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.

Adolescent synaptic pruning is thought to enable optimal cognition because it is disrupted in certain neuropathologies, yet the initiator of this process is unknown. One factor not yet considered is the α4βδ GABAA receptor (GABAR), an extrasynaptic inhibitory receptor which first emerges on dendritic spines at puberty in female mice. Here we show that α4βδ GABARs trigger adolescent pruning. Spine density of CA1 hippocampal pyramidal cells decreased by half post-pubertally in female wild-type but not α4 KO mice. This effect was associated with decreased expression of kalirin-7 (Kal7), a spine protein which controls actin cytoskeleton remodeling. Kal7 decreased at puberty as a result of reduced NMDAR activation due to α4βδ-mediated inhibition. In the absence of this inhibition, Kal7 expression was unchanged at puberty. In the unpruned condition, spatial re-learning was impaired. These data suggest that pubertal pruning requires α4βδ GABARs. In their absence, pruning is prevented and cognition is not optimal.DOI: http://dx.doi.org/10.7554/eLife.15106.001

α4βδ GABAA receptors (GABARs) have low CNS expression, but their expression is increased by 48 h exposure to the neurosteroid THP (3α-OH-5α[β]-pregnan-20-one). THP also increases the efficacy of δ-containing GABARs acutely, where GABA is a partial agonist. Thus, we examined effects of THP (100 nM) and full GABA agonists at α4β2δ (gaboxadol, 10 μM, and β-alanine, 10 μM – 1 mM), on surface expression of α4β2δ. To this end, we used an α4 construct tagged with a 3XFLAG (F) epitope or measured expression of native α4 and δ. HEK-293 cells or cultured hippocampal neurons were transfected with α4Fβ2δ and treated 24 h later with GABA agonists, THP, GABA plus THP or vehicle (0.01% DMSO) for 0.5 h – 48 h. Immunocytochemistry was performed under both non-permeabilized and permeabilized conditions to detect surface and intracellular labeling, respectively, using confocal microscopy. The high efficacy agonists and GABA (1 or 10μM) plus THP increased α4β2δ surface expression up to 3-fold after 48 h, an effect first seen by 0.5 h. This effect was not dependent upon the polarity of GABAergic current, although expression was increased by KCC2. Intracellular labeling was decreased while functional expression was confirmed by whole cell patch clamp recordings of responses to GABA agonists. GABA plus THP treatment did not alter the rate of receptor removal from the surface membrane, suggesting that THP-induced α4β2δ expression is likely via receptor insertion. Surface expression of α4β2δ was decreased by rottlerin (10 μM), suggesting a role for PKC- δ. These results suggest that trafficking of α4β2δ GABARs is regulated by high efficacy states.

SummaryGABA A receptors (GABAR) mediate most inhibition in the CNS and are also a target for neuroactive steroids such as 3α,5[α]β-THP (3αOH-5[α]β-OH-pregnan-20-one or [allo]pregnanolone). Although these steroids robustly enhance current gated by α1β2δ GABAR, we have shown that 3α,5[α]β-THP effects at recombinant α4β2δ GABAR depend on the direction of Cl-flux, where the steroid increases outward flux, but decreases inward flux through the receptor. This polarity-dependent inhibition of α4β2δ GABAR resulted from an increase in the rate and extent of rapid desensitization of the receptor, recorded from recombinant receptors expressed in HEK-293 cells with whole cell voltage clamp techniques. This inhibitory effect of 3α,5[α]β-THP was not observed at other receptor subtypes, suggesting it was selective for α4β2δ GABAR. Furthermore, it was prevented by a selective mutation of basic residue arginine 353 in the intracellular loop of the receptor, suggesting that this might be a putative chloride modulatory site. Expression of α4βδ GABAR increases markedly at extrasynaptic sites at the onset of puberty in female mice. At this time, 3α,5[α]β-THP decreased the inhibitory tonic current, recorded with perforated patch techniques to maintain the physiological Cl-gradient. By decreasing this shunting inhibition, 3α,5[α]β-THP increased the excitability of CA1 hippocampal pyramidal cells at puberty: These effects of the steroid were opposite to those observed before puberty when 3α,5[α]β-THP reduced neuronal excitability as a pre-synaptic effect. Behaviorally, the excitatory effect of 3α,5[α]β-THP was reflected as an increase in anxiety at the onset of puberty in female mice. Taken together, these findings suggest that the emergence of α4β2δ GABAR at the onset of puberty reverses the effect of a stress steroid. These findings may be relevant for the mood swings and increased response to stressful events reported in adolescence.

The aim of this study was to investigate the effect of liraglutide on Alzheimer-like learning and memory impairment in mice, which were intracerebroventricularly (i.c.v.) injected with streptozotocin (STZ). Twenty-four mice were randomly divided into three groups: control (CON), AD model (STZ), and liraglutide-treated (LIR). The results show that both hyperphosphorylated tau and neurofilament proteins had deceased protein glycosylation and the tau bound to microtubules was lower in the STZ group compared to the CON group. The expression of JNK phosphorylation was higher and the number of Fluoro-Jade-B-positive degenerative neurons was increased in the STZ group as compared to both the CON and liraglutide groups. Escape latency in the STZ group was longer than that in both the CON and LIR groups, while the number of hidden platform crossings in path length was less than that in the other two groups. Liraglutide decreased the hyperphosphorylation levels of tau and neurofilament proteins, increased protein O-glycosylation, increased tau bound to microtubules, and also significantly improved the learning and memory ability of the mice. These results suggest that the effects of liraglutide on decreasing the hyperphosphorylation of tau and neurofilament proteins by enhancing O-glycosylation of neuronal cytoskeleton protein, improving the JNK and ERK signaling pathway, and reducing neural degeneration may be related to its protective effects on AD-like learning and memory impairment induced by i.c.v. injection of STZ. Our results indicate that GLP-1 analogs represent a novel treatment strategy for Alzheimer's disease.