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BEHAVIORAL PHARMACOLOGY

Neuroactive Steroid Stereospecificity of Ethanol-Like Discriminative Stimulus Effects in Monkeys

Department of Behavioral Neuroscience, Oregon Health & Science University, Beaverton, Oregon (K.A.G.); Division of Neurosciences, Oregon National Primate Research Center, Beaverton, Oregon (K.A.G., C.M.H.); Wake Forest University School of Medicine, Winston-Salem, North Carolina (K.A.G., L.S.M.R.); and Department of Physiology and Pharmacology, the Scripps Research Institute, La Jolla, California (R.H.P.)

Received February 1, 2008; accepted April 23, 2008.

	Abstract

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Positive modulation of GABA_A and antagonism of N-methyl-D-aspartate receptors mediate the discriminative stimulus effects of ethanol. Endogenous neuroactive steroids produce effects similar to ethanol, suggesting that these steroids may modulate ethanol addiction. The four isomers of the functional esters at C-3 of the 3-hydroxy metabolites of 4-pregnene-3,20-dione (progesterone) [allopregnanolone (3,5-P), pregnanolone (3,5β-P), epiallopregnanolone (3β,5-P), and epipregnanolone (3β,5β-P)], a synthetic analog of steroids modified by endogenous sulfation [pregnanolone hemisuccinate (3,5β-P HS)], and a structurally similar, adrenally derived steroid [3-hydroxy-5-androstan-17-one (3,5-A, androsterone)] were assessed for ethanol-like discriminative stimulus effects at 30 or 60 min after administration in male (n = 9) and female (n = 8) cynomolgus monkeys (Macaca fascicularis) trained to discriminate 1.0 or 2.0 g/kg ethanol (i.g.) with a 30-min pretreatment interval. The 3-hydroxysteroids completely substituted for ethanol (80% of cases), whereas the 3β-hydroxysteroids and 3,5β-P HS rarely substituted for ethanol (6% of cases). There were no sex differences. Compared with monkeys trained to discriminate 2.0 g/kg ethanol, 3,5β-P and 3,5-A substituted more potently in monkeys trained to discriminate 1.0 g/kg ethanol. Compared with the 5β-reduced isomer (3,5β-P), the 5 isomer of pregnanolone (3,5-P) substituted for ethanol with 3 to 40-fold greater potency but was least efficacious in female monkeys trained to discriminate 2.0 g/kg ethanol. The data suggest that the discriminative stimulus effects of lower doses (1.0 g/kg) of ethanol are mediated to a greater extent by 3,5β-P- and 3,5-A-sensitive receptors compared with higher doses (2.0 g/kg). Furthermore, the discriminative stimulus effects of ethanol appear to be mediated by activity at binding sites that are particularly sensitive to 3,5-P.

Previous studies have shown that the GABA_A receptor-active progesterone derivative, allopregnanolone (3,5-P), substitutes in female cynomolgus monkeys trained to discriminate 1.0 (Grant et al., 1996, 1997) or 2.0 g/kg ethanol (Green et al., 1999). Furthermore, the sensitivity of female monkeys to the stimulus effects of 1.0 g/kg ethanol is greater when circulating progesterone levels peak (i.e., luteal phase, 4–12 ng/ml compared with follicular phase, <1 ng/ml) (Grant et al., 1997), an effect that was blunted in monkeys trained to discriminate 2.0 g/kg ethanol (Green et al., 1999). Thus, circulating levels of endogenous neuroactive steroids appear additive to exogenously administered ethanol in producing ethanol-like discriminative effects. This effect of ethanol-training dose on the substitution of neuroactive steroids is unexplored in male monkeys.

