Collapsin Response Mediator Protein 1 (CRMP1) is an intracellular phoshoprotein that mediates Semaphorin3A (Sema3A) intracellular signaling. Upon Sema3A stimulation, Fyn, a Src-type tyrosine kinase, phosphorylates and activates Cycline-dependent kinase 5 (Cdk5), which subsequently phosphorylates serine 522 of CRMP1. In addition, it has been shown that Fyn directly phosphorylates tyrosine 504 (Y504) of CRMP1 (Buel et al., 2010). Then, we investigated the functional role of this phosphorylation in Sema3A signaling. We found that Fyn phosphorylated Y504 but not other tyrosine residues of CRMP1. A dominant negative mutant of CRMP1 Y504F, substitution of tyrosine 504 to phenylalanine (F), suppressed Sema3A-induced growth cone collapse of chick E8 DRG neurons. We next tested the role of Fyn and CRMP1 in Sema3A-mediated dendritic growth in vivo. CRMP1-/- or Fyn -/- single-homozygous mice as well as Fyn^+/-; Crmp1^+/- double-heterozygous mice exhibited aberrant development of cortical layer V basal dendrites. Finally, we examined the dominant negative effect of CRMP1 Y504F on cortical dendritic morphogenesis. CRMP1 Y504F or CRMP1 WT with tdTomato was transfected in the mice cortical layer V neurons at E15 by in utero electroporation. CRMP1 Y504F-expressed layer V neurons showed poor development of basal dendrites compared with CRMP1 WT-expressed neurons at 5-weeks old mice. These results suggest that Fyn-induced phosphorylation of CRMP1 Y504 may participate in Sema3A-regulated axon pathfinding and cortical dendritic development.

Extracellular signal-regulated kinase (ERK) 5, a member of mitogen-activated protein kinase, plays important roles in the neuronal development. In our previous studies, we demonstrated that ERK5 mediates neurite/axon outgrowth and catecholamine biosynthesis in PC12 cells and sympathetic neurons. However, the regulation of membrane excitability by ERK5 remains unclear. Thus, we examined the effect of ERK5 on Ca²⁺ and K⁺ channels in PC12 cells. In order to activate ERK5 signaling selectively, ERK5 and the constitutively active MEK5 mutant were overexpressed in PC12 cells. In these cells, the gene expression of L-, P/Q- and N-type Ca²⁺ channels was not increased. In contrast, those of Kv4.2 and Kv4.3 were enhanced by ERK5 signaling. Although the protein levels of Kv4.2 were not correlated to mRNA levels, phosphorylation levels of Kv4.2 were increased by ERK5 activation. Because Kv4.2 is a pore-forming subunit of A-type K⁺ channels, which play essential roles in membrane excitability, we measured the A-type K⁺ current by a whole-cell patch clamp method. The electrophysiological data showed that ERK5 inhibits inactivation of the A-type current, which may be involved in the neural differentiation process by affecting membrane excitability.

In the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus, oxytocin and arginine-vasopressin (AVP) are synthesized to cause the lactation and reabsorption of water in the kidney, respectively. We have previously reported that M₂ muscarinic receptors in the SON, but not PVN, promote AVP synthesis. The present study was carried out to examine whether M₂ muscarinic receptors also regulate oxytocin synthesis in the hypothalamus. M₂ receptor knockout (M₂KO) mice and wild-type (WT) mice (3-4 months old) were used in the following experiments. The oxytocin neuron, AVP neuron and M₂ muscarinic receptor were identified by immunohistochemistry. c-Fos immunoreactivity was used as a marker for neuronal activity in the hypothalamus. In M₂KO mice, the number of oxytocin neurons was significantly decreased in the SON, but not in the PVN, compared with WT mice. The muscarinic agonist pilocarpine increased the number of c-fos positive cells in SON of WT mice. However, the increase of c-fos positive cells was significantly decreased in SON of M₂KO mice. Immunoreactivity of M₂ receptor was detected in the SON region, although it seemed to be not expressed in the cell body of oxytocin or AVP neurons. These results suggest that M₂ receptors may stimulate oxytocin synthesis in SON neurons as is the case of AVP, possibly through an unidentified, indirect pathway.

Dopamine plays a key role in the modulation of the circuit activity in striatum for reward behavior. We have reported that dopamine type 1 receptor (D1R) signaling in the striatum presumably regulates neuronal excitability and reward-related behaviors through PKA/Rap1/MAPK pathway. However, how D1Rs and its downstream signaling regulate neuronal excitability and behavior remain largely unknown. We focus on the post-modification of ion channels for neuronal excitability and reward behavior because protein phosphorylation of ion channels is vital for neuronal function. In this study, we identified a voltage-gated potassium channel, KCNQ2, as a phospho-candidate that is regulated by D1R signaling. Phosphorylation of KCNQ2 by MAPK cascade altered the open probability of KCNQ2/3 channels in Xenopus oocyte. The expression of phospho-defective mutants of KCNQ2 suppressed the functional modulation of KCNQ channel by MAPK. D1R agonist, SKF38393 caused a decrease in KCNQ-sensitive current in striatal slices, whereas D2R agonist, Quinpirole did not cause the effect. These results suggest that D1Rsignaling controls the channel activity of KCNQ via its phosphorylation for neuronal excitability and reward behavior.

Hippocampal sharp waves / ripples (SPW-Rs) are high-frequency oscillations emitted mainly during slow-wave sleep or quiet rest states and play a key role in memory consolidation. While SPW-Rs are initiated in the CA3 subregion and propagate to the downstream CA1 subregion, we observed that they also propagate back to the dentate gyrus. However, neither the role of CA3-to-DG SPW-Rs backpropagation nor its propagation mechanism has been fully understood. We previously demonstrated that the subthreshold membrane potentials of hilar mossy cells reflect the activity of SPW-Rs initiated in acute brain slice preparations. We thus hypothesize that mossy cells relay CA3 SPW-Rs backward to the dentate gyrus. Using in vitro whole-cell current-clamp technique, we simultaneously recorded the membrane potentials of up to five mossy cells in combination with recordings of local field potentials from the CA3 stratum pyramidale. Information theoretical analysis revealed that the activity patterns of SPW-Rs predict the combinatorial dynamics of the membrane potentials of multiple hilar mossy cells. For further confirmation, we conducted in vivo whole-cell recordings from mossy cells together with recordings of local field potential of the CA1 subregion in urethane anesthetized mice. We thus concluded that mossy cells are responsive to specific patterns of ripple information at the subthreshold level. Our research approaches further elucidation of brain information dynamics and will provide a new perspective to an information processing mechanism.

Paclitaxel (PTX) is an anticancer agent mainly used as the primary therapy for malignancies such as ovarian, breast, and stomach cancers. However, it frequently induces severe peripheral neuropathy including mechanical allodynia, numbness in a stocking-glove distribution. Recently, we developed novel and small-molecule PACAP type 1 (PAC1) receptor antagonists named PA-8 and PA-81004. In the present study, we examined the effects of novel PAC1 antagonists on PTX-induced mechanical allodynia in mice.

Repeated administration of PTX (2 mg/kg, once a day for 5 days, i.p.) induced mechanical allodynia of the hind paw and activation of spinal astrocyte. Single intrathecal injection of PA-8 (1 nmol) suppressed both PTX-induced mechanical allodynia and astrocytic activation, suggesting that spinal astrocytes activated by PACAP/PAC1 receptor signaling are involved PTX-induced mechanical allodynia.

