Oxytetracycline induces anxiety-like behavior in adult zebrafish.

Oxytetracycline (OTC) is one of the broad-spectrum antibiotics widely used for the treatment of fish-farm infection. Considering that behavior is directly related to reproduction, individual fitness, and survival, it is important to evaluate the impact of antibiotics on the behavioral repertoire in fish. Zebrafish (Danio rerio) presents a well-described behavioral repertoire to reliably demonstrate complex responses to chemical compound exposure. This work aims to identify the role of OTC in comprehensive behavioral parameters and whole-body cortisol levels in adult zebrafish. Here we report that OTC exposure (10, 20, and 100 mg/L) induces an anxiogenic-like phenotype in the novel tank test. OTC exposure also changes the behavior of social interaction with a shoal of unknown zebrafish - characterized as a stimulus group. Zebrafish exposed to OTC (10 mg/L) remains a longer period in the stimulus zone when compared to the control group. Clonazepam (0.006 mg/L) was able to reverse anxiogenic-like behavior and the changes in social behavior induced by OTC. We also demonstrated that cortisol levels were significantly decreased after exposure to OTC (10, 20, and 100 mg/L), which were not reversed by clonazepam. These findings highlight the growing utility of zebrafish as a model to understand the impact of antibiotics on behavior and their underlying mechanisms.

Anxiolytic-like effect of brominated compounds from the marine sponge Aplysina fulva on adult zebrafish (Danio rerio): Involvement of the GABAergic system.

Benzodiazepines are commonly used to treat disorders of the central nervous system, including anxiety. However, due to their adverse effects, there is a continuing interest in discovering new safe and effective drugs. Marine natural products have emerged as a prolific source of bioactive nitrogenated compounds. Aiming to discover new biologically active natural compounds, the marine sponge Aplysina fulva, a nitrogen-bearing heterocyst producer, was investigated. The main isolated compounds (4, 6, and 9) were evaluated on adult zebrafish (Danio rerio). A group of fishes (n = 6) was preliminarily subjected to acute toxicity, and open field tests using 0.1, 0.5, and 1.0 mg/mL (v. o.) of those compounds was performed. The anxiolytic effect was further investigated in the light/dark assay based on the locomotor response at zebrafish. Interactions through the GABAergic system were investigated using flumazenil, a silent modulator of GABA receptors. To improve the results, a study of molecular docking using the GABAA receptor also was performed. Based on the results, the bromotyrosine derivative compounds 4, 6, and 9 exhibited anxiolytic-like effects mediated by the GABAergic system.

How does α1Histidine102 affect the binding of modulators to α1ß2γ2 GABAA receptors? molecular insights from in silico experiments.

The activation of GABAA receptors by the neurotransmitter gamma-aminobutyric acid mediates the rapid inhibition response in the central nervous system of mammals. Many neurological and mental health disorders arise from alterations in the structure or function of these pentameric ion channels. GABAA receptors are targets for numerous drugs, including benzodiazepines, which bind to α1ß2γ2 GABAA receptors with high affinity to a site in the extracellular domain, between subunits α1 and γ2. It has been established experimentally that the binding of these drugs depends on the presence of one particular amino acid in the α1 subunit: histidine 102. However, the specific role it plays in the intermolecular interaction has not been elucidated. In this work, we applied in silico methods to understand whether certain protonation and rotamer states of α1His102 facilitate the binding of modulators. We analysed diazepam binding, a benzodiazepine, and the antagonist flumazenil to the GABAA receptor using molecular dynamics simulations and adaptive biasing force simulations. The binding free energy follows changes in the protonation state for both ligands, and rotameric states of α1His102 were specific for the different compounds, suggesting distinct preferences for positive allosteric modulators and antagonists. Moreover, in the presence of diazepam and favoured by a neutral tautomer, we identified a water molecule that links loops A, B, and C and may be relevant to the modulation mechanism.

Electrical stimulation of the posterior insula induces mechanical analgesia in a rodent model of neuropathic pain by modulating GABAergic signaling and activity in the pain circuitry.