The pharmacological effects of neuroactive steroids vary with stereochemical configuration and ionic charge of C-3 esters (Fig. 1). In vitro, GABA_A receptors are positively modulated by the 3-hydroxy metabolites of progesterone, pregnanolone (3,5β-P) and 3,5-P, in addition to androsterone (3,5-A), a metabolite of dehydroepiandrosterone (DHEA) and testosterone (Majewska et al., 1986; Paul and Purdy, 1992). In contrast, the 3β-isomers epipregnanolone (3β,5β-P) and epiallopregnanolone (3β,5-P) do not positively modulate GABA_A receptors (Park-Chung et al., 1999) but may act as GABA_A receptor antagonists (Wang et al., 2002). This study evaluated the role of C-3 steroidal stereochemistry and ionic charge of pregnanolone isomers in substituting for the discriminative stimulus effects of 1.0 or 2.0 g/kg ethanol in male and female cynomolgus monkeys. A negatively charged sulfate or hemisuccinate (HS) at C-3 abolishes GABA_A receptor-positive modulation or induces negative modulation of GABA_A or NMDA receptors (Park-Chung et al., 1994, 1999). In rats, 3,5β-P HS has sedative, hypnotic and anticonvulsant effects (Weaver et al., 1997), possibly due to inhibition of NMDA-induced Ca²⁺ currents at NMDA receptors (Park-Chung et al., 1997). Noncompetitive NMDA receptor antagonists partially substitute for ethanol in cynomolgus monkeys (Vivian et al., 2002). Thus, to address neuroactive steroid-sensitive NMDA receptor contributions to the discriminative stimulus effects of ethanol, the current study evaluated the substitution of 3,5β-P HS for ethanol. The most potent GABA_A receptor-positive modulator, 3,5-P, is generated in greater quantities from human gonads compared with adrenals (Genazzani et al., 1998), whereas DHEA is produced exclusively by the adrenals (Simard et al., 2005). By evaluating androsterone substitution for ethanol, the current study compared the ethanol-like effects of gonadal versus adrenal steroids.

View larger version (36K): [in this window] [in a new window]   Fig. 1. Structures of pregnanolone isomers. The –OH group is axial for the 5β and equatorial for the 5 steroids. Shown below is the mean (±S.E.M.) ethanol-appropriate responding (1.0 and 2.0 g/kg training groups shown separately) produced by the four neuroactive steroid isomers collapsed across the 30- and 60-min pretreatment intervals. Means include data from both male and female cynomolgus monkeys trained to discriminate 1.0 or 2.0 g/kg ethanol (i.g.) from water. Each point represents data from substitution tests in 4 to 10 monkeys.

	Materials and Methods

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Subjects
Male (n = 9, 4.9–7.1 kg) and female (n = 8, 2.9–5.1 kg) adult cynomolgus monkeys (Macaca fascicularis) were housed in stainless steel cages (7.6 x 6.0 x 7.0 m) in a temperature- (23 ± 1°C) and humidity- (40–60%) controlled colony room with a 12-h light dark cycle (lights on at 6:00 AM). The cages were modified for social housing. The monkeys were housed individually during training and feeding (3–4 h). At all other times, they were housed in groups of two, three, or four. The monkeys were fed a nutritionally complete diet (Purina Mills, St. Louis, MO) with daily fruit supplements in addition to food during experimental sessions. Water was always available except during training and testing. The females were trained with small food treats to present for external vaginal swabbing to determine the daily status of menses. Female monkeys were trained to participate in awake venipuncture to collect blood samples (3 ml) for tri-weekly assessment of menstrual cyclicity via progesterone assay (Yerkes Endocrine Core Facility, Atlanta, GA). The luteal phase was indicated by progesterone levels 4 ng/ml. The origin and housing conditions of the monkeys have been described previously (Grant et al., 1996, 2000). The study was conducted in accordance with the Wake Forest University Animal Care and Use Committee and the Institute of Laboratory Animal Resources (1996).

Apparatus
Ventilated and sound-attenuating experimental chambers (1.50 x 0.74 x 0.76 m; Med Associates, Inc., St. Albans, VT) that accommodated a primate chair (1.17 x 0.61 x 0.61 m; Plas Labs, Lansing, MI) were used for the experimental sessions. Two retractable levers were within arm's reach of a monkey sitting in a primate chair, set in a panel (0.48 x 0.69 m) 0.72 m from the bottom of the chamber. The panel had three colored lights (amber, green, and red) above each lever and a central white light. Two house lights were set at the rear of the chamber close to the ceiling. A monkey in a primate chair could access 1-g banana-flavored pellets (P. J. Noyes Company, Inc., Lancaster, NH) delivered into a food tray attached to the primate chair. The pellets were delivered from a feeder set outside of the chamber connected to vinyl tubing that led to the food tray. The apparatus and data acquisition were controlled in a PC- or Macintosh-compatible computer connected to an interface (Med Associates or National Instruments, Austin, TX) programmed with LabView software (National Instruments).

Procedure
Experimental Design. The experiment included four groups of monkeys in a 2 (sex: male or female) x 2 (training dose: 1.0 or 2.0 g/kg ethanol) design. Six male (4864, 4865, 4867, 4892, 4889, and 4995) and four female (2830, 2852, 2913, and 3123) monkeys were trained to discriminate 1.0 g/kg ethanol (20% w/v, 5 ml/kg) from water. Three male (4890, 4891, and 5496) and four female (2835, 3220, 5400, and 5999) monkeys were trained to discriminate 2.0 g/kg ethanol (20% w/v, 10 ml/kg) from water. The training conditions were the same as those described in Grant et al. (1997, 2000).

