Modulation of presynaptic GABA(A) receptors by endogenous neurosteroids

BACKGROUND AND PURPOSE

Although 3α-hydroxy, 5α-reduced pregnane steroids, such as allopregnanolone (AlloP) and tetrahydrodeoxycorticosterone, are endogenous positive modulators of postsynaptic GABA_A receptors, the functional roles of endogenous neurosteroids in synaptic transmission are still largely unknown.

EXPERIMENTAL APPROACH

In this study, the effect of AlloP on spontaneous glutamate release was examined in mechanically isolated dentate gyrus hilar neurons by use of the conventional whole-cell patch-clamp technique.

KEY RESULTS

AlloP increased the frequency of glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) in a dose-dependent manner. The AlloP-induced increase in sEPSC frequency was completely blocked by a non-competitive GABA_A receptor blocker, tetrodotoxin or Cd²⁺, suggesting that AlloP acts on presynaptic GABA_A receptors to depolarize presynaptic nerve terminals to increase the probability of spontaneous glutamate release. On the other hand, γ-cyclodextrin (γ-CD) significantly decreased the basal frequency of sEPSCs. However, γ-CD failed to decrease the basal frequency of sEPSCs in the presence of a non-competitive GABA_A receptor antagonist or tetrodotoxin. In addition, γ-CD failed to decrease the basal frequency of sEPSCs after blocking the synthesis of endogenous 5α-reduced pregnane steroids. Furthermore, γ-CD decreased the extent of muscimol-induced increase in sEPSC frequency, suggesting that endogenous neurosteroids can directly activate and/or potentiate presynaptic GABA_A receptors to affect spontaneous glutamate release onto hilar neurons.

CONCLUSIONS AND IMPLICATIONS

The modulation of presynaptic GABA_A receptors by endogenous neurosteroids might affect the excitability of the dentate gyrus-hilus-CA3 network, and thus contribute, at least in part, to some pathological conditions, such as catamenial epilepsy and premenstrual dysphoric disorder.     

Differential effects of two major neurosteroids on cerebellar and cortical GABA(A) receptor binding and function

Cerebellar and cerebrocortical A-type γ-aminobutyric acid (GABA_A) receptors were examined in mice and rats. In wild-type mouse cerebellum, the agonists GABA and gaboxadol exerted heterogeneous displacement of [³H]ethynylbicycloorthobenzoate (EBOB) binding with nanomolar and submicromolar affinities. In mouse cerebella lacking α6 subunits (α6KO), nanomolar displacement by GABA agonists was absent, while micromolar displacement was potentiated to 12-fold by 0.3 μM 5α-tetrahydrodeoxycorticosterone (5α-THDOC). In α6KO cerebellum, 60% of [³H]EBOB binding was neurosteroid-insensitive, while 5α-THDOC elicited enhancement with EC₅₀ = 150 nM instead of nanomolar displacement. In conclusion, nanomolar displacement of cerebellar [³H]EBOB binding by GABA agonists and neurosteroids can be attributed to GABA_A receptors containing α6 and δ subunits. In contrast, [³H]EBOB binding to rat cerebral cortex was affected by allopregnanolone and 5α-THDOC in bidirectional manner with nanomolar enhancement (EC₅₀ ~ 80 nM) and micromolar displacement. Nonequilibrium binding conditions with decreased incubation time tripled the maximal enhancement of [³H]EBOB binding by 5α-THDOC. 5ß-THDOC enhanced the cortical [³H]EBOB binding with EC₅₀ ~ 0.5 μM and it attenuated bidirectional modulation by 5α-THDOC. Allopregnanolone and 5α-THDOC produced biphasic enhancements of chloride currents elicited by 1 μM GABA in cerebellar granule cells, for 5α-THDOC with EC_50,1 ~ 16 nM and EC_50,2 ~ 1.3 μM. Differences in peak current enhancements in the absence minus presence of 0.1 mM furosemide corresponding to α6ßδ GABA_A receptors were augmented only by micromolar 5α-THDOC while the difference curve for allopregnanolone was polyphasic as without furosemide. Consequently, these neurosteroids differentially affected the binding and function of various GABA_A receptor populations.     

