Gunn et al. GABAA receptors, neurosteroids, and stress activates TSPO to increase the cerebral production of 5α3α- THPROG, an effect that contributes to the behavioral actions of the drug (Verleye et al., 2005). Moreover, the selective TSPO activator, XBD173 (N -benzyl-N -ethyl-2-[7,8-dihydro-7-methyl- 8-oxo-2-phenyl-9H -purin-9-yl] acetamide; Figure 1) enhances the amplitude and duration of evoked IPSCs (eIPSCs) and mIP- SCs recorded from neurons of the mouse medial prefrontal cortex (mPFC), an effect blocked by the 5α-reductase inhibitor, ﬁnas- teride (Rupprecht et al., 2009). Crucially, XBD173 is effective in tests predictive of an anxiolytic action [i.e., social isolation and ele- vated plus-maze (EPM) tests], with both actions being blocked by the TSPO antagonist PK11195 [1-(2-chlorophenylmethylpropyl)- 3-isoquinoline-carboxamide; Figure 1; Rupprecht et al., 2009]. Importantly, in humans XBD173 acts as an effective anxiolytic agent, without the sedative, or the withdrawal symptoms typi- cally associated with current therapeutic treatments for anxiety (Rupprecht et al., 2009). Certain benzodiazepines (BDZs), such as diazepam and mida- zolam not only act to enhance GABAAR function, but also to promote the synthesis of neurosteroids through the activation of TSPO (Rupprecht et al., 2010; Tokuda et al., 2010; Figure 1). For example, in a brain slice preparation, midazolam, a BDZ used clinically to facilitate the induction of anesthesia, increased neu- rosteroid levels in CA1 pyramidal neurons and inhibited LTP (Tokuda et al., 2010). Consistent with a neurosteroid involve- ment, inhibiting neurosteroid synthesis with ﬁnasteride treatment, or inhibiting the GABA-modulatory actions of neurosteroids with the selective antagonist 17PA [17-phenyl-(3α5α)-androst- 16-en-3-ol; Mennerick et al., 2004] signiﬁcantly attenuated the actions of midazolam (Tokuda et al., 2010). Furthermore, in vivo ﬁnasteride pre-treatment blocked the inhibitory effects of mida- zolam upon contextual fear learning (Tokuda et al., 2010). In contrast, clonazepam, an anxiolytic, and anticonvulsant BDZ, does not induce changes to neurosteroid synthesis and has no effect upon LTP induction. However, when administered with the TSPO agonist FGIN (2-[2-(4-ﬂuorophenyl)-1H -indol-3-yl), or together with exogenous allopregnanolone, clonazepam pro- duced similar effects to midazolam, thus inhibiting LTP induction (Tokuda et al., 2010). These observations suggest that the unique effects of certain clinically important BDZs may be mediated by the dual activation of TSPO and potentiation of GABAAR function. PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL ROLE OF NEUROSTEROIDS: A FOCUS ON STRESS INHIBITORY REGULATION OF THE STRESS CIRCUITRY The ability of an organism to adapt to conditions of stress is essential for survival and involves a number of ﬁnely regulated and highly conserved interconnected systems that maintain phys- iological homeostasis. The hypothalamic paraventricular nucleus (PVN) is a key brain region involved in initiating the neuroen- docrine and autonomic response to stressor exposure (Herman and Cullinan, 1997). Within the PVN, spinally projecting parvo- cellular neurons (located in dorsal and ventromedial parvocel- lular regions), rapidly modulate autonomic output in response to stress exposure, while neuroendocrine parvocellular neurons (located in the dorsomedial parvocellular region), project to the median eminence and initiate activation of the hypothalamo- pituitary–adrenocortical (HPA) axis (Ulrich-Lai and Herman, 2009). Regulation of the HPA axis is mediated through the inte- gration of hormonal feedback pathways, e.g., glucocorticoids, corticotrophin-releasing hormone (CRH), and a complex neuro- circuitry involving a number of mono- and polysynaptic pathways (Herman et al., 2003; de Kloet et al., 2005; Ulrich-Lai and Her- man, 2009; Figure 2A). The PVN receives considerable GABAergic innervation from local hypothalamic regions (e.g., dorsal hypo- thalamus, peri-PVN, preoptic area) and areas of the extended amygdala (e.g., bed nucleus of the stria terminalis, BST), that exert a substantial inhibitory tone upon the HPA axis (Cullinan et al., 2008). Forebrain and limbic regions (e.g., hippocampus, amyg- dala, and prefrontal cortex) do not directly innervate the PVN, but instead modulate HPA activity via projections to a number of these GABAergic nuclei surrounding the PVN (Ulrich-Lai and Herman, 2009; Figure 2A). As such, these “relay nuclei” are important sites of stress integration, located “upstream” of the PVN and it is con- ceivable that the modulation of inhibitory transmission at this level by neurosteroids, may play a role in this synaptic integration (Figure 2A). Quantitative ultrastructural analysis has revealed that CRH- releasing parvocellular neurons are the main target for GABAergic inputs to the medial parvocellular region of the PVN (Miklos and Kovacs, 2002). Furthermore, dual in situ hybridization stud- ies have demonstrated the mRNA expression of multiple GABAAR subunits (α1, α2, β1–3, and γ1, γ2) in the majority of CRH- releasing neurons (Cullinan, 2000), providing molecular evidence for a role of GABAAR-mediated inhibition at the level of the HPA axis. In agreement with this anatomical data, electrophys- iological recordings from acute hypothalamic slice preparations revealed that glutamate microinjection into regions surrounding the PVN elicited postsynaptic potentials sensitive to the GABAAR antagonist bicuculline in a signiﬁcant proportion of parvocellular neurons (Boudaba et al., 1996), while bicuculline also increased the frequency of action currents in these neurons (Hewitt et al., 2009). Furthermore, the microinjection of bicuculline into the PVN in vivo resulted in increased plasma corticosterone levels (Cullinan et al., 2008; Hewitt et al., 2009), whilst in contrast, microinjection of the GABAAR agonist muscimol reduced the cir- culating levels of this steroid (Cullinan et al., 2008). Collectively, these observations suggest that GABAAR-mediated inhibition is important for the regulation of HPA axis activity. Given this prominent role for GABA within the local stress neurocircuitry, it is conceivable that the rapid elevation of neurosteroid lev- els that occurs during stress (Purdy et al., 1991), may act as a brake upon HPA axis activity by enhancing the GABAergic inhi- bition exerted upon the PVN (Figure 2A). Indeed, the steroid synthesizing enzymes 5α-reductase and 3α-HSD are expressed and 5α3α-THPROG immunoreactivity can be detected within the hypothalamus and extended amygdala respectively (Li et al., 1997; Eechaute et al., 1999; Gao et al., 2002; Saalmann et al., 2007). There- fore, locally produced neurosteroids could potentially contribute to the control of HPA activity. The following section will discuss the physiological and patho- physiological roles of endogenous neurosteroids in acute and chronic stress. www.frontiersin.org December 2011 | Volume 5 | Article 131 | 5