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Description, D2R-NMDAR receptor heteromers are potential therapeutic targets in drug addiction., Addictive drugs increase dopamine in the nucleus accumbens (NAc), where it persistently shapes excitatory glutamate transmission and hijacks natural reward processing. Here, we provide evidence, from mice to humans, that an underlying mechanism relies on drug-evoked heteromerization of glutamate N-methyl-d-aspartate receptors (NMDAR) with dopamine receptor 1 (D1R) or 2 (D2R). Using temporally controlled inhibition of D1R-NMDAR heteromerization, we unraveled their selective implication in early phases of cocaine-mediated synaptic, morphological, and behavioral responses. In contrast, preventing D2R-NMDAR heteromerization blocked the persistence of these adaptations. Interfering with these heteromers spared natural reward processing. Notably, we established that D2R-NMDAR complexes exist in human samples and showed that, despite a decreased D2R protein expression in the NAc, individuals with psychostimulant use disorder display a higher proportion of D2R forming heteromers with NMDAR. These findings contribute to a better understanding of molecular mechanisms underlying addiction and uncover D2R-NMDAR heteromers as targets with potential therapeutic value.

Background Autism spectrum disorders (ASD) are associated with dysregulation of the microbiota-gut-brain axis, changes in microbiota composition as well as in the fecal, serum, and urine levels of microbial metabolites. Yet a causal relationship between dysregulation of the microbiota-gut-brain axis and ASD remains to be demonstrated. Here, we hypothesized that the microbial metabolite p-Cresol, which is more abundant in ASD patients compared to neurotypical individuals, could induce ASD-like behavior in mice. Results Mice exposed to p-Cresol for 4 weeks in drinking water presented social behavior deficits, stereotypies, and perseverative behaviors, but no changes in anxiety, locomotion, or cognition. Abnormal social behavior induced by p-Cresol was associated with decreased activity of central dopamine neurons involved in the social reward circuit. Further, p-Cresol induced changes in microbiota composition and social behavior deficits could be transferred from p-Cresol-treated mice to control mice by fecal microbiota transplantation (FMT). We also showed that mice transplanted with the microbiota of p-Cresol-treated mice exhibited increased fecal p-Cresol excretion, compared to mice transplanted with the microbiota of control mice. In addition, we identified possible p-Cresol bacterial producers. Lastly, the microbiota of control mice rescued social interactions, dopamine neurons excitability, and fecal p-Cresol levels when transplanted to p-Cresol-treated mice. Conclusions The microbial metabolite p-Cresol induces selectively ASD core behavioral symptoms in mice. Social behavior deficits induced by p-Cresol are dependant on changes in microbiota composition. Our study paves the way for therapeutic interventions targeting the microbiota and p-Cresol production to treat patients with ASD.

Freezing is a conserved defensive behaviour that constitutes a major stress-coping mechanism. Decades of research have demonstrated a role of the amygdala, periaqueductal grey and hypothalamus as core actuators of the control of fear responses, including freezing. However, the role that other modulatory sites provide to this hardwired scaffold is not known. Here, we show that freezing elicited by exposure to electrical foot shocks activates laterodorsal tegmentum (LDTg) GABAergic neurons projecting to the VTA, without altering the excitability of cholinergic and glutamatergic LDTg neurons. Selective chemogenetic silencing of this inhibitory projection, but not other LDTg neuronal subtypes, dampens freezing responses but does not prevent the formation of conditioned fear memories. Conversely, optogenetic-activation of LDTg GABA terminals within the VTA drives freezing responses and elicits bradycardia, a common hallmark of freezing. Notably, this aversive information is subsequently conveyed from the VTA to the amygdala via a discrete GABAergic pathway. Hence, we unveiled a circuit mechanism linking LDTg-VTA-amygdala regions, which holds potential translational relevance for pathological freezing states such as post-traumatic stress disorders, panic attacks and social phobias.

