Effects of Cardiac Glycoside Digoxin on Dendritic Spines and Motor Learning Performance in Mice.

Synapse formation following the generation of postsynaptic dendritic spines is essential for motor learning and functional recovery after brain injury. The C-terminal fragment of agrin cleaved by neurotrypsin induces dendritic spine formation in the adult hippocampus. Since the α3 subunit of sodium-potassium ATPase (Na/K ATPase) is a neuronal receptor for agrin in the central nervous system, cardiac glycosides might facilitate dendritic spine formation and subsequent improvements in learning. This study investigated the effects of cardiac glycoside digoxin on dendritic spine turnover and learning performance in mice. Golgi-Cox staining revealed that intraperitoneal injection of digoxin less than its IC50 in the brain significantly increased the density of long spines (≥2 µm) in the cerebral cortex in wild-type mice and neurotrypsin-knockout (NT-KO) mice showing impairment of activity-dependent spine formation. Although the motor learning performance of NT-KO mice was significantly lower than control wild-type mice under the control condition, low doses of digoxin enhanced performance to a similar degree in both strains. In NT-KO mice, lower digoxin doses equivalent to clinical doses also significantly improved motor learning performance. These data suggest that lower doses of digoxin could modify dendritic spine formation or recycling and facilitate motor learning in compensation for the disruption of neurotrypsin-agrin pathway.

Social Communication of Maternal Immune Activation-Affected Offspring Is Improved by Si-Based Hydrogen-Producing Agent.

Maternal immune activation (MIA) is triggered by infection or autoimmune predisposition during pregnancy, and cytokines produced by MIA are transmitted through the placenta to the fetal brain, implicating at the onset risks and vulnerability for developmental and psychiatric disorders, such as autism spectrum disorder (ASD) and schizophrenia. To address these kinds of problem in child health, we have developed a silicon (Si)-based hydrogen-producing antioxidant (Si-based agent) that continuously and effectively produces hydrogen in the body. Medical hydrogen is known to have antioxidative, anti-inflammatory, and antiapoptotic effects, therefore we applied our Si-based agent as a potential therapeutic agent to MIA. Using a MIA mouse model, we found that the Si-based agent improved the social communication of MIA offspring mice. We also found that the Si-based agent suppressed the expressions of inflammation-associated genes Ifna1 and Il-6 in the mouse brain. These results demonstrate that the Si-based agent is an effective prophylactic agent against MIA during pregnancy, suggesting that our Si-based agent may be a preventative or therapeutic agent for ASD and other disease risks in child health suppressing MIA damage.

The nitric oxide-cyclic guanosine monophosphate pathway inhibits the bladder ATP release in response to a physiological or pathological stimulus.

The release of ATP from the epithelium of the urinary bladder (urothelium) in response to mechanical/chemical stimuli contributes to the visceral sensation in the micturition reflex. The nitric oxide (NO)-mediated induction of cyclic guanosine monophosphate (cGMP) has been detected in urothelial cells and may inhibit the micturition reflex. However, the function of the NO-cGMP pathway in the regulation of urothelial ATP release remains poorly understood in contrast to its effects on smooth muscles or primary afferent nerves. Therefore, we investigated the relevance of the NO-cGMP pathway to ATP release on the mucosal side in the present study. The administration of l-arginine (NO precursor) or NOC 12 (NO donor) significantly reduced ATP release to the mucosal side at a physiologically normal urine storage pressure (5 cmH2 O). L-NAME (NO synthase inhibitor) significantly increased the distention-induced release of ATP. The phosphodiesterase-5 inhibitor, sildenafil, which increases cGMP levels, inhibited distention-induced ATP release. Furthermore, sildenafil significantly reduced ATP release in response to the administration of lipopolysaccharide. These results suggest that the NO-cGMP pathway inhibited urothelial ATP release during the storage phase under both physiological and pathological conditions.

Authentic role of ATP signaling in micturition reflex.

