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A therapeutic strategy through the gut-brain axis has been proven to be effective in treatment for depression. In our previous study, we demonstrated that Enterococcus faecalis 2001 (EF-2001) prevents colitis-induced depressive-like behavior through the gut-brain axis in mice. More recently, we found that demyelination in the prefrontal cortex (PFC) was associated with depressive-like behavior in an animal model of major depressive disorder, olfactory bulbectomized (OBX) mice. The present study investigated the effects of EF-2001 on depressive-like behaviors in OBX mice and the underlying molecular mechanisms from the perspective of myelination in the PFC. OBX mice exhibited depressive-like behaviors in the tail-suspension, splash, and sucrose preference tests, and decreased myelin and paranodal proteins along with mature oligodendrocytes in the PFC. These behavioral and biochemical changes were all prevented by treatment with EF-2001. Further, EF-2001 treatment increased brain-derived neurotrophic factor (BDNF) and leukemia inhibitory factor (LIF) in the PFC. Interestingly, an immunohistochemical analysis revealed enhanced phospho (p) -cAMP-responsive element binding protein (CREB) expression in neurons, p-nuclear factor-kappa B (NFκB) p65 (Ser536) expression in astrocytes, and p-signal transducer and activator of transcription 3 (STAT3) (Ty705) expression in mature oligodendrocytes in the PFC of OBX mice. From these results, we suggest that EF-2001 administration prevents depressive-like behaviors by regulating prefrontal cortical myelination via the enhancement of CREB/BDNF and NFκB p65/LIF/STAT3 pathways. Our findings strongly support the idea that a therapeutic strategy involving the gut microbiota may be a promising alternative treatment for alleviating symptoms of depression.

The intrathecal (i.t.) injection of substance P (SP) and N-methyl-D-aspartate (NMDA) induce transient nociceptive response by activating neurokinin (NK) 1 and NMDA receptors, respectively. We have recently reported that angiotensin (Ang) (1-7), an N-terminal fragment of Ang II, could alleviate several types of pain including neuropathic and inflammatory pain by activating spinal MAS1. Here, we investigated whether Ang (1-7) can inhibit the SP- and NMDA-induced nociceptive response. The nociceptive response induced by an i.t. injection of SP or NMDA was assessed by measuring the duration of hindlimb scratching directed toward the flank, biting and/or licking of the hindpaw or the tail for 5 min. Localization of MAS1 and either NK1 or NMDA receptors in the lumbar superficial dorsal horn was determined by immunohistochemical observation. The nociceptive response induced by SP and NMDA was attenuated by the i.t. co-administration of Ang (1-7) (0.03-3 pmol) in a dose-dependent manner. The inhibitory effects of Ang (1-7) (3 pmol) were attenuated by A779 (100 pmol), a MAS1 antagonist. Moreover, immunohistochemical analysis showed that spinal MAS1 co-localized with NK1 receptors and NMDA receptors on cells in the dorsal horn. Taken together, the i.t. injection of Ang (1-7) attenuated the nociceptive response induced by SP and NMDA via spinal MAS1, which co-localized with NK1 and NMDA receptors. Thus, the spinal Ang (1-7)/MAS1 pathway could represent a therapeutic target to effectively attenuate spinal pain transmission caused by the activation of NK1 or NMDA receptors.

Alzheimer's disease is associated with marked olfactory dysfunction observed in the early stages. Clinical studies reported that acetylcholinesterase inhibitor donepezil (DNP) attenuated this deficit; however, the underlying mechanism remains unclear. Herein, we aimed to examine the effects and underlying mechanisms of DNP on olfactory deficits in zinc sulfate (ZnSO

Several studies have proposed δ opioid receptors as influential targets for developing novel antidepressants. Deltorphin (DLT) and deltorphin II (DLT-II) have high affinity and selectivity for δ opioid receptors; thus, it is likely that DLT analogs possess antidepressant-like effects. Based on this, we evaluated the effects of four DLT analogs (DLT-related synthetic peptides) on immobility behavior in the tail suspension test. Intracerebroventricular administration of DLT or [N-isobutyl-Gly

Methylazoxymethanol (MAM)-treated pregnant rat at gestation day (GD) 17 has been shown to be a valuable developmental animal model for schizophrenia. Yet, this model remains to be established in mice. In the present study, we examined behavioral, cytoarchitectural, and neurochemical changes in the offspring of MAM-treated mice and validated the model's face, construct and predictive validities. We found that in contrast to a single injection of MAM to dams at GD 15, 16 or 17, its daily administration from GD 15 to 17 led to deficits in prepulse inhibition (PPI) of startle in the post-pubertal offspring. In addition, we observed behavioral deficits in working memory and social interactions, as well as an increase in locomotor activity induced by the NMDA antagonist MK-801 in GD15-17 MAM offspring. These animals also showed a reduction in the volume of the prefrontal cortex (PFC) and hippocampus, neuroanatomical changes such as discontinuities and heterotopias in the hippocampus, and an increase of DA level and DOPAC/DA ratio in the medial PFC. Atypical antipsychotic drugs clozapine, risperidone, and aripiprazole, but not the typical drug haloperidol, reversed the deficit in PPI and social withdrawal in the offspring of MAM-treated dams. In contrast, MK-801-induced hyperactivity in MAM mice was reversed by both and typical or atypical antipsychotic drugs. Taken together, the treatment of pregnant mice with MAM during GD 15-17 offers a new approach to study neurobiological mechanisms involved in the pathogenesis of schizophrenia.

