Neurosteroid Allopregnanolone Mediates Anxiolytic Effect of Etifoxine in Rats

BRAIN RESEARCH 1184 (2007) 193– 201

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Neurosteroid allopregnanolone mediates anxiolytic effect of etifoxine in rats

Rajesh R. Ugaleb,c, Ajaykumar N. Sharmaa,b, Dadasaheb M. Kokarea, Khemraj Hirania,d, Nishikant K. Subhedara, Chandrabhan T. Chopdea,b,⁎ aDepartment of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur-440 033, Maharashtra, India bPharmacology Division, Shrimati Kishoritai Bhoyar College of Pharmacy, New Kamptee-441 002, Nagpur, Maharashtra, India cDepartment of Psychiatry, University of Illinois at Chicago, Chicago, Illinois 60612, USA dDepartment of Anatomy, College of Medicine, University of South Florida, Health Science Centre, Tampa, FL 33612-4799, USA

ARTICLE INFO ABSTRACT

Article history: Etifoxine (6-chloro-2-ethylamino-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride), a

Accepted 17 September 2007 nonbenzodiazepine anxiolytic drug, potentiates GABAA receptor function perhaps through Available online 25 September 2007 stimulation of neurosteroid biosynthesis. However, the exact mechanism of etifoxine action is not fully understood. In this study, we have assessed the possible role of GABAergic Keywords: neurosteroid like allopregnanolone (ALLO) in the anxiolytic-like effect of etifoxine in rats

Etifoxine using elevated plus maze test. Selective GABAA receptor agonist, muscimol, ALLO or Neurosteroids neurosteroidogenic agents like progesterone, metyrapone or mitochondrial diazepam Allopregnanolone binding inhibitor receptor (MDR) agonist, FGIN 1–27 significantly heightened the etifoxine-

Anxiety induced anxiolysis. On the other hand, GABAA receptor antagonist, bicuculline or neurosteroid biosynthesis inhibitors like finasteride, indomethacin, trilostane or PBR antagonist, PK11195 significantly blocked the effect of etifoxine. Bilateral adrenalectomy did not influence anti-anxiety effect of etifoxine thereby ruling out contribution of adrenal steroids. Thus, our results provide behavioral evidence for the role of neurosteroids like ALLO in the anti-anxiety effect of etifoxine. © 2007 Elsevier B.V. All rights reserved.

1. Introduction considered to be associated with dysfunction of central γ-aminobutyric acid (GABA)ergic system (Almeida et al., 2006). Etifoxine (6-chloro-2-ethylamino-4-methyl-4-phenyl-4H-3,1- Major anxiolytic drugs including benzodiazepines, barbiturates benzoxazine hydrochloride) is a nonbenzodiazepine anxiolytic (Drafts and Fisher, 2006; Campo-Soria et al., 2006)and in rodents (Boissier et al., 1972; Schlichter et al., 2000; Micallef neurosteroids (Bitran et al., 1991, 1993; Finn et al., 1997; Akwa et al., 2001) and in humans is effective for treating adjustment et al., 1999; Palmer et al., 2002) potently and selectively disorders with anxiety (Nguyen et al., 2006). At anxiolytic facilitate the action of GABA at GABAA receptors. Interestingly, concentration, unlike classical benzodiazepines, the com- etifoxine also increases GABAergic transmission (Schlichter β β pound etifoxine has fewer side effects (Micallef et al., 2001; et al., 2000) by acting on GABAA receptors 2 or 3 subunit Nguyen et al., 2006). Anxiety and related disorders are (Hamon et al., 2003), an allosteric site distinct from that of

⁎ Corresponding author. Pharmacology Division, Shrimati Kishoritai Bhoyar College of Pharmacy, New Kamptee-441 002, Nagpur (MS), India. E-mail address: [email protected] (C.T. Chopde).

0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.09.041 194 BRAIN RESEARCH 1184 (2007) 193– 201 benzodiazepines and neurosteroids (Verleye et al., 1999, 2001, 2002; Schlichter et al., 2000). In addition, etifoxine stimulates peripheral (mitochondrial)-type benzodiazepine receptors that control synthesis of neurosteroids like allopregnanolone (3α, 5α-Tetrahydroprogesterone, ALLO), an allosteric positive mod- ulator of GABAA receptors (Harrison and Simmonds, 1984; Verleye et al., 2005). Neurosteroids are synthesized de novo in the brain inde- pendent of peripheral steroidogenic endocrine glands (Corpe- chot et al., 1981). The best-characterized neurosteroids are ALLO, dehydroepiandrosterone (DHEA), pregnanolone and their sulfates etc. They interact with ionotropic receptors and ligand-gated ion channels especially GABAA receptors at nanomolar to micromolar concentration and rapidly alter the neuronal excitability (Paul and Purdy, 1992; Lambert et al., 1995; Follesa et al., 2004; Birzniece et al., 2006). ALLO is the most potent endogenous neurosteroid and is implicated in pre- menstrual dysphoric disorder (Klatzkin et al., 2006; N-Wihl- back et al., 2006), generalized anxiety disorder (Semeniuk et al., 2001; Hirani et al., 2005; Sharma et al., 2007), post-traumatic stress disorder (Rasmusson et al., 2006), estrous cycle (Petralia et al., 2005), pregnancy (Paoletti et al., 2006), aggression (Pinna et al., 2005) and seizures (Ugale et al., 2004a). Both ALLO and etifoxine share many features namely anti-anxiety (Boissier et al., 1972; Bitran et al., 1991, 1993; Schlichter et al., 2000; Micallef et al., 2001; Verleye et al., 2005; Nguyen et al., 2006), anti-seizure properties (Verleye et al., 2001), potentiation of GABA stimulated Cl- currents (Harrison et al., 1987; Lambert et al., 1995; Schlichter et al., 2000) or inhibition of [35S]t-butyl- biclophosphorothionate ([35S]TBPS) binding (Lambert et al., 2001). The concept that etifoxine acts by stimulating neurosteroid synthesis is based on the observation that, PK11195, a compound that inhibits neurosteroid biosynthesis, partly suppresses the effect of etifoxine on GABAergic transmission or GABAA receptors (Schlichter et al., 2000; Verleye et al., 2005). Based on earlier finding that etifoxine increases brain neurosteroidal level including ALLO (Verleye et al., 2005), we explored the role of ALLO in the anxiolytic effect of etifoxine by Fig. 1 – Dose-dependent effects of etifoxine and ALLO on pharmacological manipulation of neurosteroids using elevated (A) % open arms entries, (B) % time spent in open arms and plus maze (EPM) test in Sprague–Dawley rats. We have recently (C) number of closed arms entries in EPM test in rats. Rats were demonstrated that several centrally active drugs produce their injected etifoxine (25–75 mg/kg, i.p.) or vehicle (5 ml/kg, i.p.) effects by increasing neurosteroid ALLO content in brain (Hirani 30 min prior to EPM test or ALLO (0.1–1 μg/rat, i.c.v.) or aCSF et al., 2002, 2005; Ugale et al., 2004a,b; Sharma et al., 2007). Hence (5 μl/rat, i.c.v.) 15 min prior to EPM test. Each bar represents in the present study, we investigated the anxiolytic-like effect mean±S.E.M. of data from 7 to 9 rats per group. *P<0.05 vs. of etifoxine by altering the endogenous neurosteroids using vehicle control (one-way ANOVA followed by Dunnett's test). neurosteroidogenic agents or its biosynthesis blockers.

