Membrane-Targeted palGFP Predominantly Localizes to the Plasma Membrane but not to Neurosecretory Vesicle Membranes in Rat Oxytocin Neurons.

Recent advances in viral vector technology, specifically using adeno-associated virus (AAV) vectors, have significantly expanded possibilities in neuronal tracing. We have utilized the Cre/loxP system in combination with AAV techniques in rats to explore the subcellular localization of palmitoylation signal-tagged GFP (palGFP) in oxytocin-producing neurosecretory neurons. A distinctive branching pattern of single axons was observed at the level of the terminals in the posterior pituitary. Despite challenges in detecting palGFP signals by fluorescent microscopy, immunoelectron microscopy demonstrated predominant localization on the plasma membrane, with a minor presence on the neurosecretory vesicle membrane. These findings suggest that membrane-anchored palGFP may undergo exocytosis, translocating from the plasma membrane to the neurosecretory vesicle membrane. In this study, we observed characteristic axon terminal structures in the posterior pituitary of oxytocin neurons. This study indicates the importance of understanding the plasma membrane-specific sorting system in neuronal membrane migration and encourages future studies on the underlying mechanisms.

Amyloid deposition through endocytosis in vascular endothelial cells.

No mechanistic lead is known for establishing AL amyloid deposits in organs. We here report an electron microscopic (EM) analysis in a case of intestinal AL amyloidosis before initiating treatment for amyloidosis. The dense deposits of amyloid fibrils are concentrated around the small blood vessels in the submucosal area of intestinal tissue. Surprisingly, we observed endothelial cells (ECs) of blood vessels containing plenty of endocytotic (pinocytotic) and transcytotic vesicles at the luminal side and above the basement membrane, indicating the one-way active trafficking of either the immunoglobulin (Ig) light chain or preassembled amyloid fibrils from the luminal side of ECs to the extraluminal area of ECs. Immunoelectron microscopy displayed that the immuno-gold signals were observed in the vascular cavity and the subendothelial area of amyloid deposits. However, there is no sign of an Ig light chain in pinocytotic vesicles. Therefore, the intestinal ECs may actively pump out mainly the preassembled amyloid fibrils (not light chains) from the blood stream into the subendothelial area as a physiologic function.

Function of gastrin-releasing peptide receptors in ocular itch transmission in the mouse trigeminal sensory system.

The prevalence of allergic conjunctivitis in itchy eyes has increased constantly worldwide owing to environmental pollution. Currently, anti-allergic and antihistaminic eye drops are used; however, there are many unknown aspects about the neural circuits that transmit itchy eyes. We focused on the gastrin-releasing peptide (GRP) and GRP receptor (GRPR), which are reportedly involved in itch transmission in the spinal somatosensory system, to determine whether the GRP system is involved in itch neurotransmission of the eyes in the trigeminal sensory system. First, the instillation of itch mediators, such as histamine (His) and non-histaminergic itch mediator chloroquine (CQ), exhibited concentration-dependent high levels of eye scratching behavior, with a significant sex differences observed in the case of His. Histological analysis revealed that His and CQ significantly increased the neural activity of GRPR-expressing neurons in the caudal part of the spinal trigeminal nucleus of the medulla oblongata in GRPR transgenic mice. We administered a GRPR antagonist or bombesin-saporin to ablate GRPR-expressing neurons, followed by His or CQ instillation, and observed a decrease in CQ-induced eye-scratching behavior in the toxin experiments. Intracisternal administration of neuromedin C (NMC), a GRPR agonist, resulted in dose-dependent excessive facial scratching behavior, despite the absence of an itch stimulus on the face. To our knowledge, this is the first study to demonstrate that non-histaminergic itchy eyes were transmitted centrally via GRPR-expressing neurons in the trigeminal sensory system, and that NMC in the medulla oblongata evoked facial itching.

Neuropeptidergic control circuits in the spinal cord for male sexual behaviour: Oxytocin-gastrin-releasing peptide systems.

