Address correspondence and reprint requests to Mariangela Serra, Department of Experimental Biology, Chair of Pharmacology, University of Cagliari, Cagliari 09100, Italy. E-mail: firstname.lastname@example.org
Social isolation of rats for 30 days immediately after weaning results in marked decreases in the cerebrocortical and plasma concentrations of pregnenolone, progesterone, 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG), and 3α,5α-tetrahydrodeoxycorticosterone (3α,5α-TH DOC), as well as a moderate increase in the plasma concentration of corticosterone. This mildly stressful condition has now been shown to increase the sensitivity of rats to the effect of acute ethanol administration on the cerebrocortical and plasma concentrations of neuroactive steroids. The percentage increases in the brain and plasma concentrations of pregnenolone, progesterone, 3α,5α-TH PROG, and 3α,5α-TH DOC, apparent 20 min after a single intraperitoneal injection of ethanol (1 g/kg), were thus markedly greater in isolated rats than in group-housed animals. A subcutaneous injection of isoniazid (300 mg/kg) also induced greater percentage increases in the concentrations of these steroids in isolated rats than in group-housed animals. These results suggest that mild chronic stress, such as that induced by social isolation, enhances the steroidogenic effect of ethanol, a drug abused by humans under stress or affected by neuropsychiatric disorders. Social isolation also induced hyper-responsiveness of the hypothalamic–pituitary–adrenal (HPA) axis, as was apparent after reduction of GABA-mediated inhibitory tone by isoniazid administration.
Social isolation of rats immediately after weaning results in a decrease in the brain and plasma concentrations of neuroactive steroids such as pregnenolone, progesterone, 3α-hydroxy-5α-pregnan-20-one (allopregnanolone, or 3α,5α-TH PROG), and 3α,5α-tetrahydrodeoxycorticosterone (3α,5α-TH DOC) (Serra et al. 2000). The latter two steroids are among the most potent positive allosteric modulators of GABAA receptor function (Majewska et al. 1986), and their administration in pharmacological doses elicits anxiolytic, anti-convulsant, and sedative-hypnotic effects in rodents (Majewska 1992). The molecular mechanisms that underlie the persistent decrease in the abundance of neuroactive steroids induced by social isolation of rats remain unclear. However, consistent with the notion that a ‘facilitatory trace,’ characterized by hyper-responsiveness of the hypothalamic–pituitary–adrenal (HPA) axis to new stimuli, may develop during chronic stress (Akana et al. 1992), we have previously shown that the functional response of the HPA axis to an acute stressful stimulus is increased in isolated rats (Serra et al. 2000). Thus, the increase in the brain and plasma concentrations of 3α,5α-TH PROG and 3α,5α-TH DOC induced by foot shock (Barbaccia et al. 1996a, 1997), used in this instance as a novel acute stressor, were markedly greater on a percentage basis in socially isolated rats than in group-housed animals.
This latter effect of short-term ethanol administration has recently been proposed to contribute to the central actions of ethanol associated with modulation of GABAergic transmission (Morrow et al. 2001). Indeed, the administration of finasteride, an inhibitor of 3α,5α-TH PROG synthesis, prevented both the ethanol-induced increase in the cerebral cortical content of 3α,5α-TH PROG, as well as certain pharmacological actions of ethanol in rats (Van Doren et al. 2000). Moreover, circulating 3α,5α-TH PROG has been suggested to influence ethanol reinforcement (Morrow et al. 2001). Female mice, in which the brain concentration of 3α,5α-TH PROG was higher than that in males, consumed greater amounts of ethanol than did males (Sinnott et al. 2002); furthermore, ethanol consumption in male mice was increased by the systemic administration of 3α,5α-TH PROG. Administration of 3α,5α-TH PROG also enhanced ethanol-reinforced operant responding in male rats (Janak et al. 1998). In contrast, in female rats, self-administration of ethanol was found to be lower during oestrus, when the brain concentration of 3α,5α-TH PROG is higher, than in other phases of the oestrous cycle (Roberts et al. 1998). Socially isolated rats, in which the brain concentration of 3α,5α-TH PROG is markedly reduced (Serra et al. 2000), also consume increased amounts of ethanol (Schenk et al. 1990; Wolffgramm 1990; Hall et al. 1998). The mechanisms by which 3α,5α-TH PROG influences ethanol consumption and pharmacology thus remain unclear.
We hypothesized that the increased sensitivity of the HPA axis to an acute stressful stimulus in isolated (chronically stressed) rats might enhance the ability of ethanol to increase steroidogenesis in these animals compared with its effect in group-housed (non-stressed) rats. We therefore examined the effects of systemic administration of ethanol on the brain and plasma concentrations of neuroactive steroids. In order to clarify if the mechanism underlying the increase sensitivity of HPA axis to novel stimuli is related to the GABAergic transmission we also evaluated the effect of administration of isoniazid, an inhibitor of the glutamic acid decarboxylase (Horton et al. 1979) on the brain and plasma levels of neuroactive steroids.
