The extracts of Fructus Akebiae, a preparation containing 90% of the active ingredient hederagenin: serotonin, norepinephrine and dopamine reuptake inhibitor

Fructus Akebiae is a traditional Chinese herbal extract that has been used for the treatment of depressive disorders in China. Previous studies demonstrated that Fructus Akebiae extracts (FAE) displayed a potent antidepressant-like activity in animal behavior tests and found that the specific active ingredient from the extracts of Fructus Akebiae is hederagenin. However, the underlying mechanism is unknown. Here we provide evidences that FAE enhances the signaling of central monoamines via inhibition of the reuptake of the extracellular monoamines including serotonin (5-HT), norepinephrine (NE) and dopamine (DA). In rat brain membrane preparations and HEK293 cells transfected with human serotonin transporter (SERT), NE transporter (NET) and DA transporter (DAT), we found that FAE displayed marked affinity to rat and cloned human monoamine transporters in ex vivo and in vitro experiments, using competitive radio ligand binding assay. In uptake assays using rat synaptosomes and transfected cells, FAE was found to significantly inhibit all three monoamine transporters in a dose- and time-dependent manner, with a comparable or better potency to their corresponding specific inhibitors. In contrast, FAE (10 μM), showed no significant affinity to a variety array of receptors tested from CNS. In support of our uptake data, in vivo microdialysis studies showed that administration of FAE (12.6, 25, 50 mg/kg) significantly increased extracellular concentrations of 5-HT, NE and DA in frontal cortex of freely moving rats. Taken together, our current study showed for the first time that FAE is a novel triple inhibitor of monoamine transporters, which may be one the mechanisms of its antidepressant activity.     

Antidepressant activity of methyl jasmonate, a plant stress hormone in mice

Methyl jasmonate (MJ) is a hormone released by plants in response to external stress, injury or pathogenic invasions. This present investigation evaluated the antidepressant effect of intraperitoneal doses of MJ in mice. Mice were given MJ in the doses of 10, 25 and 50 mg/kg daily for 7 days and then subjected to forced swim test (FST), tail suspension test (TST) and yohimbine lethality test (YLT). The results showed that MJ produced a significant decrease in the period of immobility in the FST and TST, indicating antidepressant activity. MJ potentiated the toxic effect of yohimbine in the YLT, which further suggests antidepressant property and also indicates facilitatory effect on both serotonergic and noradrenergic systems respectively. However, MJ did not significantly alter the spontaneous motor activity of the animals, which indicates a lack of central nervous system stimulant effect. Taken together, these findings suggest that MJ has antidepressant activity and the mechanisms underlying this effect may involve serotonergic and noradrenergic systems.     

八月札抗肝癌活性部位的筛选

目的 筛选出八月札抗肝癌的活性部位。方法 利用H22小鼠肝癌皮下移植瘤模型对八月札乙醇提取物及其石油醚萃取物、氯仿萃取物、乙酸乙酯萃取物、正丁醇萃取物和将乙醇提取后的药渣进行水提得到的水提物进行抗肝癌活性部位筛选,剂量均为生药20 g·kg^-1;采用人肝癌细胞BEL-7404裸鼠皮下移植瘤模型研究乙醇提取物的抗肝癌作用。结果 八月札乙醇提取物以及正丁醇萃取部位对H22小鼠肝癌皮下移植瘤模型具有明显的抑瘤作用,抑瘤率分别为46.87%和45.31%,其他部位无明显的抑瘤作用;八月札乙醇提取物的高剂量组（生药20 g·kg^-1）和中剂量组（生药10 g·kg^-1）对人肝癌细胞BEL-7404裸鼠移植瘤模型具有明显的抑瘤作用,抑瘤率分别为57.5%和40.0%。结论 乙醇提取物及其正丁醇萃取物部位为八月札抗肝癌活性部位。     

