Address correspondence and reprint requests to Laura Dazzi, Department of Experimental Biology ‘B. Loddo’, University of Cagliari, 09123 Cagliari, Italy. E-mail: firstname.lastname@example.org
The effect of endogenous 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) on the modulation of mesocortical dopamine extracellular concentration by ethanol was investigated by microdialysis in rats. Intraperitoneal injection of progesterone (5 mg/kg, once a day for 5 days) increased the cortical content of 3α,5α-TH PROG and potentiated the biphasic effect of acute intraperitoneal administration of ethanol on dopamine content. A dose of ethanol (0.25 g/kg) that was ineffective in naïve rats induced a 55% increase in dopamine extracellular concentration in rats pretreated with progesterone. This increase was similar to that induced by a higher dose (0.5 g/kg) of ethanol in naïve rats. Administration of ethanol at 0.5 g/kg to progesterone-pretreated rats inhibited dopamine content by an extent similar to that observed with an even higher dose (1 g/kg) in naïve rats. The administration of the 5α-reductase inhibitor finasteride (25 mg/kg, subcutaneous), together with progesterone, prevented the effects of the latter, both on the cortical concentration of 3α,5α-TH PROG and on the modulation by ethanol of dopamine content. These data suggest that 3α,5α-TH PROG contributes to the action of ethanol on the mesocortical dopaminergic system. They also suggest that physiological fluctuations in the brain concentrations of neuroactive steroids associated with the oestrous cycle, menopause, pregnancy and stress may alter the response of mesocortical dopaminergic neurons to ethanol.
The progesterone metabolite 3α-hydroxy-5α-pregnan-20-one (allopregnanolone, or 3α,5α-TH PROG), whose concentration in the brain is increased by ethanol intake, has recently been proposed to mediate various specific actions of ethanol (Morrow et al. 2001). The increase in the concentration of 3α,5α-TH PROG in rat brain thus correlates with the hypnotic, anticonvulsant and sedative effects of ethanol (VanDoren et al. 2000). Moreover, 3α,5α-TH PROG is able to substitute for ethanol in a discriminative stimulus paradigm in both primates and rats (Bowen et al. 1999; Engel and Grant 2001), and it provides protection against seizures induced by ethanol withdrawal in rats (Devaud et al. 1995; Finn et al. 1995). Given that 3α,5α-TH PROG is one of the most potent positive allosteric modulators of GABAA receptors, affecting both receptor function (Majewska 1992) and gene expression (Follesa et al. 2000, 2001), and that it induces hypnotic, anxiolytic, and anti-stress effects in animals and humans (for review see Biggio and Purdy 2001), this neuroactive steroid might mediate effects of short- or long-term administration of ethanol by promoting GABAergic transmission in specific brain regions. Indeed, the modulatory efficacy of 3α,5α-TH PROG with regard to both [3H]flunitrazepam, or t-[35S]butylbicyclophosphorothionate binding to and 36Cl– uptake mediated by GABAA receptors (Majewska et al. 1986; Gee et al. 1987), as well as its anticonvulsant effect (Belelli et al. 1989), are enhanced in the brain of ethanol-dependent rats (Devaud et al. 1995, 1996; Mehta and Ticku 1998).
Given that 3α,5αTH PROG, like other GABAergic drugs, modulates both basal and stress-activated mesocortical dopaminergic transmission (Motzo et al. 1996; Dazzi et al. 2002), and that such transmission plays an important role in the pharmacology of ethanol (Grace 2000; Weiss and Porrino 2002), we have now evaluated whether increasing the basal brain concentration of this neurosteroid affects the action of ethanol on cortical dopamine extracellular concentration. Our results are thus relevant to clarifying whether the fluctuations in the brain concentrations of neurosteroids associated with the oestrous cycle, menopause, pregnancy, stress and depression (Biggio and Purdy 2001) might alter the response of mesocortical dopaminergic neurons to ethanol intake.
Materials and methods
Male Sprague-Dawley CD rats (Charles River, Como, Italy), with initial body masses of 200–220 g, were maintained under an artificial 12-h light/12-h dark cycle (light on 0800–2000 h) at a temperature of 22° ± 2°C and 65% humidity. Food and water were freely available, and the rats were acclimatized to the animal facility for at least 8 days before experiments. Animal care and handling throughout the experimental procedures were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). The experimental protocols were approved by the Animal Ethics Committee of the University of Cagliari.