The neuropeptidergic mechanisms controlling socio-sexual behaviours consist of complex neuronal circuitry systems in widely distributed areas of the brain and spinal cord. At the organismal level, it is now becoming clear that "hormonal regulations" play an important role, in addition to the activation of neuronal circuits. The gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord is an important component of the neural circuits that control penile reflexes in rats, circuits that are commonly referred to as the "spinal ejaculation generator (SEG)." Oxytocin, long known as a neurohypophyseal hormone, is now known to be involved in the regulation of socio-sexual behaviors in mammals, ranging from social bonding to empathy. However, the functional interaction between the SEG neurons and the hypothalamo-spinal oxytocin system remains unclear. Oxytocin is known to be synthesised mainly in hypothalamic neurons and released from the posterior pituitary into the circulation. Oxytocin is also released from the dendrites of the neurons into the hypothalamus where they have important roles in social behaviours via non-synaptic volume transmission. Because the most familiar functions of oxytocin are to regulate female reproductive functions including parturition, milk ejection, and maternal behaviour, oxytocin is often thought of as a "feminine" hormone. However, there is evidence that a group of parvocellular oxytocin neurons project to the lower spinal cord and control male sexual function in rats. In this report, we review the functional interaction between the SEG neurons and the hypothalamo-spinal oxytocin system and effects of these neuropeptides on male sexual behaviour. Furthermore, we discuss the finding of a recently identified, localised "volume transmission" role of oxytocin in the spinal cord. Findings from our studies suggest that the newly discovered "oxytocin-mediated spinal control of male sexual function" may be useful in the treatment of erectile and ejaculatory dysfunction.

Aquaporin regulates cell rounding through vacuole formation during endothelial-to-hematopoietic transition.

Endothelial-to-hematopoietic transition (EHT) is crucial for hematopoietic stem cell (HSC) generation. During EHT, the morphology of hemogenic endothelial cells (HECs) changes from flat and adherent to spherical hematopoietic cells, which detach from the dorsal aorta. HECs attain a rounded shape in a mitosis-independent manner before cell adhesion termination, suggesting an atypical cell-rounding mechanism. However, the direct mechanisms underlying this change in cell morphology during EHT remain unclear. Here, we show that large vacuoles were transiently formed in avian HECs, and that aquaporin 1 (AQP1) was localized in the vacuole and plasma membranes. Overexpression of AQP1 in non-HECs induced ectopic vacuole expansion, cell rounding and subsequent cell detachment from the endothelium into the bloodstream, mimicking EHT. Loss of redundant AQP functions by CRISPR/Cas9 gene editing in HECs impeded the morphological EHT. Our findings provide the first evidence to indicate that morphological segregation of hematopoietic cells from endothelial cells is regulated by water influx into vacuoles. These findings provide important insights for further exploration of the mechanisms underlying cell/tissue morphogenesis through water-adoptive cellular responses.

Chronic corticosterone exposure evokes itch hypersensitivity and sexual dysfunction in male rats: Relationship between the two distinct gastrin-releasing peptide systems in the spinal cord.

In today's society, people are subjected to many social stressors, and excessive chronic stress causes functional disruption of the neuroendocrine system and many diseases. Although the exacerbation of atopic dermatitis with symptoms of itching and erectile dysfunction is induced by chronic stress, the details of the mechanisms are unknown. Here, we examined the effects of chronic stress on itch sensation and male sexual function at the behavioral and molecular levels, focusing on two distinct gastrin-releasing peptide (GRP) systems that independently regulate itch transmission, i.e., the somatosensory GRP system, and male sexual function, i.e., the lumbosacral autonomic GRP system, in the spinal cord. In a rat model of chronic stress induced by chronic corticosterone (CORT) administration, we observed increased plasma CORT concentrations, decreased body weight, and increased anxiety-like behavior, similar to that observed in humans. Chronic CORT exposure induced hypersensitivity to itch and increased the Grp mRNA level in the spinal somatosensory system, but there was no change in pain or tactile sensitivity. Antagonists of the somatosensory GRP receptor, an itch-specific mediator, suppressed itch hypersensitivity induced by chronic CORT exposure. In contrast, chronic CORT exposure decreased male sexual behavior, ejaculated semen volume, vesicular gland weight, and plasma testosterone levels. However, there were no effects on the expression of Grp mRNA or protein in the lumbosacral autonomic GRP system, which regulates male sexual function. In summary, chronic stress model rats showed itch hypersensitivity and impaired sexual function in males, and the involvement of the spinal GRP systems was apparent in itch hypersensitivity.