BACKGROUND Perioperative neurocognitive disorder (PND) is a postsurgical complication associated with neuroinflammation and impaired hippocampal neurogenesis, in which brain-derived neurotrophic factor (BDNF) plays a key role. Sarcopenia refers to age-related muscle loss that causes cognitive decline, muscle atrophy, and postoperative delirium. Rats with tail suspension (TS) were used to represent a low-activity model, which involves decreased hind limb function by TS. This hind limb unloading by TS can induce sarcopenia in 2 weeks. However, the relationship between PND and muscle atrophy is unclear. In this experiment, we investigated whether preoperative muscle atrophy induced by TS would affect neurogenesis and accelerate PND in rats. METHODS Sixty 21-week-old rats were assigned to 4 groups: the TS group, the TS with surgery (TS + S) group, the control group, and the control with surgery (control + S) group. After the abdominal manipulation under 3% sevoflurane anesthesia, cognitive function was assessed using the Morris water maze test and a fear-conditioning test. Neurogenesis was evaluated by checking BDNF secretion and immunohistochemical staining in the hippocampus. RESULTS The TS + S group showed impaired swimming latency (difference of means = 12.4 versus control + S; 95% confidence interval [CI], 2.0-22.7; P = .016) (difference of means = 15.2 versus TS; 95% CI, 0.4-30.1; P = .043) and path length (difference of means = 147.8 versus control + S; 95% CI, 20.7-274.9; P = .020) in the maze test and cued fear memory (difference of means = -26.0 versus TS; 95% CI, -46.4 to -5.6; P = .006) (difference of means = -22.3 versus control + S; 95% CI, -42.7 to -1.9; P = .026) in the fear-conditioning test. The postoperative levels of BDNF in the TS + S and TS groups were reduced compared with the other groups (P = .002). The number of neural precursors in the dentate gyrus was significantly lower in the TS + S group (P < .001). CONCLUSIONS We observed that preoperative hind limb muscle atrophy, indicated by TS, was associated with an increased occurrence of PND through the reduction in BDNF and neurogenesis after abdominal surgery in young adult rats. Therefore, we concluded that preoperative low skeletal muscle mass can induce PND due to impaired postoperative neurogenesis. Our findings might indicate that low-cost perioperative interventions, such as preoperative exercise, is beneficial to preventing PND.

The intrathecal (i.t.) injection of substance P (SP) and N-methyl-D-aspartate (NMDA) induce transient nociceptive response by activating neurokinin (NK) 1 and NMDA receptors, respectively. We have recently reported that angiotensin (Ang) (1-7), an N-terminal fragment of Ang II, could alleviate several types of pain including neuropathic and inflammatory pain by activating spinal MAS1. Here, we investigated whether Ang (1-7) can inhibit the SP- and NMDA-induced nociceptive response. The nociceptive response induced by an i.t. injection of SP or NMDA was assessed by measuring the duration of hindlimb scratching directed toward the flank, biting and/or licking of the hindpaw or the tail for 5 min. Localization of MAS1 and either NK1 or NMDA receptors in the lumbar superficial dorsal horn was determined by immunohistochemical observation. The nociceptive response induced by SP and NMDA was attenuated by the i.t. co-administration of Ang (1-7) (0.03-3 pmol) in a dose-dependent manner. The inhibitory effects of Ang (1-7) (3 pmol) were attenuated by A779 (100 pmol), a MAS1 antagonist. Moreover, immunohistochemical analysis showed that spinal MAS1 co-localized with NK1 receptors and NMDA receptors on cells in the dorsal horn. Taken together, the i.t. injection of Ang (1-7) attenuated the nociceptive response induced by SP and NMDA via spinal MAS1, which co-localized with NK1 and NMDA receptors. Thus, the spinal Ang (1-7)/MAS1 pathway could represent a therapeutic target to effectively attenuate spinal pain transmission caused by the activation of NK1 or NMDA receptors.