The insula has emerged as a critical target for electrical stimulation since it influences pathological pain states. We investigated the effects of repetitive electrical stimulation of the insular cortex (ESI) on mechanical nociception, and general locomotor activity in rats subjected to chronic constriction injury (CCI) of the sciatic nerve. We also studied neuroplastic changes in central pain areas and the involvement of GABAergic signaling on ESI effects. CCI rats had electrodes implanted in the left agranular posterior insular cortex (pIC), and mechanical sensitivity was evaluated before and after one or five daily consecutive ESIs (15 min each, 60 Hz, 210 µs, 1 V). Five ESIs (repetitive ESI) induced sustained mechanical antinociception from the first to the last behavioral assessment without interfering with locomotor activity. A marked increase in Fos immunoreactivity in pIC and a decrease in the anterior and mid-cingulate cortex, periaqueductal gray and hippocampus were noticed after five ESIs. The intrathecal administration of the GABAA receptor antagonist bicuculline methiodide reversed the stimulation-induced antinociception after five ESIs. ESI increased GAD65 levels in pIC but did not interfere with GABA, glutamate or glycine levels. No changes in GFAP immunoreactivity were found in this work. Altogether, the results indicate the efficacy of repetitive ESI for the treatment of experimental neuropathic pain and suggest a potential influence of pIC in regulating pain pathways partially through modulating GABAergic signaling.

Citrus limon (L.) Burm f. Essential Oil Has Anxiolytic and Sedative Properties by Modulating GABAA-Receptors

Abstract The high prevalence of anxiety disorders associated with pharmacotherapy side effects have motivated the search for new pharmacological agents. Species from Citrus genus, such as Citrus limon (sicilian lemon), have been used in folk medicine as a potential therapy to minimize emotional disorders. In order to searching for new effective treatments with fewer side effects, the present study evaluated the anxiolytic mechanism of action and the hypnotic-sedative activity from the Citrus limon fruit's peels essential oil (CLEO). Adults male Swiss mice were submitted to barbiturate-induced sleep test; elevated plus-maze (EPM) and light-dark box (LDB) (evaluation of the mechanism of action); rotarod; and catalepsy tests. CLEO oral treatment decreased latency and increased the sleep total time; moreover it induced in animals an increased the number of entries and percentage of time spent into open arms of the EPM; an increased the number of transitions and the percentage of time into light compartment in the LDB; which were only antagonized by flumazenil pretreatment, with no injury at motor function. Thus, results suggest that CLEO treatment induced an anxiolytic behavior suggestively modulated by the benzodiazepine binding site of the GABAA receptor or by an increase of GABAergic neurotransmission, without cause impairment in the motor coordination.

Efecto antagonista del flumazenil sobre el isoflurano en la emersión de la anestesia general / Antagonist effect of flumazenil on isofluran in reversion of general anesthesia

INTRODUCTION AND OBJECTIVES: Isoflurane, an inhalational general anesthetic widely used in medical practice, belonging to the group of volatile liquids together with desflurane and sevoflurane, with various properties including sedation, hypnosis and anesthesia of patients undergoing treatment. surgical acts. Volatile inhalational anesthetics (halogenated) as mechanism of action, has the property of increasing inhibitory synaptic transmission at postsynaptic level by potentiating ion channels regulated by ligand activated by alpha-aminobutyric acid (GABA). Flumazenil is a benzodiazepine antagonist belonging to the group of imidazobenzodiazepine. It is currently known that there is no specific drug capable of antagonizing the effects of halogenates that allow the rapid and complete recovery of general anesthesia, for this reason this work focuses its efforts on demonstrating whether flumazenil has the ability to reverse the actions of the patient. isoflurane and allow an early restoration of the level of consciousness. MATERIAL AND METHODS: The study to be performed is a clinical type of longitudinal, prospective, unicentric and double blind. The sample will be formed by patients who are going to be subjected to a balanced general anesthesia. The sample will be divided into 2 large groups: group C (control) and group F (Flumazenil). At the end of the surgery, the mixture will be administered according to the selected group in a random manner (Flumazenil 0.25 mg or 0.9% solution in a 20 cc syringe) and the time of extubation, recovery time of the level of consciousness, time of discharge UCPA and hemodynamic state (FC, TAM and SO2). RESULTS: The flumazenil group showed a significantly shorter time from injection to extubation than the placebo group (p = 0.007). Differences in terms of shorter times needed to achieve Aldrete of 9 points in the flumazenil group (P = 0.04) were observed as were shorter anesthetic arousal times represented by a Ramsey 2. Heart rate, mean arterial pressure and saturation they had similar values between the 2 groups. CONCLUSION: The study showed that a single dose of 0.25 mg of flumazenil administered at the end of the surgical act, just after completing all surgical stimulation was beneficial (P = 0.007) in the context of extubation times and shorter anesthetic arousal times.

