GABAB receptor-mediated increase of neurosteroids by γ-hydroxybutyric acid

Neuropharmacology 42 (2002) 782–791 www.elsevier.com/locate/neuropharm

GABAB receptor-mediated increase of neurosteroids by γ-hydroxybutyric acid M.L. Barbaccia a,∗, G. Colombo b, D. Affricano a, M.A.M. Carai c, G. Vacca c, S. Melis c, R.H. Purdy d, G.L. Gessa b,c a

Department of Neuroscience, University of Rome ‘Tor Vergata’, Via Tor Vergata 135, 00133 Rome, Italy b CNR Institute of Neurogenetics and Neuropharmacology, 09042 Cagliari, Italy c Neuroscienze Scarl, 09123 Cagliari, Italy d The Scripps Research Institute, 92037 La Jolla, CA, USA Received 1 August 2001; received in revised form 3 January 2002; accepted 4 January 2002

Abstract Among the pharmacological actions of γ-hydroxybutyric acid (GHB), some may involve GABAA receptor-mediated mechanisms. GHB, however, fails to directly interact with sites for agonists and modulators on the GABAA receptor complex. We hypothesized that, in vivo, GHB may interfere with GABAA receptor function by altering the brain concentrations of the neurosteroids 3αhydroxy-5α-pregnan-20-one (allopregnanolone, AP) and 3α,21-dihydroxy-5α-pregnan-20-one (allotetrahydrodeoxycorticosterone, THDOC), positive allosteric modulators of GABA-gated chloride currents. In male Wistar rats, GHB dose-dependently (75–1000 mg/kg, i.p.) increased AP, THDOC and their precursors pregnenolone and progesterone in brain cortex and hippocampus. The increases of AP (4–5 fold) and THDOC (3–4 fold) elicited by 300 mg/kg GHB peaked between 30 and 90 min and abated by 180 min. The selective GABAB receptor antagonist SCH 50911 (50 mg/kg, i.p.) prevented the action of GHB, while the GABAB receptor agonist baclofen (5–10 mg/kg) mimicked it. NCS-382 (50 mg/kg, i.p.), the purported selective antagonist of the GHB receptor, failed to antagonize GHB, but at 300 mg/kg increased brain cortical neurosteroids to the same extent as 300 mg/kg GHB; coadministration of GHB and NCS-382, however, failed to yield an additive effect. These results strongly suggest that GHB, via a GABAB receptor-mediated mechanism, increases the brain concentrations of neurosteroids, whose properties as amplifyers of the GABAgated chloride conductances may play a role in the GABAA receptor-mediated pharmacological actions of GHB.  2002 Elsevier Science Ltd. All rights reserved. Keywords: γ-hydroxybutyric acid (GHB); Allopregnanolone; Allotetrahydrodeoxy-corticosterone (THDOC); GABAB receptor; GHB receptor; GABAA receptor

1. Introduction It has been known for quite some time that the administration of γ-hydroxybutyric acid (GHB) to laboratory animals and humans induces anxiolytic, hypnotic and anesthetic actions (Bernasconi et al., 1999). More recent preclinical and clinical evidence shows that, on one side, GHB is effective in the pharmacotherapy of alcohol dependence while, on the other side, may have addictive properties leading to abuse (Bernasconi et al., 1999;

Corresponding author. Tel.: +39-06-72596314; fax: +39-0672596302. E-mail address: [email protected] (M.L. Barbaccia). ∗

Addolorato et al., 2000; Gallimberti et al., 2000; Gessa et al., 2000; Agabio and Gessa, 2002). It is not known, though, whether the same molecular mechanisms underpin these various aspects. Indeed, despite the fact that GHB has been characterized since long time as an endogenously occurring metabolite of γ-aminobutyric acid (GABA) that is present in relevant concentrations in brain and peripheral tissues of mammals, including man (reviewed by Maitre, 1997), the molecular mechanisms that mediate its pharmacological actions are far from being clear. Specific and high affinity GHB binding sites, presumably coupled via G-proteins to Ca++ and K+ conductances (Maitre, 1997), have been described in discrete brain regions of rodents, monkeys and humans (Maitre, 1997;

0028-3908/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 2 ) 0 0 0 2 6 - 6

