Flunitrazepam Binding Research Articles

Anxiety-like Behavior and GABAAR/BDZ Binding Site Response to Progesterone Withdrawal in a Stress-Vulnerable Strain, the Wistar Kyoto Rats

Stress susceptibility could play a role in developing premenstrual anxiety due to abnormalities in the hypothalamus–pituitary–adrenal (HPA) axis and impairments in the GABAA receptors’ benzodiazepine (BDZ) site. Hence, we studied the stress-vulnerable Wistar Kyoto rat strain (WKY) to evaluate progesterone withdrawal (PW) effects on anxiety, HPA axis response, and to explore indicators of GABAA functionality in the BDZ site. For five days, ovariectomized WKY rats were administered 2.0 mg/kg of progesterone. Twenty-four hours after the last administration, rats were tested in the anxiety-like burying behavior test (BBT) or elevated plus maze test (EPM), and corticosterone was determined. [3H]Flunitrazepam binding autoradiography served as the BDZ binding site index of the GABAA receptor in amygdala nuclei and hippocampus’s dentate gyrus (DG). Finally, different doses of diazepam in PW-WKY rats were tested in the BBT. PW induced anxiety-like behaviors in both BBT and EPM compared with No-PW rats. PW increased corticosterone, but was blunted when combined with PW and BBT. PW increased [3H]Flunitrazepam binding in the DG and central amygdala compared with No-PW rats. Diazepam at a low dose induced an anxiogenic-like response in PW rats, suggesting a paradoxical response to benzodiazepines. Overall, PW induced anxiety-like behavior, a blunted HPA axis response, and higher GABAAR/BZD binding site sensitivity in a stress-vulnerable rat strain. These findings demonstrate the role of stress-susceptibility in GABAAR functionality in a preclinical approximation of PMDD.

High Fat Diet Increases [3H] Flunitrazepam Binding in the Mouse Brain that is Dependent on the Expression of the Dopamine D2 Gene.

Dopamine is an important neuromodulator in the brain that binds to dopamine D1-like receptors (D1, D5) as well as dopamine D2-like receptors (D2, D3, D4). The D2 receptor is known to play an integral role in a variety of physiological processes including addictive behaviors, locomotion, motivation, feeding behavior, and more. It was recently reported that dopamine is a direct-acting modulator of mammalian GABA(A) receptors. To this end, we wanted to examine how the expression of the dopamine D2 gene impacts the expression of GABA(A) receptors in the brain under different dietary conditions. Adult female Drd2 wild-type (WT), heterozygous (HT), and knockout (KO) mice were given either normal or high-fat diet for a period of 30weeks. Following this, their brains were collected for [3H] Flunitrazepam binding in order to assess GABA(A) receptor expression. A high fat diet significantly increased [3H] Flunitrazepam binding in the regions of the somatosensory cortex, striatum, and various other cortical areas within WT mice. In contrast, no effect of diet was observed in HT or KO mice. As such, HT and KO mice displayed reduced [3H] Flunitrazepam binding in these areas relative to WT mice under high-fat dietary conditions. The effect of a high-fat diet on [3H] Flunitrazepam binding is consistent with recent evidence showing increases in GABA neurotransmitter levels following a high-fat diet. We demonstrate for the first time that the expression of the D2 gene plays a prominent role in the ability of a high-fat diet to impact GABA(A) receptors in the mouse brain.

Chronic neuroleptic treatment combined with a high fat diet elevated [3H] flunitrazepam binding in the cerebellum

