Modulation Of GABAA Receptor Function Research Articles

Aralia continentalis Root Enhances Non-Rapid Eye Movement Sleep by Activating GABAA Receptors.

Aralia continentalis exhibits various biological activities; however, their sleep-promoting effects have not been previously reported. In this study, we evaluated the hypnotic effects and sleep-wake profiles of A. continentalis root (KS-126) using a pentobarbital-induced sleep-acceleration test and polysomnographic recordings. Additionally, we investigated the molecular mechanism of KS-126 through patch-clamp electrophysiology. Our polysomnographic recordings revealed that KS-126 not only accelerated the onset of non-rapid eye movement sleep (NREMS) but also extends its duration. Considering the temporal dynamics of the sleep-wake stages, during the initial and subsequent periods KS-126 extended NREMS duration and decreased wakefulness, thereby enhancing sleep-promoting effects. Furthermore, the assessment of sleep quality via analysis of electroencephalogram power density indicated that KS-126 did not significantly alter sleep intensity. Finally, we found that KS-126 enhanced GABAA receptor-mediated synaptic responses in primary hippocampal neurons, leading to an increase in the percentage of the GABA current. This effect was not affected by the selective benzodiazepine receptor antagonist flumazenil, but was entirely inhibited by the GABAA receptor antagonist bicuculline. In conclusion, KS-126 extends the duration of NREMS without altering its intensity by prolonging GABAergic synaptic transmission, which modulates GABAA receptor function.

GABAA Receptor Modulators with a Pyrazolo[1,5-a]quinazoline Core: Synthesis, Molecular Modelling Studies and Electrophysiological Assays.

As a continuation of our study in the GABAA receptor modulators field, we report the design and synthesis of new 8-chloropyrazolo[1,5-a]quinazoline derivatives. Molecular docking studies and the evaluation of the 'Proximity Frequencies' (exploiting our reported model) were performed on all the final compounds (3, 4, 6a-c, 7a,b, 8, 9, 12a-c, 13a,b, 14-19) to predict their profile on the α1β2γ2-GABAAR subtype. Furthermore, to verify whether the information coming from this virtual model was valid and, at the same time, to complete the study on this series, we evaluated the effects of compounds (1-100 µM) on the modulation of GABAA receptor function through electrophysiological techniques on recombinant α1β2γ2L-GABAA receptors expressed in Xenopus laevis oocytes. The matching between the virtual prediction and the electrophysiological tests makes our model a useful tool for the study of GABAA receptor modulators.

Modulation of the mammalian GABAA receptor by type I and type II positive allosteric modulators of the α7 nicotinic acetylcholine receptor.

Positive allosteric modulators of the α7 nicotinic acetylcholine (nACh) receptor (α7-PAMs) possess promnesic and procognitive properties and have potential in the treatment of cognitive and psychiatric disorders including Alzheimer's disease and schizophrenia. Behavioural studies in rodents have indicated that α7-PAMs can also produce antinociceptive and anxiolytic effects that may be associated with positive modulation of the GABAA receptor. The overall goal of this study was to investigate the modulatory actions of selected α7-PAMs on the GABAA receptor. We employed a combination of cell fluorescence imaging, electrophysiology, functional competition and site-directed mutagenesis to investigate the functional and structural mechanisms of modulation of the GABAA receptor by three representative α7-PAMs. We show that the α7-PAMs at micromolar concentrations enhance the apparent affinity of the GABAA receptor for the transmitter and potentiate current responses from the receptor. The compounds were equi-effective at binary αβ and ternary αβγ GABAA receptors. Functional competition and site-directed mutagenesis indicate that the α7-PAMs bind to the classic anaesthetic binding sites in the transmembrane region in the intersubunit interfaces, which results in stabilization of the active state of the receptor. We conclude that the tested α7-PAMs are micromolar-affinity, intermediate- to low-efficacy allosteric potentiators of the mammalian αβγ GABAA receptor. Given the similarities in the in vitro sensitivities of the α7 nACh and α1β2γ2L GABAA receptors to α7-PAMs, we propose that doses used to produce nACh receptor-mediated behavioural effects in vivo are likely to modulate GABAA receptor function.