Our previous study showed that Enterococcus faecalis 2001 (EF-2001) suppresses colitis-induced depressive-like behavior through the enhancement of hippocampal neurogenesis in mice. In the present study, we investigated the effect of EF-2001 on the cognitive behavior of olfactory bulbectomized (OBX) mice and its molecular mechanisms. The OBX-induced cognitive dysfunction was significantly suppressed by EF-2001. Moreover, EF-2001 also recovered the reductions in p-ERK1/2, p-CREB, BDNF and DCX levels and in neurogenesis observed in the hippocampus of OBX mice. These results suggest that EF-2001-induced antidementia effects are associated with enhanced hippocampal neurogenesis through the ERK-CREB-BDNF pathway.

Olfactory bulbectomy (OBX) in rodents induces neurochemical and behavioral changes similar to those observed in individuals with depressive disorders. Our previous study suggested that OBX alters dopaminergic function in the striatum of mice; however, the effects on dopaminergic function in the hypothalamus is unknown. Therefore, in this study we examined dopaminergic system changes in the hypothalamus after OBX. Mice were administrated either the nonselective dopamine (DA) agonist apomorphine or the selective D

Etidronate is widely used as a therapeutic agent for osteoporosis. We have recently shown that intrathecal administration of etidronate into mice produces an analgesic effect against the capsaicin-induced nociceptive behavior. However, the effect of etidronate on neuropathic pain at the spinal level remains unknown. Therefore, we examined whether etidronate attenuates pain after partial sciatic nerve ligation (PSNL). We evaluated tactile allodynia 7 days after PSNL by measuring paw withdrawal with the von Frey filament test. The mRNA and protein levels of SLC17A9 in the ipsilateral lumbar dorsal spinal cord of PSNL-operated mice were determined using real-time PCR and western blotting, respectively. PSNL-induced tactile allodynia was attenuated by oral and intrathecal administration of etidronate, with maximum efficiency at 90 and 60 min after injection, respectively. The anti-allodynic effect of intrathecally administered etidronate was completely inhibited by an intrathecal administration of adenosine triphosphate (ATP). The solute carrier family, SLC17, mediates the transport of pain transmitters, like ATP and glutamate. Indeed, we detected several members of the SLC17 family in the mouse dorsal lumbar spinal cord. Among the detected mRNAs, only Slc17a9, encoding for neuronal vesicular ATP transporter, was significantly increased upon PSNL. SLC17A9 protein levels were also significantly increased. In mice subjected to PSNL, SLC17A9 was present in neurons and microglia, but not in astrocytes of the lumbar superficial dorsal horn. Collectively, our results suggest that etidronate produces its anti-allodynic effects by inhibiting SLC17A9-dependent exocytotic ATP release from the dorsal horn in mice subjected to PSNL.

Olfactory bulbectomized (OBX) mice exhibit depressive-like behaviors and memory deficits. We have reported that aripiprazole (ARI) ameliorates the behavioral hyper-responsivity to dopamine agonists and memory deficits in OBX mice; however, it is unclear whether ARI affects OBX-induced depressive-like behavior. To address this question, we evaluated the effect of ARI on depressive-like behavior in OBX mice using the forced swim test (FST). In addition, we investigated the effect of ARI on c-Fos expression in the prefrontal cortex (PFC), striatum, and hippocampus of OBX mice using western blotting. OBX mice exhibited a longer immobility duration in the FST 14 days after surgery. Depressive-like behavior in OBX mice was reversed 30 min after administration of ARI (0.01 or 0.03 mg/kg). In addition, c-Fos expression was increased in the PFC, but not the striatum or hippocampus, 30 min after acute administration of ARI. These effects were inhibited by administration of the selective 5-HT1A, D1, and D2 receptor antagonists, WAY100635, SCH23390, and L-741,626, respectively. These findings suggest that ARI produces an antidepressant effect in OBX mice that may be mediated by 5-HT1A, D1, and D2 receptors in the PFC.

Our previous study suggested that the non-competitive N-methyl-d-aspartate receptor antagonist memantine (MEM) inhibits dopamine (DA) reuptake and turnover by inhibiting brain monoamine oxidase. Clinical studies have reported that MEM may improve depressive symptoms; however, specific mechanisms underlying this effect are unclear. We performed emotional behavior, tail suspension, and forced swimming tests to examine whether MEM has antidepressant effects in olfactory bulbectomized (OBX) mice, an animal model of depression. Subsequently, we investigated the effects of MEM on the distribution of tyrosine hydroxylase (TH), altered microglia morphometry, and astrocyte and cell proliferation in the hippocampus with immunohistochemistry. We also investigated MEM effects on the levels of norepinephrine (NE), DA, and their metabolites with high performance liquid chromatography, and of neurotrophic, proinflammatory, and apoptotic molecules in the hippocampus with western blotting. Forty-two days after surgery, OBX mice showed depressive-like behaviors, as well as decreased levels of monoamines, reduced cell proliferation, and lower levels of TH, phospho(p)-TH (ser31 and ser40), p-protein kinase A (PKA), p-DARPP-32, p-ERK1/2, p-CREB, brain-derived neurotrophic factor (BDNF), doublecortin, NeuN, and Bcl-2 levels. In contrast, the number of activated microglia and astrocytes and the levels of Iba1, GFAP, p-IκB-α, p-NF-κB p65, TNF-α, IL-6, Bax, and cleaved caspase-3 were increased in the hippocampus. These changes (except for those in NE and Bax) were reversed with chronic administration of MEM. These results suggest that MEM-induced antidepressant effects are associated with enhanced hippocampal cell proliferation and neuroprotection via the PKA-ERK-CREB-BDNF/Bcl-2-caspase-3 pathway and increased DA levels.