Materials and methods
Male Sprague–Dawley CD rats at 30 days of age, immediately after weaning, were housed for 30 days, either in groups of eight per cage or individually in smaller cages. They were maintained under an artificial 12-h-light, 12-h-dark cycle (light on 08.00–20.00 h) at a constant temperature of 23° ± 2°C and 65% humidity. Food and water were freely available until the time of the experiment. Animal care and handling throughout the experimental procedures were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).
Isoniazid (isonicotinic acid hydrazide; Sigma, Milan, Italy) was dissolved in physiological saline and administered subcutaneously (300 mg per kilogram of body mass) 45 min before animals were killed. Ethanol [20% (v/v) in saline] was administered intraperitoneally (1 g/kg) 20 min before killing.
Extraction and assay of steroids
Rats were killed either with a guillotine (for measurement of plasma steroids) or by focused microwave irradiation (70 W/cm2 for 4 s) of the head (for measurement of brain steroids). This latter procedure results in a virtually instantaneous inactivation of brain enzymes (Mao et al. 1974), thus minimizing 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 were extracted and purified as previously described (Barbaccia et al. 1996a). In brief, steroids present in tissue homogenates [400 mg of protein in 4 mL of phosphate-buffered saline (pH 7.0)] were extracted three times with ethyl acetate, and the combined organic phases were dried under vacuum. The resulting residue was dissolved in 5 mL of n-hexane and applied to a SepPak silica cartridge (Waters), and components were eluted with n-hexane and 2-propanol (7 : 3, v/v). Steroids were separated and further purified by HPLC on a 5-μm Lichrosorb-diol column (250 by 4 mm; Phenomenex) with a discontinuous gradient of 2-propanol (0–30%) in n-hexane. Progesterone, which co-elutes with cholesterol, was further purified by washing the corresponding dried HPLC fractions first with 200 μL of dimethyl sulfoxide and then with 400 μL of water. Progesterone was extracted from the aqueous phase twice with 1.5-mL volumes of n-hexane. The recovery (70–80%) of steroids through the extraction and purification procedures was monitored by adding a trace amount (6000–8000 c.p.m.; 20–80 Ci/mmol) of tritiated standard to the brain homogenate.
Blood was collected from the trunk of killed rats into heparinized tubes and centrifuged at 900 g for 20 min at room temperature (22°C). The resulting plasma was frozen (− 80°C) until assayed for steroids. Steroids were extracted from plasma three times with 1.5 mL of ethyl acetate.
Steroids from both brain tissue and plasma were quantified by radioimmunoassay as previously described (Purdy et al. 1990; Barbaccia et al. 1996a) with specific antibodies to pregnenolone, progesterone, and corticosterone (ICN, Costa Mesa, CA, USA). Antibodies to 3α,5α-TH DOC and to 3α,5α-TH PROG were generated in rabbits and sheep, respectively, and characterized as previously described (Purdy et al. 1990).
Data are presented as means ± SEM and were analyzed by two-way anova. Individual means were compared by Newman–Keuls post hoc test.
Consistent with our previous data (Serra et al. 2000), social isolation for 30 days in the absence of any additional stressor induced significant decreases in the basal cerebrocortical concentrations of pregnenolone (− 28%), progesterone (− 25%), 3α,5α-TH PROG (− 42%), and 3α,5α-TH DOC (− 39%), compared with the corresponding values for group-housed animals (Table 1). Whereas social isolation also reduced the plasma concentrations of these steroids, it induced a significant increase (+ 30%) in the plasma corticosterone concentration.
Table 1. Effects of social isolation on the abundance of neuroactive steroids in the cerebral cortex and plasma of rats
Animals were housed in groups or in isolation for 30 days, after which the cerebrocortical and plasma concentrations of pregnenolone, progesterone, 3α,5α-TH PROG, 3α,5α-TH DOC, and corticosterone were measured. Data are means ±SEM of values from 24 animals. *p < 0.05, **p < 0.01 versus respective group-housed value. The data were analysed by two-way anova following Newman–Keuls post-hoc test.
