The first evidence that neurosteroids might represent endogenous anxiolytic agents was derived from behavioral changes occurring during pregnancy and PMS and from associated circulating plasma progesterone levels (). The assumption that progesterone and its neuroactive metabolites possess anxiolytic activity through their action at the GABAA receptor complex related to the estrous cycle or pregnancy was further supported by findings from animal models ().
Independently from sex-dependent anxiolytic effects, and based on the findings of Majewska et al. (), Crawley et al. () investigated the possibility that THDOC might be an endogenous anxiolytic compound. Intraperitoneally injected THDOC showed anxiolytic activity in two rat models of anxiety tested, separable from the sedative dose range (). After subcutaneous administration of progesterone to rats, D. Bitran et al. () observed anxiolytic effects, which were most probably the result of bioconversion of progesterone to allopregnanolone with subsequent augmentation of GABAA receptor-mediated function. When administered intracerebroventricularly in rats, pregnenolone and allopregnanolone were also shown to display anxiolytic properties, with sedative effects occurring at the highest doses tested (). Moreover, D. Bitran et al. () provided in vivo evidence that a 3a-hydroxyl group is necessary for neurosteroids to interact with the GABAA receptor, because the 3beta-hydroxy-epimer of allopregnanolone proved to be behaviorally inactive.
Similarly, Wieland et al. () reported that allopregnanolone produced anxiolytic effects in rats when given intraperitoneally and that anxiolytic activity of allopregnanolone was coupled to its 3α-hydroxyl group, further emphasizing the notion that modulation of the GABA/benzodiazepine receptor chloride ionophore complex by pregnane steroids follows stringent structural requirements including stereospecificity, which had already been described in vitro (). Moreover, Britton et al. () have demonstrated anxiolytic activity of the intraperitoneally administered synthetic pregnane steroid alfaxalone in rats. Interestingly, no tolerance to the antianxiety effect of alfaxalone was observed after 1 week of daily administration in contrast to the well-recognized tolerance following long-term administration of benzodiazepines (). The same group also reported on attenuation of anxiogenic behavioral effects of intracerebroventricularly administered CRH and swim stress following intraperitoneal administration of alfaxalone in rats (). Apart from adult animals, anxiolytic activity of intracerebro-ventricular allopregnanolone, as well as sedative activity at higher doses, could also be demonstrated in neonate rats ().
The in vitro results indicating that neurosteroids bind at a different site of the GABAA receptor complex than do benzodiazepines and barbiturates has been supported by studies investigating drug discrimination in rats, suggesting that allopregnanolone and allo-THDOC might possess a more desirable profile in terms of abuse liability than benzodiazepines and barbiturates (). Similar results have been obtained by S. I. Deutsch and Mastropaolo () concerning the anxiolytic efficacy of peripherally administered allo-THDOC in a drug discrimination paradigm in rats.
Further evidence concerning anxiolytic effects of neurosteroids involves its action on the prefrontal cortical dopamine system. This system has been identified as one of the neuroanatomically involved CNS areas in stress and anxiety responses, where increases in dopamine metabolism are observed following a variety of stressors (). Grobin et al. () demonstrated that intracerebroventricularly administered allo-THDOC effectively reduced dopamine metabolism in rats, thereby antagonizing stress-induced activation of the prefrontal cortical dopamine innervation.
Conflicting results with regard to previous studies have been obtained by Melchior and Ritzmann (), reporting on anxiogenic responses of mice following intraperitoneal administration of pregnenolone and mixed anxiogenic/anxiolytic responses following administration of pregnenolone sulfate that were dose-dependent. These authors have speculated that both substances that were tested participate in the initial response to stressful stimuli, which is then terminated by the action of pregnenolone and pregnenolone sulfate metabolites with anxiolytic activity. In a further study by the same authors, intraperitoneally administered DHEA and DHEAS both showed anxiolytic activity in mice, with DHEA being effective over a wide range of doses (). Moreover, ethanol enhanced the anxiolytic effect of DHEA, but at 1.0 mg/kg DHEAS blocked the anxiolytic effect of ethanol. Melchior and Ritzmann concluded that DHEA and DHEAS, in contrast to pregnenolone and pregnenolone sulfate, might be involved in the termination of a stress response.