Epipregnanolone Acts as a Partial Agonist on a Common Neurosteroid Modulatory Site of the GABAA Receptor Complex in Avian CNS

Neurosteroid modulatory sites present in the GABA_A receptor complex in chick optic lobe were investigated, in order to evaluate whether allopregnanolone and alphaxalone act through a common site of action. Results showed that either allopregnanolone or alphaxalone present a single-component enhancement of [³H]flunitrazepam binding with EC₅₀ of 1.18 ± 0.12 and 6.56 ± 0.86 M and E_max of 82.18 ± 5.80 and 62.98 ± 3.73 %, respectively. Epipregnanolone behaved as a partial agonist of these steroid modulatory sites with EC₅₀ of 0.49 ± 0.15 M and E_max 12.34 ± 1.03%. Moreover, the addition of 16 M epipregnanolone to either allopregnanolone or alphaxalone decreased EC₅₀ values to 0.54 ± 0,09 and 1.24 ± 0.25 M respectively, while E_max values were not significantly affected. On the other hand, additivity experiments disclosed that a maximal concentration (16 M) of alphaxalone in the presence of allopregnanolone failed to enhance [³H]flunitrazepam binding in excess of that produced by allopregnanolone alone. Results indicate that not only allopregnanolone and alphaxalone act through a common site of action, but such site is highly stereospeciflc with regard to the neurosteroid spatial configuration.     

Comparative Modulation by 3α,5α and 3β,5β Neurosteroids of GABA Binding Sites During Avian Central Nervous System Development

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABA_A receptor complex. The presence of a 3-hydroxyl group and a 5-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [³H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3-hydroxy,5-pregnan-20-one (3,5-P) and 3-hydroxy,5-pregnan-20-one (3,5-P). Both steroids were found able to enhance [³H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3,5-P had greater potency (EC₅₀ 0.22 M) and enhancing effect (E_max: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABA_A receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3,5-P by its 3,5 isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5-reduced progesterone metabolites present since early development, while 3,5-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5-steroids in early avian development, we propose that 3,5-P, instead of the classical potent 3,5-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.     

GABAA Receptors in Central Nervous System Disease: Anxiety,Epilepsy, and Insomnia

Brain function is based on an exquisite balance between excitatory and inhibitory neurotransmission. GABAergic neurons provide the major inhibitory control. By controlling spike timing and sculpting neuronal rhythms they play a key role in regulating behavior. GABAergic neurons are highly diverse and operate with a corresponding diversity of GABA_A receptor subtypes. In this article, the contribution of GABA_A receptor deficits to central nervous system disorders, in particular anxiety disorders, epilepsy, schizophrenia and insomnia, is reviewed.     

Hypoxia Differentially Reduces GABAA Receptor Density During Embryonic Chick Optic Lobe Development

It has been demonstrated that the CNS is severely affected by hypoxic-ischemic insults during the prenatal-perinatal period, including imbalance in excitatory and inhibitory neurotransmitter release. Using a previously developed model of acute normobaric hypoxic hypoxia on chick embryos, we studied alterations observed both on [3H]GABA binding saturation parameters and on lactate concentration on successive embryonic days (ED). While maximal density of GABA binding sites (Bmax) from the low-affinity site was significantly reduced in an age-dependent manner, earlier stages of development (ED12 and 16) proving more vulnerable (ED12: control = 5.48 +/- 0.20, hypoxia = 3.90 +/- 0.39 pmol/mg prot, P < .05; ED16: control = 3.89 +/- 0.26, hypoxia = 2.80 +/- 0.28 pmol/mg prot, P < .05), ligand affinity (Kd) values and kinetic constants of the high-affinity site remained unaltered. Not unlikely, a physiological hypoxic state prevailing from ED17 up to hatching time rendered the whole embryo less sensitive to an externally induced hypoxic state (ED17: control = 2.93 +/- 0.06, hypoxia = 2.38 +/- 0.04 pmol/mg prot, P < .05; ED18: control = 2.97 +/- 0.12, hypoxia = 2.87 +/- 0.27 pmol/mg prot). Lactate levels in chick optic lobe homogenates were constant during development. The increase observed after hypoxic treatment compared to control value was significant at all stages studied, but increased percentage changes proved similar, indicating that all days of development equally perceive externally induced hypoxia. In conclusion, the present work demonstrates that after normobaric hypoxic hypoxia at different embryonic days, the embryo senses the externally induced hypoxic state as from ED12, but the GABA(A) receptor is differentially affected. It may be speculated that a different subunit composition of GABA(A) receptor is assembled in order to build a more stable receptor capable of resisting the physiological hypoxic state observed during the last few days before hatching.     