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Castelli et al., 2000). These high affinity GHB receptors have been characterized and differentiated from GABAB receptors on the basis of their sensitivity to specific agonists and to NCS-382, the only available and purportedly selective GHB receptor antagonist (Maitre et al., 1990). The presence of synthetic pathways, Ca++dependent release and specific high affinity receptors in brain, led to suggest that endogenous GHB may act as a putative neurotransmitter/neuromodulator (Maitre, 1997; Bernasconi et al., 1999). To date, however, if and the extent to which these specific GHB receptors contribute to the various pharmacological effects of GHB needs to be established. While ‘in vitro’ GHB shows very low affinity (100– 500 µM) for the GABAB receptor(s) (Bernasconi et al., 1992; Mathivet et al., 1997) and virtually no ability to interact with the sites for agonists and modulators associated with the GABAA receptor complex (Serra et al., 1991; Snead and Liu, 1993), some of its behavioral effects appear to involve GABAergic mechanisms. For instance, GABAA and GABAB receptor-mediated interoceptive cues participate in the discriminative stimulus effects produced by GHB (Colombo et al., 1998b), GHB-induced stimulation of growth hormone release in humans (Gerra et al., 1994) and anxiolytic action in rats (Schmidt-Mutter et al., 1998) are both blocked by flumazenil, an antagonist of the benzodiazepine site associated with the GABAA receptor complex, but not by the GHB receptor antagonist NCS-382 (Schmidt-Mutter et al., 1998). To reconcile these observations with the lack of affinity for the GABAA receptor or with the poor affinity exhibited by GHB in displacing GABAB receptor ligands in vitro, the ‘GABA-mimetic’ action of GHB has been ascribed to GABA itself, either formed from the enzymatic conversion of exogenous GHB, or released by GHB through a GHB receptor-mediated mechanism (Maitre, 1997; Gobaille et al., 1999).The time courses of these processes, though, appear too slow, when compared to the rapid GABA-mimetic—i.e. anxiolytic and sedative/hypnotic—actions observed after GHB administration. The study of the effects of GHB in alcohol-preferring and alcohol-dependent rats has revealed that some of its psychotropic actions are similar to those of ethanol, suggesting that may be mediated by the same mechanisms. For instance, Sardinian alcohol-preferring rats show higher sensitivity than Sardinian alcohol non-preferring rats to the sedative/hypnotic effect of ethanol as well as of GHB (Colombo et al., 1998a, 2000), and Wistar rats exhibit cross-tolerance to the motor-impairing effects of ethanol and GHB (Gessa et al., 2000). Potentiation of the GABAA receptor-mediated inhibition of neuronal excitability plays a role in sedation/hypnosis and motor impairment induced by ethanol (Grobin et al., 1998). Two factors may synergically contribute to this mechanism: a direct interaction between ethanol and the

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GABAA receptor (Grobin et al., 1998) and an ethanolinduced increase of the brain concentrations of the neurosteroids 3α-hydroxy-5α-pregnan-20-one (allopregnanolone, AP) and 3α,21-dihydroxy-5α-pregnan20-one (allotetrahydrodeoxy-corticosterone, THDOC) (Barbaccia et al., 1999; VanDoren et al., 2000). AP and THDOC, endogenous metabolites of progesterone and deoxycorticosterone, not only are potent positive allosteric modulators of the GABA-gated chloride current intensities and are endowed with anxiolytic, anticonvulsant and sedative/hypnotic actions (Majewska et al., 1986; Turner and Simmonds, 1989; Paul and Purdy, 1992; Lambert et al., 1995), but also play a permissive action on the amplification of GABA-gated chloride currents (Criswell et al., 1999) as well as on the sedative/hypnotic and anticonvulsant effects (VanDoren et al., 2000) induced by ethanol. In order to clarify the molecular mechanisms of the GABA-mimetic and ethanol-like actions of GHB, the present study was carried out to address the working hypothesis that GHB administration may increase the brain concentrations of ‘GABAergic’ endogenous neurosteroids AP and THDOC, and to characterize the receptor (i.e. GHB and/or GABAB) that mediates this action. A preliminary report of the results shown here has been presented earlier (Barbaccia et al., 2001).