Next, we examined the systemic administration of the PAC1 receptor antagonists. Single oral administration of PA-8 or PA-81004 (3-30 mg/kg) dose-dependently alleviated PTX-induced mechanical allodynia. The effects of PA-81004 were more potent than PA-8. Repetitive treatment with PA-8 (30 mg/kg, p.o.) 30 minutes before PTX administration almost completely inhibited the induction of mechanical allodynia. These results suggest that PA-8 exerts both therapeutic and preventive effects. Our novel PAC1 receptor antagonists may become orally available analgesics against PTX-induced peripheral neuropathy.

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) in pain transmission has been well documented, but its involvement in itch transmission is entirely unclear. We recently developed novel small-molecule antagonists of PACAP type 1 (PAC1) receptor including PA-8 by in silico screening. In this study, using PA-8, we investigated the possible involvement of PACAP/PAC1 receptor signaling in itch.

Both intradermal (i.d.) and intrathecal (i.t.) injection of PACAP (1 pmol–1 nmol) dose-dependently elicited scratching/biting behaviors, and these behaviors were inhibited by subcutaneous pretreatment with the μ-opioid receptor antagonist naltrexone (1 mg/kg). The scratching/biting behaviors induced by i.d. and i.t. PACAP were inhibited by i.d. and i.t. co-injection of PA-8 (0.1–10 nmol), respectively. The application of 5-HT (200 nmol in EtOH) to the skin elicited scratching behaviors, and they were suppressed by i.t., but not i.d., pretreatment of PA-8 (0.1–10 nmol). Next, we examine the effects of PA-8 on pruritic models. In the itch model induced by cutaneous application of acetone/ether and water (AEW, a dry skin model) or 2,4-dinitrofluorobenzene (DNFB, an atopic dermatitis model), single oral administration of PA-8 (3 – 30 mg/kg) dose-dependently suppressed the itch-associated behaviors.

These results suggest that PACAP/PAC1 receptor signaling in the skin and/or spinal cord is involved in an itch sensation. The small-molecule PAC1 receptor antagonist may become an orally available antipruritic drug in the treatment of acute and chronic itch.

An endogenous sulfur, polysulfide (PS) is generated by oxidation of hydrogen sulfide. We previously reported that PS stimulated nociceptive transient receptor potential A1 (TRPA1) channel in sensory neurons. TRPA1 is also activated by reactive oxygen species (ROS). Here, we examined the effect of PS on responses to hydrogen peroxide (H₂O₂), one of ROS, using mouse sensory neurons and heterologously expressed mouse TRPA1 in HEK293 cells (mTRPA1-HEK). In mouse sensory neurons, H₂O₂ evoked two types of [Ca²⁺]_i responses, an early TRPA1-dependent and a late TRPA1-independent ones. Pretreatment with PS inhibited the H₂O₂-induced early responses in a dose-dependent manner. PS also suppressed [Ca²⁺]_i responses to PGJ₂, another endogenous TRPA1 agonist in mouse sensory neurons. In mTRPA1-HEK, PS inhibited [Ca²⁺]_i responses to not only H₂O₂ but also PS itself and PGJ₂. Simultaneous measurement of [Ca²⁺]_i and [PS]_i showed that PS did not present in the period of the inhibiting effect of PS. The removal of extracellular Ca²⁺ and calmodulin inhibitor diminished the PS-induced suppression of [Ca²⁺]_i responses to H₂O₂. When PS was administrated intraplantary prior to H₂O₂, pain-related behaviors induced by H₂O₂ significantly decreased in mouse. The present data suggest that an endogenous sulfur desensitizes TRPA1 resulting in an inhibition of subsequent activation induced by oxidative stresses via Ca²⁺ influx through TRPA1. Calmodulin signaling may be involved in PS-induced TRPA1 desensitization.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic bladder inflammation characterized by pelvic pain and urinary symptoms, such as urinary frequency and urgency. The etiology of IC/BPS is still not completely understood, and effective drug treatments have not been established. Therefore, the present study confirmed whether repeated intravesical injection of lipopolysaccharide (LPS) causes long-lasting painful and overactive bladder in rats. We further tested the effect of gabapentin on those symptoms in a rat model of LPS-induced chronic cystitis. In the histological examination, LPS-treated showed a greater inflammatory response, severe fibrosis and abnormally thick re-epithelialization. LPS revealed hyperalgesia in the region between the anus and urethral opening at 1day post-administration compared with controls, with no recovery over 21 days. In the cystometry, LPS-treated showed bladder hyperactivity at any times tested. Gabapentin showed significant analgesic effects, and significantly prevented the increased frequency of the voiding observed in the bladders of the LPS-treated. These results suggest that LPS-induced cystitis model shows long-lasting painful and overactive bladder in pathological condition, and gabapentin is effective on both symptoms in this chronic cystitis model.

Trigeminal neuropathy, caused by injury to trigeminal nerve, manifests as orofacial numbness, paresthesias, or/and pain and are refractory to treatment with commonly used analgesics. In this study, we employed distal infraorbital nerve chronic constriction injury (dIoN-CCI) model, which mimic pathology of trigeminal neuropathy, to investigate whether high mobility group box 1 (HMGB1), a kind of damage-associated molecular patterns, is involved in trigeminal neuropathy.

Under anesthesia, silk sutures were tied loosely around the dIoN of ddY male mice. Nociceptive-like behaviors were evaluated by measurement of face grooming episodes and conditioned place preference test. Microglial activity in spinal trigeminal nucleus caudalis (Sp5c) was determined by immunohistochemistry. Anti-HMGB1 neutralizing antibody (nAb) was perineurally injected right after surgery.

In dIoN-CCI mice, the mouse face grooming time was increased compared with sham mice. In addition, dIoN-CCI evoked activation of microglia in Sp5c and preference to mirogabalin-paired chamber. Moreover, the perineural treatment with anti-HMGB1 nAb blocked the dIoN-CCI-induced face grooming, microglia activation, and preference to mirogabalin. The anti-HMGB1 nAb could be a novel therapeutic reagent for inhibiting the induction of trigeminal neuropathy.

Platelet-activating factor (PAF) is a potent phospholipid mediator, which is involved in the pathology of neuropathic pain after peripheral nerve injury (PNI). In PAF receptor- and its biosynthetic enzyme lysophosphatidylcholine acyltransferase 2 (LPCAT2)- deficient mice, neuropathic hypersensitivity was significantly attenuated. However, regulation of PAF level after PNI remains to be elucidated. Here, we show that PNI increases PAF levels in the spinal cord and in the dorsal root ganglia (DRG). While PAF biosynthetic activity and mRNA expression of LPCAT2 were increased in both tissues, enzymatic activity of PAF degradation and mRNA expression of plasma type of PAF-acetylhydrolase (PAF-AH), which is one of PAF degradation enzymes, were decreased only in the DRG. These results suggest that distinct mechanisms exist between the spinal cord and the DRG to regulate PAF levels after PNI, and then increased PAF levels may contribute to neuropathic pain.