International audience; Stress has been acknowledged as one of the main risk factors for the onset of psychiatric disorders. Social stress is the most common type of stressor encountered in our daily lives. Uncovering the molecular determinants of the effect of stress on the brain would help understanding the complex maladaptations that contribute to pathological stress-related mental states. We examined molecular changes in the reward system following social defeat stress in mice, as increasing evidence implicates this system in sensing stressful stimuli. Following acute or chronic social defeat stress, the activation (i.e. phosphorylation) of extracellular signal-regulated kinases ERK1 and ERK2 (pERK1/2), markers of synaptic plasticity, was monitored in sub-regions of the reward system. We employed pharmacological antagonists and inhibitory DREADD to dissect the sequence of events controlling pERK1/2 dynamics. The nucleus accumbens (NAc) showed marked increases in pERK1/2 following both acute and chronic social stress compared to the dorsal striatum. Increases in pERK1/2 required dopamine D1 receptors and GluN2B-containing NMDA receptors. Paraventricular thalamic glutamatergic inputs to the NAc are required for social stress-induced pERK1/2. The molecular adaptations identified here could contribute to the long-lasting impact of stress on the brain and may be targeted to counteract stress-related psychopathologies.

Serotonin is a neurotransmitter involved in many psychiatric diseases. In humans, a lack of 5-HT2B receptors is associated with serotonin-dependent phenotypes, including impulsivity and suicidality. A lack of 5-HT2B receptors in mice eliminates the effects of molecules that directly target serotonergic neurons including amphetamine derivative serotonin releasers, and selective serotonin reuptake inhibitor antidepressants. In this work, we tested the hypothesis that 5-HT2B receptors directly and positively regulate raphe serotonin neuron activity. By ex vivo electrophysiological recordings, we report that stimulation by the 5-HT2B receptor agonist, BW723C86, increased the firing frequency of serotonin Pet1-positive neurons. Viral overexpression of 5-HT2B receptors in these neurons increased their excitability. Furthermore, in vivo 5-HT2B-receptor stimulation by BW723C86 counteracted 5-HT1A autoreceptor-dependent reduction in firing rate and hypothermic response in wild-type mice. By a conditional genetic ablation that eliminates 5-HT2B receptor expression specifically and exclusively from Pet1-positive serotonin neurons (Htr2b 5-HTKO mice), we demonstrated that behavioral and sensitizing effects of MDMA (3,4-methylenedioxy-methamphetamine), as well as acute behavioral and chronic neurogenic effects of the antidepressant fluoxetine, require 5-HT2B receptor expression in serotonergic neurons. In Htr2b 5-HTKO mice, dorsal raphe serotonin neurons displayed a lower firing frequency compared to control Htr2b lox/lox mice as assessed by in vivo extracellular recordings and a stronger hypothermic effect of 5-HT1A-autoreceptor stimulation was observed. The increase in head-twitch response to DOI (2,5-dimethoxy-4-iodoamphetamine) further confirmed the lower serotonergic tone resulting from the absence of 5-HT2B receptors in serotonin neurons. Together, these observations indicate that the 5-HT2B receptor acts as a direct positive modulator of serotonin Pet1-positive neurons in an opposite way as the known 5-HT1A-negative autoreceptor. Fil: Belmer, Arnauld. Universite Paris Sorbonne - Paris Iv; Francia. Inserm; Francia. Queensland University of Technology; Australia Fil: Quentin, Emily. Universite Paris Sorbonne - Paris Iv; Francia. Inserm; Francia Fil: Diaz, Silvina Laura. Inserm; Francia. Universidad de Morón; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina Fil: Guiard, Bruno P.. Université Paul Sabatier; Francia Fil: Fernandez, Sebastian P.. Inserm; Francia. Universite Paris Sorbonne - Paris Iv; Francia Fil: Doly, Stéphane. Inserm; Francia. Universite Paris Sorbonne - Paris Iv; Francia Fil: Banas, Sophie M.. Inserm; Francia. Universite Paris Sorbonne - Paris Iv; Francia Fil: Pitychoutis, Pothitos M.. Inserm; Francia. Universite Paris Sorbonne - Paris Iv; Francia Fil: Moutkine, Imane. Inserm; Francia. Universite Paris Sorbonne - Paris Iv; Francia Fil: Muzerelle, Aude. Universite Paris Sorbonne - Paris Iv; Francia. Inserm; Francia Fil: Tchenio, Anna. Universite de Lausanne; Suiza. Inserm; Francia Fil: Roumier, Anne. Inserm; Francia. Universite Paris Sorbonne - Paris Iv; Francia Fil: Mameli, Manuel. Universite de Lausanne; Suiza. Universite Paris Sorbonne - Paris Iv; Francia. Inserm; Francia Fil: Maroteaux, Luc. Inserm; Francia. Universite Paris Sorbonne - Paris Iv; Francia

