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Narcolepsy type 1 (NT1) is associated with severe loss of orexin neurons and characterized by symptoms including excessive daytime sleepiness and cataplexy. Current medications indicated for NT1 often show limited efficacy, not addressing the full spectrum of symptoms, demonstrating a need for novel drugs. We discovered a parenteral orexin receptor 2 (OX2R) agonist, danavorexton, and an orally available OX2R agonist, TAK-994; both improving NT1 phenotypes in mouse models and individuals with NT1. However, danavorexton has limited oral availability and TAK-994 has a risk of off-target liver toxicity. To avoid off-target-based adverse events, a highly potent molecule with low effective dose is preferred. Here, we show that a novel OX2R-selective agonist, TAK-861 [N-{(2S,3R)-4,4-Difluoro-1-(2-hydroxy-2-methylpropanoyl)-2-[(2,3',5'-trifluoro[1,1'-biphenyl]-3-yl)methyl]pyrrolidin-3-yl}ethanesulfonamide], activates OX2R with a half-maximal effective concentration of 2.5 nM and promotes wakefulness at 1 mg/kg in mice and monkeys, suggesting ~ tenfold higher potency and lower effective dosage than TAK-994. Similar to TAK-994, TAK-861 substantially ameliorates wakefulness fragmentation and cataplexy-like episodes in orexin/ataxin-3 and orexin-tTA;TetO DTA mice (NT1 mouse models). Compared with modafinil, TAK-861 induces highly correlated brain-wide neuronal activation in orexin-tTA;TetO DTA mice, suggesting efficient wake-promoting effects. Thus, TAK-861 has potential as an effective treatment for individuals with hypersomnia disorders including narcolepsy, potentially with a favorable safety profile., (© 2024. The Author(s).)

Pathological proteins including tau are produced in neurons and released into interstitial fluid (ISF) in a neural activity-dependent manner during wakefulness. Pathological proteins in ISF can be removed from the brain via the glymphatic pathway during nighttime. Thus, in individuals with Alzheimer's disease (AD) that have dysregulated sleep/wake rhythm, application of orexin receptor 2 (OX2R) agonists during daytime could recover the efflux of pathological proteins to ISF and indirectly promote the glymphatic pathway by improving the quality of nighttime sleep after proper daytime arousal, resulting in increased removal of these proteins from the brain. We investigated this hypothesis using OX-201, a novel OX2R-selective agonist with a 50% effective concentration of 8.0 nM. Diurnal rhythm of tau release into hippocampal ISF correlated well with neuronal activity and wakefulness in wild-type mice. In both wild-type and human P301S tau transgenic mice, OX-201 induced wakefulness and promoted tau release into hippocampal ISF. Human P301S tau transgenic mice, tested under our conditions, showed longer wakefulness time, which differs from individuals with AD. OX-201 treatment over 2 months did not alter hippocampal tau levels. Although further studies are required, at a minimum OX2R agonists may not exacerbate tau accumulation in individuals with tauopathy, including AD., (© 2024. The Author(s).)

Orexin, a neuropeptide, performs various physiological functions, including the regulation of emotion, feeding, metabolism, respiration, and sleep/wakefulness, by activating the orexin 1 receptor and orexin 2 receptor (OX2R). Owing to the pivotal role of OX2R in wakefulness and other biological functions, OX2R agonists are being developed. A detailed understanding of OX2R protein distribution is essential for determining the mechanisms of action of OX2R agonists; however, this has been hindered by the lack of selective antibodies. In this study, we first confirmed the OX2R-selective binding of [ 3 H]-EMPA in in vitro autoradiography studies, using brain slices from OX2R knockout mice and their wild-type littermates. Subsequently, OX2R protein distribution in rats was comprehensively assessed in 51 brain regions and 10 peripheral tissues using in vitro autoradiography with [ 3 H]-EMPA. The widespread distribution of OX2R protein, including that in previously unrecognized regions of the retrosplenial cortex, was identified. In contrast, OX2R protein expression was negligible/very low in peripheral tissues, suggesting that orexin exerts OX2R-dependent physiological functions primarily through activation of the central nervous system. These findings will be useful for understanding the wide range of biological functions of OX2R and the application of OX2R agonists in various disorders., (© 2022. The Author(s).)

