Long-term exposure to high glucose induces changes in the expression of AMPA receptor subunits and glutamate transmission in primary cultured cortical neurons.

Hyperglycemia, which occurs under the diabetic conditions, induces serious diabetic complications. Diabetic encephalopathy has been defined as one of the major complications of diabetes, and is characterized by neurochemical and neurodegenerative changes. However, little is known about the effect of long-term exposure to high glucose on neuronal cells. In the present study, we showed that exposure to glutamate (100 mM) for 7 days induced toxicity in primary cortical neurons using the MTT assay. Additionally, high glucose increased the sensitivity of AMPA- or NMDA-induced neurotoxicity, and decreased extracellular glutamate levels in primary cortical neurons. In Western blot analyses, the protein levels of the GluA1 and GluA2 subunits of the AMPA receptor as well as synaptophysin in neurons treated with high glucose were significantly increased compared with the control (25 mM glucose). Therefore, long-term exposure to high glucose induced neuronal death through the disruption of glutamate homeostasis.

Fluorescein Permeability of the Blood-Brain Barrier Is Enhanced in Juvenile- but Not Young Adult-Onset Type 1 Diabetes in Rats.

Clinically, neurological disorders, such as cognitive impairments and dementia, have been reported as diabetic complications, which are remarkable, especially in children with diabetes. The blood-brain barrier (BBB) is a physiologically dynamic regulatory barrier that maintains the consistency of the fluid microenvironment composition of the brain. However, the differences in BBB conditions between children and adults and the contribution of the BBB to the severity of cognitive impairments remain unclear. We generated adult-onset diabetes mellitus (DM) and juvenile-onset diabetes mellitus (JDM) diabetic rat models and investigated BBB functions in these models during the early stages of type 1 diabetes. We performed a BBB permeability assay using sodium fluorescein, a small-molecule fluorescent dye, to evaluate endothelial transport from the blood to the central nervous system. One week after diabetes onset, BBB permeability increased in the hippocampus and striatum of JDM rats, but no changes were observed in the frontal cortex and hypothalamus of JDM rats or for any region of DM rats. The double staining of tight junction proteins and astrocytes revealed no changes in the hippocampus and striatum of JDM rats. These results suggested that the observed increase in BBB permeability during early-stage diabetes onset in JDM rats, which did not depend on the expression of the interendothelial tight junction protein, claudin-5, may affect stylized neural development and cognitive function.

Oxytocin reverses Aß-induced impairment of hippocampal synaptic plasticity in mice.

AIM: Oxytocin, a peptide hormone synthesized in the hypothalamic paraventricular nucleus, has been reported to participate in the regulation of learning and memory performance. However, no report has demonstrated the effect of oxytocin on the amyloid-beta (Aß)-induced impairment of synaptic plasticity. In this study, we examined the effects of oxytocin on the Aß-induced impairment of synaptic plasticity in mice. METHODS: To investigate the effect of oxytocin on synaptic plasticity, we prepared acute hippocampal slices for extracellular recording and assessed long-term potentiation (LTP) with perfusion of the Aß active fragment (Aß25-35) in the absence and presence of oxytocin. RESULTS: We found that oxytocin reversed the impairment of LTP induced by Aß25-35 perfusion in the mouse hippocampus. These effects were blocked by pretreatment with the selective oxytocin receptor antagonist L-368,899. Furthermore, the treatment with the ERK inhibitor U0126 and selective Ca2+-permeable AMPA receptor antagonist NASPM completely antagonized the effects of oxytocin. CONCLUSION: This is the first report to demonstrate that oxytocin could reverse the effects of Aß on hippocampal LTP in mice. We propose that ERK phosphorylation and Ca2+-permeable AMPA receptors are involved in this effect of oxytocin.

Systemic administration of a delta opioid receptor agonist, KNT-127, facilitates extinction learning of fear memory in rats.

