Benzodiazepine Site Research Articles

Triphlorethol A, a Dietary Polyphenol from Seaweed, Decreases Sleep Latency and Increases Non-Rapid Eye Movement Sleep in Mice.

In our previous studies, we have demonstrated that marine polyphenol phlorotannins promote sleep through the benzodiazepine site of the gamma-aminobutyric acid type A (GABAA) receptors. In this follow-up study, the sleep-promoting effects of triphlorethol A, one of the major phlorotannin constituents, were investigated. The effect of triphlorethol A on sleep-wake architecture and profiles was evaluated based on electroencephalogram and electromyogram data from C57BL/6N mice and compared with the well-known hypnotic drug zolpidem. Oral administration of triphlorethol A (5, 10, 25, and 50 mg/kg) dose-dependently decreased sleep latency and increased sleep duration during pentobarbital-induced sleep in imprinting control region mice. Triphlorethol A (50 mg/kg) significantly decreased sleep latency and increased the amount of non-rapid eye movement sleep (NREMS) in C57BL/6N mice, without affecting rapid eye movement sleep (REMS). There was no significant difference between the effects of triphlorethol A at 50 mg/kg and zolpidem at 10 mg/kg. Triphlorethol A had no effect on delta activity (0.5–4 Hz) of NREMS, whereas zolpidem significantly decreased it. These results not only support the sleep-promoting effects of marine polyphenol phlorotannins, but also suggest that the marine polyphenol compound triphlorethol A is a promising structure for developing novel sedative hypnotics.

Behavioral Effects of α2/α3 Subtype‐Selective GABAA Receptor Positive Allosteric Modulators

Millions of individuals suffer from chronic pain. Opioids are effective for treating chronic pain but their use is limited due to undesirable side‐effects. Previous studies have shown that different subunits of the GABAA receptor are associated with different actions of the benzodiazepine site. While α2/α3 subunit‐containing GABAA receptors mediate antinociceptive effects, α1 subunit‐containing GABAA receptors mediate sedative and abuse‐related effects. This study sought to examine the behavioral effects of two α2/α3‐subtype selective PAMs: KRM‐II‐81 and NS16085. In a rat model of inflammatory pain (complete Freund's adjuvant) and a rat model of neuropathic pain (chronic constriction injury), both PAMs and the classical benzodiazepine midazolam attenuated mechanical hyperalgesia (as measured by von Frey test). In the procedure of food‐maintained operant responding, KRM‐II‐81 and NS16085 did not significantly decrease the response rate at doses that produced maximal antinociception. Contrastingly, midazolam significantly reduced the response rate at doses that attenuated mechanical hyperalgesia. In a horizontal wire test aimed to measure muscle relaxation, within the dose range that produced antinociception, only midazolam dose‐dependently increased the percentage of rats unable to grasp the wire, indicating muscle‐relaxant activity. These behavioral effects can be attenuated by benzodiazepine receptor antagonist flumazenil, confirming that the behavioral effects of these subtype‐selective GABAA PAMs are mediated through the benzodiazepine site of GABAA receptors. Taken together, while midazolam produced antinociceptive, rate‐suppressing, and muscle‐relaxant activity at similar doses, both subtype‐selective GABAA receptor PAMs selectively produced antinocicpetive effects. Collectively, these data support the notion of α2/α3‐subtype selective GABAA PAMs as novel analgesics.Support or Funding InformationR01DA034806This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Functional properties and mechanism of action of PPTQ, an allosteric agonist and low nanomolar positive allosteric modulator at GABAA receptors