The G-protein-coupled receptor MrgprA3 (MAS-related GPR family member A3) is expressed specifically in a subpopulation of dorsal root ganglion (DRG) sensory neurons. Recently, MrgprA3⁺ DRG neurons are identified as itch-selective neurons. While MrgprA3 responds to the anti-malaria drug chloroquine and causes strong itch, chloroquine requires high concentrations to activate MrgprA3 and also displays non-selective effects. Therefore, it is necessary to accurately evaluate the ability of MrgprA3 to cause itch sensation. In this study, we screened a series of small molecule compounds to search for agonists that activate MrgprA3 by high-throughput Ca²⁺ imaging. We identified papaverine, an opium alkaloid, that specifically evoked Ca²⁺ responses in cells expressing MrgprA3. Papaverine also increased Ca²⁺ level in primary cultured DRG neurons that were responded to chloroquine. Furthermore, we found that intradermal injection of papaverine to the cheek produced scratching behavior but not wiping behavior in mice. Papaverine-evoked scratching was resistant to the histamine H1 receptor antagonist chlorpheniramine. We further found that intradermal papaverine caused phosphorylation of extracellular signal-regulated kinases (ERK) in the superficial dorsal horn. Finally, we showed that mice lacking gastrin-releasing peptide receptors (GRPR) that are required for itch transmission in the spinal cord exhibited reduction of the papaverine-evoked scratching compared with wild-type mice. In this study, we found that papaverine potently activates MrgprA3 and may cause itch sensation via activation of MrgprA3.

High mobility group box 1 (HMGB1), one of damage-associated molecular patterns (DAMPs), once released to the extracellular space, aggravates inflammation and pain. We have reported that macrophage (Mφ)-derived HMGB1 is involved in chemotherapy-induced peripheral neuropathy (CIPN) following paclitaxel (PCT) treatment. Given our recent clinical evidence for a higher risk for CIPN in PCT-treated breast cancer women over menopause age than younger women, we examined the effect of ovariectomy (OVX) on the development of CIPN in mice treated with PCT. In naïve mice, repeated i.p. administration of PCT at 2 and 4 mg/kg, but not 1 mg/kg, developed CIPN. In contrast, treatment with PCT even at 1 mg/kg caused CIPN on OVX mice. The CIPN in OVX mice treated with PCT at 1 mg/kg was prevented by an anti-HMGB1-neutralizing antibody, recombinant human soluble thrombomodulin capable of promoting HMGB1 degradation, or β-estradiol. Together, our data suggest that estrogen deficiency aggravates CIPN after PCT treatment, and the underlying mechanisms involve HMGB1.

Migraine is neurological disorder that includes unilateral headache. Although migraine is highly prevalent, its pathophysiology is unclear. In humans, intravenous administration of pituitary adenylate cyclase-activating polypeptide (PACAP), but not vasoactive intestinal polypeptide (VIP), induces migraine-like attacks, suggesting that selective PACAP type 1 (PAC1) receptor antagonist could be a new anti-migraine drug. Previously, we have developed novel small-molecule antagonists of the PAC1 receptor. In the present study, we investigated the effect of PA-8, one of novel PAC1 receptor antagonist, on pain-related behaviors induced by nitroglycerin (NTG) which is widely studied and accepted as an animal model of migraine.

Single or repeated administration of NTG (5 mg/kg, i.p.) induced transient and long-lasting mechanical allodynia of the hind paw, respectively. NTG also induced the increase in the number of c-fos-positive cells in the trigeminal nucleus caudalis (TNC), light-aversive behavior, and anxiety-like behavior in mice. Single or repeated administration of PA-8 (10-30 mg/kg, i.p.) reduced NTG-induced mechanical allodynia, the number of c-fos-positive cells, and anxiety-like behavior. Single administration of PA-8 (30 mg/kg, i.p.) had a tendency to reduce the light-aversive behavior induced by NTG.

The present results suggest that PAC1 receptor is deeply involved in the NTG-induced migraine-related aversive responses and that PAC1 receptor antagonists may become novel anti-migraine drug.

We have shown that activation of the receptor for advanced glycation end-products (RAGE) by all-thiol (at)-HMGB1 participates in pathological pain. Interestingly, low molecular weight heparin (LMWH) blocks RAGE and reduces the HMGB1-dependent pain, although it has potent anti-Xa and moderate anti-IIa activities. In the present study, we assessed the anti-RAGE, anti-Xa and anti-IIa activity of middle molecular weight heparinylphenylalanine (MMWH-F), in comparison with LMWH, MMWH and heparin (HP), and tested whether it prevented the at-HMGB1-induced allodynia, oxaliplatin-induced peripheral neuropathy (OIPN) and butyrate (Bu)-induced colonic pain/hypersensitivity in mice. The RAGE-binding affinity of heparinoids was LMWH ≈ MMWH ≈ HP << MMWH-F. The potency of anti-Xa and anti-IIa activities was MMWH-F << LMWH ≈ MMWH < HP and MMWH-F (not detectable) << LMWH < MMWH < HP, respectively. LMWH, MMWH or MMWH-F, preadministered i.p. at 2.5 mg/kg, partially blocked the intraplantar at-HMGB1-induced allodynia in mice. MMWH-F as well as LMWH, but not MMWH, at 2.5 mg/kg, almost completely prevented OIPN. LMWH, MMWH or MMWH-F at 2.5 mg/kg largely prevented or reversed the Bu-induced colonic pain and hypersensitivity. Together, MMWH-F is considered a potent RAGE blocker, and useful to prevent or treat neuropathic and visceral pain without causing hemorrhage.

Conditioned Place Preference (CPP) is a test to evaluate the rewarding effects related to psychological dependence induced by drugs, and is used to clarify the mechanism of drug dependence and to search for the effects of dependence of newly developing drugs with central nervous system effects. In the conditioning of CPP in rats, intraperitoneal or subcutaneous administration is generally used, but oral administration is also required to test article that cannot be dissolved. However, very few reports exist on CPP using oral administration. Therefore, we performed CPP evaluation following oral administration. In our tests, rewarding effects were noted using morphine. Therefore, etizolam, which have been reported to cause dependence in humans, but for which few reports on experiments in animals are available, was evaluated using similarly method and rewarding effects were noted. In this presentation, we report the results of our experiment and propose a new method to analysis data, taking into consideration the effects of individual differences, based on the results obtained so far.

MAM domain glycosylphosphatidylinositol anchor 1 (MDGA1) is one of the extracellular anchor proteins which belong to immunoglobulin superfamily. MDGA1 negatively regulates inhibitory synapse via selective interaction with Neuroligin-2. Neuroligin-2 is expressed in inhibitory GABAergic neurons, and contacts synaptic terminals to form synapses including dopaminergic synapses which play a key role in rewarding systems. We previously reported that MDGA1 knockout mice actually show enhancement of inhibitory perisomatic synaptogenesis in hippocampus and impairment of cognitive functions. In this study, we investigated the characteristics of morphine-induced dependence in MDGA1 knockout mice. Acquisition of morphine-induced conditioned place preference (CPP) in MDGA1 knockout mice was slightly increased while extinction of morphine CPP was strongly impaired. In contrast, naloxone-precipitated morphine withdrawal signs were decreased in MDGA1 knockout mice. These results demonstrate that MDGA1 can help the extinction of morphine-induced reward but make the expression of somatic withdrawal signs worse.

Pretreatment of mice with pitolisant, a histamine H₃ receptor antagonist, showed a significant reduction of the hyperlocomotion induced by METH, as compared with vehicle (saline)-pretreated subjects. The pitolisant action on METH-induced hyperlocomotion was completely abolished by a histamine H₁ receptor antagonist pyrilamine resulting in hyperlocomotion, but not by a peripherally acting histamine H₁ receptor antagonist fexofenadine, a brain-penetrating histamine H₂ receptor antagonist zolantidine, or a brain-penetrating histamine H₄ receptor antagonist JNJ-7777120. Pretreatment with a histamine H₃ receptor agonist immepip rather augmented METH (3 mg/kg)-induced behavioral abnormalities from hyperlocomotion to stereotyped biting, and, combined pretreatment of pitolisant with immepip significantly attenuated the stereotyped behaviors. Pretreatment with JNJ-10181457 or conessine, other histamine H₃ receptor antagonists, showed inhibitory effects on METH-induced hyperlocomotion similar to that of pitolisant. No significant change in locomotion was observed in mice pretreated with pitolisant, JNJ-10181457, or conessine alone. Pretreatment with pitolisant prior to a high-dose METH (10 mg/kg) significantly decreased the intensity of stereotyped behaviors and increased its latency to onset in a dose-dependent manner. JNJ-1018145, but not conessine, mimicked the inhibitory action on METH-induced stereotyped behavior.

