Gunn et al. GABAA receptors, neurosteroids, and stress ACUTE STRESS Early behavioral studies demonstrated the anxiolytic properties of pregnane steroids in a variety of animal models of anxiety (Crawley et al., 1986; Bitran et al., 1995; Carboni et al., 1996). In addition, the levels of 5α3α-THPROG and 5α3α-THDOC were rapidly elevated in the rodent hypothalamus and cortex following exposure to an acute swim stress (Purdy et al., 1991). Interestingly, adrenalectomy prevented the stress-induced increase in brain lev- els of 5α3α-THDOC, but not 5α3α-THPROG demonstrating the de novo synthesis of the latter neurosteroid in response to stress (Purdy et al., 1991). Similarly, elevated levels of 5α3α-THDOC, following exposure to an acute swim stress, raised the thresh- old of pentylenetetrazol (PTZ)-induced seizures in a ﬁnasteride- and indometacin-sensitive manner (Reddy and Rogawski, 2002). Collectively, these observations suggest that neurosteroids synthe- sized de novo within the CNS together with those derived from peripheral sources (i.e., 5α3α-THDOC from the adrenal cortex; Eser et al., 2005) may play a physiological role in modulating the stress response. In electrophysiological recordings made from neonatal mouse [postnatal (PN) days 18–24] hypothalamus, we ﬁnd low concen- trations (10–100 nM) of 5α3α-THPROG to enhance the synaptic inhibition of neuroendocrine parvocellular neurons of the PVN and to inhibit their output (Belelli et al., 2009; Figure 2B). In com- mon, the output of spinally projecting parvocellular neurons is similarly reduced by low concentrations of 5α3α-THDOC (Wom- ack et al., 2006). Neuroendocrine parvocellular neurons are known to activate the HPA axis through the release of CRH (Vale et al., 1981) and i.c.v. injection of this neuropeptide induces behaviors closely resembling those associated with stress and anxiety (Owens and Nemeroff, 1991). Interestingly, the anxiogenic effects of CRH in rats exposed to the EPM were inhibited in a dose-dependent manner by pre-treatment with 5α3α-THPROG (Patchev et al., 1994). Additionally, although having no effect upon basal CRH release in vitro, 5α3α-THPROG did suppress the methoxamine [an α1-adrenoreceptor (α1-AR) agonist]-induced release of this neuropeptide (Patchev et al., 1994). This in vitro observation is intriguing given that the parvocellular region of the PVN receives considerable noradrenergic inputs from the nucleus of the soli- tary tract and the locus coeruleus (Cunningham and Sawchenko, 1988). Both of these structures are believed to trigger responses to “physical” stressors, an effect that is possibly mediated via a contribution by α1-ARs (Daftary et al., 2000; Han et al., 2002). The magnitude and polarity of GABAAR-mediated neurotrans- mission is critically dependent upon the transmembrane Cl− gradient. Thus, generally, an increase in the intracellular Cl− con- centration can shift the GABA-mediated response from neuronal hyperpolarization (inhibitory) to depolarization (excitatory). The transmembrane Cl− gradient is maintained by cation-chloride co-transporters (CCC), with Na+–K+–2Cl− co-transporter 1 (NKCC1) and K+–Cl− transporter 2 (KCC2) being responsible for the majority of neuronal Cl− uptake and extrusion respec- tively within the CNS. Crucially the relative functional expression of these transporters appears not to be static, but dynamically reg- ulated during certain physiological (e.g., development) and patho- physiological states (reviewed in Blaesse et al., 2009). Interestingly, a recent study has suggested that exposure to acute restraint stress functionally down-regulates KCC2 such that the action of GABA in neuroendocrine parvocellular neurons shifts from being hyperpolarizing to depolarizing, thus profoundly weaken- ing synaptic inhibitory transmission (Hewitt et al., 2009). The authors postulate that activation of α1-ARs underlies the depo- larizing shift in the reversal potential for GABA (EGABA), most likely via the activation of protein kinase C (PKC). The ability of secondary messenger systems to indirectly and dynamically modulate the inhibitory tone exerted upon the HPA axis through changes in KCC2 function may be an important mechanism in the initiation and termination of the physiological response trig- gered by stress. Intriguingly, GABAergic transmission (measured by [35S] t -butylbicyclophosphorothionate (TBPS) binding and 36Cl− uptake), has long been known to be rapidly (within 5 min) decreased following various forms of acute stress (Sanna et al., 1992; Barbaccia et al., 1996; reviewed in Biggio et al., 2007), whereas the subsequent increases in brain and plasma neuros- teroid levels peaked 30 min after the onset of stress and was correlated with the restoration of GABAergic transmission and reduced anxiety-like behavior (Barbaccia et al., 2001). These ﬁnd- ings support the proposal that the neuroendocrine and autonomic responses to a stressor may be modulated by these endogenous neurosteroids, which in this scenario would act to terminate the stress response. Nevertheless, although as noted above the enzymes are expressed within this region (Eechaute et al., 1999), it remains to be determined whether these neurosteroids are actually pro- duced de novo, speciﬁcally in the PVN and nearby surrounding regions in response to a stressful challenge. DOC mediates many of the physiological responses to acute stress (Reddy, 2006). Thus, it is conceivable that metabolites of this steroid, e.g., 5α3α-THDOC may contribute to other stress- induced alterations in inhibitory transmission via their actions on the GABAAR (Reddy, 2006). Indeed, the exposure of mice to a sin- gle acute hypoxic challenge increases expression of the GABAAR δ subunit in the hippocampus and enhances the GABAAR-mediated tonic conductance of DGGCs (Maguire and Mody, 2007). The incubation of brain slices for 30 min with 5α3α-THDOC followed by a 30- to 120-min washout period of the steroid, induced a similar increase in the tonic conductance of DGGCs (Maguire and Mody, 2007). The mechanism(s) responsible for this 5α3α- THDOC-induced up-regulation of δ-GABAARs remain to be determined. However, the previously reported “glucocorticoid- like” effects of endogenous neurosteroids (Patchev et al., 1994, 1996) and the GABAAR-dependent modulation of neuropeptide expression (Cole and Sawchenko, 2002; Bali and Kovacs, 2003; Brunton et al., 2009) within the PVN may be relevant in this respect. CHRONIC STRESS Exposure to chronic or repeated stress induces signiﬁcant struc- tural and functional alterations within the stress neurocircuitry, which are primarily and initially involved in promoting adap- tation by allostasis, but in the long term can have deleterious effects upon brain function (Joels et al., 2007; McEwen, 2010). Indeed, dysregulation of the HPA axis has been implicated in the pathogenesis of a number of psychiatric and neurological conditions including depression, post-traumatic stress disorder Frontiers in Neuroscience | Neuroendocrine Science December 2011 | Volume 5 | Article 131 | 6