Evolutionary differentiation of androgen receptor is responsible for sexual characteristic development in a teleost fish.

Teleost fishes exhibit complex sexual characteristics in response to androgens, such as fin enlargement and courtship display. However, the molecular mechanisms underlying their evolutionary acquisition remain largely unknown. To address this question, we analyse medaka (Oryzias latipes) mutants deficient in teleost-specific androgen receptor ohnologs (ara and arb). We discovered that neither ar ohnolog was required for spermatogenesis, whilst they appear to be functionally redundant for the courtship display in males. However, both were required for reproductive success: ara for tooth enlargement and the reproductive behaviour eliciting female receptivity, arb for male-specific fin morphogenesis and sexual motivation. We further showed that differences between the two ar ohnologs in their transcription, cellular localisation of their encoded proteins, and their downstream genetic programmes could be responsible for the phenotypic diversity between the ara and arb mutants. These findings suggest that the ar ohnologs have diverged in two ways: first, through the loss of their roles in spermatogenesis and second, through gene duplication followed by functional differentiation that has likely resolved the pleiotropic roles derived from their ancestral gene. Thus, our results provide insights into how genome duplication impacts the massive diversification of sexual characteristics in the teleost lineage.

Endolysosomal TPCs regulate social behavior by controlling oxytocin secretion.

Oxytocin (OT) is a prominent regulator of many aspects of mammalian social behavior and stored in large dense-cored vesicles (LDCVs) in hypothalamic neurons. It is released in response to activity-dependent Ca2+ influx, but is also dependent on Ca2+ release from intracellular stores, which primes LDCVs for exocytosis. Despite its importance, critical aspects of the Ca2+-dependent mechanisms of its secretion remain to be identified. Here we show that lysosomes surround dendritic LDCVs, and that the direct activation of endolysosomal two-pore channels (TPCs) provides the critical Ca2+ signals to prime OT release by increasing the releasable LDCV pool without directly stimulating exocytosis. We observed a dramatic reduction in plasma OT levels in TPC knockout mice, and impaired secretion of OT from the hypothalamus demonstrating the importance of priming of neuropeptide vesicles for activity-dependent release. Furthermore, we show that activation of type 1 metabotropic glutamate receptors sustains somatodendritic OT release by recruiting TPCs. The priming effect could be mimicked by a direct application of nicotinic acid adenine dinucleotide phosphate, the endogenous messenger regulating TPCs, or a selective TPC2 agonist, TPC2-A1-N, or blocked by the antagonist Ned-19. Mice lacking TPCs exhibit impaired maternal and social behavior, which is restored by direct OT administration. This study demonstrates an unexpected role for lysosomes and TPCs in controlling neuropeptide secretion, and in regulating social behavior.

Spinal Transection Switches the Effect of Metabotropic Glutamate Receptor Subtype 7 from the Facilitation to Inhibition of Ejaculation.

Metabotropic glutamate receptor subtype 7 (mGluR7) is a member of the group III mGluRs, which localize to presynaptic active zones of the central nervous system. We previously reported that mGluR7 knockout (KO) mice exhibit ejaculatory disorders, although they have normal sexual motivation. We hypothesized that mGluR7 regulates ejaculation by potentiating the excitability of the neural circuit in the lumbosacral spinal cord, because administration of the mGluR7-selective antagonist into that region inhibits drug-induced ejaculation. In the present study, to elucidate the mechanism of impaired ejaculation in mGluR7 KO mice, we eliminated the influence of the brain by spinal transection (spinalization). Unexpectedly, sexual responses of male mGluR7 KO mice were stronger than those of wild-type mice after spinalization. Histological examination indicated that mGluR7 controls sympathetic neurons as well as parasympathetic neurons. In view of the complexity of its synaptic regulation, mGluR7 might control ejaculation by multi-level and multi-modal mechanisms. Our study provides insight into the mechanism of ejaculation as well as a strategy for future therapies to treat ejaculatory disorders in humans.

