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Rehabilitation from alcohol addiction or abuse is challenging due to alcohol withdrawal symptoms. Headache is a severe alcohol withdrawal symptom that frequently contributes to rehabilitation failure. Despite the need for treating alcohol withdrawal-induced headache, there is no appropriate therapeutic option available. Development of improved therapeutics will depend on obtaining a clearer understanding of alcohol withdrawal-induced headache pain mechanisms. Here, we show that the mast cell-specific receptor MrgprB2 controls development of alcohol withdrawal-induced headache. Withdrawing alcohol from alcohol-acclimated mice induces strong headache behaviors, including facial allodynia, facial pain expressions, and reduced walking movement, symptoms often observed in humans suffering from headache. Observed pain behaviors were abolished in MrgprB2-deficient mice. We observed in vivo spontaneous activation and hypersensitization of trigeminal ganglia neurons in alcohol withdrawal mice, but not in MrgprB2-deficient mice. Corticotropin-releasing factor (CRF) was increased in dura mater after alcohol withdrawal. Injection of CRF into dura mater resulted in activation of trigeminal ganglia neurons and vasodilation, which was accompanied by headache behavior. In cells, CRF evoked Ca2+ transients via MrgprB2 or human MrgprX2. The results indicate that alcohol withdrawal causes headache via mast cell degranulation in dura mater. The process is under control of MrgprB2/MrgprX2, which would appear to represent a potential target for treating alcohol withdrawal-related headache.

Trigeminal (TG), dorsal root (DRG), and nodose/jugular (NG/JG) ganglia each possess specialized and distinct functions. We used RNA sequencing of two-cycle sorted Pirt-positive neurons to identify genes exclusively expressing in L3–L5 DRG, T10-L1 DRG, NG/JG, and TG mouse ganglion neurons. Transcription factor Phox2b and Efcab6 are specifically expressed in NG/JG while Hoxa7 is exclusively present in both T10-L1 and L3–L5 DRG neurons. Cyp2f2, Krt18, and Ptgds, along with pituitary hormone prolactin (Prl), growth hormone (Gh), and proopiomelanocortin (Pomc) encoding genes are almost exclusively in TG neurons. Immunohistochemistry confirmed selective expression of these hormones in TG neurons and dural nerves; and showed GH expression in subsets of TRPV1+ and CGRP+ TG neurons. We next examined GH roles in hypersensitivity in the spinal versus trigeminal systems. Exogenous GH produced mechanical hypersensitivity when injected intrathecally, but not intraplantarly. GH-induced thermal hypersensitivity was not detected in the spinal system. GH dose-dependently generated orofacial and headache-like periorbital mechanical hypersensitivity after administration into masseter muscle and dura, respectively. Periorbital mechanical hypersensitivity was reversed by a GH receptor antagonist, pegvisomant. Overall, pituitary hormone genes are selective for TG versus other ganglia somatotypes; and GH has distinctive functional significance in the trigeminal versus spinal systems.

Chronic immobilization stress (CIS) induces low levels of glutamate (Glu) and glutamine (Gln) and hypoactive glutamatergic signaling in the mouse prefrontal cortex (PFC), which is closely related to the Glu-Gln cycle. A Gln-supplemented diet ameliorates CIS-induced deleterious changes. Here, we investigated the effects of CIS and Gln supplementation on Glu-Gln cycle-related proteins to characterize the underlying mechanisms. Using the CIS-induced depression mouse model, we examined the expression of 11 proteins involved in the Glu-Gln cycle in the PFC. CIS decreased levels of glutamate transporter 1 (GLT1) and sodium-coupled neutral amino acid transporter (SNAT) 1, SANT2, SNAT3, and SNAT5. Gln supplementation did not affect the non-stressed group but significantly increased GLT1 and SNATs of the stressed group. By immunohistochemical analysis, we confirmed that SNAT1 and SNAT2 were decreased in neurons and GLT1, SNAT3, and SNAT5 were decreased in astrocytes in the medial PFC of the stressed group, but Gln-supplemented diet ameliorated these decrements. Collectively, these results suggest that CIS may cause depressive-like behaviors by decreasing Glu and Gln transportation in the PFC and that a Gln-supplemented diet could prevent the deleterious effects of CIS., Graphical Abstract

