BACKGROUND AND PURPOSEThe ω-3 polyunsaturated fatty acids exert antinociceptive effects in inflammatory and neuropathic pain; however, the underlying mechanisms remain unclear. Docosahexaenoic acid-induced antinociception may be mediated by the orphan GPR40, now identified as the free fatty acid receptor 1 (FFA1 receptor). Here, we examined the involvement of supraspinal FFA1 receptor signalling in the regulation of inhibitory pain control systems consisting of serotonergic and noradrenergic neurons. EXPERIMENTAL APPROACHFormalin-induced pain behaviours were measured in mice. Antinociception induced by FFA1 receptor agonists was examined by intrathecal injections of a catecholaminergic toxin, 5-HT lowering drug or these antagonists. The expression of FFA1 receptor protein and c-Fos was estimated by immunohistochemistry, and the levels of noradrenaline and 5-HT in the spinal cord were measured by LC-MS/MS. KEY RESULTSFFA1 receptors colocalized with NeuN (a neuron marker) in the medulla oblongata and with tryptophan hydroxylase (TPH; a serotonergic neuron marker) and dopamine β-hydroxylase (DBH; a noradrenergic neuron marker). A single i.c.v. injection of GW9508, a FFA1 receptor agonist, increased the number of c-Fos-positive cells and the number of neurons double-labelled for c-Fos and TPH and/or DBH. It decreased formalin-induced pain behaviour. This effect was inhibited by pretreatment with 6-hydroxydopamine, DL-p-chlorophenylalanine, yohimbine or WAY100635. Furthermore, GW9508 facilitated the release of noradrenaline and 5-HT in the spinal cord. In addition, GW1100, a FFA1 receptor antagonist, significantly increased formalin-induced pain-related behaviour. CONCLUSION AND IMPLICATIONSActivation of the FFA1 receptor signalling pathway may play an important role in the regulation of the descending pain control system. Abbreviations

We previously reported that levels of long-chain fatty acids (FAs) including docosahexaenoic acids (DHA) increase in the hypothalamus of inflammatory pain model mice. However, the precise mechanisms underlying the increment of free fatty acids (FFAs) in the brain during inflammation remains unknown. In this study, we characterized FFAs released by inflammatory stimulation in rat primary cultured astrocytes, and tested the involvement of phospholipase A 2 (PLA 2 ) on these mechanisms. Lipopolysaccharide (LPS) stimulation significantly increased the levels of several FAs in the astrocytes. Under these conditions, mRNA expression of cytosolic PLA 2 (cPLA 2 ) and calcium-independent PLA 2 (iPLA 2 ) in LPS-treated group increased compared with the control group. Furthermore, in the culture media, the levels of DHA and arachidonic acid (ARA) significantly increased by LPS stimuli compared with those of a vehicle-treated control group whereas the levels of saturated FAs (SFAs), namely palmitic acid (PAM) and stearic acid (STA), did not change. In summary, our findings suggest that astrocytes specifically release DHA and ARA by inflammatory conditions. Therefore astrocytes might function as a regulatory factor of DHA and ARA in the brain.Key words astrocyte; free fatty acid (FFA); inflammation; docosahexaenoic acid (DHA); phospholipase A 2 (PLA 2 ) Lipid is one of the necessary nutrients to keep homeostasis. Fatty acids (FAs) are the smallest unit that makes up Lipids. FAs are currently generating considerable attention as a major source of energy in the brain. In particular, a continuous supply of FAs is required for cellular metabolism, differentiation, and development of neuron.1-4) FAs served not only as cellular nutrients and cell membrane components but also as lipidsoluble signal molecules, suggesting that FAs play vital roles in a wide variety of physiological function. 5,6) The central nervous system (CNS) is enriched in lipids [7][8][9][10] and, in particular, brain consist of 20% polyunsaturated FAs (PUFAs) such as docosahexaenoic acid (DHA) and arachidonic acid (ARA). Furthermore, these PUFAs mainly relate the physiological function of the brain.Astrocytes, one of the glial cells, assume a crucial role to maintain brain and neuronal function via releasing glutamate or neurotrophic factors. In CNS, astrocytes but not neurons are the main source of DHA and ARA. DHA and ARA are released from astroglial membranes under basal and stimulated conditions such as neurotransmitters, bladykinin, glutamate, ATP and thrombin and are supplied to the neurons. [11][12][13][14] In our previous study, we demonstrated that hypothalamic long chain fatty acid (FA) levels increase in the early phase of complete Freund's adjuvant (CFA)-induced inflammatory pain.15) The DHA level, in particular, is significantly increased compared with the control group. These increases correlated with an increase in glial fibrillary acidic protein (GFAP) expression in the hypothalamus of CFA-treated mice and were inhibited by the suppression of as...

