Life Scicq Vol 61,No 4, pp 3%402,19!37 CC+ght*1997EktViCISdarelae PliotcdintheusA Allrightclescmd w24-mm 517.00t a0 ELSEVIER zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA PI1 soo24-3205(97)00396-2 MAJONOSIDE432 REVERSES SOCIAL ISOLATION STRESS-INDUCED DECREASE IN PENTOBARBITAL SLEEP IN MICE: POSSIBLE INVOLVEMENT OF NEUROACTIVE STEROIDS Nguyen Thi Thu Huong ‘, Kinzo Matsumoto ‘, Kazuo Yamasaki2 and Hiroshi Watanabe’ 1) Department of Pharmacology, Research Institute for Wakan-Yaku (Oriental Medicines), Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan 2) Department of Biological Active Substances, Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, l-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan (Received in final form April‘24, 1997) Majonoside-R2 (MR2) is a major ocotillol-type saponin constituent of Vietnamese ginseng We investigated the effect of MR2 on the social isolation stress-induced decrease in pentobarbital sleep in mice, and elucidated the possible involvement of neurosteroidal sites of the GABA,, receptor complex in the pharmacological activity of MR2 MR2 (3.1-6.2 mg/kg, i.p or 5-10 ug, i.c.v.) dose-dependently reversed the decrease in pentobarbital sleep caused by social isolation stress to the level of sleep in the group-housed mice, but it had no effect on pentobarbital sleep in group-housed mice Allotetrahydrodeoxycorticosterone (5a-pregnane3a,21diol-20-one, allo-THDOC; 12.5 pg, i.c.v.), the positive allosteric modulator of the GABA, receptor, and a-helical CRFgdl (ahCRF; 25 pg, i.c.v.), the corticotropinreleasing factor (CRF) antagonist, also reversed the decrease in pentobarbital sleep caused by social isolation stress The reversing effects of i.c.v MIX2 and i.c.v allo-TI-IDOC on the decrease in pentobarbital sleep in isolated mice were significantly attenuated by pregnenolone sulfate (10 ug, i.c.v.), the steroidal negative allosteric modulator of the GABA,, receptor In contrast, when injected i.c.v., MR2, as well as allo-THDOC and ahCRF, significantly reversed the decrease in pentobarbital sleep induced by pregnenolone sulfate (10 pg, i.c.v.) and CRF (10 pg, i.c.v.) in group-housed mice These results suggest that the reversing effect of MR2 on the social isolation stress-induced decrease in pentobarbital sleep is mediated by the neurosteroid site on the GABA,+ receptor complex in mice Corresponding Author: Hiroshi Watanabe, Ph.D., Department of Pharmacology, Research Institute for Wakan-Yaku (Oriental Medicines), Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan, FAX: +81-764-34-5056 3% Majonoside-R2 and Neurosteroids Vol 61, No 4, 1997 Majonoside-R2 (MR2, Fig 1) is a major ocotillol-type saponin constituent of Vietnamese ginseng (Punax vietnamensiv Ha et Grushv Araliaceae) that accounts for over 50 % of the total saponins This compound has not been isolated from zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ Panux ginseng, American ginseng or Sanchi ginseng (1) In a previous study, we demonstrated that a systemic administration of MR2 reversed the psychological stress-induced decrease in pentobarbital sleep to the level of sleep shown by unstressed control mice, and that this effect of MR2 was abolished by flumazenil, a selective benzodiazepine receptor antagonist (2) MR2 also attenuated p- or K-opioid receptor agonistinduced antinociception through the supraspinal GABA,-ergic systems (3, 4) The exact mechanism underlying the pharmacological activity of MR2 remains to be clarified, but the neurosteroid-like structure of MR2 suggests that it may have activity like that of neuroactive steroids, with an ability to positively modulate GABA, receptor function Various stressful manipulations are known to increase the levels of neuroactive steroids in the brain (5) Neuroactive steroids have been suggested to directly regulate the function of the GABA, receptor complex in a bimodal fashion (6,7), and the levels of these steroids in the brain change following acute stress exposure (8, 9) Pregnenolone sulfate, at concentrations achievable during stress exposure, reportedly decreases the density of the GABA, receptor, and this decrease contributes to the heightened anxiety and arousal induced