time spent [F(3,29)=85.45; P<0.0001] in the open arms by 2. Results 55.84% and 90.03% respectively. The effect of lower dose of etifoxine (25 mg/kg, i.p.) or ALLO (0.1 μg/rat, i.c.v.) on open arm 2.1. Anxiolytic effects of etifoxine and ALLO parameters was not significantly different from vehicle control groups (PN0.05). None of the doses of etifoxine and ALLO As shown in Fig. 1, etifoxine (50 and 75 mg/kg, i.p.) or ALLO (0.5 altered the entries into the closed arms as compared to vehicle and 1 μg/rat, i.c.v.) significantly increased the preference for controls (PN0.05). open arms in the plus maze test. Etifoxine at doses 50 and 75 mg/kg increased entries [F(3,29)=42.38; P<0.0001] by 38.65% 2.2. ALLO or neurosteroidogenic agents potentiated the and 54.78% and time spent [F(3,29)=76.01; P<0.0001] by 47.84% anxiolytic effect of etifoxine and 80.01% respectively into the open arms. Similarly, ALLO (0.5 and 1 μg/rat, i.c.v.) significantly increased entries [F(3,29)= Administration of subeffective dose of etifoxine (25 mg/kg, i.p.) 68.86; P<0.0001] into the open arms by 47.64% and 81.56% and to animals pretreated with subeffective dose of ALLO (0.1 μg/ BRAIN RESEARCH 1184 (2007) 193– 201 195 rat, i.c.v.) showed significantly greater effect on entries [F(3,31)= 29.335; P<0.0001] and time spent in open arms [F(3,31)=41.221; P<0.0001] (Fig. 2). The open arms entries were increased by 38.27% and the time spent in the open arms was increased by 78.18%. These treatments did not change the closed arms entries (PN0.05) (Fig. 2). However, the etifoxine (25 mg/kg, i.p.) and ALLO (0.1 μg/rat, i.c.v.) per se did not alter the open arm activity (PN0.05). Similarly, metyrapone (50 mg/rat, i.p.), progesterone (5 mg/ kg, i.p.) or FGIN 1–27 (0.5 μg/rat, i.c.v.) at the doses used here per se did not exert any significant effect on open arm parameters

Fig. 3 – Effect of etifoxine (75 mg/kg, i.p.) in the presence of neurosteroid biosynthesis inhibitors in the EPM test on (A) % open arms entries, (B) % time spent in open arms and (C) number of closed arms entries. Rats were injected with finasteride (50×2 mg/kg, s.c. at −4 and −1.5 h) or indomethacin (5 mg/kg, i.p. at −20 min) or trilostane (30 mg/kg, i.p. at −2 h) or PK11195 (15 mg/kg, i.p. at −0.5 h) before etifoxine (75 mg/kg, i.p.) or vehicle (5 ml/kg, i.p.) treatment. Thirty minutes thereafter, rats were subjected to EPM test. Each bar represents mean ±S.E.M. of data from 7 rats per group. *P < 0.05 vs. vehicle; #P <0.05 vs. etifoxine (one-way ANOVA followed by Student–Newman–Keuls test). Fig. 2 – Influence of ALLO and neurosteroidogenic drugs on effect of etifoxine (25 mg/kg, i.p.) in EPM test on (A) % open arms entries, (B) % time spent in open arms and (C) number of as compared to vehicle control groups (Fig. 2). However, at the closed arms entries. Rats were injected with metyrapone given doses, all three drugs significantly potentiated the (50 mg/kg, i.p.) or progesterone (5 mg/kg, i.p.) 30 before action of a subeffective dose of etifoxine (25 mg/kg, i.p.) on etifoxine (25 mg/kg, i.p) or vehicle (5 ml/kg, i.p.) or ALLO the preference for open arms activities in the plus maze test. (0.1 μg/rat, i.c.v.) or FGIN 1–27 (0.5 μg/rat, i.c.v.) 15 min before Progesterone, metyrapone or FGIN 1–27 pretreatment in- etifoxine (25 mg/kg, i.p) or aCSF (5 μl/rat, i.c.v.) treatment. creased open arms entries by 31.27% [F(3,31)=22.193; Thirty minutes thereafter rats were subjected to EPM test. P<0.0001], 42.38% [F(3,31)=28.024; P<0.0001] or 41.15% [F(3,31)= Each bar represents mean±S.E.M. of data from 8 rats per 22.636; P<0.0001] and time spent in the open arms by 80.90% group. *P<0.05 vs. etifoxine (25 mg/kg, i.p.) (one-way ANOVA [F(3,31)=32.724; P<0.0001], 87.27% [F(3,31)=63.574; P<0.0001] followed by Student–Newman–Keuls test). or 84.54% [F(3,31)=33.956; P<0.0001] respectively. The closed 196 BRAIN RESEARCH 1184 (2007) 193– 201 arms entries were not affected by any of these treatments [F(9,79)=0.8993; P=0.5308].