Neurosteroid Modulation of GABAA Receptor Function by Independent Action at Multiple Specific Binding Sites.

Neurosteroids are endogenous modulators of GABAA receptors that mediate anxiety, pain, mood and arousal. The 3-hydroxyl epimers, allopregnanolone (3α-OH) and epiallopregnanolone (3β-OH) are both prevalent in the mammalian brain and produce opposite effects on GABAA receptor function, acting as positive and negative allosteric modulators, respectively. This Perspective provides a model to explain the actions of 3α-OH and 3β-OH neurosteroids. The model is based on evidence that the neurosteroid epimers bind to an overlapping subset of specific sites on GABAA receptors, with their net functional effect on channel gating being the sum of their independent effects at each site.

Humulone Modulation of GABAA Receptors and Its Role in Hops Sleep-Promoting Activity.

Humulus lupulus L. (hops) is a major constituent of beer. It exhibits neuroactive properties that make it useful as a sleeping aid. These effects are hypothesized to be mediated by an increase in GABAA receptor function. In the quest to uncover the constituents responsible for the sedative and hypnotic properties of hops, recent evidence revealed that humulone, a prenylated phloroglucinol derivative comprising 35–70% of hops alpha acids, may act as a positive modulator of GABAA receptors at low micromolar concentrations. This raises the question whether humulone plays a key role in hops pharmacological activity and potentially interacts with other modulators such as ethanol, bringing further enhancement in GABAA receptor-mediated effects of beer. Here we assessed electrophysiologically the positive modulatory activity of humulone on recombinant GABAA receptors expressed in HEK293 cells. We then examined humulone interactions with other active hops compounds and ethanol on GABA-induced displacement of [3H]EBOB binding to native GABAA receptors in rat brain membranes. Using BALB/c mice, we assessed humulone’s hypnotic behavior with pentobarbital- and ethanol-induced sleep as well as sedation in spontaneous locomotion with open field test. We demonstrated for the first time that humulone potentiates GABA-induced currents in α1β3γ2 receptors. In radioligand binding to native GABAA receptors, the inclusion of ethanol enhanced humulone modulation of GABA-induced displacement of [3H]EBOB binding in rat forebrain and cerebellum as it produced a leftward shift in [3H]EBOB displacement curves. Moreover, the additive modulatory effects between humulone, isoxanthohumol and 6-prenylnaringenin were evident and corresponded to the sum of [3H]EBOB displacement by each compound individually. In behavioral tests, humulone shortened sleep onset and increased the duration of sleep induced by pentobarbital and decreased the spontaneous locomotion in open field at 20 mg/kg (i.p.). Despite the absence of humulone effects on ethanol-induced sleep onset, sleep duration was increased dose-dependently down to 10 mg/kg (i.p.). Our findings confirmed humulone’s positive allosteric modulation of GABAA receptor function and displayed its sedative and hypnotic behavior. Humulone modulation can be potentially enhanced by ethanol and hops modulators suggesting a probable enhancement in the intoxicating effects of ethanol in hops-enriched beer.

Methylene blue inhibits GABAA receptors by interaction with GABA binding site

Methylene blue (MB) is commonly used in diagnostic procedures and is also used to treat various medical conditions. Neurological effects of MB have been reported in clinical observations and experimental studies. Thus the modulation of GABAA receptor function by MB was investigated. Whole-cell GABA-activated currents were recorded from HEK293 cells expressing various GABAA receptor subunit configurations. MB inhibition of GABA currents was apparent at 3 μM, and it had an IC50 of 31 μM in human α1β2γ2 receptors. The MB action was rapid and reversible. MB inhibition was not mediated via the picrotoxin site, as a mutation (T6′F of the β2 subunit) known to confer resistance to picrotoxin had no effect on MB-induced inhibition. Blockade of GABAA receptors by MB was demonstrated across a range of receptors expressing varying subunits, including those expressed at extrasynaptic sites. The sensitivity of α1β2 receptors to MB was similar to that observed in α1β2γ2 receptors, indicating that MB's action via the benzodiazepine or Zn2+ site is unlikely. MB-induced inhibition of GABA response was competitive with respect to GABA. Furthermore, mutation of α1 F64 to A and β2 Y205 to F in the extracellular N-terminus, both residues which are known to comprise GABA binding pocket, remarkably diminished MB inhibition of GABA currents. These data suggest that MB inhibits GABAA receptor function by direct or allosteric interaction with the GABA binding site. Finally, in mouse hippocampal CA1 pyramidal neurons, MB inhibited GABA-activated currents as well as GABAergic IPSCs. We demonstrate that MB directly inhibits GABAA receptor function, which may underlie some of the effects of MB on the CNS.