Serotonin (5-HT) deficiency occurs in a number of brain disorders that affect cognitive function. However, a direct causal relationship between 5-HT hypo-transmission and memory and underlying mechanisms has not been established. We used mice with a constitutive depletion of 5-HT brain levels (Pet1KO mice) to analyze the contribution of 5-HT to different forms of learning and memory. Pet1KO mice exhibited a striking deficit in novel object recognition memory, a hippocampal-dependent task. No alterations were found in tasks for social recognition, procedural learning, or fear memory. Viral delivery of designer receptors exclusively activated by designer drugs was used to selectively silence the activity of 5-HT neurons in the raphe. Inhibition of 5-HT neurons in the median raphe, but not the dorsal raphe, was sufficient to impair object recognition in adult mice. In vivo electrophysiology in behaving mice showed that long-term potentiation in the hippocampus of 5-HT-deficient mice was altered, and administration of the 5-HT1A agonist 8-OHDPAT rescued the memory deficits. Our data suggest that hyposerotonergia selectively affects declarative hippocampal-dependent memory. Serotonergic projections from the median raphe are necessary to regulate object memory and hippocampal synaptic plasticity processes, through an inhibitory control mediated by 5-HT1A receptors.

Addiction is a maladaptive pattern of behavior following repeated use of reinforcing drugs in predisposed individuals, leading to lifelong changes. Common among these changes are alterations of neurons releasing dopamine in the ventral and dorsal territories of the striatum. The serotonin 5-HT2B receptor has been involved in various behaviors, including impulsivity, response to antidepressants, and response to psychostimulants, pointing toward putative interactions with the dopamine system. Despite these findings, it remains unknown whether 5-HT2B receptors directly modulate dopaminergic activity and the possible mechanisms involved. To answer these questions, we investigated the contribution of 5-HT2B receptors to cocaine-dependent behavioral responses. Male mice permanently lacking 5-HT2B receptors, even restricted to dopamine neurons, developed heightened cocaine-induced locomotor responses. Retrograde tracing combined with single-cell mRNA amplification indicated that 5-HT2B receptors are expressed by mesolimbic dopamine neurons. In vivo and ex vivo electrophysiological recordings showed that 5-HT2B-receptor inactivation in dopamine neurons affects their neuronal activity and increases AMPA-mediated over NMDA-mediated excitatory synaptic currents. These changes are associated with lower ventral striatum dopamine activity and blunted cocaine self-administration. These data identify the 5-HT2B receptor as a pharmacological intermediate and provide mechanistic insight into attenuated dopamine tone following exposure to drugs of abuse.SIGNIFICANCE STATEMENT Here we report that mice lacking 5-HT2B receptors totally or exclusively in dopamine neurons exhibit heightened cocaine-induced locomotor responses. Despite the sensitized state of these mice, we found that associated changes include lower ventral striatum dopamine activity and lower cocaine operant self-administration. We described the selective expression of 5-HT2B receptors in a subpopulation of dopamine neurons sending axons to the ventral striatum. Increased bursting in vivo properties of these dopamine neurons and a concomitant increase in AMPA synaptic transmission to ex vivo dopamine neurons were found in mice lacking 5-HT2B receptors. These data support the idea that the chronic 5-HT2B-receptor inhibition makes mice behave like animals already exposed to cocaine with higher cocaine-induced locomotion associated with changes in dopamine neuron reactivity. This work was supported by the Ministerio de Economía y Competitividad (#SAF2014-9648-P to R.M., MINECO/FEDER, UE); the Instituto de Salud Carlos III (RD06/0001/0001 to R.M.; PI14/00210 to P.R.), the FEDER funds; the Generalitat de Catalunya ICREA (Institució Catalana de Recerca i Estudis Avançats) Academia (2015) to R.M. The studies on Htr2b-/- mice were supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Université Pierre et Marie Curie, and by grants from the Fondation pour la Recherche sur le Cerveau, the Fondation de France, the Fondation pour la Recherche Médicale "Equipe FRM DEQ2014039529", the French Ministry of Research (Agence Nationale pour la Recherche ANR-12-BSV1-0015-01 and the Investissements d'Avenir programme ANR-11-IDEX-0004-02). S.D. was supported by a Lefoulon-Lalande fellowship and E.Q. by a PhD fellowship from the Region Ile de France DIM Cerveau et Pensée. L.M.'s team is part of the École des Neurosciences de Paris Ile-de-France network and of the Bio-Psy Labex.