The former sedative-hypnotic and recreational drug methaqualone (Quaalude) is a moderately potent, non-selective positive allosteric modulator (PAM) at GABAA receptors (GABAARs) (Hammer et al., 2015). In the present study, we have identified a novel methaqualone analog, 2-phenyl-3-(p-tolyl)quinazolin-4(3H)-one (PPTQ), in a screening of 67 analogs at five αβ2γ2S GABAAR subtypes and delineated its functional properties and mechanism of action at wild-type and mutant GABAARs expressed in Xenopus laevis oocytes by two-electrode voltage clamp electrophysiology. PPTQ was found to be an allosteric agonist and a PAM (ago-PAM) at human α1β2γ2S and α4β2δ GABAARs, exhibiting intrinsic activity at micromolar concentrations and potentiating the GABA-evoked signaling through the receptors at concentrations down to the low-nanomolar range. Whereas PPTQ exclusively increased the potency of GABA at the α1β2γ2S receptor, it increased both GABA potency and efficacy at α4β2δ and displayed modest potency-based preference for α4β2δ over α1β2γ2S. In elaborate mutagenesis and competition experiments PPTQ was found to act through the same or an overlapping site as etomidate in the transmembrane β(+)/α(−) subunit interfaces, whereas it did not seem to target the other three transmembrane interfaces in the GABAAR. Finally, the PPTQ site was shown to be allosterically linked with sites targeted by neurosteroids and barbiturates but not with the high-affinity benzodiazepine site in the α1β2γ2S receptor. In conclusion, the development of a highly potent, bioavailable GABAAR ago-PAM by subtle modifications to the methaqualone scaffold demonstrates that derivatization of this infamous drug from the past can lead to modulators with distinct functional characteristics at the receptors.

Anticonvulsant effects of Senna spectabilis on seizures induced by chemicals and maximal electroshock

Senna spectabilis (Fabaceae) is one of the medicinal plants used in Cameroon by traditional healers to treat epilepsy, constipation, insomnia, anxiety. The present study aimed to investigate the anticonvulsant effects of Senna spectabilis decoction on seizures induced by maximal electroshock (MES), pentylenetetrazole (PTZ), pilocarpine (PC) and its possible action mechanisms in animal models using flumazenil (FLU), methyl-ß-carboline-3-carboxylate (BC) and bicuculline (BIC). Senna spectabilis decoction (106.5 and 213.0mg/kg) antagonized completely tonic-clonic hind limbs of mice induced by MES. The lowest plant dose (42.6mg/kg) provided 100% of protection against seizures induced by PTZ (70mg/kg). Administration of different doses of the plant decoction antagonized seizures induced by PC up to 75%, causing a dose dependent protection and reduced significantly the mortality rate induced by this convulsant. Both FLU and BC antagonize strongly the anticonvulsant effects of this plant and are unable to reverse totally diazepam or the plant decoction effects on inhibiting seizures. The animals did not present any sign of acute toxicity even at higher doses of the plant decoction. In conclusion, Senna spectabilis possesses an anticonvulsant activity. We showed that its decoction protects significantly mice against seizures induced by chemicals and MES, delays the onset time and reduces mortality rate in seizures-induced. It also appears that the oral administration of the decoction of S. spectabilis is more active than the intraperitoneal administration of the ethanolic extract on inhibiting seizures induced by MES and PTZ. Moreover, the plant decoction could interact with GABAA complex receptor probably on the GABA and benzodiazepines sites.

Anxiolytic-like effect of Bifidobacterium adolescentis IM38 in mice with or without immobilisation stress.

To better understand the role of gut microbiota in the anxiety, we isolated bifidobacteria and lactobacilli from the human faecal microbiota, investigated their inhibitory effects on the expression of interleukin (IL)-6 and tumour necrosis factor (TNF)-α in lipopolysaccharide-stimulated macrophages, and examined the anxiolytic-like effect of Bifidobacterium adolescentis IM38 in mice treated with or without immobilisation stress using the elevated plus maze (EPM) task. Oral administration of IM38 at a dose of 1×109 cfu/mouse showed a significant anxiolytic-like effect both in mice exposed to immobilisation stress and in control mice using the EPM test (P<0.05). Moreover, IM38 treatment significantly increased the amount of time spent on open arms and open arm entries. The anxiolytic-like effect of IM38 was comparable to that of buspirone (1 mg/kg). Moreover, this anxiolytic-like effect was blocked by treatment with flumazenil (3 mg/kg, i.p.), a benzodiazepine receptor antagonist, but was not affected by treatment with bicuculine or WAY-100635. IM38 treatment also reduced the blood levels of corticosterone and IL-6 in mice with or without immobilisation stress, whereas this effect was abolished by treatment with flumazenil. IM38 treatment also reduced the blood TNF-α level in mice subjected to immobilisation stress but not in normal control mice. Treatment with flumazenil also significantly increased TNF-α and IL-6 levels in immobilisation stress-free mice treated with IM38. These findings suggest that IM38 may attenuate anxiety through modulation of the benzodiazepine site on the GABAA receptor and modulate stress-related cytokine expression.