Synergistic Modulation of γ-Aminobutyric Acid Type A Receptor-Mediated Synaptic Inhibition in Cortical Networks by Allopregnanolone and Propofol

The neuroactive steroid allopregnanolone (ALLO) is an endogenous allosteric modulator of γ-aminobutyric acid type A (GABAA) receptors. There is evidence that ALLO, at physiologically relevant concentrations, modulates GABAA receptor function in the cerebral cortex. The widely used anesthetic agent propofol and ALLO share a similar mode of molecular action. Here, we ask how GABAA receptor-mediated synaptic inhibition and action potential firing of neurons in cultured cortical slices are altered by either ALLO or propofol or by coapplying both agents. We explored the effects of ALLO and propofol on spontaneous action potential activity of neocortical neurons in organotypic slices cultured from C57BL6 mice by performing extracellular multiunit recordings. Furthermore, we carried out whole-cell voltage-clamp experiments to quantify the drug effects on GABAA receptor-mediated tonic and phasic currents. We found that ALLO (100 nM) decreased multiunit action potential firing of neocortical neurons by approximately 21%. Moreover, the duration of GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) was prolonged (mean Δdecay time prolongation: 12.9 ± 2.2 milliseconds; n = 23), and a bicuculline-sensitive tonic current was induced (mean Δbaseline shift: -24.6 ± 13.6 pA; P = .002; n = 6). A subanesthetic concentration of propofol (250 nM) decreased the discharge rates of cortical neurons to a similar degree as ALLO (100 nM). ALLO and propofol administered in combination acted in an additive manner to reduce action potential firing. However, during ALLO administration, propofol was significantly more effective in enhancing GABAergic synaptic transmission. Propofol (250 nM) prolonged the inhibitory postsynaptic currents decay times by 10.4 ± 6.1 milliseconds (n = 9) with ALLO added to the bathing solution; in the absence of ALLO, however, propofol prolonged the decay time by only 3.8 ± 2 milliseconds (n = 13). In cortical neurons, GABAA receptor-mediated synaptic transmission is potentiated by ALLO and propofol in a synergistic manner, whereas the effects on spontaneous action potential activity appear additive. A coapplication of neurosteroids and propofol in general anesthesia and intensive care medicine may open new ways to reduce anesthetic dose requirements and, thus, avoid undesired anesthetic-induced side effects.

Synthesis and pharmacological evaluation of functionalized isoindolinones on GABA-activated chloride currents in rat cerebellum granule cells in culture

A focused N-substituted 3-(2-piperazin-1-yl-2-oxoethyl)-2-(pyridin-2-yl)iso-indolin-1-ones small library was synthesized for modulation of GABA-A receptor function and compared to Zopiclone for the ability to increase GABA-activated chloride currents. All compounds were tested for their effects on GABA-activated chloride currents in rat cerebellar granule cells by use of the whole-cell patch clamp technique. Electrophysiological studies on cultured cerebellar granule cells revealed 3-[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-2-(5-nitropyridin-2-yl)iso-indolin-1-one (Id) as a partial agonist displaying 34% increase of the 10μM GABA evoked peak chloride currents, antagonized by flumazenil. Moreover, a second group of compounds, with bulky functional groups at N-4 position of piperazine, have shown inverse agonist effects.The simple synthetic procedure and the possibility of modulating the efficacy of this class of ligands through additional structural modifications pave the way for further development of new molecules as a novel class of compounds able to interfere with benzodiazepine receptors.