Summary: There is a growing consensus that Alzheimer's disease (AD) involves failure of the homeostatic machinery, which underlies the firing stability of neural circuits. What are the culprits leading to neuron firing instability? The amyloid precursor protein (APP) is central to AD pathogenesis, and we recently showed that its intracellular domain (AICD) could modify synaptic signal integration. We now hypothesize that AICD modifies neuron firing activity, thus contributing to the disruption of memory processes. Using cellular, electrophysiological, and behavioral techniques, we show that pathological AICD levels weaken CA1 neuron firing activity through a gene-transcription-dependent mechanism. Furthermore, increased AICD production in hippocampal neurons modifies oscillatory activity, specifically in the γ-frequency range, and disrupts spatial memory task. Collectively, our data suggest that AICD pathological levels, observed in AD mouse models and in human patients, might contribute to progressive neuron homeostatic failure, driving the shift from normal aging to AD. : Pousinha et al. report that increased AICD levels, a peptide representing amyloid precursor protein (APP) C-terminal domain, render CA1 pyramidal neurons hypoexcitable by altering Ca+ and K+ channel conductances. Modeling this alteration predicts decreased gamma oscillations. They show that increasing AICD levels in vivo weakens gamma-oscillation-dependent spatial memory. Keywords: AICD, APP, Alzheimer’s disease, neuron firing, brain oscillations, memory encoding

Defeat, Distress, and Glucocorticoids Understanding how individuals control emotions and cope with stressful events is a major clinical concern and of importance for the treatment of psychiatric illnesses (see the Perspective by McEwen ). Barik et al. (p. 332 ) discovered that aggressive defeat stress in mice caused glucocortioid release and increased activity in the dopamine system. Deleting the glucocorticoid receptors in dopaminoceptive neurons completely prevented the social avoidance that usually follows aggressive defeat. How the combination of genetic factors and environmental stressors during adolescence determines adult behavior and how their disturbance results in neuropsychiatric disorders is poorly understood. Niwa et al. (p. 335 ) found that isolation stress during adolescence, which does not cause any long-lasting changes in wild-type mice, induced significant neurochemical and behavioral alterations in mutant mice expressing a dominant-negative variant of the disrupted in schizophrenia 1 gene under the control of the prion protein promoter. These deficits could be reversed by a glucocorticoid receptor antagonist.

International audience; Serotonergic neurons of the raphe nuclei exhibit anatomical, neurochemical and elecrophysiological heterogeneity that likely underpins their specific role in multiple behaviors. However, the precise organization of serotonin (5-HT) neurons to orchestrate 5-HT release patterns throughout the brain is not well understood. We compared the electrophysiological and neurochemical properties of dorsal and median raphe 5-HT neurons projecting to the medial prefrontal cortex (mPFC), amygdala (BLA) and dorsal hippocampus (dHP), combining retrograde tract tracing with brain slice electrophysiology and single-cell RT-PCR in Pet1-EGFP mice. Our results show that 5-HT neurons projecting to the dHP and the mPFC and the BLA form largely non-overlapping populations and that BLA-projecting neurons have characteristic excitability and membrane properties. In addition, using an unbiased clustering method that correlates anatomical, molecular and electrophysiological phenotypes, we find that 5-HT neurons with projections to the mPFC and the dHP segregate from those projecting to the BLA. Single-cell gene profiling showed a restricted expression of the peptide galanin in the population of 5-HT neurons projecting to the mPFC. Finally, cluster analysis allowed identifying an atypical subtype of 5-HT neuron with low excitability, long firing delays and preferential expression of the vesicular glutamate transporter type 3. Overall, these findings allow to define correlated anatomical and physiological identities of serotonin raphe neurons that help understanding how discrete raphe cells subpopulations account for the heterogeneous activities of the midbrain serotonergic system.

