Neural Basis of Benzodiazepine Reward: Requirement for α2 Containing GABAA Receptors in the Nucleus Accumbens

Despite long-standing concerns regarding the abuse liability of benzodiazepines, the mechanisms underlying properties of benzodiazepines that may be relevant to abuse are still poorly understood. Earlier studies showed that compounds selective for α1-containing GABA_A receptors (α1GABA_ARs) are abused by humans and self-administered by animals, and that these receptors may underlie a preference for benzodiazepines as well as neuroplastic changes observed in the ventral tegmental area following benzodiazepine administration. There is some evidence, however, that even L-838, 417, a compound with antagonistic properties at α1GABA_ARs and agonistic properties at the other three benzodiazepine-sensitive GABA_A receptor subtypes, is self-administered, and that the α2GABA_ARs may have a role in benzodiazepine-induced reward enhancement. Using a two-bottle choice drinking paradigm to evaluate midazolam preference and an intracranial self-stimulation (ICSS) paradigm to evaluate the impact of midazolam on reward enhancement, we demonstrated that mice carrying a histidine-to-arginine point mutation in the α2 subunit which renders it insensitive to benzodiazepines (α2(H101R) mice) did not prefer midazolam and did not show midazolam-induced reward enhancement in ICSS, in contrast to wild-type controls, suggesting that α2GABA_ARs are necessary for the reward enhancing effects and preference for oral benzodiazepines. Through a viral-mediated knockdown of α2GABA_ARs in the nucleus accumbens (NAc), we demonstrated that α2 in the NAc is necessary for the preference for midazolam. Findings imply that α2GABA_ARs in the NAc are involved in at least some reward-related properties of benzodiazepines, which might partially underlie repeated drug-taking behavior.     

SB-205384: a GABAA receptor modulator with novel mechanism of action that shows subunit selectivity

1. SB-205384, and its (+) enantiomer (+)-SB-205384 were tested for their modulatory effects on human GABA_A receptor subunit combinations expressed in Xenopus oocytes by electrophysiological methods.
2. The slowing of the decay rate induced by SB-205384 on native GABA-activated currents in rat neurones was also seen on GABA_A currents in oocytes expressing human GABA_A subunits. This temporal effect was observed for the α3β2γ2 subunit combination with little effect in subunit combinations containing either α1 or α2.
3. Potentiation of the peak amplitude of the GABA-activated currents by SB-205384 or (+)-SB-205384 was less specific for a particular subunit combination, although the greatest effect at 10 μM drug was seen on the α3β2γ2 subunit combination.
4. In contrast, zolpidem, a benzodiazepine site modulator, did not significantly slow decay rates of GABA_A currents in oocytes expressing the α3β2γ2 subunit combination. Zolpidem, as expected, did selectively potentiate GABA-activated currents on oocytes expressing the γ2 subunit compared to those containing the γ1.
5. The results show that the novel kinetic modulatory profile of SB-205384 is selective for the α3β2γ2 subunit combination. This suggests that the compound is binding to a novel regulatory site on the subunit complex.

     

Flavan-3-ol esters: new agents for exploring modulatory sites on GABA(A) receptors

BACKGROUND AND PURPOSE

Enhancement of GABAergic function is the primary mechanism of important therapeutic agents such as benzodiazepines, barbiturates, neurosteroids, general anaesthetics and some anticonvulsants. Despite their chemical diversity, many studies have postulated that these agents may bind at a common or overlapping binding site, or share an activation domain. Similarly, we found that flavan-3-ol esters act as positive modulators of GABA_A receptors, and noted that this action resembled the in vitro profile of general anaesthetics. In this study we further investigated the interactions between these agents.

EXPERIMENTAL APPROACH

Using two-electrode voltage clamp electrophysiological recordings on receptors of known subunit composition expressed in Xenopus oocytes, we evaluated positive modulation by etomidate, loreclezole, diazepam, thiopentone, 5α-pregnan-3α-ol-20-one (THP) and the flavan-3-ol ester 2S,3R-trans 3-acetoxy-4′-methoxyflavan (Fa131) on wild-type and mutated GABA_A receptors.