As expected (Barbaccia et al. 1999; Van Doren et al. 2000), a single injection of ethanol markedly increased both the cerebral cortical (Fig. 1) and plasma (Fig. 2) concentrations of neuroactive steroids in both group-housed and isolated rats. However, the percentage increases in the brain concentrations of steroids induced by ethanol in isolated rats (pregnenolone, + 406%; progesterone, + 230%; 3α,5α-TH PROG, + 308%; 3α,5α-TH DOC, + 545%) were much greater than were those induced in group-housed rats (pregnenolone, + 163%; progesterone, + 83%; 3α,5α-TH PROG, + 96%; 3α,5α-TH DOC, + 138%). anova revealed a significant interaction between factors (social isolation and ethanol treatment) for progesterone (F1,60 = 4.911, p = 0.0305), 3α,5α-TH PROG (F1,58 = 5.649, p = 0.0208), and 3α,5α-TH DOC (F1,58 = 9.009, p = 0.004). The ability of ethanol to increase the amounts of neuroactive steroids and of corticosterone in plasma was also more pronounced in isolated rats than in group-housed animals; progesterone (F1,34 = 35,0, p = 0.001), 3α,5α-TH PROG (F1,36 = 4,535, p = 0.04), 3α,5α-TH DOC (F1,28 = 6,840, p = 0.005) and corticosterone (F1,34 = 50.503, p < 0.001).
To evaluate further the sensitivity of the HPA axis in socially isolated rats, we examined the effects of a single injection of isoniazid, an inhibitor of GABA synthesis (Horton et al. 1979), on the brain and plasma concentrations of neuroactive steroids. As previously demonstrated (Barbaccia et al. 1996b), the reduction in the brain content of GABA induced by systemic injection of isoniazid resulted in an increase in the concentrations of neuroactive steroids in both the cerebral cortex (Fig. 1) and plasma (Fig. 2) of group-housed rats. However, the effects of isoniazid on steroid concentrations in the brain were greater in isolated rats (pregnenolone, + 749%; progesterone, + 785%; 3α,5α-TH PROG, + 170%; 3α,5α-TH DOC, + 386%) than in group-housed rats (pregnenolone, + 276%; progesterone, + 397%; 3α,5α-TH PROG, + 97%; 3α,5α-TH DOC, + 256%); anova revealed a significant interaction between factors for pregnenolone (F1,22 = 13.378, p = 0.0014), 3α,5α-TH PROG (F1,25 = 5.280, p = 0.0302), and 3α,5α-TH DOC (F1,24 = 4.890, p = 0.0480). Similar differences in the effects of isoniazid between isolated and group-housed rats were apparent in plasma.
We have shown that acute administration of ethanol increased the cerebrocortical and plasma concentrations of pregnenolone, progesterone, 3α,5α-TH PROG, and 3α,5α-TH DOC to a markedly greater extent in socially isolated rats than in group-housed animals. Given that ethanol injection represents a stressful condition, the greater effects of this drug on the cerebrocortical and plasma concentrations of steroids in isolated rats may reflect the greater sensitivity of the HPA axis in these animals to stress. An acute administration of ethanol able to stimulate the HPA axis in control rats (Rivier et al. 1984; Rivier 1996) was shown to have no effect on the concentrations of neuroactive steroids in the cerebral cortex and plasma of adrenalectomized and orchietomized animals (Khisti et al. 2002a, 2002b). These results are thus consistent with the observations that the large and rapid increases in the brain and plasma concentrations of neuroactive steroids induced by various acute stressful stimuli in rats (Purdy et al. 1991; Barbaccia et al. 1996a, 1997) are abolished by removal of the adrenals and gonads (Barbaccia et al. 1997). Our present data therefore suggest that the ‘facilitatory trace,’ characterized by hyper-responsiveness of the HPA axis to new stimuli, contributes to the enhanced sensitivity of socially isolated rats to the effects of ethanol on the brain and plasma concentrations of neuroactive steroids.
The stimulatory effect of acute ethanol administration on ACTH secretion is thought to be mediated by an increased release of CRF and AVP (Sarnyai et al. 2001). Given that neurons that synthesize CRF are activated by various types of chronic stress, including that associated with social isolation (Rivier and Vale 1987; Ojima et al. 1995), the enhanced effects of ethanol on the brain and plasma concentrations of steroids in isolated rats may result from a greater increase in CRF release. GABAA receptor-mediated transmission modulates the HPA axis directly, as revealed by the observation that GABA inhibits the release of CRF from the hypothalamus (Calogero et al. 1988). However, because CRF neurons are modulated by several neurotransmitters, as are inhibited by GABAergic and oppioidergic neurons and stimulated by serotoninergic and catecolaminergic systems, and ethanol alters the function of most of the neurotransmitter systems, it is hard to reconcile the capability of this drug to stimulate GABAergic transmission with its excitatory action on HPA axis. Nevertheless, the reduction in GABAA receptor function induced by the chronic stress associated with social isolation (Serra et al. 2000) may be an important determinant of the enhanced sensitivity of the HPA axis to ethanol in isolated rats. Consistent with this conclusion, socially isolated rats manifest an increased level of anxiety (Parker and Morinan 1986; Wongwitdecha and Marsden 1996; Serra et al. 2000). Furthermore, we have now shown that the administration of isoniazid, which induces a transient reduction in GABAA receptor function and increases the brain and plasma concentrations of steroids (Barbaccia et al. 1996b, 1997) by inhibiting glutamic acid decarboxylase (Horton et al. 1979), also increased the abundance of pregnenolone, progesterone, 3α,5α-TH PROG, and 3α,5α-TH DOC in the cerebral cortex and plasma to a greater extent in isolated rats than in group-housed animals. This enhanced response to isoniazid shown by isolated animals might be due to the further decrease of GABAergic transmission induced by this drug, an effect consistent with the capability of the benzodiazepine receptor inverse agonist β-CCE to enhance the levels of neuroactive steroids in rats (Barbaccia et al. 1997).