Brain receptor binding of analogues of gamma-aminobutyric acid (GABA)

This paper reports the synthesis of some 4-(beta-hydroxyalkyl)-gamma-aminobutyric acid analogues (I a-h). The compounds were biologically tested as inhibitors of 3H-GABA binding to rat cortex synaptosomal membranes. The most potent compounds were (I c,d) which exhibited a linear alkyl chain at C4.     

The influence of subunit composition on the interaction of neurosteroids with GABA(A) receptors

The influence of the subunit composition of human GABA(A) receptors upon the GABA-modulatory properties of 5alpha-pregnan-3alpha-ol-20-one (5alpha,3alpha) has been examined using the Xenopus laevis oocyte expression system and the two electrode voltage-clamp technique. Steroid potency (EC(50)) is modestly influenced by the alpha-isoform (alpha(x)beta(1)gamma(2L); x=1-6). alpha(2)-, alpha(4)- and alpha(5)-containing receptors are significantly less sensitive to the action of low concentrations of 5alpha,3alpha (10-100 nM) when compared to alpha(1,3,6)beta(1)gamma(2L) receptors. Additionally, the maximal effect of the steroid is favoured at alpha(6)-containing receptors. The beta-isoform (alpha(1)beta(y)gamma(2L); y=1-3) has little influence on the GABA-modulatory effect of the neurosteroid. The EC(50) of 5alpha,3alpha is only modestly influenced by the omission of the gamma(2) subunit (alpha(1)beta(1)gamma(2L) vs alpha(1)beta(1)): while the maximal effect is favoured by the binary complex. However, the identity of the gamma subunit influences the GABA(A)-modulatory potency of 5alpha,3alpha with gamma(2)- and gamma(1)-containing receptors being the most and the least sensitive to 5alpha,3alpha, respectively. Finally, incorporation of the epsilon, or delta subunit dramatically reduces and augments the GABA-enhancing actions of the steroid, respectively. These findings provide evidence that 5alpha,3alpha discriminates amongst recombinant receptors of varied subunit composition. Furthermore, this selectivity may contribute to their neuronal specificity and behavioural profile.     

谷氨酸和γ-氨基丁酸对原代培养星形胶质细胞神经甾体合成释放的影响

目的研究氨基酸类神经递质对原代培养大鼠大脑皮质星形胶质细胞神经甾体合成释放的影响。方法采用原代培养的大鼠大脑皮质星形胶质细胞,分别加入不同浓度的谷氨酸和γ-氨基丁酸处理48h;采用固相萃取结合高效液相色谱质-谱联用分析方法提取分离和测定细胞培养液中游离型(脱氢表雄酮,DHEA;孕烯醇酮,PREG;别孕烯醇酮,AP)及结合型神经甾体(脱氢表雄酮硫酸酯,DHEAS;孕烯醇酮硫酸酯,PREGS)。结果与生理盐水对照组比较,谷氨酸处理使PREG和PREGS水平明显下降,DHEAS水平明显升高;γ-氨基丁酸处理使PREG水平明显降低,AP水平增加。结论谷氨酸和γ-氨基丁酸两种神经递质对原代培养的星形胶质细胞PREG合成释放均呈抑制作用;谷氨酸对DHE-AS、γ-氨基丁酸对AP的合成释放分别呈现明显促进作用;高剂量的谷氨酸还可以抑制PRGES的合成和释放。     

Mercury interaction with the GABA(A) receptor modulates the benzodiazepine binding site in primary cultures of mouse cerebellar granule cells.

Mercury compounds are neurotoxic compounds with a great specificity for cerebellar granule cells. The interaction of mercury compounds with proteins in the central nervous system may underlie some of their effects on neurotransmission. In this work we study the interaction of mercuric chloride (HgCl2) and methylmercury (MeHg) with the GABA(A) receptor in primary cultures of cerebellar granule cells. Both compounds increased, dose dependently, the binding of [3H]flunitrazepam to the benzodiazepine recognition site. EC50 values for this effect were 3.56 and 15.24 microM for HgCl2 and MeHg, respectively, after 30 min exposure of intact cultured cerebellar granule cells. The increase of [3H]flunitrazepam binding by mercury compounds was completely inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxinin, and by the organochlorine pesticide alpha-endosulfan. It was also partially inhibited by the anion transporter blocker DIDS, however this effect could be due to a possible chelation of mercury by DIDS. Intracellular events, like intracellular calcium, kinase activation/inactivation or antioxidant conditions did not affect [3H]flunitrazepam binding or its increase induced by mercury compounds. The sulfhydryl alkylating agent N-ethylmaleimide mimicked the effect of mercury compounds on [3H]flunitrazepam binding suggesting a common mechanism. We conclude that mercury compounds interact with the GABA(A) receptor by the way of alkylation of SH groups of cysteinyl residues found in GABA(A) receptor subunit sequences.     