Ethanol (20%, w/v) was administered intraperitoneally at doses of 0.25–1 g/kg of body mass. Progesterone was dissolved in olive oil by sonication for 4 h and administered intraperitoneally once a day for 5 days in a volume of 3 mL/kg. Finasteride was extracted from tablets of Prostide (SigmaTau, Milan, Italy), each containing 5 mg of drug, as described previously (Trapani et al. 2002); it was dissolved in 20% 2-hydroxypropyl-β-cyclodextrin by sonication for 4 h and injected subcutaneously at the same time as progesterone administration, as well as 24 h after the last progesterone injection (1 h before the onset of microdialysis). Control animals received an identical volume of vehicle.
Rats were anaesthetized by intraperitoneal injection of chloral hydrate (0.4 g/kg), and a concentric dialysis probe was inserted at the level of the medial prefrontal cortex (A +3.2, ML +0.8, V −5.3 relative to the bregma) according to the Paxinos atlas (Paxinos and Watson 1982). The active length of the dialysis membrane (Hospal Dasco, Bologna, Italy) was restricted to 2 mm. Surgery was performed at ∼0900 h. Experiments were performed in freely moving rats ∼24 h after probe implantation, between 0900 and 1800 h to allow recovery from surgery procedures. Ringer's solution [3 mm KCl, 125 mm NaCl, 1.3 mm CaCl2, 1 mm MgCl2, 23 mm NaHCO3, 1.5 mm potassium phosphate (pH 7.3)] was pumped through the dialysis probe at a constant rate of 2 µL/min. Samples of dialysate were collected every 20 min and immediately analyzed for dopamine by HPLC with coulometric detection, as previously described (Dazzi et al. 1997); the detection limit for dopamine was 2 fmol per injection. The average dopamine concentration of the last three samples before treatment was taken as 100%, and all post-treatment values were expressed as a percentage relative to the basal value. Stable values of dopamine were achieved ∼3–4 h after the onset of microdialysis; ethanol was therefore administered ∼28–29 h after the last progesterone injection. The in vitro recovery value for the probes was 24 ± 3% (mean ± SEM); all probes were tested before implantation, and those with a recovery value outside of this range were not used. The absolute concentration of dopamine was not corrected for recovery value. At the end of each experiment, the placement of the probe was verified histologically. All rats in which the probe was detected outside of the prefrontal cortex were excluded from the analysis.
For steroids measurements, rats were killed by focused microwave irradiation (70 W/cm2 for 4 s) to the head, which results in almost instantaneous inactivation of brain enzymes and thus minimizes post-mortem steroid metabolism. The brain was rapidly (< 1 min) removed from the skull, and the cerebral cortices were dissected and then frozen at −20°C until steroid extraction. Steroids present in cortical homogenates [40 mg of protein in 4 mL of phosphate-buffered saline (pH 7.0)] were extracted three times with ethyl acetate, as previously described (Serra et al. 2000).
The recovery (70–80%) of steroids through the extraction and purification procedures was monitored by adding a trace amount (6000–8000 cpm; 20–80 Ci/mmol) of 3H-labelled standards to the brain homogenate. Steroids were quantified by radioimmunoassay (Serra et al. 2000), with specific antibodies to progesterone (ICN, Costa Mesa, CA, USA) to AP and to allotetrahydrodeoxycorticosterone (THDOC), the latter of which were generated in sheep and characterized as described previously (Serra et al. 2000).
Data are presented as means ± SEM. Comparisons between groups were performed by two-way anova for repeated measures. Comparison among groups of data expressed as a percentage of baseline release is confounded when basal concentrations of neurotransmitter vary among groups; we therefore used the raw baseline values for statistical analysis. Post-hoc comparisons were performed by Neuman–Keuls test. A p-value of < 0.05 was considered statistically significant.
A single intraperitoneal administration of ethanol induced a dose-dependent biphasic effect on the extracellular concentration of dopamine in the rat prefrontal cortex (Fig. 1). Although it had no significant effect at a dose of 0.25 g/kg, at the dose of 0.5 g/kg ethanol elicited an increase (+ 65%) in dopamine content that was maximal 40 min after injection and remained significant for ∼100 min. In contrast, at the dose of 1 g/kg, ethanol induced a decrease (−45%) in the extracellular concentration of dopamine in this brain area, that was maximal 40 min after injection and lasted for 80 min (inset to Fig. 1; F1,20 = 11.028, p < 0.001).