预知子不同部位中常春藤皂苷元含量的比较

目的 建立预知子中常春藤皂苷元的HPLC-ELSD含量测定方法,并比较果实、种子和果皮3个不同部位中常春藤皂苷元含量差异。方法 色谱条件：色谱柱为Ultimate XB柱(4.6 mm×250 mm,5 μm),流动相为乙腈-水-甲酸(70∶30∶0.1),流速为1.0 mL·min^-1,柱温为35 ℃,ELSD漂移管温度为90 ℃,载气流速为2.5 L·min^-1。结果 常春藤皂苷元在0.36~3.60 μg内峰面积积分值常用对数与进样量常用对数有良好的线性关系(r=0.999 4);平均回收率为99.04 %,RSD为2.0 %。预知子果实、种子、果皮中常春藤皂苷元含量范围分别为24.28~35.18,37.21~58.38,7.83~19.44 mg·g^-1。结论 本研究所建立的含量测定方法简便、准确、重复性强,可用于预知子药材的质量控制。预知子不同部位中常春藤皂苷元的含量存在显著差异,在临床应用中应根据使用部位确定药材用量。     

人参总皂苷对小鼠的抗抑郁作用

目的：初步探讨人参总皂苷对小鼠的抗抑郁作用。方法：选取健康雄性昆明种小鼠,随机分为5组,空白对照组、阳性药组、人参总皂苷125、250、500 mg.kg^-1剂量组。通过小鼠自主活动实验、小鼠强迫游泳实验和小鼠悬尾实验,观察人参总皂苷对小鼠抗抑郁作用的影响。结果：各给药组小鼠自主活动行为与空白对照组比较均无明显差异;人参总皂苷125、250、500 mg.kg-1均可以显著缩短小鼠强迫游泳及小鼠悬尾不动时间。结论：实验结果表明人参总皂苷在小鼠＂行为绝望＂模型中有一定的抗抑郁作用。     

越鞠丸及各单味药醇提物对小鼠的抗抑郁作用研究

目的:探讨越鞠丸全方及各单味药(香附、川芎、栀子、苍术、神曲)醇提物的抗抑郁活性。方法:采用小鼠悬尾和强迫小鼠游泳实验2种行为绝望法复制小鼠抑郁模型,对越鞠丸全方及各单味药分别进行抗抑郁活性研究。结果:除神曲外,越鞠丸全方及各单味药醇提物均能不同程度地缩短小鼠悬尾不动时间和小鼠强迫游泳不动时间,具有抗抑郁样活性;越鞠丸全方醇提物、苍术和川芎可显著缩短小鼠悬尾不动时间和游泳不动时间。结论:越鞠丸全方及香附、苍术、川芎、栀子醇提物均有不同程度的抗抑郁活性,其抗抑郁活性部位/成分可能主要存在于苍术、川芎2味药材之中。     

绿萼梅提取物对小鼠的抗抑郁作用

目的：观察绿萼梅提取物对小鼠的抗抑郁作用。方法采用悬尾实验（ TFT）、强迫游泳（ FST）等体内药效评价方法观察绿萼梅醇提取物和水提取物对抑郁模型小鼠的治疗作用。结果绿萼梅醇提物能明显缩短小鼠悬尾和强迫游泳不动时间（ P＜0．05）,且对自主活动无影响（ P＞0．05）;而水提物对小鼠悬尾和强迫游泳不动时间无显著影响（ P＞0．05）。结论绿萼梅乙醇提取物具有抗小鼠抑郁作用。     

Sex differences in response to oral amitriptyline in three animal models of depression in C57BL/6J mice

Rationale Knockout and transgenic mice provide a tool for assessing the mechanisms of action of antidepressants. The effectiveness of oral administration of the tricyclic antidepressant amitriptyline (AMI) was assessed in C57BL/6J (B6) mice, a common genetic background on which knockout and transgenic mice are maintained.Objectives We determined whether oral AMI would have antidepressant-like effects in B6 mice and whether these effects varied according to sex, duration of treatment, and the depression model utilized.Methods Male and female B6 mice were administered AMI (200 g/ml) in the drinking water as the sole source of fluid, along with 2% saccharin to increase palatability. Control mice were administered 2% saccharin alone. Mice were assessed for responsiveness to AMI in the tail suspension test (TST), the forced swim test (FST), and the learned helplessness (LH) paradigm.Results In the TST, AMI decreased immobility time regardless of sex or duration of treatment. AMI also decreased immobility time in the FST, but chronic treatment was necessary for full efficacy in both sexes. In the LH paradigm, both subchronic and chronic AMI treatment decreased escape latencies in female mice, but AMI was effective only after chronic treatment in males. The antidepressant-like effects of AMI could not be explained by differences in locomotor activity because activity levels were not altered by antidepressant treatment.Conclusions Overall, oral AMI administration provides a valid model for behavioral assessment of antidepressant-like effects in knockout and transgenic mice maintained on a B6 background, but the effectiveness of oral AMI varies depending on sex, duration of treatment, and the depression model used.     