INTRODUCCIÓN Y OBJETIVOS: El isoflurano un anestésico general inhalatorio usado ampliamente en la práctica médica, perteneciente al grupo de los líquidos volátiles junto con el desflurano y sevoflurano, con variadas propiedades entre las que se encuentran la sedación, hipnosis y anestesia de los pacientes sometidos a actos quirúrgicos. Los anestésicos inhalatorios volátiles (halogenados) como mecanismo de acción, tiene la propiedad de aumentar la transmisión sináptica inhibidora a nivel postsináptico potenciando los canales iónicos regulados por ligando activados por ácido alfa-aminobutírico (GABA). El flumazenil es un antagonista benzodiazepínico perteneciente al grupo de los imidazobenzodiazepina. Se conoce actualmente que no existe un fármaco específico capaz de antagonizar los efectos de los halogenados que permitan la recuperación rápida y completa de la anestesia general, por tal motivo este trabajo centra sus esfuerzos en demostrar si el flumazenil tiene la capacidad para revertir las acciones del isoflurane y permitir un restablecimiento temprano del nivel de conciencia. MATERIALES Y MÉTODOS: El estudio a realizar es de tipo clínico de corte longitudinal, prospectivo, unicéntrico y doble ciego. La muestra se conformará por pacientes que vayan a ser sometidos a anestesia general balanceada. Se procederá a dividir la muestra en 2 grandes grupos: grupo C (control) y grupo F (flumazenil). Al final de la cirugía se administrará la mezcla según grupo seleccionado de manera al azar (flumazenil 0,25 mg o solución 0,9% en una jeringa de 20 cc) y se valorará el tiempo de extubación, tiempo de recuperación del nivel de conciencia, tiempo de alta de la UCPA y estado hemodinámico (FC, TAM y SO2). RESULTADOS: El grupo de flumazenil presentó un tiempo desde la inyección hasta la extubación significativamente más bajo que el grupo placebo (p = 0,007). Se observaron diferencias en términos de tiempos más bajos necesario para alcanzar Aldrete de 9 puntos en el grupo flumazenil (P = 0,04) al igual que tiempos de despertar anestésico más cortos representados por un Ramsey 2. La frecuencia cardíaca, presión arterial media y la saturación tuvieron valores similares entre los 2 grupos. CONCLUSIÓN: El estudio demostró que una única dosis de 0,25 mg de flumazenil administrado al final del acto quirúrgico, justo después de culminar toda estimulación quirúrgica fue beneficiosa (P = 0,007) en el contexto de tiempos de extubación y tiempos de despertar anestésico más cortos.

GABAA receptors modulate sympathetic vasomotor outflow and the pressor response to skeletal muscle metaboreflex activation in humans.