Adiponectin can act in the brain to increase energy expenditure and reduce body weight by mechanisms not entirely understood. We found that adiponectin type 1 and type 2 receptors (AdipoR1 and AdipoR2) are expressed in warm sensitive neurons of the hypothalamic preoptic area (POA) which play a critical role in the regulation of core body temperature (CBT) and energy balance. Thus, we tested the ability of adiponectin to influence CBT in wild-type mice and in mice deficient for AdipoR1 or AdipoR2. Local injection of adiponectin into the POA induced prolonged elevation of core body temperature and decreased respiratory exchange ratio (RER) indicating that increased energy expenditure is associated with increased oxidation of fat over carbohydrates. In AdipoR1 deficient mice, the ability of adiponectin to raise CBT was significantly blunted and its ability to decrease RER was completely lost. In AdipoR2 deficient mice, adiponectin had only diminished hyperthermic effects but reduced RER similarly to wild type mice. These results indicate that adiponectin can contribute to energy homeostasis by regulating CBT by direct actions on AdipoR1 and R2 in the POA., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: Temperature and nutrient homeostasis are two interdependent components of energy balance regulated by distinct sets of hypothalamic neurons. The objective is to examine the role of the metabolic signal insulin in the control of core body temperature (CBT)., Research Design and Methods: The effect of preoptic area administration of insulin on CBT in mice was measured by radiotelemetry and respiratory exchange ratio. In vivo 2-[(18)F]fluoro-2-deoxyglucose uptake into brown adipose tissue (BAT) was measured in rats after insulin treatment by positron emission tomography combined with X-ray computed tomography imaging. Insulin receptor-positive neurons were identified by retrograde tracing from the raphe pallidus. Insulin was locally applied on hypothalamic slices to determine the direct effects of insulin on intrinsically warm-sensitive neurons by inducing hyperpolarization and reducing firing rates., Results: Injection of insulin into the preoptic area of the hypothalamus induced a specific and dose-dependent elevation of CBT mediated by stimulation of BAT thermogenesis as shown by imaging and respiratory ratio measurements. Retrograde tracing indicates that insulin receptor-expressing warm-sensitive neurons activate BAT through projection via the raphe pallidus. Insulin applied on hypothalamic slices acted directly on intrinsically warm-sensitive neurons by inducing hyperpolarization and reducing firing rates. The hyperthermic effects of insulin were blocked by pretreatment with antibodies to insulin or with a phosphatidylinositol 3-kinase inhibitor., Conclusions: Our findings demonstrate that insulin can directly modulate hypothalamic neurons that regulate thermogenesis and CBT and indicate that insulin plays an important role in coupling metabolism and thermoregulation at the level of anterior hypothalamus.

Galanin, a neuropeptide widely expressed in the central and peripheral nervous systems and in the endocrine system, has been shown to regulate numerous physiological and pathological processes through interactions with three G-protein-coupled receptors, GalR1 through GalR3. Over the past decade, some of the receptor subtype-specific effects have been elucidated through pharmacological studies using subtype selective ligands, as well as through molecular approaches involving knockout animals. In this chapter, we summarize the current data which constitute the basis of targeting GalR1, GalR2, and GalR3 for the treatment of various human diseases and pathological conditions, including seizure, Alzheimer's disease, mood disorders, anxiety, alcohol intake in addiction, metabolic diseases, pain and solid tumors.