Clinical and preclinical studies have shown dysfunctions in genetic expression and neurotransmission of γ-Aminobutyric acid (GABA), GABAA receptor subunits, and GABA-synthesizing enzymes GAD67 and GAD65 in schizophrenia. It is well documented that there is significant weight gain after chronic neuroleptic treatment in humans. While there are limited studies on the effects of diet on GABA signaling directly, a change in diet has been used clinically as an adjunct to treatment for schizophrenic relief. In this study, rats chronically consumed either a chow diet (CD) or a 60% high-fat diet (HFD) and drank from bottles that contained one of the following solutions: water, haloperidol (1.5 mg/kg), or olanzapine (10 mg/kg) for four weeks. Rats were then euthanized and their brains were processed for GABAA in-vitro receptor autoradiography using [3H] flunitrazepam. A chronic HFD treatment yielded significantly increased [3H] flunitrazepam binding in the rat cerebellum independent of neuroleptic treatment. The desynchronization between the prefrontal cortex and the cerebellum is associated with major cognitive and motor dysfunctions commonly found in schizophrenic symptomatology, such as slowed reaction time, motor dyscoordination, and prefrontal activations related to speech fluency and cognitive alertness. These data support the notion that there is a dietary effect on GABA signaling within the cerebellum, as well as the importance of considering nutritional intervention methods as an adjunct treatment for patients chronically treated with neuroleptics. Finally, we indicate that future studies involving the analysis of individual patient's genetic profiles will further assist towards a precision medicine approach to the treatment of schizophrenia.

An innovative anticonvulsant - a GABAA receptor modulator with an alternative mechanism of action and enzyme-inducing detoxifying properties

IntroductionThe development of original drugs - new generation GABAA receptor modulators (GABAAR), with an anti-alcohol orientation, non-addictive and stimulating detoxification processes, makes it possible to increase the effectiveness of therapy and reduce the cost of treatment.ObjectivesStudy the mechanism of interaction between m-Cl-BHU and GABAA - receptorMethodsMolecular docking was performed to study the molecular docking of m-Cl-BHU with at the binding site of the target protein GABAAR.Radioreceptor studies were carried out using [3H] flunitrazepam binding with synaptosomal receptors in the cerebral cortex of Wistar rats in experimental alcoholism under the influence of therapy with m-CL-BHU. Kinetic parameters (T1/2, Clt, MRT, MET, AUC) of a model substrate - antipyrine were determined in the saliva of healthy volunteers and alcoholic patients.Results IResults of molecular docking (Schrödinger program (Glide) showed: m-CL-BHU (meta-chlorobenzhydryl urea) is complementary to the benzodiazepine GABAAR. Binding energy is low) (scoring (GScore) -11.14 kKal/mol); m-CL-BHU interacts with key amino acids at the α1γ2 interface: Tyr159, Tyr209, H101 Phe77 and is characterized by a high degree of model fit - dG insert: 0.741 Binding of [3H] flunitrazepam to the benzodiazepine site of GABAAR in rat brain in experimental alcoholism, who received 14 days of m-CL-BHU at 100 mg/kg /day, increased in receptor affinity. Changes in the kinetic parameters (T1/2, Clt, MRT, MET, AUC) of a model substrate - antipyrine in the saliva of healthy volunteers and alcoholic patients using Galodif (m-CL-BHU) at 300 mg/day 21 daysConclusionsm-CL-BHU - GABAA receptor modulator with an alternative mechanism of actionDisclosureNo significant relationships.

Positive allosteric modulation of GABAA receptors by a novel antiepileptic drug cenobamate

Cenobamate is a novel antiepileptic drug under investigation for use in patients with focal (partial-onset) seizures. To understand its potential molecular mechanism of action, the effects of cenobamate on GABAA-mediated currents and GABAA receptors in rodent hippocampal neurons were examined. Cenobamate potentiated GABA-induced currents (IGABA) in acutely isolated CA3 pyramidal cells in a concentration-dependent manner (EC50, 164 μM), which was not affected by flumazenil, a benzodiazepine receptor antagonist. Cenobamate enhanced tonic GABAA currents (Itonic), which is defined as a holding current shift by the GABAA receptor antagonist bicuculline (EC50, 36.63 μM). At therapeutically relevant concentrations, cenobamate induced minimal changes in the frequency, amplitudes, and decay time of spontaneous inhibitory postsynaptic currents in the CA1 neurons. Flumazenil failed to affect cenobamate-potentiated Itonic and Iphasic in CA1 neurons. Cenobamate showed positive allosteric modulation of GABA-induced IGABA mediated by GABAA receptors. This effect was similar for all tested hGABAA receptors containing six different alpha subunits (α1β2γ2 or α2-6β3γ2), with EC50 values ranging from 42 to 194 μM. Cenobamate did not displace the binding of flunitrazepam, a benzodiazepine derivative, or flumazenil to GABAA receptors. The results showed that cenobamate, a novel antiepileptic drug, acts as a positive allosteric modulator of high-affinity GABAA receptors, activated by GABA at a site independent of the benzodiazepine binding site and efficiently enhances Itonic inhibition in hippocampal neurons, which could be an underlying molecular mechanism stabilizing neural circuits of the epileptic hippocampus.