S.15.02 Modulation of GABA-A receptor function for the treatment of anxiety disorders

Clinical inertia has been defined as lack of change in medication treatment at visits where a medication adjustment appears to be indicated. This paper seeks to identify the extent of clinical inertia in medication treatment of bipolar disorder. A second goal is to identify patient characteristics that predict this treatment pattern.Data describe 23,406 visits made by 1815 patients treated for bipolar disorder during the STEP-BD practical clinical trial. Visits were classified in terms of whether a medication adjustment appears to be indicated, and also whether or not one occurred. Multivariable regression analyses were conducted to find which patient characteristics were predictive of whether adjustment occurred.36% of visits showed at least 1 indication for adjustment. The most common indications were non-response to medication, side effects, and start of a new illness episode. Among visits with an indication for adjustment, no adjustment occurred 19% of the time, which may be suggestive of clinical inertia. In multivariable models, presence of any indication for medication adjustment was a predictor of receiving one (OR=1.125, 95% CI =1.015, 1.246), although not as strong as clinical status measures.The associations observed are not necessarily causal, given the study design. The data also lack information about physician-patient communication.Many patients remained on the same medication regimen despite indications of side effects or non-response to treatment. Although lack of adjustment does not necessarily reflect clinical inertia in all cases, the reasons for this treatment pattern merit further examination.

Analgesic strategies aimed at stimulating the endogenous production of allopregnanolone

A growing number of studies indicate that 3-alpha reduced neurosteroids are remarkable analgesics in various pain states. This is the case for allopregnanolone (AP), one of the most potent endogenous positive allosteric modulators of GABAA receptor function. From the pioneering work of Hans Selye, who described the sedative properties of steroids, synthetic compounds resembling the progesterone metabolite AP have been developed. If some of them have been used as anesthetics, it seems difficult to propose them as a therapeutic option for pain since they display several adverse side effects such as sedation, amnesia and functional tolerance. An alternative strategy, chosen by few laboratories around the world, is aimed at stimulating the local production of 3-alpha reduced neurosteroids in order to limit these well-known side effects. This pharmacological approach has the advantage of targeting specific structures, fully equipped with the necessary biosynthetic enzymatic machinery, where neurosteroids already act as endogenous pain modulators. The various pharmacological trials which attempted to treat pain symptoms by stimulating the production of 3-alpha reduced neurosteroids are reviewed here, as well as novel neurotransmitter systems possibly regulating their endogenous production.

Neurosteroid, GABAergic and hypothalamic pituitary adrenal (HPA) axis regulation: what is the current state of knowledge in humans?

A robust epidemiological literature suggests an association between chronic stress and the development of affective disorders. However, the precise biological underpinnings of this relationship remain elusive. Central to the human response and adaptation to stress, activation and inhibition of the hypothalamic pituitary adrenal (HPA) axis involves a multi-level, multi-system, neurobiological stress response which is as comprehensive in its complexity as it is precarious. Dysregulation in this complex system has implications for human stress related illness. The pioneering research of Robert Purdy and colleagues has laid the groundwork for advancing our understanding of HPA axis regulation by stress-derived steroid hormones and their neuroactive metabolites (termed neurosteroids), which are potent allosteric modulators of GABAA receptor function in the central nervous system. This review will describe what is known about neurosteroid modulation of the HPA axis in response to both acute and chronic stress, particularly with respect to the current state of our knowledge of this process in humans. Implications of this research to the development of human stress-related illness are discussed in the context of two human stress-related psychiatric disorders - major depressive disorder and premenstrual dysphoric disorder. Neurosteroid-mediated HPA axis dysregulation is a potential pathophysiologic mechanism which may cross traditional psychiatric diagnostic classifications. Future research directions are identified.

