Biomedical subjects

Motoharu Hayashi

Publications and source records attributed to Motoharu Hayashi.

At least 19 recordsLinked to original sources

PubMed2006

Expression of protein kinase C-substrate mRNAs in the basal ganglia of adult and infant macaque monkeys.

We performed in situ hybridization histochemistry on the monkey basal ganglia to investigate the mRNA localization of three protein kinase C substrates (GAP-43, MARCKS, and neurogranin), of which expression plays a role in structural changes in neurites and synapses. Weak hybridization signals for GAP-43 mRNA and intense signals for both MARCKS and neurogranin mRNAs were observed in the adult neostriatum. All three of the mRNAs were expressed in both substance P-positive direct pathway neurons and enkephalin-positive indirect pathway neurons. In the nucleus accumbens, the hybridization signals for the three mRNAs were weaker than those in the neostriatum. Double-label in situ hybridization histochemistry in the neostriatum revealed that GAP-43 and neurogranin mRNAs were expressed in a subset of MARCKS-positive neurons. While intense hybridization signals for MARCKS mRNA were observed in all of the other basal ganglia regions such as the globus pallidus, substantia innominata, subthalamic nucleus, and substantia nigra, intense signals for GAP-43 mRNA were restricted to the substantia innominata and substantia nigra pars compacta. No signal for neurogranin mRNA was observed in the basal ganglia regions outside the neostriatum and the nucleus accumbens. These results indicate that the protein kinase C substrates are abundant in some specific connections in cortico-basal ganglia circuits. Developmental analysis showed that the expression level in the putamen and nucleus accumbens, but not in the caudate nucleus, was higher in the infant than in the adult, suggesting that synaptic maturation in the caudate nucleus occurs earlier than that in the putamen and nucleus accumbens.

Age Factors↗

PubMed2006

Northern blot and in situ hybridization analyses for the neurogranin mRNA in the developing monkey cerebral cortex.

Neurogranin is a postsynaptic substrate for protein kinase C, and its expression is related to dendritic spine development and postsynaptic plasticity. Using both Northern blot analysis and in situ hybridization techniques, we investigated the developmental changes of neurogranin expression in the monkey cerebral cortex. In each of four neocortical areas examined, i.e., the prefrontal area (area FD of von Bonin and Bailey), the temporal association area (TE), the primary somatosensory area (PB), and the primary visual area (OC), the Northern blot analysis showed that the amount of neurogranin mRNA was low during the prenatal and perinatal periods until postnatal day 8. It increased during postnatal development and reached its peak value at postnatal day 70 (in area OC) or postnatal month 6 (in area FD, TE, and PB). After that, the amount of neurogranin mRNA in the cerebral neocortex decreased gradually until postnatal years 2-3. The in situ hybridization experiments also showed a transient increase of neurogranin mRNA in the neocortex during postnatal day 70 to postnatal month 6. The transient increase was prominent in layers II and III of areas FD and TE; deep in layer III of area PB; and in layers II, III, and IV of area OC. In the hippocampus, in contrast to the results in the neocortex, the expression of neurogranin mRNA was decreased almost continuously during the postnatal period. The transiently increased expression of neurogranin in the postnatal neocortex may be a molecular basis for the postsynaptic modification of afferent inputs possibly from subcortical structures.

Age Factors↗

PubMed2006

TrkB-T1 regulates the RhoA signaling and actin cytoskeleton in glioma cells.

Recently, the truncated TrkB receptor, T1, has been reported to be involved in the control of cell morphology via the regulation of Rho proteins, through which T1 binds Rho guanine nucleotide dissociation inhibitor (Rho GDI) 1 and dissociates it in a brain-derived neurotrophic factor (BDNF)-dependent manner. However, it is unclear whether T1 signaling regulates the downstream of Rho signaling and the actin cytoskeleton. In this study, we investigated this question using C6 rat glioma cells, which express T1 endogenously. Rho GDI1 was dissociated from T1 in a BDNF-dependent manner, which also causes decreases in the activities of Rho-signaling molecules such as RhoA, Rho-associated kinase, p21-activated kinase, and extracellular-signal regulated kinase1/2. Moreover, BDNF treatment resulted in the disappearance of stress fibers in the cells treated with lysophosphatidic acid, an activator of RhoA, and in morphological changes in cells. Furthermore, a competitive assay with cyan fluorescent protein fusion proteins of T1-specific sequences reduced the effects of BDNF. These results suggest that T1 regulates the Rho-signaling pathways and the actin cytoskeleton.