2.3. Neurosteroid biosynthesis inhibitors attenuated anxiolytic effect of etifoxine

As shown in Fig. 3, neurosteroid biosynthesis inhibitors like finasteride (50×2 mg/kg, s.c.), trilostane (30 mg/kg, i.p.), indomethacin (5 mg/kg, i.p.) or PK11195 (15 mg/kg, i.p.) at the doses used here per se did not change the entries into and the time spent in the open arms. However, these drugs injected prior to an anxiolytic dose of etifoxine (75 mg/kg, i.p.) significantly reduced the preference for open arms parameters and were not different from vehicle treated control groups. Finasteride, trilostane, indomethacin or PK11195 reduced open arms entries by 25.34% [F(3,27)=15.877; P <0.0001],

Fig. 5 – Effect of adrenalectomy on etifoxine (75 mg/kg, i.p.)-induced anxiolysis on (A) % open arms entries, (B) % time spent in open arms and (C) number of closed arms entries. Rats were injected etifoxine (75 mg/kg, i.p.) or vehicle (5 ml/kg, i.p.) in sham-ADX or ADX rats. Rats were subjected to EPM test after 30 min of etifoxine injection. Each bar represents mean±S.E.M. of data from 7 rats per group (unpaired t test).

Fig. 4 – Influence of GABAergic drugs on effect of etifoxine (25 21.24% [F(3,27)=13.345; P <0.0001], 22.27% [F(3,27)=11.685; or 75 mg/kg, i.p.) in EPM test on (A) % open arms entries, (B) % P <0.0001] or 29.46% [F(3,27)=15.877; P <0.0001] and time time spent in open arms and (C) number of closed arms entries. spent in the open arms by 35.61% [F(3,27)=31.055; P<0.0001], Rats were injected with muscimol (0.5 mg/kg, i.p.) 30 min 25.57% [F(3,27)=28.390; P <0.0001], 28.77% [F(3,27)=31.197; before etifoxine (25 mg/kg, i.p.) or vehicle (5 ml/kg, i.p.). P<0.0001] or 23.27% [F(3,27)=43.264; P<0.0001] respectively Separate groups were injected with bicuculline (0.5 mg/kg, i.p.) compared to that of etifoxine (75 mg/kg, i.p.). There was no 30 min before etifoxine (75 mg/kg, i.p.) or vehicle (5 ml/kg, i.p.). marked effect of any of these treatments on number of closed Rats were subjected to EPM test after 30 min of etifoxine arms entries [F(9,69)=0.5614; P=0.8230]. administration. Each bar represents mean±S.E.M. of data from 7 rats per group. *P<0.05 vs. vehicle control (5 ml/kg, i.p.) 2.4. Effect of GABAergic drugs on anxiolytic effect of etifoxine or etifoxine (25 mg/kg, i.p.) or muscimol (0.5 mg/kg, i.p.) or bicuculline (0.5 mg/kg, i.p.); #P<0.05 vs. etifoxine (75 mg/kg, i.p.) Muscimol (0.5 mg/kg, i.p.) or bicuculline (0.5 mg/kg, i.p.) at the (one-way ANOVA followed by Student–Newman–Keuls test). doses used here per se did not alter any of EPM parameters BRAIN RESEARCH 1184 (2007) 193– 201 197 including entries and time spent into open arms as compared to drial membrane and transform into pregnanolone and thereby vehicle controls. However, the effect of etifoxine was much facilitate neurosteroidogenesis (Romeo et al., 1993) which greater in muscimol pretreated rats or attenuated in rats that subsequently modulates GABAA receptor function (Romeo received bicuculline injection. Etifoxine (25 mg/kg, i.p.) in the et al., 1992). Moreover, indirectly acting peripheral benzodiaz- presence of muscimol increased open arms entries by 45.28% epine receptor (PBR) antagonist PK11195 (Schlichter et al., 2000) [F(3,27)=11.620; P<0.0001] and time spent into the open arms by administered prior to etifoxine significantly attenuated its 85.85% [F(3,27)=25.241; P<0.0001] as compared to only etifoxine anxiolytic effect. In fact, etifoxine enhances the inhibitory treated rats. On the other hand, in bicuculline pretreated rats, GABAergic system involved in the anxiety regulation (Schlich- the effect of etifoxine (75 mg/kg, i.p.) on open arms entries was ter et al., 2000; Verleye et al., 2001, 2002; Hamon et al., 2003). It β β declined by 25.79% [F(3,27)=18.138; P< 0.0001] and time spent acts on GABAA receptors at 2 or 3 subunit, a site distinct from into open arms was reduced by 24.78% [F(3,27)=67.129; benzodiazepines (Sigel and Buhr, 1997; Korpi et al., 2002)or P<0.0001] as compared to etifoxine alone (Fig. 4). neurosteroids (Sigel and Buhr, 1997; Hamon et al., 2003). Thus the increased anxiolytic effect of etifoxine in presence of ALLO 2.5. Anxiolytic effect of etifoxine in adrenalectomized rats or other neurosteroidogenic drugs could be due to enhanced