Discrimination Training. For each session, the monkey was seated in a primate chair and wheeled into the chamber. After learning to respond on the levers for banana pellets, the response requirement was increased from fixed ratio (FR)-1 to a final schedule of FR-15 to FR-100. A final FR was selected that resulted in each subject receiving the maximal number of pellets in approximately 5 min. Males received 20 to 25 pellets, and females received 10 to 15 pellets per session. A session ended either after all of the pellets were delivered or 30 min had elapsed, whichever occurred first. With each response, the center colored light above the lever shut off and reilluminated (<1 s). Upon completion of the FR requirement and delivery of a banana pellet, the central white light had shut off and re-illuminated (<1 s).

After response rates on the final FR schedule were >0.25 responses/s for three consecutive sessions, the monkeys were habituated to nasogastric (i.g.) gavage. An infant feeding tube (5 French, 1.7 x 381 mm) was passed down one nostril through the esophagus and into the stomach. For five consecutive sessions, the monkeys received tap water (5 ml/kg i.g.) and were wheeled into the chamber, and 30 min later, only the "water-appropriate" lever was extended. For the next five sessions, the monkeys were administered a training dose of ethanol (1.0 or 2.0 g/kg i.g.) and wheeled into the chamber, and 30 min later, only the "ethanol-appropriate" lever extended into the chamber. After the 30-min pretreatment interval, the house lights and the central white light were illuminated. After these 10 sessions, either ethanol or water was administered, and 30 min later, both the water- and ethanol-appropriate levers extended into the chamber. A reinforcer was delivered after the monkey completed the number of responses in its final FR on the substance-appropriate lever consecutively, i.e., responding uninterrupted by a switch to the substance-inappropriate lever. Responding on the substance-inappropriate lever reset the response count for the substance-appropriate lever to zero.

A double-alternating pattern (i.e., ethanol, ethanol, water, water) characterized discrimination training sessions. The training session condition was changed if 70% of responses in the first FR and 90% of total session responses were to the substance-appropriate lever. The discrimination was acquired when, for five consecutive sessions, 70% of the responses in the first FR and 90% of total session responses were directed to the substance-appropriate lever.

Substitution Testing. During twice-weekly test sessions, a single dose of 3,5-P (range of doses tested, 0.17–10 mg/kg s.c.), 3,5β-P (1.0–30 mg/kg s.c.), 3,5β-P HS (3.9–39.5 mg/kg s.c.), 3β,5β-P (10–30 mg/kg s.c.), 3β,5-P (10–30 mg/kg s.c.), or 3,5-A (3.0–56 mg/kg s.c.) was administered, and 30 or 60 min later, both the water- and ethanol-appropriate levers extended into the chamber. In previous studies, 3,5-P substituted for ethanol between 15 and 60 min after administration, with no substitution at 90 min (Grant et al., 1996). Tests were conducted after both 30- and 60-min pretreatment intervals to evaluate the time course of ethanol-like effects for the steroids tested. Responding on either lever was reinforced according to the final FR of the monkey. Between test sessions, training sessions occurred. If responding did not meet discrimination criteria during a training session, training was continued until the criteria were met for three consecutive sessions. In a majority of cases, each test drug dose was administered both after an ethanol-training session and after a water-training session. The range of doses tested was selected for each monkey to encompass the dose range resulting in substitution in prior studies (Grant et al., 1996, 1997; Bowen et al., 1999). Monkeys were first tested with intermediate doses, followed by an equal distribution of higher and lower doses. If a monkey did not eat its entire next meal, indicating reduced appetite, or exhibited tremors after administration of a test drug, a higher dose was not tested. Visible signs of tremors included slight shaking of the body and slowed ambulation when returned to the home cage. Tremors were typically evident for approximately 2 h.

Drugs
Anhydrous ethanol (1.0 or 2.0 g/kg; Warner-Graham Co., Cockeysville, MD) was diluted to 20% w/v with tap water and administered in a 5 to 10 ml/kg (i.g.) volume followed by a 5-ml flush of water 30 min before each training session. The neuroactive steroids, 3,5-P, 3,5β-P, 3β,5β-P, 3β,5-P, and 3,5-A (Sigma-Aldrich, St. Louis, MO) were diluted in concentrations 10 mg/ml and administered in injection volumes of 0.5 to 5 ml s.c. All drugs were prepared fresh daily. All steroids were purified by R. Purdy for the configurations listed, and 3,5-P was synthesized as in Purdy et al. (1990). All steroids were suspended in hydroxypropyl β-cyclodextrin (4% w/v; Cerestar, Hammond, IN). The synthetic analog 3,5β-P HS was tested instead of 3,5β-P sulfate because 3,5β-P HS is unaffected by endogenous sulfatases, has a higher pK_a, and, therefore, crosses the blood-brain barrier more freely. Similar to 3,5β-P sulfate, 3,5β-P HS negatively modulates NMDA receptor function (Weaver et al., 1997).

