Research at Tsutsui's Lab
We are conducting comprehensive studies for understanding of neuronal control systems such as memory, learning, instinctive control, biological regulation etc in vertebrate brains. We analyze functional, biochemical and behavioral analyses at molecular, cellular, neural circuits, and individual levels. Typically, we first identify new molecules important for brain functions. Then, we elucidate what kind of brain cells, and how these molecules relate to memory, learning, instinctive control, biological regulation. What kinds and how neural networks are formed. Based on these results, we will form hypotheses and approaches. Specifically, we study syntheses and functions of novel brain molecules and neurosteroids with a focus on memory, learning and instinctive control. We identify and functional analyses of novel neuropeptides with a focus on biomodulation. Our studies are briefly outlined below.
1. Studies on syntheses and functions of neurosteroids in the brain
Syntheses of steroids-the discovery of neurosteroids
The brain is an information center; however, has been considered as a target organ of steroids synthesized by the peripheral endocrine glands such as gonads. Studies in Baulieu's lab using mammalian models and our studies in birds, amphibians, reptiles, and fish have revealed that vertebrate brains can also synthesize steroids from cholesterol. Synthesizing and functions of steroids in the brain is a discovery that challenges dogmas. This new concept of active brain molecule is therefore referred as "neurosteroids" to distinct from the "classical steroid" synthesized by the peripheral endocrine glands. Follow-up studies in my lab utilizing molecular or biochemical tools have demonstrated that vertebrate brain synthesizes pregnenolone and its sulfate ester, progesterone and its metabolic steroids, estradiol and other neurosteroids from cholesterol. We have also elucidated major steroid synthesizing pathways in vertebrate brains.
Neurons synthesizes steroids
To understand the effects of neurosteroids, it was necessary to identify neural cells that can synthesize neurosteroids. We have discovered that cerebellar Purkinje cells, known as memory neurons responsible for motor learning, is actively synthesizing neurosteroids based results of immunohistochemistry and in situ hybridization. For the first time, we revealed specific type of neurons that synthesizes steroids. The fact of synthesizing neurosteroids in the Purkinje cells is an important discovery that can also be generalized in the vertebrates. In this neuron, the syntheses of progesterone and estradiol increases during the neonatal period, after which pregnenolone and its sulfate esters are synthesized constantly.
Motor learning and regulation of synaptic plasticity by neurosteroids
Syntheses of various neurosteroids is time-specific in Purkinje cells, which have become an excellent cell model for studying actions of neurosteroids. Our ultrastructural and electrophysiological analyses have demonstrated that neurosteroids have genomic actions during neuronal development such as synapse formation, and nongenomic actions acutely regulating synaptic signal transduction. We study functions of progesterone synthesized in the brain, development of neurons and synapse formation during the neonatal period and formation of cerebellar cortex in rat model. Our results are briefly outlined below: 1) Progesterone promotes dendritic elongation of Purkinje cells and an increase in barbed synapse, (2) These actions of neuronal steroids are via a genomic pathway and the nuclear progesterone receptor presented in the nucleus of Purkinje cells. A similar genomic effect of estradiol was also identified. Neurocircuitry responsible for cerebellar motor learning is affected by these genomic actions of progesterone and estradiol.
Our series of studies have demonstrated the syntheses and functions of neurosteroids in Purkinje cells, a memory neuron responsible for motor learning. Motor learning shows that the flow of information at the synapses of the cerebellar nerve circuit has plasticity and changes over time. Our studies show neurosteroid as active brain molecules play important roles in the motor learning.
Physiological variations and instinctive behavioral control of neurosteroids
Neurosteroids are also synthesized in the prenatal prefrontal cortex and the hypothalamus, which is the center for instinctive behaviors. Pregnenolone and its sulfate esters and progesterone fluctuate seasonally in the brains of wild birds and amphibians. This variation of neurosteroids has physiological significance. We found: (1) Activities of the neurosteroid synthesizing enzymes located in the preoptic prefrontal / hypothalamus fluctuates, (2) Photoperiod regulates the fluctuation of neurosteroids, which increase from the nonbreeding period to the breeding season. From the behavioral analyses, we also found: (1) Increase synthesis of progesterone during breeding season promotes the activities of preoptic neurons and leads to the development of bird's parental behavior, (2) Pregnenolone sulfate controls activities of preoptic neuron in amphibians, which is important for arousal from hibernation and the development of mating behaviors.