The evidence that isolated animals are more sensitive to acute foot-shock stress (Serra et al. 2000) and to ethanol is consistent with the established relations among stress, glucocorticoids, and vulnerability to drugs of abuse (Piazza and Le Moal 1996). Indeed, plasma glucocorticoid concentrations appear to be related to the vulnerability to self-administration of ethanol. Thus, adrenalectomy-reduced ethanol consumption in alcohol-preferring rats to the level apparent in non-alcohol-preferring animals, whereas administration of corticosterone to the adrenalectomized rats restored their intake of ethanol to pre-operative levels (Fahlke et al. 1994). Furthermore, glucocorticoids not only interact with the reinforcing properties of drugs of abuse (Piazza and Le Moal 1996), but also have intrinsic reinforcing effects, as revealed by the demonstration of corticosterone self-administration in rats (Piazza et al. 1993). In contrast to the other steroids measured, the plasma concentration of corticosterone was significantly increased in isolated rats compared with that in group-housed animals. This higher basal level of corticosterone may account for the increase in ethanol intake induced by social isolation (Schenk et al. 1990; Wolffgramm 1990; Hall et al. 1998). Given that social isolation represents a model of mild chronic stress (Serra et al. 2000), that 3α,5α-TH PROG and 3α,5α-TH DOC are among the most potent endogenous positive modulators of GABAA receptors (Majewska 1992), and that the concentrations of these steroids are reduced in socially isolated animals (Serra et al. 2000), the increased rate of ethanol intake in isolated rats may also result from a rewarding effect of this drug that is attributable to the marked increase in the concentrations of endogenous anxiolytic steroids induced by its consumption. Thus, the natural preference for ethanol may be related to the effect of this drug on the levels of neuroactive steroids, an effect that is enhanced by social isolation.
Given that, in studies of ethanol preference, rats are usually housed individually, it is possible that the isolation-induced decrease in the concentrations of neuroactive steroids and the reversal of this effect by ethanol might contribute to the voluntary consumption of this drug. Consistent with this notion, the acute administration of ethanol induced a greater increase in the brain concentrations of 3α,5α-TH PROG and 3α,5α-TH DOC in Sardinian alcohol-preferring rats, a strain of rats selectively bred for ethanol preference that exhibits a high level of anxiety-related behaviour (Colombo 1997), than in non-alcohol-preferring rats (Barbaccia et al. 1999). This enhanced response to ethanol may be due to a hyper-responsiveness of the HPA axis, to which housing conditions may contribute. The concentration of CRF in several brain regions and the electro-encephalographic response to CRF have been shown to be increased in ethanol-preferring rats, compared with those in control animals (George et al. 1990; Ehlers et al. 1992).
At the molecular level, the increased sensitivity to ethanol in isolated rats may be also correlated to changes in the GABAA-receptor gene expression induced by the persistent reduction of brain progesterone metabolites. Accordingly, in vivo and in vitro studies demonstrated that the persistent or sudden reduction in the concentration of 3α,5α-TH PROG, such as that elicited by chronic administration of oral contraceptives (Follesa et al. 2002), or by progesterone withdrawal (Smith et al. 1998; Follesa et al. 2000) resulted in a selective changes in the abundance of GABAA-receptor subunits. Moreover, very recently, after progesterone withdrawal, Sundstrom-Poromaa et al. (2002) have found an overexpression of GABAA receptor containing δ and α4 subunit which are preferentially activated by very low doses of ethanol.
In conclusion, the previous demonstration that isolated rats exhibit increased self-administration of ethanol, and our present observation that these animals are more sensitive to the effects of ethanol on the brain and plasma concentrations of 3α,5α-TH PROG and 3α,5α-TH DOC, both of which posses anxiolytic properties (Engel and Grant 2001) and potentiate the central actions of ethanol (Vanover et al. 1999), suggest that chronic stress may induce plastic adaptation of neuronal systems that contributes to a vulnerability to alcohol abuse.