Benzodiazepine exposure induces transcriptional down-regulation of GABAA receptor α1 subunit gene via L-type voltage-gated calcium channel activation in rat cerebrocortical neurons

GABAA receptors are targets of different pharmacologically relevant drugs, such as barbiturates, benzodiazepines, and anesthetics. In particular, benzodiazepines are prescribed for the treatment of anxiety, sleep disorders, and seizure disorders. Benzodiazepines potentiate GABA responses by binding to GABAA receptors, which are mainly composed of α (1–3, 5), β2, and γ2 subunits. Prolonged activation of GABAA receptors by endogenous and exogenous modulators induces adaptive changes that lead to tolerance. For example, chronic administration of benzodiazepines produces tolerance to most of their pharmacological actions, limiting their usefulness. The mechanism of benzodiazepine tolerance is still unknown. To investigate the molecular basis of tolerance, we studied the effect of sustained exposure of rat cerebral cortical neurons to diazepam on the GABAA receptor. Flunitrazepam binding experiments showed that diazepam treatment induced uncoupling between GABA and benzodiazepine sites, which was blocked by co-incubation with flumazenil, picrotoxin, or nifedipine. Diazepam also produced selective transcriptional down-regulation of GABAA receptor α1 subunit gene through a mechanism dependent on the activation of L-type voltage-gated calcium channels. These findings suggest benzodiazepine-induced stimulation of calcium influx through L-type voltage-gated calcium channels triggers the activation of a signaling pathway that leads to uncoupling and an alteration of receptor subunit expression. Insights into the mechanism of benzodiazepine tolerance will contribute to the design of new drugs that can maintain their efficacies after long-term treatments.

Hops compounds modulatory effects and 6-prenylnaringenin dual mode of action on GABAA receptors

Hops (Humulus lupulus L.), a major component of beer, contain potentially neuroactive compounds that made it useful in traditional medicine as a sleeping aid. The present study aims to investigate the individual components in hops acting as allosteric modulators in GABAA receptors and bring further insight into the mode of action behind the sedative properties of hops. GABA-potentiating effects were measured using [3H]ethynylbicycloorthobenzoate (EBOB) radioligand binding assay in native GABAA receptors. Flumazenil sensitivity of GABA-potentiating effects, [3H]Ro 15–4513, and [3H]flunitrazepam binding assays were used to examine the binding to the classical benzodiazepines site. Humulone (alpha acid) and 6-prenylnaringenin (prenylflavonoid) were the most potent compounds displaying a modulatory activity at low micromolar concentrations. Humulone and 6-prenylnaringenin potentiated GABA-induced displacement of [3H]EBOB binding in a concentration-dependent manner where the IC50 values for this potentiation in native GABAA receptors were 3.2 μM and 3.7 μM, respectively. Flumazenil had no significant effects on humulone- or 6-prenylnaringenin-induced displacement of [3H]EBOB binding. [3H]Ro 15–4513 and [3H]flunitrazepam displacements were only minor with humulone but surprisingly prominent with 6-prenylnaringenin despite its flumazenil-insensitive modulatory activity. Thus, we applied molecular docking methods to investigate putative binding sites and poses of 6-prenylnaringenin at the GABAA receptor α1β2γ2 isoform. Radioligand binding and docking results suggest a dual mode of action by 6-prenylnaringenin on GABAA receptors where it may act as a positive allosteric modulator at α+β- binding interface as well as a null modulator at the flumazenil-sensitive α+γ2- binding interface.