Actins↗

PubMed2006

Distribution and pharmacological characterization of primate NK-1 and NK-3 tachykinin receptors in the central nervous system of the rhesus monkey.

Much attention has focused on tachykinin receptors as therapeutic targets for neuropsychiatric disorders, although their expressional distributions in the primate central nervous system (CNS) remain unclear. We cloned the genes encoding the NK-1 and NK-3 tachykinin receptors (referred to as rmNK-1 and rmNK-3) from the rhesus monkey (Macaca mulatta) brain and examined their pharmacological profiles and regional distributions in the CNS. The deduced rmNK-1 amino-acid sequence differed by only two amino acids from the human NK-1 (hNK-1). The deduced rmNK-3 amino-acid sequence was two amino acids shorter than human NK-3 (hNK-3), with a seven-amino-acid difference in sequence. Ligand binding studies revealed that the affinity of rmNK-1 to substance P (SP) was comparable to that of hNK-1 in cell lines that expressed individual receptors stably. Nonpeptide antagonists had similar effects on the binding of rmNK-1 and hNK-1. Affinity of rmNK-3 for NKB was stronger than for SP and the IC50 value was comparable with that of hNK-3. Ca2+ imaging showed that activations of both rmNK-1 and rmNK-3 by specific ligands, SP and senktide, induced increased intracellular Ca2+ in cell lines that stably expressed individual primate tachykinin receptors. The amounts of rmNK-1 and rmNK-3 mRNAs were quantitatively determined in the monkey CNS. The expression of rmNK-1 was observed in all of the cortical and subcortical regions, including the hippocampus and the amygdala. The putamen contained the most NK-1 mRNA in the brain, with less rmNK-3 mRNA found in the cortex compared to rmNK-1 mRNA. In the monkey hippocampus and amygdala, rmNK-1 mRNA was present at markedly higher concentrations than rmNK-3 mRNA. The present results provide an insight into the distinct physiological nature and significance of the NK-1 and NK-3 tachykinin systems in the primate CNS. These findings are indispensable for establishing model systems in the search for a subtype-specific tachykinin receptor agonist and antagonist for the treatment of neuropsychiatric disorders.

Amino Acid Sequence↗

PubMed2006

Decreases of calcium and phosphorus in monkey cardiac walls with development and aging.

To elucidate compositional changes of the cardiac walls with development and aging, the authors investigated changes of elements in the atrial and ventricular walls of monkeys. The left and right atrial walls, left and right ventricular walls, and interatrial and interventricular septa were resected from the subjects. The subjects consisted of 17 rhesus and 13 Japanese monkeys, ranging in age from 10 d to 33 yr. The element content of the cardiac walls was analyzed by inductively coupled plasma-atomic emission spectrometry. The Ca and P contents decreased in all of the left and right atrial and ventricular walls, interatrial septa, and interventricular septa with development, whereas the S and Mg contents decreased in the left and right ventricular walls with development. Regarding the relationships among elements, significant direct correlations were found among Ca, P, Mg, and Zn in all of the left and right atrial walls, left and right ventricular walls, and interatrial and interventricular septa, with some exceptions. As Ca decreased in the cardic walls, P, Mg, and Zn decreased simultaneously in the cardiac walls. The mass ratio of Ca/P decreased gradually with Ca decrease in both the atrial and ventricular walls, but it was not constant.

Aging↗

PubMed2005

Heterogeneity of the developmental patterns of neurotrophin protein levels among neocortical areas of macaque monkeys.