Behavioural osmoregulation during land invasion in fish: Prandial drinking and wetting of the dry skin.

Osmoregulatory behaviours should have evolutionarily modified for terrestrialisation of vertebrates. In mammals, sensations of buccal food and drying have immediate effects on postprandial thirst to prevent future systemic dehydration, and is thereby considered to be 'anticipatory thirst'. However, it remains unclear whether such an anticipatory response has been acquired in the non-tetrapod lineage. Using the mudskipper goby (Periophthalmus modestus) as a semi-terrestrial ray-finned fish, we herein investigated postprandial drinking and other unique features like full-body 'rolling' over on the back although these behaviours had not been considered to have osmoregulatory functions. In our observations on tidal flats, mudskippers migrated into water areas within a minute after terrestrial eating, and exhibited rolling behaviour with accompanying pectoral-fin movements. In aquarium experiments, frequency of migration into a water area for drinking increased within a few minutes after eating onset, without systemic dehydration. During their low humidity exposure, frequency of the rolling behaviour and pectoral-fin movements increased by more than five times to moisten the skin before systemic dehydration. These findings suggest anticipatory responses which arise from oral/gastrointestinal and cutaneous sensation in the goby. These sensation and motivation seem to have evolved in distantly related species in order to solve osmoregulatory challenges during terrestrialisation.

Mating experiences with the same partner enhanced mating activities of naïve male medaka fish.

Mating experience shapes male mating behavior across species, from insects, fish, and birds, to rodents. Here, we investigated the effect of multiple mating experiences on male mating behavior in "naïve" (defined as sexually inexperienced) male medaka fish. The latency to mate with the same female partner significantly decreased after the second encounter, whereas when the partner was changed, the latency to mate was not decreased. These findings suggest that mating experiences enhanced the mating activity of naïve males for the familiar female, but not for an unfamiliar female. In contrast, the mating experiences of "experienced" (defined as those having mated > 7 times) males with the same partner did not influence their latency to mate. Furthermore, we identified 10 highly and differentially expressed genes in the brains of the naïve males after the mating experience and revealed 3 genes that are required for a functional cascade of the thyroid hormone system. Together, these findings suggest that the mating experience of naïve male medaka fish influences their mating behaviors, with neural changes triggered by thyroid hormone activation in the brain.

Footedness for scratching itchy eyes in rodents.

The neural bases of itchy eye transmission remain unclear compared with those involved in body itch. Here, we show in rodents that the gastrin-releasing peptide receptor (GRPR) of the trigeminal sensory system is involved in the transmission of itchy eyes. Interestingly, we further demonstrate a difference in scratching behaviour between the left and right hindfeet in rodents; histamine instillation into the conjunctival sac of both eyes revealed right-foot biased laterality in the scratching movements. Unilateral histamine instillation specifically induced neural activation in the ipsilateral sensory pathway, with no significant difference between the activations following left- and right-eye instillations. Thus, the behavioural laterality is presumably due to right-foot preference in rodents. Genetically modified rats with specific depletion of Grpr-expressing neurons in the trigeminal sensory nucleus caudalis of the medulla oblongata exhibited fewer and shorter histamine-induced scratching movements than controls and eliminated the footedness. These results taken together indicate that the Grpr-expressing neurons are required for the transmission of itch sensation from the eyes, but that foot preference is generated centrally. These findings could open up a new field of research on the mechanisms of the laterality in vertebrates and also offer new potential therapeutic approaches to refractory pruritic eye disorders.

Nanobody-based RFP-dependent Cre recombinase for selective anterograde tracing in RFP-expressing transgenic animals.

Transgenic animals expressing fluorescent proteins are widely used to label specific cells and proteins. By using a split Cre recombinase fused with mCherry-binding nanobodies or designed ankyrin repeat proteins, we created Cre recombinase dependent on red fluorescent protein (RFP) (Cre-DOR). Functional binding units for monomeric RFPs are different from those for polymeric RFPs. We confirmed selective target RFP-dependent gene expression in the mouse cerebral cortex using stereotaxic injection of adeno-associated virus vectors. In estrogen receptor-beta (Esr2)-mRFP1 mice and gastrin-releasing peptide receptor (Grpr)-mRFP1 rats, we confirmed that Cre-DOR can be used for selective tracing of the neural projection from RFP-expressing specific neurons. Cellular localization of RFPs affects recombination efficiency of Cre-DOR, and light and chemical-induced nuclear translocation of an RFP-fused protein can modulate Cre-DOR efficiency. Our results provide a method for manipulating gene expression in specific cells expressing RFPs and expand the repertory of nanobody-based genetic tools.