Dopamine (DA) modulates cognitive functions in the prefrontal cortex (PFC) and hippocampus. Olfactory bulbectomy (OBX) in mice induces cognitive dysfunctions. Recently, we reported that aripiprazole (ARI) normalizes the behavioral hyper-responsivity to DA agonists in OBX mice. However, it remains unclear whether ARI affects OBX-induced cognitive dysfunctions. To address this question we evaluated ARI-treated and untreated OBX mice in a passive avoidance test. Then, we investigated the effects of ARI on cell proliferation in the hippocampal dentate gyrus by immunohistochemistry, and on c-fos levels in the PFC and hippocampus, as well as nerve growth factor (NGF) levels in the hippocampus by western blotting. On the 14th day after surgery OBX mice showed an alteration in passive avoidance and decreases in both cell proliferation and levels of p-ERK, p-CREB and NGF in the hippocampus. The cognitive dysfunctions in OBX mice improved 30min to 24h after the administration of ARI (0.01mg/kg). C-fos levels in the PFC but not in the hippocampus was increased 30min after the administration (early response). This early response was inhibited by the selective D1 receptor antagonist SCH23390. Cell proliferation and NGF levels in the hippocampus increased 24h after ARI administration (late response), and these effects were also inhibited by SCH23390. The MEK1/2 inhibitor U0126 prevented ARI from improving the behavioral impairment as well as enhancing NGF levels in OBX mice. These findings revealed the potential of ARI to improve cognitive dysfunctions via D1 receptors with the PFC and hippocampus being affected sequentially.

Background Patients with inflammatory bowel disease (IBD), including those with ulcerative colitis and Crohn’s disease, have higher rates of psychiatric disorders, such as depression and anxiety; however, the mechanism of psychiatric disorder development remains unclear. Mice with IBD induced by dextran sulfate sodium (DSS) in drinking water exhibit depressive-like behavior. The presence of Lactobacillus in the gut microbiota is associated with major depressive disorder. Therefore, we examined whether Enterococcus faecalis 2001 (EF-2001), a biogenic lactic acid bacterium, prevents DSS-induced depressive-like behavior and changes in peripheral symptoms. Methods We evaluated colon inflammation and used the tail suspension test to examine whether EF-2001 prevents IBD-like symptoms and depressive-like behavior in DSS-treated mice. The protein expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), X-linked inhibitor of apoptosis protein (XIAP), and cleaved caspase-3 in the rectum and hippocampus was assessed by western blotting. Hippocampal neurogenesis, altered nuclear factor-kappa B (NFκB) p65 morphometry, and the localization of activated NFκB p65 and XIAP were examined by immunohistochemistry. Results Treatment with 1.5% DSS for 7 days induced IBD-like pathology and depressive-like behavior, increased TNF-α and IL-6 expression in the rectum and hippocampus, activated caspase-3 in the hippocampus, and decreased hippocampal neurogenesis. Interestingly, these changes were reversed by 20-day administration of EF-2001. Further, EF-2001 administration enhanced NFκB p65 expression in the microglial cells and XIAP expression in the hippocampus of DSS-treated mice. Conclusion EF-2001 prevented IBD-like pathology and depressive-like behavior via decreased rectal and hippocampal inflammatory cytokines and facilitated the NFκB p65/XIAP pathway in the hippocampus. Our findings suggest a close relationship between IBD and depression.

Neonatal ventral hippocampal-lesioned (NVHL) rats have been shown to display neurochemical and behavioral abnormalities at adulthood, analogous to some of those seen in schizophrenia. Serotonergic neurotransmission is implicated the pathophysiology and treatment of schizophrenia. In this study, we evaluated possible role of serotonergic transmission is the behaviors of NVHL-lesioned rats. Bilateral lesions to the ventral hippocampus (VH) in rat pups were made using the excitotoxin ibotenic acid. We investigated 5-HT