Strategies to facilitate extinction of fear memory have attracted increasing attention for enhancing the effectiveness of exposure therapy for anxiety disorders. Previously, we demonstrated that systemic administration of a delta opioid receptor agonist, KNT-127, has clear anxiolytic-like effects in rats, without impairing memory. These observations led us to hypothesize that KNT-127 might be an appropriate therapeutic agent for anxiety disorders when combined with exposure therapy. In the present study, we demonstrate that KNT-127 (3 mg/kg) facilitates extinction learning of fear memory using the contextual fear conditioning test. As expected, a partial agonist at the glycine-binding site on the glutamatergic N-methyl-d-aspartate receptor, d-cycloserine (15 mg/kg), facilitated extinction learning of contextual fear in rats. In contrast, a benzodiazepine anxiolytic, diazepam (1 mg/kg), impaired the fear extinction learning. Interestingly, the facilitatory effect of KNT-127 on extinction learning was observed not only after a 10-min re-exposure, but also after a much shorter (2-min) re-exposure to the context, while d-cycloserine was ineffective at facilitating extinction when a short-duration exposure was given. Our findings may suggest that administration of a delta opioid receptor agonist might have therapeutic efficacy when combined with exposure therapy for treating a range of anxiety disorders.

[The effect of lunch box provision and mass feeding on energy and nutrient supply at emergency shelters after the Great East Japan Earthquake].

Objectives　Improvement in the quality of meals provided after a disaster is an important issue. We reanalyzed the data obtained from the dietary survey of emergency shelters in Miyagi Prefecture 2 and 3 months after the Great East Japan Earthquake in 2011 to improve the quality of meals at emergency shelters.Methods　We performed a secondary analysis of the data from the dietary survey conducted by the Miyagi prefectural government. Two hundred and forty-one and 49 emergency shelters participated in the 1-day dietary survey in May 2011 and June 2011, respectively. We targeted emergency shelters that had no missing data and provided 3 meals a day. As a result, we targeted 216 emergency shelters in May and 49 in June. We examined the amounts of nutrients (i.e. energy, protein, vitamin B1, vitamin B2, and vitamin C) and food groups (i.e. cereal, potatoes, meat, seafood, eggs, dairy, vegetables, fruits, and fats) in meal portions provided in lunch boxes, rationing, and mass feeding.Results　We found significant differences in the amounts of energy and nutrients in the meals provided at the emergency shelters 2 months after the Great East Japan Earthquake but found no significant differences 3 months after the disaster. The amounts of energy, protein, seafood, and fats were high, and those of vitamins B1 and C, potatoes, and vegetables were low, at the emergency shelters where lunch boxes were provided. The amounts of potatoes, meat, and vegetables were high at the emergency shelters where mass feeding was conducted.Conclusion　Two months after the Great East Japan Earthquake, the provision of lunch boxes at emergency shelters may have increased the amounts of energy, protein, and seafood in meals served to survivors, whereas the amounts of vitamins B1 and C have remained low. These results indicate that providing lunch boxes at an early stage in the event of a disaster can improve energy and protein supply. We believe a combination of lunch box and mass feeding will improve the nutrient supply at emergency shelters.

A delta opioid receptor agonist, KNT-127, in the prelimbic medial prefrontal cortex attenuates glial glutamate transporter blocker-induced anxiety-like behavior in mice.

We previously reported that systemic administration of a delta opioid receptor (DOP) agonist, KNT-127, produced a potent anxiolytic-like effect in rats. Interestingly, DOPs are highly distributed in the prelimbic medial prefrontal cortex (PL-PFC). In the present study, we investigated the effect of KNT-127 co-perfusion in the PL-PFC on anxiety-like behavior in mice, induced by a glial glutamate transporter inhibitor, (3S)-3-[[3-[[4-(Trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartic acid (TFB-TBOA). Extracellular glutamate levels were measured in male C57BL/6N mice by in vivo microdialysis high-performance liquid chromatography/electrochemical detection, with behavior simultaneously assessed in the open field test. As expected, extracellular glutamate levels were significantly increased, and anxiety-like behavior was induced after local perfusion of TFB-TBOA in the PL-PFC. Uniquely, co-perfusion of KNT-127 in the PL-PFC diminished anxiety-like behavior induced by TFB-TBOA without affecting extracellular glutamate levels. Further, the effect of KNT-127 on anxiety-like behavior was antagonized by a selective DOP antagonist, naltrindole, suggesting that KNT-127 acts via DOPs. These findings do not support our preconceived hypothesis that KNT-127 in PL-PFC produces an anxiolytic-like effect via suppression of glutamatergic transmission. Hence, further studies are necessary to understand the mechanisms of DOP agonist-induced anxiolytic-like effects in the PL-PFC.