Data Analysis
Percentage of total responding on the ethanol-appropriate lever (total responses on the ethanol-appropriate lever/total responses) and response rate (total responses/session time) were calculated for each monkey and session if at least one FR was completed. For a given dose, mean ethanol-appropriate responding was calculated from test sessions occurring after ethanol- and water-training sessions. Complete and partial substitution were operationally defined, respectively, as 80 and 20–79% of responses on the ethanol-appropriate lever, as described in Grant et al. (1997, 2000). When a drug completely substituted for ethanol, the ED₅₀ was computed via linear interpolation between the two doses that encompassed the 50% effect (Holtzman et al., 1991). Baseline response rate was calculated by averaging response rates during each ethanol- and water-training session immediately preceding a test session. Ethanol-appropriate responding and percentage of baseline response rate were analyzed with mixed-factor ANOVAs for each test drug, with pretreatment interval as the within-subjects factor and with sex, training dose, and drug dose as between-subjects factors. Including drug dose as within-subjects factors would have omitted some subjects from the analysis because not all of the same doses were tested in each subject. Mean ED₅₀ for each test drug and pretreatment interval was analyzed with 2 (sex: male and female) x 2 (training dose: 1.0 and 2.0 g/kg) ANOVAs. To compare ED₅₀ values, test drug was treated as a within-subjects factor, although this analysis omitted subjects not tested with each drug. The ED₅₀ values were log-transformed before analysis to normalize skewed distributions. Huynh-Feldt corrections were used when repeated factors were included; adjusted degrees of freedom are cited throughout the article. Interactions were evaluated with Bonferroni-corrected pairwise comparisons. For all tests, was 0.05.

	Results

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The 3-hydroxy steroids, 3,5-P, 3,5β-P, and 3,5-A, completely substituted for ethanol in 29 of 32, 28 of 34, and 24 of 36 dose-response determinations, respectively. For all four groups and each pretreatment interval, percentage of ethanol-appropriate responding increased as a function of dose for 3,5-P [F(8, 48) = 7.42, p < 0.001] (Fig. 2), 3,5β-P [F(7, 60) = 8.14, p < 0.001] (Fig. 3), and 3,5-A [F(6, 59) = 8.94, p < 0.001] (Fig. 4). The potency (ED₅₀) of 3,5-P to substitute for ethanol after either pretreatment interval did not differ between the training doses or between males and females. Despite the absence of a training dose effect, Fig. 2 suggests a trend for 3,5-P to substitute less potently for the discriminative stimulus effects of 2.0 g/kg ethanol compared with 1.0 g/kg ethanol in male and female monkeys at both pretreatment intervals. Figure 2 also suggests that 3,5-P was less efficacious in female monkeys trained to discriminate 2.0 g/kg ethanol compared with female monkeys trained to discriminate 1.0 g/kg ethanol. Indeed, the only group showing partial, rather than full, substitution after 3,5-P administration were females trained to discriminate 2.0 g/kg ethanol. However, as shown in Supplemental Fig. 1, the difference between full (1.0 g/kg ethanol females) and partial (2.0 g/kg ethanol females) substitution was due to 1 less monkey showing substitution in the 2.0 g/kg ethanol-trained group. In every male tested, 3,5-P completely substituted for ethanol.

View larger version (32K): [in this window] [in a new window]   Fig. 2. Mean (±S.E.M.) percentage of total session responding on the ethanol-appropriate lever 30- (closed circles) or 60-min (open circles) after administration of 3,5-pregnanolone (P) in cynomolgus monkeys trained to discriminate 1.0 (female, n = 2–4; male, n = 1–5) or 2.0 g/kg (female, n = 2–4; male, n = 1–3) ethanol from water with a 30-min pretreatment interval. Additional doses were tested, but are not shown, as indicated by the dose-response curves for individual monkeys shown in Supplemental Fig. 1.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Mean (±S.E.M.) percentage of total session responding on the ethanol-appropriate lever 30- (closed circles) or 60-min (open circles) after administration of 3,5β-pregnanolone (P) in cynomolgus monkeys trained to discriminate 1.0 (female, n = 1–4; male, n = 4–6) or 2.0 (female, n = 2–4; male, n = 2–3) g/kg ethanol from water with a 30-min pretreatment interval. Additional doses were tested, but are not shown, as indicated by the dose-response curves for individual monkeys shown in Supplemental Fig. 2.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Mean (±S.E.M.) percentage of total session responding on the ethanol-appropriate lever 30- (closed circles) or 60-min (open circles) after administration of 3,5-androsterone (A) in female and male cynomolgus monkeys trained to discriminate 1.0 (female, n = 3–4; male, n = 2–6) or 2.0 g/kg ethanol (female, n = 3–4; male, n = 2–3) from water with a 30-min pretreatment interval. Dose-response curves for individual monkeys are shown in Supplemental Fig. 3.