Emerging evidence has shown the low levels of glutamate (Glu) and glutamine (Gln) and the hypoactivity in the cortex of patients with depression. The hypoactivity is closely related with low frequency of glutamatergic signaling that is affected by the levels of Glu and Gln. Thus, we hypothesized that there might be a causality among low levels of Glu and Gln, hypoactive glutamatergic neurotransmissions, and depressive behaviors. Here, we found low Glu and Gln levels and low frequency of spontaneous excitatory postsynaptic current (sEPSC) of glutamatergic neurons in the medial prefrontal cortex (mPFC) of chronic immobilization stress (CIS)-induced depressed mice. The depressed mice also showed hypoactive Gln synthetase (GS). Inhibition of GS by methionine sulfoximine (MSO) decreased Glu and Gln levels and increased depressive behaviors with low frequency of sEPSC in the mPFC, indicating that Glu and Gln decrements cause hypoactive glutamatergic neurotransmissions and depressive behaviors. Both Glu and Gln could increase sEPSC of glutamatergic neurons in the mPFC on slice patch, but only Gln overcame MSO to increase sEPSC, suggesting that exogenous Gln would recover CIS-induced low frequency of sEPSC caused by hypoactive GS and act as an antidepressant. Expectedly, Gln supplementation showed antidepressant effects against CIS; it increased glutamatergic neurotransmissions with Glu and Gln increment in the mPFC and attenuated depressive behaviors. Moreover, selective glutamatergic activation in the mPFC by optogenetics decreased depressive behavior. In conclusion, depressive behaviors evoked by chronic stress were due to hypoactive glutamatergic neurons in the mPFC caused by low levels of Glu and Gln, and exogenous Gln can be used as an alternative antidepressant to increase glutamatergic neurotransmission.

Ketone bodies can be increased in the blood under certain physiological conditions, but their role under such conditions remains to be clarified. In the present study, we found the increment and usage of β-hydroxybutyrate (BHB) in the prefrontal cortex (PFC) during acute stress. BHB levels increased in the blood and PFC after 30-min acute immobilization stress, and BHB dehydrogenase 1 increased in the PFC simultaneously, but not in the hippocampus. Moreover, increased levels of acetyl-CoA, pyruvate carboxylase, and glutamate dehydrogenase 1 were found in the PFC, implicating the metabolism of increased BHB in the brain. Thus, we checked the levels of glutamate, glutamine, and GABA and found increased levels of glutamate and glutamine in the stressed group compared with that in the control group in the PFC. Exogenous administration of BHB enhanced struggling behaviors under stressful conditions. Our results suggest that the metabolism of BHB from peripheral blood in the PFC may contribute to acute stress responses to escape stressful conditions.

Rehabilitation from alcohol addiction or abuse is challenging due to alcohol withdrawal symptoms. Headache is a severe alcohol withdrawal symptom that frequently contributes to rehabilitation failure. Despite the need for treating alcohol withdrawal-induced headache, there is no appropriate therapeutic option available. Development of improved therapeutics will depend on obtaining a clearer understanding of alcohol withdrawal-induced headache pain mechanisms. Here, we show that the mast cell-specific receptor MrgprB2 controls development of alcohol withdrawal-induced headache. Withdrawing alcohol from alcohol-acclimated mice induces strong headache behaviors, including facial allodynia, facial pain expressions, and reduced walking movement, symptoms often observed in humans suffering from headache. Observed pain behaviors were abolished in MrgprB2-deficient mice. We observed in vivo spontaneous activation and hypersensitization of trigeminal ganglia neurons in alcohol withdrawal mice, but not in MrgprB2-deficient mice. Corticotropin-releasing factor (CRF) was increased in dura mater after alcohol withdrawal. Injection of CRF into dura mater resulted in activation of trigeminal ganglia neurons and vasodilation, which was accompanied by headache behavior. In cells, CRF evoked Ca2+ transients via MrgprB2 or human MrgprX2. The results indicate that alcohol withdrawal causes headache via mast cell degranulation in dura mater. The process is under control of MrgprB2/MrgprX2, which would appear to represent a potential target for treating alcohol withdrawal-related headache.

