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 hormone progesterone is readily converted to 3alpha-OH-5alpha-pregnan-20-one (3alpha,5alpha-THP) in the brains of males and females. In the brain, 3alpha,5alpha-THP acts like a sedative, decreasing anxiety and reducing seizure activity, by enhancing the function of GABA (gamma-aminobutyric acid), the brain's major inhibitory neurotransmitter. Symptoms of premenstrual syndrome (PMS), such as anxiety and seizure susceptibility, are associated with sharp declines in circulating levels of progesterone and, consequently, of levels of 3alpha,5alpha-THP in the brain. Abrupt discontinuation of use of sedatives such as benzodiazepines and ethanol can also produce PMS-like withdrawal symptoms. Here we report a progesterone-withdrawal paradigm, designed to mimic PMS and post-partum syndrome in a rat model. In this model, withdrawal of progesterone leads to increased seizure susceptibility and insensitivity to benzodiazepine sedatives through an effect on gene transcription. Specifically, this effect was due to reduced levels of 3alpha,5alpha-THP which enhance transcription of the gene encoding the alpha4 subunit of the GABA(A) receptor. We also find that increased susceptibility to seizure after progesferone withdrawal is due to a sixfold decrease in the decay time for GABA currents and consequent decreased inhibitory function. Blockade of the alpha4 gene transcript prevents these withdrawal properties. PMS symptoms may therefore be attributable, in part, to alterations in expression of GABA(A) receptor subunits as a result of progesterone withdrawal.

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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 ...

In the present study, we have characterized properties of steroid withdrawal using a pseudopregnant rat model. This paradigm results in increased production of endogenous progesterone from ovarian sources and as such is a useful physiological model. "Withdrawal" from progesterone induced by ovariectomy on day 12 of pseudopregnancy resulted in increased anxiety, as determined by a decrease in open arm entries on the elevated plus maze compared to control rats and pseudopregnant animals not undergoing withdrawal. Similar findings were obtained 24 hr after administration of a 5␣-reductase blocker to a pseudopregnant animal, suggesting that it is the GABA Amodulatory 3␣-OH-5␣-pregnan-20-one (3␣,5␣-THP) that produces anxiogenic withdrawal symptoms. Twenty-four hours after steroid withdrawal, the time constant for decay of GABA Agated current was also reduced sixfold, assessed using wholecell patch-clamp procedures on pyramidal neurons acutely dissociated from CA1 hippocampus. Thus, 3␣,5␣-THP withdrawal results in a marked decrease in total GABA A current, a possible mechanism for its anxiogenic, proconvulsant sequelae. In addition, 3␣,5␣-THP withdrawal resulted in insensitivity to the normally potentiating effect of the benzodiazepine lorazepam (LZM) on GABA A -gated Cl Ϫ current. This withdrawal profile is similar to that reported for other GABA A -modulatory drugs such as the benzodiazepines (BDZs), barbiturates, and ethanol. These changes were also associated with significant two and threefold increases in both the mRNA and protein for the ␣4 subunit of the GABA A receptor, respectively, in hippocampus. The pseudopregnancy paradigm may be a useful model for periods of endogenous 3␣,5␣-THP withdrawal such as premenstrual syndrome and postpartum or postmenopausal dysphoria, when increased emotional lability and BDZ insensitivity have been reported.

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.

Previous work from this laboratory has demonstrated that withdrawal from the neuroactive steroid 3alpha,5alpha-THP (3alpha-hydroxy-5alpha-pregnan-20-one) after 3-week exposure to its parent compound, progesterone (P), increases anxiety and produces benzodiazepine (BDZ) insensitivity in female rats. These events were linked to upregulation of the alpha4 subunit of the GABA(A) receptor (GABAR) in the hippocampus [Brain Res. 507 (1998) 91; Nature 392 (1998) 926; J. Neurosci. 18 (1998) 5275]. The present study investigates the role of shorter term hormone treatment on alpha4 subunit levels as well as relevant behavioral and pharmacological end-points related to GABAR function. After 2-3 days of P exposure, two- to threefold increases in alpha4 protein levels were observed, which declined to control values after 5-6 days of hormone exposure. This effect was due to the GABA-modulatory metabolite of P, 3alpha,5alpha-THP. alpha4 upregulation was inversely correlated with BDZ potentiation of GABA-gated current, assessed using whole cell patch clamp techniques on acutely isolated hippocampal pyramidal cells. A near total BDZ insensitivity was observed by 2-3 days of hormone exposure in association with the maximal increase in alpha4 levels. Up-regulation of the alpha4 GABAR subunit was also reflected by an increase in anxiety in the elevated plus maze. A significant decrease in open arm entries was observed after 72-h exposure to P, an effect which recovered by 6 days of P treatment. As demonstrated in vitro, alpha4 upregulation also resulted in a relative insensitivity to the anxiolytic actions of BDZ. These results suggest that short-term exposure to 3alpha,5alpha-THP produces changes in GABAR subunit composition similar to those that occur after chronic exposure and withdrawal from the steroid.

Rationale-3α-OH-5α[β]-pregnan-20-one (THP) is a positive modulator of the GABA A receptor (GABAR), which underlies its reported anxiolytic effect. However, there are conditions such as premenstrual dysphoric disorder (PMDD) where increases in THP levels can be associated with adverse mood.Objectives-In order to test for conditions where THP might be anxiogenic, we developed a mouse model of THP withdrawal. Because δ-containing GABAR are highly sensitive to THP modulation, results were compared in wild-type and δ knockout mice.Methods-Finasteride, a 5α-reductase blocker, was administered for 3 days to female wild-type or δ knockout mice. Then, animals were tested in the elevated plus maze, following acute administration of THP, lorazepam, flumazenil, or 4,5,6,pyridin-3-ol (THIP), and results compared to vehicle-injected controls. CA1 hippocampal GABAR α4 subunit levels were assessed by Western blot.Results-After THP withdrawal, THP produced anxiogenic effects, decreasing open arm entries on the elevated plus maze, following a brief shock, in contrast to its expected anxiolytic effects. As we have shown in rats, THP withdrawal also resulted in increased expression of the α4 subunit in mouse CA1 hippocampus. As expected for increases in α4-containing GABAR, THP withdrawn mice were relatively insensitive to the benzodiazepine (BDZ) lorazepam and had atypical responses to the BDZ antagonist flumazenil when tested on the plus maze. In contrast, they showed a greater anxiolytic response to THIP, which has greater efficacy at α4βδ than other GABAR. Although THP Correspondence to: Sheryl S. Smith, Sheryl.smith@downstate.edu. NIH Public AccessAuthor Manuscript Psychopharmacology (Berl) withdrawal in δ knockout mice also increased the α4 GABAR subunit, the anxiogenic effects of THP and the anxiolytic effects of THIP were not observed, implicating α4βδ GABAR in these effects.Conclusions-Based on these behavioral and pharmacological findings, we suggest that THP withdrawal in the mouse may serve as a rodent model of PMDD.