2. Materials and methods 2.1. Animals and treatment Male Wistar rats, 300–350 g of body mass, were housed under standard laboratory conditions with 12-h light and 12-h dark periods, at a constant temperature of 22 ± 2°C, 60% relative humidity, with water and food ad libitum. GHB (75, 150, 300 and 1000 mg/kg, i.p.) was dissolved in double-distilled water and injected at the volume of 29.4 ml/kg. This large volume was chosen to minimize tissue irritation at the injection site. Control rats received an equal volume of saline. Baclofen (0, 2.5, 5 and 10 mg/kg, i.p.), NCS-382 (0, 50 and 300 mg/kg, i.p.) and SCH 50911 (0, 25 and 50 mg/kg, i.p.) were dissolved in saline. SCH 50911 and NCS-382, whose dose range is comparable to that previously shown to antagonize GABAB or GHB receptor-mediated events (Maitre et al., 1990; Maitre, 1997; Erhardt et al., 1998), were injected 30 min prior to GHB or saline, in a volume of 5 ml/kg. With the exception of the time-course experiments in which the rats were killed at the indicated time points, the rats, habituated to the handling that preceeds sacrifice for at least 4 days prior to the experiment, were killed 30 min after saline, GHB or baclofen administration. In each experiment, the rats were killed by guillotine between 11.00 and 13.00 h, alternating the rats from each group to minimize the inter-group variability

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due to circadian fluctuations of circulating steroid levels. The brain cortices and hippocampi from each rat were rapidly (⬍3 min) dissected and immediately frozen on dry ice. Blood was collected in buffered 0.1 M ethylendiamino-tetraacetic acid (EDTA, 100 µl/⬇2 ml of blood)-containing test-tubes and centrifuged at 2000g for 10 min at room temperature. Brain tissues and plasma were frozen at ⫺70 °C, until steroid assays. All procedures involving animal care and treatments were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and all efforts were made to minimize animal suffering. 2.2. Brain and plasma steroid extraction and measurement The procedures were carried out as described previously (Barbaccia et al., 1996). Briefly, the brain tissue (cortex and hippocampus) from each rat were homogenized with an Ultra-Turrax 125, in phosphate bufferedsaline (pH 7.0), additioned with 1 g/l bovine serum albumine and 1 g/l sodium-azide. After extraction of the steroids with ethyl-acetate (1:1, vol:vol, three times), the organic phases from each sample were pooled and dried in a vacuum centrifuge (Speed-vac, Savant). Following reconstitution of the extracts in 100% n-hexane, the steroids were separated by HPLC (Beckman, System Gold) on a 5 µm Lichrosphere-diol column, 4×250 mm, (Phenomenex), developed with a discontinuous gradient of 2-propanol, .from 0 to 30%, in n-hexane (in 60 min, flow rate 0.5 ml/min), preceded by a 10-min isocratic washing (flow rate 1.5 ml/min) with 100% n-hexane. The elution time of each steroid was consistently: 32– 34 min (progesterone), 38–40 min (AP), 44–46 min (pregnenolone), and 58–60 min (THDOC). The recovery (70–80%) of each steroid through the extraction and chromatographic procedures was monitored by adding, prior to the extraction, trace amounts (9000–10,000 dpm) of each tritiated steroid to every sample. Plasma (1 ml) was diluted 1:3 (vol/vol) with phosphate-buffered saline and extracted three times with ethyl-acetate (1:1, vol:vol). The three organic phases were pooled, dried and resuspended in n-hexane/2-propanol (70/30%). Appropriate fractions (1/1000 for corticosterone and 1/20 for all other steroids) of this solution were aliquoted for steroid RIA. The steroid recovery from plasma, monitored by adding trace amounts of 3 H-corticosterone (8–10,000 dpm), amounted to 85– 95%. The fractions eluted from the HPLC in the corresponding radioactive peak for each steroid were combined by washing three times the test tubes with 2propanol/n-hexane (30/70%). Appropriate aliquots, according to the relative abundance of each steroid, were tested by radioimmunoassay performed with specific antisera. The detection limit for each steroid was 0.01 ng/sample and the standard curves ranged from 0.005

Fig. 1. Dose-reponse of GHB on brain cortical neurosteroids. GHB (dissolved in double-distilled water) was injected i.p., in a volume of 29.4 ml/kg for each dose (75-150-300-1000 mg/kg). Veh ⫽ vehicle (saline, injected i.p. at the volume of 29.4 ml/kg). The rats were sacrificed 30 min after treatment. Each bar represents the mean ± SEM of 10 rats, and each determination was run in duplicate. ∗P ⬍ 0.05 and ∗∗P ⬍ 0.01, versus the respective value in vehicle (Veh)-treated rats.