Sigma (sigma) receptor and neurosteroids

Studies on the sigma receptor and related compounds are becoming more attractive since they were found to be closely related to higher brain functions such as memory, learning, depression, anxiety, schizophrenia and neuroprotection. Along with these pharmacological findings, the single transmembrane-type, non-metabotropic sigma binding protein has been cloned, while the presence of metabotropic sigma receptor has been also claimed. Thus, various pharmacological findings are now ready to be characterized on the molecular basis of receptor mechanisms. On the other hand, neurosteroids have higher brain functions such as non-genomic fast actions, which are similar to the actions of sigma compounds. Indeed some neurosteroids were revealed to behave as sigma agonists while others behave like antagonists of metabotropic sigma receptors. Pharmacological studies to determine if sigma compounds can be used to cure various central symptoms related to neurosteroids or steroid hormones can be expected.     

GABA(B) receptor-mediated increase of neurosteroids by gamma-hydroxybutyric acid

Among the pharmacological actions of gamma-hydroxybutyric acid (GHB), some may involve GABA(A) receptor-mediated mechanisms. GHB, however, fails to directly interact with sites for agonists and modulators on the GABA(A) receptor complex. We hypothesized that, in vivo, GHB may interfere with GABA(A) receptor function by altering the brain concentrations of the neurosteroids 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone, AP) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone, THDOC), positive allosteric modulators of GABA-gated chloride currents. In male Wistar rats, GHB dose-dependently (75-1000 mg/kg, i.p.) increased AP, THDOC and their precursors pregnenolone and progesterone in brain cortex and hippocampus. The increases of AP (4-5 fold) and THDOC (3-4 fold) elicited by 300 mg/kg GHB peaked between 30 and 90 min and abated by 180 min. The selective GABA(B) receptor antagonist SCH 50911 (50 mg/kg, i.p.) prevented the action of GHB, while the GABA(B) receptor agonist baclofen (5-10 mg/kg) mimicked it. NCS-382 (50 mg/kg, i.p.), the purported selective antagonist of the GHB receptor, failed to antagonize GHB, but at 300 mg/kg increased brain cortical neurosteroids to the same extent as 300 mg/kg GHB; coadministration of GHB and NCS-382, however, failed to yield an additive effect. These results strongly suggest that GHB, via a GABA(B) receptor-mediated mechanism, increases the brain concentrations of neurosteroids, whose properties as amplifiers of the GABA-gated chloride conductances may play a role in the GABA(A) receptor-mediated pharmacological actions of GHB.     

Effects of GABA(A) receptor partial agonists in primary cultures of cerebellar granule neurons and cerebral cortical neurons reflect different receptor subunit compositions

Based on an unexpected high maximum response to piperidine-4-sulphonic acid (P4S) at human alpha1alpha6beta2gamma2 GABA(A) receptors expressed in Xenopus oocytes attempts to correlate this finding with the pharmacological profile of P4S and other GABA(A) receptor ligands in neuronal cultures from rat cerebellar granule cells and rat cerebral cortex were carried out. GABA and isoguvacine acted as full and piperidine-4-sulphonic acid (P4S) as partial agonists, respectively, at alpha1beta2gamma2, alpha6beta2gamma2 and alpha1alpha6beta2gamma2 GABA receptors expressed in Xenopus oocytes with differences in potency. Whole-cell patch-clamp recordings were used to investigate the pharmacological profile of the partial GABA(A) receptor agonists 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP), P4S, 5-(4-piperidyl)isoxazol-3-ol (4-PIOL), and 3-(4-piperidyl)isoxazol-5-ol (iso-4-PIOL), and the competitive GABA(A) receptor antagonists Bicuculline Methbromide (BMB) and 2-(3-carboxypropyl)-3-amino-6-methoxyphenyl-pyridazinium bromide (SR95531) on cerebral cortical and cerebellar granule neurons. In agreement with findings in oocytes, GABA, isoguvacine and P4S showed similar pharmacological profiles in cultured cortical and cerebellar neurones, which are known to express mainly alpha1, alpha2, alpha3, and alpha5 containing receptors and alpha1, alpha6 and alpha1alpha6 containing receptors, respectively. 4-PIOL and iso-4-PIOL, which at GABA(A) receptors expressed in oocytes were weak antagonists, showed cell type dependent potency as inhibitors of GABA mediated responses. Thus, 4-PIOL was slightly more potent at cortical neurones than at granule neurones and iso-4-PIOL was more potent in inhibiting isoguvacine-evoked currents at cortical than at granule neurons. Furthermore the maximum response to 4-PIOL corresponded to that of a partial agonist, whereas that of iso-4-PIOL gave a maximum response close to zero. It is concluded that the pharmacological profile of partial agonists is highly dependent on the receptor composition, and that small structural changes of a ligand can alter the selectivity towards different subunit compositions. Moreover, this study shows that pharmacological actions determined in oocytes are generally in agreement with data obtained from cultured neurons.     