Post-reexposure administration of D-cycloserine facilitates reconsolidation of contextual conditioned fear memory in rats.

The effect of D-cycloserine, a N-methyl-D-aspartate receptor partial agonist, on the reconsolidation of conditioned fear memory is not precisely understood. In this study, we clarified the effects of D-cycloserine on the reconsolidation in rats, by performing contextual fear conditioning with a mild fear-conditioning procedure and with post-reexposure administration of the drug. The D-cycloserine (15 mg/kg subcutaneously)-treated rats showed a persistent and greater fear response during the test session compared with the control group. In conclusion, we have confirmed that post-reexposure administration of D-cycloserine facilitates the reconsolidation of fear memory in rats.

Neuroprotection by chotosan, a Kampo formula, against glutamate excitotoxicity involves the inhibition of GluN2B-, but not GluN2A-containing NMDA receptor-mediated responses in primary cultured cortical neurons.

Chotosan (CTS), a traditional herbal formula called Kampo medicine, was shown to be effective in the treatment of vascular dementia in a clinical study, and exerted protective effects against transient cerebral ischemia-induced cognitive impairment in mice. In the present study, we investigated the neuroprotective effects of CTS using primary cultured rat cortical neurons. CTS (250-1000 µg/mL) inhibited neuronal death induced by 100 µM glutamate. This glutamate-induced neuronal death was blocked by a GluN2B-, but not GluN2A-containing NMDA receptor antagonist. Therefore, the neuroprotective effects of CTS were related to an inhibition of GluN2B-containing NMDA receptor-mediated responses.

Serotonergic and dopaminergic systems are implicated in antidepressant-like effects of chotosan, a Kampo formula, in mice.

We previously demonstrated that chotosan (CTS), a traditional herbal formula called Kampo medicine, improves diabetes-induced cognitive deficits. In the present study, we investigated the antidepressant-like effects of CTS in mice. The administration of CTS (1.0 g/kg, for 3 days) decreased the immobility time in the forced-swim test, and this decrease was prevented by the prior administration of sulpiride (an antagonist of D2/3 receptors) and WAY100635 (an antagonist of 5-HT1A receptors). None of the treatments tested altered the locomotor activity of mice. These results suggest that CTS exerts antidepressant-like effects through changes in the serotonergic and dopaminergic systems.

The effects of changes in the meal providing system on emergency shelter menus following the Great East Japan Earthquake.

Objectives　To assess the improvement of the menus by the meal providing system in emergency shelters, we reanalyzed the data obtained from the dietary survey at emergency shelters in the Miyagi Prefecture one month after the Great East Japan Earthquake in 2011.Methods　We performed secondary use of the dietary survey conducted by the Miyagi prefectural government. In total, 386 emergency shelters participated in the dietary survey of one day in April 2011. We examined the meal providing system (the frequency of meals per day, the frequency of mass feeding for evacuees per day, and menu creators) and the menus at each emergency shelter (260). We classified menus into the following five groups: staple foods, main dishes, side dishes, dairy products, and fruits, and calculated the frequency of provision per day.Results　There was no emergency shelter that provided zero or one meal per day. Compared with the emergency shelters that provided two meals per day, shelters that provided three had a significantly higher provision frequency of staple foods. However, there were no significant differences in the provision frequencies of the other four food groups between the shelters with two or three meals per day. In emergency shelters with a higher frequency of mass feeding for evacuees, the provision frequency of four food groups (staple foods, main dishes, side dishes, and fruits) was significantly higher compared to shelter with a lower frequency of mass feeding for evacuees. Furthermore, in the emergency shelters where dietitians created menus, the provision frequency for two food groups (dairy products and fruits) was significantly higher compared to shelters without dietitians.Conclusion　A high frequency of mass feeding for evacuees resulted in a high provision frequency of main dishes, side dishes, and fruits, which can be limited in supply after a disaster. Also, in the emergency shelters where dietitians created menus, the provision frequency of dairy products and fruits was higher than in those without dietitians. These results suggested that diets in the emergency shelters may be improved by mass feeding and the involvement of dietitians.

Changes in hippocampal synaptic functions and protein expression in monosodium glutamate-treated obese mice during development of glucose intolerance.

