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- Materials and methods
Previously we have demonstrated that social isolation of rats reduces both the cerebrocortical and plasma concentrations of 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG), and potentiates the positive effects of acute ethanol administration on the concentrations of this neurosteroid. We now show that the ethanol-induced increase in 3α,5α-TH PROG is more pronounced in the brain than in the plasma of isolated rats. The ability of ethanol to inhibit isoniazid-induced convulsions is greater in isolated rats than in group-housed animals and this effect is prevented by treatment with finasteride. Social isolation modified the effects of ethanol on the amounts of steroidogenic regulatory protein mRNA and protein in the brain. Moreover, ethanol increased the amplitude of GABAA receptor-mediated miniature inhibitory postsynaptic currents recorded from CA1 pyramidal neurones with greater potency in hippocampal slices prepared from socially isolated rats than in those from group-housed rats, an effect inhibited by finasteride. The amounts of the α4 and δ subunits of the GABAA receptor in the hippocampus were increased in isolated rats as were GABAA receptor-mediated tonic inhibitory currents in granule cells of the dentate gyrus. These results suggest that social isolation results in changes in GABAA receptor expression in the brain, and in an enhancement of the stimulatory effect of ethanol on brain steroidogenesis, GABAA receptor function and associated behaviour.
Social isolation of rats after weaning results in a decrease in the brain and plasma concentrations of neuroactive steroids such as 3α-hydroxy-5α-pregnan-20-one (allopregnanolone, 3α,5α-TH PROG) and 3α,5α-tetrahydrodeoxycorticosterone (Serra et al. 2000). The molecular mechanisms that underlie this effect remain unclear. However, consistent with the hypothesis that ethanol (EtOH) activates the hypothalamic–pituitary–adrenal (HPA) axis (Ellis 1966; Rivier et al. 1984; Rivier 1996), we have previously shown that the increases in both the activity of the HPA axis and in the plasma and brain concentrations of neuroactive steroids induced by an acute injection of EtOH are potentiated by social isolation (Serra et al. 2003). EtOH increases the abundance of 3α,5α-TH PROG and 3α,5α-tetrahydrodeoxycorticosterone in the brain and plasma of control rats (Barbaccia et al. 1999; Van Doren et al. 2000), an effect thought to be dependent predominantly on stimulation of the HPA axis, given that it is largely abolished after adrenalectomy (O'Dell et al. 2004).
We have shown recently that EtOH promotes brain steroidogenesis by a local action independent of the HPA axis. EtOH increased both the amount of 3α,5α-TH PROG in isolated hippocampal tissue of control rats and the amplitude of GABAA receptor-mediated spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded from CA1 pyramidal neurones, two effects prevented by finasteride (Sanna et al. 2004). These observations are consistent with evidence that the steroidogenic machinery, including steroidogenic regulatory protein (StAR), is present in specific regions of the brain (Furukawa et al. 1998; King et al. 2002; Sierra 2004). StAR is responsible for the delivery of cholesterol to the cytochrome P450 side-chain cleavage enzyme (P450scc), which catalyses the conversion of cholesterol to pregnenolone in mitochondria.
To examine the mechanism responsible for the reduction in the basal concentrations of neuroactive steroids and the increased sensitivity of the production of these steroids to EtOH induced by social isolation, we have now investigated possible changes in the expression of StAR in the cerebral cortex of isolated rats under basal conditions and after administration of EtOH. To investigate further whether the greater increase in brain concentration of 3α,5α-TH PROG induced by EtOH in isolated rats is of functional significance in terms of GABAA receptor activity, we also examined the effects of EtOH both on isoniazid-induced seizures in group-housed and isolated rats as well as on mIPSCs in hippocampal slices prepared from such animals. Moreover, given that fluctuations in brain and plasma concentrations of neuroactive steroids associated with physiological conditions are functionally correlated with changes in the expression of genes for specific GABAA receptor subunits (Concas et al. 1998; Herbison 2001; Koksma et al. 2005; Maguire et al. 2005), the effect of social isolation on the abundance of α4 and δ subunits of the GABAA receptor was also studied. Our results provide insight into the functional link between 3α,5α-TH PROG and GABAA receptor expression and function, and the relevance of this system to the pharmacology of EtOH.