Biphasic Modulation of GABAA Receptor Binding by Steroids Suggests Functional Correlates

Neuroactive steroids and other positive modulators of GABA_A receptors showed regional variation in both the efficacy and potency for modulation of [³⁵S]TBPS binding to rat brain membrane homogenates, with biphasic concentration-dependence. GABA present in the binding assays prevented the enhancement phase of the steroid concentration-dependence plot while the antagonists bicuculline and RU5135 prevented the inhibition phase. Using recombinant GABA_A receptors, expressed in insect cell line Sf9 using baculovirus, enhancement by steroids of [³⁵S]TBPS binding was sensitive to the presence of the 2 subunit and the nature of the subunit (122S > 12, 62, 622S, and 62). As in cerebellum, addition of RU5135 reduced the inhibitory phase and revealed a small enhancement of TBPS binding by neuroactive steroids. The subunit-dependent interactions of steroid and GABA site ligands are consistent with a three-state model in which the receptor mono-liganded by GABA or steroid has a different affinity for TBPS than the resting state, and the receptor biliganded by GABA, steroid, or both has little affinity for TBPS.     

The GABAA Receptor Complex as a Target for Fluoxetine Action

The clinically important antidepressant fluoxetine is established as a selective serotonin reuptake inhibitor. This study demonstrates that fluoxetine also interacts with the GABA_A receptor complex. At concentrations above 10 M fluoxetine inhibited the binding of both [³H]GABA (IC₅₀ = 2 mM) and [³H]flunitrazepam (IC₅₀ = 132 M ) to the GABA_A receptor complex in brain cortical membranes. Low fluoxetine concentrations (1 nM) enhanced GABA-stimulated Cl^– uptake by a rat cerebral cortical vesicular preparation. At higher concentrations (100 M and 1 mM), however, fluoxetine inhibited GABA-stimulated Cl^– uptake, an effect related to a reduction in E_max. These observations might assist in an explanation of the basis of the antidepressant action of fluoxetine.     

Differential Drug Responses on Native GABAA Receptors Revealing Heterogeneity in Extrasynaptic Populations in Cultured Hippocampal Neurons

Hippocampal pyramidal neurons potentially express multiple subtypes of GABA_A receptors at extrasynaptic locations that could therefore respond to different drugs. We activated extrasynaptic GABA_A receptors in cultured rat hippocampal pyramidal neurons and measured single-channel currents in order to compare the actions of two drugs that potentially target different GABA_A receptor subtypes. Despite the possible difference in receptor targets of etomidate and diazepam, the two drugs were similar in their actions on native extrasynaptic GABA_A receptors. Each drug produced three distinct responses that differed significantly in current magnitude, implying heterogeneous GABA_A receptor populations. In the majority of patches, drug application increased both the single-channel conductance (>40 pS) and the open probability of the channels. By contrast, in the minority of patches, drug application caused an increase in open probability only. In the third group high-conductance channels were observed upon GABA activation and drug application increased their open probability only. The currents potentiated by etomidate or diazepam were substantially larger in patches displaying high-conductance GABA channels compared to those displaying only low-conductance channels. Factors contributing to the large magnitude of these currents were the long mean open time of high-conductance channels and the presence of multiple channels in these patches. In conclusion, we suggest that the local density of extrasynaptic GABA_A receptors may influence their single-channel properties and may be an additional regulating factor for tonic inhibition and, importantly, differential drug modulation. This work is dedicated to the memory of Professor P. W. Gage.     