GABA(A) receptor channel pharmacology

GABA(A) receptor channels are ubiquitous in the mammalian central nervous system mediating fast inhibitory neurotransmission by becoming permeant to chloride ions in response to GABA. The emphasis of this review is on the rich chemical diversity of ligands that influence GABA(A) receptor function. Such diversity provides many avenues for the design and development of new chemical entities acting on GABA(A) receptors. There is also a significant diversity of GABA(A) receptor subtypes composed of different protein subunits. The discovery of subtype specific agents is a major challenge in the continuing development of GABA(A) receptor pharmacology. Leads for the discovery of new chemical entities that influence GABA(A) receptors come from using recombinant GABA(A) receptors of known subunit composition as has been elegantly demonstrated by the refining of benzodiazepine actions with alpha1 subunit preferring agents showing sedative properties but not anxiolytic properties. The most recent advances in the therapeutic use of agents acting on GABA(A) receptors concern the promotion of sound sleep. Many herbal medicines are used to promote sleep and many of their active ingredients include flavonoids and terpenoids known to modulate GABA(A) receptor function.     

GABA(A) receptor epilepsy mutations

Idiopathic generalized epilepsy (IGE) syndromes are diseases that are characterized by absence, myoclonic, and/or primary generalized tonic-clonic seizures in the absence of structural brain abnormalities. Although it was long hypothesized that IGE had a genetic basis, only recently have causative genes been identified. Here we review mutations in the GABA(A) receptor alpha1, gamma2, and delta subunits that have been associated with different IGE syndromes. These mutations affect GABA(A) receptor gating, expression, and/or trafficking of the receptor to the cell surface, all pathophysiological mechanisms that result in neuronal disinhibition and thus predispose affected patients to seizures.     

Interaction between cyclodextrin and neuronal membrane results in modulation of GABA(A) receptor conformational transitions

Cyclodextrins (CDs) are nanostructures widely applied in biotechnology and chemistry. Owing to partially hydrophobic character, CDs interact with biological membranes. While the mechanisms of CDs interactions with lipids were widely studied, their effects on proteins are less understood. In the present study we investigated the effects of beta cyclodextrin (betaCD) on GABA(A) receptor (GABA(A)R) gating. To reliably resolve the kinetics of conformational transitions, currents were elicited by ultrafast gamma-aminobutyric acid (GABA) applications to outside-out patches from rat cultured hippocampal neurons. betaCD increased the amplitude of responses to saturating GABA concentration ([GABA]) in a dose-dependent manner and this effect was accompanied by profound alterations in the current kinetics. Current deactivation was slowed down by betaCD but this effect was biphasic with a maximum at around 0.5 mM betaCD. While the fast deactivation time constant was monotonically slowed down within considered betaCD concentration range, the slow component first increased and then, at millimolar betaCD concentration, decreased. The rate and extent of desensitization was decreased by betaCD in a dose-dependent manner. The analysis of current responses to nonsaturating [GABA] indicated that betaCD affected the GABA(A)R agonist binding site by slowing down the unbinding rate. Modulation of GABA(A)R desensitization and binding showed different concentration-dependence suggesting different modualtory sites with higher affinity of the latter one. All the betaCD effects were fully reversible indicating that cholesterol uptake into betaCD was not the primary mechanism. We conclude that betaCD is a strong modulator of GABA(A)R conformational transitions.     