KEY RESULTS

The newly identified flavan, 2S,3S-cis 3-acetoxy-3′,4′-dimethoxyflavan (Fa173), antagonized the potentiating actions of Fa131, etomidate and loreclezole at α1β2 and α1β2γ2L GABA_A receptors. Furthermore, Fa173 blocked the potentiation of GABA responses by high, but not low, concentrations of diazepam, but did not block the potentiation induced by propofol, the neurosteroid THP or the barbiturate thiopental. Mutational studies on ‘anaesthetic-influencing’ residues showed that, compared with wild-type GABA_A receptors, α1M236Wβ2γ2L and α1β2N265Sγ2L receptors are resistant to potentiation by etomidate, loreclezole and Fa131.

CONCLUSIONS AND IMPLICATIONS

Fa173 is a selective antagonist that can be used for allosteric modulation of GABA_A receptors. Flavan-3-ol derivatives are potential ligands for etomidate/loreclezole-related binding sites at GABA_A receptors and the low-affinity effects of diazepam are mediated via the same site.     

Alterations of the benzodiazepine site of rat α6β2γ2-GABAAreceptor by replacement of several divergent amino-terminal regions with the α1 counterparts

1. The benzodiazepine site of the α6β2γ2 subtype of γ-aminobutyric acid_A (GABA_A) receptors is distinguishable from that of the α1β2γ2 subtype by its inability to interact with classical benzodiazepines (i.e., diazepam) and its agonistic response to Ro 15-1788, which behaves as an antagonist in the α1β2γ2 subtype.
2. The point mutation of Arg 100 of the α6 subunit to histidine (the corresponding residue in α1) has been shown to enable the α6β2γ2 subtype to interact with diazepam but failed in this study to abolish the ability of Ro 15-1788 to enhance GABA-induced Cl⁻ currents.
3. Here we identified the segment of P161 to L187 of α6 to contain the functional region responsible for the agonistic action of Ro 15-1788. Its replacement with the corresponding α1 sequence abolished the ability of Ro 15-1788 to enhance GABA currents without appreciable effects on its binding affinity to the benzodiazepine site or on the functionality of the other benzodiazepine site ligands such as diazepam, U-92330 and 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate. These data support the evidence that the functionality of a given ligand could arise from a single region of the benzodiazepine site, not shared by others.
4. In addition we have learned that several residues in the N-terminal region of α6, such as R100, V142 and N143, have the ability to influence GABA-dependent Cl⁻ current induction probably by allosterically modulating low affinity GABA sites.

     

A Novel α5GABAAR-Positive Allosteric Modulator Reverses Hyperactivation of the Dopamine System in the MAM Model of Schizophrenia

We have shown previously that aberrant hippocampal (HPC) output underlies the dopamine (DA) dysfunction observed in the methylazoxymethanol acetate (MAM) developmental model of schizophrenia in the rodent. This alteration of HPC activity was proposed to result from a reduction in parvalbumin (PV)-expressing GABAergic interneurons and consequent destabilization of the output of pyramidal neurons, as well as disrupted activation across a broad neural network. In vivo extracellular recordings were performed in the ventral tegmental area (VTA) and ventral HPC of saline- (SAL) and MAM-treated animals. A novel benzodiazepine-positive allosteric modulator (PAM), selective for the α5 subunit of the GABA_A receptor, SH-053-2′F-R-CH3, was tested for its effects on the output of the HPC, leading to dopamine system hyperactivity in MAM-treated animals. In addition, the effect of SH-053-2′F-R-CH3 on the hyperactive locomotor response to amphetamine in MAM animals was examined. We demonstrate that treatment with the α5GABA_AR PAM reduced the number of spontaneously active DA neurons in the VTA of MAM animals to levels observed in SAL rats, both when administered systemically and when directly infused into the ventral HPC. Moreover, HPC neurons in both SAL and MAM animals showed diminished cortical-evoked responses following α5GABA_AR PAM treatment. In addition, the increased locomotor response to amphetamine observed in MAM rats was reduced following α5GABA_AR treatment. This study supports a novel treatment of schizophrenia that targets abnormal HPC output, which in turn normalizes dopaminergic neuronal activity.     