Neurosteroid modulation of native and recombinant GABAA receptors

Summary 1. The pioneering work of Hans Selye over 50 years ago demonstrated that certain steroid metabolites can produce a rapid depression of central nervous system activity.2. Research during the last 10 years has established that such effects are mediated by a nongenomic and specific interaction of these steroids with the brain's major inhibitory receptor, the GABA_A receptor.3. Here we describe the molecular mechanism of action of such steroids and review attempts to define the steroid binding site on the receptor protein. The therapeutic potential of such neurosteroids is discussed.     

Methylmercury modulates GABAA receptor complex differentially in rat cortical and cerebellar membranes in vitro

Effects of methylmercury (MetHg) on the specific [³H]flunitrazepam binding were studied in rat cortical and cerebellar P₂-fractions in vitro. MetHg did not affect significantly the specific [³H]flunitrazepam binding in unwashed P₂-fraction but increased it marginally (by 16%) at 100 M in washed P₂-fraction, in both brain regions.Muscimol (3 M), a GABA_A agonist, stimulated the [³H]flunitrazepam binding by 30% to 50% depending on the brain region. In washed cerebellar membranes the enhancing response of muscimol was 10 to 14% lower after preincubation of the tissue with MetHg but in cerebral cortex MetHg did not modulate the muscimol response at all. The results indicate that Met-Hg may have region specific effects on GABA_A receptors in vitro and the effect may depend on the occupational state of the GABA binding domain of the receptor complex.     

Effects of GABA antagonists,SR 95531 and bicuculline,on GABAA receptor-regulated chloride flux in rat cortical synaptoneurosomes

Synaptoneurosomes isolated from cerebral cortices of male Sprague-Dawley rats were used for studying GABA_A receptor-regulated chloride influx. The in vitro effects of GABA antagonists, SR 95531 (a pyridazinyl GABA derivative) and bicuculline, on pentobarbital-stimulated, muscimol-stimulated or flunitrazepam-enhanced, muscimol-stimulated chloride uptake were studied. The chloride uptake was determined at 30°C, for 5 sec. Pentobarbital and muscimol produced a maximal stimulation of chloride uptake in cortical synaptoneurosomes at 500 M and 50M, respectively. SR 95531 as well as bicuculline had no effect on the basal uptake of chloride. Whereas, SR 95531 (0.3–30 M) and bicuculline (0.1–100 M), when added 5 min before muscimol (50 M), produced a significant concentration-dependent inhibition of muscimol (50 M)-stimulated chloride uptake (IC₅₀ s of 0.89±0.11 M and 13.45±2.10M, respectively). In studies of the inhibitory effects of SR 95531 and bicuculline on pentobarbital (500 M)-stimulated chloride uptake, the IC₅₀ s were 0.81±0.12 M and 3.86±1.14 M, respectively. SR 95531 exhibited a more potent inhibitory effect than bicuculline on flunitrazepam-enhanced, muscimol-stimulated chloride uptake. The results revealed that SR 95531 has a more potent antagonistic effect than bicuculline on GABA_A-regulated chloride flux.     

Evidence for a Reduction of Coupling between GABA<Subscript>A</Subscript> Receptor Agonist and Ionophore Binding Sites by Inorganic Phosphate

[³⁵S]TBPS binding to the GABA_A receptor ionophore binding site is anion dependent. Using autoradiography on rat brain sections, we show that permeabilities of anions through the receptor channel correlate with their efficiencies to promote basal [³⁵S]TBPS binding. Phosphate made an exception as it induced more binding than expected from its permeability. Well-permeable anions (chloride, nitrate, formate) allowed [³⁵S]TBPS binding to be effectively displaced by 1 mM GABA, whereas low-permeable anions (acetate, phosphate, propionate) markedly prevented this GABA effect, especially in the thalamus, the transition from the high to the low GABA effect being between formate and acetate. In the presence of phosphate, GABA enhanced [³H]flunitrazepam binding to benzodiazepine site of recombinant α1β2γ2 receptors with the same efficacy but lower potency as compared to the presence of chloride, whereas [³⁵S]TBPS binding was abnormally modulated by GABA. These results suggest that inorganic phosphate affects coupling between agonist and ionophore sites in GABA_A receptors. Special issue dedicated to Simo S. Oja     

Effects of Bisphenol A and its Derivatives on the Response of GABAA Receptors Expressed in Xenopus Oocytes