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Table 1 GHB dose-dependently increases the concentrations of AP and THDOC in hippocampus Treatment (mg/kg)

AP (pmol/g)

THDOC (pmol/g)

Vehicle GHB (75) (150) (300) (1000)

1.23±0.13

5.0±0.53

2.3±0.42 2.8±0.58∗ 5.9±0.89∗∗ 7.0±1.1∗∗

8.4±1.26 10.3±1.0∗ 22±2.8∗∗ 20±3.1∗∗

The hippocampi were dissected from the brain of each rat whose brain cortical neurosteroid values are shown in Fig. 1. AP=allopregnanolone; THDOC=allotetrahydrodeoxy-corticosterone. The rats were killed 30 min after GHB administration. Each value represents the mean ± SEM of 10 individual determinations, each run in duplicate. ∗P ⬍ 0.05, ∗∗P ⬍ 0.01, when compared to the respective value in vehicle-treated rats.

to 1.0 ng. Raw data from the scintillation spectroscopy counting of each sample were analyzed by the RIA program of Munson P., Rodbard D. and Jaffe M.L. (N.I.H., Bethesda, MD). The intra- and inter-assay variation amounted to 5–7% and 15–20%, respectively. Proteins were quantified by the method of Lowry et al. (1951). Data are expressed as picomoles of steroid per gram of tissue (brain), considering a 1:10 ratio between protein and wet weight of tissue, or picomoles per milliliter (plasma). 2.3. Materials GHB was obtained from Laboratorio Farmaceutico CT, Sanremo, IM, Italy, and L-baclofen was purchased from RBI, Natick, MA, USA. NCS-382 (6,7,8,9-tetrahydro-5(H)-5-ol-ylidene acetic acid) was synthesized as described previously by Maitre et al. (1990) and SCH 50911 [(+)-5,5-dimethyl-2-morpholineacetic acid hyd-

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rochloride] was synthesized as described previously by Blythin et al. (1996). Pregnenolone, progesterone, AP, THDOC and corticosterone were purchased from Sigma–Aldrich (Milan, Italy). [7-3H]Pregnenolone (20 Ci/mmol), [1,2,6,7-3H]Progesterone (92 Ci/mmol), [9,11,12-3H]AP (50 Ci/mmol), [9,11,12-3H]THDOC (61 Ci/mmol) and [1,2,6,7-3H]Corticosterone (88 Ci/mmol) were purchased from New England Nuclear (Milan, Italy). Antisera to pregnenolone, progesterone and corticosterone were purchased from ICN (Milan, Italy). Antisera to AP and THDOC have been obtained by immunization of sheep and rabbit, respectively, and characterized as described previously (Purdy et al., 1990). Organic solvents (HPLC grade) were from Labscan (Dublin, Ireland). 2.4. Statistical analysis Data are expressed as the mean±SEM. Significant differences among groups means were analyzed by ANOVA followed by post hoc Scheffe’s and Dunnett’s T3 tests.

3. Results 3.1. Dose- and time-dependent GHB-induced increase of brain and plasma neurosteroid concentration Fig. 1(A)–(D) shows that 30 min following intraperitoneal administration, GHB (75-150-300-1000 mg/kg, i.p.) induced a dose-dependent increase in the concentrations of pregnenolone, progesterone, AP and THDOC in rat brain cortex. The sedative/hypnotic dose of 1000 mg/kg yielded the maximal increases (approximately 14, 13, 11 and seven fold for pregnenolone, progesterone, AP and THDOC, respectively). On the other hand, the dose of 150 mg/kg produced a significant increase of

Table 2 GHB and baclofen dose-dependently increase steroid plasma concentrations Treatment (mg/kg) Vehicle GHB (75) (150) (300) (1000) Baclofen (2.5) (5.0) (10.0)

PRO (pmol/ml)

AP (pmol/ml)

THDOC (pmol/ml)

CORT (pmol/ml)

4.4±0.73

3.1±0.33

8.5±1.1

305±72

3.9±0.51 10.7±3.9 36±4.7∗∗ 46±4.1∗∗

2.6±0.03 5.2±1.8 21±4.1∗∗ 17±2.3∗∗

12±2.2 20±5.4∗ 35±3.9∗∗ 42±5.5∗∗

370±51 500±90 760±46∗∗ 721±52∗∗

5.7±0.88 15±4.4∗ 35±2.4∗∗

4.0±1.4 8.3±2.1∗ 21±1.6∗∗

12.5±3.0 13.1±3.5 38±3.0∗∗

356±65 325±63 663±88∗∗

PRO=progesterone; AP=allopregnanolone; THDOC=allotetrahydrodeoxycorticosterone; CORT=corticosterone. The rats were treated i.p. with either saline (Vehicle), GHB or baclofen and killed 30 min thereafter. Each value represents the mean ± SEM of seven (baclofen) and 10 (saline and GHB) individual determination, each run in duplicate. ∗P ⬍ 0.05 and ∗∗P ⬍ 0.01, versus the respective value in vehicle-treated rats.