Differential modulation of GABAA receptor function by aryl pyrazoles

Several aryl pyrazoles characterized by a different molecular structure (flexible vs constrained), but chemically related to rimonabant and AM251, were tested for their ability to modulate the function of recombinant α1β2γ2L GABAA receptors expressed in Xenopus laevis oocytes. The effects of 6Bio-R, 14Bio-R, NESS 0327, GP1a and GP2a (0.3–30μM) were evaluated using a two-electrode voltage-clamp technique. 6Bio-R and 14Bio-R potentiated GABA-evoked Cl− currents. NESS 0327, GP1a and GP2a did not affect the GABAA receptor function, but they acted as antagonists of 6Bio-R. Moreover, NESS 0327 inhibited the potentiation of the GABAA receptor function induced by rimonabant. The benzodiazepine site seems to participate in the action of these compounds. In fact, flumazenil antagonized the potentiation of the GABAA receptor induced by 6Bio-R, and NESS 0327 reduced the action of lorazepam and zolpidem. On the contrary, NESS 0327 did not antagonize the action of “classic” GABAergic modulators (propanol, anesthetics, barbiturates or steroids). In α1β2 receptors 6Bio-R potentiated the GABAergic function, but flumazenil was still able to antagonize the potentiation induced by 6Bio-R. Aryl pyrazole derivatives activity at the GABAA receptor depends on their molecular structure. These compounds bind to both an αβγ binding site, and to an α⧸β site which do not require the γ subunit and that may provide structural leads for drugs with potential anticonvulsant effects.

Allopregnanolone modulation of HPA axis function in the adult rat

GABAergic neuronal circuits regulate neuroendocrine stress response, and the most potent positive endogenous modulator of GABAA receptor function is allopregnanolone. This neurosteroid acts in a nongenomic manner to selectively increase the inhibitory signal meditated by GABAA receptors; in addition, it also induces long-lasting changes in the expression of specific GABAA receptor subunits in various brain regions, with consequent changes in receptor function. The objective of this review is to summarize our findings on emotional state and stress responsiveness in three animal models in which basal brain concentrations of allopregnanolone differ. It is postulated that individual differences in allopregnanolone levels can influence general resilience. The results showed that there is an apparent correlation between endogenous levels of brain allopregnanolone and basal and stress-stimulated HPA axis activity. The relationship between endogenous brain levels of allopregnanolone and HPA axis activity and function sustains the therapeutic potential of this neurosteroid for the treatment of stress-associated disorders.

Phosphatidylinositol 4,5-bisphosphate depletion fails to affect neurosteroid modulation of GABAA receptor function

Neurosteroids and likely other lipid modulators access transmembrane sites on the GABAA receptor (GABAAR) by partitioning into and diffusing through the plasma membrane. Therefore, specific components of the plasma membrane may affect the potency or efficacy of neurosteroid-like modulators. Here, we tested a possible role for phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that governs activity of many channels and transporters, in modulation or function of GABAARs. In these studies, we sought to deplete plasma-membrane PIP2 and probe for a change in the strength of potentiation by submaximal concentrations of the neurosteroid allopregnanolone (3α5αP) and other anesthetics, including propofol, pentobarbital, and ethanol. We also tested for a change in the behavior of negative allosteric modulators pregnenolone sulfate and dipicrylamine. We used Xenopus oocytes expressing the ascidian voltage-sensitive phosphatase (Ci-VSP) to deplete PIP2. Voltage pulses to positive membrane potentials were used to deplete PIP2 in Ci-VSP-expressing cells. GABAARs composed of α1β2γ2L and α4β2δ subunits were challenged with GABA and 3α5αP or other modulators before and after PIP2 depletion. KV7.1 channels and NMDA receptors (NMDARs) were used as positive controls to verify PIP2 depletion. We found no evidence that PIP2 depletion affected modulation of GABAARs by positive or negative allosteric modulators. By contrast, Ci-VSP-induced PIP2 depletion depressed KV7.1 activation and NMDAR activity. We conclude that despite a role for PIP2 in modulation of a wide variety of ion channels, PIP2 does not affect modulation of GABAARs by neurosteroids or related compounds.