KEY POINTS: The activation of the group III/IV skeletal muscle afferents is one of the principal mediators of cardiovascular responses to exercise; however, the neuronal circuitry mechanisms that are involved during the activation of group III/IV muscle afferents in humans remain unknown. Recently, we showed that GABAergic mechanisms are involved in the cardiac vagal withdrawal during the activation of mechanically sensitive (predominantly mediated by group III fibres) skeletal muscle afferents in humans. In the present study, we found that increases in muscle sympathetic nerve activity and mean blood pressure during isometric handgrip exercise and postexercise ischaemia were significantly greater after the oral administration of diazepam, a benzodiazepine that increases GABAA activity, but not after placebo administration in young healthy subjects. These findings indicate for the first time that GABAA receptors modulate sympathetic vasomotor outflow and the pressor responses to activation of metabolically sensitive (predominantly mediated by group IV fibres) skeletal muscle afferents in humans. ABSTRACT: Animal studies have indicated that GABAA receptors are involved in the neuronal circuitry of the group III/IV skeletal muscle afferent activation-induced neurocardiovascular responses to exercise. In the present study, we aimed to determine whether GABAA receptors modulate the neurocardiovascular responses to activation of metabolically sensitive (predominantly mediated by group IV fibres) skeletal muscle afferents in humans. In a randomized, double-blinded, placebo-controlled and cross-over design, 17 healthy subjects (eight women) performed 2 min of ischaemic isometric handgrip exercise at 30% of the maximal voluntary contraction followed by 2 min of postexercise ischaemia (PEI). Muscle sympathetic nerve activity (MSNA), blood pressure (BP) and heart rate (HR) were continuously measured and trials were conducted before and 60 min after the oral administration of either placebo or diazepam (10 mg), a benzodiazepine that enhances GABAA activity. At rest, MSNA was reduced, whereas HR and BP did not change after diazepam administration. During ischaemic isometric handgrip, greater MSNA (pre: ∆13 ± 9 bursts min-1 vs. post: ∆29 ± 15 bursts min-1 , P < 0.001), HR (pre: ∆23 ± 11 beats min-1 vs. post: ∆31 ± 17 beats min-1 , P < 0.01) and mean BP (pre: ∆33 ± 12 mmHg vs. post: ∆37 ± 12 mmHg, P < 0.01) responses were observed after diazepam. During PEI, MSNA and mean BP remained elevated from baseline before diazepam (∆10 ± 8 bursts min-1 and ∆25 ± 14 mmHg, respectively) and these elevations were increased after diazepam (∆17 ± 12 bursts min-1 and ∆28 ± 13 mmHg, respectively) (P ≤ 0.05). Importantly, placebo pill had no effect on neural, cardiac and pressor responses. These findings demonstrate for the first time that GABAA receptors modulate MSNA and the pressor responses to skeletal muscle metaboreflex activation in humans.

Ginkgo biloba treatments reverse the impairment of conditioned suppression acquisition induced by GluN2B-NMDA and 5-HT1A receptor blockade: Modulatory effects of the circuitry of the dorsal hippocampal formation.

To improve our understanding of the effects of standardized extract of Ginkgo biloba (EGb) as a cognitive enhancer, we investigated the conditioned lick suppression-induced expression (mRNA and protein) of the GluN2B-containing N-methyl-D-aspartic acid receptor (GluN2B-NMDAR), serotonin (5-HT) 1A receptor (5-HT1AR), gamma-aminobutyric acid type A receptor (GABAAR) and glial fibrillary acidic protein (GFAP) in the dorsal hippocampal formation (dHF) of untreated and EGb-treated (0.25, 0.5 and 1.0 g.kg-1) groups of rats. To substantiate our data, we analysed the molecular changes in dHF following treatment with vehicle, with agonists or antagonists of GABAAR, GluN2B-NMDAR and 5-HT1AR or with one of these antagonists prior to EGb and fear memory acquisition. Additionally, we performed a pharmacological analysis of the drug-receptor-receptor interactions and their supplemental role in fear memory by blocking individual receptors and analysed the possible changes in expression level with each of the other receptors in the study as well as astrocytes. Our data show for the first time that EGb treatment not only upregulated GluN2B, GABAAR-α5, and GFAP compared with the control but also differentially upregulated GABAAR-α1 in the dHF and 5HT1AR in the CA3. We found that the activation of GABAARs (diazepam) and the inactivation of GluN2B-NMDARs (Ro25-6981) or 5-HT1AR ((S)-WAY100135) resulted in memory impairment. Further, higher doses of EGb treatment reversed the effect of blocking GluN2B (P < 0.001) and 5-HT1AR (P < 0.001). Here, treatment with Ro25-6981 + EGb or (S)-WAY100135 + EGb prevented the impairment of the acquisition of lick suppression in association with the upregulation or prevention of the downregulation of Grin2b expression as well as the expression of GluN2B-NMDA and/or α1 and α5 subunit-containing GABAAR in the CA1 (P < 0.0001). Our data are in line with previous findings concerning the necessity of GluN2B for fear memory formation and add to the current knowledge of the role of the GABAAR-α1 and -α5 subunits and of GluN2B as a target of cognitive enhancers. Furthermore, our data show that these receptors play a complementary role in controlling the neural circuitry in the dHF that seems to be essential to conditioned lick suppression and the modulatory effects of EGb.