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progesterone and THDOC (+298 and +136%, respectively, P ⬍ 0.05), but failed to significantly affect pregnenolone and AP (+82 and +77%, respectively, P ⬎ 0.1). GHB increased the concentrations of AP and THDOC, as well as of their precursors pregnenolone and progesterone (not shown), also in the hippocampus of the same rats (Table 1). At variance with cerebral cortex, both neurosteroids were significantly enhanced (+128 and 106%, respectively, P ⬍ 0.05) by the dose of 150 mg/kg, while the two higher GHB doses were equally effective on hippocampal AP and THDOC concentrations. The plasma concentrations of pregnenolone (not shown), progesterone, AP, THDOC and corticosterone were also dose-dependently increased by GHB (Table 2). However, the time course (Fig. 2(A) and (B)) of the GHB effect on steroid rate of synthesis/metabolism was

different in plasma and brain; in brain cortex the AP and THDOC enhancement by 300 mg/kg GHB was apparent after 30 min and persisted up to 90 min, while in plasma, not only was less marked, but also showed a peak at 30 min and abated by 90 min. The brain cortical concentrations of the precursors pregnenolone and progesterone were still higher than control values, but already started to decline at 90 min, with respect to the values at 30 min. Moreover, a correlation analysis between the plasma corticosterone values (an index of HPA axis activation) and the brain cortical AP and THDOC values, measured 30 min post-administration of either vehicle or 300 mg/kg GHB, revealed a positive and significant correlation [Pearsons correlation coefficients (r) ⫽ 0.734 (n ⫽ 23, P ⬍ 0.01) and 0.747 (n ⫽ 23, P ⬍ 0.01), for corticosterone versus AP and THDOC, respectively] in vehicle-treated rats, but not in GHBtreated rats [Pearsons correlation coefficients (r) ⫽ 0.289 (n ⫽ 22, P ⫽ 0.204) and 0.298 (n ⫽ 22, P ⫽ 0.157), for corticosterone versus AP and THDOC, respectively]. The concentrations of dehydroepiandrosterone, a neurosteroid that positively modulates the NMDA-type of glutamate receptor (Paul and Purdy, 1992; Lambert et al., 1995), were not affected by GHB either in brain or in plasma (data not shown). 3.2. Effect of GABAB and GHB receptor antagonists on the GHB-induced brain neurosteroid increase

Fig. 2. Time-dependent effect of GHB on brain and plasma steroid concentration. Rats were treated with GHB (300 mg/kg, i.p.) and killed after 15, 30, 90, 180 min (n ⫽ 6, for each time point). Control rats (n ⫽ 16) were treated with vehicle (Veh) and killed in groups of four at each time point. The steroid values in vehicle-treated rats, pooled because they were not significantly different in the four groups, were: in brain cortex (pmol/g), pregnenolone 25.3 ± 3.5, progesterone 9.9 ± 1.8, AP 0.65 ± 0.058, THDOC 5.6 ± 0.92; in plasma (pmol/ml), pregnenolone 3.2 ± 0.31, progesterone 3.5 ± 0.71, AP 4.2 ± 0.29, corticosterone 318 ± 58. THDOC in plasma showed a time course similar to that of the other steroids (not shown). N ⫽ 6, for each GHB time point. ∗P ⬍ 0.05 and ∗∗P ⬍ 0.01, versus the respective vehicle value.

SCH 50911, a selective GABAB receptor antagonist (Bolser et al., 1995; Blythin et al., 1996) which fails to compete for 3H-GHB binding to GHB receptors (Snead, 1996), administered at the dose of 50 mg/kg, i.p. 30 min prior to GHB (300 mg/kg), completely blocked the GHB-induced increase of pregnenolone (not shown), progesterone, AP and THDOC in rat brain cortex (Fig. 3(A)–(C)) and corticosterone in plasma, as well (Table 3). The lower SCH 50911 dose (25 mg/kg) only marginally reduced the GHB-elicited THDOC increase in brain cortex (Fig. 3(C)). Both SCH 50911 doses were per se ineffective on the brain and plasma neurosteroid concentrations. In contrast, NCS-382, a selective antagonist of the GHB receptor (Maitre et al., 1990; Gobaille et al., 1999) which does not compete for 3H-GABA binding to GABAB receptors (Snead, 1996), administered at the dose of 50 mg/kg, per se ineffective, failed to prevent the neurosteroid increase induced by GHB, both in brain (Fig. 3(A)–(C)) and in plasma (Table 3). At 300 mg/kg, NCS-382 injected to vehicle-treated rats induced a marked increase of pregnenolone (not shown), progesterone, AP and THDOC in brain cortex (Fig. 3AC) and plasma (Table 3). This effect, however, was not additive to that of GHB, whose enhancement of brain neurosteroid concentrations was occluded in the presence of 300 mg/kg NCS-382. Interestingly, the GHBinduced stimulation of plasma corticosterone concen-