Role of neurosteroids in experimental 3-nitropropionic acid induced neurotoxicity in rats

Huntington's disease is an autosomal dominant, progressive, and fatal neurodegenerative disease characterized by motor and non-motor symptoms. Systemic administration of 3-nitropropionic acid, a complex II inhibitor of the electron transport chain induces selective striatal lesions in rodents. Neurosteroids are synthesized in central nervous system, able to modulate GABAA receptor function and has been reported to have neuroprotective action. The present study has been designed to investigate the role of neurosteroids such as progesterone and pregnenolone which are positive and negative modulators of GABA respectively against 3-nitropropionic acid induced experimental Huntington's disease. Systemic administration of 3-nitropropionic acid (10mg/kg i.p.) for 14 days significantly reduced body weight, locomotor activity, motor coordination, balance beam walk performance, antioxidant defense enzymes (reduced glutathione and catalase) and significantly increase oxidative stress markers (lipid peroxidation and nitrite level) in striatum and cortex. 3-Nitropropionic acid treatment also increases pro-inflammatory cytokines (TNF-α and IL-1β) level in striatum. Progesterone (10, 20mg/kg/day i.p.) treatments for 14 days significantly reversed the behavioral, antioxidant defense enzymes, oxidative stress marker and pro-inflammatory cytokines as compared to the 3-Nitropropionic acid treated group. Pregnenolone (1 and 2mg/kg i.p.), a negative modulator of GABAA pretreatment significantly reversed the protective effect of progesterone on behavioral and biochemical parameters. The results of the present study suggest that the positive GABAergic modulation may be beneficial for the treatment of motor disorder.

Postsynaptic GABABReceptors Enhance Extrasynaptic GABAAReceptor Function in Dentate Gyrus Granule Cells

Ambient GABA in the brain tonically activates extrasynaptic GABA(A) receptors, and activity-dependent changes in ambient GABA concentration can also activate GABA(B) receptors. To investigate an interaction between postsynaptic GABA(B) and GABA(A) receptors, we recorded GABA(A) currents elicited by exogenous GABA (10 μm) from dentate gyrus granule cells (DGGCs) in adult rat hippocampal slices. The GABA(B) receptor agonist baclofen (20 μm) enhanced GABA(A) currents. This enhancement was blocked by the GABA(B) receptor antagonist CGP 55845 and intracellular solutions containing the GTP analog GDP-β-s, indicating that baclofen was acting on postsynaptic GABA(B) receptors. Modulation of GABA(A) currents by postsynaptic GABA(B) receptors was not observed in CA1 pyramidal cells or layer 2/3 cortical pyramidal neurons. Baclofen reduced the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) but did not alter sIPSC amplitude or kinetics. Thus, GABA(A) receptors activated at synapses were not modulated by postsynaptic GABA(B) receptors. In contrast, tonic GABA currents and currents activated by the GABA(A) receptor δ subunit-selective agonist THIP (10 μm) were potentiated by baclofen. Our data indicate that postsynaptic GABA(B) receptors enhance the function of extrasynaptic GABA(A) receptors, including δ subunit-containing receptors that mediate tonic inhibition in DGGCs. The modulation of GABA(A) receptor function by postsynaptic GABA(B) receptors is a newly identified mechanism that will influence the inhibitory tone of DGGCs when GABA(B) and GABA(A) receptors are both activated.

In vitro neuropharmacological evaluation of penitrem-induced tremorgenic syndromes: Importance of the GABAergic system

The effects of the fungal neurotoxin penitrem A on the GABAergic and glutamatergic systems in rat brain were evaluated. Penitrem A inhibited binding of the GABA A-receptor ligand [ 3H]TBOB to rat forebrain and cerebellar membrane preparations with IC 50 (half maximal inhibitory concentration) values of 11 and 9 μM, respectively. Furthermore, penitrem A caused a concentration-dependent increase of [ 3H]flunitrazepam and [ 3H]muscimol binding in rat forebrain, but not in cerebellar preparations. The stimulation of [ 3H]flunitrazepam binding by penitrem A was abolished by the addition of GABA. In cerebellar preparations, a different pharmacological profile was found, with penitrem A allosterically inhibiting [ 3H]TBOB binding by interacting with a bicuculline-sensitive site. Moreover, penitrem A inhibited the high affinity uptake of GABA and glutamate into cerebellar synaptosomes with IC 50 values of 20 and 47 μM, respectively. The toxin showed no effect on NMDA or AMPA glutamate receptor binding. In conclusion, our results suggest that penitrem A exerts region-specific effects in the brain, leading to positive modulation of GABA A-receptor function in forebrain. Conversely, penitrem A may act as a bicuculline-like convulsant in cerebellum.