Recently, we found actively syntheses of a neurosteroid named 7alpha-hydroxypregnenolone in the brains of wild amphibian. The presence of this steroid has been found in the brains of many vertebrates, but no one has reported physiological functions of this steroid until we did. This neurosteroid may be important for the brain functions in wild animals. we analyzed physiological functions of 7alpha-hydroxypregnenolone in a newt. We found an increase of syntheses of 7alpha-hydroxypregnenolone during the active period (breeding season), likely leading to increase of activities in animals. We also found that 7alpha-hydroxypregnenolone acts on dopamine neurons to increase activities in animals by promoting the release of dopamine. Furthermore, there was sex difference in the synthesis of this neurosteroid. We found male brain synthesized more 7alpha-hydroxypregnenolone than the female brain. During the breeding season, the activities of the male newt increases markedly, is likely due to the actions of 7alpha-hydroxypregnenolone (Matsunaga et al. (2004) Proc. Natl. Acad. Sci. USA, 101: 17282 -17287).
2. Identification and functional analyses of novel neuropeptides
Based on our studies of the identification and functional analyses of novel neuropeptides involved in biological control, we have also made the following discoveries. These discoveries have challenged dogmas and reversed some commonly accepted mis-concepts in the field. These new peptides found have been registered in hormonal handbooks.
Novel hypothalamic peptide control the release of pituitary hormone
In the 1970s, hypothalamic hormones (peptides) that control release of pituitary hormone such as gonadotropin-releasing hormone (GnRH) and thyrotropin-releasing hormone (TRH) from the hypothalamus of mammals were first discovered by Schally and Guillemin. Subsequently, similar hypothalamic hormones were found in other vertebrate species ranging from fish to birds, which significantly advanced studies and our knowledge in neuroendocrinology. Since then, there are few findings of new hypothalamic hormones during the last decade. We introduced a new hypothesis and searched for novel hypothalamic hormones controlling the release of pituitary hormone. As a result, we have discovered a novel hypothalamic peptide group (LPXRFa peptide family) described below.
Gonadotropin releasing inhibitory peptide (hormone, GnIH)
Gonadotropin release hormone (GnRH; BBRC 43: 1334-1339, 1971) discovered by Schally is a well-known hypothalamic hormone that controls the release of gonadotropin. The presence of GnRH is found in vertebrates, protozoa and mollusk. On the other hand, the presence of the gonadotropin release-inhibiting hormone was unknown. In 2000, we identified a novel hypothalamic peptide that suppresses gonadotropin release in quails and named it as gonadotropin inhibiting hormone (GnIH; BBRC 275: 661-667, 2000). GnIH is a novel peptide consisting of 12 amino acid residues that is localized in the paravertebral nucleus ending in the midline uplift outer layer. Cloning of the GnIH precursor cDNA revealed that GnIH and several more GnIH related peptides. GnIH and GnIH-related peptides were defined as LPXRFa (X = L or Q) on the C-terminal side, and were classified as LPXRFa peptides based on their structure.
Growth hormone releasing peptide (fGRP)
Following identification of GnIH in birds, LPXRFa peptides with high homology to GnIH were identified in the hypothalamus of amphibian. This peptide has role in promoting the release of growth hormone and therefore named as growth hormone releasing peptide (fGRP). The precursor of fGRP cDNA also encodes fGRP and multiple fGRP related peptides, which are indeed belong to LPXRFa peptide family.
LPXRFa peptide family
In addition to birds and amphibians, presence of LPXRFa peptides were also identified in the hypothalamus in other vertebrate species ranging from fish to mammals. These LPXRFa peptides also control the release of pituitary hormones in vertebrates. LPXRFa peptide is a novel peptide that controls the release of pituitary hormone in vertebrates.
Spawning-inducing peptides-mechanisms of neural regulation of spawning
The spawning of birds is a process in which eggs in the fallopian tubes are released outside the body. This process is guided by violent muscle contraction of the uterine tube. Not limited to birds, vertebrate other than mammals commonly lay eggs. Endocrinological studies have shown that avian egg laying is regulated by hormones. On the other hand, nerve control in spawning was also suggested based on presence of many nerve fibers in the egg laying tendon layer. Existence and identities of neuropeptides was unknown. We identified an oviposition-inducing peptide consisting of 29 amino acid residues and found a neural regulation mechanism for oviposition by this peptide. Our discoveries are: (1) Sympathetic ganglion neurons project on the oviduct producing spawning inducing peptides, (2) Egg-induced peptide receptor is induced in the oviduct by the action of the sex steroid in the ovary, (3) Release of spawning-induced-peptide from the sympathetic ganglion neurons leads to muscle contraction of the fallopian tube signaling via its receptor. (4) Finally, eggs in the uterine tube are released outside the body. This neuronal control mechanism of egg laying may also exist in other non-mammalian vertebrates.
