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

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

A Benzodiazepine Ligand with Improved GABAA Receptor α5-Subunit Selectivity Driven by Interactions with Loop C.

The family of GABAA receptors is an important drug target group in the treatment of sleep disorders, anxiety, epileptic seizures, and many others. The most frequent GABAA receptor subtype is composed of two α-, two ß-, and one γ2-subunit, whereas the nature of the α-subunit critically determines the properties of the benzodiazepine binding site of those receptors. Nearly all of the clinically relevant drugs target all GABAA receptor subtypes equally. In the past years, however, drug development research has focused on studying α5-containing GABAA receptors. Beyond the central nervous system, α5-containing GABAA receptors in airway smooth muscles are considered as an emerging target for bronchial asthma. Here, we investigated a novel compound derived from the previously described imidazobenzodiazepine SH-053-2'F-R-CH3 (SH53d-ester). Although SH53d-ester is only moderately selective for α5-subunit-containing GABAA receptors, the derivative SH53d-acid shows superior (>40-fold) affinity selectivity and is a positive modulator. Using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes and radioligand displacement assays with human embryonic kidney 293 cells, we demonstrated that an acid group as substituent on the imidazobenzodiazepine scaffold leads to large improvements of functional and binding selectivity for α5ß3γ2 over other αxß3γ2 GABAA receptors. Atom level structural studies provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABAA receptor α-subunit is the dominant molecular determinant of drug selectivity. Thus, we characterize a promising novel α5-subunit-selective drug candidate. SIGNIFICANCE STATEMENT: In the current study we present the detailed pharmacological characterization of a novel compound derived from the previously described imidazobenzodiazepine SH-053-2'F-R-CH3. We describe its superior (>40-fold) affinity selectivity for α5-containing GABAA receptors and show atom-level structure predictions to provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABAA receptor α-subunit is the dominant molecular determinant of drug selectivity.

Effect of flunitrazepam as an oral hypnotic on 24-hour blood pressure in healthy volunteers.

PURPOSE: The present study was carried out in order to assess the effects of chronic administration of flunitrazepam (as an oral hypnotic) on 24-h blood pressure (BP) and heart rate (HR) in healthy young adults. MATERIALS AND METHODS: Following a 2-week placebo run-in period, 28 healthy volunteers (13 males and 15 females) between 21 and 30 years were randomized to receive either flunitrazepam 1 mg or placebo (both administered once a day in the evening) for 4 weeks in two cross-over periods; each separated by a 2-week placebo period. At the end of each study period, non-invasive 24-h BP and HR ambulatory monitoring was performed. RESULTS: Flunitrazepam produced a significant decrease in nighttime systolic blood pressure (SBP) (- 6.4 mmHg) and diastolic blood pressure (DBP) (- 4.1 mmHg) (both P < 0.05 vs placebo) without affecting nocturnal HR. During the morning hours, significantly higher values of SBP (+ 7.4 mmHg, P < 0.01), DBP (+ 3.4 mmHg, P < 0.05) and HR (+ 3.9 beats/min, P < 0.05) were observed in the flunitrazepam group compared to the placebo-treated group. No significant differences were noted between the two groups during afternoon and evening hours. CONCLUSIONS: These results suggest that chronic oral administration of 1 mg flunitrazepam as a hypnotic agent causes a significant nocturnal fall in BP and a transient rebound increase of both BP and HR at awakening in the morning. Mechanisms underlying these cardiovascular effects remain unclear, although the direct vasodilatory effect, which is typical of flunitrazepam (with consequent reflex counter-regulatory responses), and the attenuation of baroreflex sensitivity are likely to play a major role.

Prevalence and clinical correlates of flunitrazepam-related complex sleep behaviors.