GABAA receptor function by their direct allosteric effects Bilateral adrenalectomy of rats neither changed the entries (Verleye et al., 2001). Alternatively, etifoxine is also reported (unpaired t test, t=0.1905, df=12, P=0.8521) and time spent in to increase GABAergic neurotransmission by stimulating the open arms (unpaired t test, t=1.07, df=12, P=0.3041) nor mitochondrial-type benzodiazepine receptors leading to aug- did it modify closed arms entries (P N0.05) in EPM test. mented neurosteroid synthesis (Schlichter et al., 2000; Verleye Anxiolytic dose of etifoxine (75 mg/kg, i.p.) significantly et al., 2005). Thus, there appeared to be a correlation between increased the open arm activities in ADX as well as sham- etifoxine treatment and neurosteroid in the brain. ADX rats. However, no significant difference in open arms To strengthen the above hypothesis, we administered behavior (% open arms entries, t=0.1231, df=12, P=0.9041; % several neurosteroidogenic enzyme inhibitors prior to etifox- time spent in open arms, t=0.2129, df=12, P=0.8350) and ine. For example, trilostane inhibits 3β-hydroxysteroid dehy- closed arms entries (PN0.05) was observed following etifoxine drogenase (3β-HSD) to prevent the conversion of pregnanolone treatment in sham-ADX and ADX groups (Fig. 5). to progesterone in the brain (Potts et al., 1978; Korneyev et al., 1993), finasteride, a 5α-reductase inhibitor, that prevents the conversion of progesterone to 5α-dihydroprogesterone (5α- 3. Discussion DHP) (Blurton-Jones et al., 1999; Concas et al., 1998; Kokate et al., 1999; VanDoren et al., 2000) and indomethacin, that In agreement with previous reports (Boissier et al., 1972; inhibits enzyme 3α-hydroxysteroid oxidoreductase (3α-HSOR), Verleye and Gillardin, 2004; Verleye et al., 2005), etifoxine thereby preventing conversion of 5α-DHP to ALLO (Beyer et al., produced a behavioral profile consistent with anxiety reduc- 1999). Interestingly, the anxiolytic effect of etifoxine was tion. It dose dependently increased the time spent and number significantly inhibited by these agents. Moreover, PK11195, of entries into the open arms of EPM. In view of the an antagonist of PBRs that restrict the transport of cholesterol demonstrable anxiolytic property of etifoxine and neuroster- to inner membrane of mitochondria (Schlichter et al., 2000), oid ALLO (Bitran et al., 1993; Verleye and Gillardin, 2004; also significantly blocked the anxiolytic effect of etifoxine. Verleye et al., 2005) and since the neurosteroid biosynthesis Thus, in agreement with the previous report (Verleye et al., inhibitor molecule PK11195 is known to attenuate the etifoxine 2005), our results suggest that ALLO plays a significant role in effect (Schlichter et al., 2000), we focused on determining the the anxiolytic-like effect of etifoxine. However, etifoxine and potential role of ALLO in anxiolytic-like property of etifoxine. ALLO bind to distinct recognition sites (Verleye et al., 2001), ALLO in combination with etifoxine heightened anxiolytic their enhancing action may be an additive effect. Furthermore, effect than its individual effect. Similarly, neurosteroidogenic the involvement of other neurosteroids in the etifoxine- molecules like progesterone, metyrapone and FGIN 1–27 induced anxiolysis cannot be ignored. augmented the anxiolytic effect of etifoxine. Metyrapone has In addition, GABAA receptor agonist, muscimol potentiated, been demonstrated to amplify the levels of neurosteroids like its antagonist bicuculline inhibited the anxiolytic action of pregnenolone, progesterone, 11-deoxycortisol, 5α-DHDOC (5α- etifoxine. This finding supports the earlier studies which α α α α pregnan-21-ol-3, 20-dione), 3 ,5 -THDOC (5 -pregnan-3 ,21- suggest that etifoxine potentiates GABAA receptor function diol-20-one), 5α-DHP (5α-pregnan-3,20-dione), 3α,5α-THP and facilitates GABAergic transmission (Verleye et al., 1999, (ALLO) and DHEA (Jain et al., 1994; Rupprecht et al., 1998). 2001, 2002; Schlichter et al., 2000). We may recall that the These subsequently might contribute to anxiolytic-like effect anxiolytic effect of etifoxine was attenuated by antagonist of of etifoxine. FGIN 1–27, an agonist at DBI (diazepam binding PBRs and neurosteroid biosynthesis inhibitors, indicating the inhibitor) receptor, stimulates neurosteroidogenesis indepen- indirect action of etifoxine on GABAA receptors via production of dent of peripheral endocrine sources (Korneyev et al., 1993). It neurosteroids (Schlichter et al., 2000; Verleye et al., 2001, 2005). increases the levels of pregnanolone (Korneyev et al., 1993; Furthermore, to assess the absolute contribution of central Romeo et al., 1993), the progenitor hormone for the biosynthe- neurosteroids and not the adrenal steroids in the etifoxine sis of range of endogenous neurosteroids. Furthermore, FGIN induced anxiolysis, the experiments were also carried out in – 1 27 does not have any direct effect on GABAA receptor or any bilateral adrenalectomized rats. Surprisingly, the anxiolytic-like other receptor systems (Romeo et al., 1992). FGIN 1–27 is known effect of etifoxine was not altered by adrenalectomy, thus ruling to enhance cholesterol influx from outer to inner mitochon- out the involvement of steroids from peripheral source, at least 198 BRAIN RESEARCH 1184 (2007) 193– 201 adrenal steroids. However, this must be interpreted with aseptically into the right lateral ventricle (coordinates from caution since gonads were not ablated. Thus, it is possible that Paxinos and Watson, 1998; posterior −0.8 mm; lateral from increased production of neurosteroids particularly ALLO by midline +1.2 mm and ventral −3.5 mm; relative to bregma). The etifoxine may contribute to its anxiolytic effect. guide cannula was secured in place by dental cement (Dental Products of India, Mumbai) affixed to two mounting screws (Plastics One). A stainless-steel dummy cannula (28 gauge, 4. Experimental procedures C316DC/Spc, o.d., 0.25 mm) (Plastics One) was used to occlude the guide cannula when not in use. Animals were then housed 4.1. Subjects singly and allowed for a week to recover from surgery before any experimentation. During this 7-day period, animals were Adult Sprague–Dawley male rats (220–260 g body weight and also habituated to testing environment by transferring to 90–110 days old) were housed five per cage (cage size experimental room and twice-daily handling. Injections were 640×410×250 mm, acrylic) before surgical procedures, there- made using microliter syringe (Hamilton, Nevada, USA) after they were housed individually. Animals were main- connected to stainless-steel internal cannula (31 gauge, C316I/ tained at 24±1 °C under 12:12 h light/dark cycle (lights on Spc, i.d., 0.12 mm; o.d., 0.25 mm) (Plastics One) by polyethylene 07:00–19:00 h) with rodent chow (Lipton, India) and water ad tubing (PE-10, i.d., 0.28 mm; o.d., 0.61 mm). A volume of 5 μlwas libitum. Rats were housed individually and brought to the administered over a period of 1 min into the right lateral experimental room 12 h prior treatments to minimize non- ventricle. The injection cannula was left in place for further specific stress-induced steroid fluctuations. The experimental 1 min before being slowly withdrawn to avoid back flow. On procedures were in strict accordance with the guidelines completion of the experiments, 5 μl dilute India ink was approved by the Institutional Experimental Animal Ethical injected through the cannula and animals were immediately Committee of Rashtrasant Tukadoji Maharaj Nagpur Univer- euthanized by overdose of thiopentone sodium. Post-mortem sity, Department of Pharmaceutical Sciences, Nagpur, India. examination of the brain sections was undertaken to confirm Animals were naive to drug treatment and experimentation at the correct placement of cannula and the data of only those the beginning of each study. Each experimental group had a animals with uniform distribution of ink into the ventricles separate set of seven to nine animals. were used for statistical analysis. The animals that showed incorrect placement of guide cannula or neurological deficits 4.2. Drugs and administration (<15%) were excluded from the study.