(3) Originality and meaning of our studies
Our studies have reversed some conventional wisdom and mis-concepts. Our discoveries have also greatly impacted related fields including endocrinology, neuroendocrinology, neuroscience, and behavioral science. Our results have advanced and opened new research fields in the last 10 years. We are pioneers in the new research fields.
1. Studies on syntheses and functions of neurosteroids in the brain
Syntheses of steroids-the discovery of neurosteroids
The brain is an information center; however, has been considered as a target organ of steroids synthesized by the peripheral endocrine glands such as gonads. Studies in Baulieu's lab using mammalian models and our studies in birds, amphibians, reptiles, and fish have revealed that vertebrate brains can also synthesize steroids from cholesterol. Synthesizing and functions of steroids in the brain is a discovery that challenges dogmas. This new concept of active brain molecule is therefore referred as "neurosteroids" to distinct from the "classical steroid" synthesized by the peripheral endocrine glands. Follow-up studies in my lab utilizing molecular or biochemical tools have demonstrated that vertebrate brain synthesizes pregnenolone and its sulfate ester, progesterone and its metabolic steroids, estradiol and other neurosteroids from cholesterol. We have also elucidated major steroid synthesizing pathways in vertebrate brains.
Neurons synthesizes steroids
To understand the effects of neurosteroids, it was necessary to identify neural cells that can synthesize neurosteroids. We have discovered that cerebellar Purkinje cells, known as memory neurons responsible for motor learning, is actively synthesizing neurosteroids based results of immunohistochemistry and in situ hybridization. For the first time, we revealed specific type of neurons that synthesizes steroids. The fact of synthesizing neurosteroids in the Purkinje cells is an important discovery that can also be generalized in the vertebrates. In this neuron, the syntheses of progesterone and estradiol increases during the neonatal period, after which pregnenolone and its sulfate esters are synthesized constantly.
Motor learning and regulation of synaptic plasticity by neurosteroids
Syntheses of various neurosteroids is time-specific in Purkinje cells, which have become an excellent cell model for studying actions of neurosteroids. Our ultrastructural and electrophysiological analyses have demonstrated that neurosteroids have genomic actions during neuronal development such as synapse formation, and nongenomic actions acutely regulating synaptic signal transduction. We study functions of progesterone synthesized in the brain, development of neurons and synapse formation during the neonatal period and formation of cerebellar cortex in rat model. Our results are briefly outlined below: 1) Progesterone promotes dendritic elongation of Purkinje cells and an increase in barbed synapse, (2) These actions of neuronal steroids are via a genomic pathway and the nuclear progesterone receptor presented in the nucleus of Purkinje cells. A similar genomic effect of estradiol was also identified. Neurocircuitry responsible for cerebellar motor learning is affected by these genomic actions of progesterone and estradiol.
Our series of studies have demonstrated the syntheses and functions of neurosteroids in Purkinje cells, a memory neuron responsible for motor learning. Motor learning shows that the flow of information at the synapses of the cerebellar nerve circuit has plasticity and changes over time. Our studies show neurosteroid as active brain molecules play important roles in the motor learning.
Physiological variations and instinctive behavioral control of neurosteroids
Neurosteroids are also synthesized in the prenatal prefrontal cortex and the hypothalamus, which is the center for instinctive behaviors. Pregnenolone and its sulfate esters and progesterone fluctuate seasonally in the brains of wild birds and amphibians. This variation of neurosteroids has physiological significance. We found: (1) Activities of the neurosteroid synthesizing enzymes located in the preoptic prefrontal / hypothalamus fluctuates, (2) Photoperiod regulates the fluctuation of neurosteroids, which increase from the nonbreeding period to the breeding season. From the behavioral analyses, we also found: (1) Increase synthesis of progesterone during breeding season promotes the activities of preoptic neurons and leads to the development of bird's parental behavior, (2) Pregnenolone sulfate controls activities of preoptic neuron in amphibians, which is important for arousal from hibernation and the development of mating behaviors.
Recently, we found actively syntheses of a neurosteroid named 7alpha-hydroxypregnenolone in the brains of wild amphibian. The presence of this steroid has been found in the brains of many vertebrates, but no one has reported physiological functions of this steroid until we did. This neurosteroid may be important for the brain functions in wild animals. we analyzed physiological functions of 7alpha-hydroxypregnenolone in a newt. We found an increase of syntheses of 7alpha-hydroxypregnenolone during the active period (breeding season), likely leading to increase of activities in animals. We also found that 7alpha-hydroxypregnenolone acts on dopamine neurons to increase activities in animals by promoting the release of dopamine. Furthermore, there was sex difference in the synthesis of this neurosteroid. We found male brain synthesized more 7alpha-hydroxypregnenolone than the female brain. During the breeding season, the activities of the male newt increases markedly, is likely due to the actions of 7alpha-hydroxypregnenolone (Matsunaga et al. (2004) Proc. Natl. Acad. Sci. USA, 101: 17282 -17287).
