Ninjin'yoeito, a traditional Japanese medicine, attenuates age-related deficits of muscle performance, self-care motivation, and body temperature in C57BL/6 mice.

As both physiological and psychological factors influence age-associated declines in older people, the development of drug therapy with multifaceted effects is required. To investigate the utility of ninjin'yoeito (NYT) against geriatric syndromes, we evaluated the effects of NYT on age-related declines in old C57BL/6 mice (88-week-old) as a preclinical model of frailty progression. Here, we showed that NYT reversed the decline of rectal temperature in old mice and also improved forelimb grip strength compared with that in the old control group without affecting skeletal muscle loss. Moreover, NYT significantly increased the duration of grooming after a sucrose solution was sprayed, which reflected self-care motivation. Finally, we revealed the antioxidant effects of NYT using a cell-free assay. These results suggest that NYT can improve both physiological and psychological declines associated with aging, and the mechanism may include antioxidant effects. NYT may have potential utility for maintaining the health of older people.

Analysis of the Effects of Ninjin'yoeito on Physical Frailty in Mice.

Physical frailty is an aging-related clinical syndrome involving decreases in body weight, mobility, activity, and walking speed that occurs in individuals with sarcopenia and is accelerated by increased oxidative stress. Ninjin'yoeito, a traditional Japanese Kampo medicine, is used for treating conditions, including anemia and physical weakness. Here, we investigated whether ninjin'yoeito could improve physical frailty by controlling oxidative stress in the senescence-accelerated mouse prone 8 (SAMP8) model. First, SAMP8 mice were divided into two groups, ninjin'yoeito treated and untreated, with the former consuming a diet containing 3% ninjin'yoeito from 3 months of age. At 7 months of age, body weight, motor function, locomotor activity, and mean walking speed were measured. Subsequently, mice were euthanized and measured for muscle weight, 8-hydroxy-2'-deoxyguanosine levels in muscle and brain, and cleaved caspase-3 expression in brain. The results showed reductions in weight, locomotor function, locomotion, and average walking speed in the untreated group, which were significantly improved by ninjin'yoeito. Furthermore, 8-hydroxy-2'-deoxyguanosine levels were reduced in muscle and brain from ninjin'yoeito-treated mice, compared with the levels in untreated mice; cleaved caspase-3 expression was similarly reduced in brain from the treated mice, indicating reduced apoptosis. Our findings suggest that ninjin'yoeito inhibits sarcopenia-based physical frailty through its antioxidant effects.

Ninjin'yoeito, a traditional Japanese medicine, increases dopamine content in PC12 cells.

Dementia is exacerbated by loss of appetite and amotivation, and recent studies have indicated that ninjin'yoeito improves anorexia and amotivation. Previous studies suggest that ninjin'yoeito inhibits dopamine-metabolizing enzymes and enhances dopamine signaling. However, whether ninjin'yoeito increases dopamine content in living cells remains unclear. Here, PC12 cells were used to examine whether ninjin'yoeito affects the dopamine metabolic pathway. Dopamine content significantly increased 3 h after treatment ninjin'yoeito extract. Concomitantly, the levels of 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid were significantly reduced. The effects of components of ninjin'yoeito on the dopamine metabolic pathway were also assessed. Treatment with onjisaponin B, nobiletin, and schisandrin, and the ingredients of Polygalae Radix, Citri Unshiu Pericarpium, and Schisandrae Fructus increased dopamine content and decreased its metabolite content in the culture media. Our findings suggest that ninjin'yoeito improves anorexia and amotivation by inhibiting metabolic enzyme and increasing the dopamine content in cells.

Yokukansankachimpihange, a traditional Japanese (Kampo) medicine, enhances the adaptation to circadian rhythm disruption by increasing endogenous melatonin levels.