Deficits in olfaction are associated with neurodegenerative disorders such as Alzheimer's disease. A recent study reported that intranasal zinc sulfate (ZnSO4)-treated mice show olfaction and memory deficits. However, it remains unknown whether olfaction deficit-induced learning and memory impairment is associated with the cholinergic system in the brain. In this study, we evaluated olfactory function by the buried food find test, and learning and memory function by the Y-maze and passive avoidance tests in ZnSO4-treated mice. The expression of choline acetyltransferase (ChAT) protein in the olfactory bulb (OB), prefrontal cortex, hippocampus, and amygdala was assessed by western blotting. Moreover, we observed the effect of the acetylcholinesterase inhibitor physostigmine on ZnSO4-induced learning and memory deficits. We found that intranasal ZnSO4-treated mice exhibited olfactory dysfunction, while this change was recovered on day 14 after treatment. Both short-term and long-term learning and memory were impaired on days 4 and 7 after treatment with ZnSO4, whereas the former, but not the latter, was recovered on day 14 after treatment. A significant correlation was observed between olfactory function and short-term memory, but not long-term memory. Treatment with ZnSO4 decreased the ChAT level in the OB on day 4, and increased and decreased the ChAT levels in the OB and hippocampus on day 7, respectively. Physostigmine improved the ZnSO4-induced deficit in short-term, but not long-term, memory. Taken together, the present results suggest that short-term memory may be closely associated with olfactory function via the cholinergic system.

Olfactory bulbectomy (OBX) in rodents induces neurochemical and behavioral changes similar to those observed in individuals with depressive disorders. Our previous study suggested that OBX alters dopaminergic function in the striatum of mice; however, the effects on dopaminergic function in the hypothalamus is unknown. Therefore, in this study we examined dopaminergic system changes in the hypothalamus after OBX. Mice were administrated either the nonselective dopamine (DA) agonist apomorphine or the selective D2 agonist quinelorane, or pretreated with the selective D1 antagonist SCH23390 in combination with the selective D2 antagonist sulpiride or D3 antagonist SB277011A. Body temperature, which is regulated by the hypothalamic dopaminergic system, was monitored to evaluate changes in the dopaminergic system of the hypothalamus. DA D2 receptor (D2DR), tyrosine hydroxylase (TH), and phosphorylated (p)- DA- and cAMP-regulated phosphoprotein-32 (DARPP-32) levels in the hypothalamus were evaluated by western blotting. OBX mice exhibited significantly enhanced apomorphine-induced or quinelorane-induced hypothermia. The apomorphine-induced hypothermic response was reversed by the administration of sulpiride, but not SCH23390 or SB277011A. Moreover, TH and p-DARPP-32 levels were reduced and D2DR increased in the hypothalamus of OBX mice. These findings revealed that the OBX mice display enhanced DA receptor responsiveness associated with the hypothalamus, which may relate to some of the behavioral and neurochemical alterations reported in this animal model. Identification of changes in the hypothalamic dopaminergic system of OBX mice may provide useful information for the development of novel antidepressant treatments.

We have previously reported that the carborane compound BE360, a novel selective estrogen receptor modulator, has a therapeutic potential against dementia. This study aimed to explore the effects and underlying mechanisms of BE360 on depression-like behaviors in ovariectomized (OVX) mice subjected to subchronic stress, which are postmenopausal depression models. BE360 was subcutaneously administrated using a mini-osmotic pump, for 2 weeks. Depression-like behaviors were evaluated using the forced swimming test. Neurogenesis in the hippocampal dentate gyrus (DG) was measured by analyzing cells expressing doublecortin (DCX) following 5-bromo-2’-deoxyuridine (BrdU) uptake. The levels of phosphorylated cyclic-AMP response element-binding protein (p-CREB), brain-derived neurotrophic factor (BDNF), and Bcl-2 were measured using immunohistochemistry or immunoblotting. Depression-like behaviors in OVX + Stress-exposed mice improved after chronic treatment with BE360. BE360 treatment in OVX + Stress-exposed mice increased p-CREB, BDNF, and Bcl-2 expressions in the hippocampus. Immunohistochemistry showed that the number of BrdU/DCX double-positive cells in the DG of the hippocampus, which decreased significantly in OVX + Stress-exposed mice, increased after subchronic treatment with BE360. The present study demonstrates that BE360 exerts antidepressant effects via hippocampal neurogenesis, potentially activated through CREB/BDNF, Bcl-2 signaling pathways. These results indicate that BE360 may have therapeutic potential against postmenopausal depression.