The neuropeptide galanin has been shown to play a role in psychiatric disorders as well as in other biological processes including regulation of pain threshold through interactions with three G-protein coupled receptors, galanin receptor subtypes 1-3 (GalR1-3). While most of the pharmacological studies on galanin in stress-related disorders have been done with rats, the continuous development of genetically engineered mice involving galanin or its receptor subtype(s) validates the importance of mouse pharmacological studies. The present study on mice examined the homeostatic, endocrinological and neuroanatomical effects of the galanin, injected intracerebroventricularly (i.c.v.), in regulation of stress responses after restraint stress. Furthermore, the roles of GalR1 on these effects were studied using GalR1 knockout (KO) mice. The core body temperature and the locomotor activity were monitored with radio telemetry devices. Galanin (i.c.v.) decreased locomotor activity and exerted a bidirectional effect on the restraint stress-induced hyperthermia; a high dose of galanin significantly attenuated the stress-induced hyperthermic response, while a low dose of galanin moderately enhanced this response. The bidirectional effect of galanin was correlated with changes in stress hormone levels (adrenocorticotropic hormone and corticosterone). To neuroanatomically localize the effects of galanin on stress response, cFos immunoreactivity was assessed in galanin receptor rich areas; paraventricular nucleus (PVN) of the hypothalamus and the locus coeruleus (LC), respectively. A high dose of galanin significantly induced cFos activity in the LC but not in the PVN. In GalR1KO mice, a high dose of galanin failed to induce any of the above effects, suggesting the pivotal role of GalR1 in decreased locomotor activity and stress-resistant effects caused by galanin i.c.v. injection studied here.

Aim: To investigate the effect of recombinant human lactoferrin (rh-LF) on the expression of matrix metalloproteinase as a marker of cervical maturation, using a rabbit preterm delivery model in which preterm labor was induced by bacteria., Methods: We used cervical tissues that had been excised in a previous study in which rabbits were randomly assigned to receive either inoculation with Escherichia coli (E. coli) or saline solution and to receive pretreatment with or without rh-LF inserted into the cervix two hours before bacterial inoculation (Condition A: saline + saline; Condition B: rh-LF + E. coli; Condition C: saline +E. coli). E. coli, saline solution, and rh-LF were inserted into the cervix using a hysteroscope and a sterile polyethylene cannula. Both cervices of the rabbit uterus, which is bicorpus-bicolli, were taken out and preserved, and expression of matrix metalloproteinases MMP-2,-3, and -9 in the cervix was evaluated using Western blot., Results: MMP-2,-3, and -9 levels in the cervix under Conditions A and B were significantly lower than that under Condition C., Conclusions: These results suggest that the prevention of preterm delivery by rh-LF in a rabbit model has been achieved through inhibition of cervical maturation promoted by matrix metalloproteinase activity.

Galanin, a neuropeptide widely expressed in the central and peripheral nervous systems and in the endocrine system, has been shown to regulate numerous physiological and pathological processes through interactions with three G-protein-coupled receptors, GalR1 through GalR3. Over the past decade, some of the receptor subtype-specific effects have been elucidated through pharmacological studies using subtype selective ligands, as well as through molecular approaches involving knockout animals. In the present review, we summarize the current data which constitute the basis of targeting GalR1, GalR2 and GalR3 for the treatment of various human diseases and pathological conditions, including seizure, Alzheimer's disease, mood disorders, anxiety, alcohol intake in addiction, metabolic diseases, pain and solid tumors.

Purpose: The purpose of this study was to predict developmental competence of human oocytes during ICSI via analysis of connexin 43 (Cx43) in cumulus cells surrounding mature oocytes., Materials and Methods: Human cumulus cells were manually separated from the oocyte-cumulus complex under a microscope. Cx43 mRNA was expressed by real-time quantitative polymerase chain reaction (RT-PCR) measurement in cumulus cells., Results: There was no significant relationship between expression of Cx43 and fertilisation or cleavage rate. However, Cx43 expression was lower in the good morphology group (blastomeres>7 cells with fragmentation<10% on day 3) when compared to the other groups (p=0.035)., Conclusions: These results suggest that full reduction of Cx43 expression on cumulus cells at the time of oocyte collection during ICSI is essential for developmental competence of human oocytes.