Delayed creatine supplementation counteracts reduction of GABAergic function and protects against seizures susceptibility after traumatic brain injury in rats

Traumatic brain injury (TBI) is a devastating disease frequently followed by behavioral disabilities including post-traumatic epilepsy (PTE). Although reasonable progress in understanding its pathophysiology has been made, treatment of PTE is still limited. Several studies have shown the neuroprotective effect of creatine in different models of brain pathology, but its effects on PTE is not elucidated. Thus, we decided to investigate the impact of delayed and chronic creatine supplementation on susceptibility to epileptic seizures evoked by pentylenetetrazol (PTZ) after TBI. Our experimental data revealed that 4 weeks of creatine supplementation (300 mg/kg, p.o.) initiated 1 week after fluid percussion injury (FPI) notably increased the latency to first myoclonic and tonic-clonic seizures, decreased the time spent in tonic-clonic seizure, seizure intensity, epileptiform discharges and spindle oscillations induced by a sub-convulsant dose of PTZ (35 mg/kg, i.p.). Interestingly, this protective effect persists for 1 week even when creatine supplementation is discontinued. The anticonvulsant effect of creatine was associated with its ability to reduce cell loss including the number of parvalbumin positive (PARV+) cells in CA3 region of the hippocampus. Furthermore, creatine supplementation also protected against the reduction of GAD67 levels, GAD activity and specific [3H]flunitrazepam binding in the hippocampus. These findings showed that chronic creatine supplementation may play a neuroprotective role on brain excitability by controlling the GABAergic function after TBI, providing a possible new strategy for the treatment of PTE.

Flavonoids isolated from Tibetan medicines, binding to GABAA receptor and the anticonvulsant activity

Background: Our previous studies on Asterothamnus centrali-asiaticus Novopokr. (ACN) and Arenaria kansuensis Maxim. (AKM) had led to the isolation of some phytochemical constituents and evaluation of anticonvulsant effect based on their extracts. ACN and AKM have been widely used in traditional Tibetan herbs for neuropsychiatric diseases and cardiopulmonary disorders. Purpose: The purpose is to investigate structure-activity relationships of flavonoids isolated from ACN and AKM, for binding to the benzodiazepine site (BZ-S) of γ-aminobutyric acid type A (GABAA) receptor complex, and to search for anticonvulsant compounds without undesirable effects such as myorelaxation and sedation. Study design and methods: The affinities of these flavonoids for the BZ-S of GABAA receptors were determined by [3H]flunitrazepam binding to mouse cerebellum membranes in vitro. And the anticonvulsant, myorelaxant and sedative effects were determined by pentylenetetrazol (PTZ)-induced seizure and electrogenic seizure protection, rotarod test and locomotor activity test, respectively. Results: Fifteen and thirteen flavonoids were isolated from ACN and AKM, respectively. Structure-activity relationships analysis indicated that 6-and/or 8-OMe flavones exhibited the most potent binding affinity to GABAA receptors. Furthermore, 2′,4′,5,7-tetrahydroxy-5′,6-dimethoxyflavone (DMF, IC50 value of 0.10 μM), a flavone isolated from ACN, presented high anticonvulsant activity against chemical-induced seizures and electrogenic seizures, without myorelaxation and sedation. Conclusion: This study suggested that these flavones, especially DMF, are new BZ receptor ligands and prospective therapeutic candidates for seizures.