Non-alcoholic steatosis and insulin resistance are critical health problems and cause metabolic complications worldwide. In this study, we investigated the molecular mechanism of Polygonum multiflorum Thunb. (PM) against hepatic lipid accumulation and insulin resistance by using in vitro and in vivo models. PM extract significantly attenuated the accumulation of lipid droplets and hepatic triglyceride in free fatty acid (FFA)-exposed HepG2 cells. PM extract increased the AMPK and ACC phosphorylation and GLUT4 expression, whose levels were downregulated in FFA-exposed cells. PM extract also decreased precursor and mature forms of SREBP-1 in FFA-exposed cells. C57BL/6 mice fed with normal diet (ND) or high-fat diet (HFD) were administered PM extract (100 mg/kg) or vehicle orally for 16 weeks. PM extract attenuated the increases of the epididymal and perirenal fats on HFD feeding. PM extract markedly reduced hepatic lipid accumulation and fasting glucose levels, and improved glucose and insulin sensitivity in HFD-fed mice. HFD-fed mice decreased the AMPK and ACC phosphorylation and GLUT4 expression, and increased precursor and mature forms of SREBP-1, these changes were significantly restored by PM extract. In conclusion, PM extract alleviates non-alcoholic steatosis and insulin resistance through modulating the expression of proteins on lipid metabolism and glucose transport in the liver.

Glutamatergic synapses constitute a major excitatory neurotransmission system and are regulated by glutamate/glutamine (Gln) cycling between neurons and astrocytes. Gln synthetase (GS) produced by astrocytes plays an important role in maintaining the cycle. However, the significance of GS during synaptogenesis has not been clarified. GS activity and expression significantly increase from postnatal day (PD) 7 to 21, and GS is expressed prior to glial fibrillary acidic protein (GFAP) and is more abundant than GFAP throughout synaptogenesis. These observations suggest that GS plays an important role in synaptogenesis. We investigated this by inhibiting GS activity in neonatal mice and assessed the consequences in adult animals. Lower expression levels of GS and GFAP were found in the CA3 region of the hippocampus but not in the CA1 region. Moreover, synaptic puncta and glutamatergic neurotransmission were also decreased in CA3. Behaviorally, mice with inhibited GS during synaptogenesis showed spatial memory-related impairment as adults. These results suggest that postnatal GS activity is important for glutamatergic synapse development in CA3.

We recently reported that glutamine (Gln) supplementation protected glutamatergic neurotransmission from the harmful effects of chronic stress. Altered glutamatergic neurotransmission is one of the main causes of cognitive disorders. However, the cognitive enhancer function of Gln has not been clearly demonstrated thus far. Here, we evaluated whether and how Gln supplementation actually affects chronic stress-induced cognitive impairment. Using a chronic immobilization stress (CIS) mouse model, we confirmed that chronic stress induced mild cognitive impairment (MCI) and neuronal damage in the hippocampus. In contrast, Gln-supplemented mice did not show evidence of MCI. To investigate possible underlying mechanisms, we confirmed that CIS increased plasma corticosterone levels as well as brain and plasma levels of reactive oxygen/nitrogen species. CIS also increased levels of inducible nitric oxide synthase and NADPH oxidase subunits (p47phox and p67phox) in both the prefrontal cortex and CA1 region of the hippocampus. CIS decreased the number of synaptic puncta in the prefrontal cortex and hippocampus, but these effects were inhibited by Gln supplementation. Taken together, the present results suggest that Gln is an effective agent against chronic stress-induced MCI.

Non-alcoholic steatosis is a common health problem worldwide due to altered food habits and life styles, and it is intimately linked with various metabolic disorders. In the present study, we investigated the molecular mechanism of Ganoderma lucidum (GL) against the development of non-alcoholic steatosis using in vivo and in vitro settings. C57BL/6 mice fed with normal diet (ND) or high fat diet (HFD) were administered GL extract or vehicle for 16 weeks. HFD feeding increased serum alanine aminotransferase level and hepatic lipid droplet, but these increases were significantly attenuated by GL. GL inhibited the increases in epididymal and perirenal adipose tissue weights and serum cholesterol and LDL levels in HFD-fed mice. Fasting blood glucose levels were elevated in HFD-fed mice compared to ND-fed mice, and glucose and insulin sensitivities were deteriorated. These changes were markedly improved by GL. GL restored the reduction of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in the liver of HFD-fed mice, and increased AMPK and ACC phosphorylation in HepG2 and 3T3-L1 cells. GL induced GLUT4 protein expression in 3T3-L1 cells. Finally, GL attenuated lipid accumulation induced by free fatty acid in HepG2 cells. Taken together, our results indicate that GL has a potential to improve non-alcoholic steatosis and the associated complicated disorders via the induction of energy metabolizing enzymes.