A study of subunit selectivity,mechanism and site of action of the delta selective compound 2 (DS2) at human recombinant and rodent native GABAA receptors

Background and Purpose

Most GABA_A receptor subtypes comprise 2α, 2β and 1γ subunit, although for some isoforms, a δ replaces a γ-subunit. Extrasynaptic δ-GABA_A receptors are important therapeutic targets, but there are few suitable pharmacological tools. We profiled DS2, the purported positive allosteric modulator (PAM) of δ-GABA_A receptors to better understand subtype selectivity, mechanism/site of action and activity at native δ-GABA_A receptors.

Experimental Approach

Subunit specificity of DS2 was determined using electrophysiological recordings of Xenopus laevis oocytes expressing human recombinant GABA_A receptor isoforms. Effects of DS2 on GABA concentration–response curves were assessed to define mechanisms of action. Radioligand binding and electrophysiology utilising mutant receptors and pharmacology were used to define site of action. Using brain-slice electrophysiology, we assessed the influence of DS2 on thalamic inhibition in wild-type and δ^0/0 mice.

Key Results

Actions of DS2 were primarily determined by the δ-subunit but were additionally influenced by the α, but not the β, subunit (α4/6βxδ > α1βxδ >> γ2-GABA_A receptors > α4β3). For δ-GABA_A receptors, DS2 enhanced maximum responses to GABA, with minimal influence on GABA potency. (iii) DS2 did not act via the orthosteric, or known modulatory sites on GABA_A receptors. (iv) DS2 enhanced tonic currents of thalamocortical neurones from wild-type but not δ^0/0 mice.

Conclusions and Implications

DS2 is the first PAM selective for α4/6βxδ receptors, providing a novel tool to investigate extrasynaptic δ-GABA_A receptors. The effects of DS2 are mediated by an unknown site leading to GABA_A receptor isoform selectivity.     

Analogues of γ-aminobutyric acid (GABA) and trans-4-aminocrotonic acid (TACA) substituted in the 2 position as GABAC receptor antagonists

1. γ-Aminobutyric acid (GABA) and trans-4-aminocrotonic acid (TACA) have been shown to activate GABA_C receptors. In this study, a range of C2, C3, C4 and N-substituted GABA and TACA analogues were examined for activity at GABA_C receptors.
2. The effects of these compounds were examined by use of electrophysiological recording from Xenopus oocytes expressing the human ρ₁ subunit of GABA_C receptors with the two-electrode voltage-clamp method.
3. trans-4-Amino-2-fluorobut-2-enoic acid was found to be a potent agonist (K_D=2.43 μM). In contrast, trans-4-amino-2-methylbut-2-enoic acid was found to be a moderately potent antagonist (IC₅₀=31.0 μM and K_B=45.5 μM). These observations highlight the possibility that subtle structural substitutions may change an agonist into an antagonist.
4. 4-Amino-2-methylbutanoic acid (K_D=189 μM), 4-amino-2-methylenebutanoic acid (K_D=182 μM) and 4-amino-2-chlorobutanoic acid (K_D=285 μM) were weak partial agonists. The intrinsic activities of these compounds were 12.1%, 4.4% and 5.2% of the maximal response of GABA, respectively. These compounds more effectively blocked the effects of the agonist, GABA, giving rise to K_B values of 53 μM and 101 μM, respectively.
5. The sulphinic acid analogue of GABA, homohypotaurine, was found to be a potent partial agonist (K_D=4.59 μM, intrinsic activity 69%).
6. It was concluded that substitution of a methyl or a halo group in the C2 position of GABA or TACA is tolerated at GABA_C receptors. However, there was dramatic loss of activity when these groups were substituted at the C3, C4 and nitrogen positions of GABA and TACA.
7. Molecular modelling studies on a range of active and inactive compounds indicated that the agonist/competitive antagonist binding site of the GABA_C receptor may be smaller than that of the GABA_A and GABA_B receptors. It is suggested that only compounds that can attain relatively flat conformations may bind to the GABA_C receptor agonist/competitive antagonist binding site.