A single administration with morphine induced a long-lasting hyperlocomotion in mice. Pretreatment of mice with tetrabenazine (TBZ; a reversible vesicular monoamine transporter-2 inhibitor) significantly attenuated the hyperlocomotion induced by morphine, as compared with vehicle-pretreated mice. No significant change in locomotion was observed in mice pretreated with TBZ alone. Mice treated with TBZ showed an increase in immobility time in a tail suspension test, as compared with saline-treated mice. Pretreatment with TBZ had no effect on morphine-induced alterations in expression levels of dopamine transporter in brain. TBZ inhibited dopamine turnover (the ratio of DOPAC/dopamine) and 5-HT turnover (the ratio of 5-HIAA/5-HT) in the cerebral cortex of mice challenged with morphine, as compared with saline-pretreated mice challenged with morphine. No stereotyped behavior was observed in mice treated with morphine in combination with TBZ, so that the reduction in observed locomotion did not result from induction of stereotypy. Moreover, TBZ pretreatment had no effect on stereotypy in methamphetamine-treated mice. These data support the potential antagonistic actions of TBZ on some opiate actions, and encourage further exploration of potential effects on morphine reinforcement.

Methamphetamine (METH) is a highly addictive psychostimulant with reinforcing properties. We previously found that the cannabinoid CB₁ receptors drive reinstatement of METH-seeking behaviors. The purpose of this study was to determine whether the activation of endocannabinoids regulates the reinstatement of METH-seeking behaviors. Rats were tested for reinstatement of METH-seeking behaviors following METH self-administration and extinction. We investigated the effects of JZL184 or URB597, inhibitors of endocannabinoid hydrolysis, on the reinstatement of METH-seeking behaviors. JZL184 (40 mg/kg, i.p.), an inhibitor of monoacylglycerol lipase, significantly attenuated both the cue- and footshock-induced reinstatement of METH-seeking behaviors. URB597 (3.2 mg/kg, i.p.), an inhibitor of fatty acid amide hydrolase, attenuated only cue-induced reinstatement. On the other hands, we also investigated the effect of the inhibitors of endocannabinoid hydrolysis on cognitive function using the novel object recognition task in mice. The recognition index level in the test did not change in JZL184-treated mice. However, URB597 significantly decreased the recognition index level. These findings suggested that JZL184 might have potential as a new therapeutic agent with anti-craving effect, without amnestic effects, in METH addiction.

Some men sometimes invite an interested woman to bar for alcohol drinking. Alcohol makes them cheerful and improves their relationship occasionally. The man expects alcohol could decrease the threshold to open her mind, and, in some cases, lose accurate judgments about him as a sexual partner. In this research, we are trying to reveal a part of the neural activities and behavioral characteristics in male and female using a mouse model in such situation.

We have established the behavioral model for male preference of female mice, in which we found that attractiveness of male mice to female mice is dependent on their appearance, not voice nor smell. Additionally, we performed in vivo microdialysis analyses under the experimental conditions, and found that dopamine levels in the nucleus accumbens of female mice responded to attractive male mice, but not to unattractive male mice.

Using this model, we examined the effects of alcohol on the male preference and dopamine response of female mice.

Q1. Can alcohol-administered female mice recognize attractiveness of male mice?

Q2. Can dopamine levels in the nucleus accumbens of female mice increase at the timing to meet with an unattractive male mouse after alcohol administration?

Q3. When alcohol-administered female mouse was set in rectangular box with an unattractive male mouse in a transparent small box, does she stay close to him or opposite area of him?

Q4. After repeated conditioning with alcohol aministration like as question 3, does the preference of female mice to the unattractive male mouse increase or not?

We will try to answer these questions in this presentation. These results will present the hint for how alcohol affects the relationship between male and female.

Mating-inexperienced male mice show aggressive behavior toward pup. However, they show parental behavior after the social experiences such as mating and seeing pups. Previously, we found that the GABAergic projection in the medial preoptic area (MPOA) is suppressed in the father mice in gestation experience (FGE) which experienced mating and staying with late pregnant female. This synaptic change was reversed by using G protein signaling inhibitor GDPβ. However, the mechanism of plastic changes in Me → MPOA pathway synapses induced by cohabitation with females remains unclear. In this study, we carried out the electrophysiological recordings from MPOA neurons of sexually inexperienced male mice and FGE. It was newly found that the amplitude of inhibitory post-synaptic potential of MPOA was significantly increased by U0126 in FGE mice. Furthermore, in order to validate the effects of U0126 on the behavioral pattern, U0126, was microinjected into MPOA of FGE mice, and a behavioral test in response to pup exposure was tested. As a result, in the group administered with U0126, the proportion of individuals exhibiting aggressive behavior toward pups increased significantly. These results suggest that signaling through MEK1/2 is involved in GABAergic synaptic plastic changes in MPOA that stop attacks and induce parenting behavior.

Alcohol exposure impairs the retention of spatial memory. Consistently, previous reports have demonstrated widespread changes in a variety of receptor functions and gene expressions in the hippocampus, a brain region involved in spatial memory. However, it remains unknown how these molecular mechanisms are integrated to alter neuronal spike patterns. Hippocampal neurons consist of place cells as they fire preferentially when an animal visits a specific area (a place field), which are considered to play a crucial role in spatial memory. Here, we recorded spatial spike patterns of hippocampal neuronal ensembles from freely moving rats running on familiar linearized tracks. The rats were tested in two 20-min sessions of running and during a 10-min inter-session interval, they were injected intraperitoneally with 1.5 g/kg ethanol, a dose comparable to those generally consumed by humans. The alcohol administration triggered the emergence of a subset of place cell populations, while abolishing place-selective firing of the other place cell populations. Moreover, a subset of place cells altered their place fields. These results demonstrate that hippocampal spatial maps are dynamically reorganized by ethanol administration. The neuronal mechanism may underlie alcohol-induced impairments in hippocampus-dependent memory.

In the central nervous system, glycine plays an excitatory role via binding to the NMDA receptors (NMDARs) and an inhibitory role via binding to the glycine receptors (GlyRs). Recently, it has been found that upon the increase of glycine concentration in the synaptic cleft, glycine binds to the GlyRs, mainly expressed in the extrasynapse, and introduces LTD. In addition, NMDARs and AMPA receptors (AMPARs) involve in this type of LTD. To elucidate the mechanisms underlying glycine-dependent LTD, we examined the phosphorylation of AMPARs under excess amount of glycine. Furthermore, we hypothesized that the administration of high-dose glycine would attenuate hippocampal- dependent fear memory, known to require the phosphorylation of AMPARs.

We performed biochemical analysis to examine the phosphorylation status of AMPARs in the hippocampus under different doses of glycine. Glycine decreased the phosphorylation of serine 845 of GluA1 subunit of AMPARs dose-dependently. Furthermore, high-dose glycine co-incubated with strychnine, AP5 and FK506 did not show the reduction in the phosphorylation of serine 845 of GluA1, suggesting that this effect of glycine was mediated by not only GlyRs but also NMDARs. Interestingly, the high-dose administration of glycine to the rats before fear conditioning reduced the freezing behavior during the test. Moreover, this effect was eliminated by co-administration of strychnine.