Objective: To investigate the effect of recombinant human lactoferrin (rhLF) on cervical ripening using a rabbit model in which preterm labor was induced by bacterial endotoxin lipopolysaccharide (LPS)., Study Design: Timed pregnant rabbits (New Zealand White, 3-4 kg, day 14) were randomly assigned to the following treatment groups: Group A, LPS + rhLF (n = 4); Group B, LPS (n = 4); and Group C, control (n = 4). Recombinant human lactoferrin (10 microg) was administrated to pregnant rabbits in Group A and not in Group B. Lipopolysaccharide (100 microg) was given to the rabbits in both groups for 3 days (days14-16). Drugs were administered as a vaginal suppository. On day 18, the rabbits were anesthetized with intramuscular ketamine hydrochloride (20 mg/kg) and diazepam (4 mg/kg). Both cervices of the rabbit uterus, which is bicorpus-bicolli, were taken out. One cervix was placed in 10% formalin solution for a histological study with standard hematoxylin-eosin staining. The other was used for an extension test to assess the grade of ripening. Extension was measured after a 5-mm length of cervical tissue was loaded with 5.8 g., Results: The histological study showed remarkably loose and edematous connective tissue in Group B cervices. Cervical tissues in Group A was not different from those in Group C. Extension lengths were 2.2 +/- 0.2 mm in Group A, 7.0 +/- 2.7 mm in Group B, and 1.7 +/- 0.3 mm in Group C., Conclusion: These results suggest that rhLF inhibits cervical maturation induced by LPS in a rabbit model and may have a potential to prevent preterm delivery caused by cervical infection and ripening.

Objective: To investigate the level of lactoferrin (LF) in follicular fluid (FF), as well as the correlation between the LF concentration of FF, fertilization rate, and embryo quality., Design: Retrospective study., Setting: In vitro fertilization program at the Showa University School of Medicine, Tokyo, Japan., Patient(s): Follicular fluid was obtained from 35 IVF patients, aged 25-34 years., Intervention(s): In total, 70 FF and serum samples were collected at the time of oocyte harvesting (44 of the oocytes were fertilized, and 26 were not). The LF concentration in FF was assayed via ELISA., Result(s): The mean LF concentration in FF of the fertilized group (500.2 +/- 35.5 ng/mL) was significantly higher than that in the unfertilized group (379.7 +/- 45.1 ng/mL). A positive correlation between follicular size and LF concentration was not found (r = 0.214). However, a high LF concentration in FF was indicative of good embryo quality., Conclusion(s): High LF concentrations in FF correlate with both a high fertilization rate and good embryo quality.

Regulation of neurotransmission via group-III metabotropic glutamate receptors (mGluR4, -6, -7, and -8) has recently been implicated in the pathophysiology of affective disorders, such as major depression and anxiety. For instance, mice with a targeted deletion of the gene for mGluR7 (mGluR7-/-) showed antidepressant and anxiolytic-like effects in a variety of stress-related paradigms, including the forced swim stress and the stress-induced hyperthermia tests. Deletion of mGluR7 reduces also amygdala- and hippocampus-dependent conditioned fear and aversion responses. Since the hypothalamic-pituitary-adrenal (HPA) axis regulates the stress response we investigate whether parameters of the HPA axis at the levels of selected mRNA transcripts and endocrine hormones are altered in mGluR7-deficient mice. Over all, mGluR7-/- mice showed only moderately lower serum levels of corticosterone and ACTH compared with mGluR7+/+ mice. More strikingly however, we found strong evidence for upregulated glucocorticoid receptor (GR)-dependent feedback suppression of the HPA axis in mice with mGluR7 deficiency: (i) mRNA transcripts of GR were significantly upregulated in the hippocampus of mGluR7-/- animals, (ii) similar increases were seen with 5-HT1A receptor transcripts, which are thought to be directly controlled by the transcription factor GR and finally (iii) mGluR7-/- mice showed elevated sensitivity to dexamethasone-induced suppression of serum corticosterone when compared with mGluR7+/+ animals. These results indicate that mGluR7 deficiency causes dysregulation of HPA axis parameters, which may account, at least in part, for the phenotype of mGluR7-/- mice in animal models for anxiety and depression. In addition, we present evidence that protein levels of brain-derived neurotrophic factor are also elevated in the hippocampus of mGluR7-/- mice, which we discuss in the context of the antidepressant-like phenotype found in those animals. We conclude that genetic ablation of mGluR7 in mice interferes at multiple sites in the neuronal circuitry and molecular pathways implicated in affective disorders.