The traditional Japanese (Kampo) medicines yokukansan (YKS) and yokukansankachimpihange (YKSCH) have similar formulas and the same indications. In animals or cultured cells, the neuropharmacological actions of YKS are sometimes more beneficial than those of YKSCH. Since both drugs are used to treat sleep disorders in Japan, we examined the ameliorative effects of YKS and YKSCH on circadian rhythm disturbance and compared their efficacy using a mouse model of circadian rhythm disruption. Ramelteon was used as the positive control. Ramelteon treatment significantly reversed decreased running wheel activity during the advanced dark phase, indicating facilitation of circadian adaptation. YKS treatment also reversed the activity in the early period of drug treatment; however, it was not statistically significant. YKSCH treatment significantly reversed the decreased activity during the advanced dark phase. Plasma melatonin (MT) levels were significantly increased in the YKSCH but not in the YKS group. The ameliorative effect of YKSCH on rhythm disruption was significantly inhibited by coadministration of the MT2 receptor antagonist. Therefore, the therapeutic effect of YKSCH on circadian rhythm disruption would be attributable, to elevated endogenous MT levels. Taken together, YKS and YKSCH have different pharmacological properties and may be more precisely prescribed depending on patients' psychological symptoms.

[6]-gingerol and [6]-shogaol, active ingredients of the traditional Japanese medicine hangeshashinto, relief oral ulcerative mucositis-induced pain via action on Na+ channels.

The traditional Japanese herbal medicine hangeshashinto (HST) has beneficial effects for the treatment of oral ulcerative mucositis (OUM) in cancer patients. However, the ingredient-based mechanism that underlies its pain-relieving activity remains unknown. In the present study, to clarify the analgesic mechanism of HST on OUM-induced pain, we investigated putative HST ingredients showing antagonistic effects on Na+ channels in vitro and in vivo. A screen of 21 major ingredients using automated patch-clamp recordings in channel-expressing cells showed that [6]-gingerol and [6]-shogaol, two components of a Processed Ginger extract, considerably inhibited voltage-activated Na+ currents. These two ingredients inhibited the stimulant-induced release of substance P and action potential generation in cultured rat sensory neurons. A submucosal injection of a mixture of [6]-gingerol and [6]-shogaol increased the mechanical withdrawal threshold in healthy rats. In a rat OUM model, OUM-induced mechanical pain was alleviated 30min after the swab application of HST despite the absence of anti-bacterial and anti-inflammatory actions in the OUM area. A swab application of a mixture of [6]-gingerol and [6]-shogaol induced sufficient analgesia of OUM-induced mechanical or spontaneous pain when co-applied with a Ginseng extract containing abundant saponin. The Ginseng extract demonstrated an acceleration of substance permeability into the oral ulcer tissue without an analgesic effect. These findings suggest that Na+ channel blockage by gingerol/shogaol plays an essential role in HST-associated analgesia of OUM-induced pain. This pharmacological mechanism provides scientific evidence supporting the use of this herbal medicine in patients suffering from OUM-induced pain.

Goshajinkigan, a traditional Japanese medicine, prevents oxaliplatin-induced acute peripheral neuropathy by suppressing functional alteration of TRP channels in rat.

The acute peripheral neuropathy induced by oxaliplatin treatment occurs very frequently and is aggravated by exposure to cold. Goshajinkigan (GJG), a traditional Japanese (kampo) medicine, was recently shown to be effective against oxaliplatin-induced acute neuropathy. However, because the effects of GJG and its mechanism in relation to those of its ingredients and its mechanism are not well understood, we examined the effects of GJG on acute neuropathy. Further, we investigated whether GJG affects the functions and gene expressions of transient receptor potential (TRP) channels using a rat model of oxaliplatin-induced neuropathy. Administration of oxaliplatin increased withdrawal responses from cold stimulation, and GJG or calcium gluconate/magnesium sulfate significantly inhibited the oxaliplatin-induced cold hypersensitivity. Application of menthol, a TRPA1/TRPM8 agonist, or allyl isothiocyanate (AITC), a selective TRPA1 agonist, to the hind paw of oxaliplatin-treated rats enhanced the nocifensive behaviors evoked by each agonist, whereas oxaliplatin had no significant effect on nocifensive behaviors evoked by capsaicin, a TRPV1 agonist. GJG treatment reduced menthol- or AITC-evoked withdrawal responses potentiated by oxaliplatin. Furthermore, GJG suppressed the increase of TRPA1 and TRPM8 mRNA expression induced by oxaliplatin in dorsal root ganglia. These findings suggest that GJG prevented oxaliplatin-induced acute peripheral neuropathy by suppressing functional alteration of TRP channels, especially TRPA1 and TRPM8.