Selective inhibition of extra-synaptic α5-GABAA receptors by S44819, a new therapeutic agent

In the mammalian central nervous system (CNS) GABAA receptors (GABAARs) mediate neuronal inhibition and are important therapeutic targets. GABAARs are composed of 5 subunits, drawn from 19 proteins, underpinning expression of 20–30 GABAAR subtypes. In the CNS these isoforms are heterogeneously expressed and exhibit distinct physiological and pharmacological properties. We report the discovery of S44819, a novel tricyclic oxazolo-2,3-benzodiazepine-derivative, that selectively inhibits α5-subunit-containing GABAARs (α5-GABAARs). Current α5-GABAAR inhibitors bind to the “benzodiazepine site”. However, in HEK293 cells expressing recombinant α5-GABAARs, S44819 had no effect on 3H-flumazenil binding, but displaced the GABAAR agonist 3H-muscimol and competitively inhibited the GABA-induced responses. Importantly, we reveal that the α5-subunit selectivity is uniquely governed by amino acid residues within the α-subunit F-loop, a region associated with GABA binding. In mouse hippocampal CA1 neurons, S44819 enhanced long-term potentiation (LTP), blocked a tonic current mediated by extrasynaptic α5-GABAARs, but had no effect on synaptic GABAARs. In mouse thalamic neurons, S44819 had no effect on the tonic current mediated by δ-GABAARs, or on synaptic (α1β2γ2) GABAARs. In rats, S44819 enhanced object recognition memory and reversed scopolamine-induced impairment of working memory in the eight-arm radial maze. In conclusion, S44819 is a first in class compound that uniquely acts as a potent, competitive, selective antagonist of recombinant and native α5-GABAARs. Consequently, S44819 enhances hippocampal synaptic plasticity and exhibits pro-cognitive efficacy. Given this profile, S44819 may improve cognitive function in neurodegenerative disorders and facilitate post-stroke recovery.

Evidence for the involvement of the GABA-ergic pathway in the anticonvulsant activity of the roots bark aqueous extract of Anthocleista djalonensis A. Chev. (Loganiaceae).

The root bark of Anthocleista djalonensis A. Chev. (Loganiaceae) is widely used in traditional medicine in Northern Cameroon to treat epilepsy and related conditions, such as migraine, insomnia, dementia, anxiety, and mood disorders. To investigate the anticonvulsant effects and the possible mechanisms of this plant, an aqueous extract of Anthocleista djalonensis (AEAD) was evaluated by using animal models of bicuculline-, picrotoxin-, pilocarpine-, and pentylenetetrazole-induced convulsions. Their effects on brain γ-aminobutyric acid (GABA) concentration and GABA-T activity were also determined. This extract significantly protected mice against bicuculline-induced motor seizures. It provided 80% protection against picrotoxin-induced tonic-clonic seizures, and strongly antagonized convulsions induced by pilocarpine. AEAD also protected 100% of mice against pentylenetetrazole-induced seizures. Flumazenil, a central benzodiazepine receptor antagonist and FG7142, a partial inverse agonist in the benzodiazepine site of the GABAA receptor complex, were found to have an inhibitory effect on the anticonvulsant action of AEAD in pentylenetetrazole test. Finally, the brain GABA concentration was significantly increased and GABA-T activity was inhibited by AEAD. The effects of Anthocleista djalonensis suggested the presence of anticonvulsant properties that might involve an action on benzodiazepine and/or GABA sites in the GABAA receptor complex or by modulating GABA concentration in the central nervous system (CNS).