     

Design,Synthesis, and Pharmacological Evaluation of Fluorescent and Biotinylated Antagonists of ρ1 GABAC Receptors

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Flunitrazepam rapidly reduces GABAA receptor subunit protein expression via a protein kinase C-dependent mechanism

Acute flunitrazepam (1 μM) exposure for 1 h reduced GABA_A receptor α1 (22±4%, mean±s.e.mean) and β2/3 (21±4%) subunit protein levels in cultured rat cerebellar granule cells. This rapid decrease in subunit proteins was completely prevented by bisindolymaleimide 1 (1 μM), an inhibitor of protein kinase C, but not by N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89, 4.8 μM), an inhibitor of protein kinases A and G. These results suggest the existence of a benzodiazepine-induced mechanism to rapidly alter GABA_A receptor protein expression, that appears to be dependent on protein kinase C activity.     

The orthosteric GABAA receptor ligand Thio-4-PIOL displays distinctly different functional properties at synaptic and extrasynaptic receptors

BACKGROUND AND PURPOSE

Explorations into the heterogeneous population of native GABA type A receptors (GABA_ARs) and the physiological functions governed by the multiple GABA_AR subtypes have for decades been hampered by the lack of subtype-selective ligands.

EXPERIMENTAL APPROACH

The functional properties of the orthosteric GABA_A receptor ligand 5-(4-piperidyl)-3-isothiazolol (Thio-4-PIOL) have been investigated in vitro, ex vivo and in vivo.

KEY RESULTS

Thio-4-PIOL displayed substantial partial agonist activity at the human extrasynaptic GABA_AR subtypes expressed in Xenopus oocytes, eliciting maximal responses of up to ∼30% of that of GABA at α₅β₃γ_2S, α₄β₃δ and α₆β₃δ and somewhat lower efficacies at the corresponding α₅β₂γ_2S, α₄β₂δ and α₆β₂δ subtypes (maximal responses of 4–12%). In contrast, it was an extremely low efficacious agonist at the α₁β₃γ_2S, α₁β₂γ_2S, α₂β₂γ_2S, α₂β₃γ_2S, α₃β₂γ_2S and α₃β₃γ_2S GABA_ARs (maximal responses of 0–4%). In concordance with its agonism at extrasynaptic GABA_ARs and its de facto antagonism at the synaptic receptors, Thio-4-PIOL elicited robust tonic currents in electrophysiological recordings on slices from rat CA1 hippocampus and ventrobasal thalamus and antagonized phasic currents in hippocampal neurons. Finally, the observed effects of Thio-4-PIOL in rat tests of anxiety, locomotion, nociception and spatial memory were overall in good agreement with its in vitro and ex vivo properties.

CONCLUSION AND IMPLICATIONS

The diverse signalling characteristics of Thio-4-PIOL at GABA_ARs represent one of the few examples of a functionally subtype-selective orthosteric GABA_AR ligand reported to date. We propose that Thio-4-PIOL could be a useful pharmacological tool in future studies exploring the physiological roles of native synaptic and extrasynaptic GABA_ARs.     

Selective antagonism of the GABAA receptor by ciprofloxacin and biphenylacetic acid

1. Previous studies have shown that ciprofloxacin and biphenylacetic acid (BPAA) synergistically inhibit γ-aminobutyric acid (GABA)_A receptors. In the present study, we have investigated the actions of these two drugs on other neuronal ligand-gated ion channels.
2. Agonist-evoked depolarizations were recorded from rat vagus and optic nerves in vitro by use of an extracellular recording technique.
3. GABA (50 μM)-evoked responses, in the vagus nerve in vitro, were inhibited by bicuculline (0.3–10 μM) and picrotoxin (0.3–10 μM), with IC₅₀ values and 95% confidence intervals (CI) of 1.2 μM (1.1–1.4) and 3.6 μM (3.0–4.3), respectively, and were potentiated by sodium pentobarbitone (30 μM) and diazepam (1 μM) to (mean±s.e.mean) 168±18% and 117±4% of control, respectively. 5-Hydroxytryptamine (5-HT; 0.5 μM)-evoked responses were inhibited by MDL 72222 (1 μM) to 10±4% of control; DMPP (10 μM)-evoked responses were inhibited by hexamethonium (100 μM) to 12±5% of control, and αbMeATP (30 μM)-evoked responses were inhibited by PPADS (10 μM) to 21±5% of control. Together, these data are consistent with activation of GABA_A, 5-HT₃, nicotinic ACh and P2_X receptors, respectively.
4. Ciprofloxacin (10–3000 μM) inhibited GABA_A-mediated responses in the vagus nerve with an IC₅₀ (and 95% CI) of 202 μM (148–275). BPAA (1–1000 μM) had little or no effect on the GABA_A-mediated response but concentration-dependently potentiated the effects of ciprofloxacin by up to 33,000 times.
5. Responses mediated by 5-HT₃, nicotinic ACh and P2_X receptors in the vagus nerve and strychnine-sensitive glycine receptors in the optic nerve were little or unaffected by ciprofloxacin (100 μM), BPAA (100 μM) or the combination of these drugs (both at 100 μM).
6. GABA (1 mM)-evoked responses in the optic nerve were inhibited by bicuculline with an IC₅₀ of 3.6 μM (2.8–4.5), a value not significantly different from that determined in the vagus nerve. Ciprofloxacin also inhibited the GABA-evoked response with an IC₅₀ of 334 μM (256–437) and BPAA (100 μM) potentiated these antagonist effects. However, the magnitude of the synergy was 48 times less than that seen in the vagus nerve.
7. These data indicate that ciprofloxacin and BPAA are selective antagonists of GABA_A receptors, an action that may contribute to their excitatory effects in vivo. Additionally, our data suggest that the molecular properties of GABA_A receptors in different regions of the CNS influence the extent to which these drugs synergistically inhibit the GABA_A receptor.