The potency (ED₅₀) of 3,5β-P to substitute for ethanol did not vary with sex or training dose after a 30-min pretreatment interval. With a 60-min pretreatment interval, however, 3,5β-P more potently substituted for ethanol in male and female monkeys trained to discriminate 1.0 g/kg compared with monkeys trained to discriminate 2.0 g/kg ethanol [main effect of training dose, F(1, 11) = 16.1, p = 0.002] (Fig. 3). In terms of efficacy, mean ethanol-appropriate responding met the criterion for complete substitution for 1.0 g/kg ethanol at both pretreatment intervals in males and at the 30-min pretreatment interval in females. In females trained to discriminate 2.0 g/kg ethanol, mean ethanol-appropriate responding 30 and 60 min after 3,5β-P administration met the criterion for partial substitution. However, three of four and four of four female monkeys, respectively, showed complete substitution for 2.0 g/kg ethanol at 30 and 60 min after administration, but only with the highest dose tested in six of seven cases (Supplemental Fig. 2). In males trained to discriminate 2.0 g/kg ethanol, 3,5β-P partially substituted for ethanol 30 min after administration and completely substituted for ethanol after a 60-min pretreatment. All cases of complete substitution of 3,5β-P in males trained to discriminate 2.0 g/kg ethanol were observed with the highest dose tested (Supplemental Fig. 2). In general, however, ethanol-appropriate responding in cases of partial substitution was >70%, suggesting minor variation in the efficacy of 3,5β-P to substitute for ethanol across sex, training dose, and pretreatment interval. Supplemental Fig. 2 shows partial substitution of 3,5β-P for ethanol in two female monkeys (one trained to 1.0 g/kg ethanol and one trained to 2.0 g/kg ethanol) and three male monkeys (one trained to discriminate 1.0 g/kg and two trained to discriminate 2.0 g/kg ethanol).

Main effects of training dose indicated that 3,5-A substituted more potently for 1.0 g/kg compared with 2.0 g/kg ethanol, with both the 30-min [F(1, 7) = 9.0, p = 0.02] and the 60-min [F(1, 11) = 7.9, p = 0.03] pretreatment intervals for both sexes (Fig. 4). The potency of 3,5-A to substitute for ethanol did not differ between the sexes. In males trained to discriminate 1.0 or 2.0 g/kg ethanol, 3,5-A completely substituted after each pretreatment interval. In females trained to discriminate 1.0 g/kg ethanol, 3,5-A showed complete substitution after a 30-min pretreatment and partial substitution after a 60-min pretreatment. In contrast, group means showed that 3,5-A only partially substituted in females trained to discriminate 2.0 g/kg ethanol, indicating reduced efficacy. Across both pretreatment intervals, 3,5-A completely substituted for 2.0 g/kg ethanol in females in five cases, with four of these cases occurring with the highest dose tested. All cases of complete substitution in males trained to discriminate 2.0 g/kg ethanol occurred with the highest dose of 3,5-A tested (Supplemental Fig. 3). Supplemental Fig. 3 shows that partial substitution was observed in four male monkeys (two trained to 1.0 g/kg ethanol and two trained to 2.0 g/kg ethanol) and three female monkeys (one trained to 1.0 g/kg ethanol and two trained to 2.0 g/kg ethanol).

A main effect of test drug in a 3 (test drug: 3,5-P, 3,5β-P, 3,5-A) x 2 (sex) x 2 (training dose) x 2 (pretreatment interval) mixed factor ANOVA [F(2, 24) = 155.75, p < 0.001] indicated that the potency to substitute for the discriminative stimulus effects of ethanol differed among the 3-hydroxy steroids, with rank order potency corresponding to 3,5-P > 3,5β-P > 3,5-A (Fig. 1, Table 1).

As predicted, 3,5β-P HS, 3β,5-P, and 3β,5β-P rarely (5 of 84 dose-response determinations) substituted for ethanol. For the 30- and 60-min pretreatment intervals, respectively, complete substitution for ethanol was observed in 1 of 11 and 2 of 11 monkeys tested with 3,5β-P HS, in 0 of 15 and 1 of 15 monkeys tested with 3β,5-P, and in 0 of 16 and 1 of 16 monkeys tested with 3β,5β-P (Table 1; Supplemental Figs. 4–6).