In this study, we revealed that LTD required the dephosphorylation of serine 845 of GluA1 and this type of LTD could attenuate contextual fear memory.

Background: In general, hypoxia can suppress neural activities and cause cognitive impairment. Hypoxia inducible factor-1α (HIF-1α) is known as a transcription factor expected to play pivotal roles in the response to hypoxia in various tissues. HIF-1 a is hydroxylated by prolylhydroxylase (PHD) in an oxygen-dependent manner, and then it is degraded in ubiquitin system. Preceding studies have revealed that PHD inhibitors increase HIF-1α and induce hypoxia-like responses. In this study, we investigated the effects of PHD inhibitors on cognitive performance in mice.

Methods: Male 5-weeks-old ddY mice were subjected to novel object recognition test for evaluation of long-term memory performance. Dimethyloxalylglycine (DMOG) and roxadustat, PHD inhibitors, are subcutaneously injected.

Results: Single administration of DMOG or roxadustat 30 min before the learning phase of novel object recognition test significantly lowered discrimination index. The suppressive effect on memory performance of 5 consecutive daily administration of DMOG was similar to that of a single administration.

Conclusion: The present results suggest the possibility that an increase of HIF-1α causes cognitive impairment. Since PHD inhibitors are candidates for the treatment of anemia, attention should be paid to the side effects on cognitive performance.

Previously, we found that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) model mice (PD mice) show facilitation of hippocampal memory extinction via reduced cyclic adenosine monophosphate (cAMP)/cAMP-dependent response element-binding protein (CREB) signaling, which may cause cognitive impairment in PD. Serotonergic neurons in the median raphe nucleus (MnRN) project to the hippocampus, and functional abnormalities have been reported. In the present study, we investigated the effects of the serotonin 5-HT₄ receptor (5-HT₄R) agonists prucalopride and velusetrag on the facilitation of memory extinction in PD mice, because 5-HT₄R, which is a Gs protein-coupled receptor that activates adenylate cyclase, is highly expressed in the hippocampus. We found that 5-HT₄R agonists restored facilitation of contextual fear extinction in PD mice by stimulating the cAMP/CREB pathway in the dentate gyrus (DG) of the hippocampus. These findings suggest that 5-HT₄R agonists could be potentially useful as therapeutic drugs for treating cognitive deficits in PD by improving the cAMP/CREB signaling pathway in the hippocampal DG.

We previously reported that intracerebroventricular administration of calcitonin gene-related peptide (CGRP) effect on hippocampus-dependent fear memory in mice. Although CGRP plays an important role in central nervous system, it seems to be degraded before reaching the brain by intravenous administration. The intranasal route offers an alternative approach for drug delivery to the brain without the interference of the blood-brain-barrier. In this study, we evaluated the nose-to-brain delivery of CGRP to investigate the effects on fear memory retention by contextual learning test. 8-week-old male C57BL6J mice were examined to contextual fear learning test. Mice were given a 0.3 mA foot shock. After fear conditioning, mice were given saline or CGRP (0.5 nmol) by intranasal administration (i.n.). CGRP injections shortened the freezing time when compared to saline. Next we also evaluated Bdnf or Npas4 mRNA in mice hippocampus. As same as intracerebroventricular administration, CGRP i.n. significantly increased the level of Npas4 rather than saline treatment. Bdnf level were also significantly increased. These results suggest that nose-to-brain delivery of CGRP alleviate the fear memory with increases Npas4 and Bdnf in mice hippocampus.

Hippocampus is crucial for episodic-like memory formation. The dynamics of hippocampal neuronal ensembles during learning has not been well understood. To investigate the hippocampal circuit property during learning, we designed a new spatial learning task and performed multiunit recording from CA1 neurons. Analyses for hippocampal place cells revealed that hippocampus captured not only the structure of environments, but also the structure of behavior task. Further analyses for hippocampal neuronal replays, the neuronal ensemble reactivations followed by sharp-wave ripples, showed rate increases in the frequency of replays with dynamic changes in replay patterns, reflecting the newly learned pathways. Real-time neuro-feedback experiments confirmed that such prioritized experience replay supports efficient learning. These results indicate that hippocampal neurons represent the self-state in the environment with multiple axes, enabling the hippocampus to flexibly emit experience replays to support the reinforcement of a specific behavior pattern.

Adenosine deaminase (ADA) is a widely expressed enzyme that catabolizes adenosine and deoxyadenosine into inosine and deoxyinosine, respectively. Adenosine is known to play a protective role by interacting with adenosine receptors when its extracellular concentration is increased. Therefore, ADA has been considered toxic to the central nervous system under ischemia, hypoxia and tissue damage. However, ADA-deficiency results in neurological disorders. We were interested in determining whether ADA is protective or harmful in the striatum, which is especially vulnerable during cerebral ischemia. In order to determine the effects of ADA on transient ischemic stress of the striatum, we used acute rat corticostriatal slices. We found that ADA has substantial neuroprotective effects in the striatum. In addition, we examined whether the neuroprotective effect of ADA is due to the removal of deoxyadenosine. The deoxyadenosine administration does not show acute cytotoxicity, suggesting that the neuroprotective effect of ADA may be due to other mechanisms, such as the recently reported direct effect of ADA on the adenosine A_2A receptors.

[Introduction] Subarachnoid hemorrhage (SAH) is a life-threatening type of stroke and can be frequently caused by a ruptured aneurysm of cerebrovascular blood vessels. Although one third of patients could survive with good recovery; one-third will survive with a disability; and one-third will die.

It is well accepted that lowering blood cholesterol level is mandatory in prevention of cerebral circulatory disorder. However, the relationship between cholesterol and cerebral aneurysm is still controversial.

In this study, we elucidate the above relationship by monitoring aneurysm and SAH in 1) aneurysm model of LDL receptor/ Apobec 1 double knock out (LA-/-) mice and that of control mice. 2) Reducing cholesterol intake by administering Cholestyramine, cholesterol lowering cationic resin, to LA -/- aneurysm model mice together with daily food.

[Method] Experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Hamamatsu University School of Medicine, Hamamatsu, Japan.

Hashimoto model of animal cerebral aneurysms was performed.

Briefly, left kidney was excised one week before the experiment. Elastase was administered to the subarachnoid space to damage cerebral artery and sustained-release deoxycorticosterone was placed subcutaneously. Drinking water was substituted with 1% salt solution. Three weeks later, the brain tissue was harvested for evaluation of cerebral aneurysm and subarachnoid hemorrhage.

[Results] 1) lesser amount of cerebral aneurysm and SAH were detected in aneurysm model of LA-/- mice compared to control mice.

2) Increasing trend of SAH was observed in LA-/- mice with cholestyramine administered group.

Vasogenic edema is a severe condition resulted from disruption of blood-brain barrier (BBB) after traumatic brain injury (TBI). Endothelin (ET) is an aggravating factor for the BBB disruption. In this study, we confirmed the effects of ambrisentan, an ETA receptor antagonist for TBI-induced vasogenic edema. As a model of TBI, mice (male ddY, 6 to 7 weeks) were given fluid percussion injury (FPI) by hydraulic impact on dura mater. Ambrisentan (0.02, 0.1 and 0.5 mg/kg/day) were repeatedly administrated from mouse tail vein in 6 hour to 2 days after FPI. To evaluate vasogenic edema, the BBB disruption and brain edema were evaluated by Evans blue extravasation and brain water content, respectively. Expressions of vascular endothelial growth factor-A (VEGF-A) as an aggravating factor for the BBB disruption and angiopoietin-1 (ANG-1) as a protective factor were measured by Real-time PCR. Expression of ETA receptors was observed by immunohistochemistry. The i.v. administrations of ambrisentan ameliorated the BBB disruption and decreased in brain water content in 2 day after FPI. Ambrisentan decreased in VEGF-A and increased in ANG-1 after FPI. The immunochemical observations indicated that ETA receptors were distributed in brain endothelial cells in mouse cerebrum. In vitro experiments, treatment with ET-1 (100 nM) increased in VEGF-A and decreased in ANG-1 in brain endothelial cells (bEnd.3 cells). Treatment with ambrisentan (1 μM) inhibited the effects of ET-1. These results suggest that ambrisentan is expected to be a novel therapeutic drug for vasogenic edema.