Patients with inflammatory bowel disease (IBD) have higher rates of psychiatric pathology, including anxiety and depression. The dextran sulfate sodium (DSS)-treated mouse is a well-characterized animal model of colitis that exhibits IBD-like and depressive-like changes. A recent study found that phosphorylated (p-) adenosine monophosphate-activated protein kinase (AMPK) was associated with anti-inflammatory and antidepressant effects. Our previous research in an animal model of major depression suggests that liver hydrolysate (LH) has an antidepressant effect and combats physical fatigue by enhancement via the hippocampal or peripheral p-AMPK pathway. In this study, we examined whether or not LH has antidepressant and anti-inflammatory effects in mice with DSS-induced changes. We evaluated colon inflammation in DSS-treated mice and used the tail suspension and forced swimming tests to confirm whether or not LH prevents IBD-like symptoms and depressive-like behavior. Hippocampal expression of AMPK, brain-derived neurotrophic factor (BDNF), doublecortin, and neuronal nuclear antigen proteins was assessed by Western blotting. Hippocampal neurogenesis and morphometric changes in the microglia and astrocytes were examined by immunohistochemistry. DSS-treated mice showed IBD-like pathology and depressive-like behavior, a reduction in the hippocampal neuronal nuclear antigen level and neurogenesis, and increased hippocampal activation of microglia and astrocytes. These changes were reversed by LH. DSS-treated mice showed enhanced hippocampal expression of p-AMPK and BDNF after administration of LH. LH prevented depressive-like behavior by enhancing hippocampal neurogenesis through the AMPK/BDNF pathway and anti-neuroinflammation in the hippocampus. LH may be a therapeutic option for patients with IBD and depression.

We have previously reported that the spinal angiotensin (Ang) system is involved in the modulation of streptozotocin (STZ)-induced diabetic neuropathic pain in mice. An important drawback of this model however is the fact that the neuropathic pain is independent of hyperglycemia and produced by the direct stimulation of peripheral nerves. Here, using the leptin deficient ob/ob mouse as a type 2 diabetic model, we examined whether the spinal Ang system was involved in naturally occuring diabetic neuropathic pain. Blood glucose levels were increased in ob/ob mice at 5–15 weeks of age. Following the hyperglycemia, persistent tactile and thermal hyperalgesia were observed at 11–14 and 9–15 weeks of age, respectively, which was ameliorated by insulin treatment. At 12 weeks of age, the expression of Ang-converting enzyme (ACE) 2 in the spinal plasma membrane fraction was decreased in ob/ob mice. Spinal ACE2 was expressed in neurons and microglia but the number of NeuN-positive neurons was decreased in ob/ob mice. In addition, the intrathecal administration of Ang (1-7) and SB203580, a p38 MAPK inhibitor, attenuated hyperalgesia in ob/ob mice. The phosphorylation of spinal p38 MAPK was also attenuated by Ang (1-7) in ob/ob mice. These inhibitory effects of Ang (1-7) were prevented by A779, a Mas receptor antagonist. In conclusion, we revealed that the Ang (1-7)-generating system is downregulated in ob/ob mice and is accompanied by a loss of ACE2-positive neurons. Furthermore, Ang (1-7) decreased the diabetic neuropathic pain through inhibition of p38 MAPK phosphorylation via spinal Mas receptors.

We have previously demonstrated that the phosphorylation of p38 MAPK, through spinal AT1 receptor activation, is involved in formalin-induced nociception and follows accompanied by the increase in spinal angiotensin (Ang) II levels. We have also found that Ang (1-7), an N-terminal fragment of Ang II generated by ACE2, prevents the Ang II-induced nociceptive behavior via spinal MAS1 and the inhibition of p38 MAPK phosphorylation. Here, we examined whether the ACE2 activator diminazene aceturate (DIZE) can prevent the formalin-induced nociception in mice. The i.t. administration of DIZE attenuated the second, but not the first phase of formalin-induced nociceptive response. An increase in the activity of spinal ACE2 was measured following DIZE administration. The inhibitory effect of DIZE on nociception was abolished by the i.t. co-administration of the MAS1 antagonist A779. The i.t. administration of Ang (1-7) showed a similar effect on the second phase of the response which was also attenuated by A779. Furthermore, DIZE and Ang (1-7) each inhibited the formalin-induced phosphorylation of p38 MAPK on the dorsal lumbar spinal cord. This inhibition was again prevented by A779. ACE2 was expressed in neurons and microglia but absent from astrocytes in the superficial dorsal horn. Our data show that the i.t.-administered DIZE attenuates the second phase of the formalin-induced nociception which is accompanied by the inhibition of p38 MAPK phosphorylation. They also suggest the involvement of MAS1 activation on spinal neurons and microglia in response to the increase in Ang (1-7) following ACE2 activation.