To study the effects of bisphenol-A (BPA) known to have estrogenic actions, and its derivatives, 3,5-dimethylphenol (DMP) and p-t-butylphenol (TBP), on ionotropic γ-aminobutyric acid (GABA) receptors, GABA_A receptors were expressed in Xenopus oocytes by injecting both poly(A)⁺RNA prepared from rat whole brain and cRNAs synthesized from cloned cDNAs of α₁ and β₁ subunit of the bovine receptors, and their electrical responses were measured by the voltage clamping method. BPA caused the potentiation and inhibition of the former receptor-responses, while it caused only inhibition of the latter ones. In the presence of low concentrations of GABA, DMP and TBP potentiated the responses of both receptors. DMP and TBP also increased the rate of decay of the response, possibly by desensitization of the receptors when GABA solution was continuously bath-applied. Diethyl terephthalate (DTP), which is also known to have estrogenic actions, had little effect on both the responses and the decay of both receptors.     

Biphasic action of midazolam on GABAA receptor-mediated responses in rat sacral dorsal commissural neurons

The effect of the benzodiazepine agonist midazolam on gamma-aminobutyric acid(A) (GABA(A)) receptor-mediated currents was investigated in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. Midazolam displayed a biphasic effect on GABA responses. Low concentrations of midazolam (1nM-10 microM) reversibly potentiated GABA (3 microM)-activated Cl(-) currents (I(GABA)) in a bell-shaped manner, with the maximal facilitary effect at 0.1 microM; whereas at higher concentrations (above 10 microM), midazolam had an antagonistic effect on I(GABA). Our further study indicated that midazolam changed GABA(A) receptor affinity to GABA and the effects of midazolam on I(GABA) were voltage-independent. The benzodiazepine receptor antagonist, flumazenil, abolished the facilitary effect of low concentrations of midazolam rather than the antagonism of I(GABA) induced by high doses of midazolam. In addition, activation of protein kinase C prevented the inhibitory effect of midazolam at higher concentrations, but did not influence the effect of midazolam at low concentrations. These results indicate that midazolam interacts with another distinct site other than the central benzodiazepine receptors on GABA(A) receptors as an antagonist at higher concentrations in SDCN neurons.     

Complex Role of Collybistin and Gephyrin in GABAA Receptor Clustering

Gephyrin and collybistin are key components of GABA_A receptor (GABA_AR) clustering. Nonetheless, resolving the molecular interactions between the plethora of GABA_AR subunits and these clustering proteins is a significant challenge. We report a direct interaction of GABA_AR α2 and α3 subunit intracellular M3–M4 domain (but not α1, α4, α5, α6, β1–3, or γ1–3) with gephyrin. Curiously, GABA_AR α2, but not α3, binds to both gephyrin and collybistin using overlapping sites. The reciprocal binding sites on gephyrin for collybistin and GABA_AR α2 also overlap at the start of the gephyrin E domain. This suggests that although GABA_AR α3 interacts with gephyrin, GABA_AR α2, collybistin, and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABA_AR α2 and collybistin or GABA_AR α2 and gephyrin are strengthened in the presence of gephyrin or collybistin, respectively. Collybistin and gephyrin also compete for binding to GABA_AR α2 in co-immunoprecipitation experiments and co-localize in transfected cells in both intracellular and submembrane aggregates. Interestingly, GABA_AR α2 is capable of “activating ” collybistin isoforms harboring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABA_AR α2-collybistin interaction was disrupted by a pathogenic mutation in the collybistin SH3 domain (p.G55A) that causes X-linked intellectual disability and seizures by disrupting GABA_AR and gephyrin clustering. Because immunohistochemistry in retina revealed a preferential co-localization of collybistin with α2 subunit containing GABA_ARs, but not GlyRs or other GABA_AR subtypes, we propose that the collybistin-gephyrin complex has an intimate role in the clustering of GABA_ARs containing the α2 subunit.     