ALLO, progesterone (ALLO precursor), muscimol (GABAA 4.4. Anxiety test receptor agonist), bicuculline (GABAA receptor antagonist), FGIN 1–27 (mitochondrial diazepam binding inhibitor receptor Rats were tested on an elevated plus maze that consisted of (MDR) agonist), PK11195 (PBR antagonist) and metyrapone two Plexiglas (painted black) opposite facing open arms (11β-hydroxylase inhibitor) were purchased from RBI, USA. (50×10 cm, L×W) and two enclosed arms (50×10×40 cm, Etifoxine (Biocodex Laboratories, France), finasteride (Dr. Red- L×W×H) connected by a central platform (10×10 cm, L×W). dy's Laboratories, India), trilostane (Sanofi Winthrop Develop- The whole maze was raised 60 cm above the floor and ment Center, UK) and indomethacin (Zim Laboratories, India) illuminated by 100-W lamp fixed 2 m above the maze floor. were received as gift samples. Etifoxine, ALLO, progesterone, On the day of experiment animals were shifted to behavioral metyrapone, FGIN 1–27, muscimol, bicuculline, finasteride, room 1 h prior to testing to facilitate adaptation. Rats were trilostane, indomethacin and PK11195 were dissolved in 2- placed singly in the center square of the maze, head facing one hydroxypropyl-β-cyclodextrin (45% w/v) solution and diluted of the open arms and allowed to explore for 5 min. The number with 0.9% saline or for intracerebroventricular (i.c.v.) admin- of entries into and time spent in each arm were recorded by an istration with artificial cerebrospinal fluid (aCSF, composition observer blind to the treatment. Arm entry was registered as in double-distilled water: 0.2 M NaCl, 0.02 M NaH2CO3,2mM placing all four paws of the animal on it. The maze was wiped

KCl, 0.5 mM KH2PO4, 1.2 mM CaCl2, 1.8 mM MgCl2, 0.5 mM clean with damp cotton and dried after testing each rat.

Na2SO4 and 5.8 mM D-glucose). Other drugs were either Anxiogenic or anxiolytic effects were assessed based on the dissolved in or diluted with 0.9% saline. Except ALLO, FGIN 1– frequency of entries as well as time spent in the open arms 27, muscimol (i.c.v.) and finasteride (s.c.) all drugs were (Pellow et al., 1985). Increase in time spent as well as frequency injected intraperitoneally. Control groups of rats treated with of entries in the open arms relative to control animals was corresponding vehicle were maintained simultaneously. considered as an anxiolytic behavior. Separate groups of animals were used for each treatment and each subject tested 4.3. Intracerebroventricular (i.c.v.) administration was given a single 5 min trial or tested once only to avoid ‘one trial tolerance’ to drug effect (Bertoglio and Carobrez, 2002). As reported from our laboratory earlier (Khisti et al., 2002; Ugale et al., 2004b; Hirani et al., 2005; Kokare et al., 2006), rats were 4.5. Drug treatments anesthetized with thiopentone sodium (45 mg/kg, i.p.) (Abbott Pharmaceuticals, Mumbai, MS, India) and fixed in a stereotaxic 4.5.1. Dose related effect of etifoxine and ALLO in EPM test frame (David Kopf, USA). A stainless-steel guide cannula (24 Separate groups (n=7–9) of rats were injected with different gauge, C316G/Spc, internal diameter 0.29 mm; outer diameter doses of etifoxine (25–75 mg/kg, i.p.) or ALLO (0.1–1.0 μg/rat, i.c.v.) 0.56 mm) (Plastics One Inc., Roanoke, VA, USA) was implanted and 30 min after i.p. or 15 min after i.c.v. injection, individual rat BRAIN RESEARCH 1184 (2007) 193– 201 199 was subjected to EPM test for 5 min as described above. The 4.6. Statistical analysis control animals were given an equivalent volume (5 ml/kg, i.p. or 5 μl/rat, i.c.v.) of vehicle. Data are expressed as means±S.E.M. and analyzed by one-way analysis of variance (ANOVA). Significant interactions were 4.5.2. Influence of ALLO and neurosteroidogenic drugs on assessed by post hoc Dunnett's or Student–Newman–Keuls anxiolytic effect of etifoxine test. Unpaired t test was used to analyze the data of adrenal- In these experiments, the effect of ALLO and the drugs that ectomy experiment. A value of P<0.05 was considered as increase endogenous brain neurosteroid levels on etifoxine- statistically significant for all the treatments. induced anxiolysis was assessed. Different groups (n=8)ofrats were injected with progesterone (5 mg/kg, i.p.) or 11-β- hydroxylase inhibitor metyrapone (50 mg/kg, i.p.) 30 min prior Acknowledgments to etifoxine (25 mg/kg, i.p.) or vehicle (5 ml/kg, i.p.) treatment. Other groups of rats were injected with neurosteroid ALLO The authors express gratitude to Departement de Pharmaco- μ – μ (0.1 g/rat, i.c.v.) or MDR agonist FGIN 1 27 (0.5 g/rat, i.c.v.) logie, Laboratoires Biocodex, Zac de Mercieres, Chemin d'Ar- μ 15 min prior to etifoxine (25 mg/kg, i.p.) or vehicle (5 l/rat, i.c.v.) mancourt, 60200 Compiegne, France for providing the gift treatment and 30 min thereafter, rats were subjected individ- sample of etifoxine. ually to EPM test for 5 min.

