Puberty is characterized by mood swings and anxiety, often produced by stress. Here, we show that THP (allopregnanolone), a steroid released by stress, increases anxiety in pubertal female mice, a reversal of its well-known anxiety-reducing effect in adults. Anxiety is regulated by GABAergic inhibition in limbic circuits. Although this inhibition is increased by THP before puberty and in adults, THP reduced tonic inhibition of CA1 hippocampal pyramidal cells at puberty, leading to increased excitability. This paradoxical effect of THP was due to inhibition of α4βδ GABA A receptors. These receptors are normally expressed at very low levels, but at puberty, their expression was increased in CA1 hippocampus where they generated outward currents. THP also decreased outward current at recombinant α4β2δ receptors, an effect dependent on arginine 353 in the α4 subunit, a putative Cl − modulatory site. Thus, inhibition of α4β2δ GABA A receptors by THP provides a mechanism for anxiety at puberty.The onset of puberty is associated with increases in emotional reactivity and anxiety 1,2 . Responses to stressful events are amplified 3 , and anxiety and panic disorder first emerge at this time 2 , twice as likely to occur in girls than in boys 2 . Few studies have addressed the biological basis of this important issue, although suicide risk increases in adolescence, despite the use of adult-based medical strategies 2 .The GABA A receptor plays a pivotal role in the generation of anxiety 4 . This receptor is the target for endogenous steroids such as THP (3α-OH-5α [β]-pregnan-20-one or [allo] pregnanolone), which increase GABA-gated currents at physiological concentrations 5 of the steroid. THP is a metabolite of the ovarian/adrenal steroid progesterone, but is also formed in the brain as a compensatory response to stress 6 . In adults, THP potently reduces anxiety in humans 7 , an effect seen in animal models with direct administration into the dorsal CA1 hippocampus 8 , part of the limbic system that regulates emotion. It is generally accepted that * Correspondence and requests for materials should be addressed to S.S.Smith, Dept. of Physiology and Pharmacology, SUNY Downstate Medical Center, 450 Clarkson Ave., Brooklyn, NY 11203 USA; phone: 718-270-2226; FAX: 718-270-3103; email: Sheryl.smith@downstate NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the GABA-enhancing action of THP underlies its well-known anxiety-reducing effect in adults, which is similar to other GABA-enhancing drugs such as the benzodiazepines.GABA A receptors are pentamers formed predominantly of 2α, 2β and 1γ subunits 9 which gate a Cl − current and produce most fast synaptic inhibition in the brain. Substitution of the δ subunit for γ2 yields a receptor with the highest sensitivity to steroids such as THP 10-12 . These highly sensitive δ-GABA A receptors are extrasynaptic 13 , and mediate tonic rather than synaptic inhibition in areas such as dentate gyrus 14 . Thus, THP and related steroids enhance inhibition h...

The onset of puberty defines a developmental stage when some learning processes are diminished, but the mechanism for this deficit remains unknown. We found that, at puberty, expression of inhibitory α4βδ γ-aminobutyric acid type A (GABA A ) receptors (GABAR) increases perisynaptic to excitatory synapses in CA1 hippocampus. Shunting inhibition via these receptors reduced Nmethyl-D-aspartate receptor activation, impairing induction of long-term potentiation (LTP). Pubertal mice also failed to learn a hippocampal, LTP-dependent spatial task that was easily acquired by δ−/− mice. However, the stress steroid THP (3αOH-5α[β]-pregnan-20-one), which reduces tonic inhibition at puberty, facilitated learning. Thus, the emergence of α4βδ GABARs at puberty impairs learning, an effect that can be reversed by a stress steroid.Certain learning and cognitive processes decline at the onset of puberty (1-3). The pubertal process that shapes this developmental decline is unknown but is likely to involve the hippocampus, which is widely regarded as the site for learning (4-6). In addition to excitatory input, the inhibitory GABAergic (GABA, γ-aminobutyric acid) system plays a pivotal role in shaping developmental plasticity, as in the visual cortex (7), where drugs that target the γ-aminobutyric acid type A (GABA A ) receptor (GABAR) alter the timing of the critical period. The GABAR mediates most central nervous system inhibition and consists of diverse subtypes with distinct properties. Of these, α4βδ GABARs increase at pubertal onset in the mouse hippocampus (8), suggesting that they may shape plasticity here.We employed immunocytochemical, electron microscopic techniques (9) to localize and quantify α4 and δ GABAR subunits on CA1 hippocampal pyramidal cells across the pubertal state of female mice, because females exhibit greater deficits in learning at puberty than males † To whom correspondence should be addressed. sheryl.smith@downstate.edu. * These authors contributed equally to this work. (10,11). We detected immunostaining of both subunits perisynaptic to asymmetric synapses on the plasma membrane of spines of the apical dendrite, which increased up to 700% at puberty ( Fig. 1, A to C, and fig. S1; α4, P = 0.0048; δ, P = 0.00091) (9). In contrast, α4 and δ immunoreactivity on the dendritic shaft increased by less than 100% at puberty ( fig. S2). Functional expression of δ-containing GABAR at puberty was demonstrated by robust responses of pyramidal cells at puberty to 100 nM gaboxadol, which, at this concentration, is selective for this receptor (Fig. 1, D and E) (12). Gaboxadol had no effect before puberty and only a modest effect in the adult hippocampus (Fig. 1, D and E), where α4 and δ expression is lower than at puberty ( fig. S3).Extrasynaptic α4β2δ GABARs on spines could impair voltage-triggered Mg++ unblock of Nmethyl-D-aspartate (NMDA) receptors. Thus, we used whole-cell voltage clamp techniques with blockade of synaptic GABARs (13) to record evoked NMDA excitatory postsynaptic currents (EPSCs) from CA1 ...