     

Catecholamine synthesis and metabolism in the central nervous system of mice lacking α2-adrenoceptor subtypes

Background and purpose:

This study investigates the role of α₂-adrenoceptor subtypes, α_2A, α_2B and α_2C, on catecholamine synthesis and catabolism in the central nervous system of mice.

Experimental approach:

Activities of the main catecholamine synthetic and catabolic enzymes were determined in whole brains obtained from α_2A-, α_2B- and α_2C-adrenoceptor knockout (KO) and C56Bl\7 wild-type (WT) mice.

Key results:

Although no significant differences were found in tyrosine hydroxylase activity and expression, brain tissue levels of 3,4-dihydroxyphenylalanine were threefold higher in α_2A- and α_2C-adrenoceptor KO mice. Brain tissue levels of dopamine and noradrenaline were significantly higher in α_2A and α_2CKOs compared with WT [WT: 2.8 ± 0.5, 1.1 ± 0.1; α_2AKO: 6.9 ± 0.7, 1.9 ± 0.1; α_2BKO: 2.3 ± 0.2, 1.0 ± 0.1; α_2CKO: 4.6 ± 0.8, 1.5 ± 0.2 nmol·(g tissue)⁻¹, for dopamine and noradrenaline respectively]. Aromatic L-amino acid decarboxylase activity was significantly higher in α_2A and α_2CKO [WT: 40 ± 1; α_2A: 77 ± 2; α_2B: 40 ± 1; α_2C: 50 ± 1, maximum velocity (V_max) in nmol·(mg protein)⁻¹·h⁻¹], but no significant differences were found in dopamine β-hydroxylase. Of the catabolic enzymes, catechol-O-methyltransferase enzyme activity was significantly higher in all three α₂KO mice [WT: 2.0 ± 0.0; α_2A: 2.4 ± 0.1; α_2B: 2.2 ± 0.0; α_2C: 2.2 ± 0.0 nmol·(mg protein)⁻¹·h⁻¹], but no significant differences were found in monoamine oxidase activity between all α₂KOs and WT mice.

Conclusions and implications:

In mouse brain, deletion of α_2A- or α_2C-adrenoceptors increased cerebral aromatic L-amino acid decarboxylase activity and catecholamine tissue levels. Deletion of any α₂-adrenoceptor subtypes resulted in increased activity of catechol-O-methyltransferase. Higher 3,4-dihydroxyphenylalanine tissue levels in α_2A and α_2CKO mice could be explained by increased 3,4-dihydroxyphenylalanine transport.     