Diazepam blocks 50 kHz ultrasonic vocalizations and stereotypies but not the increase in locomotor activity induced in rats by amphetamine.

RATIONALE: We have recently shown that the benzodiazepine diazepam inhibits dopamine release in the NAc and blocks the increased release of dopamine induced by DL-amphetamine. Rewarding stimuli and many drugs of abuse can induce dopamine release in the nucleus accumbens as well as 50-kHz ultrasonic vocalizations (USVs) in rats. OBJECTIVES: In the present study, we tested the hypothesis that diazepam can also block the increase in locomotor activity and USVs elicited by amphetamine. METHODS: Fifty-kilohertz USVs, stereotypy, and locomotor behavior were scored in adult male Wistar rats treated with i.p. injections of saline, 3 mg/kg DL-amphetamine, 2 mg/kg diazepam, 0.2 mg/kg haloperidol, or a combination of these drugs. RESULTS: In agreement with previous studies, amphetamine caused significant increases in the number of USV calls, stereotypies, and locomotor activity. The D2 dopamine receptor antagonist haloperidol blocked the effects of amphetamine on USVs, stereotypy, and locomotor activity. Diazepam blocked the effect of amphetamine on USV and stereotypy, but not on horizontal locomotion. CONCLUSIONS: These results suggest that diazepam blocks the rewarding effect of amphetamine. This finding is promising for basic research regarding treatments of substance use disorders and evaluation of the impact of benzodiazepines on motivation.

Is flumazenil an alternative for the treatment of hepatic encephalopathy? / ¿Es flumazenil una alternativa para el tratamiento de la encefalopatía hepática?

INTRODUCTION: Flumazenil is an antagonist of the GABA/benzodiazepines receptor complex that might play a role in the treatment of hepatic encephalopathy. However, its efficacy and safety are a matter of debate. METHODS: To answer this question we used Epistemonikos, the largest database of systematic reviews in health, which is maintained by screening multiple information sources, including MEDLINE, EMBASE, Cochrane, among others. We extracted data from the systematic reviews, reanalyzed data of primary studies, conducted a meta-analysis and generated a summary of findings table using the GRADE approach. RESULTS AND CONCLUSIONS: We identified two systematic reviews including fourteen randomized trials. We concluded flumazenil does not reduce mortality in patients with hepatic encephalopathy and it is not clear whether it leads to any clinical improvement because the certainty of the evidence is very low.

INTRODUCCIÓN: El flumazenil es un antagonista del complejo receptor GABA/benzodiacepinas que podría tener un rol en el manejo de la encefalopatía hepática. Sin embargo, existe controversia sobre su eficacia y seguridad. MÉTODOS: Para responder esta pregunta utilizamos Epistemonikos, la mayor base de datos de revisiones sistemáticas en salud, la cual es mantenida mediante búsquedas en múltiples fuentes de información, incluyendo MEDLINE, EMBASE, Cochrane, entre otras. Extrajimos los datos desde las revisiones identificadas, reanalizamos los datos de los estudios primarios, realizamos un metanálisis y preparamos tablas de resumen de los resultados utilizando el método GRADE. RESULTADOS Y CONCLUSIONES: Identificamos dos revisiones sistemáticas que en conjunto incluyen catorce estudios aleatorizados. Concluimos que el uso de flumazenil no disminuye la mortalidad en pacientes con encefalopatía hepática, y no está claro si produce alguna mejoría clínica porque la certeza de la evidencia es muy baja.