Adenosine monophosphate-activated protein kinase (AMPK) is critical for whole-body energy metabolism regulation. Recent studies have suggested that physical exercise ameliorates depressive-like behaviors via AMPK activation; however, the underlying mechanism is unclear. Here, we examined the effects and underlying mechanisms of AMPK activation on depressive-like behavior in olfactory bulbectomized (OBX) mice. We treated OBX mice with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleotide (AICAR) on the 7th or 14th day after bilateral bulbectomy and evaluated depressive-like behavior using the tail-suspension test (TST) and forced swimming test (FST) on the 21st day. The expression of phosphorylated AMPK, protein kinase C ζ (PKCζ), nuclear factor-kappa B (NF-κB), brain-derived neurotrophic factor (BDNF), and cAMP response element-binding protein (CREB) in the hippocampus was assessed by western blotting. Hippocampal neurogenesis and localization of AMPK and phosphorylated NF-κB were examined by immunohistochemistry. Chronic AICAR treatment suppressed the prolonged immobility of OBX mice in the TST and FST, and increased the levels of phosphorylated AMPK, PKCζ, NF-κB, CREB, and BDNF. Hippocampal neurogenesis in OBX mice was promoted by chronic AICAR treatment. Co-administration of AICAR with the PKCζ inhibitor or the neurotrophic tyrosine kinase receptor type 2 (TrkB) antagonist, ANA-12, inhibited these effects. Phosphorylated AMPK was detected in mature and immature hippocampal neurons and microglia, while phosphorylated NF-κB was detected only in neurons in AICAR-treated OBX mice. These data indicate that AMPK activation produces anti-depressant effects, which are mediated by elevated hippocampal neurogenesis potentially via PKCζ/NF-κB/BDNF/TrkB/CREB signaling in neurons.

Olfactory bulbectomized (OBX) mice are characterized by impaired performance in the passive avoidance test and decreased number of cholinergic neurons in the hippocampus. Several studies have reported that κ-opioid receptor agonists improve cognitive function in mice. However, their influence on OBX-induced cognitive dysfunction remains unclear. To address this question, we evaluated the effects of the endogenous κ-opioid receptor agonist dynorphin A (Dyn A) and the selective agonist trans-(-)-U-50488 on the behavior of OBX mice in the passive avoidance test. The cognitive dysfunction of OBX mice was significantly recovered by the intracerebroventricular administration of Dyn A or trans-(-)-U-50488. The effects of these two agonists were counteracted by the selective κ-opioid receptor antagonist nor-binaltorphimine or the inhibitor of acetylcholine release β-bungarotoxin. These findings suggest that κ-opioid receptor agonists produce anti-dementia effects through activation of cholinergic neurons in OBX mice.

Background We have recently reported that the spinal angiotensin (Ang) converting enzyme (ACE)/Ang II/AT1 receptor axis and downstream p38 MAPK phosphorylation are activated in streptozotocin (STZ)-induced diabetic mice and lead to tactile hypersensitivity. Moreover, our previous results suggested that the intrathecal (i.t.) administration of Ang (1-7), an N-terminal fragment of Ang II, may attenuate the Ang II-induced nociceptive behaviour through the inhibition of p38 MAPK phosphorylation via Mas receptors. Here, we investigated whether the i.t. administration of Ang (1-7) can attenuate STZ-induced diabetic neuropathic pain. Methods Tactile and thermal hypersensitivities were determined using the von Frey filament and Hargreaves tests, respectively. The protein expression of ACE2, Mas receptors and phospho-p38 MAPK was measured by western blotting. Spinal ACE2 activity was determined using ACE2 activity assay kit. Results The i.t. administration of Ang (1-7) significantly reduced the tactile and thermal hypersensitivities on day 14 after STZ injection, and these effects were significantly prevented by the Mas receptor antagonist A779. The expression of ACE2 and Mas receptors in the plasma membrane fraction of the lumbar dorsal spinal cord was both significantly decreased in STZ mice. Spinal ACE2 activity was also decreased while p38 MAPK phosphorylation was increased in the lumbar dorsal region of these mice. This phosphorylation was attenuated by the injection of Ang (1-7), whose effect was reversed by A779. Conclusions Our data demonstrate that Ang (1-7) attenuates STZ-induced diabetic neuropathic pain and that this occurs through a mechanism involving spinal Mas receptors and he inhibition of p38 MAPK phosphorylation. Significance The ACE2/Ang (1-7)/Mas receptor axis was down-regulated in the spinal cord of STZ mice and the i.t. administration of Ang (1-7) attenuated the STZ-induced diabetic neuropathic pain via Mas receptors. Therefore, the activation of this axis could be an effective therapeutic target to alleviate the neuropathic pain in diabetic patients.

We have recently reported that an intrathecal (i.t.) administration of angiotensin II (Ang II) into mice induces a nociceptive behavior accompanied by the activation of p38 MAPK signaling via AT1 receptors (Nemoto et al., 2013, Mol. Pain 9, 38). These results suggested that Ang II participates in the facilitation of nociceptive transmission in the spinal cord. In the present study, we used formalin test to examine the effect of i.t.-administered losartan, an AT1 receptor antagonist, and determine whether Ang II acts as a neurotransmitter and/or neuromodulator in the spinal transmission of nociceptive information. When administered i.t. 5 min before the injection of a 2% formalin solution into the plantar surface of the hindpaw, losartan (30-100 nmol) produced a dose-dependent and significant antinociceptive effect during both the first and second phases of the test. In the superficial dorsal horn of the spinal cord (laminae I and II), the fluorescence intensities for Ang II and phospho-p38 MAPK were both significantly increased on the ipsilateral side 3 min after the injection of formalin compared to saline-treated controls. Moreover, the increase of phospho-p38 MAPK fluorescence intensity was significantly inhibited by the i.t. administration of losartan (54.8 nmol) 5 min prior to formalin. These results indicate that losartan produces an antinociceptive effect through the inhibition of p38 MAPK phosphorylation in the mouse formalin test and that Ang II may act as a neurotransmitter and/or neuromodulator in the spinal transmission of nociceptive information.