Synthesis and pharmacological evaluation of pyrazolo[4,3-c]quinolinones as high affinity GABAA-R ligands and potential anxiolytics

The synthesis, in vitro ligand binding study and in vivo Elevated Plus Maze test (EPM) of a series of pyrazolo[4,3-c]quinolin-3-ones (PQs) are reported. Multistep synthesis of PQs started from anilines and diethyl 2-(ethoxymethylene)malonate to give the quinolin-4-one nucleus, via the Gould-Jacobs reaction. These quinolinones were transformed to 4-chloroquinolines, which react with aryl-hydrazines affording the final compounds. PQs exhibited different potency in displacing specific [3H]Flunitrazepam binding from the benzodiazepine binding site at the γ-aminobutyric acid receptor (GABAA-R) depending on the substitution of the pyrazoloquinolone nucleus. PCA helped determine how different substituents contributed to the differential behavior of the PQs studied. Compounds with high affinity for the GABAA-R were tested regarding their anxiolytic properties in Wistar adult male rats using the Elevated Plus Maze (EPM). Thus, PQs with a p-methoxy phenyl group at N-1 (7b-ii and 7c-ii) displayed a remarkable anxiolytic activity at low doses (0.5–1.0 mg/kg). Meanwhile, PQs featuring an unsubstituted phenyl (7b-i) or p-fluoro phenyl group (7b-iii) at the N-1 showed anxiogenic effects in the EPM test.

N-propyl-2,2-diphenyl-2-hydroxyacetamide, a novel α-hydroxyamide with anticonvulsant, anxiolytic and antidepressant-like effects that inhibits voltage-gated sodium channels

In patients with epilepsy, anxiety and depression are the most frequent psychiatric comorbidities but they often remain unrecognized and untreated.We report herein the antidepressant-like activity in two animal models, tail suspension and forced swimming tests, of six anticonvulsants α-hydroxyamides. From these, N-propyl-2,2-diphenyl-2-hydroxyacetamide (compound 5) emerged not only as the most active as anticonvulsant (ED50 = 2.5mg/kg, MES test), but it showed the most remarkable antidepressant-like effect in the tail suspension and forced swimming tests (0.3–30mg/kg, i.p.); and, also, anxiolytic-like action in the plus maze test (3–10mg/kg, i.p.) in mice. Studies of its mechanism of action, by means of its capacity to act via the GABAA receptor ([3H]-flunitrazepam binding assay); the 5-HT1A receptor ([3H]-8-OH-DPAT binding assay) and the voltage-gated sodium channels (either using the patch clamp technique in hNav 1.2 expressed in HEK293 cell line or using veratrine, in vivo) were attempted. The results demonstrated that its effects are not likely related to 5-HT1A or GABAAergic receptors and that its anticonvulsant and antidepressant-like effect could be due to its voltage-gated sodium channel blocking properties.

Cerebral Malaria Causes Enduring Behavioral and Molecular Changes in Mice Brain Without Causing Gross Histopathological Damage

Malaria, parasitic disease considered a major health public problem, is caused by Plasmodium protozoan genus and transmitted by the bite of infected female Anopheles mosquito genus. Cerebral malaria (CM) is the most severe presentation of malaria, caused by P. falciparum and responsible for high mortality and enduring development of cognitive deficits which may persist even after cure and cessation of therapy. In the present study we evaluated selected behavioral, neurochemical and neuropathologic parameters after rescue from experimental cerebral malaria caused by P. berghei ANKA in C57BL/6 mice. Behavioral tests showed impaired nest building activity as well as increased marble burying, indicating that natural behavior of mice remains altered even after cure of infection. Regarding the neurochemical data, we found decreased α2/α3 Na+,K+-ATPase activity and increased immunoreactivity of phosphorylated Na+,K+-ATPase at Ser943 in cerebral cortex after CM. In addition, [3H]-Flunitrazepam binding assays revealed a decrease of benzodiazepine/GABAA receptor binding sites in infected animals. Moreover, in hippocampus, dot blot analysis revealed increased levels of protein carbonyls, suggesting occurrence of oxidative damage to proteins. Interestingly, no changes in the neuropathological markers Fluoro-Jade C, Timm staining or IBA-1 were detected. Altogether, present data indicate that behavioral and neurochemical alterations persist even after parasitemia clearance and CM recovery, which agrees with available clinical findings. Some of the molecular mechanisms reported in the present study may underlie the behavioral changes and increased seizure susceptibility that persist after recovery from CM and may help in the future development of therapeutic strategies for CM sequelae.