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 GABAA 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 GABAA 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 GABAA 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 GABAA 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 GABAA receptors. Flavan-3-ol derivatives are potential ligands for etomidate/loreclezole-related binding sites at GABAA receptors and the low-affinity effects of diazepam are mediated via the same site.

Emotional disorders such as depression, panic attacks, generalized anxiety, phobias and post-traumatic stress have been associated to decreased serotonin (5-HT) function, based on the positive effects of treatments that enhance 5-HT neurotransmission. However, it has been difficult to establish a primary role for 5-HT deficiency in these diseases, making preclinical models particularly useful. Over the last ten years a variety of genetic mouse models of 5-HT depletion have been produced, complementing previous pharmacologically-based models. Initial models hindered the differentiation of the raphe 5-HT neurons, while more recently produced models suppressed 5-HT production or incapacitated 5-HT vesicular packaging and release in normally developed raphe neurons. Here, we provide an overview of 11 genetic mouse models with lowered 5-HT transmission and summarize the available behavioural investigations concerning their anxiety and depression phenotypes. Although these studies are still ongoing, some common anxiety-related traits and behavioural phenotypes have emerged. Most studies have reported decreased innate anxiety to novelty but heightened fear responses to conditioned aversive cues. This complex phenotype is in general agreement with the proposed dual function of 5-HT in modulating different defensive behaviours. Surprisingly, the depressive-like behaviours have been less studied and, so far, did not yield a consistent phenotype in standard tests. Future studies should be conducted using more ethological relevant models to conclude on the causal role of 5-HT depletion in depression. This review also describes the differences in level and regional distribution of 5-HT depletion among the available mouse models, which could contribute to the diverse phenotypes observed. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Heterogeneity of central serotonin (5-HT) raphe neurons is suggested by numerous lines of evidence, but its genetic basis remains elusive. The transcription factor Pet1 is required for the acquisition of serotonergic identity in a majority of neurons in the raphe nuclei. Nevertheless, a subset of 5-HT neurons differentiates in Pet1 knock-out mice. We show here that these residual 5-HT neurons outline a unique subpopulation of raphe neurons with highly selective anatomical targets and characteristic synaptic differentiations. In Pet1 knock-out mice, 5-HT innervation strikingly outlines the brain areas involved in stress responses with dense innervation to the basolateral amygdala, the paraventricular nucleus of the hypothalamus, and the intralaminar thalamic nuclei. In these regions, 5-HT terminals establish asymmetric synaptic junctions. This target selectivity could not be related to altered growth of the remaining 5-HT neurons, as indicated by axon tracing and cell culture analyses. The residual 5-HT axon terminals are functional with maintained release propertiesin vitroandin vivo. The functional consequence of this uneven distribution of 5-HT innervation on behavior was characterized. Pet1 knock-out mice showed decreased anxiety behavior in novelty exploration and increased fear responses to conditioned aversive cues. Overall, our findings lead us to propose the existence of Pet1-dependent and Pet1-resistant 5-HT neurons targeting different brain centers that might delineate the anatomical basis for a dual serotonergic control on stress responses.

The consumption of flavonoid-rich foods, in particular fruits and vegetables, has been epidemiologically associated with a reduced risk of heart disease, neurodegenerative disease, cancer and other chronic diseases. Flavonoid glycosides, the main class of flavonoids, have been shown to exert CNS-mediated activities, particularly as sedative-hypnotics, analgesics or both, nevertheless no studies have evaluated these agents in anxiety. This study assessed the potential anxiolytic effect of three flavonoid glycosides, myrcitrin, naringin and gossypin, in the elevated plus maze test (EPM). Myricitrin (1 mg/kg) was effective on the EPM showing a clear anxiolytic effect with no signs of sedation. However, higher doses showed possible sedative and myorelaxation effects. Gossypin and naringin both shared a similar profile, with low doses (1 mg/kg) inducing a robust anxiolytic effect which diminished with increasing doses of the flavonoids. Higher doses of these two flavonoids showed a dramatic increase in the open arm exploration accompanied by a decrease in locomotor activity. Hence, naringin (30 mg/kg) and gossypin (30 mg/kg) induce both anxiolytic and sedative effects. These results suggest that flavonoid glycosides have the potential to exert a range of CNS-mediated biological activities.