Based on morphological and physiological characteristics, the mammalian neocortex is divided into various neocortical areas and its diversity is prominent in the primates including humans. These neocortical areas are constructed during development, but the details of the developmental events remain unclear, especially at the molecular level. We measured the mRNA and protein levels of neurotrophins in various neocortical areas of developing rhesus monkeys. The expression patterns of both the neurotrophin-3 (NT-3) mRNA and the protein showed area differences. In the sensory and motor areas, NT-3 mRNA and protein levels had started to decline by a week after birth. In contrast, the levels declined after the third postnatal week in the association neocortical areas. The level of brain-derived neurotrophic factor (BDNF) protein changed in an area-dependent manner during development, but that of mRNA did not. The decline of the BDNF protein level started earlier in the sensory and motor neocortical areas than in the association neocortical areas, suggesting that sensory and motor neocortical areas develop earlier than the association areas in terms of the developmental changes in neurotrophins.

Animals↗

PubMed2005

Developmental changes in concentrations and distributions of neurotrophins in the monkey cerebellar cortex.

Neurotrophins are involved in the survival, differentiation, migration and neurite outgrowth of various neuronal populations. Neurotrophins and their receptors are widely expressed in the developing cerebellum of various experimental animals. To gain some insight into the possible roles played by these molecules in monkey cerebellum, we examined the protein levels of BDNF, NT-4/5 and NT-3 and distributions of those neurotrophins and TrkC, a high affinity receptor for NT-3, in the cerebellum of developing macaque monkeys using ELISAs and immunohistochemical methods. We found that the level of BDNF increased during development, while the level of NT-3 was higher during embryonic stages and decreased toward adulthood. The level of NT-4/5 increased from embryonic stages to infant stages and gradually declined with age. Among the three neurotrophins, BDNF immunoreactivity was found in all kinds of cerebellar neurons, including all inhibitory interneurons, throughout the postnatal periods examined, indicating that BDNF may be an essential factor for the maintenance of cerebellar neural functions. The Bergmann glial fibers and the internal part of the external granule cell layer were strongly NT-3 immunopositive at the early postnatal stages, and more weakly immunoreactive toward adulthood. In addition, we found that the premigratory precursors of the granule cells were TrkC immunopositive at early postnatal stages. These findings suggest that NT-3 in Bergmann glial fibers may be involved in the migration of the premigratory granule cells.

Animals↗

PubMed2005

Differential expression of the truncated TrkB receptor, T1, in the primary motor and prefrontal cortices of the adult macaque monkey.

A truncated TrkB receptor, T1, which is one of the receptors for brain-derived neurotrophic factor, has been shown to regulate the morphology of neurons and glial cells in primary cultures and/or slices overexpressing T1 in the recent past. However, in vivo localization of T1 at protein level remains unclear. In the present study, we examined the localization of T1 in the primary motor and prefrontal cortices of adult monkeys by using immunohistochemistry. In the primary motor cortex, T1 immunoreactivity was observed mainly in the pyramidal neurons of layers II-VI, especially Betz cells of layer V. The apical and basal dendrites and cell bodies of Betz cells were strongly stained. In addition, we found that the interneurons were also T1-immunopositive and that there were no T1-positive astrocytes. In the prefrontal cortex, we observed strong immunoreactivity of T1 in astrocytes as well as pyramidal neurons of layer V. The pyramidal neurons and interneurons in layers II/III were faintly immunoreactive for T1. Thus, these findings, together with the fact that T1 is involved in morphological control of neurons and glial cells, suggest that the prefrontal cortex might possess a different degree of morphological plasticity than the primary motor cortex in the adult monkey.

Animals↗

PubMed2005

A truncated tropomyosin-related kinase B receptor, T1, regulates glial cell morphology via Rho GDP dissociation inhibitor 1.