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Fig. 3. The GABAB receptor antagonist SCH 50911, but not the GHB receptor antagonist NCS-382, antagonizes the neurosteroid stimulation induced by GHB in brain cortex. SCH 50911 (SCH) and NCS-382 (NCS) were dissolved in vehicle (Veh ⫽ saline) and administered i.p., at the doses indicated, 30 min prior to GHB (300 mg/kg). The rats were killed 30 min after saline or GHB administration. Each bar represents the mean ± SEM of eight rats. ∗∗P ⬍ 0.01, versus the respective value in vehicle-treated rats.

trations was potentiated by 50 mg/kg NCS-382, while at 300 mg/kg NCS-382, either alone or in combination with GHB, stimulated plasma corticosterone levels to a greater extent than GHB alone (Table 3).

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Fig. 4. Baclofen mimics GHB effect on brain cortical neurosteroid. Baclofen (2.5–5 and 10 mg/kg, i.p.), dissolved in saline, and GHB (300 mg/kg, i.p.) dissolved in double-distilled water, were administered i.p. 30 min prior to sacrifice. Vehicle-treated rats (Veh) received an equivalent volume of saline. Values for each steroid in vehicle-treated rats represent a pool of the two respective groups. Each bar represents the mean ± SEM of seven rats. ∗∗P ⬍ 0.01, versus the respective value in vehicle-treated rats.

3.3. Effect of baclofen on brain and plasma neurosteroid concentrations The selective GABAB receptor agonist baclofen (2.55-10 mg/kg, i.p.) dose-dependently increased the con-

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Table 3 SCH 50911, a GABAB receptor antagonist, but not NCS-382, a GHB receptor antagonist, inhibits the GHB-induced increase of plasma steroid concentrations Treatment (mg/kg)

PRO (pmol/ml)

AP (pmol/ml)

Vehicle GHB (300) SCH (25) +GHB SCH (50) +GHB NCS (50) +GHB NCS (300) +GHB

4.2±0.38 31±5.3∗ 4.6±0.44 32±4.8∗ 4.0±0.35 5.2±0.48 3.7±0.62 27±2.5∗ 19.5±2.6∗ 21±1.9∗

3.9±0.51 20±5.0∗ 4.8±0.47 21±3.4∗ 4.8±0.53 4.5±0.5 4.4±0.93 22±2.9∗ 18±2.6∗ 19±1.5∗

THDOC (pmol/ml) 6.9±0.65 30±6.1∗ 7.0±0.33 31±4.5∗ 7.9±0.93 7.7±0.84 8.6±1.8 28±3.6∗ 28±3.0∗ 29±3.3∗

CORT (pmol/ml) 292±58 615±54∗ 324±35 648±102∗ 264±40 371±84 457±117 924±124∗∗ 1400±191∗∗ 1533±74∗∗

The rats were treated with vehicle (saline), SCH 50911 or NCS-382 (at the indicated doses) and 30 min later received either saline or GHB (300 mg/kg). The animals were killed 30 min after GHB injection. Each value represents the mean ± SEM of eight individual determination, each run in duplicate. ∗P ⬍ 0.05, versus the respective value in vehicle-treated rats; ∗∗P ⬍ 0.05, versus the respective value in GHB-treated rats.

centrations of pregnenolone (not shown), progesterone, AP and THDOC in brain cortex (Fig. 4(A)–(C)). The increase induced by the highest dose tested (10 mg/kg) was comparable to that induced by 300 mg/kg GHB, while the enhancement of progesterone, AP and THDOC induced by 5 mg/kg baclofen approached to significance. The same dose-related effect of baclofen was observed also on the plasma concentrations of all steroids, with the exception of corticosterone, that was clearly, and significantly, increased only by 10 mg/kg baclofen, but not at any other dose tested (Table 2).