Cerebral ischemia is one of the main causes of disability. Although many studies report that exercise improves prognosis of cerebral ischemia, the mechanisms are not revealed. The aim of this study is to reveal the underlying mechanisms of exercise on functional recovery after cerebral ischemia. We occluded the middle cerebral artery in C.B-17/Icr-^+/+Jcl mice to obtain reproducible size of cerebral infarction. Mice were divided into 4 groups: Sham, Sham + exercise, Middle cerebral artery occlusion (MCAO) and MCAO + exercise. Exercise groups had free access to a running wheel. Wire hang test was performed on day 14 post-ischemia. Grid walking test was performed daily for 14 days after cerebral ischemia. Then, we visualized the neuronal processes and dendritic spines of pyramidal cells in the layer 5 by microinjection with Lucifer yellow. As a result, voluntary exercise contributed to functional recovery after cerebral ischemia and affected the number of dendritic spines. Our data suggest that functional recovery is caused by the change in dendritic spine density following voluntary exercise.

Aging is one of the major risk factors to worse mortality rate and neurobehavioral deficits after intracerebral hemorrhage (ICH). Previous research has demonstrated that aged ICH rodents show exacerbated ICH pathology, and recently, mid-life cardiovascular factors are implied to be an independent predictive factor for later-life mild cognitive impairment and dementia. However, no study has ever compared ICH pathology between young vs middle-aged mice. In this study, therefore, we used 2-month and 8-month old mice to examine whether 8-month old mice show different patterns of glial responses after ICH. ICH was induced by unilateral microinjection of 0.025 U type VII collagenase (0.5 uL) into the right side of brains of male C57Bl/6J mice. One or 8 days after collagenase injection, animals were sacrificed, and the brains were subjected to immunostaining using an astrocyte maker GFAP antibody. In the peri-lesion area, the number of GFAP-positive cells in 8-month old mice was decreased compared to the one in 2-month old mice. These data may suggest that glial responses after brain injury are already changed even in the middle-aged mice. Future studies are warranted to examine how these mid-life glial changes affect later-life neurological and cognitive dysfunction after ICH.

Stroke-prone spontaneously hypertensive rat (SHRSP) is widely accepted as an animal model of hypertension and cerebrovascular disease. SHRSP occurs BBB dysfunction even if chronic hypertension and onset of stroke are not well-established. The properties of BBB are maintained by cross-talk between brain endothelial cells and surrounded adjacent cells, such as astrocytes and pericytes. To reveal the detailed mechanism underlay the BBB dysfunction of SHRSP, we constructed an in vitro BBB model using brain endothelial cells, pericytes, and astrocytes. Isolated brain capillary endothelial cells (BECs) from SHRSP showed leaky barrier function and altered composition of tight junction proteins (claudin-5 and occludin). The co-culture method of BECs and astrocytes indicated that SHRSP astrocytes had less ability to induce barrier function on BECs than did astrocytes derived from normotensive control rat (WKY/Izm). In comparison using the triple co-culture model, SHRSP model showed a weak barrier function than WKY model. These results suggest that BBB-relate cells in SHRSP have different properties and the defective interactions among these cells. This altered cross-talk may be related to occurrences of cerebrovascular diseases in SHRSP.

Intracerebral hemorrhage (ICH), a bleeding into the brain parenchyma, is a devastating neurologic disease with the highest mortality among all stroke subtypes. In ICH brain, thrombin induces activation of microglia/macrophages followed by neuroinflammation. Furthermore, ICH leads to infiltration of numerous leukocytes. Recent report shows the arachidonic acid metabolite, leukotriene B₄ (LTB₄), participates pathological progression of ICH (Hijioka et al., 2017). In this study, we focused on lipoxin A₄ (LXA₄), synthesized from arachidonic acid as same as LTB₄. Treatment of murine microglial cell line BV-2 cells with thrombin (30 U/mL) increased mRNA expression level of inducible NO synthase (iNOS) and interleukin-6 (IL-6). Pretreatment with LXA₄ (100 µM) suppressed thrombin-induced increases in iNOS and IL-6 mRNA expression. Moreover, immunocytochemical analysis revealed the translocation of nuclear factor-κB (NF-κB) into the nucleus induced by thrombin, and thrombin-induced nuclear translocation of NF-κB was suppressed by LXA₄. Finally, daily intravenous administration of LXA₄ receptor agonist, BML-111 (1 mg/kg) attenuated the motor dysfunction of mouse model of ICH. These data suggest that LXA₄ may be the novel therapeutic agent for ICH.

Stroke is a global health problem and leads to disability. Efficacy of current treatment in both acute and chronic phase of stroke is limited. To accelerate individualized approach to primary prevention for stroke, identification of biological parameter, which is able to predict the risk of stroke development, is an indispensable component. Stroke-prone spontaneously hypertensive rat (SHRSP) is genetic animal model of chronic hypertension that progresses to stroke. Most of SHRSP spontaneously develop stroke including cerebral infarction and cerebral hemorrhage. Hematological tests readily provide health condition information. In this study, we investigated the time course of hematological parameters in Wistar rats and SHRSP. SHRSP develop stroke-related symptoms including onset of neurological symptoms, decreased body weight and blood brain barrier leakage between 12 and 14 weeks of age. Lymphocyte counts were gradually decreased at 3 weeks before development of stoke-related symptoms and then were further decreased after the development of stroke-related symptoms. The platelet volume, as shown by the mean platelet volume and large platelet ratio, gradually increased at 3 weeks before the development of stoke-related symptoms. However, although SHRSP showed more microcytic red cells than Wistar rats, the trajectories of change in erythrocyte-related parameters were similar between Wistar rats and SHRSP. Our findings suggest that alterations of lymphocyte count and platelet volume may be predictive indicators for stroke development in SHRSP.

Neurogenesis is transiently enhanced by cerebral ischemia, but the number of newly generated neurons are not enough to compensate for damage of brain tissue. Furthermore, it seems that the majority of newly generated endogenous neurons after cerebral ischemia fail to survive. Therefore, acceleration of endogenous neurogenesis in the brain has a potential to become a new therapeutic approach for stroke. Progranulin (PGRN) is a cysteine-rich protein which is implicated in cell proliferation and tumorigenesis. In this study, we investigated the effect of PGRN on proliferation and differentiation in cultured neural stem cells. The sphere diameter of neural stem cells used in this study increased with time and these cells had multipotency. Treatment with recombinant PGRN (rPGRN) enhanced the ability of neural stem cells to proliferate and differentiate into neurons. Proliferation of neural stem cells was promoted by oxygen-glucose deprivation (OGD) treatment, and PGRN was co-localized with nestin in these cells. These results suggest that PGRN contributes to the proliferation and differentiation of neural stem cells after cerebral ischemia.

