Neurosteroids Ameliorate Conditioned Fear Stress: An Association with Sigma1 Receptors Yukihiro Noda, Ph.D., Hiroyuki Kamei, Ph.D., Yukie Kamei, M.S., Taku Nagai, M.S., Mikio Nishida, Ph.D., and Toshitaka Nabeshima, Ph.D. Mice exhibited a marked suppression of motility (25 mg/kg) was co-administered with (ϩ)-SKF-10,047 (3 (conditioned fear stress) when placed in an environment in mg/kg) at doses that do not affect the conditioned fear stress which they had previously received an electric footshock. response by themselves, motor suppression was This conditioned fear stress response was dose-dependently significantly attenuated. In mice showing the conditioned attenuated by neurosteroids such as dehydroepiandrosterone fear stress response, the serum concentration of DHEAS sulfate (DHEAS; 25 and 50 mg/kg, s.c.) and pregnenolone was lower than that in non-shocked mice. These results sulfate (PREGS; 10–50 mg/kg, s.c.), and by a putative suggest that the attenuating effects of DHEAS and PREGS ϩ ϩ sigma1 receptor agonist, ( )-N-allylnormetazocine (( )- on the conditioned fear stress response are mediated via SKF-10,047; 3 and 6 mg/kg, s.c.). However, progesterone sigma1 receptors and that PROG has a sigma1 receptor (PROG; 10–50 mg/kg, s.c.) and allopregnanolone (5 and 20 antagonistic property. Further, the endogenous DHEAS mg/kg, s.c.) had no effect on this stress response. The may be involved in the expression of conditioned fear stress attenuating effects of DHEAS (50 mg/kg, s.c.), PREGS (50 response in mice. [Neuropsychopharmacology 23:276– mg/kg, s.c.), and (ϩ)-SKF-10,047 (6 mg/kg, s.c.) were 284, 2000] © 2000 American College of reversed by NE-100 (5 mg/kg, i.p.), a sigma1 receptor Neuropsychopharmacology. Published by Elsevier Science antagonist and PROG (5 or 10 mg/kg, i.p.). When DHEAS Inc. All rights reserved. KEY WORDS: Neurosteroids; Sigma1 receptors; Conditioned progesterone (PROG), pregnenolone (PREG), dehy- fear stress; DHEAS droepiandrosterone (DHEA), and their respective sul- fate ester (PREGS or DHEAS), are involved in regulat- Neurosteroids are synthesized in the brain, either de ing the imbalance between excitation and inhibition in novo from cholesterol or from steroid hormone precur- the CNS (Wu et al. 1991). The neurosteroids, allopreg- sors, and accumulate in the nervous system to levels nanolone, allotetrahydrodeoxycorticosterone, PREGS, that are at least in part independent of steroidogenic and DHEAS have been shown to possess antistress, gland secretion (Baulieu 1981). Neurosteroids, such as anxiolytic, and antiamnesic properties in experimental animal models (Bitran et al. 1991; Brot et al. 1997; Mau- rice et al. 1997, 1999; Reddy and Kulkarni 1998; Reddy From the Department of Neuropsychopharmacology and Hospi- tal Pharmacy, Nagoya University Graduate School of Medicine et al. 1998; Urani et al. 1998; Wieland et al. 1991). Recent (YN, HK, YK, TN, TN), Nagoya, Japan; and Department of Clinical evidence suggests that DHEAS and PREGS also play an Pharmacy, Faculty of Pharmacy, Meijo University (YK, TN, MN), important role in depression. Interestingly, decreased Nagoya, Japan. Address correspondence to: Dr. Toshitaka Nabeshima, Depart- levels of DHEA, DHEAS, and PREGS have been associ- ment of Neuropsychopharmacology and Hospital Pharmacy, ated with depression, cognitive dysfunction, aging, and Nagoya University Graduate School of Medicine, Nagoya 466–8560, other neurological conditions (Orentreich et al. 1984; Japan. Received October 18, 1999; revised February 2, 2000; accepted Vallée et al. 1997), and DHEA improves the depression February 18, 2000. score in patients (Wolkowitz et al. 1997). However, the NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 3 © 2000 American College of Neuropsychopharmacology Published by Elsevier Science Inc. All rights reserved. 0893-133X/00/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(00)00103-2 NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 3 Neurosteroids and Sigma1 Receptors 277 mechanism underlying the beneficial effects of neuro- METHODS steroids is not yet known. Animals Neurosteroids have been shown to affect the activity of neurotransmitter systems, which are involved in a Male mice of the ddY strain (Japan SLC, Inc., Shizuoka, variety of neuropsychiatric illnesses. DHEAS and Japan), at 7–8 weeks of age were used. The animals were PREGS are negative allosteric modulators of the ␥-amino- housed in plastic cages and were kept in a regulated Ϯ Њ Ϯ butyric acid (GABA)A receptors (Majewska 1992), and environment (23 1 C, 50 5% humidity), with a 12/12 positive modulators of N-methyl-D-aspartate (NMDA) hr light-dark cycle (lights on at 7:00 A.M.). Food (CE2; receptor-mediated responses (Bowlby 1993; Irwin et al. Clea Japan Inc., Tokyo, Japan) and tap water were avail- 1992, 1994). Evidence is mounting that DHEAS and able ad libitum. PREGS act as agonists at central sigma receptors, espe- All experiments were performed in accordance with cially sigma1 sites, and exert facilitatory actions on the Guidelines for Animal Experiments