槟榔壳总酚类提取物抗抑郁作用研究

目的:探讨槟榔壳总酚类提取物对抑郁模型小鼠的抗抑郁作用。方法:采用小鼠悬尾、强迫游泳等抑郁模型,以小鼠行为绝望的不动时间作为指标,考察槟榔壳总酚类抗抑郁活性。结果:槟榔壳总酚类320,160 mg.kg-1剂量组均能显著减少小鼠悬尾和强迫游泳的不动时间。结论:槟榔壳总酚类可以改善小鼠的绝望行为,具有明显的抗抑郁作用。     

Activation of the mGlu7 receptor elicits antidepressant-like effects in mice

Rationale Broad evidence indicates that modulation of the glutamatergic system could be an efficient way to achieve antidepressant activity. Metabotropic glutamate receptor (mGlu receptor) ligands seem to be promising agents to treat several central nervous system disorders, including psychiatric ones. Objectives The aim of our study was to investigate potential antidepressant-like activity of the first, selective, and bio-available mGlu7 receptor agonist, AMN082 (N,N′-dibenzyhydryl-ethane-1,2-diamine dihydrochloride), in wild-type (WT) and mGlu7 receptor knock-out (KO) mice. Materials and methods The forced swim test (FST) and the tail suspension test (TST) in mice were used to assess antidepressant-like activity of AMN082. Results We found that AMN082, administered IP, induced a dose-dependent decrease in the immobility time of WT animals in the FST and TST, suggesting antidepressant-like potency of an mGlu7 receptor agonist. Moreover, AMN082 did not change the behaviour of mGlu7 receptor KO mice compared to WT littermates in the TST, while imipramine, used as a reference control, significantly reduced their immobility, indicating an mGlu7 receptor-dependent mechanism of the antidepressant-like activity of AMN082. However, at high doses, AMN082 significantly decreased spontaneous locomotor activity of both mGlu7 receptor KO mice and WT control animals, suggesting off-target activity of AMN082 resulting in hypo-locomotion. Conclusions These results strongly suggest that activation of the mGlu7 receptor elicits antidepressant-like effects.     

Folic acid administration produces an antidepressant-like effect in mice: evidence for the involvement of the serotonergic and noradrenergic systems

Clinical studies have shown that folic acid plays a role in the pathophysiology of depression. However, very few studies have investigated its effect in behavioral models of depression. Hence, this study tested its effect in the forced swimming test (FST) and the tail suspension test (TST), two models predictive of antidepressant activity, in mice. Folic acid administered by oral route (p.o.) produced a reduction in the immobility time in the FST (50-100mg/kg) and in the TST (10-50mg/kg). The administration of folic acid by i.c.v. route also reduced the immobility time in the FST (10nmol/site) and in the TST (1-10nmol/site). Both folic acid administered by oral and i.c.v. route produced no psychostimulant effect, which indicates that its antidepressant-like effect is specific. Pretreatment of mice with p-chlorophenylalanine methyl ester (PCPA; 100mg/kg, i.p., an inhibitor of serotonin (5-HT) synthesis, for 4 consecutive days), ketanserin (5mg/kg, i.p., a 5-HT(2A/2C) receptor antagonist), prazosin (1mg/kg, i.p., an alpha(1)-adrenoceptor antagonist) or yohimbine (1mg/kg, i.p., an alpha(2)-adrenoceptor antagonist) prevented the anti-immobility effect of folic acid (50mg/kg, p.o.) in the FST. Moreover, the pretreatment of mice with WAY100635 (0.1mg/kg, s.c., a selective 5-HT(1A) receptor antagonist) blocked the decrease in immobility time in the FST elicited by folic acid (50mg/kg, p.o.), but produced a synergistic effect with a subeffective dose of folic acid (10mg/kg, p.o.). In addition, a subeffective dose of folic acid (10mg/kg, p.o.) produced a synergistic antidepressant-like effect with fluoxetine (10mg/kg, p.o.) in the FST. Overall, the results firstly indicate that folic acid produced an antidepressant-like effect in FST and in TST and that this effect appears to be mediated by an interaction with the serotonergic (5-HT(1A) and 5-HT(2A/2C) receptors) and noradrenergic (alpha(1)- and alpha(2)-adrenoceptors) systems.     