Adenosine triphosphate (ATP) is a signaling molecule that regulates cellular processes. Based on previous studies of bladder function over the past decade, bladder ATP signaling was thought to have an essential role in the normal micturition reflex. In this study, we performed detailed analyses of bladder function in purinergic receptor-deficient mice using the automated voided stain on paper method and video-urodynamics. Unexpectedly, a lack of P2X2 or P2X3 receptors did not affect bladder function under normal physiological conditions, indicating that bladder ATP signaling is not essential for normal micturition reflex. In contrast, we found that lipopolysaccharide (LPS) induced markedly high levels of ATP release from the urothelium. In addition, LPS-induced rapid bladder hyperactivity was attenuated in P2X2(-/-) and P2X3(-/-) mice. Contrary to the previous interpretation, our present findings indicate that bladder ATP signaling has a fundamental role in the micturition reflex, especially in bladder dysfunction, under pathological conditions. Therefore, the bladder ATP signaling pathway might be a highly promising therapeutic target for functional bladder disorders. This study newly defines an authentic role for bladder ATP signaling in the micturition reflex.

Serotonergic regulation of distention-induced ATP release from the urothelium.

Serotonin [5-hydroxytryptamine (5-HT)] is involved in both motor and sensory functions in hollow organs, especially in the gastrointestinal tract. However, the involvement of 5-HT in visceral sensation of the urinary bladder remains unknown. Because distention-induced ATP release from the urothelium plays an essential role in visceral sensation of the urinary bladder, we investigated the regulation of urothelial ATP release by the 5-HT signaling system. RT-PCR and immunohistochemical analyses of the urothelium revealed specific expression of 5-HT1D and 5-HT4 receptors. The addition of 5-HT did not affect urothelial ATP release without bladder distention, but it significantly reduced distention-induced ATP release by physiological pressure during urine storage (5 cmH2O). The inhibitory effect of 5-HT on distention-elicited ATP release was blocked by preincubation with the 5-HT1B/1D antagonist GR-127935 but not by the 5-HT4 antagonist SB-204070. mRNA encoding tryptophan hydroxylase 1 was detected in the urinary bladder by nested RT-PCR amplification, and l-tryptophan or the selective serotonin reuptake inhibitor citalopram also inhibited ATP release, indicating that 5-HT is endogenously synthesized and released in the urinary bladder. The addition of GR-127935 significantly enhanced the distention-elicited ATP release 40 min after distention, whereas SB-204070 reduced the amount of ATP release 20 min after distention. These data suggest that 5-HT4 facilitates the distention-induced ATP release at an earlier stage, whereas 5-HT1D inhibits ATP release at a later stage. The net inhibitory effect of 5-HT indicates that the action of 5-HT on the urothelium is mediated predominantly by 5-HT1D.

Regulatory Effects of 5-Hydroxytryptamine Receptors on Voiding Function.

UNLABELLED: A growing body of evidence suggests that 5-hydroxytryptamine (5-HT; serotonin) has both physiological and pathological functions in the lower urinary tract. A wide variety of 5-HT receptor subtypes are variably expressed in different organs, both peripheral and central. On urinary bladder smooth muscle, 5-HT1A, 5-HT2, 5-HT3, and 5-HT7 subtypes could function as postjunctional receptors. Postjunctional 5-HT2 receptors induce detrusor contraction of the bladder body. 5-HT1A is suggested to have a similar effect to 5-HT2, while 5-HT3 might suppress detrusor contraction evoked by direct muscle stimulation. Postjunctional 5-HT7 is reported to induce relaxation of the bladder neck, which might be required for efficient voiding. 5-HT1A, 5-HT2A, 5-HT2C, 5-HT3, 5-HT4, and 5-HT7 subtypes also could act as prejunctional receptors in autonomic excitatory nerve terminals. 5-HT2A, 5-HT2C, 5-HT3, 5-HT4, and 5-HT7 subtypes facilitate the neurogenic contraction of the detrusor by enhancing cholinergic or purinergic transmission, whereas 5-HT1A receptors might inhibit the release of acetylcholine in the detrusor. Furthermore, 5-HT1D could be involved in the suppression of ATP release from the urothelium, aiding visceral sensation of the urinary bladder. In the central pathways controlling the micturition reflex, 5-HT1A, 5-HT2A, and 5-HT7 are involved in regulation of bladder and urethral sphincter activities. Their functions, especially that of 5-HT1A, vary in a species- and site (spinal or supraspinal)- dependent manner. In addition to urinary bladder, 5-HT could be involved in prostate contraction and cell proliferation. Evidence indicates that 5-HT receptor subtypes may be novel therapeutic targets for lower urinary tract symptoms. FUNDING: Grants-in-Aid for Scientific Research (C) (KAKENHI 23590707, 24590722, and 26460694) from the Japan Society for the Promotion of Science.