4.5.3. Effect of neurosteroid biosynthesis inhibitors on REFERENCES etifoxine-induced anxiolysis These experiments were conducted to determine the influence of reduced neurosteroid brain content on etifoxine-induced Akwa, Y., Purdy, R.H., Koob, G.F., Britton, K.T., 1999. The amygdala mediates the anxiolytic-like effect of the neurosteroid in the anxiolysis. We administered various neurosteroid biosynthesis rat. Behav. Brain Res. 106, 119–125. inhibitors before etifoxine injection. The doses of inhibitors Almeida, R.M., Giovenardi, M., Damra, A., Frey, R.M., Rasia-Filho, used and their injection intervals were selected on the basis of A.A., 2006. Plus-maze following the microinjection of muscimol existing literature. Neurosteroid biosynthesis inhibitors such as into the dorsal periaqueductal gray of the rat. Rev. Bras. 5α-reductase types I and II inhibitor (Blurton-Jones et al., 1999; Psiquiatr. 28, 80–82. Concas et al., 1998; Kokate et al., 1999; VanDoren et al., 2000) Bertoglio, L.J., Carobrez, A.P., 2002. Prior maze experience required finasteride (50×2 mg/kg, s.c. at −4and−1.5 h), 3α-hydroxyster- to alter midazolam effects in rats submitted to the elevated plus-maze. Pharmacol. Biochem. Behav. 72, 449–455. oid oxidoreductase (3α-HSOR) inhibitor (Beyer et al., 1999) Beyer, C., Gonzalez-Flores, O., Ramirez-Orduna, J.M., − β indomethacin (5 mg/kg, i.p. at 20 min), 3 -hydroxysteroid Gonzalez-Mariscal, G., 1999. Indomethacin inhibits lordosis β dehydrogenase (3 -HSD) inhibitor (Potts et al., 1978; Korneyev induced by ring—a reduced progestins: possible role of et al., 1993) trilostane (30 mg/kg, i.p. at −2 h) or PBR antagonist 3α-oxoreduction in progestin-facilitated lordosis. Horm. (Schlichter et al., 2000) PK11195 (15 mg/kg, i.p. at −0.5 h) were Behav. 35, 1–8. injected before etifoxine (75 mg/kg, i.p.) or vehicle (5 ml/kg, i.p.; Birzniece, V., Türkmen, S., Lindblad, C., Zhu, D., Johansson, I.M., n=7 per group). Individual rat was then subjected for 5 min to Bäckström, T., Wahlström, G., 2006. GABA(A) receptor changes in acute allopregnanolone tolerance. Eur. J. Pharmacol. 535, 125–134. EPM test after 30 min of etifoxine or vehicle treatment. Bitran, D., Hilvers, R.J., Kellogg, C.K., 1991. Ovarian endocrine status modulates the anxiolytic potency of diazepam and the efficacy 4.5.4. Effect of GABAergic drugs on anxiolytic effect of etifoxine of γ-aminobutyric acid-benzodiazepine receptor mediated – Rats (n=7) were injected with the GABAA receptor agonist chloride ion transport. Behav. Neurosci. 105, 651 660. muscimol (0.5 mg/kg, i.p.) 30 min prior to a fixed subeffective Bitran, D., Purdy, R., Kellogg, C., 1993. Anxiolytic effect of progesterone is associated with increases in cortical 3α, dose (25 mg/kg, i.p.) of etifoxine. On the other hand, GABAA 5α-THP, pregnanolone and GABAA receptor function. receptor antagonist bicuculline (0.5 mg/kg, i.p.) was adminis- Pharmacol. Biochem. Behav. 45, 423–428. tered at the same time interval prior to effective dose (75 mg/ Blurton-Jones, M.M., Roberts, J.A., Tuszynski, M.H., 1999. Estrogen kg, i.p.) of etifoxine. Control groups treated with vehicle (5 ml/ receptor immunoreactivity in the adult primate brain: neuronal kg, i.p.) were also assessed during these studies. Following an distribution and association with P75, trkA, and choline interval of 30 min, individual rat was allowed to explore EPM acetyltransferase. J. Comp. Neurol. 405, 529–542. for a 5 min test session. Boissier, J.R., Simon, P., Zaczinska, M., Fichelle, J., 1972. Experimental psychopharmacologic study of a new psychotropic drug, 4.5.5. Etifoxine anxiolysis in adrenalectomized rats 2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine. These experiments were performed to investigate the contri- Therapie 27, 325–338. bution of adrenal steroids in the anxiolytic effect of etifoxine. Campo-Soria, C., Chang, Y., Weiss, D.S., 2006. Mechanism of action Rats were surgically, bilaterally adrenalectomized (ADX) or of benzodiazepines on GABAA receptors. Br. J. Pharmacol. 148, sham-operated (sham-ADX) under thiopentone sodium 984–990. (45 mg/kg, i.p.) anesthesia using dorsal approach. After Concas, A., Mostallino, M.C., Porcu, P., Barbaccia, M.L., Trabucchi, surgery, rats were provided with 1% sucrose solution in 0.9% M., Purdy, R.H., Grisenti, P., Biggio, G., 1998. Role of allopregnanolone in the plasticity of γ-aminobutyric acid saline daily and were allowed to recover for 1 week before being type-A receptor in rat brain during pregnancy and after subjected to experiments. ADX or sham-ADX rats (n=7) were delivery. Proc. Natl. Acad. Sci. U. S. A. 95, 13284–13289. treated with vehicle (5 ml/kg, i.p.) or etifoxine (75 mg/kg, i.p.) Corpechot, C., Robel, P., Axelson, M., Sjovall, J., Baulieu, E.E., 1981. and 30 min later subjected to the EPM test. Characterization and measurement of 200 BRAIN RESEARCH 1184 (2007) 193– 201