Glucose is the sole neural fuel for the brain and is essential for cognitive function. Abnormalities in glucose tolerance may be associated with impairments in cognitive function. Experimental obese model mice can be generated by an intraperitoneal injection of monosodium glutamate (MSG; 2 mg/g) once a day for 5 days from 1 day after birth. MSG-treated mice have been shown to develop glucose intolerance and exhibit chronic neuroendocrine dysfunction associated with marked cognitive malfunctions at 28-29  weeks old. Although hippocampal synaptic plasticity is impaired in MSG-treated mice, changes in synaptic transmission remain unknown. Here, we investigated whether glucose intolerance influenced cognitive function, synaptic properties and protein expression in the hippocampus. We demonstrated that MSG-treated mice developed glucose intolerance due to an impairment in the effectiveness of insulin actions, and showed cognitive impairments in the Y-maze test. Moreover, long-term potentiation (LTP) at Schaffer collateral-CA1 pyramidal synapses in hippocampal slices was impaired, and the relationship between the slope of extracellular field excitatory postsynaptic potential and stimulus intensity of synaptic transmission was weaker in MSG-treated mice. The protein levels of vesicular glutamate transporter 1 and GluA1 glutamate receptor subunits decreased in the CA1 region of MSG-treated mice. These results suggest that deficits in glutamatergic presynapses as well as postsynapses lead to impaired synaptic plasticity in MSG-treated mice during the development of glucose intolerance, though it remains unknown whether impaired LTP is due to altered inhibitory transmission. It may be important to examine changes in glucose tolerance in order to prevent cognitive malfunctions associated with diabetes.

Lysophosphatidic acid induces anxiety-like behavior via its receptors in mice.

Lysophosphatidic acid (LPA) is a potent bioactive lipid mediator with diverse biological properties. We previously found altered expression of the LPA-related genes in rodents after treatment with sertraline, which is widely used to treat anxiety disorders and depression. However, little is known about the behavioral effects of LPA. In the present study, we investigated the behavioral effects of intracerebroventricular injection of LPA in adult mice. LPA did not significantly affect spontaneous locomotor activity, suggesting that LPA does not induce hyperactivity, ataxia, or sedation. We next investigated the emotional effects of LPA via the hole-board test. LPA significantly increased the number of head-dips in a dose- and time-related manner. A significant induction of head-dip counts occurred 15 and 30 min after LPA administration. To clarify the involvement of LPA receptors, we examined the effect of the non-selective LPA1-4 receptor antagonist, 1-bromo-3(S)-hydroxy-4-(palmitoyloxy)butyl-phosphonate (BrP-LPA) co-administered with LPA. BrP-LPA dose-dependently inhibited LPA-induced head-dip counts. We next investigated anxiety-like behavior via the elevated plus-maze test. LPA significantly reduced the percentage of time spent in the open arms and BrP-LPA dose-dependently inhibited this anxiety-like behavior. In conclusion, LPA induced anxiety-like behavior in mice via LPA receptors. Our results suggest that LPA signaling plays an important role in regulating anxiety in mice.

Induction of c-Fos immunoreactivity in the amygdala of mice expressing anxiety-like behavior after local perfusion of veratrine in the prelimbic medial prefrontal cortex.

Local perfusion of the sodium channel activator veratrine in mouse prelimbic medial prefrontal cortex (PL) induced c-Fos immunoreactivity in the sub-regions of amygdala. Co-perfusion of the NMDA receptor antagonist MK-801 diminished the c-Fos expression. Significant correlations were observed between c-Fos immunoreactivity and behavioral measures in the open-field test. The PL stimulation activates a neural network projecting to the amygdala via NMDA receptor-mediated glutamatergic neurotransmission. Anxiety-like behavior induced after the PL stimulation may be partly mediated through the activation of amygdala.

Glucagon-like peptide-1 protects synaptic and learning functions from neuroinflammation in rodents.