The influence of midazolam on heart rate arises from cardiac autonomic tones alterations in Burmese pythons, Python molurus.

The GABAA receptor agonist midazolam is a compound widely used as a tranquilizer and sedative in mammals and reptiles. It is already known that this benzodiazepine produces small to intermediate heart rate (HR) alterations in mammals, however, its influence on reptiles' HR remains unexplored. Thus, the present study sought to verify the effects of midazolam on HR and cardiac modulation in the snake Python molurus. To do so, the snakes' HR, cardiac autonomic tones, and HR variability were evaluated during four different experimental stages. The first stage consisted on the data acquisition of animals under untreated conditions, in which were then administered atropine (2.5mgkg-1; intraperitoneal), followed later by propranolol (3.5mgkg-1; intraperitoneal) (cardiac double autonomic blockade). The second stage focused on the data acquisition of animals under midazolam effect (1.0mgkg-1; intramuscular), which passed through the same autonomic blockade protocol of the first stage. The third and fourth stages consisted of the same protocol of stages one and two, respectively, with the exception that atropine and propranolol injections were reversed. By comparing the HR of animals that received midazolam (second and fourth stages) with those that did not (first and third stages), it could be observed that this benzodiazepine reduced the snakes' HR by ~60%. The calculated autonomic tones showed that such cardiac depression was elicited by an ~80% decrease in cardiac adrenergic tone and an ~620% increase in cardiac cholinergic tone - a finding that was further supported by the results of HR variability analysis.

A diagnostic and therapeutic approach to primary burning mouth syndrome.

Primary burning mouth syndrome (BMS) is an oral mucosal disorder that is characterized by a chronic and often debilitating intraoral burning sensation for which no localized or systemic cause can be found. BMS most commonly affects postmenopausal women. The pathophysiology of primary BMS is not well understood. Diagnosing BMS can prove to be challenging. BMS patients can also pose a therapeutic challenge to clinicians who are consulted to evaluate these patients. Most commonly used therapies include tricyclic antidepressants, α-lipoic acid, clonazepam, and cognitive-behavioral therapy. Clinical judgment, patient counseling, and monitoring of pain are important. Further research is required to assess the effectiveness of serotonin and newer serotonin-noradrenalin reuptake inhibitors.

GABAergic signaling within the Basolateral Amygdala Complex modulates resistance to the labilization/reconsolidation process.

It is well known that stress can affect mnemonic processes. In particular, stress before contextual fear conditioning induces a memory which exhibits resistance to being interfered with by Midazolam (MDZ) when applied after memory retrieval. Moreover, stress exposure strongly affects GABAergic transmission within the Basolateral Amygdala Complex (BLA), a brain structure critically involved in fear memory processing. The present study evaluated the involvement of GABAergic signaling within the BLA on the induction of resistance to memory reconsolidation interference. Results showed that MDZ administered intra-BLA before stress prevented the induction of resistance to the interfering effect of systemic administration of both MDZ and Propranolol on fear memory reconsolidation, when both applied after memory retrieval. The blockade of amygdala GABA-A receptors by the antagonist Bicuculline (BIC) before memory encoding induced resistance to interference by post-recall MDZ administration, similarly to that observed with stress exposure. Additionally, the systemic administration of d-cycloserine, a positive allosteric modulator of NMDA receptor, reverted the BIC-induced resistance to the MDZ interfering effect, in the same manner as that reported with stress-induced resistance. In summary, these results suggest that the GABAergic signaling in the BLA at the moment of memory encoding is determinant for the induction of fear memory resistance to the onset of the labilization/reconsolidation process.

(+)-Dehydrofukinone modulates membrane potential and delays seizure onset by GABAa receptor-mediated mechanism in mice.