The free fatty acid receptor 1 (GPR40/FFAR1) is a G protein-coupled receptor, which is activated by long chain fatty acids. We have previously demonstrated that activation of brain GPR40/FFAR1 exerts an antinociceptive effect that is mediated by the modulation of the descending pain control system. However, it is unclear whether brain GPR40/FFAR1 contributes to emotional function. In this study, we investigated the involvement of GPR40/FFAR1 in emotional behavior using GPR40/FFAR1 deficient (knockout, KO) mice. The emotional behavior in wild and KO male mice was evaluated at 9-10 weeks of age by the elevated plus-maze test, open field test, social interaction test, and sucrose preference test. Brain monoamines levels were measured using LC-MS/MS. The elevated plus-maze test and open field tests revealed that the KO mice reduced anxiety-like behavior. There were no differences in locomotor activity or social behavior between the wild and KO mice. In the sucrose preference test, the KO mice showed reduction in sucrose preference and intake. The level of noradrenaline was higher in the hippocampus, medulla oblongata, hypothalamus and midbrain of KO mice. Therefore, these results suggest that brain GPR40/FFAR1 is associated with anxiety- and depression-related behavior regulated by the increment of noradrenaline in the brain.

Nausea, vomiting, and renal injury are the common adverse effects associated with cisplatin.Cisplatin is excreted via the multidrug and toxin release (MATE) transporter, and the involvement of the MATE transporter in cisplatin-induced kidney injury has been reported. The MATE transporter is also involved in the excretion of ondansetron, but the effects of 5-HT 3 receptor antagonists used clinically for cisplatin-induced renal injury have not been elucidated. Therefore, the aim of this study was to investigate the effects of 5-HT 3 receptor antagonists in a mouse model of cisplatin-induced kidney injury and to validate the results using medical big data analysis of more than 1.4 million reports and a survey of 3,000 hospital medical records. The concomitant use of a first-generation 5-HT 3 receptor antagonist (ondansetron, granisetron, or ramosetron) significantly increased cisplatin accumulation in the kidney and worsened renal damage.Conversely, the concomitant use of palonosetron had no effect on renal function compared with the use of cisplatin alone. Furthermore, an analysis of data from the US Food and Drug Administration Adverse Event Reporting System and retrospective medical records revealed that the combination treatment of cisplatin and a first-generation 5-HT 3 receptor antagonist significantly increased the number of reported renal adverse events compared with the combination treatment of cisplatin and a second-generation 5-HT 3 receptor antagonist. These results suggest that compared with the first-generation antagonists, second-generation 5-HT 3 receptor antagonists do not worsen cisplatin-induced acute kidney injury. The findings should be validated in a prospective controlled trial before implementation in clinical practice.