Nerve Growth Factor and Estrogen Receptor mRNA Expression in Paravertebral Muscles of Patients With Adolescent Idiopathic Scoliosis: A Preliminary Study.

STUDY DESIGN: Comparison of nerve growth factor (NGF) and estrogen receptor (ER)α messenger ribonucleic acid (mRNA) expression in bilateral paravertebral muscles in adolescent idiopathic scoliosis (AIS). This expression in AIS was compared with that of normal control subjects. OBJECTIVES: To investigate NGF and ERα mRNA expression in bilateral paravertebral muscles in AIS and control subjects to clarify its association with the development and progression of spinal curvature. SUMMARY OF BACKGROUND DATA: Paravertebral muscle abnormalities in AIS patients have been investigated through various methods. Despite the roles of NGF and ER in human skeletal muscles, the association with idiopathic scoliosis is still unclear. METHODS: A total of 14 AIS patients (average age, 15.9 ± 2.2 years; average Cobb angle, 48.2° ± 8.9°) and 8 controls (average age, 27.3 ± 9.3 years) were included. Muscle samples were harvested from bilateral paravertebral muscles at the apical vertebral level. Nerve growth factor and ERα mRNA expression was evaluated by the real-time polymerase chain reaction. The researchers compared expression levels in bilateral paravertebral muscles in each group. The expression ratio, the expression at the convex side relative to the concave side, was compared between groups and the correlation between Cobb angle and expression ratio was analyzed. RESULTS: Nerve growth factor and ERα mRNA expression on the convex side was higher than on the concave side in the AIS group (p = .024 and .007, respectively) and the expression ratio of NGF and ERα in the AIS group was higher than that of control subjects (p = .004 and .017, respectively). The expression ratio of NGF and the Cobb angle were significantly correlated (r = -0.5728; p = .0323). CONCLUSIONS: In the AIS group, both NGF and ERα mRNA expression was asymmetric. The AIS group had higher expression ratios than control group and the NGF expression ratio was positively correlated to the Cobb angle.

Activity-controlled proteolytic cleavage at the synapse.

Activity-controlled enzymatic cleavage of proteins on the surface of synaptic membranes or in the synaptic or perisynaptic interstitial compartment represents a direct way to regulate synaptic structure, function, and number. Extracellular proteolysis at synapses was initially understood to be plasticity enabling by freeing synapses from the constraints provided by the extracellular matrix. However, recent observations indicate that at least part of the extracellular protein cleavage results in activation of previously cryptic functions that regulate adaptive changes of synapses and neuronal circuits. Here, we focus on peptidases with distinct localization and function at synapses combined with regulation by neuronal and synaptic activity, and evaluate their function in the context of developmental and/or adult synaptic plasticity.

Synergistic effects of loxoprofen and glycine on the micturition reflex in conscious rats.