Opiate receptors in neuronal primary cultures

The occurrence and characteristics of mu-, delta- and kappa-receptors were studied as effects of the respective agonists on forskolin-stimulated accumulation of cAMP in neuronal enriched primary cultures from the cerebral cortex of foetal rats. Morphine or [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO) were used as mu-receptor agonists. [D-Ala2,D-Leu5]-Enkephalin (DADLE) or [D-Pen2,D-Pen5]-enkephalin (DPDPE) were used as delta-receptor agonists and dynorphin 1-13 (Dyn) or U-50,488H were used as kappa-receptor agonists. In the presence of 10(-8)-10(-5) M morphine or 10(-8)-10(-5) M DAGO, there was a dose-related inhibition of the 10(-5) M forskolin-stimulated accumulation of cAMP. The inhibitory action of morphine or DAGO was reversed by naloxone. In the presence of 10(-9)-10(-6) M DADLE or 10(-9)-10(-6) M DPDPE, there was also a dose-related inhibition of the forskolin-stimulated accumulation of cAMP and a similar result was obtained in the presence of 10(-9)-10(-5) M Dyn or 10(-9)-10(-5) M U-50,488 H. These findings indicate that neurones from the cerebral cortex in culture express mu-, delta- and kappa-receptors, that inhibit the forskolin-stimulated accumulation of cAMP. Administration of 10(-5) M morphine and 10(-6) M DADLE or 10(-6) M DPDPE together, resulted in a non-additive inhibition of the forskolin-stimulated accumulation of cAMP, indicating the presence of both mu- and delta-receptors on the same population of cells.     

Interaction of steroids with the GABA(A) receptor

Over the last two decades there has been a resurgence of interest in steroids as potential therapeutics for central nervous system disorders. This interest followed the discovery that neurosteroids and neuroactive steroids are potent modulators of GABA(A) receptor function. This article traces those developments focussing particularly on the structure-activity relationships that have been identified through synthetic modification of established ligands, but also examines the influence of GABA(A) receptor subunit composition for steroid modulation. The review then covers some of the physiological effects such steroids are liable to exert and their therapeutic potential for treating central nervous system disorders including epilepsy, anxiety and insomnia.     

Cyclohexanol analogues are positive modulators of GABA(A) receptor currents and act as general anaesthetics in vivo

GABA(A) receptors meet all the pharmacological criteria required to be considered important general anaesthetic targets. In the following study, the modulatory effects of various commercially available and novel cyclohexanols were investigated on recombinant human γ-aminobutyric acid (GABA(A), α(1)β(2)γ(2s)) receptors expressed in Xenopus oocytes, and compared to the modulatory effects on GABA currents observed with exposures to the intravenous anaesthetic agent, propofol. Submaximal EC(20) GABA currents were typically enhanced by co-applications of 3-300 μM cyclohexanols. For instance, at 30 μM 2,6-diisopropylcyclohexanol (a novel compound) GABA responses were increased ~3-fold (although similar enhancements were achieved at 3 μM propofol). As regards rank order for modulation by the cyclohexanol analogues at 30 μM, the % enhancements for 2,6-dimethylcyclohexanol~2,6-diethylcyclohexanol~2,6-diisopropylcyclohexanol~2,6-di-sec-butylcyclohexanol ?2,6-di-tert-butylcyclohexanol~4-tert-butylcyclohexanol>cyclohexanol~cyclopentanol~2-methylcyclohexanol. We further tested the potencies of the cyclohexanol analogues as general anaesthetics using a tadpole in vivo assay. Both 2,6-diisopropylcyclohexanol and 2,6-dimethylcyclohexanol were effective as anaesthetics with EC(50)s of 14.0 μM and 13.1 μM respectively, while other cyclohexanols with bulkier side chains were less potent. In conclusion, our data indicate that cyclohexanols are both positive modulators of GABA(A) receptors currents and anaesthetics. The positioning and size of the alkyl groups at the 2 and 6 positions on the cyclohexanol ring were critical determinants of activity.     