dehydroepiandrosterone sulfate in rat brain. Proc. Natl. Acad. placebo controlled comparison of sedative and amnesic effects Sci. U. S. A. 78, 4704–4707. of etifoxine and lorazepam in healthy subjects. Fundam. Clin. Drafts, B.C., Fisher, J.L., 2006. Identification of structures within Pharmacol. 15, 209–216. GABAA receptor alpha subunits that regulate the agonist Nguyen, N., Fakra, E., Pradel, V., Jouve, E., Alquier, C., Le Guern, M.E., action of pentobarbital. J. Pharmacol. Exp. Ther. 318, 1094–1101. Micallef, J., Blin, O., 2006. Efficacy of etifoxine compared to Finn, D.A., Phillips, T.J., Okorn, D.M., Chester, J.A., Cunningham, C.L., lorazepam monotherapy in the treatment of patients with 1997. Rewarding effect of the neuroactive steroid 3 adjustment disorders with anxiety: a double-blind controlled alpha-hydroxy-5 alpha-pregnan-20-one in mice. Pharmacol. study in general practice. Hum. Psychopharmacol. Clin. Exp. 21, Biochem. Behav. 56, 261–264. 139–149. Follesa, P., Biggio, F., Mancuso, L., Cabras, S., Caria, S., Gorini, G., N-Wihlback, A.C., Sundstrom-Poromaa, I., Backstrom, T., 2006. Manca, A., Orru, A., Biggio, G., 2004. Ethanol withdrawal-induced Action by and sensitivity to neuroactive steroids in up-regulation of the alpha2 subunit of the GABAA receptor and menstrual cycle related CNS disorders. Psychopharmacology its prevention by diazepam or gamma-hydroxybutyric acid. Mol. 186, 388–401. Brain Res. 120, 130–137. Palmer, A.A., Miller, M.N., McKinnon, C.S., Phillips, T.J., 2002. Hamon, A., Morel, A., Hue, B., Verleye, M., Gillardin, J.M., 2003. The Sensitivity to the locomotor stimulant effects of ethanol and modulatory effects of the anxiolytic etifoxine on GABA(A) allopregnanolone shares common genetic influence. Behav. receptors are mediated by the beta subunit. Neuropharmacology Neurosci. 116, 126–137. 45, 293–303. Paoletti, A.M., Romagnino, S., Contu, R., Orru, M.M., Marotto, M.F., Harrison, N.L., Simmonds, M.A., 1984. Modulation of GABA Zedda, P., Lello, S., Biggio, G., Concas, A., Melis, G.B., 2006. receptor complex by a steroid anesthetic. Brain Res. 323, Observational study on the stability of the psychological status 284–293. during normal pregnancy and increased blood levels of Harrison, N.L., Majewska, M.D., Harrington, J.W., Barkes, J.L., 1987. neuroactive steroids with GABA-A receptor agonist activity. Structure activity relationships for steroid interaction with the Psychoneuroendocrinology 31, 485–492. gamma aminobutyric acid receptor complex. J. Pharmacol. Exp. Paul, S.M., Purdy, R.H., 1992. Neuroactive steroids. FASEB J. 6, Ther. 241, 346–353. 2311–2322. Hirani, K., Khisti, R.T., Chopde, C.T., 2002. Behavioral action of Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic ethanol in Porsolt's forced swim test: modulation by 3 Coordinates. Academic Press, San Diego. alpha-hydroxy-5 alpha-pregnan-20-one. Neuropharmacology Pellow, S., Chopin, P., File, S.E., Briley, M., 1985. Validation of open: 43, 1339–1350. closed arm entries in an elevated plus-maze as a measure of Hirani, K., Sharma, A.N., Jain, N.S., Ugale, R.R., Chopde, C.T., 2005. anxiety in the rat. J. Neurosci. Methods 14, 149–167. Evaluation of GABAergic neuroactive steroid 3alpha-hydroxy-5 Petralia, S.M., Jahagirdar, V., Frye, C.A., 2005. Inhibiting biosynthesis alpha-pregnane-20-one as a neurobiological substrate for the and/or metabolism of progestins in the ventral tegmental area anti-anxiety effect of ethanol in rats. Psychopharmacology 180, attenuates lordosis of rats in behavioural oestrus. 267–278. J. Neuroendocrinol. 17, 545–552. Jain, M.R., Khan, F.A., Krishna, N.S., Subhedar, N., 1994. Pinna, G., Costa, E., Guidotti, A., 2005. Changes in brain Intracranial metyrapone stimulates CRF-ACTH axis in the testosterone and allopregnanolone biosynthesis elicit teleost, Clarias batrachus: possible role of neurosteroids. aggressive behavior. Proc. Natl. Acad. Sci. U. S. A. 102, 2135–2140. NeuroReport 5, 2093–2096. Potts, G.O., Creange, J.E., Hardomg, H.R., Schane, H.P., 1978. Khisti, R.T., Deshpande, L.S., Chopde, C.T., 2002. The neurosteroid Trilostane, a orally active inhibitor of steroid biosynthesis. 3α-hydroxy-5α-pregnan-20-one affects dopamine-mediated Steroids 32, 257–267. behavior in rodents. Psychopharmacology 161, 120–128. Rasmusson, A.M., Pinna, G., Paliwal, P., Weisman, D., Gottschalk, Klatzkin, R.R., Morrow, A.L., Light, K.C., Pedersen, C.A., Girdler, S.S., C., Charney, D., Krystal, J., Guidotti, A., 2006. Decreased 2006. Associations of histories of depression and PMDD cerebrospinal fluid allopregnanolone levels in women with diagnosis with allopregnanolone concentrations following the posttraumatic stress disorder. Biol. Psychiatry 60, 704–713. oral administration of micronized progesterone. Romeo, E., Auta, J., Kozikowski, A.P., Ma, D., Papadopoulos, V., Psychoneuroendocrinology 31, 1208–1219. Puia, G., Costa, E., Guidotti, A., 1992. 2-Aryl-3-indoleacetamides Kokare, D.M., Chopde, C.T., Subhedar, N.K., 2006. Participation of (FGIN-1): a new class of potent and specific ligands for the alpha-melanocyte stimulating hormone in ethanol-induced mitochondrial DBI receptor (MDR). J. Pharmacol. Exp. Ther. 262, anxiolysis and withdrawal anxiety in rats. Neuropharmacology 971–978. 51, 336–345. Romeo, E., Cavallaro, S., Korneyev, A., Kozikowski, A.P., Ma, D., Kokate, T.G., Banks, M.K., Magee, T., Yamaguchi, S., Rogawski, M.A., Polo, A., Costa, E., Guidotti, A., 1993. Stimulation of brain 1999. Finasteride, a 5α-reductase inhibitor, blocks the steroidogenesis by 2-aryl-indole-3-acetamide derivatives anticonvulsant activity of progesterone in mice. J. Pharmacol. acting at the mitochondrial diazepam-binding inhibitor Exp. Ther. 288, 679–684. receptor complex. J. Pharmacol. Exp. Ther. 287, 462–471. Korneyev, A., Pan, B.S., Polo, A., Romeo, E., Guidotti, A., Costa, E., Rupprecht, R., Stohle, A., Hermann, B., di Michele, F., Spaletta, G., 1993. Stimulation of brain pregnenolone synthesis by Pasini, A., Holsboer, F., Romeo, E., 1998. Neuroactive steroid mitochondrial diazepam binding inhibitor receptor ligands in concentrations following metyrapone administration in vivo. J. Neurochem. 61, 1515–1524. depressed patients and healthy volunteers. Biol. Psychiatry 44, Korpi, E.R., Grunder, G., Luddens, H., 2002. Drug interactions at 912–914. GABAA receptors. Prog. Neurobiol. 67, 113–159. Schlichter, R., Rybalchenko, V., Poisbeau, P., Verleye, M., Gillardin, Lambert, J.J., Belelli, D., Hill-Venning, C., Peters, J.A., 1995. J.M., 2000. Modulation of GABAergic synaptic transmission by Neurosteroids and GABAA receptor function. Trends Pharmacol. the non-benzodiazepine anxiolytic etifoxine. Sci. 16, 295–303. Neuropharmacology 39, 1523–1535. Lambert, J.J., Belelli, D., Harney, S.C., Peters, J.A., Frenguelli, B.G., Semeniuk, T., Jhangri, G.S., Le Guern, M.E., Melledo, J.M., 2001. 2001. Modulation of native and recombinant GABAA receptors Neuroactive steroid levels in patients with generalized anxiety by endogenous and synthetic neuroactive steroids. Brains Res. disorder. J. Neuropsychiatry Clin. Neurosci. 13, 396–398. Rev. 37, 68–80. Sharma, A.N., Chopde, C.T., Hirani, K., Kokare, D.M., Ugale, R.R., Micallef, J., Soubrouillard, C., Guet, F., Le Guern, M.E., Alquier, C., 2007. Chronic progesterone treatment augments while Bruguerolle, B., Blin, O., 2001. A double blind parallel group dehydroepiandrosterone sulphate prevents tolerance to BRAIN RESEARCH 1184 (2007) 193– 201 201