Background:  The concentrations of the iron-binding protein lactoferrin (LF) and interleukin-6 (IL-6) were measured in the cervical mucus of patients being treated for infertility throughout the menstrual cycle. Methods:  A total of 251 cervical mucus samples were obtained from the patients throughout the menstrual cycle. One hundred and fifty samples were from primary infertility patients with unexplained infertility and 101 samples were from secondary infertility patients as a control. The concentrations of LF and IL-6 were measured by enzyme immunoassays. The standard curve of LF concentrations ranged from 1.6 to 50 ng/mL. Results:  The mean LF and IL-6 concentrations in the cervical mucus of primary infertility patients were higher than that of the control patients ( P = 0.04, P = 0.032, respectively) The LF and IL-6 concentrations were highly correlated ( P < 0.0001). Conclusion:  Elevated levels of IL-6 and LF in the cervical mucus were obtained from primary infertility patients. We speculate that LF might also be one of the causes of infertility and might play an important role in reproductive processes in the cervix. (Reprod Med Biol 2006; 5 : 105-109).

Objective: To evaluate concentrations of interleukin (IL)-6, IL-8, lactoferrin (LF), and alpha defencine (alpha-DF) in the cervical mucus of pregnant women and analyze their relation to cervicitis and bacterial vaginosis (BV)., Methods: Cervical mucus samples were obtained from August 2003 through May 2004 from 157 women who were between the 6th and 36th week of an uncomplicated singleton pregnancy. All women were delivered at term, 69 without BV or cervicitis, 9 with BV, and 79 with cervicitis., Results: Interleukin 8, LF, and mu-DF concentrations were higher in women with cervicitis (0.81 +/- 0.36 pg/mL, 14.8+/-12.3 microg/mL, and 0.60 +/- 0.49 microg/mL) than in women without BV or cervicitis (0.35 +/- 0.34 pg/mL, 8.0 +/- 11.0 microg/mL, and 0.15 +/- 0.12 microg/mL). Interleukin 6 concentration was higher in women with BV (0.26 +/- 0.32 pg/mL) than in women without BV or cervicitis (0.09 +/- 0.15 pg/mL) or in women with cervicitis (0.12 +/- 0.18 pg/mL)., Conclusions: Higher levels of inflammatory cytokines in the cervical mucus of pregnant women may lead to early detection of lower genital tract infection.

Metabotropic glutamate receptor (mGluR) subtypes (mGluR1 to mGluR8) act as important pre- and postsynaptic regulators of neurotransmission in the CNS. These receptors consist of two domains, an extracellular region containing the orthosteric agonist site and a transmembrane heptahelical domain involved in G protein activation and recognition of several recently synthesized pharmacological modulators. The presynaptic receptor mGluR7 shows the highest evolutionary conservation within the family, but no selective pharmacological tool was known. Here we characterize an mGluR7-selective agonist, N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082), which directly activates receptor signaling via an allosteric site in the transmembrane domain. At transfected mammalian cells expressing mGluR7, AMN082 potently inhibits cAMP accumulation and stimulates GTPgammaS binding (EC50-values, 64-290 nM) with agonist efficacies comparable with those of L-2-amino-4-phosphonobutyrate (L-AP4) and superior to those of L-glutamate. AMN082 (< or = 10 microM) failed to show appreciable activating or inhibitory effects at other mGluR subtypes and selected ionotropic GluRs. Chimeric receptor studies position the binding site of AMN082 in the transmembrane region of mGluR7, and we demonstrate that this allosteric agonist has little, if any, effect on the potency of orthosteric ligands. Here we provide evidence for full agonist activity mediated by the heptahelical domain of family 3 G protein-coupled receptors (which have mGluR-like structure) that may lead to drug development opportunities. Further, AMN082 is orally active, penetrates the blood-brain barrier, and elevates the plasma stress hormones corticosterone and corticotropin in an mGluR7-dependent fashion. Therefore, AMN082 is a valuable tool for unraveling the role of mGluR7 in stress-related CNS disorders.

