Hirotaka Sakamoto

Platelet-rich plasma enhances the initial mobilization of circulation-derived cells for tendon healing.

Circulation‐derived cells play a crucial role in the healing processes of tissue. In early phases of tendon healing processes, circulation‐derived cells temporarily exist in the wounded area to initiate the healing process and decrease in number with time. We assumed that a delay of time‐dependent decrease in circulation‐derived cells could improve the healing of tendons. In this study, we injected platelet‐rich plasma (PRP) containing various kinds of growth factors into the wounded area of the patellar tendon, and compared the effects on activation of circulation‐derived cells and enhancement of tendon healing with a control group (no PRP injection). To follow the circulation‐derived cells, we used a green fluorescent protein (GFP) chimeric rat expressing GFP in the circulating cells and bone marrow cells. In the PRP group, the numbers of GFP‐positive cells and heat‐shock protein (HSP47; collagen‐specific molecular chaperone)‐positive cells were significantly higher than in the control group at 3 and 7 days after injury. At the same time, the immunoreactivity for types I and III collagen was higher in the PRP group than in the control group at early phase of tendon healing. These findings suggest that locally injected PRP is useful as an activator of circulation‐derived cells for enhancement of the initial tendon healing process. J. Cell. Physiol. 215: 837–845, 2008.

Novel brain function: biosynthesis and actions of neurosteroids in neurons.

Peripheral steroid hormones act on brain tissues through intracellular receptor-mediated mechanisms to regulate several important brain neuronal functions. Therefore, the brain is considered to be a target site of steroid hormones. However, it is now established that the brain itself also synthesizes steroids de novo from cholesterol. The pioneering discovery of Baulieu and his colleagues, using mammals, and our studies with non-mammals have opened the door of a new research field. Such steroids synthesized in the brain are called neurosteroids. Because certain structures in vertebrate brains have the capacity to produce neurosteroids, identification of neurosteroidogenic cells in the brain is essential to understand the physiological role of neurosteroids in brain functions. Glial cells are generally accepted to be the major site for neurosteroid formation, but the concept of neurosteroidogenesis in brain neurons has up to now been uncertain. We recently demonstrated neuronal neurosteroidogenesis in the brain and indicated that the Purkinje cell, a typical cerebellar neuron, actively synthesizes several neurosteroids de novo from cholesterol in both mammals and non-mammals. Pregnenolone sulfate, one of neurosteroids synthesized in the Purkinje neuron, may contribute to some important events in the cerebellum by modulating neurotransmission. Progesterone, produced as a neurosteroid in this neuron only during neonatal life, may be involved in the promotion of neuronal and glial growth and neuronal synaptic contact in the cerebellum. More recently, biosynthesis and actions of neurosteroids in pyramidal neurons of the hippocampus were also demonstrated. These serve an excellent model for the study of physiological roles of neurosteroids in the brain, because both cerebellar Purkinje neurons and hippocampal neurons play an important role in memory and learning. This paper summarizes the advances made in our understanding of neurosteroids, produced in neurons, and their actions.

Mode of Action and Functional Significance of Estrogen-Inducing Dendritic Growth, Spinogenesis, and Synaptogenesis in the Developing Purkinje Cell

Neurosteroids are synthesized de novo from cholesterol in the brain. To understand neurosteroid action in the brain, data on the regio- and temporal-specific synthesis of neurosteroids are needed. Recently, we identified the Purkinje cell as an active neurosteroidogenic cell. In rodents, this neuron actively produces several neurosteroids including estradiol during neonatal life, when cerebellar neuronal circuit formation occurs. Estradiol may be involved in cerebellar neuronal circuit formation through promoting neuronal growth and neuronal synaptic contact, because the Purkinje cell expresses estrogen receptor-β (ERβ). To test this hypothesis, in this study we examined the effects of estradiol on dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell using neonatal wild-type (WT) mice or cytochrome P450 aromatase knock-out (ArKO) mice. Administration of estradiol to neonatal WT or ArKO mice increased dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell. In contrast, WT mice treated with tamoxifen, an ER antagonist, or ArKO mice exhibited decreased Purkinje dendritic growth, spinogenesis, and synaptogenesis at the same neonatal period. To elucidate the mode of action of estradiol, we further examined the expression of brain-derived neurotrophic factor (BDNF) in response to estrogen actions in the neonate. Estrogen administration to neonatal WT or ArKO mice increased the BDNF level in the cerebellum, whereas tamoxifen decreased the BDNF level in WT mice similar to ArKO mice. BDNF administration to tamoxifen-treated WT mice increased Purkinje dendritic growth. These results indicate that estradiol induces dendritic growth, spinogenesis, and synaptogenesis in the developing Purkinje cell via BDNF action during neonatal life.