Изменение специфического связывания [N-метил-<sup>3</sup>H]флунитразепама при воздействии анксиогенами

Изучено влияние лекарственных препаратов и фармакологических соединений, обладающих анксиогенными свойствами: йохимбин, кофеин, производное β-карболинов — соединение β-ССВ на специфическое связывание полного агониста бензодиазепинового участка ГАМКА рецептора [N-метил-3H]флунитразепама. Кофеин в дозе 100 мг/кг, n-бутил-β-карболин-3 карбоксилат (β-ССВ) в дозе 3 мг/кг и йохимбин в дозе 5 мг/кг вызывали анксиогенный эффект у мышей линии Balb/c и C57Bl/6. Анксиогенная активность кофеина, β-ССВ и йохимбина сопряжена с падением уровня бензодиазепиновой рецепции.

High Resolution UHPLC-MS Metabolomics and Sedative-Anxiolytic Effects of Latua pubiflora: A Mystic Plant used by Mapuche Amerindians.

Latua pubiflora (Griseb) Phil. Is a native shrub of the Solanaceae family that grows freely in southern Chile and is employed among Mapuche aboriginals to induce sedative effects and hallucinations in religious or medicine rituals since prehispanic times. In this work, the pentobarbital-induced sleeping test and the elevated plus maze test were employed to test the behavioral effects of extracts of this plant in mice. The psychopharmacological evaluation of L. pubiflora extracts in mice determined that both alkaloid-enriched as well as the non-alkaloid extracts produced an increase of sleeping time and alteration of motor activity in mice at 150 mg/Kg. The alkaloid extract exhibited anxiolytic effects in the elevated plus maze test, which was counteracted by flumazenil. In addition, the alkaloid extract from L. pubiflora decreased [3H]-flunitrazepam binding on rat cortical membranes. In this study we have identified 18 tropane alkaloids (peaks 1–4, 8–13, 15–18, 21, 23, 24, and 28), 8 phenolic acids and related compounds (peaks 5–7, 14, 19, 20, 22, and 29) and 7 flavonoids (peaks 25–27 and 30–33) in extracts of L. pubiflora by UHPLC-PDA-MS which are responsible for the biological activity. This study assessed for the first time the sedative-anxiolytic effects of L. pubiflora in rats besides the high resolution metabolomics analysis including the finding of pharmacologically important tropane alkaloids and glycosylated flavonoids.

Effects of acute and chronic administration of neurosteroid dehydroepiandrosterone sulfate on neuronal excitability in mice.

BackgroundNeurosteroid dehydroepiandrosterone sulfate (DHEAS) has been associated with important brain functions, including neuronal survival, memory, and behavior, showing therapeutic potential in various neuropsychiatric and cognitive disorders. However, the antagonistic effects of DHEAS on γ-amino-butyric acidA receptors and its facilitatory action on glutamatergic neurotransmission might lead to enhanced brain excitability and seizures and thus limit DHEAS therapeutic applications. The aim of this study was to investigate possible age and sex differences in the neuronal excitability of the mice following acute and chronic DHEAS administration.MethodsDHEAS was administered intraperitoneally in male and female adult and old mice either acutely or repeatedly once daily for 4 weeks in a 10 mg/kg dose. To investigate the potential proconvulsant properties of DHEAS, we studied the effects of acute and chronic DHEAS treatment on picrotoxin-, pentylentetrazole-, and N-methyl-D-aspartate-induced seizures in mice. The effects of acute and chronic DHEAS administration on the locomotor activity, motor coordination, and body weight of the mice were also studied. We also investigated the effects of DHEAS treatment on [3H]flunitrazepam binding to the mouse brain membranes.ResultsDHEAS did not modify the locomotor activity, motor coordination, body weight, and brain [3H]flunitrazepam binding of male and female mice. The results failed to demonstrate significant effects of single- and long-term DHEAS treatment on the convulsive susceptibility in both adult and aged mice of both sexes. However, small but significant changes regarding sex differences in the susceptibility to seizures were observed following DHEAS administration to mice.ConclusionAlthough our findings suggest that DHEAS treatment might be safe for various potential therapeutic applications in adult as well as in old age, they also support subtle interaction of DHEAS with male and female hormonal status, which may underline observed sex differences in the relationship between DHEAS and various health outcomes.