During baseline sessions, female monkeys trained to discriminate 1.0 and 2.0 g/kg ethanol averaged 2.98 ± 0.24 and 1.73 ± 0.15 responses/s, respectively; male monkeys trained to discriminate 1.0 and 2.0 g/kg ethanol averaged 1.84 ± 0.12 and 1.20 ± 0.10 responses/s, respectively. Baseline response rate was significantly greater for females compared with males [F(1, 15) = 6.12, p = 0.03] and for monkeys trained to discriminate 1.0 g/kg ethanol compared with monkeys trained to discriminate 2.0 g/kg ethanol [F(1, 15) = 4.65, p = 0.048]. Response rates during substitution tests were therefore expressed as percentage of baseline (Supplemental Fig. 7). Percentage of baseline response rate was lower after the 60-min pretreatment intervals compared with the 30-min pretreatment intervals. This trend was statistically significant only for 3,5β-P [F(1, 79) = 4.54, p = 0.04], 3,5β-P HS [F(1, 22) = 26.78, p < 0.001], and 3β,5β-P [F(1, 49) = 10.01, p = 0.003] according to main effects of pretreatment interval. The 3-hydroxysteroids generally increased response rate above baseline in monkeys trained to discriminate 1.0 g/kg [mean ± S.E.M.: 3,5-P, 105.2 ± 5.5; 3,5β-P, 110.6 ± 4.9; 3,5-A, 111.8 ± 4.0] but decreased response rates below baseline in monkeys trained to discriminate 2.0 g/kg ethanol [mean ± S.E.M.: 3,5-P, 79.2 ± 7.3; 3,5β-P, 87.4 ± 6.9; 3,5-A, 86.2 ± 5.6]. This training dose difference was not shared with the 3β-hydroxysteroids and 3,5β-P HS, which decreased response rates below baseline in monkeys trained to discriminate 1.0 g/kg ethanol [3,5β-P HS, 88.3 ± 11.3; 3β,5β-P, 83.5 ± 4.8; 3β,5-P, 86.2 ± 6.8] and 2.0 g/kg ethanol [3,5β-P HS, 81.0 ± 10.3; 3β,5β-P, 74.6 ± 6.6; 3β,5-P, 70.4 ± 7.2]. Males showed greater decreases in response rate compared with females for all of the drugs tested, but this effect was significant only for 3,5β-P [F(1, 79) = 5.08, p = 0.03; 3,5-P, F(1, 69) = 0.57; 3,5-A, F(1, 80) = 3.53, p = 0.06; 3,5β-P HS, F(1, 22) = 0.55; 3β,5β-P, F(1, 49) = 0.51; 3β,5-P, F(1, 46) = 0.76].

	Discussion

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Humans produce four neuroactive isomers of pregnanolone (Havlíková et al., 2006), with distinct pharmacological properties in vitro (Park-Chung et al., 1997, 1999). The data show distinct differences in efficacy of 3 versus 3β isomers of pregnanolone to produce ethanol-like discriminative stimulus effects in nonhuman primates. In contrast to the 3β isomers, the 3 isomers of pregnanolone and androsterone showed ethanol-like effects in a majority of tests across sex, training dose, and pretreatment times. Furthermore, 3,5β-P but not 3,5β-P HS substituted for ethanol. Thus, addition of a negatively charged group at C-3 prevents the ethanol-like discriminative stimulus effects of 3,5β-P. A main implication from the data are that individual differences in bioconversion of the precursors progesterone or DHEA/testosterone to 3- or 3β-neuroactive steroids or sulfate derivatives could underlie individual differences in subjective effects following ethanol consumption. An individual predisposed to synthesize relatively more 3β compared with 3-pregnanolone or sulfated compared with nonsulfated pregnanolone may be less sensitive to the effects of consumed ethanol, perhaps a mechanism for innate or acquired tolerance. This, in turn, could affect the choice to continue drinking ethanol on a given occasion resulting in binge-levels of intake. For example, 3,5β-P HS reduces ethanol self-administration in rats (O'Dell et al., 2005), but this has not been explored in nonhuman primates. How endogenous levels of 3β- or sulfated hydroxysteroids influences ethanol self-administration or subjective effects remains to be explored.