Expression and intracellular distribution of the G protein-coupled receptor 30 in rat hippocampal formation

Although the expression and distribution of nuclear estrogen receptors in the hippocampus has been described, it has been proposed that the nuclear receptors may not explain all aspects of estrogen function in the hippocampus. Recently, a G protein-coupled receptor for estrogen, GPR30, was identified as a membrane-localized estrogen receptor in several cancer cell lines. In this study, we examined the expression and intracellular distribution of GPR30 in the rat hippocampal formation. We found expression of GPR30 in pyramidal cells of CA1-3 and granule cells of the dentate gyrus at both mRNA and protein levels. Specific markers for intracellular organelles and immunoelectron microscopy revealed that GPR30 was mainly localized to the Golgi apparatus and partially in the endoplasmic reticulum of the neuron but could not detect the protein at the cell surface. Expression levels were not different among male, female in proestrus and female in estrus at the adult stage, but were higher in newborn male than newborn female.

Activity and localization of 3β‐hydroxysteroid dehydrogenase/ Δ5‐Δ4‐isomerase in the zebrafish central nervous system

Little information is available for neurosteroidogenesis in the central nervous system (CNS) of lower vertebrates. Therefore, in the present study, we examined the enzymatic activity and localization of 3β‐hydroxysteroid dehydrogenase/Δ5‐Δ4‐isomerase (3βHSD), a key steroidogenic enzyme, in the CNS of adult male zebrafish to clarify central progesterone biosynthesis. Biochemical studies together with HPLC analysis revealed that the zebrafish brain converted pregnenolone to progesterone, suggesting the enzymatic activity of 3βHSD. This conversion was significantly reduced by trilostane, a specific inhibitor of 3βHSD. By using Western immunoblotting with the polyclonal antiserum directed against purified bovine adrenal 3βHSD, a 3βHSD‐like substance was found in homogenates of the zebrafish brain. Immunocytochemical analysis was then undertaken to investigate the localization of the 3βHSD‐like substance in the zebrafish brain and spinal cord. Clusters of immunoreactive cell bodies were localized in the dorsal telencephalic areas (D), central posterior thalamic nucleus (CP), preoptic nuclei (NPO), posterior tuberal nucleus (PTN), paraventricular organ (PVO), and nucleus of medial longitudinal fascicle (NMLF). 3βHSD‐like immunoreactivity was also observed in somata of cerebellar Purkinje neurons. A widespread distribution of immunoreactive fibers was found throughout the brain and spinal cord. In addition, positively stained cells were restricted to other organs, such as the pituitary and retina. Preabsorbing the antiserum with purified bovine adrenal microsome resulted in a complete absence of 3βHSD‐like immunoreactivity. These results suggest that the fish CNS possesses steroidogenic enzyme 3βHSD and produces progesterone. The present study further provides the first immunocytochemical mapping of the site of 3βHSD expression in the fish CNS. J. Comp. Neurol. 439:291–305, 2001.

Sexually dimorphic gastrin releasing peptide system in the spinal cord controls male reproductive functions.

Neurons in the upper lumbar spinal cord project axons containing gastrin-releasing peptide (GRP) to innervate lower lumbar regions controlling erection and ejaculation. This system is vestigial in female rats and in males with genetic dysfunction of androgen receptors, but in male rats, pharmacological stimulation of spinal GRP receptors restores penile reflexes and ejaculation after castration. GRP offers new avenues for understanding potential therapeutic approaches to masculine reproductive dysfunction.

GFP chimeric models exhibited a biphasic pattern of mesenchymal cell invasion in tendon healing

The healing of an injured musculoskeletal system requires an influx of mesenchymal cells that can differentiate into osteoblasts, fibroblasts, chondroblasts, and skeletal myoblasts. However, whether these mesenchymal cells arise from the circulation (bone marrow) or the injured tissues themselves has been controversial. To reveal the spatiotemporal characteristics of the reparative mesenchymal cells, we investigated the healing process after patellar tendon injury using two types of green fluorescent protein (GFP) chimeric rats; one expressing GFP in the circulating cells, and the other expressing it in the patellar tendon. We analyzed the behavior of GFP‐positive cells after experimental tendon injury in both chimeric rats to clarify the origin of reparative cells. At 24 h after the injury, the wound contained circulation‐derived cells but not tendon‐derived cells. Tendon‐derived cells first appeared in the wounded area at 3 days after the injury, and had significantly increased in number with time and had maintained a high level of proliferative activity until 7 days after the injury, whereas the circulation‐derived cells had decreased in number and had been replaced by the tendon‐derived cells. These findings suggest that circulation‐derived and tendon‐derived cells contribute to the healing of tendons in different periods as part of a biphasic process. J. Cell. Physiol. 210: 684–691, 2007.