Discovery of a novel general anesthetic chemotype using high-throughput screening.

The development of novel anesthetics has historically been a process of combined serendipity and empiricism, with most recent new anesthetics developed via modification of existing anesthetic structures. Using a novel high-throughput screen employing the fluorescent anesthetic 1-aminoanthracene and apoferritin as a surrogate for on-pathway anesthetic protein target(s), we screened a 350,000 compound library for competition with 1-aminoanthracene-apoferritin binding. Hit compounds meeting structural criteria had their binding affinities for apoferritin quantified with isothermal titration calorimetry and were tested for γ-aminobutyric acid type A receptor binding using a flunitrazepam binding assay. Chemotypes with a strong presence in the top 700 and exhibiting activity via isothermal titration calorimetry were selected for medicinal chemistry optimization including testing for anesthetic potency and toxicity in an in vivo Xenopus laevis tadpole assay. Compounds with low toxicity and high potency were tested for anesthetic potency in mice. From an initial chemical library of more than 350,000 compounds, we identified 2,600 compounds that potently inhibited 1-aminoanthracene binding to apoferritin. A subset of compounds chosen by structural criteria (700) was successfully reconfirmed using the initial assay. Based on a strong presence in both the initial and secondary screens the 6-phenylpyridazin-3(2H)-one chemotype was assessed for anesthetic activity in tadpoles. Medicinal chemistry efforts identified four compounds with high potency and low toxicity in tadpoles, two were found to be effective novel anesthetics in mice. The authors demonstrate the first use of a high-throughput screen to successfully identify a novel anesthetic chemotype and show mammalian anesthetic activity for members of that chemotype.

Synaptosomal membrane-based Langmuir-Blodgett films: a platform for studies on γ-aminobutyric acid type A receptor binding properties.

In this work we used Langmuir-Blodgett films (LB) as model membranes to study the effect of molecular packing on the flunitrazepam (FNZ) accessibility to the binding sites at the GABAA receptor (GABAA-R). Ligand binding data were correlated with film topography analysis by atomic force microscopy images (AFM) and SDS-PAGE. Langmuir films (LF) were prepared by the spreading of synaptosomal membranes (SM) from bovine brain cortex at the air-water interface. LBs were obtained by the transference, at 15 or 35 mN/m constant surface pressure (π), of one (LB15/1c and LB35/1c) or two (LB35/2c) LFs to a film-free hydrophobic alkylated substrate (CONglass). Transference was performed in a serial manner, which allowed the accumulation of a great number of samples. SDS-PAGE clearly showed a 55 kDa band characteristic of GABAA-R subunits. Detrended fluctuation analysis of topographic data from AFM images exhibited a single slope value (self-similarity parameter α) in CONglass and a discontinuous slope change in the α value at an autocorrelation length of ∼100 nm in all LB samples, supporting the LF transference to the substrate. AFM images of CONglass and LB15/1c exhibited roughness and average heights that were similar between measurements and significantly lower than those of LB35/1c and LB35/2c, suggesting that the substrate coverage in the latter was more stable than in LB15/1c. While [(3)H]FNZ binding in LB15/1c did not reach saturation, in LB35/1c the binding kinetics became sigmoid with a binding affinity lower than in the SM suspension. Our results highlight the π dependence of both binding and topological data and call to mind the receptor mechanosensitivity. Thus, LB films provide a tool for bionanosensing GABAA-R ligand binding as well as GABAA-R activity modulation induced by the environmental supramolecular organization.

