Qi Hua Gong

Hormonally regulated α4β2δ GABAA receptors are a target for alcohol

Here we report that low concentrations of alcohol (1–3 mM) increased Cl− currents gated by a recombinant GABAA receptor, α4β2δ, by 40–50% in Xenopus laevis oocytes. We also found greater hippocampal expression of receptors containing α4 and δ subunits, using a rat model of premenstrual syndrome (PMS) in which 1–3 mM alcohol preferentially enhanced GABA-gated currents, and low doses of alcohol attenuated anxiety and behavioral reactivity. The alcohol sensitivity of δ-containing receptors may underlie the reinforcing effects of alcohol during PMS, when eye saccade responses to low doses of alcohol are increased.

Short-term exposure to a neuroactive steroid increases α4 GABAA receptor subunit levels in association with increased anxiety in the female rat

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.

Reversal of neurosteroid effects at α4β2δ GABAA receptors triggers anxiety at puberty

Puberty is characterized by mood swings and anxiety, which are often produced by stress. Here we show that THP (allopregnanolone), a steroid that is released as a result of stress, increases anxiety in pubertal female mice, in contrast to its anxiety-reducing effect in adults. Anxiety is regulated by GABAergic inhibition in limbic circuits. Although this inhibition is increased by THP administration before puberty and in adults, during puberty THP reduces the tonic inhibition of pyramidal cells in hippocampal region CA1, leading to increased excitability. This paradoxical effect of THP results from inhibition of α4βδ GABAA receptors. These receptors are normally expressed at very low levels, but at puberty, their expression is increased in hippocampal area CA1, where they generate outward currents. THP also decreases the outward current at recombinant α4β2δ receptors, and this effect depends on arginine 353 in the α4 subunit, a putative site for modulation by Cl−. Therefore, inhibition of α4β2δ GABAA receptors by THP provides a mechanism for the generation of anxiety at puberty.

Short-term steroid treatment increases δ GABAA receptor subunit expression in rat CA1 hippocampus: Pharmacological and behavioral effects

In this study, 48 h administration of 3alpha-OH-5beta-pregnan-20-one (3alpha,5beta-THP) or 17beta-estradiol (E2)+progesterone (P) to female rats increased expression of the delta subunit of the GABA(A) receptor (GABAR) in CA1 hippocampus. Coexpression of alpha4 and delta 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 microM lanthanum (La3+). Because alpha4betadelta GABAR are extrasynaptic, we also recorded pharmacologically isolated GABAergic holding current from CA1 hippocampal pyramidal cells in the slice. The La3+-sensitive THIP current, representative of current gated by alpha4betadelta GABAR, was measurable only following 48 h steroid treatment. In contrast, the bicuculline-sensitive 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 microM) and zolpidem (100 nM). These results suggest that 48 h steroid treatment increases expression of alpha4betadelta 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.

Progesterone withdrawal increases the α4 subunit of the GABAA receptor in male rats in association with anxiety and altered pharmacology — a comparison with female rats

Withdrawal from the neurosteroid 3alpha,5alpha-allopregnanolone after chronic administration of progesterone increases anxiety in female rats and up-regulates the alpha4 subunit of the GABA(A) receptor (GABA(A)-R) in the hippocampus. We investigated if these phenomena would also occur in male rats. Progesterone withdrawal (PWD) induced higher alpha4 subunit expression in the hippocampus of both male and female rats, in association with increased anxiety (assessed in the elevated plus maze) comparable to effects previously reported. Because alpha4-containing GABA(A)-R are insensitive to the benzodiazepine (BDZ) lorazepam (LZM), and are positively modulated by flumazenil (FLU, a BDZ antagonist), we therefore tested the effects of these compounds following PWD. Using whole-cell patch clamp techniques, LZM-potentiation of GABA ((EC20))-gated current was markedly reduced in CA1 pyramidal cells of male rats undergoing PWD compared to controls, whereas FLU had no effect on GABA-gated current in control animals but increased it in PWD animals. Behaviorally, both male and female rats were significantly less sensitive to the anxiolytic effects of LZM. In contrast, FLU demonstrated significant anxiolytic effects following PWD. These data suggest that neurosteroid regulation of the alpha4 GABA(A)-R subunit may be a relevant mechanism underlying anxiety disorders, and that this phenomenon is not sex-specific.

Neurosteroid administration and withdrawal alter GABAA receptor kinetics in CA1 hippocampus of female rats

Withdrawal from the GABA‐modulatory steroid 3α‐OH‐5α‐pregnan‐20‐one (3α,5α‐THP) following exposure of female rats to the parent compound progesterone (P) produces a syndrome characterized by behavioural excitability in association with up‐regulation of the α4 subunit of the GABAA receptor (GABAR) in the hippocampus. Similar changes are seen after 48 h exposure to its stereoisomer, 3α,5β‐THP. Here, we further characterize the effects of P withdrawal on GABAR kinetics, using brief (1 ms) application of 5–10 mm GABA to outside‐out patches from acutely isolated CA1 hippocampal pyramidal cells. Under control conditions, GABA‐gated current deactivated biexponentially, with τfast= 12–19 ms (45–60% of the current), and τslow= 80–140 ms. P withdrawal resulted in marked acceleration of deactivation (τfast= 3–7 ms and τslow= 30–100 ms), as did 48 h exposure to 3α,5β‐THP (τfast= 5–8 ms; τslow= 40–120 ms). When recombinant receptors were tested in HEK‐293 cells, a similar acceleration in τfast was observed for α4β2δ and α4β2γ2 GABARs, compared to α1β2γ2 and α5β2γ2 receptors. In addition, τslow was also accelerated for α4β2δ receptors, which are increased following steroid withdrawal. As predicted by the Jones‐Westbrook model, this change was accompanied by reduced receptor desensitization as well as an acceleration of the rate of recovery from rapid desensitization. A theoretical analysis of the data suggested that steroid treatment leads to receptors with a greater stability of the bound, activatable state. This was achieved by altering multiple parameters, including desensitization and gating rates, within the model. These results suggest that fluctuations in endogenous steroids result in altered GABAR kinetics which may regulate neuronal excitability.