Glucagon-like peptide-1 (GLP-1) is derived from the processing of proglucagon in intestinal L-cells and releases insulin from pancreatic ß-cells as an incretin. The GLP-1 receptor has been proposed as a possible therapeutic target for the treatment of Alzheimer's disease, in which neuroinflammation is critical in the pathogenesis. The present study investigates whether GLP-1 (7-36) amide, an active fragment of GLP-1, protected against synaptic impairments induced by inflammation-related injurious agents (lipopolysaccharide [LPS], interleukin-1ß [IL-1ß], and H2 O2). In the Y-maze test, LPS (10 µg/mouse, i.c.v) significantly decreased the percentage alternation. Pretreatment with GLP-1 (7-36) amide (0.09-0.9 nmol/mouse, i.c.v.) prevented an impairment in spontaneous alternation performance. Pretreatment with LPS (10 µg/ml, 2 hr) impaired LTP induction but not paired-pulse facilitation in the CA1 region of rat hippocampal slices. This impairment was prevented by cotreatment with GLP-1 (7-36) amide (50 nM). IL-1ß (0.57 nM) or H2 O2 (50 µM) also impaired LTP induction. This impairment was prevented by GLP-1 (7-36) amide (50 nM). These results suggest that GLP-1 (7-36) amide improves the synaptic impairments induced by inflammation-related injurious agents in the CA1 region of the hippocampus.

Activation of the prelimbic medial prefrontal cortex induces anxiety-like behaviors via N-Methyl-D-aspartate receptor-mediated glutamatergic neurotransmission in mice.

We investigated the possible roles of the prelimbic medial prefrontal cortex (PL) in the regulation of anxiety-like behaviors by pharmacologically activating the terminals of neuronal inputs or postsynaptic efferent neurons with a sodium channel activator veratrine. The extracellular glutamate levels were measured by in vivo microdialysis, and the behaviors were assessed with the open field (OF) test in mice simultaneously. The samples were collected every 10 min for 60 min, as basal levels of glutamate. The medium containing drugs were perfused for 30 min. The OF test was performed in the last 10 min of drug perfusion. After the drug treatments, the perfusion medium containing drugs was switched back to perfusion medium without drugs, and then samples were collected for another 90 min. The extracellular glutamate levels were significantly elevated after local perfusion of veratrine in the PL. At the same time, perfusion of veratrine in the PL produced anxiety-like behaviors in mice. Local coperfusion of a sodium channel blocker, lamotrigine, completely diminished the veratrine-induced elevated extracellular glutamate levels and the behavioral changes. Local coperfusion of an NMDA receptor antagonist, MK-801, but not a non-NMDA (AMPA/kainate) receptor antagonist, CNQX, completely diminished the behavioral changes without any effects on the veratrine-induced elevated extracellular glutamate levels. This study demonstrates that the activation of the PL with veratrine induces anxiety-like behaviors via NMDA receptor-mediated glutamatergic neurotransmission in mice.

Polysulfides are possible H2S-derived signaling molecules in rat brain.

Accumulating evidence shows that hydrogen sulfide (H2S) has a variety of physiological functions. H2S is produced from cysteine by 3 sulfurtransferases. H2S, in turn, generates polysulfides, the functions of which are not well understood. H2S induces Ca(2+) influx in astrocytes, a type of glia. However, the receptor that mediates the response has not been identified. Here, we have shown that polysulfides induce Ca(2+) influx by activating transient receptor potential (TRP)A1 channels in rat astrocytes (EC50 91 nM, Hill coefficient value 1.77±0.26) and that the maximum response was induced at 0.5 µM, which is 1/320 of the concentration of H2S required to achieve a response of similar magnitude (160 µM, EC50 116 µM). TRPA1-selective agonists, allyl isothiocyanate and cinnamaldehyde, induced Ca(2+) influx, and responses to polysulfides were suppressed by TRPA1-selective inhibitors, HC-030031 and AP-18, as well as by siRNAs selective to TRPA1. The present study suggests that polysulfides are possible H2S-derived signaling molecules that stimulate TRP channels in the brain.

Hippocampal acetylcholine receptor activation-dependent long-term depression in streptozotocin-induced diabetic rats.