Through tropomyosin-related kinase B (TrkB) receptors, brain-derived neurotrophic factor (BDNF) performs many biological functions such as neural survival, differentiation, and plasticity. T1, an isoform of TrkB receptors that lacks a tyrosine kinase, predominates in the adult mammalian CNS, yet its role remains controversial. In this study, to examine whether T1 transduces a signal and to determine its function, we first performed an affinity purification of T1-binding protein with the T1-specific C-terminal peptide and identified Rho GDP dissociation inhibitor 1 (GDI1), a GDP dissociation inhibitor of Rho small G-proteins, as a signaling protein directly associated with T1. The binding of BDNF to T1 caused Rho GDI1 to dissociate from the C-terminal tail of T1. Astrocytes cultured for 30 d expressed only endogenous T1 among the BDNF receptors. In 30 d cultured astrocytes, Rho GDI1, when dissociated in a BDNF-dependent manner, controlled the activities of the Rho GTPases, which resulted in rapid changes in astrocytic morphology. Furthermore, using 2 d cultured astrocytes that were transfected with T1, a T1 deletion mutant, or cyan fluorescent protein fusion protein of the T1-specific C-terminal sequence, we demonstrated that T1-Rho GDI1 signaling was indispensable for regulating the activities of Rho GTPases and for the subsequent morphological changes among astrocytes. Therefore, these findings indicate that the T1 signaling cascade can alter astrocytic morphology via regulation of Rho GTPase activity.

Amino Acid Motifs↗

PubMed2005

Comparison of calcium accumulation between the arteries of human and monkey.

To examine whether an accumulation of elements in the arteries was affected by the way of walking, the authors investigated age-related changes of elements in the arteries of the Japanese, Thai, and Japanese monkeys. After the ordinary dissections by medical students were finished, the subclavian, axillary, brachial, radial, common iliac, external iliac, femoral, and posterior tibial arteries were resected from the subjects of the Japanese and Thai over 60 yr of age and they were also resected from the Japanese monkeys over 20 yr of age. The element content was determined by inductively coupled plasma-atomic emission spectrometry. It was found that in an comparison between the arteries of anatomically corresponding regions, the average content of Ca was eight times or four times higher in the arteries of the lower limb than in the arteries of the upper limb in the Japanese or Thai over 60 yr of age, respectively. In the Japanese monkeys over 20 yr of age, the average content of Ca was 1.2 times higher in the arteries of the lower limb than in the arteries of the upper limb. The result suggests that an accumulation of Ca in the arteries of the lower limb with aging is affected by the way of walking.

Adult↗

PubMed2005

Age-dependent decreases of calcium, phosphorus, sulfur, and zinc in the cardiac valves of monkeys.

To elucidate compositional changes of the cardiac valves in monkey with aging, the authors investigated age-related changes of elements in the four cardiac valves of rhesus and Japanese monkeys and the relationships among elements by inductively coupled plasma-atomic emission spectrometry (ICP-AES). The four cardiac valves of the aortic, pulmonary, mitral, and tricuspid valves were resected from 19 rhesus and 11 Japanese monkeys, ranging in age from 10 d to 33 yr. The element contents were determined by ICP-AES. It was found that the Ca, P, S, and Zn contents were high in the four cardiac valves of the monkeys below 1 yr and thereafter decreased significantly with aging, except for Ca in the mitral valve, for which no significant correlation was found between age and Ca content. The Ca and P contents did not increase in the four cardiac valves at old age. This result revealed that calcification scarcely occurred in the four cardiac valves at old age. With regard to the relationships among elements, it was found that there were significant direct correlations among the Ca, P, S, and Zn contents in all of the four cardiac valves of the monkeys, with two exceptions between P and Zn contents in both the aortic and pulmonary valves. Therefore, as Ca decreased in the four cardiac valves, P, S, and Zn decreased simultaneously in the same cardiac valves.

Aging↗

PubMed2005

Age-related attenuation in the elements in monkey sino-atrial node.

Changes in trace elements of the sino-atrial (SA) node with aging was investigated using 24 hearts of the Japanese and rhesus monkeys of ages ranging from 27 d to 30 yr. With aging, sympathetic activity decreases and SA nodal function deteriorates. The SA nodal tissue was removed from the anatomical position and was confirmed by means of histological observation. The elements, such as Ca, P, S, Mg, Na, Fe, and Zn, were analyzed using inductively coupled plasma-atomic emission spectrometry (ICP-AES). Advancing age never increased the contents of the trace elements, but decreased them. The correlation coefficients for the age-dependent attenuations were -0.561 (n = 24, p < 0.01) in Ca and -0.482 (n = 24, p < 0.05) in P. The correlations for the attenuations induced by other trace elements were not significant. Furthermore, close relationships of the elements between Ca and P, S, Zn, or Na contents, between P and Zn or Na contents, and between Zn and Na contents were observed. These results indicate that the elements in the monkey SA node are attenuated with an increase in age, presumably suggesting the age-related suppression of cardiac functions as a result of the histological alterations of the SA nodal cells.