The pharmacological effects on the central nervous system (CNS) of a range of available flavonoid glycosides were explored and compared to those of the glycosides 2S-hesperidin and linarin, recently isolated from valeriana. The glycosides 2S-neohesperidin, 2S-naringin, diosmin, gossipyn and rutin exerted a depressant action on the CNS of mice following i.p. injection, similar to that found with 2S-hesperidin and linarin. We demonstrate in this work that these behavioural actions, as measured in the hole board, thiopental induced sleeping time and locomotor activity tests, are unlikely to involve a direct action on gamma-aminobutyric acid type A (GABA(A)) receptors. The corresponding aglycones were inactive, pointing to the importance of the sugar moieties in the glycosides in their CNS depressant action following systemic administration. The pharmacological properties of the flavonoid glycosides studied here, in addition to our previous results with hesperidin and linarin, opens a promising new avenue of research in the field.

The aim of the present work is to demonstrate the putative sedative and anxiolytic-like effects of a hydro-ethanolic extract obtained from the aerial parts of Aloysia polystachya (Verbenaceae) in male mice using several behavioural assays. Groups of male mice orally treated with doses of 1.0, 10.0 and 100.0 mg/kg of the extract did not show any significant alteration of their locomotor activity, body temperature or motor coordination. The same treatment increased the duration of the sleeping time induced by 30.0 mg/kg i.p. of sodium pentobarbital. However, the sleeping time induced by ethyl ether was not modified by the oral administration of the extract, not confirming the putative sedative effect of the plant. The ethanolic extract also significantly increased the percentage of both entries (1.0 and 100.0 mg/kg) and the time spent (10.0 and 100.0 mg/kg) into the open arms of the elevated plus maze (EPM). Nevertheless, the binding of 3H-flunitrazepam (3H-FNZ) to the benzodiazepine binding site (BDZ-bs), in washed crude synaptosomal membranes from rat cerebral cortex, was not affected by the semi-purified components from Aloysia polystachya. These results indicate an anxiolytic-like profile of action for the extract of Aloysia polystachya without sedative side effect, being this activity probably mediated by other mechanism than BDZ-bs modulation at the GABAA receptors.

We have recently reported the presence of the anxiolytic flavone 6-methylapigenin (MA) and of the sedative and sleep-enhancing flavanone glycoside 2S (−) hesperidin (HN) in Valeriana officinalis and Valeriana wallichii . MA, in turn, was able to potentiate the sleep-inducing properties of HN. The present paper reports the identification in V. officinalis of the flavone glycoside linarin (LN) and the discovery that it has, like HN, sedative and sleep-enhancing properties that are potentiated by simultaneous administration of valerenic acid (VA). These effects should be taken into account when considering the pharmacological actions of valeriana extracts.

Valerian is an ancient tranquillizing drug obtained from the underground organs of several Valeriana species. Its active principles were assumed to be terpenoids in the form of valepotriates and/or as components of the essential oil. However, unknown active compounds were not discarded and synergic effects were suspected. We have recently isolated 6-methylapigenin (MA) from Valeriana wallichii and proved that it is a benzodiazepine binding site (BDZ-bs) ligand [Planta Med. 68 (2002) 934]. The present paper is the first report of the presence of 2S(−)-hesperidin in valeriana and describes that it has sedative and sleep-enhancing properties. MA, in turn, was found to have anxiolytic properties and was able to potentiate the sleep-enhancing properties of hesperidin (HN). MA and HN are new members of the growing family of natural flavonoids with activity on the CNS, and their properties suggest that they are promising drug leads in the field.