by stress (10) The levels of allopregnanolone (3a-hydroxy-5 a-pregnane-20-one), allo-THDOC and tetrahydrodeoxycorticosterone (5p-pregnane-3a,21-diol-20-one, THDOC) that are capable of producing GABA,agonistic and anxiolytic activities and enhancing GABA-induced 36C1-uptake into brain tissue, are also changed by acute stress exposure (11-13) Ojima et al (14) demonstrated that social isolation stress decreases the duration of pentobarbital-induced sleep in mice and that this decrease appears to depend on the duration of the social isolation period Moreover, it has been suggested that the social isolation stress-induced decrease in pentobarbital sleep is mediated by not only the hyperactivity of central noradrenergic and corticotropin-releasing factor systems, but also by changes in the level of neuroactive steroids with ago&tic or antagonistic activity at the GABA, receptor complex (14, 15) Based on these findings, we attempted in the present study to clarify whether neuroactive steroids were involved in the effect of MR2 on the social isolation stress-induced decrease in pentobarbital sleep in mice Methods Animals Male ddY mice (Japan SLC, Shizuoka, Japan) were obtained at the age of weeks They were housed in groups of 8-10 per cage (21 x 32 x 13 cm) or socially isolated by being housed individually for 5-7 weeks before the start of the experiments Food and water were given ad libitum Housing conditions were thermostatically maintained at 24 r “C and a relative humidity of 55 + 5% with a 12 h 1ight:dark cycle (lights on: 08:00-20:OO) The present studies were conducted in accordance with the standards established by the Guide for the Care and Use of Laboratory Animals of Toyama Medical and Pharmaceutical University Measurement of pentobarbital-induced sleep Pentobarbital-induced sleep in group-housed and isolated mice was measured as previously reported (2, 14) Pentobarbital sodium (50 mg/kg; Tokyo Kasei Co., Tokyo, Japan) was injected intraperitoneally, and the sleeping time zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJ was taken as the period between the loss of the righting reflex and its return Vol 61, No 4, 1997 Major&de-W and Neurosteroids Majonoside-R2 (MR2) was purified from the saponin fraction of Vietnamese ginseng (yield: 5.29% of dry material, purity: over 85%) as previously described (1, 4) The following drugs were obtained from commercial sources: 5a-pregnane-3a,21-diol-20-one (allotetrahydrodeoxycorticosterone, allo-THDOC), 5-pregnen-3fl-ol-20-one sulfate (pregnenoione sulfate), CRF and ahelical CRF,,, (ahCRF) (Sigma Chem., Co., St Louis, MO) For intraperitoneal injection (i.p.), drugs were dissolved in saline and administered in a constant volume of 0.1 ml/10 g body weight For intracerebroventricular (i.c.v.) injection, drugs except pregnenolone sulfate and allo-THDOC were dissolved in artificial cerebrospinal fluid (aCSF) and administered in a constant volume of pi/mouse according to the method of Haley and McCormick (16) Pregnenolone sulfate and alloTHDOC were suspended in aCSF containing 40% propylene glycol All drugs were administered 30 before the start of the experiment When testing antagonism, test drugs were coadministered i.c.v in a total volume of pi/mouse The doses of test drugs and the time schedule for drug administration were chosen based on the results reported in the previous studies (2, 4, 14, 15) Statistical analysis The data were analyzed with two-way or three-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparison among groups Differences with p < 0.05 were considered significant q Z HO o-Gh?- Xyl Fig Chemical structure of majonoside-R2 Effect of majonoside-R2 on the social isolation-induced decrease in pentobarbital sleep As shown in Fig 2, pentobarbital-induced sleep in socially isolated mice was significantly shorter than that in group-housed mice An intraperitoneal (3.1-6.2 mg/kg) or intracerebroventricular (5-10 ug) injection of MR2 exhibited no effect on pentobarbital sleep in group-housed mice, but it dose-dependently reversed the duration of pentobarbital sleep, which had been shortened by social isolation stress, to the level of group-housed mice (Fhousingconditionx MR2 (2,45)=3.293, p