Epithelial transient receptor potential ankyrin 1 (TRPA1)-dependent adrenomedullin upregulates blood flow in rat small intestine.

The functional roles of transient receptor potential (TRP) channels in the gastrointestinal tract have garnered considerable attention in recent years. We previously reported that daikenchuto (TU-100), a traditional Japanese herbal medicine, increased intestinal blood flow (IBF) via adrenomedullin (ADM) release from intestinal epithelial (IE) cells (Kono T et al. J Crohns Colitis 4: 161-170, 2010). TU-100 contains multiple TRP activators. In the present study, therefore, we examined the involvement of TRP channels in the ADM-mediated vasodilatatory effect of TU-100. Rats were treated intraduodenally with the TRP vanilloid type 1 (TRPV1) agonist capsaicin (CAP), the TRP ankyrin 1 (TRPA1) agonist allyl-isothiocyanate (AITC), or TU-100, and jejunum IBF was evaluated using laser-Doppler blood flowmetry. All three compounds resulted in vasodilatation, and the vasodilatory effect of TU-100 was abolished by a TRPA1 antagonist but not by a TRPV1 antagonist. Vasodilatation induced by AITC and TU-100 was abrogated by anti-ADM antibody treatment. RT-PCR and flow cytometry revealed that an IEC-6 cell line originated from the small intestine and purified IE cells expressed ADM and TRPA1 but not TRPV1. AITC increased ADM release in IEC cells remarkably, while CAP had no effect. TU-100 and its ingredient 6-shogaol (6SG) increased ADM release dose-dependently, and the effects were abrogated by a TRPA1 antagonist. 6SG showed similar TRPA1-dependent vasodilatation in vivo. These results indicate that TRPA1 in IE cells may play an important role in controlling bowel microcirculation via ADM release. Epithelial TRPA1 appears to be a promising target for the development of novel strategies for the treatment of various gastrointestinal disorders.

Hepcidin expression in the trigeminal ganglion and the oral mucosa in an oral ulcerative mucositis rat model.

Severe intraoral pain induces difficulty in eating and speaking, leading to a decline in the quality of life. However, the molecular mechanisms underlying intraoral pain remain unclear. Here, we investigated gene modulation in the trigeminal ganglion and intraoral pain-related behavior in a rat model of acetic acid-induced oral ulcerative mucositis. Oral ulceration was observed on day 2 after acetic acid treatment to the oral mucosa of male Wistar rats, causing spontaneous pain and mechanical allodynia. Deoxyribonucleic acid microarray analysis of trigeminal ganglion tissue indicated that Hamp (a hepcidin gene that regulates cellular iron transport) was the most upregulated gene. In the oral ulcerative mucositis model, the upregulation of Hamp was also induced in the ulcer region but not in the liver, with no increase in hepcidin levels in the plasma and saliva, indicating that hepcidin was produced locally in the ulcer region in the model. Systemic antibiotic pretreatment did not increase the mRNA levels of Hamp in the trigeminal ganglion and ulcer regions. Hepcidin injection into the oral mucosa enhanced neuronal excitability in response to noxious mechanical stimulation of the oral mucosa in trigeminal spinal subnucleus interpolaris/caudalis neurons. These results imply that oral ulcerative mucositis induces oral mucosal pain because of infectious inflammation of the ulcerative area and potentiates Hamp, which represents anti-bacterial and anti-peptidase gene expression in the ulcer region and trigeminal ganglion. The regulation of cellular iron transport by hepcidin is likely involved in oral ulcerative mucositis-induced pain.

The effects of ninjin'yoeito on the electrophysiological properties of dopamine neurons in the ventral tegmental area/substantia nigra pars compacta and medium spiny neurons in the nucleus accumbens.