4. Discussion The major finding of this study is that the systemic administration of GHB significantly increases the brain concentrations of AP and THDOC, two potent endogenous positive allosteric modulators of the GABA-gated chloride currents (Majewska et al., 1986; Turner and Simmonds, 1989; Paul and Purdy, 1992; Lambert et al., 1995) endowed with anxiolytic, sedative/hypnotic and anticonvulsant actions. Furthermore, the antagonism by the selective GABAB receptor antagonist SCH 50911 (Bolser et al., 1995; Blythin et al., 1996) and the GHBmimetic action of baclofen on brain neurosteroids strongly suggest that this GHB effect is mediated by an activation of GABAB receptors. Indeed, the active doses of GHB (present results) would yield brain GHB concentrations (Gobaille et al., 1999) compatible with the affinity (high micromolar) exhibited by GHB at GABAB receptors (Bernasconi et al., 1992; Mathivet et al., 1997). The concomitant and SCH 50911-sensitive increases of plasma corticosterone, progesterone, AP and THDOC, suggest that the GHB-induced increase in brain neurosteroids is associated with the activation of the HPA axis. This inference is supported by the fact that baclofen also

increases plasma corticosterone upon systemic administration, though fails to elicit corticosterone release when directly applied to adrenal cells (Hausler et al., 1993), and is consistent with the existence of presynaptic GABAB receptors on GABAergic afferents to neurons of the hypothalamic paraventricular nucleus (Cui et al., 2000). By reducing GABA release, the activation of these GABAB receptors would hinder the tonic inhibitory control, operated by GABA through GABAA receptors (Boudaba et al., 1996), on corticotropic releasing hormone secretion and thereby on HPA axis (Fig. 5). Accordingly, pharmacological manipulations leading to a reduction of GABAA receptor-mediated synaptic transmission increase plasma corticosterone and brain AP concentrations, while an anxiolytic dose of abecarnil, a positive allosteric modulator of GABAA receptors, fails to affect the HPA axis (Barbaccia et al., 1997). Nevertheless, the present results show a lack of correlation between plasma corticosterone and brain AP and THDOC concentrations in GHB-treated rats, as well as a greater and outlasting effect of GHB on brain neurosteroids, with respect to that on corticosterone, pregnenolone, progesterone, AP and THDOC in plasma. In addition, we found a marked and early peaking increase of the precursors—pregnenolone and progesterone—in brain, and a different dose-responsiveness of AP and THDOC to GHB in brain cortex and hippocampus. These findings indicate that rate of synthesis/metabolism of these steroids is differently affected by GHB in different brain regions and in plasma, with respect to brain, and suggest that the GHB-induced enhancement of AP and THDOC in brain may partially depend on the local expression or activation of steroidogenic enzymes (Mensah-Nyagan et al., 1999). The failure of NCS-382 in inhibiting GHB in our experiments rules out an involvement of high affinity GHB receptors in this action of GHB. On the other hand,

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Fig. 5. Hypothetical mechanisms involved in the GHB-induced increase of brain neurosteroid concentrations. Corticotropic releasing hormone (CRH)-neurons of the hypothalamic paraventricular nucleus (PVN) receive GABAergic inputs from the surrounding hypothalamic regions (Boudaba et al., 1996). The latter, through GABAA receptors, hyperpolarize CRH neurons. As we are not aware of a direct GABAB-mediated control of ACTH release in the pituitary, we postulate that presynaptic GABAB receptors (target of GHB and baclofen) on the GABAergic afferents to the PVN (Cui et al., 2000) would reduce GABA release, which in turn may result in an increased release of CRH and activation of the HPA axis, as indicated by the increase of plasma corticosterone. The concomitant enhancement of circulating pregnenolone, progesterone, AP and THDOC may increase the availability of these neuroactive steroids either directly or indirectly, by supplying brain tissue with greater concentrations of the precursors (pregnenolone, progesterone and, presumably, deoxycorticosterone), which may be locally metabolized to AP and THDOC.