Glutamine synthetase (GS), an astrocytic protein in the brain, mediates the process by which glutamate (Glu) is transformed into glutamine (Gln) during Glu and gamma-aminobutyric acid (GABA) de novo synthesis. There are many types of neural complications related with those neurotransmitters in type 1 diabetes (T1D) patients, but there is little information about the change GS. Therefore, we examined changes in GS activity and expression, as well as the amount of Glu, Gln, and GABA in the brain of a T1D animal model. Using primary culture we found that glucose fluctuation caused glial fibrillary acidic protein (GFAP) and GS changes but constant high glucose level didn׳t. In T1D mouse, GS expression increased in the prefrontal cortex (PFC) and hippocampus (HI), but decreased GS activity was only observed in the HI whereas GFAP expression decreased in both regions. Gln increased in both regions, but Glu and GABA were only increased in the HI of T1D animals where GS activity decreased with higher reactive oxygen/nitrogen species. Collectively, low GS activity may be closely related with high levels of Glu and GABA in the HI of T1D brain, and this would result in abnormal neurotransmissions.

Regulators of G-protein signaling (RGS) proteins regulate certain G-protein-coupled receptor (GPCR)-mediated signaling pathways. The GABAB receptor (GABABR) is a GPCR that plays a role in the stress response. Previous studies indicate that acute immobilization stress (AIS) decreases RGS4 in the prefrontal cortex (PFC) and hypothalamus (HY) and suggest the possibility of a signal complex composed of RGS4 and GABABR. Therefore, in the present study, we tested whether RGS4 associates with GABABR in these brain regions. We found the co-localization of RGS4 and GABABR subtypes in the PFC and HY using double immunohistochemistry and confirmed a direct association between GABAB2R and RGS4 proteins using co-immunoprecipitation. Furthermore, we found that AIS decreased the amount of RGS4 bound to GABAB2R and the number of double-positive cells. These results indicate that GABABR forms a signal complex with RGS4 and suggests that RGS4 is a regulator of GABABR. [BMB Reports 2014; 47(6): 324-329]

The changes in telomere length and mitochondrial DNA copy number (mtDNAcn) are considered to be aging markers. However, many studies have provided contradictory or only fragmentary information about changes of these markers in animal models, due to inaccurate analysis methods and a lack of objective aging standards. To establish chronological aging standards for these two markers, we analyzed telomere length and mtDNAcn in 12 tissues&mdash, leukocytes, prefrontal cortex, hippocampus, pituitary gland, adrenal gland, retina, aorta, liver, kidney, spleen, skeletal muscle, and skin&mdash, from a commonly used rodent model, C57BL/6 male mice aged 2&ndash, 24 months. It was found that at least one of the markers changed age-dependently in all tissues. In the leukocytes, hippocampus, retina, and skeletal muscle, both markers changed age-dependently. As a practical application, the aging marker changes were analyzed after chronic immobilization stress (CIS) to see whether CIS accelerated aging or not. The degree of tissue-aging was calculated using each standard curve and found that CIS accelerated aging in a tissue-specific manner. Therefore, it is expected that researchers can use our standard curves to objectively estimate tissue-specific aging accelerating effects of experimental conditions for least 12 tissues in C57BL/6 male mice.

Spinach is one of the most widely consumed vegetables, and is known as for both physical and mental health maintenance. However, there is little information about how spinach protects one from stress. In the present study, we created three extracts from Spinach oleracea L., (frozen powder (FP), water extract (WE), and ethanol extract (EE)), and examined their anti-stress and anti-depressive effects on mouse using a chronic immobilization stress (CIS) regimen. FP, WE, and EE showed different free amino acid constituents. Calorie-balanced diets derived from each extract were tested for their ability to reduce blood corticosterone (CORT) levels in naïve mice. Diets supplemented with FP or EE induced lower blood CORT levels than a normal diet, but the WE diet did not. Mobility duration and sucrose preference were increased by FP and EE supplementation in the CIS-induced depression animal models. Moreover, FP and EE increased glutamate and glutamine levels in the medial prefrontal cortex (mPFC) compared with CIS-induced depressed group. These results suggest that spinach has anti-stress and anti-depressive properties by lowering CORT and increasing glutamate and glutamine levels in the mPFC.

A healthy acute stress response requires both rapid increase and rapid clearance of blood corticosteroids. We previously showed that regulators of G-protein signaling 4 (RGS4), which decreases in the paraventricular nucleus (PVN) during acute stress, forms a complex with the GABAB receptor. In the present study, we show that this decrease in RGS4 levels in the PVN during an acute stress response facilitates the return of blood corticosteroids to basal levels. Moreover, the effect of RGS4 decrease is attenuated by a GABAB receptor antagonist. These results suggest that RGS4 in the PVN regulates blood corticosteroid-related GABAB receptor signaling during the acute stress response.

