TR-1 cells, which stably expressed TREK-1 channel and were grown on a small cover glass (3 18 mm), were placed in a recording chamber with an internal volume of 200 l

TR-1 cells, which stably expressed TREK-1 channel and were grown on a small cover glass (3 18 mm), were placed in a recording chamber with an internal volume of 200 l. showed antidepressive effects in the forced swim and tail suspension tests, although previous studies reported that inhibition of TREK-1 channels resulted in an antidepressive effect. The anxiolytic and antidepressive effect was diminished by co-administration of a TREK-1 blocker, amlodipine, McMMAF indicating the involvement of TREK-1 channels. Administration of ostruthin suppressed the stress-induced increase in anti-c-Fos immunoreactivity in the lateral septum, without affecting immunoreactivity in other mood disorder-related nuclei, e.g. the amygdala, paraventricular nuclei, and dorsal raphe nucleus. Ostruthin may exert its anxiolytic and antidepressive effects through a different mechanism from current drugs. Introduction Anxiety and depression are common mental disorders, for which most patients are treated with medication. However, anxiolytic medicines can lead to tolerance and dependence [1]. In addition, approximately one-third of patients with depression are resistant to current antidepressants, such as serotonin reuptake inhibitors and serotonin-norepinephrine reuptake inhibitors [2]. Therefore, new medicines, for which the mechanism of action is different from current ones, are desired for the treatment of these mental disorders. Potassium (K+) channels play a pivotal role in the regulation of excitability of the central neurons. Among the broad range of K+ channel families, the most recently identified family is the two-pore domain K+ (K2P) channels responsible for background K+ currents, which are also Pou5f1 known as leak K+ currents [3]. Mammalian K2P channels now include 15 members, one of which is the TWIK-related K+ channel, TREK-1. These channels are highly expressed in the central nervous system [4, 5] and are suggested to be involved in mental diseases, i.e. anxiety and depression [6, 7]. For instance, TREK-1-deficient mice showed a depression-resistance phenotype through activation of the dorsal raphe nucleus (DRN), which provides serotonergic innervation [6]. Riluzole, which activates TREK-1 channels in addition to Na+ channels and McMMAF glutamate receptor blockade, showed anxiolytic effects [8]. Therefore, TREK-1 channel activators and blockers are possible candidate for anxiolytic and antidepressive drugs, respectively. TREK-1 channels can be activated or inhibited McMMAF by several chemical compounds. For example, TREK-1 channels are activated by arachidonic acid, volatile anesthetic (chloroform, diethyl ether, halothane, and isoflurane), and riluzole [9], and inhibited by fluoxetine [10] and bupivacaine [11]. However, TREK-1 modulating activities are only side effects of these compounds, and they have major activities elsewhere, e.g. serotonin uptake inhibition and blockade of Na+ channels. Currently, there seems to be no TREK-1-specific compound that can regulate the pharmacological activity of this channel. Plants cells express K+ channels, the structures of which are similar to those of mammalian K+ channels [12, 13]. In addition, tropical and semitropical plants also produce compounds that modify K+ channel function [14, 15]; therefore, botanical compounds are a promising resource for K+ channel modifiers. In this study, we screened a library of botanical compounds, which were isolated from plants in Vietnam, for a modulator of TREK-1 channel activity using whole-cell patch clamp recordings. We identified a TREK-1 activator, ostruthin, which had anxiolytic and antidepressive activities in mice. Ostruthin suppressed stress-induced increases in c-Fos expression in the lateral septum without affecting that in the amygdala or DRN, suggesting a possible difference in the mechanism of action from current drugs. Materials and methods Purification of ostruthin The roots of were collected in Khanh Hoa province Vietnam in 2014 and dried. The material (200 g) was powdered and extracted with methanol at room temperature, and the methanol was evaporated under reduced pressure at 45C. The crude extract was dissolved in CH2Cl2 at room temperature with sonication. After solvent evaporation at 40C, the sample was separated to 7 fractions by silica gel column chromatography, and the fourth fraction was again chromatographed on a silica gel column with an increasing concentration of ethyl acetate mixed with n-hexane (5.0C6.7%). Ostruthin was purified to homogeneity (> 99.1%) according to the chromatogram of A330 nm. Patch-clamp recordings For recordings of K+ channel currents, we prepared stable cell lines for TREK-1, TWIK-related acid-sensitive K+ channel (TASK-1), strongly inwardly rectifying K+ channel (Kir2.1), and human ether-a-go-go-related gene (HERG-1) channels and transiently expressed other channels in 293T cells using a calcium-phosphate transfection method. For the establishment of the stable lines, we used lentiviral vectors, and the preparation methods for the lentiviral vectors were described in our previous report [16]. TR-1 cells, which stably expressed TREK-1 channel and were grown on a small cover glass (3 18 mm), were placed in a recording chamber with an internal volume of 200 l. Whole-cell currents were recorded in Tyrode solution using an.