the marked increase of neurosteroids in brain and of corticosterone in plasma elicited by the high NCS-382 dose suggests that NCS-382 either increases the levels of endogenous GHB by inhibiting its metabolism (Maitre, 1997), or that this molecule might have an, as yet, undescribed (partial) agonist action at GABAB receptors. Both these hypotheses, consistent with the potentiation of the GHB-induced corticosterone response by NCS382, cannot be ruled out because it was not tested whether SCH 50911 would inhibit NCS-382 effect. However, in line with the latter hypothesis, a GHB-like constipating effect of NCS-382, blocked by SCH 50911, was recently observed in mouse small intestine (Carai et al., unpublished results). Could the neurosteroid increases in brain bear any relevance to the pharmacological effects of GHB? Assuming that AP and THDOC are evenly distributed throughout brain cortex and hippocampus, their respective actual concentrations after GHB reach 3–5 and 15– 40 nM. Though they may appear low, when compared to those that have been added to in vitro preparations to amplify GABAA receptor function (Turner and Simmonds, 1989; Paul and Purdy, 1992; Lambert et al., 1995), a reduction of the endogenous brain cortical AP

concentration from approximately 3 to less than 1 nM results in a decreased potency of muscimol and GABA at native GABAA receptors and in a reduction of muscimol-induced sleeping time (Pinna et al., 2000). Vice versa, the acute stress-induced increase in brain AP, by an extent similar to that observed in the present experiments, was found to correlate with the recovery of GABAA receptor function and with the waning of stresselicited anxiety-related behavior (Barbaccia et al., 1996). Thus, it is reasonable to suggest that the GHB-induced changes in brain AP and THDOC concentrations may affect the efficiency of transmission at GABAA receptors and that, through this mechanism, these neurosteroids may modulate/mediate the psychotropic—i.e. anxiolytic and sedative/hypnotic—actions of GHB. In line with this inference, not only SCH 50911 (50– 100 mg/kg) blocks the sedative/hypnotic actions of GHB—while NCS-382 up to 500 mg/kg does not (Carai et al., 2001), but also the GHB doses that increase brain neurosteroids are in the range of those that also produce GABAA receptor-mediated pharmacological effects. Indeed, 150–250 mg/kg GHB induce an anxiolytic effect, insensitive to NCS-382 but blocked by the selective antagonist of the GABAA receptor-coupled benzodi-

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azepine site flumazenil, in rats exposed to the elevated plus maze (Schmidt-Mutter et al., 1998). Flumazenil also blocks the anxiolytic effect of AP when assessed in the burying behavior test (Fernandez-Guasti and Picazo, 1995), though not in the conflict test of anxiety (Brot et al., 1997). Moreover, GHB (300 mg/kg), similar to AP and THDOC (Bowen et al., 1999), produces ethanol-like discriminative stimulus effects, involving GABAA receptor-mediated interoceptive cues (Colombo et al., 1998b; Gessa et al., 2000). At higher dose (1000 mg/kg), GHB induces sleep (Colombo et al., 1998a) and counteracts ethanol withdrawal symptoms, including spontaneous convulsions, in alcohol dependent rats (Gessa et al., 2000). Interestingly, ethanol-withdrawing rats are cross-tolerant to the anticonvulsant effect of barbiturates and benzodiazepines, but show increased sensitivity to AP and THDOC (Devaud et al., 1996). Also, the time course of the GHB-elicited increase in brain neurosteroid concentrations (present results) is consistent with the time courses of its sedative/hypnotic and antialcohol actions, which show a peak between 30 and 60 min postdrug administration and last for at least 2 h (Colombo et al., 1998a; Gessa et al., 2000). These results, which represent the first demonstration that GHB, presumably via a GABAB receptor-mediated mechanism, markedly increases the brain concentrations of ‘GABAergic’ neurosteroids AP and THDOC, open a new perspective to understand the molecular mechanisms underpinning the GABA-mimetic psychotropic actions of GHB. As GHB activates HPA axis also in humans (Van Cauter et al., 1997), endogenous neurosteroids might have relevance also for some clinical aspects of GHB pharmacology, in particular for the induction of slow wave sleep (Mamelak et al., 1977), regularization of sleep pattern in narcoleptic patients (Lammers et al., 1993) and the anti-alcohol effects in alcohol-dependent subjects (Addolorato et al., 2000; Gallimberti et al., 2000; Gessa et al., 2000). Moreover, the observation that CGP 35348, another selective GABAB receptor antagonist, blocks the discriminative stimulus effect of GHB (Colombo et al., 1998b, 2000) calls for additional experiments to evaluate whether endogenous neurosteroids may be implicated also in the addictive properties of GHB.

Acknowledgements

This work was supported by grants (no. 2001055342—001 and no. 2001055342—002) from M.I.U.R. (Ministry of Instruction, University and Research, Italy) to G.L.G. and M.L.B.

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