Synthesis and Pharmacological Evaluation of Novel GABAA Subtype Receptor Ligands with Potential Anxiolytic‐like and Anti‐hyperalgesic Effect

The identification of selective benzodiazepine site ligands, endowed with anxiolytic and anti‐hyperalgesic action, is a relevant opportunity for the treatment of pain syndromes. Previously, we selected a compound with a promising anti‐hyperalgesic profile, the 3‐iodo‐8‐benzylaminopyrazolo [5,1‐c][1,2,4]benzotriazine 5‐oxide. Aimed to verify the structure–activity relationship, the corresponding 7‐arylakylamino derivatives were synthesized. Compounds were tested for their affinity at GABAA‐receptor subtype; the compound 12 was further investigated in animal models of anxiety and persistent pain.

Investigation of the Anxiolytic and Antidepressant Effects of Curcumin, a Compound From Turmeric (Curcuma longa), in the Adult Male Sprague-Dawley Rat.

As the use of herbal medications continues to increase in America, the potential interaction between herbal and prescription medications necessitates the discovery of their mechanisms of action. The purpose of this study was to investigate the anxiolytic and antidepressant effects of curcumin, a compound from turmeric (Curcuma longa), and its effects on the benzodiazepine site of the γ-aminobutyric acid receptor A (GABAA) receptor. Utilizing a prospective, between-subjects group design, 55 male Sprague-Dawley rats were randomly assigned to 1 of the 5 intraperitoneally injected treatment groups: vehicle, curcumin, curcumin + flumazenil, midazolam, and midazolam + curcumin. Behavioral testing was performed using the elevated plus maze, open field test, and forced swim test. A 2-tailed multivariate analysis of variance and least significant difference post hoc tests were used for data analysis. In our models, curcumin did not demonstrate anxiolytic effects or changes in behavioral despair. An interaction of curcumin at the benzodiazepine site of the GABAA receptor was also not observed. Additional studies are recommended that examine the anxiolytic and antidepressant effects of curcumin through alternate dosing regimens, modulation of other subunits on the GABAA receptor, and interactions with other central nervous system neurotransmitter systems.

Curcumol allosterically modulates GABA(A) receptors in a manner distinct from benzodiazepines

Inhibitory A type γ-aminobutyric acid receptors (GABAARs) play a pivotal role in orchestrating various brain functions and represent an important molecular target in neurological and psychiatric diseases, necessitating the need for the discovery and development of novel modulators. Here, we show that a natural compound curcumol, acts as an allosteric enhancer of GABAARs in a manner distinct from benzodiazepines. Curcumol markedly facilitated GABA-activated currents and shifted the GABA concentration-response curve to the left in cultured hippocampal neurons. When co-applied with the classical benzodiazepine diazepam, curcumol further potentiated GABA-induced currents. In contrast, in the presence of a saturating concentration of menthol, a positive modulator for GABAAR, curcumol failed to further enhance GABA-induced currents, suggesting shared mechanisms underlying these two agents on GABAARs. Moreover, the benzodiazepine antagonist flumazenil did not alter the enhancement of GABA response by curcumol and menthol, but abolished that by DZP. Finally, mutations at the β2 or γ2 subunit predominantly eliminated modulation of recombinant GABAARs by curcumol and menthol, or diazepam, respectively. Curcumol may therefore exert its actions on GABAARs at sites distinct from benzodiazepine sites. These findings shed light on the future development of new therapeutics drugs targeting GABAARs.