We examined the inhibitory effects of loxoprofen, a cyclooxygenase inhibitor, and glycine, a major inhibitory neurotransmitter, on the micturition reflex in conscious rats and hypothesized that these drugs would interact synergistically to inhibit micturition. Voiding behaviors were assessed using a metabolic cage. Oral loxoprofen decreased the urinary frequency, and only a high dose(10 mg/kg) significantly reduced the voided volume. With cystometry, intravenous loxoprofen(0.1-3 mg/kg) and glycine (30 and 100 mg/kg) prolonged the intercontraction intervals (ICI) in adose-dependent manner, but did not change the maximum voiding pressure (MVP) in conscious rats. The combination of loxoprofen (3 mg/kg) and glycine (100 mg/kg) strongly prolonged the ICI more than with either drug alone. The lowest dose of loxoprofen (0.1 mg/kg) and glycine(30 mg/kg) did not affect either the ICI or the MVP, but their combination resulted in a significant increase in the ICI. These results suggest that the combined administration of loxoprofen and glycine produced a synergistic inhibitory effect on the micturition reflex.

The regulation of distention-induced ATP release from urothelium by the adenylyl cyclase-cyclic AMP pathway.

Distention of the bladder during urine storage induces ATP release from urothelium, thereby facilitating transmission of visceral sensory signals to afferent nerve fibers. An excess of urothelial ATP release was found in interstitial cystitis, a condition accompanied by hyperesthesia of the urinary bladder; it remains unclear which signals are involved in this upregulation. The present study demonstrated that the adenylyl cyclase pathway enhances distention-induced ATP release in mouse bladder. In the absence of distention, adenylyl cyclase activation by forskolin or cyclic AMP increases by rolipram did not induce significant ATP release. However, forskolin or rolipram significantly enhanced ATP release from urothelium by a physiologically normal urine storage pressure (5 cmH(2)O). Blockade of adenylyl cyclases did not alter pressure-induced ATP release in normal condition. Thus, the adenylyl cyclase-cAMP pathway might be activated in pathological conditions and cause an excess of ATP release.

Pulse Diode Laser Irradiation (830 nm) of Lumbosacral Spinal Roots Diminished Hyperreflexia-Induced by Acetic Acid or Prostaglandin E2 Infusion in Rat Urinary Bladder.

OBJECTIVES: Low power diode Iaser (830 nm) irradiation is a useful analgesic tool in superficial pain. Pulse laser irradiation allows us to increase the laser power because the non-irradiation time reduces heating effects and/or direct tissue damage at the irradiation area. This new irradiation device using pulse laser was applied to the dorsal skin to investigate the effects on the micturition reflex in the rat by targeting underlying sacral spinal roots. METHODS: Vesical pressure measurement during the continuous infusion of the urinary bladder with saline, acetic acid (AA, 0.1%) or prostaglandin E2 (PGE2 , 10(-5) M) were performed in un-anesthetized rats. Multi-unit recording from bladder afferent nerves preformed under urethane anesthesia. Laser irradiation, either continuously at 1 W or in 10 W-pulse mode, was delivered at 830 nm from 1.5 cm above the skin at the lumbosacral joint. RESULTS: During continuous saline infusion to the urinary bladder, neither continuous (1 W) nor pulse (10 W) laser irradiation altered the intercontraction interval and nerve firing during distention of the bladder. Pulse laser, but not continuous laser irradiation, increased the intercontraction interval with AA or PGE2 infusion and diminished nerve firing during distention of the bladder with AA or PGE2 infusion. CONCLUSION: These data indicate that pulse laser could diminish inflammation related nerve firing from the bladder. Since this laser irradiation did not affect the normal bladder distention elicited nerve firing, it appears capable of reducing urgency sensation without loss of the basic micturition reflex.

Store-operated Ca2+ entry suppresses distention-induced ATP release from the urothelium.

Epithelial cells in the urinary bladder (urothelium) trigger sensory signals in micturition by releasing ATP in response to distention of the bladder wall. Our previous study revealed the distinct roles of extracellular Ca(2+) and the Ca(2+) stores in the endoplasmic reticulum (ER) in urothelial ATP release. In the present study, we investigated the regulation of urothelial ATP release by Ca(2+) influx from the extracellular space and Ca(2+) release from the ER using a distention assay of the mouse bladder wall in a small Ussing chamber. Stimulation of Ca(2+) release from the ER in the mucosal side of the bladder induced significant ATP release without distention. Blockade of the inositol 1,4,5-triphosphate receptor reduced distention-induced ATP release, suggesting that Ca(2+) release from the ER is essential for the induction of urothelial ATP release. On the other hand, blockade of store-operated Ca(2+) entry (SOCE) from the extracellular space significantly enhanced distention-induced ATP release. Thus Ca(2+) release from the ER causes urothelial ATP release and depletion of Ca(2+) stores in the ER, which in turn causes the depletion-inducing SOCE to suppress the amount of urothelial ATP released.