Prior treatment with progesterone resulted in a significant potentiation of the effect of ethanol on cortical dopamine extracellular concentration (Fig. 2). At the dose of 0.25 g/kg, which was ineffective in naïve or vehicle-pretreated rats, ethanol thus induced a significant increase (+ 55%) in the extracellular concentration of dopamine in progesterone-pretreated animals (F1,24 = 9.124, p < 0.001). The extent of this increase was similar to that induced by the administration of a higher dose (0.5 g/kg) of ethanol in naïve or vehicle-pretreated rats. In contrast, pretreatment of animals with progesterone reversed the increase in cortical dopamine content normally elicited by ethanol at 0.5 g/kg; ethanol at this dose thus inhibited dopamine content in progesterone-pretreated rats by an extent similar to that induced by the dose of 1 g/kg in naïve rats (F1,20 = 12.548, p < 0.001).
To clarify the mechanism by which progesterone modulates the effect of ethanol on cortical dopamine release, we measured the cerebrocortical concentration of 3α,5α-TH PROG. Administration of progesterone for 5 days induced a significant increase in the cerebrocortical concentration of 3α,5α-TH PROG (15.86 ± 1.2 vs. 10.79 ± 1.0 ng/g protein for progesterone- or vehicle-pretreated rats, respectively; F1,22 = 8.067, p = 0.0095) measured 25–30 h after the last injection. Moreover, subcutaneous injection of the selective 5α-reductase inhibitor finasteride, together with progesterone treatment, prevented the increase in the cortical concentration of 3α,5α-TH PROG induced by the latter (10.12 ± 0.9 ng/g protein). Similarly, finasteride prevented the potentiating effect of progesterone on the increase in cortical dopamine extracellular concentration induced by ethanol at a dose of 0.25 g/kg (Fig. 3). Furthermore, at the dose of 0.5 g/kg, ethanol induced an increase (+ 55%) in the extracellular concentration of dopamine in rats pretreated with both progesterone and finasteride (data not shown). Finally, pretreatment with progesterone in the absence or presence of finasteride did not significantly affect the basal extracellular concentration of dopamine in the prefrontal cortex (Fig. 3; p = 0.685). In fact, the basal extracellular concentration of dopamine in the prefrontal cortex of control rats was 15.24 ± 1.95 fmol per 40-µL sample. Subchronic treatment with progesterone (5 mg/kg, once a day for 5 days) or finasteride (25 mg/kg, once a day for 5 days) did not significantly affect this parameter. In fact, basal extracellular concentration of dopamine was 14.02 ± 2.10 fmol per 40 µL sample in progesterone treated rats (p = 0.725 vs. control rats) and 15.56 ± 1.95 fmol in 40 µL sample in finasteride treated rats (p = 0.685 vs. control rats). Similarly, acute administration of either drug had no effect on cortical dopamine content (Fig. 1).
Pretreatment of rats with progesterone shifted the dose–response relation for the effect of acute intraperitoneal administration of ethanol on dopamine content in the prefrontal cortex to higher ethanol concentrations. In rats pretreated with progesterone, which alone had no effect on the basal extracellular concentration of dopamine, a low dose (0.25 g/kg) of ethanol that was ineffective in naïve or vehicle pretreated animals thus induced a marked increase in cortical dopamine extracellular concentration. In contrast, a higher dose (0.5 g/kg) of ethanol that significantly increased dopamine content in naïve or vehicle-pretreated rats induced a pronounced decrease in the cortical extracellular concentration of this monoamine in progesterone-pretreated animals. This latter effect was similar to that elicited by an even higher dose (1 g/kg) of ethanol in naïve rats. Moreover, similar progesterone treatment significantly increased the cortical concentration of the progesterone metabolite 3α,5α-TH PROG.
Subchronic progesterone treatment has been shown to increase also the brain content of THDOC (+ 38%; Serra, unpublished results), a neuroactive steroid able to elicit anxiolytic and anticonvulsant effects (Crawley et al. 1986; Kokate et al. 1996; Pick et al. 1996) and to modulate basal and stress-induced dopamine extracellular concentration (Grobin et al. 1992). Given that brain content of THDOC is increased by ethanol administration (Barbaccia et al. 1999), a possible role of this progesterone metabolite in the modulation of the effect of ethanol on cortical dopamine neurons cannot be excluded.
The modulation by progesterone of the effects of both low and high doses of ethanol on cortical dopamine content was abolished by concomitant administration of finasteride. Given that the inhibition of 5α-reductase (Rasmusson et al. 1986), by repeated administration of finasteride, results in a marked decrease in both plasma and brain concentrations of 3α,5α-TH PROG (Concas et al. 1998; Dazzi et al. 2002), our results suggest that this progesterone metabolite plays an important role in determining the efficacy with which ethanol affects mesocortical dopamine content. This conclusion is supported by the observation that finasteride prevented the increase in the cortical concentration of 3α,5α-TH PROG induced by progesterone in the present study. Moreover, our results are also consistent with the previous demonstration that a dose- and time-dependent increase in the cortical content of 3α,5α-TH PROG elicited by systemic ethanol administration contributes both to the actions of this drug at GABAA receptors as well as to electrophysiological and behavioural effects of ethanol in vivo (Morrow et al. 2001).