Bayesian parameter estimation from dispersion relation observation data with Poisson process.

In this study, we estimate the distribution of lattice model parameters based on Bayesian estimation using the dispersion relation spectral data of lattice vibration. The dispersion relation of lattice vibration is observed using inelastic scattering of neutrons or x rays and is used to analyze the speed of sound and interatomic force. However, the current analysis method of dispersion relation observation data in the field of experimental physics requires manually fitting parameters, so the analysis is costly and cannot effectively handle high-dimensional data and large amounts of data. Moreover, it is impossible to discuss the estimation accuracy with the conventional method. Therefore, we solve these problems by estimating the distribution of parameters using Bayesian inference. We propose a lattice model parameter estimation method that uses Bayesian inference with a physical observation stochastic process and determine the method's effectiveness using artificial data.

Lattice-patterned collagen fibers and their dynamics in axolotl skin regeneration.

The morphology of collagen-producing cells and the structure of produced collagen in the dermis have not been well-described. This lack of insights has been a serious obstacle in the evaluation of skin regeneration. We succeeded in visualizing collagen-producing cells and produced collagen using the axolotl skin, which is highly transparent. The visualized dermal collagen had a lattice-like structure. The collagen-producing fibroblasts consistently possessed the lattice-patterned filopodia along with the lattice-patterned collagen network. The dynamics of this lattice-like structure were also verified in the skin regeneration process of axolotls, and it was found that the correct lattice-like structure was not reorganized after simple skin wounding but was reorganized in the presence of nerves. These findings are not only fundamental insights in dermatology but also valuable insights into the mechanism of skin regeneration.

Characterization of the expression of gastrin-releasing peptide and its receptor in the trigeminal and spinal somatosensory systems of Japanese macaque monkeys: Insight into humans.

Gastrin-releasing peptide (GRP) and its receptor (GRPR) have been identified as itch mediators in the spinal and trigeminal somatosensory systems in rodents. In primates, there are few reports of GRP/GRPR expression or function in the spinal sensory system and virtually nothing is known in the trigeminal system. The aim of the present study was to characterize GRP and GRPR in the trigeminal and spinal somatosensory system of Japanese macaque monkeys (Macaca fuscata). cDNA encoding GRP was isolated from the macaque dorsal root ganglion (DRG) and exhibited an amino acid sequence that was highly conserved among mammals and especially in primates. Immunohistochemical analysis demonstrated that GRP was expressed mainly in the small-sized trigeminal ganglion and DRG in adult macaque monkeys. Densely stained GRP-immunoreactive (ir) fibers were observed in superficial layers of the spinal trigeminal nucleus caudalis (Sp5C) and the spinal cord. In contrast, GRP-ir fibers were rarely observed in the principal sensory trigeminal nucleus and oral and interpolar divisions of the spinal trigeminal nucleus. cDNA cloning, in situ hybridization, and Western blot revealed substantial expression of GRPR mRNA and GRPR protein in the macaque spinal dorsal horn and Sp5C. Our Western ligand blot and ligand derivative stain for GRPR revealed that GRP directly bound in the macaque Sp5C and spinal dorsal horn as reported in rodents. Finally, GRP-ir fibers were also detected in the human spinal dorsal horn. The spinal and trigeminal itch neural circuits labeled with GRP and GRPR appear to function also in primates.

Neuronal pentraxin 2 is required for facilitating excitatory synaptic inputs onto spinal neurons involved in pruriceptive transmission in a model of chronic itch.

