed that the mediobasal hypothalamus (MBH) and the adjacent pars tuberalis (PT) of the pituitary gland constitute key components of the photoperiodic signal transduction machinery. In the present study, we generated a polyclonal antibody against the chicken circadian clock protein BMAL1 to examine BMAL1 distribution in the Japanese quail brain by using immunohistochemistry. BMAL1-like immunoreactivity (lir) was confirmed in the pineal gland and the medial SCN, which are critical circadian pacemakers. We also observed strong immunoreactivity in the MBH, including the ependymal cells (ECs), the infundibular nucleus (IN), the median eminence (ME), and the adjacent PT. Furthermore, semiquantitative analysis suggested that BMAL1-lir shows daily fluctuation in these regions. It is possible that circadian clocks in the photoperiodic signal transduction machinery such as the PT and the EC may be involved in the regulation of photoperiodism.
CS mice show unique properties of circadian rhythms: unstable free-running periods and distinct b... more CS mice show unique properties of circadian rhythms: unstable free-running periods and distinct bimodal rhythms (similar to rhythm splitting, but hereafter referred to as bimodal rhythms) under constant darkness. In the present study, we compared clock-related gene expression (mPer1, mBmal1 and Dbp) in the SCN and peripheral tissues (liver, adrenal gland and heart) between CS and C57BL/6J mice. In spite of normal robust oscillation in the SCN of both mice, behavioral rhythms and peripheral rhythms of clock-related genes were significantly different between these mice. However, when daytime restricted feeding was given, no essential differences between the two strains were observed. These results indicate that unusual circadian behaviors and peripheral gene expression in CS mice do not depend on the SCN but rather mechanisms outside of the SCN.
In most organisms living in temperate zones, reproduction is under photoperiodic control. Althoug... more In most organisms living in temperate zones, reproduction is under photoperiodic control. Although photoperiodic time measurement has been studied in organisms ranging from plants to vertebrates, the underlying molecular mechanism is not well understood. The Japanese quail (Coturnix japonica) represents an excellent model to study this problem because of the rapid and dramatic photoperiodic response of its hypothalamic-pituitary-gonadal axis. Recent investigations of Japanese quail show that long-day-induced type 2 deiodinase (Dio2) expression in the mediobasal hypothalamus (MBH) plays an important role in the photoperiodic gonadal regulation by catalyzing the conversion of the prohormone thyroxine (T(4)) to bioactive 3,5,3'-triiodothyronine (T3). The T3 content in the MBH is approximately 10-fold higher under long than short days and conditions, and the intracerebroventricular infusion of T3 under short days and conditions mimics the photoperiodic gonadal response. While Dio2 generates active T3 from T4 by outer ring deiodination, type 3 deiodinase (Dio3) catalyzes the conversion of both T3 and T4 into inactive forms by inner ring deiodination. In contrast to Dio2 expression, Dio3 expression in the MBH is suppressed under the long-day condition. Photoperiodic changes in the expression of both genes during the photoinduction process occur before the changes in the level of luteinizing hormone (LH) secretion, suggesting that the reciprocal changes in Dio2 and Dio3 expression act as gene switches of the photoperiodic molecular cascade to trigger induction of LH secretion.
In temperate zones, animals use changes in day length as a calendar to time their breeding season... more In temperate zones, animals use changes in day length as a calendar to time their breeding season. However, the photoreceptive and neuroendocrine mechanisms of seasonal reproduction are considered to differ markedly between birds and mammals. This can be understood from the fact that the eye is the only photoreceptive organ, and melatonin mediates the photoperiodic information in mammals, whereas in birds, photoperiodic information is directly received by the deep brain photoreceptors and melatonin is not involved in seasonal reproduction. Recent molecular and functional genomics analysis uncovered the gene cascade regulating seasonal reproduction in birds and mammals. Long day-induced thyroid stimulating hormone in the pars tuberalis of the pituitary gland regulates thyroid hormone catabolism within the mediobasal hypothalamus. Further, this local thyroid hormone catabolism appears to regulate seasonal gonadotropin-releasing hormone secretion. These findings suggest that although the light input pathway is different between birds and mammals (i.e. light or melatonin), the core mechanisms are conserved in these vertebrates.
Administration of vitamin B12 (VB12) has been reported to normalize human sleep-wake rhythm disor... more Administration of vitamin B12 (VB12) has been reported to normalize human sleep-wake rhythm disorders such as non-24-h sleep-wake syndrome (HNS), delayed sleep phase syndrome (DSPS) or insomnia. However, the mechanisms of the action of VB 12 on the ...
Appropriate timing of various seasonal processes is crucial to the survival and reproductive succ... more Appropriate timing of various seasonal processes is crucial to the survival and reproductive success of animals inhabiting temperate regions. When seasonally breeding animals are subjected to annual changes in daylength, dramatic changes in neuroendocrine-gonadal ...
It is known that retinally degenerate C57BL/6J (rd/rd) mice have unattenuated circadian photosens... more It is known that retinally degenerate C57BL/6J (rd/rd) mice have unattenuated circadian photosensitivity. However, the authors have previously found that CBA/J (rd/rd) mice that carry the same rd mutation have attenuated circadian photosensitivity compared to normal CBA/N (+/+) mice. In the present study, a quantitative trait locus (QTL) analysis using C57BL/6J (rd/rd) and CBA/J (rd/rd) mice was conducted in order to identify the genes affecting circadian photosensitivity of the rd mice. As a result, several putative QTLs onthree separate chromosomes (8, 12, 17) were detected, which indicates that circadian photosensitivity in rd mice is altered by multiple genes. Identification of these genes may provide new insights into the understanding of regulation of circadian photoentrainment and sleep-wake disorders.