- Top of page
- Materials and methods
We have shown that social isolation results in changes in GABAA receptor expression in the brain as well as in enhancement of the stimulatory effect of EtOH on brain steroidogenesis, GABAA receptor function and associated behaviour. The increases in the abundance of 3α,5α-TH PROG in brain and plasma induced by systemic injection of EtOH were greater in isolated rats than in group-housed animals. Acute administration of EtOH to socially isolated rats increased the concentration of 3α,5α-TH PROG to a markedly greater extent in the cerebral cortex than in plasma (Fig. 1) (Serra et al. 2003). This finding, which is consistent with our previous observation that EtOH increases local neurosteroid synthesis in the brain independently of the HPA axis (Sanna et al. 2004), suggests that a hyper-responsiveness of the HPA axis due to chronic stress (Akana et al. 1992) is not the only mechanism responsible for the enhanced effect of EtOH on neuroactive steroid concentrations in the brain of isolated rats. This greater efficacy of EtOH in isolated rats might also be attributable to functional changes induced by social isolation in the brain steroidogenic machinery, including enzymes, peripheral benzodiazepine receptors (PBRs) and StAR.
We previously showed that, although social isolation had no significant effect on the binding kinetics of PBRs in the cerebral cortex, this housing condition increased the steroidogenic response to the selective PBR agonist CB34, a response more pronounced in the cerebral cortex than in the plasma of isolated rats (Serra et al. 2004). Given that EtOH is not known to interact with PBRs, it is likely that the greater effects of EtOH and CB34 on the cerebrocortical versus plasma concentrations of neuroactive steroids in isolated rats are due to a change in another component of the neuronal steroidogenic system induced by isolation.
The present data show that the acute administration of EtOH induced a greater increase in the amount of StAR mRNA in the cerebral cortex of isolated rats than in group-housed rats. EtOH treatment reduced the amount of the 37-kDa form and increased that of the 32-kDa form in the cerebral cortex of isolated rats, whereas the abundance of both StAR forms was increased by EtOH in the cortex of group-housed rats. Given that the molecular mechanism of StAR function remains unclear (Sierra 2004), it is not yet possible to explain the opposite effects of EtOH on the abundance of the two forms of the protein in the cerebral cortex of isolated rats. An increase in the amount of the 32- (or 30-) kDa form of StAR has previously been shown to be accompanied by a decrease in the amount of the 37-kDa form (Kimoto et al. 2001; Shibuya et al. 2003). Our data suggest that the rate of proteolytic conversion of the full-length StAR protein to the 32-kDa form may be increased by EtOH in the cerebral cortex of isolated rats, although the levels of StAR mRNA in isolated animals was increased to an greater extent than in group-housed animals.
The increase in the amount of 3α,5α-TH PROG in the brain induced by EtOH is thought to contribute to its anticonvulsant effect (Van Doren et al. 2000). We have now shown that the greater efficacy of EtOH in increasing 3α,5α-TH PROG synthesis in the brain of socially isolated rats is associated with an increase in the ability of EtOH to antagonize convulsions induced by isoniazid, an effect prevented by pretreatment with finasteride, a 5α-reductase inhibitor that blocks the synthesis of 3α,5α-TH PROG. Finasteride reduced the delay in the onset of isoniazid-induced seizures in group-housed animals but not in isolated rats, consistent with their reduced basal level of 3α,5α-TH PROG in the brain. The reduction in the brain level of 3α,5α-TH PROG induced by social isolation is thus probably responsible for the increased vulnerability to seizures apparent in isolated animals. Accordingly, isolated mice were more susceptible to picrotoxin-induced seizures (Matsumoto et al. 2003).
The enhanced anticonvulsant action of EtOH in isolated rats is consistent with our finding that EtOH induced a concentration-dependent increase in the amplitude of GABAA receptor-mediated mIPSCs in CA1 pyramidal neurones with a greater potency in socially isolated rats than in group-housed animals. Moreover, the observation that the effect of EtOH on mIPSC amplitude was inhibited by finasteride supports the idea that this action of EtOH is mediated by an increased production of 3α,5α-TH PROG. This hypothesis is also consistent with the acute modulatory effect of 3α,5α-TH PROG on eGABAA receptor-mediated IPSCs recorded from CA1 pyramidal neurones. The lack of a difference in this effect of 3α,5α-TH PROG between isolated and group-housed rats suggests that the greater potency of EtOH with regard to increasing the amplitude of GABAA receptor-mediated mIPSCs in the hippocampus of isolated rats might be due to the greater production of 3α,5α-TH PROG induced by EtOH in these animals.