An excitatory neuron subset in the spinal dorsal horn (SDH) that expresses gastrin-releasing peptide receptors (GRPR) is critical for pruriceptive transmission. Here, we show that glutamatergic excitatory inputs onto GRPR+ neurons are facilitated in mouse models of chronic itch. In these models, neuronal pentraxin 2 (NPTX2), an activity-dependent immediate early gene product, is upregulated in the dorsal root ganglion (DRG) neurons. Electron microscopy reveals that NPTX2 is present at presynaptic terminals connected onto postsynaptic GRPR+ neurons. NPTX2-knockout prevents the facilitation of synaptic inputs to GRPR+ neurons, and repetitive scratching behavior. DRG-specific NPTX2 expression rescues the impaired behavioral phenotype in NPTX2-knockout mice. Moreover, ectopic expression of a dominant-negative form of NPTX2 in DRG neurons reduces chronic itch-like behavior in mice. Our findings indicate that the upregulation of NPTX2 expression in DRG neurons contributes to the facilitation of glutamatergic inputs onto GRPR+ neurons under chronic itch-like conditions, providing a potential therapeutic target.

A spinal microglia population involved in remitting and relapsing neuropathic pain.

Neuropathic pain is often caused by injury and diseases that affect the somatosensory system. Although pain development has been well studied, pain recovery mechanisms remain largely unknown. Here, we found that CD11c-expressing spinal microglia appear after the development of behavioral pain hypersensitivity following nerve injury. Nerve-injured mice with spinal CD11c+ microglial depletion failed to recover spontaneously from this hypersensitivity. CD11c+ microglia expressed insulin-like growth factor-1 (IGF1), and interference with IGF1 signaling recapitulated the impairment in pain recovery. In pain-recovered mice, the depletion of CD11c+ microglia or the interruption of IGF1 signaling resulted in a relapse in pain hypersensitivity. Our findings reveal a mechanism for the remission and recurrence of neuropathic pain, providing potential targets for therapeutic strategies.

Vasopressin-oxytocin-type signaling is ancient and has a conserved water homeostasis role in euryhaline marine planarians.

Vasopressin/oxytocin (VP/OT)-related peptides are essential for mammalian antidiuresis, sociosexual behavior, and reproduction. However, the evolutionary origin of this peptide system is still uncertain. Here, we identify orthologous genes to those for VP/OT in Platyhelminthes, intertidal planarians that have a simple bilaterian body structure but lack a coelom and body-fluid circulatory system. We report a comprehensive characterization of the neuropeptide derived from this VP/OT-type gene, identifying its functional receptor, and name it the "platytocin" system. Our experiments with these euryhaline planarians, living where environmental salinities fluctuate due to evaporation and rainfall, suggest that platytocin functions as an "antidiuretic hormone" and also organizes diverse actions including reproduction and chemosensory-associated behavior. We propose that bilaterians acquired physiological adaptations to amphibious lives by such regulation of the body fluids. This neuropeptide-secreting system clearly became indispensable for life even without the development of a vascular circulatory system or relevant synapses.

Sexual Experience Induces the Expression of Gastrin-Releasing Peptide and Oxytocin Receptors in the Spinal Ejaculation Generator in Rats.

Male sexual function in mammals is controlled by the brain neural circuits and the spinal cord centers located in the lamina X of the lumbar spinal cord (L3-L4). Recently, we reported that hypothalamic oxytocin neurons project to the lumbar spinal cord to activate the neurons located in the dorsal lamina X of the lumbar spinal cord (dXL) via oxytocin receptors, thereby facilitating male sexual activity. Sexual experiences can influence male sexual activity in rats. However, how this experience affects the brain-spinal cord neural circuits underlying male sexual activity remains unknown. Focusing on dXL neurons that are innervated by hypothalamic oxytocinergic neurons controlling male sexual function, we examined whether sexual experience affects such neural circuits. We found that >50% of dXL neurons were activated in the first ejaculation group and ~30% in the control and intromission groups in sexually naïve males. In contrast, in sexually experienced males, ~50% of dXL neurons were activated in both the intromission and ejaculation groups, compared to ~30% in the control group. Furthermore, sexual experience induced expressions of gastrin-releasing peptide and oxytocin receptors in the lumbar spinal cord. This is the first demonstration of the effects of sexual experience on molecular expressions in the neural circuits controlling male sexual activity in the spinal cord.