Investigating the potential of the anti-epileptic drug imepitoin as a treatment for co-morbid anxiety in dogs with idiopathic epilepsy

BackgroundBehavioural changes associated with idiopathic epilepsy (IE) have been identified in dogs, with fear and anxiety-related problems seen in both drug-naïve dogs and dogs treated with anti-epileptic drugs (AEDs). Treating anxiety-related behaviour in dogs with IE may be challenging, as seizures are a contraindication for many conventional anxiolytic drugs. In addition, many dogs with IE are already treated with AEDs to reduce their seizure frequency, which may have negative effects if used in polytherapy. Imepitoin is low-affinity partial agonist at the benzodiazepine (BDZ) site of the GABAA receptor, and has been demonstrated to have both anticonvulsant and anxiolytic effects in laboratory rodents. Imepitoin has been developed for the treatment of IE in dogs, with demonstrated anticonvulsant effects and high tolerability and safety. To date, imepitoin’s potential to reduce anxiety in dogs with IE has not been investigated. An online survey was conducted to investigate the effect of imepitoin on fear and anxiety-related behaviours in dogs with IE. Eighty-five valid responses were received from owners of dogs with IE currently treated with imepitoin. Anxiety-related behaviour was quantified before and during imepitoin treatment using a validated questionnaire tool (C-BARQ).ResultsNo differences were observed in the five fear/anxiety-related measures between the two time periods (before vs. during treatment) for dog directed fear, stranger directed fear, non-social fear, pain sensitivity and separation related behaviour. A median 45% reduction in seizure frequency/month was observed following imepitoin treatment; however, imepitoin did not appear effective in reducing seizure frequency in a minority of cases. Polyphagia was the most common chronic side effect, and more side effects were reported in polytherapy cases.ConclusionsImepitoin does not appear to improve anxiety-related behaviour in dogs with IE treated with this medication for its anti-epileptic effects. Investigating the effects of imepitoin upon the behaviour of dogs with recognised behavioural anxiety-related problems (e.g. specific fears and phobias, separation related behaviours), in both healthy dogs and dogs with epilepsy is required to further explore any potential anxiolytic effects of this medication.

Pharmacological and antihyperalgesic properties of the novel α2/3 preferring GABAA receptor ligand MP-III-024.

γ-Aminobutyric acid type A (GABAA) receptors are located in spinal nociceptive circuits where they modulate the transmission of pain sensory signals from the periphery to higher centers. Benzodiazepine-type drugs bind to GABAA receptors containing α1, α2, α3, and α5 subunits (α1GABAA, α2GABAA, α3GABAA and α5GABAA receptors, respectively) through which they inhibit the transmission of these signals. In the present study we describe the novel benzodiazepine site positive allosteric modulator modulator methyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate (MP-III-024). MP-III-024 displayed preference for α2GABAA and α3GABAA receptors relative to α1GABAA and α5GABAA receptors as well as an improved metabolic profile relative to subtype-selective positive modulators that are available currently. Administration of MP-III-024 resulted in a dose- and time-dependent reversal of mechanical hyperalgesia. On locomotor activity and schedule-controlled responding, MP-III-024 was ineffective across the doses tested. These data provide further evidence that α2GABAA and α3GABAA receptors play an important role in the antihyperalgesic effects and may not be involved in some of the undesired effects of benzodiazepine-like drugs. Further, these findings suggest that MP-III-024 is a suitable research tool for investigating the role of α2GABAA and α3GABAA receptors in the behavioral properties of benzodiazepine-like drugs in mice.