Aging↗

PubMed2004

Differential expression patterns of TrkB ligands in the macaque monkey brain.

We measured the concentrations of brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) in the brains of mature and developing macaque monkeys. In the adult macaque brain, the highest level of BDNF was found in the hippocampus. Comparisons between cortical regions showed higher levels of BDNF in the prefrontal, temporal, and parietal association cortices than elsewhere. In contrast, NT-4 levels were 3-30 times lower than those of BDNF. During development, the contents of BDNF and NT-4 protein in cerebral cortices were highest at the postnatal second month and at embryonic day 140, respectively. These results suggest that the two TrkB ligands, BDNF and NT-4, may be differently regulated and might play separate roles in monkey cortical development.

Animals↗

PubMed2004

Cell type- and region-specific expression of neurogranin mRNA in the cerebral cortex of the macaque monkey.

Neurogranin is a postsynaptic substrate for protein kinase C (PKC). It has been identified in the central nervous system, and the expression has been related to postsynaptic plasticity. Using non-radioactive in situ hybridization histochemistry, we investigated whether mRNA expression of neurogranin varied among the cerebral region and cell types. In most areas of the neocortex excluding area OC (the primary visual area), intense signals were observed in the pyramidal cells in layers III, V and VI. In area OC, intense signals were observed in layers IV as well as layers III and VI. We previously showed that intense signals for GAP-43, a presynaptic PKC substrate, were observed in relay neurons of the lateral geniculate nucleus. From this result and the present result in area OC, we conclude that both pre- and postsynaptic PKC substrates (GAP-43 and neurogranin) are abundant in the geniculocortical synapses. In the hippocampus, intense signals were observed in the pyramidal cells in the subiculum. Taken together with our previous study showing intense signals for GAP-43 in Ammon's horn, the result indicates that both PKC substrates are abundant in the connections between neurons in Ammon's horn and in the subiculum.

Animals↗

PubMed2004

Expression of BDNF and TrkB receptor subtypes in the postnatal developing Purkinje cells of monkey cerebellum.

In the previous study, we have shown the complementary expression of TrkB subtypes (TK+ and T1) in the adult monkey cerebellar cortex. In this study, to clarify when that expression pattern appeared, we examined the expressions of TrkB subtypes and its ligand brain-derived neurotrophic factor (BDNF) by immunohistochemistry and Western blot analysis. At the newborn stage, both TK+ and T1 were expressed uniformly in the cerebellar cortex. At postnatal month 3.5, the uneven expression of TrkB subtypes was observed, while the BDNF immunoreactivity was strongly detected in all regions of the cerebellar cortex. The expression patterns of TrkB subtypes and BDNF at both postnatal month 6 and year 7 were the same as those at postnatal month 3.5. Western blot analysis demonstrated that TK+ and T1 were expressed at high levels in the synaptic membrane from newborn to adult stages. Furthermore, the dimerization of TrkB subtypes changed at postnatal month 3, which was similar to the adult pattern: at the newborn stage, the TK+ and TK- homodimers; after postnatal month 3.5, the TK+ and TK- homodimers, and the TK+/TK- heterodimer. These findings suggest that the localization of TrkB subtypes in each Purkinje would be changed at postnatal month 3.5, resulting in the uneven expression of TrkB subtypes and the change of TrkB dimerization.

Animals↗

PubMed2004

[Molecular mechanisms for the development and aging of the primate central nervous system].