Progranulin (PGRN) is implicated in neuronal protection and anti-inflammation. On the other hand, granulin (GRN), which is cleaved from PGRN by neutrophil elastase, has pro-inflammatory effects. However, pathophysiological roles of PGRN and GRN after cerebral ischemia have not been fully determined. In this study, we examined time-course of changes in the levels of PGRN and GRN and their cellular sources after cerebral ischemia using a rat microsphere-embolism (ME) model and rat primary cultured microglia. Protein and mRNA levels of PGRN in activated microglia were increased in the ischemic region of cerebral cortex on day 3 after ME. Elastase activity was increased on day 1 after ME. GRN was increased on days 1 and 3 after ME. Next, we examined effect of sivelestat, a selective neutrophil elastase inhibitor, on the levels of PGRN and GRN after ME. The level of PGRN was increased in the cerebral cortex, whereas elastase activity and GRN level were decreased in sivelestat-treated rats on day 1 after ME compared with those of vehicle-treated rats. Thus, the increase in PGRN level and inhibition of GRN production after ME caused by siverestat treatment would prevent ischemic brain injury.

Minoxidil opens ATP-sensitive potassium channel (KATP) which is an inwardly rectifying potassium channel, which expressed in the heart, kidney, blood vessel and brain. Minoxidil was developed as an anti-hypertension agent and is now clinically used as a hair restorer. Minoxidil is also attracting attention as a treatment aimed at protecting against heart and brain ischemic damage. However, the underlying mechanism is not clear. We have reported that administration of minoxidil suppressed the damage of nerve tissue after cerebral ischemia, while it was unclear whether this was a direct action of nerve tissue or a cause of lowering blood pressure. Here, we examined whether another drug administration that produces the same blood pressure lowering effect as minoxidil suppresses damage due to cerebral ischemia. Losartan alone and doxazosin alone showed a transient decrease in blood pressure, but the combined use of both drugs resulted in a sustained decrease in blood pressure equivalent to minoxidil. Damage caused by transient ischemia model of middle cerebral artery occlusion in C57/BL mice was suppressed by minoxidil administration, but the combination of losartan and doxazosin did not protect. These results suggested that the neuroprotective effect of minoxidil was not related to the blood pressure lowering effect.

We investigated the effect of gadolinium trichloride (Gd) on microglial polarization and neuronal injury after intracerebral hemorrhage (ICH). An in vivo mouse ICH model was prepared by intrastriatal microinjection of collagenase type VII. One day after ICH, the mRNA level of proinflammatory M1 microglial markers, such as inducible nitric oxide synthase (iNOS), increased. Anti-inflammatory M2 microglial markers arginase1 (M2a, c), Ym1 (M2a), and transforming growth factor-beta (M2c) increased 1 day after ICH, and chemokine CCL1 (M2b) increased after 3 days. Gd administration decreased these M1 and M2 markers. Arginase1 and iNOS protein levels also increased 3 days after ICH, and Gd decreased them because of the decrease of cell number due to apoptosis. Next, we investigated whether Gd had an anti-inflammatory effect in an ICH model. Three days after ICH, brain edema was formed, and the number of NeuN-positive cells, which indicates neuronal nuclei, decreased in the peripheral region inside the hematoma. Gd improved the edema, neuron loss, and behavioral abnormality, without affecting the hematoma size. Furthermore, Gd improved the mortality rate by ICH. Overall, Gd evoked M1 and M2 microglial apoptosis and had an acute protective effect after ICH.

Low-density lipoprotein receptor-related protein-1 (LRP1), a member of the LDL receptor family, plays important roles in endocytosis and intracellular signaling. LRP1 is processed by furin in the trans-Golgi network (TGN), becomes mature LRP1 and moves to the cell surface. Previous studies have shown that LRP1 suppresses glutamate excitotoxicity in primary cultured retinal ganglion cells. However, the pathophysiological role of LRP1 in the brain after cerebral ischemia is unclear. The purpose of this study was to investigate the pathophysiological role of LRP1 after cerebral ischemia and N-methyl-D-aspartate (NMDA)-induced neuronal injury. First, we demonstrated that LRP1 was significantly cleaved after cerebral ischemia and in NMDA-induced neuronal injury. The LRP1-intracellular domain (ICD) produced by neuronal injury was co-localized with TGN and furin. In addition, we found that furin inhibitors inhibited LRP1 cleavage and suppressed co-localization with TGN or furin. Theses findings suggest that furin-mediated LRP1 cleavage and LRP1-ICD localization are involved in ischemic neuronal injury.

The pathological hallmark of Parkinson disease (PD) is a widespread distribution of the aggregated α-synuclein (α-Syn) proteins in the inclusions known as Lewy bodies. Exogenous α-Syn secreted from neurons could induce inflammatory responses including microglial activation. This activated microglia was observed in the substantia nigra of patients with PD. We previously reported that pericytes, one of the blood-brain barrier (BBB) constituent cells released various inflammatory mediators in response to monomeric α-Syn. Here, we investigated whether Toll-like receptors (TLRs) mediated the α-Syn-induced production of inflammatory mediators in pericytes. In response to monomeric α-Syn, pericytes released the highest levels of IL-1β, IL-6 and MMP-9 than the other cell types of the BBB (brain endothelial cells and astrocytes). TAK242 (a TLR4 inhibitor, 5 μM) but not CU CPT22 (a TLR1/2 inhibitor, 5 μM) attenuated the increased mRNA levels of IL-1β, IL-6, MMP-9 and TNF-α induced by a 24-hr exposure of α-Syn (50 μg/mL). The initial uptake of α-Syn by pericytes was inhibited by CU CPT22 and TAK242. These results suggest that TLR4 may mediate α-Syn uptake and subsequent production of inflammatory mediators in pericytes.

Idiopathic basal ganglia calcification (IBGC) is an intractable disease characterized by bilateral calcification in basal ganglia and other regions. The causative genes have been identified. Among them, the variant frequency of PDGFB in familial IBGC is about 10%. PDGFB encode platelet-derived growth factor B (PDGF-B). Previous studies showed PDGF-B is expressed in endothelial cells and neurons in the brain and PDGF-BB, a homodimer of PDGF-B, stimulates pericytes which are abundant in the brain and the Pi transport in the vascular smooth muscle cells. In this study, variant analyses of PDGFB for IBGC patients showed four novel pathogenic variants, c.160 + 2T > A, c.457−1G > T, c.33_34delCT and c.342_343insG. The iPS cells (iPSCs) from three patients with novel PDGFB variant were established and endothelial cells were induced. Enzyme-linked immunoassay analysis showed that the levels of PDGF-BB in the blood sera of patients with PDGFB variants were decreased to 34.0% of that of the control levels. Those in the culture media of the endothelial cells derived from iPSCs of patients decreased to 58.6% of the control levels. As the endothelial cells developed from iPSCs of the patients showed a phenotype of the disease, IBGC-specific iPSCs will give us more information on the pathophysiology and the therapy of IBGC in the future.