Inhibition of neuronal GABA uptake and glial β-alanine uptake by synthetic GABA analogues

Thirty three synthetic analogues of GABA were tested for their ability to act as inhibitors of neuronal or glial uptake sites for GABA and β-alanine, using [³H]GABA uptake by synaptosome preparations from rat cerebral cortex, and [³H]β-alanine uptake by cortical slices as test systems for neuronal and glial uptake sites, respectively. The results confirm that both uptake processes favour a substantially folded conformation. Neuronal uptake sites were significantly inhibited by analogues with long alkyl side chain substituents, such as dodecyl GABA-amide which was of comparable potency to GABA.     

Determinants of amentoflavone interaction at the GABA(A) receptor

We investigated the recognition properties of different GABA(A) receptor subtypes and mutant receptors for the biflavonoid amentoflavone, a constituent of St. John's Wort. Radioligand binding studies showed that amentoflavone recognition paralleled that of the classical benzodiazepine diazepam in that it had little or no affinity for alpha4- or alpha6-containing receptors. Lysine and alanine substitutions at position 101 of the rat alpha1 subunit resulted in a complete loss of competitive amentoflavone binding, but functional analysis of the alanine mutant expressed with beta2 and gamma2 subunits in Xenopus oocytes revealed no significant difference in the negative modulation of GABA-induced currents brought about by amentoflavone. Furthermore, elimination of the gamma subunit had no effect on the negative modulation of these currents. This negative modulation was also observed at alpha1beta1gamma2 GABA(A) receptors and is therefore not likely mediated by the loreclezole site. These results suggest a complex mechanism of amentoflavone interaction at GABA(A) receptors.     

GABA(A) receptor changes in acute allopregnanolone tolerance

To study acute tolerance, rats were anesthetized with interrupted i.v. allopregnanolone infusions where the "silent second" in the electroencephalogram (EEG) was the target. Animals were killed either directly at the first silent second or at the silent second level after 30 or 90 min of anaesthesia. Acute tolerance was demonstrated at 90 min of anaesthesia as earlier shown. In situ hybridization showed a decreased expression of the gamma-aminobutyric acid(A) (GABA(A)) receptor subunit alpha4mRNA amount in the thalamus ventral-posteriomedial nucleus of the tolerant rats. A parallel change in the abundance of the alpha4 subunit was detected with immunohistochemistry. The increase in maintenance dose rate (MDR) was significantly negatively correlated with the alpha4mRNA in the thalamus ventral-posteriomedial nucleus, and positively correlated with alpha2mRNA in different hippocampal subregions. There was also a positive relationship between the alpha1mRNA amounts in the different hippocampal subregions, with significant differences between groups. These changes in GABA(A) receptor subunits mRNA expression and protein (alpha4) might be of importance for the development of acute tolerance to allopregnanolone.     

GABA(A) receptor subtype selective cognition enhancers

Currently the treatment of Alzheimer's disease (AD) and Mild Cognitive Impairment (MCI) is largely unrealised, with no preventive or curative therapies. The marketed acetylcholinesterase inhibitors (eg. donepezil, Aricept) are directed toward temporary symptomatic relief from impaired cognition, but have prominent adverse effects with minimal efficacy. In pursuit of novel cognition enhancers, the observation that classical benzodiazepines (BZ, eg. diazepam) are amnesic, coupled with the preservation of GABA(A) receptors in brain areas most affected by AD, highlighted the GABA(A) receptor as a potential therapeutic target. In contrast to the amnesic BZ agonists, the BZ inverse agonists (eg. DMCM) which attenuate GABA(A) receptor function, have been shown to improve performance in animal models of learning and memory. Unfortunately, such non-selective ligands also induce anxiety and convulsions. More recently, novel ligands have been developed (eg. 6,6-dimethyl-3-(2-hydroxyethyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one) that demonstrate binding selectivity and high inverse agonism for the alpha5 GABA(A) receptor subtype, which is preferentially located in the hippocampus, a region of the brain associated with learning and memory. Pre-clinical results are encouraging, since these alpha5 selective inverse agonists enhance memory in animal models, such as spatial learning in the Morris water-maze, but are devoid of the adverse effects associated with activity at other GABA(A) receptor subtypes in other brain regions. If the efficacy and safety profiles of alpha5 inverse agonists in humans prove to be similar to those seen in pre-clinical studies, these compounds would offer significant benefit to AD and MCI patients.