of the Nagoya NMDA-mediated glutamatergic and noradrenergic University School of Medicine. The procedures involv- neurotransmission (Maurice and Lockhart 1997; Mon- ing animals and their care conformed with the interna- net et al. 1995). Some neurosteroids such as PREGS and tional guidelines set out in ‘Principles of Laboratory PROG have been shown to have an affinity for sigma1 Animal Care’ (NIH publication no. 85–23; revised 1985). receptors (Su et al. 1988; Walker et al. 1990). Further, Maurice et al. (1996) have reported that exogenous ad- Drugs ministration of neurosteroids leads to dose-dependent inhibition of in vivo binding of [3H]-(ϩ)-N-allyl- The following drugs were used: (ϩ)-N-allylnormetazo- ϩ ϩ normetazocine (( )-SKF-10,047) to sigma1 receptors. cine hydrochloride (( )-SKF-10,047; Research Biochem- Thus, it is possible that neurosteroids are the main en- icals Inc., Natick, MA, USA), dehydroepiandrosterone dogenous modulator of sigma1 receptors, and that the sulfate (DHEAS; Sigma, St. Louis MO, USA), preg- pharmacological effects of neurosteroids described nenolone sulfate (PREGS; Sigma), allopregnanolone above are mediated, at least partly, through the sigma1 (Sigma), and progesterone (PROG; Sigma). NE-100 (N, receptors. N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]- To date, we have tried to clarify the functional role of ethylamine monohydrochloride) was kindly supplied sigma1 receptors in a stressful situation by using the by Taisho Pharmaceutical Co. Ltd. (Saitama, Japan). conditioned fear stress response defined by Fanselow DHEAS radioimmunoassay kit was obtained from (1980). Rats and mice exhibit a marked suppression of Japan DPC Co. (Chiba, Japan). motility when they are re-placed in an environment in (ϩ)-SKF-10,047 and NE-100 were dissolved in saline which they have previously received an electric foot- and distilled water, respectively. DHEAS, PREGS, allo- shock (Kameyama and Nagasaka 1982, 1983; Yamada pregnanolone, and PROG were solubilized in 7% di- and Nabeshima 1995). This motor suppression is re- methylsulfoxide (Sigma). All compounds were admin- garded as a conditioned emotional response to the envi- istered in a volume of 0.1 ml/10 g body weight. ronment associated with previous footshock (Kameyama and Nagasaka 1982, 1983; Yamada and Nabeshima Schedule for Conditioned Fear Stress 1995). We have found that (ϩ)-SKF-10,047 and dex- tromethorphan, prototype sigma1 receptor agonists, The experiments were carried out according to the dose dependently attenuate the conditioned fear stress method of Kamei et al. (1994, 1996a,b), with a minor (Kamei et al. 1994, 1996a) and that this effect was antag- modification. A transparent acrylic rectangular test onized by NE-100, a sigma1 receptor antagonist stress cage (25.5 x 31.0 x 14.0 cm) equipped with a metal wire (Kamei et al. 1994, 1996a), indicating that the activation floor was used. The test cage was located in a sound- of sigma1 receptors is responsible for the attenuation of attenuated room and was illuminated with a 20-W conditioned fear stress response. These observations bulb. suggest that the sigma1 receptors play an important role Each mouse was placed in the test cage and received in stress responses. However, the functional role of electric shocks (0.1 Hz, 200 ms, 1 mA) for 6 min through neurosteroids having affinity for sigma1 receptors, in a shock generator-scrambler (Neuroscience Inc., Tokyo, the conditioned fear stress response is unclear. Japan). The test trial was then carried out 24 h after In the present study, therefore, we examined the ef- shock treatment; the animals were again placed into the fects of neurosteroids on the conditioned fear stress re- same test cage, but no electric footshocks were given sponse in mice, in comparison with those of the sigma1 (shocked group). The spontaneous motility of the ani- receptor ligand, (ϩ)-SKF-10,047 and discussed the rela- mal was determined for 6 min in the same test cage by tionship between neurosteroids and sigma1 receptors in using Scanet SV-10 (Toyo Sangyo, Toyama, Japan) the stress response. equipped with photosensors. The non-shocked control 278 Y. Noda et al. NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 3 group was prepared exactly the same way except with- apparatus in which they had been given an electric out electric footshock (non-shocked group). shock (Figure 1 and Table 1A), in agreement with our All test drugs were administered before measuring previous results (Kamei et al. 1994, 1996a,b). The motility in the test trial; (ϩ)-SKF-10,047, DHEAS, shocked mice mostly froze on the floor and crouched, PREGS, allopregnanolone, and PROG were adminis- while the non-shocked mice showed typical explor- tered subcutaneously (s.c.) 30 min before the test trial. atory behavior such as ambulation, sniffing and rearing. In the antagonistic experiments, NE-100 and PROG Figure 1 and Table 1A show the effects of neuroste- were administered intraperitoneally (i.p.) 45 min before roids and (ϩ)-SKF-10,047, respectively, on motor sup- the test trial.