醒神开郁方抗抑郁作用的实验研究

目的：研究醒神开郁方的抗抑郁作用及其可能的作用机制。方法：采用小鼠悬尾、强迫游泳实验观察小鼠悬尾、游泳不动时间;自主活动实验测定小鼠自主活动次数;利血平诱导小鼠体温下降实验考察小鼠肛温、眼睑下垂度以及脑内单胺类神经递质含量等指标。采用SPSS 13.0软件对上述观察指标进行统计分析。结果：醒神开郁方对小鼠自主活动行为无显著影响;在小鼠悬尾和强迫游泳实验中,醒神开郁方低、中、高剂量组可明显缩短小鼠悬尾和强迫游泳不动时间（P〈0.05,P〈0.01,P〈0.01）。利血平诱导小鼠体温下降实验中,与模型组比较,醒神开郁方中、高剂量组可显著拮抗利血平所致小鼠的体温下降和眼睑下垂（P〈0.05,P〈0.01）;与模型组比较,醒神开郁方中、高剂量组均能显著增加小鼠脑组织内NE、5-HT含量（P〈0.01）以及DA含量（P〈0.05,P〈0.01）。结论：醒神开郁方具有显著的抗抑郁作用,其作用机制与增强脑组织内NE、5-HT、DA调节神经系统作用有关,且醒神开郁方无中枢兴奋作用。     

HPLC-Q-TOF-MS技术在预知子配方颗粒指纹图谱建立和化学成分归属中的应用

目的 建立预知子配方颗粒的HPLC指纹图谱,并使用Q-TOF-MS技术鉴定共有峰成分。方法 采用Agilent Poroshell 120 EC-C₁₈色谱柱（4.6 mm×50 mm,2.7 mm）,以乙腈-0.05%甲酸水溶液为流动相进行梯度洗脱,检测波长210 nm,柱温30℃,流速0.5 mL·min^-1。采用相似度分析软件对指纹图谱进行分析。以标准物质和文献数据为参照,使用Q-TOF-MS负离子检测模式对特征峰进行质谱分析并指认。结果 建立了预知子配方颗粒的HPLC指纹图谱,以4,5-O-二咖啡酰基奎宁酸峰为参照峰,标定了14个共有峰,样品间相似度范围为0.943~0.994,样品与对照指纹图谱相似度范围为0.976~0.988。使用Q-TOF-MS对共有峰进行了指认,确定了8个峰成分,分别为新绿原酸、绿原酸、木通苯乙醇苷B、3,4-O-二咖啡酰基奎宁酸、3,5-O-二咖啡酰基奎宁酸、4,5-O-二咖啡酰基奎宁酸、川续断皂苷Ⅵ、α-常春藤皂苷。结论 该方法灵敏、准确、可靠,可用于预知子配方颗粒的质量评价。     

Antidepressant Effect of Baicalin Extracted from the Root of Scutellaria baicalensis. in Mice and Rats

Abstract

The flavonoid baicalin, isolated from the dried root of Scutellaria baicalensis. G. (Labiatae), is widely used in traditional Chinese herbal medicine. In the present study, baicalin, at doses of 20, 40, and 80 mg/kg (p.o.), reduced immobility time in tail suspension test (TST) and the forced swimming test (FST) in mice. Baicalin also decreased immobility time at 12.5, 25, and 50 mg/kg (p.o.) in FST in rats. Furthermore, baicalin (25 mg/kg), as well as fluoxetine (FLU; 20 mg/kg), showed a significant recovery in sucrose intake compared with the vehicle-treated stressed animals for 5 weeks treatment in a chronic mild stress (CMS) model in rats. The effect of baicalin at the dose of 25 mg was as potent as that of reference antidepressant FLU (20 mg/kg) in the CMS model. With the monoamine oxidase (MAO A and B) assay, oral administration of baicalin at the doses of 12.5, 25, and 50 mg/kg significantly inhibited MAO A activity in a dose-dependent manner in rats. However, only baicalin at the doses of 25 and 50 mg/kg markedly inhibited MAO B activity. Neither baicalin nor FLU, at the doses tested, produced a significant effect on locomotor activity in mice. These results suggest that baicalin had a specific antidepressant-like effect in vivo.. The antidepressant activity of baicalin may be mediated in part through MAO A and B inhibition in rat brain.     