ethanol anxiolysis and withdrawal anxiety in rats. Eur. J. Verleye, M., Gillardin, J.-M., 2004. Effects of etifoxine on Pharmacol. 567, 211–222. stress-induced hyperthermia, freezing behavior and colonic Sigel, E., Buhr, A., 1997. The benzodiazepine binding site of GABAA motor activation in rats. Physiol. Behav. 82, 891–897. receptors. Trends Pharmacol. Sci. 18, 425–429. Verleye, M., Schlichter, R., Gillardin, J.-M., 1999. Interaction of Ugale,R.R.,Mittal,N.,Hirani,K.,Chopde,C.T.,2004a.Essentiality etifoxine with the chloride channel coupled to the GABAA of central GABAergic neuroactive steroid allopregnanolone receptor complex. NeuroReport 10, 3207–3210. for anticonvulsant action of fluoxetine against Verleye, M., Schlichter, R., Neliat, G., Pansart, Y., Gillardin, J.M., pentylenetetrazole-induced seizures in mice. Brain Res. 1023, 2001. Functional modulation of gamma-amino acid A receptor 102–111. by etifoxine and allopregnanolone in rodents. Neurosci. Lett. Ugale, R.R., Hirani, K., Morelli, M., Chopde, C.T., 2004b. Role of 301, 191–194. neuroactive steroid allopregnanolone in antipsychotic-like Verleye, M., Pansart, Y., Gillardin, J.M., 2002. Effects of etifoxine on action of olanzapine in rodents. Neuropsychopharmacology ligand binding to GABAA receptors in rodents. Neurosci. Res. 29, 1597–1609. 44, 167–172. VanDoren, M.J., Matthews, D.B., Janis, G.C., Grobin, A.C., Devaud, Verleye, M., Akwa, Y., Liere, P., Ladurelle, N., Pianos, A., Eychenne, L.L., Morrow, A.L., 2000. Neuroactive steroid B., Schumacher, M., Gillardin, J.-M., 2005. The anxiolytic 3alpha-hydroxy-5alpha-pregnan-20-one modulates etifoxine activates the peripheral benzodiazepine receptor and electrophysiological and behavioral actions of ethanol. increases the neurosteroid levels in rat brain. Pharmacol. J. Neurosci. 20, 1982–1989. Biochem. Behav. 82, 712–720.