Sex Steroids Effects on the Content of GAD, TH, GABAA, and Glutamate Receptors in the Olfactory Bulb of the Male Rat

Sex steroids exert multiple functions in the central nervous system. They modulate responses to olfactory information in mammals but their participation in the regulation of neurotransmission in the olfactory bulb is unknown. We studied by Western blot the effects of estradiol (E2), progesterone (P4), and allopregnanolone (Allo) on the content of glutamic acid decarboxylase (GAD), γ-aminobutyric acid A receptor α-2 subunit (GABA_AR α-2), glutamate receptor 2/3 (GlutR 2/3), and tyrosine hydroxylase (TH) in the olfactory bulb of gonadectomized male rats. GAD content was increased by all steroids administered alone. Interestingly, progestins reduced E2 effects on GAD content. Steroids increased the content of TH and GABA_AR α-2. In contrast, GlutR 2/3 content was decreased by E2 and P4, whereas Allo did not modify it. These results suggest that estrogens and progestins regulate olfactory bulb functions in the male rat by modulating the expression of key proteins involved in several neurotransmission systems. Special issue article in honor of Dr. Ricardo Tapia.     

The GABAA receptor is an FMRP target with therapeutic potential in fragile X syndrome

Previous research indicates that the GABA_Aergic system is involved in the pathophysiology of the fragile X syndrome, a frequent form of inherited intellectual disability and associated with autism spectrum disorder. However, the molecular mechanism underlying GABA_Aergic deficits has remained largely unknown. Here, we demonstrate reduced mRNA expression of GABA_A receptor subunits in the cortex and cerebellum of young Fmr1 knockout mice. In addition, we show that the previously reported underexpression of specific subunits of the GABA_A receptor can be corrected in YAC transgenic rescue mice, containing the full-length human FMR1 gene in an Fmr1 knockout background. Moreover, we demonstrate that FMRP directly binds several GABA_A receptor mRNAs. Finally, positive allosteric modulation of GABA_A receptors with the neurosteroid ganaxolone can modulate specific behaviors in Fmr1 knockout mice, emphasizing the therapeutic potential of the receptor.     

Prolonged Treatment with Ligands Affects Ligand Binding to the Human Serotonin1A Receptor in Chinese Hamster Ovary Cells

SUMMARY 1. The serotonin_1A receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins, and appear to be involved in several behavioral and cognitive functions.2. We monitored the effect of prolonged treatment of the human serotonin_1A receptor expressed in Chinese hamster ovary (CHO) cells with pharmacologically well-characterized ligands on its binding to the agonist 8-hydroxy-2(di-N-propylamino)tetralin (8-OH-DPAT) and antagonist 4-(2′-methoxy)-phenyl-1-[2′-(N-2″-pyridinyl)-p-fluorodobenzamido]ethyl-piperazine (p-MPPF).3. Our results indicate that prolonged treatment with the specific agonist (8-OH-DPAT) differentially affects subsequent binding of the agonist and antagonist to the receptor in a manner independent of receptor-G-protein coupling. Importantly, our results show that prolonged treatment with the commonly used antagonist p-MPPF, and its iodinated analogue 4-(2′-methoxy)-phenyl-1-[2′-(N-2″-pyridinyl)-p-iodobenzamido]ethyl-piperazine (p-MPPI), which have earlier been reported to display similar binding properties to serotonin_1A receptors, induces significantly different effects on the ligand binding function of serotonin_1A receptors.     

Effect of Chronic Administration of Ethanol on the Regulation of Tyrosine Kinase Phosphorylation of the GABA<Subscript>A</Subscript> Receptor Subunits in the Rat Brain

One of the many pharmacological targets of ethanol is the GABA inhibitory system, and chronic ethanol (CE) is known to alter the polypeptide levels of the GABA_A receptor subunits in rat brain regions. In the present study, we investigated the regulation of the tyrosine kinase phosphorylation of the GABA_A receptor α₁-, β₂- and γ₂-subunits in the rat cerebellum, cerebral cortex and hippocampus following chronic administration of ethanol to the rats. We observed either down-regulation or no change in the tyrosine kinase phosphorylation of the α₁ subunit, whereas there was an up-regulation or no change in the case of β₂- and γ₂-subunits of the GABA_A receptors depending on the brain region following chronic administration of ethanol to the rats. These changes reverted back to the control level following 48 h of ethanol-withdrawal. These results suggest that tyrosine kinase phosphorylation of GABA_A receptors may play a significant role in ethanol dependence.