Depletion of serotonin and catecholamines block the acute behavioral response to different classes of antidepressant drugs in the mouse tail suspension test

RATIONALE: Few studies have investigated whether the behavioral effects elicited by different types of antidepressant drugs are mediated by either serotonin (5-HT) or the catecholamines norepinephrine (NE) and dopamine (DA). OBJECTIVES: By depleting 5-HT, or NE and DA, the present study investigated the contributions of these monoamines to the acute behavioral effects of selective serotonin reuptake inhibitors (SSRIs; fluoxetine and citalopram) and norepinephrine reuptake inhibitors (NRIs; desipramine and reboxetine) in the mouse tail suspension test (TST). RESULTS: Depletion of 5-HT tissue content by para-chlorophenylalanine (PCPA), an inhibitor of tryptophan hydroxylase, completely blocked reductions of immobility by the SSRIs in the TST. In contrast, PCPA did not alter the behavioral effects of the NRIs. Inhibition of catecholamine synthesis by alpha-methyl-para-tyrosine (AMPT) reduced brain NE and DA tissue content, whereas disruption of vesicular storage with reserpine decreased brain NE, DA and 5-HT tissue content. However, neither treatment completely prevented responses to desipramine, fluoxetine, or citalopram in the TST. Depleting both newly synthesized and vesicular components of NE and DA transmission with a combination of reserpine and AMPT completely prevented the behavioral effects of desipramine, reboxetine, and fluoxetine and attenuated those of citalopram. Although PCPA did not alter baseline immobility, AMPT and reserpine increased baseline values in the TST. CONCLUSIONS: These studies demonstrated that endogenous 5-HT synthesis mediates the behavioral effects of SSRIs, but not NRIs, in the TST. In contrast, disruption of the behavioral effects of NRI and SSRI antidepressants required disruption of both catecholamine synthesis and vesicular storage and release mechanisms.     

Stressors can affect immobility time and response to imipramine in the rat forced swim test

We subjected Wistar rats to the forced swim test (FST) to compare the effects of two doses of imipramine in physically stressed rats (P: unavoidable electric footshocks), emotionally stressed rats (E: odors), or non-stressed rats (C). Stress or control sessions lasted 35 days. Drug treatments began on day 21 and continued for the next 14 days. E rats were placed for 10 min, once per day for 35 days, in a small non-movement-restricting cage impregnated with urine collected from a P rat. E and P rats exhibited opposite changes in locomotion. After 21 days of stress sessions, P rats displayed the longest immobility times in the FST, followed by E rats. In the P group, on day 7 of treatment (day 28 of the study), imipramine (2.5 mg/kg) reduced immobility time to baseline values. In the E group, immobility time decreased only after 14 days of treatment with the low imipramine dose. The high dose of imipramine (5.0 mg/kg) reduced immobility time at day 7 of treatment in all groups. In conclusion, physical and emotional stress similarly increased immobility time in the FST, but emotional stress appears to be more resistant to imipramine treatment.     

Toluene has antidepressant-like actions in two animal models used for the screening of antidepressant drugs

Rationale  Many abused solvents share a profile of effects with classical antidepressants. For example, toluene, which is a representative and widely abused solvent, has been reported to increase both serotonin and noradrenaline levels in several brain areas after an acute exposure and to act as a noncompetitive antagonist of the glutamatergic N-methyl-d-aspartic acid (NMDA) receptor subtype. Therefore, it is possible that toluene could possess antidepressant-like actions. Objective  To provide an initial screening of toluene’s antidepressant-like actions in the forced swimming test (FST) and the tail suspension test (TST) in mice and to analyze its possible mechanism of action. Materials and methods  Two series of experiments were performed. In the first one, male animals were exposed to toluene (0, 500, 1,000, 2,000, or 4,000 ppm) in a static exposure chamber for 30 min, and immediately after, evaluated for antidepressant-like effects. The results were compared with those obtained from mice treated with the serotonergic antidepressant clomipramine (CMI), the noradrenergic antidepressant desipramine (DMI), and the glutamatergic antidepressants, ketamine and MK-801. In the second part, we analyzed the effect of a combined administration of a subeffective concentration of toluene with a suboptimal dose of the various antidepressants acting at different neurotransmitter systems. Results  Toluene produced a concentration-dependent antidepressant-like action in the FST and TST and facilitated both MK-801 and ketamine antidepressant-like effects, but not those of DMI or CMI. Conclusions  Toluene has antidepressant-like effects that are synergized with NMDA receptor antagonists.     