Cholinergic innervation of the hippocampus correlates with memory formation. In a well-established animal model of type 1 diabetes mellitus, obtained by injecting young adult rats with streptozotocin (STZ), reductions have been reported in the expression of acetylcholine receptors and choline acetyltransferase. In this study, we showed that long-term synaptic depression (LTD) induced by carbachol (CCh), a nonselective cholinergic receptor agonist, at Schaffer collateral-CA1 synapses in hippocampal slices was significantly weaker in streptozotocin-induced diabetic rats (STZ rats) than in age-matched control rats. No significant change was observed in the paired-pulse ratio between before and 80 min after the application of CCh in control and STZ rats. Moreover, CCh-induced LTD in control and STZ rats was not affected by an NMDA receptor antagonist. Although the application of CCh down-regulated the surface expression of GluA2 in the hippocampus of control rats, but not STZ rats. Therefore, the present results suggest that acetylcholine receptor-mediated LTD in STZ rats requires the internalization of AMPA receptors on the postsynaptic surface and their intracellular effects in the hippocampus.

Nighttime snacking reduces whole body fat oxidation and increases LDL cholesterol in healthy young women.

The increase in obesity and lipid disorders in industrialized countries may be due to irregular eating patterns. Few studies have investigated the effects of nighttime snacking on energy metabolism. We examined the effects of nighttime snacking for 13 days on energy metabolism. Eleven healthy women (means ± SD; age: 23 ± 1 yr; body mass index: 20.6 ± 2.6 kg/m(2)) participated in this randomized crossover trial for a 13-day intervention period. Subjects consumed a specified snack (192.4 ± 18.3 kcal) either during the daytime (10:00) or the night time (23:00) for 13 days. On day 14, energy metabolism was measured in a respiratory chamber without snack consumption. An oral glucose tolerance test was performed on day 15. Relative to daytime snacking, nighttime snacking significantly decreased fat oxidation (daytime snacking: 52.0 ± 13.6 g/day; nighttime snacking: 45.8 ± 14.0 g/day; P = 0.02) and tended to increase the respiratory quotient (daytime snacking: 0.878 ± 0.022; nighttime snacking: 0.888 ± 0.021; P = 0.09). The frequency of snack intake and energy intake, body weight, and energy expenditure were not affected. Total and low-density lipoprotein (LDL) cholesterol significantly increased after nighttime snacking (152 ± 26 mg/dl and 161 ± 29 mg/dl; P = 0.03 and 76 ± 20 mg/dl and 83 ± 24 mg/dl; P = 0.01, respectively), but glucose and insulin levels after the glucose load were not affected. Nighttime snacking increased total and LDL cholesterol and reduced fat oxidation, suggesting that eating at night changes fat metabolism and increases the risk of obesity.

Kampo formulations, chotosan, and yokukansan, for dementia therapy: existing clinical and preclinical evidence.

Cognitive deficits and behavioral and psychological symptoms of dementia (BPSD) are typical features of patients with dementia such as Alzheimer's disease (AD), vascular dementia (VD), and other forms of senile dementia. Clinical evidence has demonstrated the potential usefulness of chotosan (CTS) and yokukansan (YKS), traditional herbal formulations called Kampo medicines, in the treatment of cognitive disturbance and BPSD in dementia patients, although the indications targeted by CTS and YKS in Kampo medicine differ. The availability of CTS and YKS for treating dementia patients is supported by preclinical studies using animal models of dementia that include cognitive/emotional deficits caused by aging and diabetes, dementia risk factors. These studies have led not only to the concept of a neuronal basis for the CTS- and YKS-induced amelioration of cognitive function and emotional/psychiatric symptom-related behavior in animal models, but also to a proposal that ingredient(s) of Uncariae Uncis cum Ramulus, a medicinal herb included in CTS and YKS, may play an important role in the actions of these formulae in dementia patients. Further studies are needed to clarify the active ingredients of these formulae and their target endogenous molecules implicated in the anti-dementia drug-like actions.

Riluzole does not affect hippocampal synaptic plasticity and spatial memory, which are impaired by diazepam in rats.

We have previously demonstrated that riluzole has anxiolytic-like effects in rats, without affecting spontaneous alternation performance in the Y-maze test. However, the effects of riluzole on hippocampal synaptic plasticity were still unclear. In this study, we showed that bath application of riluzole did not impair long-term potentiation and long-term depression, whereas a benzodiazepine anxiolytic, diazepam, significantly impaired them. Furthermore, the acquisition of spatial memory in the Morris water maze test was impaired in diazepam-treated but not riluzole-treated rats. We thus provide further evidence for the potential usefulness of riluzole as an anxiolytic that does not cause amnesia.