Neurosteroids, such as the progesterone metabolite 3α-OH-5α[β]-pregnan-20-one (THP or [allo] pregnanolone), function as potent positive modulators of the GABA A receptor (GABAR) when acutely administered. However, fluctuations in the circulating levels of this steroid at puberty, across endogenous ovarian cycles, during pregnancy or following chronic stress produce periods of prolonged exposure and withdrawal, where changes in GABAR subunit composition may occur as compensatory responses to sustained levels of inhibition. A number of laboratories have demonstrated that both chronic administration of THP as well as its withdrawal transiently increase expression of the α4 subunit of the GABAR in several areas of the central nervous system (CNS) as well as in in vitro neuronal systems. Receptors containing this subunit are insensitive to benzodiazepine (BDZ) modulation and display faster deactivation kinetics, which studies suggest underlie hyperexcitability states. Similar increases in α4 expression are triggered by withdrawal from other GABA-modulatory compounds, such as ethanol and BDZ, suggesting a common mechanism. Other studies have reported puberty or estrous cycle-associated increases in δ-GABAR, the most sensitive target of these steroids which underlies a tonic inhibitory current. In the studies reported here, the effect of steroids on inhibition, which influence anxiety state and seizure susceptibility, depend not only on the subunit composition of the receptor but also on the direction of Cl -current generated by these target receptors. The effect of neurosteroids on GABAR function thus results in behavioral outcomes relevant for pubertal mood swings, premenstrual dysphoric disorder and catamenial epilepsy, which are due to fluctuations in endogenous steroids.

In this study, 48 h administration of 3α-OH-5β-pregnan-20-one (3α,5β-THP) or 17β-estradiol (E 2 )+progesterone (P) to female rats increased expression of the δ subunit of the GABA A receptor (GABAR) in CA1 hippocampus. Coexpression of α4 and δ subunits was suggested by an increased response of isolated pyramidal cells to the GABA agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), following 48 h steroid treatment, and nearly complete blockade by 300 μM lanthanum (La 3+ ). Because α4βδ GABAR are extrasynaptic, we also recorded pharmacologically isolated GABAergic holding current from CA1 hippocampal pyramidal cells in the slice. The La 3+ -sensitive THIP current, representative of current gated by α4βδ GABAR, was measurable only following 48 h steroid treatment. In contrast, the bicucullinesensitive current was not altered by steroid treatment, assessed with or without 200 nM gabazine to block synaptic current. However, 48 h steroid treatment resulted in a tonic current insensitive to the benzodiazepine agonists lorazepam (10 μM) and zolpidem (100 nM). These results suggest that 48 h steroid treatment increases expression of α4βδ GABAR which replace the ambient receptor population. Increased anxiolytic effects of THIP were also observed following 48 h steroid treatment. The findings from the present study may be relevant for alterations in mood and benzodiazepine sensitivity reported across the menstrual cycle.