(+)-Dehydrofukinone (DHF), isolated from Nectandra grandiflora (Lauraceae) essential oil, induces sedation and anesthesia by modulation of GABAa receptors. However, no study has addressed whether DHF modulates other cellular events involved in the control of cellular excitability, such as seizure behavior. Therefore, the aim of the present study was to investigate the effect of DHF on cellular excitability and seizure behavior in mice. For this purpose, we used isolated nerve terminals (synaptosomes) to examine the effect of DHF on the plasma membrane potential, the involvement of GABAa receptors and the downstream activation of Ca2+ mobilization. Finally, we performed an in vivo assay in order to verify whether DHF could impact on seizures induced by pentylenetetrazole (PTZ) in mice. The results showed that DHF induced a GABA-dependent sustained hyperpolarization, sensitive to flumazenil and absent in low-[Cl-] medium. Additionally, (1-100µM) DHF decreased KCl-evoked calcium mobilization over time in a concentration-dependent manner and this effect was prevented by flumazenil. DHF increased the latency to myoclonic jerks (10mg/kg), delayed the onset of generalized tonic-clonic seizures (10, 30 and 100mg/kg), and these effects were also blocked by the pretreatment with flumazenil. Our data indicate that DHF has anticonvulsant properties and the molecular target underlying this effect is likely to be the facilitation of GABAergic neuronal inhibition. The present study highlights the therapeutic potential of the natural compound DHF as a suppressor of neuronal excitability.

The role of GABAA in the expression of updated information through the reconsolidation process in humans.

Consolidated memory can be again destabilized by the presentation of a memory cue (reminder) of the previously acquired information. During this process of labilization/restabilization memory traces can be either impaired, strengthened or updated in content. Here, we study if a consolidated memory can be updated by linking one original cue to two different outcomes and whether this process was modulated by the GABAergic system. To aim that, we designed two experiments carried out in three consecutive days. All participants learned a list of non-sense syllable pairs on day 1. On day 2 the new information was introduced after the reminder or no-reminder presentation. Participants were tested on day 3 for the updated or original list (Exp. 1). In Exp. 2 we tested whether this new information was incorporated by an inhibitory process mediated by the GABAergic system. For that, participants retrieved the original information before being taken Clonazepam 0.25mg (GABAA agonist) or Placebo pill. We found that the groups that received the reminder correctly recalled the old and new information. However, the no reminder groups only correctly recalled the original information. Furthermore, when testing occurred in the presence of Clonazepam, the group that received the reminder plus the new information showed an impaired original memory performance compared to the group that received only Clonazepam (without reminder) or the reminder plus Placebo pill. These results show that new information can be added to a reactivated declarative memory in humans by linking one cue to two different outcomes. Furthermore, we shed light on the mechanisms of memory updating being the GABAergic system involved in the modulation of the old and new information expression.

The anxiolytic-like effect of rutin in rats involves GABAA receptors in the basolateral amygdala.

Rutin is a bioflavonoid found in medicinal plants used to reduce anxiety. Evidence is lacking of rutin's anxiolytic-like activity, putative mechanism(s) of action, and neural sites of effects. The basolateral amygdala (BLA) is the main brain region that regulates anxiety, through GABAA/benzodiazepine (BDZ) receptors, which are modulated by flavonoids. Therefore, the main aim of this study was to investigate whether the anxiolytic-like effect of rutin involves GABAA/BDZ receptors in the BLA. Rutin was administered systemically (30-1000 mg/kg, intraperitoneal) or microinjected into the BLA (16 nmol/4 µl, intracerebral), and its effects were assessed in the elevated plus-maze and open-field tests. Diazepam (1 mg/kg, intraperitoneal, or 7 nmol/4 µl, respectively) was used as a positive control. The mechanism of action was studied using flumazenil (BDZ antagonist, 5 mg/kg, intraperitoneal, or 7 nmol/4 µl, intracerebral) or picrotoxin (chloride channel GABAA antagonist, 0.3 nmol/4 µl, intracerebral). Rutin, administered systemically or intra-amygdala, induced anxiolytic-like responses, similar to those of diazepam. The effect of diazepam was completely blocked by flumazenil, which also partly antagonized the effects of systemic rutin. By contrast, flumazenil exerted no effect and picrotoxin had only a partial action when rutin was infused in the BLA. These results suggest that the anxiolytic-like effect of rutin in the BLA involves GABAergic neurotransmission that is not associated with BDZ receptors.