Objective: Lactoferrin, an iron-binding glycoprotein found in cervical mucus and amniotic fluid, plays a defensive role against mucosal infections. This study examined the effect of recombinant human lactoferrin on preterm delivery in a rabbit model., Study Design: Anesthetized rabbits were randomly assigned to receive either inoculation with Escherichia coli or saline solution and to receive treatment with or without recombinant human lactoferrin inserted into the cervix 2 hours before bacterial inoculation (condition A: saline + saline; condition B: E coli + saline; condition C: E coli + recombinant human lactoferrin). E coli , saline solution, and recombinant human lactoferrin were inserted into the cervix using a hysteroscope and a sterile polyethylene cannula. Fetus survival rate and days to delivery after inoculation were monitored and tumor necrosis factor-alpha concentrations were measured in maternal serum and amniotic fluid., Results: Fetus survival for conditions A, B, and C were 95.7%, 0%, and 32.6%, respectively, whereas pregnancy continuation was 7.00 +/- 0 days, 3.25 +/- 0.43 days, and 4.85 +/- 1.77 days, respectively., Conclusion: Cervical recombinant human lactoferrin administration increased fetal survival and extended pregnancy. Lactoferrin has an anti-inflammatory action as well as an antibacterial action, suggesting that recombinant human lactoferrin has the potential to prevent preterm delivery originating from cervical infection in the clinical setting.

Objective: The following animal studies have been conducted to investigate whether recombinant human lactoferrin (rh-LF) has the same effect as bovine lactoferrin (b-LF) in the prevention of preterm delivery., Study Design: Female C3H/HeNCrj mice were pair-mated with male Crj:B6D2F1 mice. On day 15 of gestation, as a model of preterm delivery, a 50 microg/kg intraperitoneal injection of lipopolysaccharide (LPS) was administered twice with a 3-h interval between injections (2:00 and 5:00 PM). One hour prior to each LPS injection (1:00 and 4:00 PM), an intraperitoneal injection of saline, b-LF, or rh-LF (1 mg/body) was administered. In non-LPS-treated controls, an intraperitoneal injection of saline was administered 4 times (1:00, 2:00, 4:00 and 5:00 PM). Body weights and delivery times were recorded. To compare plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) between experimental and other pregnant mice, prepared as above, were sacrificed 6 h after the second LPS injection, and then blood samples were obtained and analyzed., Results: Preterm delivery occurred (16.2+/-0.4 days of gestation) in all LPS-treated mice that were not administered LF. LF significantly prolonged gestation of LPS-treated mice: b-LF+LPS, 17.8+/-0.3 days; rh-LF+LPS, 18.0+/-0.8 days (P<0.05). LF (1 mg/body) significantly suppressed plasma IL-6 in LPS-treated mice:b-LF+LPS, 1060+/-154; rh-LF+ LPSF, 244+/-59; LPS without LF, 1628+/-115 pg/mL (P<0.05). As well, LF (1 mg/body) significantly suppressed plasma TNF-alpha in LPS-treated mice: b-LF+LPS, 88+/-36; rh-LF+LPS, 37+/-30; LPS without LF, 114+/-49 pg/mL (P<0.05)., Conclusions: Rh-LF may prolong gestation in LPS-induced preterm delivery in mice, by suppressing LPS-induced plasma IL-6 and TNF-alpha augmentation.

Colchicine, a potent microtubule-depolymerizing agent, is well known to selectively kill dentate granule cells in the hippocampal formation in vivo. Using organotypic cultures of rat entorhino-hippocampal slices, we confirmed that in vitro exposure to 1 microM and 10 microM of colchicine reproduced a specific degeneration of the granule cells after 24 h. Similar results were obtained with other types of microtubule-disrupting agents, i.e., nocodazole, vinblastine, and Taxol. Interestingly, the actin-depolymerizing agents cytochalasin D and latrunculin A also elicited selective neurotoxicity in the dentate gyrus without affecting survival of hippocampal pyramidal cells. The selective pattern of degeneration was observable 24 h after a brief treatment with the toxins as short as 5 min, but this delayed neuronal death was unlikely to be a result of excitotoxicity because it was virtually unaffected by glutamate receptor antagonists, tetrodotoxin, or extracellular Ca(2+)-free conditions. The damaged tissues contained a large number of TUNEL-positive neurons and exhibited an increased level in caspase-3-like activity, suggesting that cytoskeleton disruption triggers an apoptosis-like process in dentate granule cells. Thus, this study may provide a basis for understanding the distinctive mechanism that supports granule cell survival.