2. Identification and functional analyses of novel neuropeptides
Based on our studies of the identification and functional analyses of novel neuropeptides involved in biological control, we have also made the following discoveries. These discoveries have challenged dogmas and reversed some commonly accepted mis-concepts in the field. These new peptides found have been registered in hormonal handbooks.
Novel hypothalamic peptide control the release of pituitary hormone
In the 1970s, hypothalamic hormones (peptides) that control release of pituitary hormone such as gonadotropin-releasing hormone (GnRH) and thyrotropin-releasing hormone (TRH) from the hypothalamus of mammals were first discovered by Schally and Guillemin. Subsequently, similar hypothalamic hormones were found in other vertebrate species ranging from fish to birds, which significantly advanced studies and our knowledge in neuroendocrinology. Since then, there are few findings of new hypothalamic hormones during the last decade. We introduced a new hypothesis and searched for novel hypothalamic hormones controlling the release of pituitary hormone. As a result, we have discovered a novel hypothalamic peptide group (LPXRFa peptide family) described below.
Gonadotropin releasing inhibitory peptide (hormone, GnIH)
Gonadotropin release hormone (GnRH; BBRC 43: 1334-1339, 1971) discovered by Schally is a well-known hypothalamic hormone that controls the release of gonadotropin. The presence of GnRH is found in vertebrates, protozoa and mollusk. On the other hand, the presence of the gonadotropin release-inhibiting hormone was unknown. In 2000, we identified a novel hypothalamic peptide that suppresses gonadotropin release in quails and named it as gonadotropin inhibiting hormone (GnIH; BBRC 275: 661-667, 2000). GnIH is a novel peptide consisting of 12 amino acid residues that is localized in the paravertebral nucleus ending in the midline uplift outer layer. Cloning of the GnIH precursor cDNA revealed that GnIH and several more GnIH related peptides. GnIH and GnIH-related peptides were defined as LPXRFa (X = L or Q) on the C-terminal side, and were classified as LPXRFa peptides based on their structure.
Growth hormone releasing peptide (fGRP)
Following identification of GnIH in birds, LPXRFa peptides with high homology to GnIH were identified in the hypothalamus of amphibian. This peptide has role in promoting the release of growth hormone and therefore named as growth hormone releasing peptide (fGRP). The precursor of fGRP cDNA also encodes fGRP and multiple fGRP related peptides, which are indeed belong to LPXRFa peptide family.
LPXRFa peptide family
In addition to birds and amphibians, presence of LPXRFa peptides were also identified in the hypothalamus in other vertebrate species ranging from fish to mammals. These LPXRFa peptides also control the release of pituitary hormones in vertebrates. LPXRFa peptide is a novel peptide that controls the release of pituitary hormone in vertebrates.
Spawning-inducing peptides-mechanisms of neural regulation of spawning
The spawning of birds is a process in which eggs in the fallopian tubes are released outside the body. This process is guided by violent muscle contraction of the uterine tube. Not limited to birds, vertebrate other than mammals commonly lay eggs. Endocrinological studies have shown that avian egg laying is regulated by hormones. On the other hand, nerve control in spawning was also suggested based on presence of many nerve fibers in the egg laying tendon layer. Existence and identities of neuropeptides was unknown. We identified an oviposition-inducing peptide consisting of 29 amino acid residues and found a neural regulation mechanism for oviposition by this peptide. Our discoveries are: (1) Sympathetic ganglion neurons project on the oviduct producing spawning inducing peptides, (2) Egg-induced peptide receptor is induced in the oviduct by the action of the sex steroid in the ovary, (3) Release of spawning-induced-peptide from the sympathetic ganglion neurons leads to muscle contraction of the fallopian tube signaling via its receptor. (4) Finally, eggs in the uterine tube are released outside the body. This neuronal control mechanism of egg laying may also exist in other non-mammalian vertebrates.
(3) Originality and meaning of our studies
Our studies have reversed some conventional wisdom and mis-concepts. Our discoveries have also greatly impacted related fields including endocrinology, neuroendocrinology, neuroscience, and behavioral science. Our results have advanced and opened new research fields in the last 10 years. We are pioneers in the new research fields.