The direct actions of cannabidiol and 2-arachidonoyl glycerol at GABAA receptors

Cannabidiol (CBD) is a major non-intoxicating component of cannabis and possesses anti-epileptic, anxiolytic and anti-hyperalgesic properties. The mechanism of action of CBD in producing such effects remains unclear. Despite evidence that some endogenous and synthetic cannabinoids interact with GABAA receptors, no-one has yet investigated the effects of CBD. Here we used two-electrode voltage clamp electrophysiology to compare the actions of CBD with those of the major central endocannabinoid, 2-arachidonoyl glycerol (2-AG) on human recombinant GABAA receptors (synaptic α1-6βγ2 and extrasynaptic α4β2δ) expressed on Xenopus oocytes. CBD and 2-AG were positive allosteric modulators at α1-6βγ2 receptors, with low micromolar potencies. The maximal level of enhancement seen with either CBD or 2-AG were on α2-containing GABAA receptor subtypes, with approximately a 4-fold enhancement of the GABA EC5 evoked current, more than twice the potentiation seen with other α-subunit receptor combinations. Further we observed β-subunit selectivity, whereby modulatory activity was higher at β2/β3 over β1 subunits. The β1-subunit homologous mutant β2(V436T) substantially diminished the efficacy of both drugs to a third of that obtained with wild-type β2 subunit combinations, but without changing potency. The potency of CBD increased and efficacy preserved in binary α1/α2β2 receptors indicating that their effects do not involve the classic benzodiazepine site. Exploration of extrasynaptic α4β2δ receptors revealed that both compounds enhanced GABA EC5 evoked currents at concentrations ranging from 0.01–1μM. Taken together these results reveal a mode of action of CBD on specifically configured GABAA receptors that may be relevant to the anticonvulsant and anxiolytic effects of the compound.

GABAA receptor subtypes in the mouse brain: Regional mapping and diazepam receptor occupancy by in vivo [18F]flumazenil PET

Classical benzodiazepines, which are widely used as sedatives, anxiolytics and anticonvulsants, exert their therapeutic effects through interactions with heteropentameric GABAA receptors composed of two α, two β and one γ2 subunit. Their high affinity binding site is located at the interface between the γ2 and the adjacent α subunit. The α-subunit gene family consists of six members and receptors can be homomeric or mixed with respect to the α-subunits. Previous work has suggested that benzodiazepine binding site ligands with selectivity for individual GABAA receptor subtypes, as defined by the benzodiazepine-binding α subunit, may have fewer side effects and may even be effective in diseases, such as schizophrenia, autism or chronic pain, that do not respond well to classical benzodiazepines. The distributions of the individual α subunits across the CNS have been extensively characterized. However, as GABAA receptors may contain two different α subunits, the distribution of the subunits does not necessarily reflect the distribution of receptor subtypes with respect to benzodiazepine pharmacology. In the present study, we have used in vivo [18F]flumazenil PET and in vitro [3H]flumazenil autoradiography in combination with GABAA receptor point-mutated mice to characterize the distribution of the two most prevalent GABAA receptor subtypes (α1 and α2) throughout the mouse brain. The results were in agreement with published in vitro data. High levels of α2-containing receptors were found in brain regions of the neuronal network of anxiety. The α1/α2 subunit combinations were predictable from the individual subunit levels. In additional experiments, we explored in vivo [18F]flumazenil PET to determine the degree of receptor occupancy at GABAA receptor subtypes following oral administration of diazepam. The dose to occupy 50% of sensitive receptors, independent of the receptor subtype(s), was 1–2mg/kg, in agreement with published data from ex vivo studies with wild type mice. In conclusion, we have resolved the quantitative distribution of α1- and α2-containing homomeric and mixed GABAA receptors in vivo at the millimeter scale and demonstrate that the regional drug receptor occupancy in vivo at these GABAA receptor subtypes can be determined by [18F]flumazenil PET. Such information should be valuable for drug development programs aiming for subtype-selective benzodiazepine site ligands for new therapeutic indications.

Low Expression in Xenopus Oocytes and Unusual Functional Properties of α1β2γ2 GABAA Receptors with Non-Conventional Subunit Arrangement.