We examined whether chondroitin sulfate (CS), a compound used to treat osteoarthritis and joint pain, is effective against partial sciatic nerve ligation (PSNL)-induced neuropathic pain. Repeated oral administration of CS (300 mg/kg, b.i.d. for 20 days) resulted in inhibition of tactile allodynia observed 21 days after PSNL. On day 21, phosphorylation of spinal p38 mitogen-activated protein kinase (MAPK) was attenuated by CS. CS also inhibited c-Fos upregulation in ipsilateral deep dorsal horn (laminae III–IV) neurons, which receive Aβ-fiber afferent inputs. These findings suggest that CS attenuates PSNL-induced tactile allodynia by inhibiting spinal p38 MAPK phosphorylation and Aβ-fiber activation. Keywords: Chondroitin sulfate, Partial sciatic nerve ligation, Spinal cord

Background We have recently demonstrated that intrathecal (i.t.) administration of angiotensin II (Ang II) induces nociceptive behaviour in mice accompanied by a phosphorylation of p38 mitogen-activated protein kinase (MAPK) mediated through Ang II type 1 (AT1) receptors. The N-terminal fragment of Ang II, Ang (1–7), plays a pivotal role in counterbalancing many of the well-established actions induced by Ang II. However, the role of Ang (1–7) in spinal nociceptive transmission remains unclear. Therefore, we examined whether i.t. administration of Ang (1–7) can inhibit the Ang II-induced nociceptive behaviour in mice. Methods In the behavioural experiments, the accumulated response time of nociceptive behaviour consisting of scratching, biting and licking in conscious mice was determined during a 25-min period starting after i.t. injection. The distribution and localization of AT1 or Mas receptors were analysed using a MapAnalyzer and confocal microscope, respectively. Phosphorylation of p38 MAPK in the dorsal spinal cord was measured by Western blotting. Results The nociceptive behaviour induced by Ang II was dose-dependently inhibited by the co-administration of Ang (1–7). The inhibitory effect of Ang (1–7) was reversed by the co-administration of A779, a Mas receptor antagonist. Western blot analysis showed that the increase in spinal p38 MAPK phosphorylation following the i.t. administration of Ang II was also inhibited by Ang (1–7), and the Ang (1–7) induced-inhibition was prevented by A779. Conclusions Our data show that the i.t. administration of Ang (1–7) attenuates an Ang II-induced nociceptive behaviour and is accompanied by the inhibition of p38 MAPK phosphorylation mediated through Mas receptors.

We have previously shown that intracerebroventricular (i.c.v.) administration of cysteine protease inhibitors suppresses naloxone-precipitated withdrawal jumping in morphine-dependent mice, presumably through the inhibition of dynorphin degradation (see (Tan-No, K., Sato, T., Shimoda, M., Nakagawasai, O., Niijima, F., Kawamura, S., Furuta, S., Sato, T., Satoh, S., Silberring, J., Terenius, L., Tadano, T., 2010. Suppressive effects by cysteine protease inhibitors on naloxone-precipitated withdrawal jumping in morphine-dependent mice. Neuropeptides 44, 279-283)). In the present study, we examined the effect of phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor, on naloxone-precipitated withdrawal jumping in morphine-dependent mice. The doses of morphine (mg/kg per injection) were subcutaneously given twice daily for 2 days [day 1 (30) and day 2 (60)]. On day 3, naloxone (8 mg/kg) was intraperitoneally administered 3h after the final injection of morphine (60 mg/kg), and the number of jumps was immediately recorded for 20 min. Naloxone-precipitated withdrawal jumping was significantly suppressed by i.c.v. administration of PMSF (4 nmol), given 5 min before each morphine treatment during the induction phase, with none given on the test day. The expression of tissue plasminogen activator (tPA), a serine protease that converts plasminogen to plasmin, in the prefrontal cortex was significantly increased in morphine-dependent and -withdrawal mice, as compared with saline-treated mice. Moreover, trans-4-(aminomethyl)-cyclohexanecarboxylic acid (300 pmol), an antiplasmin agent, and (Tyr(1))-thrombin receptor activating peptide 7 (0.45 and 2 nmol), an antagonist of protease activated receptor-1 (PAR-1), significantly suppressed naloxone-precipitated withdrawal jumping. The present results suggest that PMSF suppresses naloxone-precipitated withdrawal jumping in morphine-dependent mice, presumably through the inhibition of activities of tPA and plasmin belonging to the serine proteases family, which subsequently activates PAR-1.