The ventral tegmental area (VTA), substantia nigra pars compacta (SNpc) and nucleus accumbens (NAc) are involved in the regulation of appetite and motivational behaviors. A traditional Japanese (Kampo) medicine, ninjin'yoeito (NYT), has been reported to improve decreased motivation and anorexia in patients with Alzheimer's disease and apathy-like model mice. Thus, NYT may affect the activities of neurons in the VTA, SNpc and NAc. However, little is known about the underlying mechanisms of NYT. Here, we investigated the effects of NYT on the electrophysiological properties of dopaminergic neurons in the VTA and SNpc, as well as on those of medium spiny neurons (MSNs) in the NAc (core and shell subregions), by applying the patch-clamp technique in the brain slices. NYT reduced the resting membrane potential of VTA and SNpc dopaminergic neurons. In contrast, NYT increased the firing frequency of NAc MSNs accompanied by shortened first spike latency and interspike interval. Furthermore, NYT attenuated the inward rectification and sustained outward currents. In conclusion, NYT may directly influence the excitability of dopaminergic neurons in the VTA and SNpc, as well as MSNs in the NAc (core and shell). NYT may modulate dopamine signals in appetite and motivational behaviors.

Isoliquiritigenin, an active ingredient of Glycyrrhiza, elicits antinociceptive effects via inhibition of Nav channels.

Glycyrrhiza extract has been used for the treatment of oral and gastric ulcers, but the analgesic mechanism remains unknown. In the present study, we investigated the effects of isoliquiritigenin, an active ingredient of Glycyrrhiza, on Nav channels in vitro and nociceptive behaviors in vivo. In an autopatch-clamp study, isoliquiritigenin inhibited the currents of Nav1.1, Nav1.3, Nav1.6, Nav1.7, and Nav1.8 in a channel expression system. In small- and medium-sized cultured trigeminal ganglion neurons, the compound suppressed Nav currents in many neurons (78%) and Kv currents in all neurons, dose-dependently. In current-clamp mode, isoliquiritigenin blocked action potential generation in many neurons (64%), but it conversely accelerated action potential generation in the remaining neurons. The opposing effects on action potentials were reproduced in a computational simulation of a modified Hodgkin-Huxley-based model, based on the electrophysiological data. In behavioral experiments, local treatment with isoliquiritigenin suppressed nociceptive behaviors in response to oral ulcer development or nociceptive TRP channel agonists in the oral mucosa and hind paw. These results suggest that isoliquiritigenin exerts an analgesic effect predominantly via inhibitory action on Nav channels on sensory nociceptive fibers. This pharmacological mechanism indicates that isoliquiritigenin is useful for pain relief and provides scientific evidence for Glycyrrhiza at the ingredient level.

Ninjin'yoeito, a traditional Japanese Kampo medicine, suppresses the onset of anhedonia induced by dysfunction in the striatal dopamine receptor type 2-expressing medium spiny neurons.

OBJECTIVE: Recent studies have suggested that ninjin'yoeito (NYT), a traditional Japanese Kampo medicine, improves diminished motivation in humans and animals, rendering it a novel therapeutic option for impaired motivation. To better characterize the effect of NYT on motivation, we examined its effect on motivated behaviors in mice. METHODS: Mouse models of neurodegeneration-related apathy, in which striatal dopamine receptor type 2-expressing medium spiny neurons (D2-MSNs) were progressively damaged by diphtheria toxin expression, were chosen. RESULTS: The decrease in effort-based operant responding for rewards (sucrose pellets), indicative of the mouse's motivated behavior, in the affected mice was not suppressed by chronic treatment with NYT suspended in drinking water at 1% (w/v). Mice were then subjected to a sucrose preference test, wherein they freely chose to ingest tap water and a sucrose solution without being required to exert effort. The affected mice showed a decline in preference for sucrose over tap water, relative to nonaffected controls, indicating anhedonia-like traits. In contrast to the diminished operant behavior, the anhedonic behavior in the affected mice was prevented by the NYT administration. Furthermore, NYT did not affect the size of Drd2 mRNA disappearance in the striatum of affected mice, suggesting that the NYT effect was unrelated to DTA-mediated neurodegeneration. CONCLUSION: These results demonstrate that the beneficial effect of NYT on motivation is mediated, at least in part, through the potentiation of hedonic capacity by certain neuromodulatory pathways.

Evaluation of organ glucose metabolism by 18F-FDG accumulation with insulin loading in aged mice compared with young normal mice.