Diazepam Inhibits Electrically Evoked and Tonic Dopamine Release in the Nucleus Accumbens and Reverses the Effect of Amphetamine.

Diazepam is a benzodiazepine receptor agonist with anxiolytic and addictive properties. Although most drugs of abuse increase the level of release of dopamine in the nucleus accumbens, here we show that diazepam not only causes the opposite effect but also prevents amphetamine from enhancing dopamine release. We used 20 min sampling in vivo microdialysis and subsecond fast-scan cyclic voltammetry recordings at carbon-fiber microelectrodes to show that diazepam caused a dose-dependent decrease in the level of tonic and electrically evoked dopamine release in the nucleus accumbens of urethane-anesthetized adult male Swiss mice. In fast-scan cyclic voltammetry assays, dopamine release was evoked by electrical stimulation of the ventral tegmental area. We observed that 2 and 3 mg of diazepam/kg reduced the level of electrically evoked dopamine release, and this effect was reversed by administration of the benzodiazepine receptor antagonist flumazenil in doses of 2.5 and 5 mg/kg, respectively. No significant effects on measures of dopamine re-uptake were observed. Cyclic voltammetry experiments further showed that amphetamine (5 mg/kg, intraperitoneally) caused a significant increase in the level of dopamine release and in the half-life for dopamine re-uptake. Diazepam (2 mg/kg) significantly weakened the effect of amphetamine on dopamine release without affecting dopamine re-uptake. These results suggest that the pharmacological effects of benzodiazepines have a dopaminergic component. In addition, our findings challenge the classic view that all drugs of abuse cause dopamine release in the nucleus accumbens and suggest that benzodiazepines could be useful in the treatment of addiction to other drugs that increase the level of dopamine release, such as cocaine, amphetamines, and nicotine.

The Effect of Flunitrazepam (Rohypnol(®) ) on the Development of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) and its Implications for Forensic Entomology.

This study investigated the potential effects of flunitrazepam (known as "date rape drug") on the developmental cycle of Chrysomya megacephala, an important forensic species, and their possible implications for the calculation of the PMI. A 1050 C. megacephala eggs were divided into five groups with seven replications each. The eggs were placed on artificial diet prepared with four drug concentrations of flunitrazepam (4, 8, 16, and 32 ng/g), besides the control group (prepared with water). Were evaluated the potential effects on development time, weight gain, and mortality during the cycles. The drug had no significant effect on development time or mortality although it did affect the weight of the pupae and adults (Kruskal-Wallis, p < 0.05). The result can be deduced that the determination of the postmortem interval is not affected.

Effect of (+)-dehydrofukinone on GABAA receptors and stress response in fish model.

(+)-Dehydrofukinone (DHF) is a major component of the essential oil of Nectandra grandiflora (Lauraceae), and exerts a depressant effect on the central nervous system of fish. However, the neuronal mechanism underlying DHF action remains unknown. This study aimed to investigate the action of DHF on GABAA receptors using a silver catfish (Rhamdia quelen) model. Additionally, we investigated the effect of DHF exposure on stress-induced cortisol modulation. Chemical identification was performed using gas chromatography-mass spectrometry and purity was evaluated using gas chromatography with a flame ionization detector. To an aquarium, we applied between 2.5 and 50 mg/L DHF diluted in ethanol, in combination with 42.7 mg/L diazepam. DHF within the range of 10-20 mg/L acted collaboratively in combination with diazepam, but the sedative action of DHF was reversed by 3 mg/L flumazenil. Additionally, fish exposed for 24 h to 2.5-20 mg/L DHF showed no side effects and there was sustained sedation during the first 12 h of drug exposure with 10-20 mg/L DHF. DHF pretreatment did not increase plasma cortisol levels in fish subjected to a stress protocol. Moreover, the stress-induced cortisol peak was absent following pretreatment with 20 mg/L DHF. DHF proved to be a relatively safe sedative or anesthetic, which interacts with GABAergic and cortisol pathways in fish.