Novel Cyclic Phosphinic Acids as GABAC ρ Receptor Antagonists: Design,Synthesis, and Pharmacology

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PNU-107484A with α isoform-dependent functional changes in αxβ2γ2 subtypes of rat recombinant GABAA receptors

1. We discovered a novel γ-aminobutyric acid_A (GABA_A) receptor ligand displaying seemingly opposite functionalities, depending on the α isoform of the αxβ2γ2 subtypes. PNU-107484A enhanced GABA-induced Cl⁻ currents in the α1β2γ2 subtype, but inhibited the currents in the α3β2γ2 and α6β2γ2 subtypes, and its half-maximal concentrations in the subtypes were 3.1±0.5, 4.2±1, and 3.5±0.2 μM, respectively, without showing much dependency on α isoforms.
2. In the α1β2 subtype, the drug at concentrations up to 40 μM showed no effect on GABA-induced Cl⁻ currents, suggesting the requirement of the γ subunit for its action.
3. PNU-107484A behaved like a positive allosteric modulator of the α1β2γ2 subtype with its binding site distinct from those for benzodiazepines, barbiturates and neurosteroids. With the α3β2γ2 subtype, the drug behaved like a non-competitive inhibitor of GABA, thus blocking Cl⁻ currents by GABA alone or in the presence of pentobarbitone and neurosteroids.
4. It appears that PNU-107484A is a unique GABA_A receptor ligand with α isoform-dependent functionalities, which may provide a basis for development of α isoform-selective ligands, and it could be useful as a probe to investigate the physiological roles of the various α isoform subtypes.

     

Retrochalcone derivatives are positive allosteric modulators at synaptic and extrasynaptic GABA(A) receptors in vitro

BACKGROUND AND PURPOSE

Flavonoids, important plant pigments, have been shown to allosterically modulate brain GABA_A receptors (GABA_ARs). We previously reported that trans-6,4′-dimethoxyretrochalcone (Rc-OMe), a hydrolytic derivative of the corresponding flavylium salt, displayed nanomolar affinity for the benzodiazepine binding site of GABA_ARs. Here, we evaluate the functional modulations of Rc-OMe, along with two other synthetic derivatives trans-6-bromo-4′-methoxyretrochalcone (Rc-Br) and 4,3′-dimethoxychalcone (Ch-OMe) on GABA_ARs.

EXPERIMENTAL APPROACH

Whole-cell patch-clamp recordings were made to determine the effects of these derivatives on GABA_ARs expressed in HEK-293 cells and in hippocampal CA1 pyramidal and thalamic neurones from rat brain.

KEY RESULTS

Rc-OMe strongly potentiated GABA-evoked currents at recombinant α_1–4β₂γ_2s and α₄β₃δ receptors but much less at α₁β₂ and α₄β₃. Rc-Br and Ch-OMe potentiated GABA-evoked currents at α₁β₂γ_2s. The potentiation by Rc-OMe was only reduced at α₁H101Rβ₂γ_2s and α₁β₂N265Sγ_2s, mutations known to abolish the potentiation by diazepam and loreclezole respectively. The modulation of Rc-OMe and pentobarbital as well as by Rc-OMe and the neurosteroid 3α,21-dihydroxy-5α-pregnan-20-one was supra-additive. Rc-OMe modulation exhibited no apparent voltage-dependence, but was markedly dependent on GABA concentration. In neurones, Rc-Br slowed the decay of spontaneous inhibitory postsynaptic currents and both Rc-OMe and Rc-Br positively modulated synaptic and extrasynaptic diazepam-insensitive GABA_ARs.

CONCLUSIONS AND IMPLICATIONS

The trans-retrochalcones are powerful positive allosteric modulators of synaptic and extrasynaptic GABA_ARs. These novel modulators act through an original mode, thus making them putative drug candidates in the treatment of GABA_A-related disorders in vivo.     

Valerenic acid derivatives as novel subunit-selective GABAA receptor ligands �Cin vitro and in vivo characterization

BACKGROUND AND PURPOSE

Subunit-specific modulators of γ-aminobutyric acid (GABA) type A (GABA_A) receptors can help to assess the physiological function of receptors with different subunit composition and also provide the basis for the development of new drugs. Valerenic acid (VA) was recently identified as a β_2/3 subunit-specific modulator of GABA_A receptors with anxiolytic potential. The aim of the present study was to generate VA derivatives as novel GABA_A receptor modulators and to gain insight into the structure–activity relation of this molecule.