It is important to determine the functional changes of organs that occur as a result of aging, the understanding of which may lead to the maintenance of a healthy life. Glucose metabolism in healthy bodies is one of the potential markers used to evaluate the changes of organ function. Thus, information about normal organ glucose metabolism may help to understand the functional changes of organs. [18F]-Fluoro-2-deoxy-2-D-glucose (18F-FDG), a glucose analog, has been used to measure glucose metabolism in various fields, such as basic medical research and drug discovery. However, glucose metabolism changes in aged animals have not yet been fully clarified. The aim of this study is to evaluate changes in glucose metabolism in organs and brain regions by measuring 18F-FDG accumulation and 18F-FDG autoradiography with insulin loading in aged and young wild-type mice. In the untreated groups, the levels of 18F-FDG accumulation in the blood, plasma, muscle, lungs, spleen, pancreas, testes, stomach, small intestine, kidneys, liver, brain, and brain regions, namely, the cortex, striatum, thalamus, and hippocampus, were all significantly higher in the aged mice. The treated group showed lower 18F-FDG accumulation levels in the pancreas and kidneys, as well as in the cortex, striatum, thalamus, and hippocampus in the aged mice than the untreated groups, whereas higher 18F-FDG accumulation levels were observed in those in the young mice. These results demonstrate that insulin loading decreases effect on 18F-FDG accumulation levels in some organs of the aged mice. Therefore, aging can increase insulin resistance and lead to systemic glucose metabolism dysfunction.

Evaluation of Effect of Ninjin'yoeito on Regional Brain Glucose Metabolism by 18F-FDG Autoradiography With Insulin Loading in Aged Mice.

Introduction: A recent clinical study revealed that Ninjin'yoeito (NYT) may potentially improve cognitive outcome. However, the mechanism by which NYT exerts its effect on elderly patients remains unclear. The aim of this study is to evaluate the effect of Ninjin'yoeito on regional brain glucose metabolism by 18F-FDG autoradiography with insulin loading in aged wild-type mice. Materials and Methods: After 12 weeks of feeding NYT, mice were assigned to the control and insulin-loaded groups and received an intraperitoneal injection of human insulin (2 U/kg body weight) 30 min prior to 18F-FDG injection. Ninety minutes after the injection, brain autoradiography was performed. Results: After insulin loading, the 18F-FDG accumulation showed negative changes in the cortex, striatum, thalamus, and hippocampus in the control group, whereas positive changes were observed in the NYT-treated group. Conclusions: Ninjin'yoeito may potentially reduce insulin resistance in the brain regions in aged mice, thereby preventing age-related brain diseases.

Kamikihito Enhances Cognitive Functions and Reward-Related Behaviors of Aged C57BL/6J Mice in an Automated Behavioral Assay System.

The cognitive and psychological domains of frailty in the elderly have drawn increasing attention given the aging of society. However, therapeutics to treat minor deficits in cognition and mental state in the elderly remain an unmet need. Kamikihito (KKT), a traditional Japanese Kampo medicine indicated for neuroses, anxiety, and insomnia, is effective for treating cognitive dysfunction and depressive-like behaviors in animal models, suggesting that it may have therapeutic potential for treating cognitive and/or mental frailty. In this study, we first validated the known anxiolytic effects of KKT in a conventional maze test. We then introduced an automated behavioral assay system, IntelliCage, to evaluate the therapeutic potential of KKT for age-related and diverse central functions by performing sequential behavioral tasks in young and aged mice to assess basal activities, cognitive functions, perseveration, and hedonic-related behaviors. Although young mice treated with KKT did not exhibit changes in diurnal variation, KKT-administered aged mice exhibited an accelerated decline in voluntary activity during the early part of the light period, implying that KKT may promote sleep onset in aged mice. Neither place learning acquisition for gaining rewards nor subsequent behavioral flexibility performance was altered by KKT in the young group, whereas the aged KKT group exhibited significantly enhanced performance in both phases of learning relative to age-matched controls. Conversely, perseverative nose-pokes (NPs) to gain rewards observed during place learning, indicative of compulsivity, were attenuated by KKT in both age groups. Regarding hedonic processing, aged mice exhibited a decreased preference for sweet solutions compared to young mice, which was effectively reversed by KKT treatment. Furthermore, KKT elevated high-effort choices for high-value reward in an effort-based decision-making paradigm in both age groups, implying augmentation of motivational behaviors by KKT. Collectively, KKT exerted various beneficial effects in cognitive and emotional domains, several of which were more evident in aged mice than in young mice, suggesting the potential of KKT for treating cognitive and mental frailty.