The molecular mechanism underlying photoperiodism is not well understood in any organism. Long-da... more The molecular mechanism underlying photoperiodism is not well understood in any organism. Long-day-induced conversion of prohormone T(4) to bioactive T(3) within the mediobasal hypothalamus (MBH) is critical for the photoperiodic regulation of reproduction. However, because thyroidectomy does not completely block the photoperiodic response in some species, the existence of a thyroid hormone-independent regulatory mechanism appears certain. To identify this novel mechanism, differential subtractive hybridization analysis was performed using MBH of quail kept under short-day and long-day conditions. This analysis identified a gene encoding TGFalpha. Expression of TGFalpha mRNA was induced in the median eminence by the stimulus of long days, and this induction was observed at dusk on the first long day. This rapid induction of TGFalpha mRNA was similar to induction of the thyroid hormone-activating enzyme gene [Dio2 (type 2 iodothyronine deiodinase)], which is the earliest event yet determined in the photo-induction process. Expression analysis of epidermal growth factor receptors revealed strong expression of erbB4 and weak expression of erbB1 and erbB2 in the median eminence. Intracerebroventricular infusion of physiological dose of TGFalpha induced LH secretion and testicular growth under short-day conditions. Finally, we demonstrate that T(3) implantation and TGFalpha infusion into the MBH, either of which causes testicular growth, do not affect the expression of TGFalpha and Dio2, respectively. Thus, long-day-induced activation of the TGFalpha signaling pathway appears to mediate a thyroid hormone-independent pathway for the photoperiodic regulation of reproduction.
L-Glutamate is a major neurotransmitter at the excitatory synapses in the vertebrate brain. It is... more L-Glutamate is a major neurotransmitter at the excitatory synapses in the vertebrate brain. It is also the excitatory neurotransmitter at neuromuscular junctions in insects, however its functions in their brains remain to be established. We identified and characterized two different subtypes (AmGluRA and AmGluRB) of metabotropic glutamate receptors (mGluRs) from an eusocial insect, honeybee. Both AmGluRA and AmGluRB form homodimers independently on disulfide bonds, and bind [3H]glutamate with K(D) values of 156.7 and 80.7 nM, respectively. AmGluRB is specifically expressed in the brain, while AmGluRA is expressed in the brain and other body parts, suggesting that AmGluRA is also present at the neuromuscular junctions. Both mGluRs are expressed in the mushroom bodies and the brain regions of honeybees, where motor neurons are clustered. Their expression in the brain apparently overlaps, suggesting that they may interact with each other to modulate the glutamatergic neurotransmission.
Using the magnitude of phase shift of circadian locomotor rhythms induced by a single pulse of wh... more Using the magnitude of phase shift of circadian locomotor rhythms induced by a single pulse of white fluorescent light, we compared the sensitivity of the circadian system to light in retinally degenerate mice and in normal mice. In the first experiment, phase response curves (PRCs) for 10-lux white light were generated in CBA/J mice with retinal degeneration (rd/rd) and CBA/N mice with normal retinas (+/+). Although large phase delays early in the subjective night and small phase advances in the late subjective night were observed in CBA/N mice, CBA/J mice showed only small phase delays early in the subjective night. In the second experiment, we found that the magnitude of phase shifts at circadian time (CT) 16 for delays and CT 24 for advances in CBA/J mice became larger with increasing light intensity, and that CBA/J mice could show the same amount of phase shift as CBA/N mice when higher intensities were used. These findings indicate that the differences in the shapes of PRCs are not due to differences in the nature of the oscillating system, but to differences in circadian photosensitivity between these strains. Because the genetic background for the rd loci was not completely identical in the CBA/N and CBA/J mice, it was possible that genes other than the rd gene might have caused different photosensitivity in these mice. Therefore, in the last experiment, we studied the circadian photosensitivity in F1 hybrids between CBA/N and CBA/J mice and in the backcross progeny with different genotypes (+/rd and rd/rd) obtained from the crossing between F1 hybrids between CBA/N and CBA/J mice and in the backcross progeny with different genotypes (+/rd and rd/rd) obtained from the crossing between F1 and CBA/J mice. In these mice with heterogeneous genetic backgrounds as well, mice with retinal degeneration were always less sensitive to light, suggesting that reduced circadian photosensitivity is caused by retinal degeneration. These results are discussed in relation to recent findings in retinally degenerate C57BL mice, which have been found to have normal circadian sensitivity to light.
Using the mPer1::luc real-time monitoring technique, the authors observed the bimodal patterns of... more Using the mPer1::luc real-time monitoring technique, the authors observed the bimodal patterns of mPer1 bioluminescence on each side of the SCN, in parallel with maintaining synchronization between the left and right sides of the SCN under an artificial light:dark:light:dark (LDLD) 7:5:7:5 condition. In situ hybridization analysis of mPer1 and mBmal1 mRNA distribution in the SCN showed that in 1 photophase (morning photophase; M) of LDLD, the mPer1 level in the ventrolateral-like (VL-like) subdivision of the SCN was higher than that in the dorsomedial-like (DM-like) subdivision, and this regional distribution pattern was reversed in another photophase (evening photophase; E). In contrast, the mBmal1 level was higher in the DM-like subdivision than in the VL-like subdivision in the M phase, and this distribution changed in the E phase. The prokineticin 2 (PK2) mRNA that encodes an SCN output molecule that is thought to transmit the circadian locomotor rhythms was reduced in both the DM-like and VL-like SCN and did not clearly correlate with the activity under the LDLD condition. The expression of mPer1 and mPer2 in the liver was clearly bimodal, whereas the expressions of other clock genes were not synchronized to the LDLD condition. These results may provide important insights into the mechanism underlying the splitting or bimodal rhythms that may in turn facilitate the understanding of the ability to measure the seasonal day length in mammals.
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