Regulator of G-protein signaling (RGS) proteins play an important role in G-protein coupled receptor (GPCR) signaling and the activity of some GPCRs is modulated via RGS protein levels during stress response. The aim of this study was to investigate changes in RGS protein mRNA expressions in the mouse brain after 2h restraint stress. The mRNA level of 19 RGS proteins was analyzed using real-time PCR in six brain regions, which included the prefrontal cortex, amygdala, hippocampus, hypothalamus, striatum, and pituitary gland, from control and stressed mouse. We found that the level of mRNA of each RGS varied according to brain region and that two to eight RGS proteins exhibited changes in mRNA levels in each brain region by restraint stress. It was also revealed that RGS4 protein amount was consistent with mRNA level, indicating RGS4 protein may have regulatory roles in the acute stress response.

Background: The brain levels of glutamate (Glu) and glutamine (Gln) are partially regulated through the Glu‐Gln cycle. Astrocytes play a role in regulating the Glu‐Gln cycle, and loss of astrocytes has been associated with depressive disorders. We hypothesized that levels of Glu and Gln would be affected by astrocyte loss and dysregulation of the Glu‐Gln cycle and that depressive-like behaviours would be closely related to the level of changes in Glu and Gln. Methods: We used liquid chromatography to measure Glu and Gln con centrations in the prefrontal cortex of male mice infused with L- α aminoadipic acid (L-AAA), a specific astrocyte toxin, in the prelimbic cortex. Methionine sulfoximine, a Gln synthetase inhibitor, and α-methyl-amino-isobutyric acid, a blocker of neuronal Gln transporters, were used to disturb the Glu‐Gln cycle. We assessed the behavioural change by drug infusion using the forced swim test (FST) and su crose preference test. Results: The Glu and Gln levels were decreased on the fifth day after L-AAA infusion, and the infused mice showed longer durations of immobility in the FST and lower sucrose preference, indicative of depressive-like behaviour. Mice in which Gln synthetase or Gln transport were inhibited also exhibited increased immobility in the FST. Direct infusion of L-Gln reversed the in creased immobility induced by astrocyte ablation and Glu‐Gln cycle impairments. Limitations: Genetically modified animal models and diverse behavioural assessments would have been helpful to solidify our conclusions. Conclusion: Neuronal Gln deficiency in the pre frontal cortex may cause depressive behaviours.

A previous study using a mouse model of depression showed that chronic immobilization stress (CIS) reduces levels of insulin-like growth factor (IGF)-2, IGF binding protein 2 (IGFBP2), osteoglycin, and fibromodulin in the amygdala. Here, using human neuroblastoma cells, we tested whether these four proteins cooperatively modulate neuronal plasticity. We found that IGF-2 and IGFBP2 synergistically increased neurite outgrowth via enhanced early signaling through the IGF type 1 receptor. Furthermore, we found that osteoglycin, a small leucine-rich proteoglycan, significantly increased IGF-2/IGFPB2-induced neurite outgrowth, but fibromodulin had no effect. We also found that central amygdala neurons of CIS-induced depressive mouse showed a decreased total dendritic length. These findings suggest that CIS-responsive proteins modulate neuronal morphology during chronic stress.

Background The amygdala plays an essential role in controlling emotional behaviors and has numerous connections to other brain regions. The functional role of the amygdala has been highlighted by various studies of stress-induced behavioral changes. Here we investigated gene expression changes in the amygdala in the chronic immobilization stress (CIS)-induced depression model. Results Eight genes were decreased in the amygdala of CIS mice, including genes for neurotrophic factors and extracellular matrix proteins. Among these, osteoglycin, fibromodulin, insulin-like growth factor 2 (Igf2), and insulin-like growth factor binding protein 2 (Igfbp2) were further analyzed for histological expression changes. The expression of osteoglycin and fibromodulin simultaneously decreased in the medial, basolateral, and central amygdala regions. However, Igf2 and Igfbp2 decreased specifically in the central nucleus of the amygdala. Interestingly, this decrease was found only in the amygdala of mice showing higher immobility, but not in mice displaying lower immobility, although the CIS regimen was the same for both groups. Conclusions These results suggest that the responsiveness of the amygdala may play a role in the sensitivity of CIS-induced behavioral changes in mice.