Author Correction: The Japanese herbal medicine Hangeshashinto enhances oral keratinocyte migration to facilitate healing of chemotherapy-induced oral ulcerative mucositis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Japanese herbal medicine Hangeshashinto enhances oral keratinocyte migration to facilitate healing of chemotherapy-induced oral ulcerative mucositis.

Chemotherapy often induces oral ulcerative mucositis (OUM) in patients with cancer, characterized by severe painful inflammation. Mouth-washing with the Japanese herbal medicine hangeshashinto (HST) ameliorates chemotherapy-induced OUM in patients with colorectal cancer. Previously, we demonstrated that HST decreased interleukin 1ß-induced prostaglandin E2 production in human oral keratinocytes (HOKs) and OUM-induced mechanical or spontaneous pain in rats. However, HST effects on tissue repair functions in HOKs remain unclear. Here, we examined the effects of HST on scratch-induced wound healing in vitro and in vivo. In vitro, HST enhanced wound healing mainly through scratch-induced HOK migration. Screening of the seven constituent medicinal herbs and their major components revealed that Scutellaria root, processed ginger, and Glycyrrhiza components mainly induced the scratch-induced HOK migration. Pharmacokinetic analyses indicated that the active ingredient concentrations in rat plasma following oral HST administration were below the effective doses for HOK migration, suggesting direct effects of HST in OUM. Mitogen-activated protein kinase and C-X-C chemokine receptor 4 inhibitors significantly suppressed HST-induced HOK migration. Moreover, HST enhanced tissue repair in our OUM rat model. Thus, HST likely enhanced OUM tissue repair through oral keratinocyte migration upon MAPK and CXCR4 activation and may be useful in patients with cancer-associated OUM.

Comparison of the Effects of Yokukansan and Yokukansankachimpihange on Glutamate Uptake by Cultured Astrocytes and Glutamate-Induced Excitotoxicity in Cultured PC12 Cells.

The traditional Japanese Kampo medicine yokukansan (YKS) is effective for behavioral and psychological symptoms of dementia (BPSD) in patients with Alzheimer's disease. As the pharmacological mechanisms, YKS is known to protect astrocytes from thiamine-deficiency (TD)-induced decreased glutamate (Glu) uptake and neuron model cells (PC 12 cells) from Glu-induced death. Yokukansankachimpihange (YKSCH) is an alternative formula to YKS, in which Citrus unshiu peel and Pinellia tuber are added to the YKS components, and is sometimes used to treat BPSD, but its pharmacological properties remain unknown. This study aims to investigate the cellular pharmacological effects of YKS and YKSCH on glutamatergic pathways, compare their efficacy, and determine the differences and similarities in the activities between these formulations. First, we examined the effects of YKS and YKSCH on Glu uptake by cultured astrocytes under TD conditions. We observed significant ameliorative effects of YKS and YKSCH on the TD-induced decrease in Glu uptake, with a 50% effective dose of 8.9 ± 1.8 µg/mL and 45.3 ± 9.2 µg/mL, respectively. Second, using cultured PC12 cells as a model for neurons, we examined the effects of YKS and YKSCH on Glu-induced cell death. We observed that YKS and YKSCH had significant inhibitory effects on Glu-induced cell death, with a 30% effective dose of 51.4 ± 20.8 µg/mL and 49.2 ± 11.0 µg/mL, respectively. Thus, while YKSCH was less effective than YKS in ameliorating the TD-induced decrease in Glu uptake by astrocytes, the two drugs showed similar inhibitory effects on Glu-induced PC12 cell death. These findings are important for understanding the differences and similarities in pharmacological actions between these drugs.

Influence of aging on the behavioral phenotypes of C57BL/6J mice after social defeat.