Individual differences in the metabolism of progesterone and its derivatives could account for the rare cases in which 3β-hydroxysteroids substituted for ethanol in the monkeys. For example, the 3β isomer 3β,5-P is increased in women with chronic fatigue syndrome (Murphy et al., 2004), and pharmacokinetic analyses showed dramatically reduced clearance of 3,5β-P in one of nine females tested (Dale et al., 1999). The steroidal milieu resulting from enzymatic activity could affect GABA_A receptor pharmacology, ethanol sensitivity, and subsequent neuroactive steroid substitution for ethanol. For example, in vitro experiments show that 3,5-P treatment increased the expression of GABA_A receptor ₄ subunits (Zhou and Smith, 2007), which are potently modulated by ethanol (Wallner et al., 2006) and neuroactive steroids due to coexpression with subunits (Belelli and Lambert, 2005). Very little data address species differences in the role of endogenous enzymes in producing behaviorally relevant concentrations of neuroactive steroids. Drug discrimination in macaque monkeys appears to provide a pharmacologically accurate animal model, with which to study neuroactive steroid mechanisms mediating the subjective effects of ethanol in humans.

These data have implications for species differences in ethanol-like discriminative stimulus effects from neuroactive steroids that positively modulate GABA_A receptors. Specifically, the progesterone derivatives 3,5-P, 3,5β-P, and 3β,5β-P had substituted for 1.0 and 2.0 g/kg ethanol in male Long-Evans rats (Bowen et al., 1999). Substitution of the latter contrasts with the current findings in monkeys in which the isomers that do not positively modulate GABA_A receptors, 3β,5β-P, 3β,5-P, in addition to 3,5β-P HS, rarely substituted (only 6% of dose-response determinations) for ethanol. Given that the monkey data match in vitro pharmacological data of activity at the GABA_A receptor, the substitution of 3β,5β-P for ethanol in rats may be due to species differences in bioconversion of administered neuroactive steroids. Rodents appear to have minimal 5β-reductase activity (R. H. Purdy, unpublished data) and may not produce the four pregnanolone isomers present in primates. The role of endogenous isomers in GABA_A receptor mediation of ethanol-like discriminative stimulus effects in rodents compared with primates is unclear.

Individual differences in endogenous progesterone related to menstrual cycles may influence sensitivity to behavioral effects, including the discriminative stimulus effects of ethanol. For example, 12% of females with epilepsy suffered from a greater incidence of seizures during the low-progesterone perimenstrual phase (Duncan et al., 1993), which appears to be related to reduced GABA_A receptor positive modulation (Reddy et al., 2001; Herzog and Frye, 2003). Female monkeys showed reduced potency and efficacy in 3,5-P substitution for ethanol in the follicular (perimenstrual) phase of the menstrual cycle (Grant et al., 1997). In contrast to the current design, menstrual cycle effects were previously investigated by collecting a cumulative dose-response curve in a single session. The present experiment found no sex differences in neuroactive steroid substitution for ethanol, although the female monkeys had normal menstrual cycles. The monkeys were trained to discriminate ethanol across all menstrual cycle phases (2–3 times per week), perhaps training them to discriminate a range of ethanol stimulus effects due to differential circulating progesterone derivatives. For each test drug reported here, as indicated by mense records and serum progesterone, 26.4 to 52.6 and 36.9 to 69.8% of tests occurred in the luteal and follicular phases, respectively. The substitution data suggest that the potency of 3,5-P to substitute for ethanol in female monkeys reflects composite menstrual cycle phase influences.

In addition to the 3 and 3β isomers, the 5 and 5β isomers differed in potency to produce ethanol-like discriminative stimulus effects. The potency of 3,5-P was 3 to 40-fold greater compared with 3,5β-P. This is consistent with the stereospecificity of effects mediated by 5 and 5β neuroactive steroids in a variety of species and assays (e.g., Simmonds, 1991). Neuroactive steroids act as agonists at GABA_A receptors at binding sites that are distinct from the sites at which neuroactive steroids act as positive modulators. As an agonist, 3,5-P is more efficacious than 3,5β-P (Hosie et al., 2006). Given that 3,5-P substituted for ethanol with greater potency than 3,5β-P, the agonist site could be an important target for alcoholism pharmacotherapy. Although central nervous system GABA_A receptor agonism appears to be insufficient to produce ethanol-like discriminative stimulus effects, as indicated by the lack of substitution of peripherally administered muscimol (but see Hodge and Cox, 1998; Grant et al., 2000), whether agonism via a neuroactive steroid binding site is sufficient to produce ethanol-like discriminative stimulus effects remains to be determined. Synthetic steroids are currently being developed that could be used to evaluate this hypothesis (Mennerick et al., 2004).