Effect of Mimosa pudica (Linn.) extract on anxiety behaviour and GABAergic regulation of 5-HT neuronal activity in the mouse

Mimosa pudica (Linn.) (M. pudica L.) is a plant used in some countries to treat anxiety and depression. In the present study we investigated the effects of an aqueous extract of M. pudica L. on mouse anxiety-like behaviour using the elevated T maze, and on regulation of dorsal raphe nucleus (DRN) 5-hydroxytryptamine (5-HT) neuronal activity using an in-vitro mouse brain slice preparation. Acute treatment with M. pudica L. extract had an anxiolytic effect on behaviour in the elevated T maze, specifically on inhibitory avoidance behaviour. Acute application of the extract alone had no effect on the activity of DRN 5-HT neurones. However, when co-applied with the GABA(A) receptor agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), the extract enhanced the inhibitory effect of the THIP on DRN 5-HT neurones. These observed effects of M. pudica L. on both behaviour and GABA modulation of 5-HT neuronal activity are similar to the effects of diazepam, the established anxiolytic and positive modulator of the GABA(A) receptor. This study suggests that the aqueous extract of M. pudica L. contains a positive modulator of GABA(A) receptor function and provides impetus for further investigation of the neuropharmacologically active constituents of the extract.

Impaired GABAA receptor endocytosis and its correlation to spatial memory deficits

GABAA receptors mediate the majority of fast synaptic inhibition in the mammalian brain. Mechanisms that regulate GABAA function are thus of critical importance in modulating overall synaptic inhibition. Phosphorylation of GABAA receptor subunits is one such mechanism that leads to the dynamic modulation of GABAA receptor function. In particular, phosphorylation of tyrosine residues 365 and 367 (Y365, Y367) within the γ2 subunit of GABAA receptors has been shown in previous in vitro studies to negatively regulate clathrin-dependent endocytosis of GABAA receptors and to enhance the efficacy of synaptic inhibition. With the aim of investigating the impact of this phosphorylation-dependent modulation of GABAA receptors on animal behavior, we recently developed a knock-in mouse in which these critical tyrosine residues within the γ2 subunit have been mutated to phenylalanines (Y365/7F). These animals exhibited enhanced GABAA receptor accumulation at postsynaptic inhibitory synaptic specializations on pyramidal neurons within the hippocampus, primarily due to aberrant trafficking within the endocytic pathway. We found that this enhanced inhibition correlated with a specific deficit in spatial memory in these mice, without modifying a number of other behavioral paradigms. Here, we summarize our recently reported observations and further discuss their possible implications.

Steroid Interaction with a Single Potentiating Site Is Sufficient to Modulate GABA-A Receptor Function

Neuroactive steroids are efficacious potentiators of GABA-A receptors. Recent work has identified a site in the alpha1 subunit of the GABA-A receptor, that is essential for potentiation by steroids. However, each receptor contains two copies of the alpha1 subunit. We generated concatemers of subunits so that the alpha1 subunits could be mutated separately and examined the consequences of mutations that remove potentiation by most neurosteroids (alpha1 Q241L, alpha1 Q241W). Concatemers were expressed in Xenopus laevis oocytes, and activation by GABA, potentiation by neurosteroids, and the agonist activity of piperidine-4-sulfonic acid (P4S) were determined. When the alpha1 Q241L mutation is present in alpha1 subunits the EC(50) for activation by GABA is shifted to higher concentration and potentiation by neurosteroids is diminished. When the alpha1 Q241W mutation is expressed, the EC(50) for GABA is shifted to lower concentration, the relative efficacy of P4S is increased, and potentiation by neurosteroids is diminished. Mutation of only one alpha1 subunit does not produce the full effect of mutating both sites. Overall, the data demonstrate that at a macroscopic level, the presence of a single wild-type steroid-binding site is sufficient to mediate responses to steroid, but both must be mutated to completely remove the effects of steroids. Differences between the two sites seem to be relatively subtle.