Depression and anxiety are common psychiatric disorders that can occur throughout an individual's lifetime. Numerous pathways underlying the onset of these diseases have been identified in rodents using a social defeat stress protocol, whereby socially defeated individuals exhibit depression- and/or anxiety-like phenotypes that typically manifest as social avoidance behavior. However, most studies in this field have been conducted using young adult mice; therefore, information about social defeat stress-related behavioral phenotypes in older mice is limited. In this study, we exposed groups of young adult (8-16 weeks old) and aged (24 months old) C57BL/6J mice to mild social defeat stress by challenging them with aggressive CD1 mice while restricting the intensity of aggression to protect the animals from severe injuries. We then identified stress-induced behavioral changes and compared their expression between the age groups and with a non-defeated (non-stressed) control group. We found that the stressed mice in both age groups exhibited similar reduced social interactions that were indicative of increased social avoidance behavior. Moreover, unlike the young stressed and control groups, only the aged stressed group showed a reduced preference for sucrose, which was correlated with social avoidance behavior. Also, the aged stressed mice exhibited an attenuated defeat-induced increase in water intake. These findings reveal that aging alters behavioral phenotypes after social defeat and that the hedonic behavior of aged mice is more vulnerable to social defeat compared with younger mice.

Role of alpha2-adrenoceptors in enhancement of antinociceptive effect in diabetic mice.

The present studies investigated behavioral and neurochemical aspects of the noradrenergic and serotonergic nervous systems in streptozotocin-induced diabetic mice. We previously reported that intrathecal (i.t.) injection of norepinephrine significantly potentiated antinociception in diabetic mice compared to that in non-diabetic mice, and that antinociception due to norepinephrine injection was completely abolished by pretreatment with yohimbine, an alpha2-adrenoceptor antagonist. The present studies demonstrated that i.t. injection of clonidine also showed more-potent antinociceptive activity in diabetic mice than in non-diabetic mice, but that i.t. methoxamine injection did not affect diabetic or non-diabetic mice. The antinociceptive potency due to i.t. injection of 5-HT was significantly lower in diabetic than in non-diabetic mice. In a neurochemical study, we found that the density of [3H]-rauwolscine binding sites in spinal alpha2-adrenoceptors was significantly higher in diabetic than in non-diabetic mice, but that the binding affinity was unchanged. Spinal norepinephrine turnover was determined by measuring the decline in tissue norepinephrine concentration at 3 h after injection of the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine. The spinal norepinephrine concentration decreased to 43.7% from the baseline in non-diabetic mice, while it was 21.0% in diabetic mice. These results suggest that, based on the decrease of norepinephrine release in the spinal cord, up-regulation of spinal alpha2-adrenoceptors caused the increase of antinociception due to i.t. injection of an alpha2-adrenoceptor agonist in streptozotocin-induced diabetic mice, and it seemed that the stimulation of alpha2-adrenoceptors potentiated the antinociceptive effect. Thus, the spinal noradrenergic systems play an important moderating role in diabetes-induced neuropathic pain.

Yokukansan, a Traditional Japanese Medicine, Enhances the Glutamate Transporter GLT-1 Function in Cultured Rat Cortical Astrocytes.

Astrocytes carry two glutamate transporters-GLAST and GLT-1-the latter of which is responsible for >90% of glutamate uptake activity in the brain; however, under culture conditions, the GLT-1 expression in astrocytes is exceedingly low, as is the glutamate uptake activity mediated by GLT-1. This study aimed to elucidate the effects of yokukansan (YKS) in relation to the GLT-1-mediated regulation of extracellular glutamate concentrations. Thus, we treated cultured astrocytes with tumor necrosis factor-α (TNF-α) and dibutyryl-cAMP (dBcAMP) (hereinafter, referred to as "TA") to increase GLT-1 expression and then functionally examined how YKS would affect glutamate uptake ability derived from GLT-1. Contrary to expectations, although the TA treatments did not affect the uptake activity, YKS significantly augmented it. Conversely, GLAST-derived glutamate uptake was significantly reduced by TA treatments but was unaffected by YKS. Subsequently, we analyzed the GLT-1 protein and mRNA levels and found that TA treatments had significantly increased them, which were then further augmented by YKS. These findings suggest that YKS enhances GLT-1-derived glutamate transport functions in TA-treated cultured astrocytes and that this process entails increased GLT-1 protein and mRNA levels. This type of mechanism may contribute to the YKS-mediated regulation of extracellular glutamate concentrations.