EXPERIMENTAL APPROACH

The carboxyl group of VA was substituted by an uncharged amide or amides with different chain length. Modulation of GABA_A receptors composed of different subunit compositions by the VA derivatives was studied in Xenopus oocytes by means of the two-microelectrode voltage-clamp technique. Half-maximal stimulation of GABA-induced chloride currents (I_GABA) through GABA_A receptors (EC₅₀) and efficacies (maximal stimulation of I_GABA) were estimated. Anxiolytic activity of the VA derivatives was studied in mice, applying the elevated plus maze test.

KEY RESULTS

Valerenic acid amide (VA-A) displayed the highest efficacy (more than twofold greater I_GABA enhancement than VA) and highest potency (EC₅₀= 13.7 ± 2.3 µM) on α₁β₃ receptors. Higher efficacy and potency of VA-A were also observed on α₁β₂γ_2s and α₃β₃γ_2s receptors. Anxiolytic effects were most pronounced for VA-A.

CONCLUSIONS AND IMPLICATIONS

Valerenic acid derivatives with higher efficacy and affinity can be generated. Greater in vitro action of the amide derivative correlated with a more pronounced anxiolytic effect in vivo. The data give further confidence in targeting β₃ subunit containing GABA_A receptors for development of anxiolytics.     

Neurosteroid Agonist at GABAA Receptor Induces Persistent Neuroplasticity in VTA Dopamine Neurons

The main fast-acting inhibitory receptors in the mammalian brain are γ-aminobutyric acid type-A (GABA_A) receptors for which neurosteroids, a subclass of steroids synthesized de novo in the brain, constitute a group of endogenous ligands with the most potent positive modulatory actions known. Neurosteroids can act on all subtypes of GABA_A receptors, with a preference for δ-subunit-containing receptors that mediate extrasynaptic tonic inhibition. Pathological conditions characterized by emotional and motivational disturbances are often associated with perturbation in the levels of endogenous neurosteroids. We studied the effects of ganaxolone (GAN)—a synthetic analog of endogenous allopregnanolone that lacks activity on nuclear steroid receptors—on the mesolimbic dopamine (DA) system involved in emotions and motivation. A single dose of GAN in young mice induced a dose-dependent, long-lasting neuroplasticity of glutamate synapses of DA neurons ex vivo in the ventral tegmental area (VTA). Increased α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/N-methyl-D-aspartate ratio and rectification of AMPA receptor responses even at 6 days after GAN administration suggested persistent synaptic targeting of GluA2-lacking AMPA receptors. This glutamate neuroplasticity was not observed in GABA_A receptor δ-subunit-knockout (δ-KO) mice. GAN (500 nM) applied locally to VTA selectively increased tonic inhibition of GABA interneurons and triggered potentiation of DA neurons within 4 h in vitro. Place-conditioning experiments in adult wild-type C57BL/6J and δ-KO mice revealed aversive properties of repeated GAN administration that were dependent on the δ-subunits. Prolonged neuroadaptation to neurosteroids in the VTA might contribute to both the physiology and pathophysiology underlying processes and changes in motivation, mood, cognition, and drug addiction.     

Pachymic Acid Enhances Pentobarbital-Induced Sleeping Behaviors via GABAA-ergic Systems in Mice

This study was investigated to know whether pachymic acid (PA), one of the predominant triterpenoids in Poria cocos (Hoelen) has the sedative-hypnotic effects, and underlying mechanisms are mediated via γ-aminobutyric acid (GABA)-ergic systems. Oral administration of PA markedly suppressed locomotion activity in mice. This compound also prolonged sleeping time, and reduced sleep latency showing synergic effects with muscimol (0.2 mg/kg) in shortening sleep onset and enhancing sleep time induced by pentobarbital, both at the hypnotic (40 mg/kg) and sub-hypnotic (28 mg/kg) doses. Additionally, PA elevated intracellular chloride levels in hypothalamic primary cultured neuronal cells of rats. Moreover, Western blotting quantitative results showed that PA increased the amount of protein level expression of GAD_65/67 over a broader range of doses. PA increased α- and β-subunits protein levels, but decreased γ-subunit protein levels in GABA_A receptors. The present experiment provides evidence for the hypnotic effects as PA enhanced pentobarbital-induced sleeping behaviors via GABA_A-ergic mechanisms in rodents. Taken together, it is proposed that PA may be useful for the treatment of sleep disturbed subjects with insomnia.     