Effects of single-prolonged stress on neurons and their afferent inputs in the amygdala.

The amygdala modulates memory consolidation with the storage of emotionally relevant information and plays a critical role in fear and anxiety. We examined changes in neuronal morphology and neurotransmitter content in the amygdala of rats exposed to a single prolonged stress (SPS) as a putative animal model for human post-traumatic stress disorder (PTSD). Rats were perfused 7 days after SPS, and intracellular injections of Lucifer Yellow were administered to neurons of the basolateral (BLA) and central amygdala (CeA) to analyze morphological changes at the cellular level. A significant increase of dendritic arborization in BLA pyramidal neurons was observed, but there was no effect on CeA neurons. Neuropeptide Y (NPY) was abundant in BLA under normal conditions. The local concentration and number of immunoreactive fibers of NPY in the BLA of SPS-exposed rats were increased compared with the control. No differences were observed in this regard in the CeA. Double immunostaining by fluorescence and electron microscopy revealed that NPY immunoreactive terminals were closely associated with calcium/calmodulin II-dependent protein kinase (CaMKII: a marker for pyramidal neurons)-positive neurons in the BLA, which were immunopositive to glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). SPS had no significant effect on the expression of CaMKII and MR/GR expression in the BLA. Based on these findings, we suggest that changes in the morphology of pyramidal neurons in the BLA by SPS could be mediated through the enhancement of NPY functions, and this structural plasticity in the amygdala provides a cellular and molecular basis to understand for affective disorders.

Dendritic spine formation in response to progesterone synthesized de novo in the developing Purkinje cell in rats

The cerebellar Purkinje cell (PC) is a typical site for neurosteroid formation. We have demonstrated that this neuron possesses intranuclear receptor for progesterone and actively synthesizes progesterone de novo from cholesterol only during rat neonatal life, when the formation of the cerebellar cortex occurs dramatically. In this study, we therefore analyzed the effect of progesterone on dendritic spine formation of the PC. In vitro studies using cerebellar slice cultures from newborn rats showed that progesterone increases the density of PC dendritic spines in a dose-dependent manner. This effect was blocked by the progesterone receptor antagonist, RU486. Furthermore, trilostane, a specific inhibitor of progesterone synthesis, inhibited the increase of spine density. These results suggest that progesterone can promote dendritic spine formation, and endogenous progesterone synthesized de novo in the developing PC may induce such an effect.

Expression and localization of 25-Dx, a membrane-associated putative progesterone-binding protein, in the developing Purkinje cell.

Neurosteroids are synthesized de novo in the brain and the cerebellar Purkinje cell is a major site for neurosteroid formation. We have demonstrated that the rat Purkinje cell actively produces progesterone de novo from cholesterol only during neonatal life and progesterone promotes dendritic growth, spinogenesis and synaptogenesis via its nuclear receptor in this neuron. On the other hand, 25-Dx, a putative membrane progesterone receptor, has been identified in the rat liver. In this study, we therefore investigated the expression and localization of 25-Dx in the Purkinje cell to understand the mode of progesterone actions in this neuron. Reverse transcription-PCR and Western immunoblot analyses revealed the expressions of 25-Dx mRNA and 25-Dx-like protein in the rat cerebellum, which increased during neonatal life. By immunocytochemistry, the expression of 25-Dx-like protein was localized in the Purkinje cell and external granule cell layer. At the ultrastructural level, we further found that 25-Dx-like immunoreactivity was associated with membrane structures of the endoplasmic reticulum and Golgi apparatus in the Purkinje cell. These results indicate that the Purkinje cell expresses the putative membrane progesterone receptor, 25-Dx during neonatal life. Progesterone may promote dendritic growth, spinogenesis and synaptogenesis via 25-Dx as well as its nuclear receptor in the Purkinje cell in the neonate.