The slight increase in susceptibility to seizures apparent in isolated rats might also reflect changes in GABAA receptor gene expression induced by the persistent decrease in the brain concentration of neuroactive steroids. Both in vivo and in vitro studies have shown that fluctuations in the concentration of 3α,5α-TH PROG result in selective changes in the abundance of specific GABAA receptor subunits as well as in consequent changes in GABAA receptor function (Concas et al. 1998; Smith et al. 1998; Follesa et al. 2000, 2002; Herbison 2001Maguire et al. 2005). Withdrawal of progesterone or EtOH thus resulted in increased expression of the α4 subunit as well as a decreased seizure threshold and increased anxiogenic behaviour in rats (Mahmoudi et al. 1997; Smith et al. 1998; Reddy and Rogawski 2000; Gulinello et al. 2001). We have now shown that social isolation is associated with increased expression of α4 and δ subunits in hippocampal neurones. An increase in transcription of the gene for the α4 subunit in the hippocampus of isolated rats elicited by the decrease in level of 3α,5α-TH PROG might thus contribute to the increase in susceptibility to seizures as well as to anxiogenic behaviour in such animals (Serra et al. 2000). Increased expression of the α4 subunit is associated with many seizure-prone states (Brooks-Kayal et al. 1998; Peng et al. 2004). Consistent with the notion that GABAA receptors containing the α4 subunit possess high affinity for the benzodiazepine receptor antagonist flumazenil and the partial inverse agonist Ro15-4513 (Wafford et al. 1996; Whittemore et al. 1996; Sanna et al. 2003), we found that Ro15-4513 increased the time constant for mIPSC decay in CA1 pyramidal neurones from isolated rats whereas it reduced this parameter in group-housed rats, although this difference in effect did not achieve statistical significance.
Social isolation also increased the expression of the δ subunit in the rat hippocampus, probably resulting in the formation of GABAA receptors that contain both α4 and δ subunits. Similar results were obtained in rats after progesterone withdrawal (Sundstrom-Poromaa et al. 2002). The putative increase in the number of GABAA receptors containing the δ subunit as well as α4 may explain the limited potentiation of mIPSCs by Ro15-4513 in CA1 neurones of isolated rats. In fact, receptors containing both α4 and δ subunits manifest low affinity for benzodiazepines (Saxena and Macdonald 1994, 1996).
Given that δ substitutes for γ2 and that the latter subunit is essential for synaptic localization of GABAA receptors (Essrich et al. 1998), receptors containing α4 and δ would be expected to be extrasynaptic. Extrasynaptic GABAA receptors are responsible for tonic inhibition in granule cells of the cerebellum and DG (Nusser et al. 1998; Nusser and Mody 2002; Stell and Mody 2002). Our data show that GABAA receptor-mediated tonic inhibitory currents in granule cells of the DG were significantly enhanced in hippocampal slices from socially isolated rats. Consistent with the notion that tonic inhibition in CA1 pyramidal cells is not mediated by δ subunit-containing GABAA receptors, but most likely by extrasynaptic α5γ2-containing receptors (Crestani et al. 2002; Stell et al. 2003; Caraiscos et al. 2004), tonic currents recorded from these cells did not differ between isolated and group-housed rats. In agreement with the marked effect of neuroactive steroids on tonic inhibition mediated by δ subunit-containing GABAA receptors (Stell et al. 2003), we found that the current noise variation induced by 3α,5α-TH PROG in granule cells of the DG was greater for isolated rats than for group-housed animals. GABAA receptors containing the δ subunit play an important role in the regulation of neuronal excitability. Mice that lack this subunit develop epilepsy and show other signs of hyperexcitability (Mihalek et al. 1999; Spigelman et al. 2002), and expression of this subunit is decreased in the hippocampal formation in a mouse model of temporal lobe epilepsy (Peng et al. 2004) as well as in the rat kindling model of alcohol dependence (Cagetti et al. 2003). It should also be mentioned that the increased expression of GABAA receptors containing both the α4 and δ subunit associated with social isolation might also be expected to have important consequences for the ability of EtOH to directly modulate GABAergic inhibitory transmission. In fact, recombinant GABAA receptors comprising the α4 and δ subunits were shown to be selectively sensitive to extremely low concentrations of EtOH (Sundstrom-Poromaa et al. 2002; Wallner et al. 2003). In line with these data, it was also reported that the tonic inhibitory currents in DG granule cells, mediated by GABAA receptors containing the α4 and δ subunits, was potentiated by 30 mm EtOH (Wei et al. 2004). However, a recent paper (Borghese et al. 2006) failed to reproduce these observations both in recombinant receptors and in DG granule cells. Thus, the role of GABAA receptors containing α4 and δ subunits in the modulatory effects of EtOH is still controversial and requires further investigation.