2'-Methoxy-6-methylflavone: a novel anxiolytic and sedative with subtype selective activating and modulating actions at GABA(A) receptors

BACKGROUND AND PURPOSE

Flavonoids are known to have anxiolytic and sedative effects mediated via actions on ionotropic GABA receptors. We sought to investigate this further.

EXPERIMENTAL APPROACH

We evaluated the effects of 2′-methoxy-6-methylflavone (2′MeO6MF) on native GABA_A receptors in new-born rat hippocampal neurons and determined specificity from 18 human recombinant GABA_A receptor subtypes expressed in Xenopus oocytes. We used ligand binding, two-electrode voltage clamp and patch clamp studies together with behavioural studies.

KEY RESULTS

2′MeO6MF potentiated GABA at α2β1γ2L and all α1-containing GABA_A receptor subtypes. At α2β2/3γ2L GABA_A receptors, however, 2′MeO6MF directly activated the receptors without potentiating GABA. This activation was attenuated by bicuculline and gabazine but not flumazenil indicating a novel site. Mutation studies showed position 265 in the β1/2 subunit was key to whether 2′MeO6MF was an activator or a potentiator. In hippocampal neurons, 2′MeO6MF directly activated single-channel currents that showed the hallmarks of GABA_A Cl^- currents. In the continued presence of 2′MeO6MF the single-channel conductance increased and these high conductance channels were disrupted by the γ2(381–403) MA peptide, indicating that such currents are mediated by α2/γ2-containing GABA_A receptors. In mice, 2′MeO6MF (1–100 mg·kg⁻¹; i.p.) displayed anxiolytic-like effects in two unconditioned models of anxiety: the elevated plus maze and light/dark tests. 2′MeO6MF induced sedative effects at higher doses in the holeboard, actimeter and barbiturate-induced sleep time tests. No myorelaxant effects were observed in the horizontal wire test.

CONCLUSIONS AND IMPLICATIONS

2′MeO6MF will serve as a tool to study the complex nature of the activation and modulation of GABA_A receptor subtypes.     

Modulation of GABAA receptors and inhibitory synaptic currents by the endogenous CNS sleep regulator cis-9,10-octadecenoamide (cOA)

1. Cis-9,10-octadecenoamide (cOA) accumulates in the CSF of sleep-deprived cats and may represent a novel signalling molecule. Synthetic cOA has been shown to induce physiological sleep when injected into laboratory rats. Here we assess the cellular mode of action of cOA in vitro.
2. In all rat cultured cortical neurones (pyramidal cells) examined, the synthetic brain lipid (3.2–64 μM) enhanced the responses to subsaturating GABA concentrations (up to circa 2×) in a concentration-dependent manner (EC₅₀, circa 15 μM).
3. (20 μM) cOA significantly enhanced the affinity of exogenous GABA for its receptor without changing the Hill slope or the maximal response. These effects were not voltage-dependent or secondary to shifts in E_Cl.
4. In the absence of GABA, cOA directly evoked small inhibitory currents in a subpopulation (<7%) of sensitive cells.
5. 20 μM cOA reversibly enhanced the duration of spontaneous inhibitory post synaptic currents (circa 2 fold) without significantly altering their amplitude.
6. At 32–64 μM, cOA reversibly reduced the incidence and amplitude of both inhibitory post synaptic currents (i.p.s.cs) and excitatory post synaptic currents (e.p.s.cs) in the cultured neuronal circuits in common with other depressant drugs acting at the GABA_A receptor.
7. 32 μM Oleic acid did not modulate exogenous GABA currents or synaptic activity suggesting that cOAs actions are mediated through a specific receptor.
8. A specific, protein-dependent interaction with GABA_A receptors was confirmed in Xenopus oocytes. Recombinant human receptors were modulated by 10 μM cOA (and diazepam) only when a γ₂ subunit was co-expressed with α₁β₂: the cOA response was not sensitive to the specific benzodiazepine antagonist flumazenil (1 μM).
9. cOA may represent an endogenous ligand for allosteric modulatory sites on isoforms of GABA_A receptors which are crucial for the regulation of arousal and have recently been implicated in the circadian control of physiological sleep.