We previously showed that activation of G protein-coupled receptor 40/free fatty acid receptor 1 (GPR40/FFAR1) signaling modulates descending inhibition of pain. In this study, we investigated the involvement of fatty acid-GPR40/FFAR1 signaling in the transition from acute to chronic pain. We used GPR40/FFAR1-knockout (GPR40KO) mice and wild-type (WT) mice. A plantar incision was performed, and mechanical allodynia and thermal hyperalgesia were evaluated with a von Frey filament test and plantar test, respectively. Immunohistochemistry was used to localize GPR40/FFAR1, and the levels of free fatty acids in the hypothalamus were analyzed with liquid chromatography-tandem mass spectrometry. The repeated administration of GW1100, a GPR40/FFAR1 antagonist, exacerbated the incision-induced mechanical allodynia and significantly increased the levels of phosphorylated extracellular signal-regulated kinase in the spinal cord after low-threshold touch stimulation in the mice compared to vehicle-treated mice. The levels of long-chain free fatty acids, such as docosahexaenoic acid, oleic acid, and palmitate, which are GPR40/FFAR1 agonists, were significantly increased in the hypothalamus two days after the surgery compared to levels in the sham group. Furthermore, the incision-induced mechanical allodynia was exacerbated in the GPR40KO mice compared to the WT mice, while the response in the plantar test was not changed. These findings suggested that dysfunction of the GPR40/FFAR1 signaling pathway altered the endogenous pain control system and that this dysfunction might be associated with the development of chronic pain.

Our previous studies demonstrated that emotional dysfunction associated with early life stress exacerbated nerve injury-induced mechanical allodynia. Sex differences were observed in several anxiety tests, but not in mechanical allodynia. To elucidate the mechanism underlying these findings, we have now investigated the involvement of astrocytes in emotional dysfunction and enhancement of nerve injury-induced mechanical allodynia in mice subjected to maternal separation combined with social isolation (MSSI) as an early life stress. We measured expression of glial fibrillary acidic protein (GFAP), an astrocyte maker, in each brain area by immunohistochemistry. GFAP expression in the locus coeruleus (LC) of female, but not of male mice, significantly increased after MSSI, corresponding to the behavioral changes at 7 and 12 weeks of age. Lipopolysaccharide (LPS)-treated astrocyte-derived supernatant was administered to local brain regions, including LC. Intra-LC injection of conditioned medium from cultured astrocytes treated with LPS increased GFAP expression, anxiety-like behavior and mechanical allodynia in both male and female mice. Furthermore, increases in anxiety-like behavior correlated with increased mechanical allodynia. These findings demonstrate that emotional dysfunction and enhanced nerve injury-induced mechanical allodynia after exposure to MSSI are mediated, at least in part, by astrocyte activation in the LC. Male but not female mice may show resistance to MSSI stress during growth.

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The free fatty acid receptor 1 (GPR40/FFAR1) is activated by polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acids (DHA). This receptor has been the focus of many studies regarding physiological functions of the central nervous system. PUFAs are essential for neuronal development and maintenance of neuronal function; thus, the decrease of PUFAs in the brain is closely related to the induction of psychiatric diseases associated with emotional disorder, such as anxiety, depression, and schizophrenia. However, details of the mechanisms remain unclear. In this study, we investigated changes of maternal and/or emotional behavior caused by a deficiency of GPR40/FFAR1 signaling. GPR40/FFAR1 deficient (FFAR1 / ) female mice exhibited impaired maternal care such as retrieving behaviors and an increased rate of neglect and infanticide when compared to wild type (WT) female mice. Furthermore, FFAR1 / female mice showed increased time spent in the open arms in an elevated plus maze test, reduction of locomotor activity and social interaction behavior, and decreased sucrose intake, when compared to WT female mice. In conclusion, these findings suggest that PUFAs-GPR40/FFAR1 signaling might function, at least in part, as a regulatory factor of emotional and maternal behavior in mice.Key words free fatty acid receptor 1; maternal behavior; emotional damage; polyunsaturated fatty acid The dietary intake of n-3 fatty acids such as docosahexaenoic acid (DHA) has dramatically decreased with the westernization of dietary habits and food satiation.