Antidepressant-like actions of an AMPA receptor potentiator (LY392098)

LY392098 is a member of a novel class of biarylpropylsulfonamides that potentiates AMPA receptor-mediated responses both in vitro and in vivo. In this study, the effects of LY392098 were evaluated in two “behavioral despair” models (the forced swim and tail suspension tests) commonly used to identify clinically useful antidepressants. LY392098 reduced immobility in the forced swim test in both rats and mice, with a minimum effective dose of 0.5 mg/kg (i.p.) in both species. LY392098 (0.1–10 mg/kg, i.p.) did not affect motor activity of rats, indicating that the ability of this compound to reduce immobility in the forced swim test is unrelated to a motor stimulant action. LY392098 also reduced immobility in the tail suspension test in a dose-dependent manner, with a minimum effective dose of 5 mg/kg (i.p). A non-competitive AMPA antagonist (LY300168) blocked the activity of LY392098 in the forced swim test, but did not affect imipramine-induced reductions in immobility. Thus, AMPA receptor activation appears to be required for the antidepressant-like effect of LY392098, but not imipramine. These findings indicate that biarylpropylsulfonamides, exemplified by LY392098, may represent a novel class of antidepressants.     

Antidepressant-like effect of coadministration of sulpiride and fluvoxamine in mice

We have recently reported that coadministration of sulpiride, an antipsychotic drug, and fluvoxamine, a selective serotonin (5-HT) reuptake inhibitor, selectively increases in vivo dopamine release in the prefrontal cortex. This study examined the effects of coadministration of these drugs on duration of immobility in the tail suspension test using mice. Neither sulpiride (3 or 10 mg/kg) nor fluvoxamine (10 or 20 mg/kg) alone affected immobility time, whereas coadministration significantly reduced immobility time. WAY100635, a 5-HT_1A receptor antagonist, did not affect the effects of sulpiride and fluvoxamine coadministration, but reduced immobility time in combination with fluvoxamine (20 mg/kg). A high dose of fluvoxamine alone (60 mg/kg) also reduced immobility time. These results suggest that the antidepressant-like effects of fluvoxamine in combination with sulpiride or WAY100635 in the tail suspension test are mediated by the activation of dopamine or 5-HT systems, respectively.     

D-004, a lipid extract from royal palm fruit, exhibits antidepressant effects in the forced swim test and the tail suspension test in mice

D-004, a lipid extract of Roystonea regia fruits, has been shown to reduce Testosterone, but not dihydrotestosterone-induced prostate hyperplasia in rodents. Inhibition of prostate 5α-reductase seems to explain these effects of D-004. Finasteride, an inhibitor of 5α-reductase used to treat benign prostate hyperplasia (BPH), has been shown to produce drug-induced depression and to increase mouse immobility in the forced swim test (FST). In this study, therefore, we investigated the effect of D-004 on the immobility in the FST and the tail suspension test (TST) in mice. Also, its effects on other behavioural tests (grip strength, open field activity and rotarod test) were investigated. Mice were randomized into five groups: three groups orally treated with D-004 (250, 500 and 1000 mg/kg) or vehicle (control group), and a fifth group that received intraperitoneally (IP) imipramine 20 mg/kg for 30 days. In the FST, D-004 (250, 500 and 1000 mg/kg) produced a statistically significant reduction in immobility (51, 58, and 65%, respectively, versus the control group), whereas imipramine reduced FST immobility by 69%. In the TST, D-004 (250 and 500 mg/kg) significantly, but modestly (21%) reduced the immobility versus the control group, although less than imipramine (50%). The lowest dose of D-004 (50 mg/kg), however, was ineffective. D-004 did not alter the results of other behavioural tests. In conclusion, D-004 (250-1000 mg/kg) administered orally for 30 days reduced the immobility in the FST and the TST in mice, and had no effect on other behavioural tests in mice.