Our data suggest that an increase in the brain concentration of 3α,5α-TH PROG in response to ethanol may alter the functional activity of specific neuronal populations, such as the mesocortical dopaminergic neurons. Consistent with this conclusion, we have previously shown that administration of pharmacological doses of 3α,5α-TH PROG both results in a marked decrease in the basal concentration of dopamine in the rat prefrontal cortex and abolishes the stimulatory effect of stress or anxiogenic drugs on dopamine content in the same brain area (Motzo et al. 1996). Moreover, we have more recently demonstrated that the finasteride-induced reduction in the cerebrocortical content of 3α,5α-TH PROG is associated with a marked potentiation of the stimulatory effect of stress on mesocortical dopamine extracellular concentration (Dazzi et al. 2002).
The abilities of progesterone to increase the brain content of 3α,5α-TH PROG and to potentiate the action of ethanol on cortical dopamine content, together with the shared ability of ethanol and 3α,5α-TH PROG to modulate the function of GABAA receptors (Lambert et al. 1995; Grobin et al. 1998), suggest that the interaction between 3α,5α-THPROG and ethanol is important for the modulation by ethanol of mesocortical dopaminergic neurons. Although ethanol and 3α,5α-TH PROG each potentiate GABAA receptor function in rat brain, they do so by different mechanisms. Whereas 3α,5α-TH PROG activates the GABA-gated Cl– channel by interacting directly with the receptor, ethanol has been proposed to bind to small binding pocket in the α1 subunit of the GABAA receptor (Mihic et al. 1997).
GABAA receptors located on the somas of dopaminergic neurons in the ventral tegmental area regulate the activity of the mesocortical dopaminergic pathway (Tzschentke 2001). Activation of these receptors by anxiolytic benzodiazepines or their inhibition by anxiogenic compounds thus results in inhibition and activation, respectively, of the mesocortical dopaminergic pathway (Tam and Roth 1990; Imperato et al. 1994; Dazzi et al. 1995; Hutson and Barton 1997). Given the role of GABAA receptors in regulation of mesocortical dopaminergic neurons, it might be expected that changes in the brain concentrations of endogenous GABAA receptor modulators such as 3α,5α-THPROG would also affect the activity of these neurons.
The mesocortical dopamine system contributes to modulation of complex behaviours, including addiction, motor activity, sensitization, learning, cognition and emotional state (Tzschentke 2001). Moreover, these neurons mediate many central effects of ethanol, including aggressive behaviour (Miczek et al. 1994). A change in the activity of these neurons in response to fluctuations in the brain content of 3α,5α-TH PROG might thus result in behavioural changes associated with various mental disorders. Given that marked fluctuations in the plasma and CSF concentrations of 3α,5α-TH PROG are associated with physiological or pathological conditions such as menopause (Barbaccia et al. 2000), premenstrual syndrome (Rapkin et al. 1997; Girdler et al. 2001; Rasgon et al. 2001), ageing (Genazzani et al. 1998), stress (Purdy et al. 1991; Serra et al. 2000), and depression (Romeo et al. 1998; Uzunova et al. 1998), it is possible that changes in the brain content of this neurosteroid reflect an neurochemical mechanism responsible for modulation of the activity of the mesocortical dopamine system as well as for the development of anxiety and affective disorders. Indeed, administration of antidepressant drugs (Romeo et al. 1998; Uzunova et al. 1998) or hormone replacement therapy (Florio et al. 2001), treatments that relieve some of the symptoms associated with anxiety or affective disorders, changes the concentration of 3α,5α-TH PROG in the plasma (and probably in the brain) of patients with such conditions.
Finally, the synergistic action exerted by 3α,5α-TH PROG and ethanol on dopamine content in the prefrontal cortex of freely moving rats suggests that ethanol intake associated with physiological, pharmacological, or pathological changes in the brain concentration of 3α,5α-TH PROG, may result in alterations in mental activity modulated by mesocortical dopaminergic neurons. Such a scenario might reflect a biochemical mechanism involved in the physiopathology of emotional state often associated with changes in the concentration of this endogenous neurosteroid.