The training dose of ethanol influenced the potency or efficacy of 3-hydroxysteroids to substitute for ethanol (Figs. 2, 3, 4). In cases for which this effect was not statistically significant, the analysis may have been underpowered, given that only three males were trained to discriminate 2.0 g/kg ethanol. In general, 3,5-P, 3,5β-P, and 3,5-A substituted for ethanol with greater potency in male and female monkeys trained to discriminate 1.0 compared to 2.0 g/kg ethanol. Efficacy was comparatively low in the females trained to discriminate 2.0 g/kg ethanol, where ethanol-appropriate responding after administration of 3,5-P, 3,5β-P and 3,5-A met the criterion for partial substitution. For each of these test drugs, however, partial rather than complete substitution for 2.0 g/kg ethanol was primarily due to substitution patterns in a single female monkey, 5999 (Supplemental Figs. 1–3). Investigations of the role of serotonin and glutamate receptor systems indicate that multiple receptors mediate the discriminative stimulus effects of ethanol (Grant, 1999). A greater proportion of the ethanol stimulus may involve receptors sensitive to 3-hydroxysteroids in monkeys trained to discriminate lower compared with higher doses of ethanol. In support of this hypothesis, NMDA receptors appear to mediate the discriminative stimulus effects of 2.0 g/kg ethanol in female monkeys (Vivian et al., 2002), the group showing the lowest efficacy of GABA_A receptor-sensitive neuroactive steroids in this study. However, the NMDA receptor antagonist 3,5β-P HS (Park-Chung et al., 1997) did not substitute for ethanol in any group, including females trained to discriminate 2.0 g/kg ethanol. Antagonism of NMDA receptors by 3,5β-P HS is likely to be similar to its endogenous analog, 3,5β-P sulfate, which noncompetitively antagonizes NMDA receptors (Park-Chung et al., 1994). The lack of substitution of 3,5β-P HS in the current study could be due to negation of NMDA effects via inhibition of GABA_A receptors (Park-Chung et al., 1999).

The effects of 3- and 3β-neuroactive steroids and 3,5β-P HS on rates of responding were distinct from their ethanol-like discriminative stimulus effects. For example, the effect of neuroactive steroids on response rate, but not ethanol-appropriate responding, depended on the pretreatment interval. This difference could reflect an interaction between regional pharmacokinetics, steroid binding (Sapp et al., 1992), and the brain regions mediating the sedative versus discriminative stimulus effects of ethanol. Indeed, that 3,5-A substituted less potently for ethanol compared with 3,5-P and, in many cases, 3,5β-P could indicate low brain levels of 3,5-A in primates. For example, the first of three enzymes involved in the conversion of DHEA to 3,5-A, 3β-hydroxysteroid dehydrogenase/⁵-⁴ isomerase (3β-HSD) (Simard et al., 2005) is absent from the cerebral cortex, cerebellum, and cerebrum of rhesus monkeys (Martel et al., 1994). To evaluate this hypothesis, additional studies must be conducted to identify regional differences in neuroactive steroid levels following ethanol administration.

Overall, the receptors mediating the effects of neuroactive steroids represent an important avenue for future research regarding alcoholism pharmacotherapy. These data provide key information regarding structure-activity requirements, the effects of ethanol dose, and sex as a first-step toward identifying specific pharmacotherapies targeting neuroactive steroid-sensitive receptors mediating the subjective effects of ethanol. Future studies could address the relationship between endogenous steroid levels, steroidogenic enzyme activity, and sensitivity to the ethanol-like discriminative stimulus effects of neuroactive steroids.

	Acknowledgements

 
We thank Courtney A. Waters for excellent technical expertise.

	Footnotes

 
This work was funded by NIAAA, National Institutes of Health Grants AA10009, AA 13860, and RR 000163. This work was presented at the 30th Annual Meeting of the Research Society on Alcoholism; 2007 Jul 7–11; Chicago, IL. Research Society on Alcoholism, Austin, TX (Helms CM et al. (2007) Neurosteroid-like discriminative stimulus effects of ethanol in monkeys are influenced by C-3 steroidal stereochemistry and ionic charge of esters. Alcohol Clin Exp Res 31 (Suppl):80A).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.137315.

ABBREVIATIONS: NMDA, N-methyl-D-aspartate; DHEA, dehydroepiandrosterone; FR, fixed ratio; ANOVA, analysis of variance; 3,5-P, allopregnanolone; 3,5β-P, pregnanolone; 3β,5-P, epiallopregnanolone; 3β,5β-P, epipregnanolone; 3,5β-P HS, pregnanolone hemisuccinate; 3,5-A, androsterone.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Kathleen A. Grant, Oregon Health and Science University, 505 N.W. 185th Avenue, Beaverton, OR 97006-6448. E-mail grantka{at}ohsu.edu

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