The major subunit isoform of GABAA receptors is α1β2γ2. The subunits are thought to surround an ion pore with the counterclockwise arrangement α1γ2β2α1β2 as seen from the outside of the neuron. These receptors have two agonist sites and one high affinity drug binding site specific for benzodiazepines. Recently, this receptor was postulated to assume alternative subunit stoichiometries and arrangements resulting in only one agonist site and one or even two sites for benzodiazepines. In order to force a defined subunit arrangement we expressed a combination of triple and dual concatenated subunits. Here we report that these unconventional receptors express only small current amplitudes in Xenopus oocytes. We determined agonist properties and modulation by diazepam of two of these receptors that resulted in currents large enough for a characterization, that is, β2-α1-γ2/α1-γ2 and β2-α1-γ2/β2-γ2. The first pentamer predicted to have two benzodiazepine binding sites shows similar response to diazepam as the standard receptor. As expected for both receptors with a single predicted agonist site the concentration response curves for GABA were characterized by a Hill coefficient < 1. β2-α1-γ2/β2-γ2 displayed a mM apparent GABA affinity for channel opening instead of the expected μM affinity. Based on their subunit and binding site stoichiometry, that contradicts all previous observations, their unusual functional properties and their very low expression levels in oocytes, we consider it unlikely that these unconventional receptors are expressed in neurons to an appreciable extent.

Anesthesia and anesthetic action mechanism of essential oils of Aloysia triphylla and Cymbopogon flexuosus in silver catfish (Rhamdia quelen)

ObjectivesTo document the time for anesthesia induction and recovery using different concentrations of essential oils (EOs) of Cymbopogon flexuosus and Aloysia triphylla in silver catfish (Rhamdia quelen), and to determine whether the mechanism of action of either EO involves the benzodiazepine (BDZ) site of the GABAA receptor. Study designExperimental study. AnimalsA total of 144 silver catfish, length 7.5 ± 1.1 cm, weighing 3.95 ± 0.85 g. MethodsEssential oils were evaluated at concentrations of 25, 150 and 300 μL L−1, and also ethanol alone (seven groups, n = 6 per group). Induction of sedation or anesthesia and recovery were assessed. In a further six groups (n = 6 per group), fish were exposed to both EOs (25, 150 or 300 μL L−1) with diazepam 150 μm, and also diazepam (10 μm) alone. Flumazenil (5 or 10 μm) was added to the recovery water of fish exposed to diazepam (150 μm) or both EOs (150 and 300 μL L−1) (total of 10 groups = 60 fish). ResultsBoth EOs induced anesthesia at concentrations of 150 and 300 μL L−1, and sedation at 25 μL L−1. There was no significant difference between EOs for reaching deep anesthesia; there was a significantly longer recovery time for the EO of C. flexuosus. The addition of diazepam (150 μm) resulted in faster induction of anesthesia with both EOs, with no significant change in recovery times. Flumazenil (10 μm) reversed the diazepam-induced anesthesia, but not the anesthesia induced by EOs. Conclusions and clinical relevanceThe EO of C. flexuosus induced effective sedation (25 μL L−1) and anesthesia (150 and 300 μL L−1) without short-term mortality. The modulation of the BDZ site of the GABAA receptor in the anesthetic action mechanism of both EOs was not demonstrated.

Synthesis of n-alkylated derivatives of 1-(4-methoxyphenyl)-1,5-dihydro-4h-pyrazolo[3,4-d]pyrimidin-4-ones as potential anticonvulsants

High psychotropic activity of pyrimidine derivatives attracts attention and directs the creation of new pyrimidine drugs which affect the central nervous system. As psychotropic agents a special attention deserve azolopyrimidine derivatives, including pyrazolopyrimidines. Thus, among the pyrazolopyrimidine derivatives had been found compounds with antiepileptic, anticonvulsant, sedative, anxiolytic activity [1, 2], ligands of benzodiazepine site of GABA receptors [3]. In addition, the ligands of 5HT-6 receptors were identified that are promising for the treatment of central nervous system, muscle relaxants [4]. The purpose of this research was a synthesis of alkylated derivatives of 1-(4-methoxyphenyl)-1,5-dihydro-4 H -pyrazolo[3,4-d]pyrimidine-4-ones.