Progesterone withdrawal increases the anxiolytic actions of gaboxadol: role of α4βδ GABAA receptors

Hippocampal α4βδ GABA A receptors (GABA A -R) are increased following progesterone withdrawal (PWD) in a rodent model of premenstrual anxiety. This α4βδ receptor isoform uniquely responds to the GABA agonist gaboxadol (THIP) with a maximum current greater than that gated by GABA, and is potentiated more by pentobarbital than are other GABA A -R. We therefore investigated the anxiolytic effects of these drugs using the elevated plus maze. Gaboxadol (1.25 mg/kg) was markedly more anxiolytic in animals undergoing PWD than in controls. Pentobarbital (10 mg/kg) also produced a greater anxiolytic effect during PWD. These results suggest that the pharmacological properties of α4βδ GABAA-R following PWD are evident behaviorally. Alterations in the α4βδ GABA A -R population may have implications for the etiology and treatment of premenstrual syndrome.

Neurosteroid effects at α4βδ GABAA receptors alter spatial learning and synaptic plasticity in CA1 hippocampus across the estrous cycle of the mouse

Fluctuations in circulating levels of ovarian hormones have been shown to regulate cognition (Sherwin and Grigorova, 2011. Fertil. Steril. 96, 399-403; Shumaker et al., 2004. JAMA. 291, 2947-2958), but increases in estradiol on the day of proestrus yield diverse outcomes: In vivo induction of long-term potentiation (LTP), a model of learning, is reduced in the morning, but optimal in the afternoon (Warren et al., 1995. Brain Res. 703, 26-30). The mechanism underlying this discrepancy is not known. Here, we show that impairments in both CA1 hippocampal LTP and spatial learning observed on the morning of proestrus are due to increased dendritic expression of α4βδ GABAA receptors (GABARs) on CA1 pyramidal cells, as assessed by electron microscopic (EM) techniques, compared with estrus and diestrus. LTP induction and spatial learning were robust, however, when assessed on the morning of proestrus in α4-/- mice, implicating these receptors in mediating impaired plasticity. Although α4βδ expression remained elevated on the afternoon of proestrus, increases in 3α-OH-THP (3α-OH-5α-pregnan-20-one) decreased inhibition by reducing outward current through α4βδ GABARs (Shen et al., 2007. Nat. Neurosci. 10, 469-477), in contrast to the usual effect of this steroid to enhance inhibition. Proestrous levels of 3α-OH-THP reversed the deficits in LTP and spatial learning, an effect prevented by the inactive metabolite 3β-OH-THP (10 mg/kg, i.p.), which antagonizes actions of 3α-OH-THP. In contrast, administration of 3α-OH-THP (10 mg/kg, i.p.) on the morning of proestrus improved spatial learning scores 150-300%. These findings suggest that cyclic fluctuations in ovarian steroids can induce changes in cognition via α4βδ GABARs that are dependent upon 3α-OH-THP. This article is part of a Special Issue entitled SI: Brain and Memory.

Ethanol administration rapidly reverses α4 GABAA receptor subunit upregulation following steroid exposure

Both short-term (48 h) exposure to the neuroactive steroid 3alpha,5alpha[beta]-THP and its withdrawal increase expression of the benzodiazepine (BDZ)-insensitive GABAA receptor (GABAR) alpha4 subunit in hippocampus. This increase in alpha4 subunit expression was associated with a relative insensitivity of CA1 hippocampal pyramidal cells to modulation of GABA-gated current by the BDZ lorazepam (LZM), assessed using whole cell patch clamp techniques. Chronic ethanol is also known to regulate expression of the alpha4 subunit. Thus, in the present study we investigated the capacity of ethanol, administered in low doses across a 2 h period (0.5 g/kg, i.p., 3x), to suppress alpha4 expression produced by 48 h exposure to 3alpha,5 beta-THP in adult female rats. We show here that 2 h ethanol administration reverses the increase in alpha4 expression normally observed following 48 h steroid treatment. This effect was correlated with a recovery of responses recorded from CA1 hippocampal pyramidal cells to the GABA-modulatory effects of LZM. Similar effects of ethanol in suppressing alpha4 expression and restoring LZM responsiveness were seen following steroid withdrawal when alpha4 expression is normally increased. These results suggest that increases in expression of the alpha4 subunit produced by steroid exposure or withdrawal are altered by other GABA-modulatory drugs, such as ethanol.

GABAA RECEPTOR ALPHA 4 SUBUNIT SUPPRESSION PREVENTS WITHDRAWAL PROPERTIES OF AN ENDOGENOUS STEROID

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 brains 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.