Role of supraspinal and spinal alpha1-adrenergic receptor subtypes in micturition reflex in conscious rats.

α(1)-Adrenergic receptor subtypes are widely distributed in the central nervous system and are involved in autonomic functions such as micturition. We investigated the presence and the role of supraspinal and/or spinal α(1)-adrenergic receptors in modulating the micturition reflex in conscious female Wistar rats. The expression of α(1)-adrenergic receptor subtypes in rat brain and lumbosacral spinal cord was studied using RT-PCR. Continuous-infusion cystometrograms were obtained in conscious rats, and α(1)-adrenergic receptor antagonists were administered via intracerebroventricular or intrathecal routes. The mRNA expression of α(1A)-, α(1B)-, and α(1D)-adrenergic receptors was detected in rat brain (midbrain and pons) and lumbosacral spinal cord (dorsal and ventral parts of spinal cord). In addition, intracerebroventricular injection of the α(1)-adrenergic receptor antagonist tamsulosin (1-10 µg), the selective α(1A)-adrenergic receptor antagonist silodosin (1-10 µg), and the selective α(1D)-adrenergic receptor antagonist BMY 7378 (1-10 µg) significantly prolonged the intercontraction interval (ICI) but did not alter maximum voiding pressure (MVP). Although intrathecal injection of BMY 7378 (0.0001-10 µg) did not affect ICI, tamsulosin and silodosin prolonged ICI in a dose-dependent manner. MVP was significantly reduced by intrathecal injection of tamsulosin (10 µg) but not by silodosin or BMY 7378 (0.0001-10 µg). Supraspinal α(1A)- and α(1D)-adrenergic receptors are apparently important for the regulation of reflex-bladder activity in conscious rats. Noradrenergic projection from the brain stem to the lumbosacral spinal cord may promote the afferent limb rather than the efferent limb of the micturition reflex pathway via α(1A)-adrenergic receptors.

Extracellular Ca2+ regulates the stimulus-elicited ATP release from urothelium.

Accumulating evidence shows that the epithelial cells in urinary bladder (urothelium) serve as a sensory organ in micturition and/or in nociception pathway by releasing ATP in response to mechanical and/or chemical stimuli. Here, we compared the effects of capsaicin, acetylcholine, and prostaglandin E(2) receptor EP1 agonist (ONO-DI-004) on the urothelial ATP release in primary cultured mouse urothelial cells in low Ca(2+) medium. All of these chemicals induced a gradual ATP release from urothelium, implying that the downstream Ca(2+) release from endoplasmic reticulum could trigger the ATP release. Consistent with this suggestion, blockade of inositol 1,4,5-triphosphate receptor reduced the distention-induced ATP release from urothelial tissues. The distention-induced ATP release was not affected by tetrodotoxin. However, an increase in extracellular Ca(2+) diminished both chemical- and distention-induced ATP release from urothelium. Thus raising the extracellular Ca(2+) concentration was found to inhibit stimulation-evoked ATP urothelial release.

Coincident pre- and postsynaptic activation induces dendritic filopodia via neurotrypsin-dependent agrin cleavage.