Multiple sclerosis (MS) is one of the most common demyelinating diseases. Microsomal prostaglandin E synthase-1 (mPGES-1) is a key enzyme that acts downstream of cyclooxygenase and plays a major role in inflammation and immune responses by converting prostaglandin (PG) H₂ to PGE₂. PGE₂ is highly produced in the cerebrospinal fluid of patients with MS. However, the role of mPGES-1 in MS has not been fully elucidated yet. In this study, we demonstrate the role of mPGES-1 in demyelination and motor dysfunction induced by cuprizone, one of the well established models of MS. Demyelination in the brain was induced in mice lacking mPGES-1 (mPGES-1^−/− mice) and wild-type (WT) mice by feeding ad libitum with a powdered diet containing 0.2% cuprizone for 6 weeks under specific pathogen free condition. The expression of mPGES-1 in the brain was determined by real-time PCR. The cuprizone-induced demyelination was assessed by a myelin staining with coronal brain sections, and motor dysfunction was evaluated by the rotarod test. Cuprizone up-regulated the expression of mPGES-1 mRNA in the brain of WT mice. Interestingly, mPGES-1^−/− mice exhibited lower degree of demyelination compared to WT mice. In addition, mPGES-1 gene deletion or COX-2 selective inhibitor celecoxib reduced cuprizone-induced motor dysfunction. These data indicate that COX-2/mPGES-1/PGE₂ system contributes to the pathophysiology of MS and open possible novel therapeutic approaches for MS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective death of motor neurons. In previous our study, an ethanol extract of Brazilian green propolis (EBGP) prevented mutant copper–zinc superoxide dismutase 1 (SOD1^mut)-induced neurotoxicity. This paper aims to reveal the effects of p-coumaric acid (p-CA), an active ingredient contained in EBGP, against SOD1^mut-induced neurotoxicity. We found that p-CA reduced the accumulation of SOD1^mut subcellular aggregation and prevented SOD1^mut-associated neurotoxicity. Moreover, p-CA attenuated SOD1^mut-induced oxidative stress and endoplasmic reticulum stress, which are significant features in ALS pathology. To examine the mechanism of neuroprotective effects, we focused on autophagy, and we found that p-CA induced autophagy. Additionally, the neuroprotective effects of p-CA were inhibited by chloroquine, an autophagy inhibiter. Therefore, these results obtained in this paper suggest that p-CA prevents SOD1^mut-induced neurotoxicity through the activation of autophagy and provides a potential therapeutic approach for ALS.

Ubiquilin 2 (UBQLN2) has ubiquitin-like and ubiquitin-associated domains and regulates protein degradation systems. Missense mutations of UBQLN2 have been recently identified as causal genes of familial amyotrophic lateral sclerosis (ALS). ALS-associated mutant UBQLN2 has been reported to impair protein degradation via ubiquitin-proteasome system. In the present study, we investigated how mutant UBQLN2 affects protein degradation via autophagy-lysosome system, which is classified into three pathways, macroautophagy (MA), microautophagy (mA) and chaperone-mediated autophagy (CMA). We first assessed mA/CMA activity using AD293 cells stably expressing GAPDH-HT, a marker of mA/CMA activity. Overexpressed wild-type UBQLN2 decreased mA/CMA activity, and the overexpression of mutant UBQLN2 exacerbated the decrease of mA/CMA activity. Experiments using rapamycin and mycophenolic acid, activators of mA and CMA, respectively, revealed that CMA was mainly impaired by wild-type and mutant UBQLN2. As for MA, autophagic flux assay using bafilomycin A1, a lysosome inhibitor, revealed that mutant UBQLN2 decreased MA activity in AD293 cells. These findings suggest that ALS-associated mutant UBQLN2 impairs MA and CMA and disturbs protein quality control. This disturbance would be related to the pathogenesis of ALS caused by UBQLN2 mutation.

Niemann-Pick disease type C (NPC) is a genetic disorder in which patient cells have endosomal/lysosomal accumulation of cholesterol and sphingolipids. However, the relationship between sphingolipids and cholesterol accumulation in NPC cells has not been established. Here, we investigated the role of sphingomyelin (SM) on the accumulation of cholesterol in NPC cells. Reduction of SM by inhibition of the ceramide transfer protein CERT decreased the cholesterol accumulation in NPC cells. The accumulation of SM in NPC cells inhibited the transport of cholesterol to the endoplasmic reticulum. Overexpression of Rab9 in NPC cells reduced the cholesterol accumulation, which was recovered by treatment with SM. In NPC cells that overexpressed a Rab9 constitutively active mutant, SM treatment did not lead to the cholesterol accumulation. These results indicate that SM negatively regulates the Rab9-dependent vesicular trafficking of cholesterol, and a reduction in SM levels in NPC cells recovers the Rab9-dependent vesicular trafficking defect.

Bleomycin is used as an anticancer agent in the treatment of squamous cell carcinoma. Bleomycin is also used to prepare a pulmonary fibrosis model in experimental medicine due because, it has pulmonary fibrosis as a side effect. We investigated whether the gastric ulcer drug lansoprazole could also be used to suppress pulmonary fibrosis because it suppressed liver fibrosis through suppression of Tgf-β activation in a dietary nonalcoholic steatohepatitis model. Rats were subcutaneously treated with lansoprazole (LAP, 30 mg / kg / day), bleomycin (BLM, 1 mg / kg / day) or bleomycin and lansoprazole (LAP + BLM) for 28 days. The hypertrophic visceral peritoneum was strongly induced in livers of LAP+BLM group, which could be macroscopically confirmed. Histologically analysis indicated that a strong thickening of the visceral peritoneum was observed in LAP+BLM group but neither LAP or BLM group. Furthermore, in the LAP + BLM group, immunohistochemistry indicated M2 macrophages in the visceral peritoneum thickened, and the expression of Tgfβ and Col1a1 gene were increased using real-time PCR. The bleomycin interview form reported that the hypertrophic visceral peritoneum was observed in the rats treated with bleomycin continuously. Thus, lansoprazole may have promoted the hypertrophic visceral peritoneum in the liver of rats treated with bleomycin. Both bleomycin and lansoprazole are clinically used drugs, and we plan to conduct a retrospective study to investigate whether this phenomenon is confirmed.

It is estimated that the number of potential fecal incontinence patient in Japan is about 5 millions, but no fundamental treatment has been developed. Therefore, new treatments and novel drugs have being desired to be developed at present. Screening with model animals is important for developing new therapeutic agents. Therefore, we developed a measurement method of internal anal sphincter (IAS) movement in un-anesthetized and unrestrained dogs by placing the force transducers used for gastrointestinal motility measurement in the IAS and each gastrointestinal tract, and measuring the defecation movement using a telemetry method and reported the results of the research at the 92^nd Annual Meeting of the Japanese Pharmacological Society.  In the previous experiments, INS was observed to contract after dosing noradrenaline and the contraction was suppressed or enhanced by α-blocker or β-blocker, respectively, but it was different under anesthesia from that under awakening. In the present study, a cholinesterase inhibitor was administered and the INS movement was determined to confirm whether the cholinergic mechanism is involved in INS; and the effects of comparative control substances were also examined to evaluate the action of drugs on INS movement.

Inflammatory bowel disease is a group of chronic disorders characterized by damaged smooth muscle tissues in the digestive tract. We investigated the alteration of carbachol (CCh) and bradykinin (BK) -induced contractile responses in rat colon under inflammatory conditions. Inflammation was induced by an infusion to colonic lumen of the TNBS and the preparations for ring specimen of the colon which we resected were done three days after an injection. The colon tissues were isolated from saline-treated control and TNBS-treated rats. These tissues were placed in physiological organ bath system and then, isometric force were assessed. Treatment of CCh induced dose-dependent force responses in both types of tissue, but the responses were attenuated in the TNBS-treated rat. Maximal response in TNBS-treated rat was about 60 % of control. The presence of nifedipine markedly inhibited the CCh-induced contraction. This the results suggest that the contractile response to CCh of the rat colonis muscle is caused primarily the increase in the cellular Ca²⁺ concentration. On the other hand, treatment of BK responses were significantly increased in the TNBS-treated rat. These contractile response were antagonized by BK₁ receptor antagonists. These results that BK suggests that the susceptibility to BK₁ receptor increases by inflammation.