Extracts of Feretia apodanthera Del. demonstrated anticonvulsant activities against seizures induced by chemicals and maximal electroshock

Extracts of Feretia apodanthera Del. (Rubiaceae) have been extensively used in traditional Cameroonian medicine to treat a variety of diseases, including some neurological disorders. The present study was aimed to tests the anticonvulsant properties of the aqueous extract and the alkaloid fraction of the stem barks of Feretia apodanthera. The anticonvulsant investigation was carried out against bicuculline-, picrotoxin-, pentylenetetrazol-, Methyl-β-carboline-3-carboxylate-, N-Methyl-D-aspartate-, 4-aminopyridine-, and maximal electroshock-induced seizures or turning behavior in mice. The aqueous extract protected mice against bicuculline-, picrotoxin-, pentylenetetrazol-, Methyl-β-carboline-3-carboxylate-, N-methyl-D-aspartate −, 4-aminopyridine- and maximal electroshock-induced seizures or turning behavior. Also, N-Methyl-D-aspartate-, 4-aminopyridine- and maximal electroshock- induced seizures or turning behavior, were significantly antagonized by the alkaloid fraction (80mg/kg) from Feretia apodanthera. The total protection of mice provided by the aqueous extract against convulsions induced by pentylenetetrazol or picrotoxin was anagonized by flumazenil, a specific antagonist of the benzodiazepine site in the GABAA receptor complex.The aqueous extract of Feretia apodanthera (but not the alkaloid fraction) increased the brain GABA content and inhibited the GABA transaminase activity.In conclusion, Feretia apodanthera was revealed possessing anticonvulsant effects in mice, likely via the GABAergic neurotransmission.

Zolpidem is a potent stoichiometry-selective modulator of α1β3 GABAA receptors: evidence of a novel benzodiazepine site in the α1-α1 interface.

Zolpidem is not a typical GABAA receptor hypnotic. Unlike benzodiazepines, zolpidem modulates tonic GABA currents in the rat dorsal motor nucleus of the vagus, exhibits residual effects in mice lacking the benzodiazepine binding site, and improves speech, cognitive and motor function in human patients with severe brain injury. The receptor by which zolpidem mediates these effects is not known. In this study we evaluated binary α1β3 GABAA receptors in either the 3α1:2β3 or 2α1:3β3 subunit stoichiometry, which differ by the existence of either an α1-α1 interface, or a β3-β3 interface, respectively. Both receptor stoichiometries are readily expressed in Xenopus oocytes, distinguished from each other by using GABA, zolpidem, diazepam and Zn2+. At the 3α1:2β3 receptor, clinically relevant concentrations of zolpidem enhanced GABA in a flumazenil-sensitive manner. The efficacy of diazepam was significantly lower compared to zolpidem. No modulation by either zolpidem or diazepam was detected at the 2α1:3β3 receptor, indicating that the binding site for zolpidem is at the α1-α1 interface, a site mimicking the classical α1-γ2 benzodiazepine site. Activating α1β3 (3α1:2β3) receptors may, in part, mediate the physiological effects of zolpidem observed under distinct physiological and clinical conditions, constituting a potentially attractive drug target.

Novel positive allosteric modulators of GABAA receptors with anesthetic activity.

GABAA receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics and anesthetics. We previously identified novel ligands of the classical benzodiazepine binding pocket in α1β2γ2 GABAA receptors using an experiment-guided virtual screening (EGVS) method. This screen also identified novel ligands for intramembrane low affinity diazepam site(s). In the current study we have further characterized compounds 31 and 132 identified with EGVS as well as 4-O-methylhonokiol. We investigated the site of action of these compounds in α1β2γ2 GABAA receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology combined with a benzodiazepine site antagonist and transmembrane domain mutations. All three compounds act mainly through the two β+/α− subunit transmembrane interfaces of the GABAA receptors. We then used concatenated receptors to dissect the involvement of individual β+/α− interfaces. We further demonstrated that these compounds have anesthetic activity in a small aquatic animal model, Xenopus laevis tadpoles. The newly identified compounds may serve as scaffolds for the development of novel anesthetics.