The synaptic serine protease neurotrypsin is essential for cognitive function, as its deficiency in humans results in severe mental retardation. Recently, we demonstrated the activity-dependent release of neurotrypsin from presynaptic terminals and proteolytical cleavage of agrin at the synapse. Here we show that the activity-dependent formation of dendritic filopodia is abolished in hippocampal neurons from neurotrypsin-deficient mice. Administration of the neurotrypsin-dependent 22 kDa fragment of agrin rescues the filopodial response. Detailed analyses indicated that presynaptic action potential firing is necessary for the release of neurotrypsin, whereas postsynaptic NMDA receptor activation is necessary for the neurotrypsin-dependent cleavage of agrin. This contingency characterizes the neurotrypsin-agrin system as a coincidence detector of pre- and postsynaptic activation. As the resulting dendritic filopodia are thought to represent precursors of synapses, the neurotrypsin-dependent cleavage of agrin at the synapse may be instrumental for a Hebbian organization and remodeling of synaptic circuits in the CNS.

NMDA-dependent proteolysis of presynaptic adhesion molecule L1 in the hippocampus by neuropsin.

Synaptic plasticity requires an activity-dependent, rapid, and long-lasting modification of synaptic character, including morphology and coupling strength. Here we show that a serine protease, neuropsin, directly and specifically modifies the synaptic adhesion molecule L1, which was localized to the presynaptic site of the asymmetric synapse in the mouse hippocampus. Increased neural activity triggered the rapid, transient activation of the precursor form of neuropsin in an NMDA receptor-dependent manner. The activated neuropsin immediately cleaved L1 and released a neuropsin-specific extracellular 180 kDa fragment. This neuropsin-specific L1-cleaving system is involved in NMDA receptor-dependent synaptic plasticity, such as the Schaffer collateral long-term potentiation.

Distribution and densitometry mapping of L1-CAM immunoreactivity in the adult mouse brain--light microscopic observation.

BACKGROUND: The importance of L1 expression in the matured brain is suggested by physiological and behavioral studies showing that L1 is related to hippocampal plasticity and fear conditioning. The distribution of L1 in mouse brain might provide a basis for understanding its role in the brain. RESULTS: We examined the overall distribution of L1 in the adult mouse brain by immunohistochemistry using two polyclonal antibodies against different epitopes for L1. Immunoreactive L1 was widely but unevenly distributed from the olfactory bulb to the upper cervical cord. The accumulation of immunoreactive L1 was greatest in a non-neuronal element of the major fibre bundles, i.e. the lateral olfactory tract, olfactory and temporal limb of the anterior commissure, corpus callosum, stria terminalis, globus pallidus, fornix, mammillothalamic tract, solitary tract, and spinal tract of the trigeminal nerve. High to highest levels of non-neuronal and neuronal L1 were found in the grey matter; i.e. the piriform and entorhinal cortices, hypothalamus, reticular part of the substantia nigra, periaqueductal grey, trigeminal spinal nucleus etc. High to moderate density of neuronal L1 was found in the olfactory bulb, layer V of the cerebral cortex, amygdala, pontine grey, superior colliculi, cerebellar cortex, solitary tract nucleus etc. Only low to lowest levels of neuronal L1 were found in the hippocampus, grey matter in the caudate-putamen, thalamus, cerebellar nuclei etc. CONCLUSION: L1 is widely and unevenly distributed in the matured mouse brain, where immunoreactivity was present not only in neuronal elements; axons, synapses and cell soma, but also in non-neuronal elements.

Decidualization induces the expression and activation of an extracellular protease neuropsin in mouse uterus.

Uterine decidualization is accompanied by the remodeling of the cell-matrix and cell-cell interactions around the endometrial stromal cells to allow an appropriate invasion of trophoblasts. This remodeling is thought to require the proteolysis of extracellular matrix proteins or cell adhesion molecules; however, the molecular mechanism remains poorly understood. In this study, decidualization induced the expression and activation of an extracellular serine protease neuropsin in the mouse uterus. Although nonpregnant uteri contained little neuropsin, the protein content and enzymatic activity increased markedly and peaked at the midgestational period in pregnant uteri. Neuropsin expression and activity was also upregulated in artificially induced deciduomata but not in nondecidualized pseudopregnant uteri. Neuropsin is the first extracellular protease to show the evident induction of expression and activity by decidualization and might contribute to the remodeling of extracellular components after decidualization.