International audience; 5-HT neurons are topographically organized in the hindbrain, and have been implicated in the etiology and treatment of psychiatric diseases such as depression and anxiety. Early studies suggested that the raphe 5-HT neurons were a homogeneous population showing similar electrical properties, and feedback inhibition mediated by 5-HT1A autoreceptors. We utilized histochemistry techniques in ePet1-eGFP and 5-HT1A-iCre/R26R mice to show that a subpopulation of 5-HT neurons do not express the somatodendritic 5-HT1A autoreceptor mRNA. In addition, we performed patch-clamp recordings followed by single-cell PCR in ePet1-eGFP mice. From 134 recorded 5-HT neurons located in the dorsal, lateral, and median raphe, we found lack of 5-HT1A mRNA expression in 22 cells, evenly distributed across raphe subfields. We compared the cellular characteristics of these neuronal types and found no difference in passive membrane properties and general excitability. However, when injected with large depolarizing current, 5-HT1A-negative neurons fired more action potentials, suggesting a lack of autoinhibitory action of local 5-HT release. Our results support the hypothesis that the 5-HT system is composed of subpopulations of serotonergic neurons with different capacity for adaptation.

International audience; The therapeutic effects induced by serotonin-selective reuptake inhibitor (SSRI) antidepressants are initially triggered by blocking the serotonin transporter and rely on long-term adaptations of pre- and post-synaptic receptors. We show here that long-term behavioral and neurogenic SSRI effects are abolished after either genetic or pharmacological inactivation of 5-HT(2B) receptors. Conversely, direct agonist stimulation of 5-HT(2B) receptors induces an SSRI-like response in behavioral and neurogenic assays. Moreover, the observation that (i) this receptor is expressed by raphe serotonergic neurons, (ii) the SSRI-induced increase in hippocampal extracellular serotonin concentration is strongly reduced in the absence of functional 5-HT(2B) receptors and (iii) a selective 5-HT(2B) agonist mimics SSRI responses, supports a positive regulation of serotonergic neurons by 5-HT(2B) receptors. The 5-HT(2B) receptor appears, therefore, to positively modulate serotonergic activity and to be required for the therapeutic actions of SSRIs. Consequently, the 5-HT(2B) receptor should be considered as a new tractable target in the combat against depression.

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.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.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.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.

We herein describe the synthesis and positive modulatory activities of a small library of flavan-3-ol derivatives on alpha(1)beta(2)gamma(2L) GABA(A) receptors. Structure-activity relationships of various substituents on the A, B and C rings were evaluated in a functional electrophysiological assay. A trans configuration and a 3-acetoxy moiety are essential for activity. Substitution of the B ring appears to be well tolerated, with substituents on the A ring playing a major role in determining activity.

This article profiles ten examples of chemicals derivedfrom herbal preparations that influence ionotropicreceptors for the brain’s major inhibitory neurotrans-mitter g-aminobutyric acid (GABA). Many herbalmedicines are used to influence brain function inordertotreatanxiety,cognitivedisorders, andinsom-nia. Such disorders are considered likely to involveGABA receptors, particularly ionotropic receptorsthat are responsible for fast synaptic transmissionvia ion channels. The evidence of efficacy, or other-wise, in the use of herbal medicines to treat centralnervous system (CNS) disorders remains to be estab-lished unequivocally in many cases. There is nodoubt, however, that these products contain chemi-cals that in their pure form can be shown to influencebrain function in specific ways. The structural diver-sity of these chemicals provides valuable leads for thedevelopment of new therapeutic agents and toolswith which to investigate nervous system function.Herbal medicines are usually complex mixtures ofchemicals. The combined actions of these chemicalsmay contribute to the overall effects of the medicines.Thus, it is important to study interactions among theindividualchemicals.WithrespecttoionotropicGABAreceptors,thereareexamplesofsynergisticinteractionsbetweentwochemicalsandexamplesofwhathasbeentermed second-order modulation, in which a secondmodulator enhances the action of a first-order modu-lator.Thismayrepresentanewformofdrugactionandlead to the identification of new drug targets. Investi-gating the complex ways in which these chemicalsinteract could lead to a better understanding of thefunction of ionotropic GABA receptors.GABA itself is an important plant constituent, andthus it should not be surprising that plants contain arange of other chemicals that can influence GABAfunction. In addition, there are examples of plant-derived GABA mediating some form of communica-tionamonganimals,bacteria,fungi,andplants.Thus,GABA may represent a conserved and ubiquitousbiological signaling molecule.Quality control of herbal medicines is a significantproblem. Identification of key active ingredients inherbal medicines and the interactions among themmay provide the means to better quality control usingfunctional assays in addition to the currently usedchemical assays. Many of the advances in studies ofherbal products and GABA receptors have come fromligand binding studies, for example, from the bindingof benzodiazepines to brain membranes and, morerecently, from functional studies in which GABAreceptorsofknownsubunitcompositionareexpressedincellsthatdonotusuallyexpresssuchreceptors(e.g.,frog oocytes).