The central nervous system (CNS) of primates, including humans, is more complex than the CNS of other mammals. In particular, the cerebral cortex expands during evolution and this has resulted in the emergence of higher cognitive abilities in primates. Recent neurochemical and neuroanatomical methods have clarified the presence of various neuroactive substances including neuropeptides, neurotrophic factors and growth associated proteins in the developing mammalian cerebral cortex. Among these signal molecules, we have focused on somatostatin (SRIF), neurotrophins (BDNF, NT-4/5 and NT-3) and their receptors (Trk), growth associated proteins such as GAP-43 and SCG-10 during the development and aging of primate CNS. We found that although full-length TrkB, a high affinity receptor for BDNF and NT4/5, was detected from the embryonic stage to adulthood, the level of truncated TrkB which lacks tyrosine kinase domain, only increased after birth. This development of truncated TrkB correlated well with down-regulation in the gene expression of GAP-43 and SCG-10. The reductions of GAP-43 and SCG-10 may result in the elimination of callosal axons in the monkey cerebral cortex after birth. The highest levels of BDNF protein in the various cerebral cortices occurred between postnatal 1 and 6 when the number of synapses is highest. In contrast, there was no transient increase in the levels of NT4/5 or NT3 after birth. These findings suggest that BDNF is one of the candidates for the synaptic development of the primate cerebral cortex. During aging processes, we observed decreases in the levels of SRIF and BDNF mRNAs in the cerebral cortex. Since BDNF is an upstream gene expression molecule of SRIF, the decline of SRIF mRNA during aging may be due to the decrease in the gene expression of BDNF. Similar reductions of gene expressions of SRIF and BDNF in the brains of the patients with Alzheimer's disease, suggest that aged monkeys are good model animals for these neuro-degenerative diseases.

Aging↗

PubMed2003

Cell type- and region-specific expression of protein kinase C-substrate mRNAs in the cerebellum of the macaque monkey.

We performed nonradioactive in situ hybridization histochemistry in the monkey cerebellum to investigate the localization of protein kinase C-substrate (growth-associated protein-43 [GAP-43], myristoylated alanine-rich C-kinase substrate [MARCKS], and neurogranin) mRNAs. Hybridization signals for GAP-43 mRNA were observed in the molecular and granule cell layers of both infant and adult cerebellar cortices. Signals for MARCKS mRNA were observed in the molecular, Purkinje cell, and granule cell layers of both infant and adult cortices. Moreover, both GAP-43 and MARCKS mRNAs were expressed in the external granule cell layer of the infant cortex. In the adult cerebellar vermis, signals for both GAP-43 and MARCKS mRNAs were more intense in lobules I, IX, and X than in the remaining lobules. In the adult hemisphere, both mRNAs were more intense in the flocculus and the dorsal paraflocculus than in other lobules. Such lobule-specific expressions were not prominent in the infant cerebellar cortex. Signals for neurogranin, a postsynaptic substrate for protein kinase C, were weak or not detectable in any regions of either the infant or adult cerebellar cortex. The prominent signals for MARCKS mRNA were observed in the deep cerebellar nuclei, but signals for both GAP-43 and neurogranin mRNAs were weak or not detectable. The prominent signals for both GAP-43 and MARCKS mRNAs were observed in the inferior olive, but signals for neurogranin were weak or not detectable. The cell type- and region-specific expression of GAP-43 and MARCKS mRNAs in the cerebellum may be related to functional specialization regarding plasticity in each type of cell and each region of the cerebellum.

Age Factors↗

PubMed2003

Inhibin B is the major form of inhibin secreted from testes in male Japanese macaques ( Macaca fuscata).

In order to clarify the cellular source and forms of bioactive inhibin in male Japanese macaques ( Macaca fuscata), circulating concentrations of inhibin A and B, and immunohistochemical localization of inhibin subunits in testis were studied. Plasma concentrations of testosterone were also measured. The present study showed that inhibin B was clearly detected in the plasma of male Japanese macaques. Moreover, concentrations of both inhibin B and testosterone during the breeding (mating) season were significantly higher than those of the non-breeding season. On the other hand, plasma inhibin A was detected neither during the breeding seasons nor during the non-breeding seasons. Positive stainings with alpha and betaB subunit antibodies were observed in the Sertoli cells, however staining with betaA subunit antibody was not observed in the testicular samples. These results indicate that inhibin B is the major circulating inhibin and probably secreting from Sertoli cells in male Japanese macaques.

Animals↗