Effect of synthetic steroids on GABAA receptor binding in rat brain

Neuroactive steroids, like allopregnanolone (A) and pregnanolone (P), bind to specifics sites on the GABAA receptor complex and modulate receptor function. They are capable to inhibit or stimulate the binding of GABAA receptor-specific ligands, like t-butyl-bicyclophosphorothionate, flunitrazepam and muscimol. We have previously characterized a set of oxygen-bridged synthetic steroids (SS) analogs to A or P using synaptosomes. Considering that the subunit composition of the GABAA receptor throughout the central nervous system affects the magnitude of the modulation of the GABAA receptor by NAS, we evaluated the action of two selected SS, in brain sections containing the cerebral cortex (CC) and hippocampus (HC) using quantitative receptor autoradiography. Both SS affected the binding of the three ligands in a similar way to A and P, with some differences on certain CC layers according to the ligand used. One of the SS, the 3α-hydroxy-6,19-epoxypregn-4-ene-20-one (compound 5), behaved similarly to the natural neuroactive steroids. However, significant differences with compound 5 were observed on the HC CA2 region, making it steroid suitable for a specific action. Those differences may be related to structural conformation of the SS and the subunits’ composition present on the receptor complex.

GABA(A) receptor subtype-selectivity of novel bicuculline derivatives.

GABA(A) receptors are the major inhibitory neurotransmitter receptors in the central nervous system and are targets of clinically important drugs modulating GABA induced ion flux by interacting with distinct allosteric binding sites. ROD 185 is a previously investigated structural analogue of the GABA site antagonist bicuculline, and a positive allosteric modulator acting via the benzodiazepine binding site. Here, we investigated 13 newly synthesized structural analogues of ROD 185 for their interaction with rat GABA(A) receptors. Using [(3)H]flunitrazepam binding assays, we identified four compounds exhibiting a higher affinity for the benzodiazepine binding site than ROD 185. Two electrode voltage clamp electrophysiology at recombinant GABA(A) receptors indicated that most of these compounds positively modulated GABA-induced currents at these receptors. Additionally, these experiments revealed that this compound class not only interacts with the benzodiazepine binding site at αβγ receptors but also with a novel, so far unidentified binding site present in αβ receptors. Compounds with a high affinity for the benzodiazepine binding site stimulated GABA-induced currents stronger at αβγ than at αβ receptors and preferred α3β3γ2 receptors. Compounds showing equal or smaller effects at αβγ compared to αβ receptors differentially interacted with various αβ or αβγ receptor subtypes. Surprisingly, five of these compounds interacting with αβ receptors showed a strong stimulation at α6β3γ2 receptors. The absence of any direct effects at GABA(A) receptors, as well as their potential selectivity for receptor subtypes not being addressed by benzodiazepines, make this compound class to a starting point for the development of drugs with a possible clinical importance.

Inhibitory effects of carvone isomers on the GABAA receptor in primary cultures of rat cortical neurons.

Carvone is a natural terpene which can be purified as R-(-) or S-(+) enantiomers. There are many reports about its antibacterial, antifungal, and insecticide activities, and also of some effects on the nervous system, where both enantiomers showed different potencies. Considering that the GABA(A) receptor is a major insecticide target, we studied the pharmacological activity of both carvone enantiomers, and of thujone as a reference compound acting on the receptor, on native GABA(A) by determining their effects on benzodiazepine recognition sites using primary neuronal cultures. Both isomers were able to inhibit the GABA-induced stimulation of [(3)H]flunitrazepam binding, suggesting their interaction with the GABA(A) receptor as negative allosteric modulators. Their activity was comparable to that described for thujone in the present article, with the R-(-)-carvone being the more similar and potent stereoisomer. The different configuration of the isopropenyl group in position 5 thus seems to be significant for receptor interaction and the bicycle structure not to be critical for receptor recognition. The concentrations necessary to induce negative modulation of the receptor were not cytotoxic in a murine neuron culture system. These results confirm that, at least partially, the reported insecticidal activity of carvones may be explained by their interaction with the GABA(A) receptor at its noncompetitive blocker site.