The aim of this work was to evaluate if the intraperitoneal administration of the natural compound hesperidin, in a sedative dose, and neo-hesperidin, a hesperidin structural analog that exerts minor sedative effect, were able to induce changes in intracellular signaling cascades in different areas of the brain. The systemic administration of hesperidin produced a marked reduction in the phosphorylation state of extracellular signal-regulated kinases 1/2 (ERK 1/2), but not of Ca(+2)/calmodulin-dependent protein kinase II alpha subunit (alphaCaMKII), in the cerebral cortex, cerebellum and hippocampus. In contrast, neo-hesperidin did not markedly affect the activity of ERK 1/2 in both the cortex and the cerebellum. Taken together, these results demonstrated that intracellular signalling involving a selective decrease in ERK1/2 activation accompanied the depressant action of hesperidin. Even more, the low sedative action of neo-hesperidin correlates with a negligible decrease in phosphorylation state of ERK 1/2 (pERK 1/2), suggesting that low levels of pERK 1/2 in CNS could be a marker of sedative efficacy of flavonoids.

Recent genetic and pharmacological studies have demonstrated that alpha(2)-containing GABA(A) receptors mediate the anxiolytic effects of benzodiazepines, setting a new strategy in developing novel, non-sedative anxiolytic agents. In this study we show that stereoisomers of 3-acetoxy-4'-methoxyflavan are positive modulators of recombinant alpha(1,2,3,5)beta(2)gamma(2L) and alpha(1)beta(2) GABA(A) receptors expressed in Xenopus laevis oocytes. GABA(C) receptors are insensitive to modulation by these compounds. In each case, the enhancement was evident at low micromolar concentrations and occurred independently of the classical high affinity benzodiazepine site, as it could not be blocked by the antagonist flumazenil. Importantly, the compound Fa131 was significantly more efficacious at enhancing GABA-induced currents (EC(5)) at alpha(2)beta(2)gamma(2L) receptors compared to alpha(1)beta(2)gamma(2L), alpha(3)beta(2)gamma(2L) and alpha(5)beta(2)gamma(2L) receptors (E(max)=21.0+/-1.7 times, compared to 8.5+/-0.7 times at alpha(1)-, 9.5+/-0.6 times at alpha(3)- and 5.2+/-0.4 times at alpha(5)-contaning GABA(A) receptors), suggesting a potential use as an anxiolytic. In mice, this agent (1-30mg/kg i.p.) induced anxiolytic-like action in two unconditioned models of anxiety: the elevated plus maze and the light/dark paradigms. No sedative or myorelaxant effects were detected using the hole board, actimeter and horizontal wire tests, and only weak barbiturate-potentiating effects on the loss of righting reflex test. Fa131 demonstrated improved segregation of anxiolytic and sedative doses when compared to the non-selective agonist diazepam. Finally, flavan derivatives highlight the potential of targeting non-benzodiazepine allosteric sites in the search for new anxioselective drugs.

It has been recently reported the presence in Valeriana of the flavone 6-methylapigenin and the flavanone glycoside hesperidin. The apigenin derivative is a ligand for the benzodiazepine binding site in the gamma-aminobutyric acid receptor type A (GABA(A)) and has anxiolytic properties. Hesperidin has sedative and sleep-enhancing properties but is not a ligand for the benzodiazepine binding site. 6-Methylapigenin is able to potentiate the sleep-enhancing effect of hesperidin. In this work we demonstrate that this property is shared with various GABA(A) receptor ligands, among them the agonist diazepam, which was used to study the potentiation as measured in the hole board test. Isobolar analysis of the results showed the interaction being synergistic. We discarded pharmacokinetic effects or a direct action of hesperidin on the benzodiazepine binding site. A possible use of hesperidin properties to decrease the effective therapeutic doses of benzodiazepines is suggested.