AIM: Complex sleep behaviors (CSB) are often associated with the use of hypnotic drugs. This study investigated the prevalence and correlates of CSB among psychiatric patients who were given flunitrazepam. METHODS: From June 2011 to May 2012, a total of 268 psychiatric outpatients who had received flunitrazepam for at least 3 months were enrolled. Data on occurrence of CSB, demographic characteristics, flunitrazepam dosage and duration of use, psychiatric diagnoses, physical illnesses, and alcohol use were collected. Logistic regression analysis was used to examine the clinical correlates of CSB. RESULTS: Sixty-six participants (24.6%) reported experiencing CSB. Logistic regression analysis showed that a high dosage (>2 mg/day) of flunitrazepam (odds ratio [OR] = 1.941, 95% confidence interval [CI] = 1.090-3.455, P = 0.024) and alcohol use (OR = 1.948, 95%CI = 1.023-3.709, P = 0.042) were significantly associated with the occurrence of CSB. Sex, age, duration of flunitrazepam use, psychiatric diagnoses, and physical illnesses were not significantly associated with the occurrence of CSB. CONCLUSION: CSB among flunitrazepam users should be monitored routinely, especially among those receiving a high dosage who also consume alcohol.

Effects of the 5-HT(1A) Receptor Agonist Tandospirone on ACTH-Induced Sleep Disturbance in Rats.

The aim of this study was to compare the effect of the serotonin (5-HT)1A receptor agonist tandospirone versus that of the benzodiazepine hypnotic flunitrazepam in a rat model of long-term adrenocorticotropic hormone (ACTH)-induced sleep disturbance. Rats implanted with electrodes for recording electroencephalogram and electromyogram were injected with ACTH once daily at a dose of 100 µg/rat. Administration of ACTH for 10 d caused a significant increase in sleep latency, decrease in non-rapid eye movement (non-REM) sleep time, and increase in wake time. Tandospirone caused a significant decrease in sleep latency and increase in non-REM sleep time in rats treated with ACTH. The effect of tandospirone on sleep patterns was antagonized by the 5-HT1A receptor antagonist WAY-100635. In contrast, flunitrazepam had no significant effect on sleep parameters in ACTH-treated rats. These results clearly indicate that long-term administration of ACTH causes sleep disturbance, and stimulating the 5-HT1A receptor by tandospirone may be efficacious for improving sleep in cases in which benzodiazepine hypnotics are ineffective.

Neurotoxic effects induced by flunitrazepam and its metabolites in zebrafish: Oxidative stress, apoptosis, and histone hypoacetylation.

Benzodiazepines (BZDs) have been widely detected in aquatic environments, but their neurotoxic effects and potential mechanisms are still unclear. This study focuses on flunitrazepam (FLZ) and its metabolite, 7-aminoflunitrazepam (7-FLZ), as representative psychotropic BZD. We investigated their neurotoxic effects on adult zebrafish following a 30-day exposure to environmentally relevant concentrations. The findings reveal that exposure to these drugs induces anxiety-like and aggressive behaviors in zebrafish. Additionally, notable morphological damage to brain tissue and mitochondrial structures was observed. Through TUNEL staining, an increase in apoptotic cells was detected in the brain tissue of the exposed group, accompanied by marked elevations in ROS and caspase-3/9 levels. The upregulation of apoptosis-related genes Bax, p53, and Bcl-2 confirmed the occurrence of apoptosis. Furthermore, exposure to the drugs resulted in decreased acetylation levels of brain histones H3 and H4. The upregulation of histone deacetylation enzyme genes (HDAC1, HDAC3, HDAC4, and HDAC6) supported this result. Molecular docking results suggest that compared to 7-FLZ, FLZ has a higher binding affinity with HDAC3 and HDAC4, explaining why it causes lower histone acetylation levels. This study in zebrafish elucidates the neurotoxicity and molecular mechanisms induced by FLZ and 7-FLZ, which is significant for further understanding the impact of BZDs on human health and assessing their ecological risks.

Potentiation of the depressant effect of alcohol by flunitrazepam in rats: an electrocorticographic, respiratory and electrocardiographic study.

Alcohol, a widely commercialized psychotropic drug, and the benzodiazepine Flunitrazepam, an anxiolytic widely prescribed for patients with anxiety and insomnia problems, are well known drugs and both act on the central nervous system. The misuse and the association of these two drugs are public health concerns in several countries and could cause momentary, long-lasting and even lethal neurophysiological problems due to the potentiation of their adverse effects in synergy. The present study observed the result of the association of these drugs on electrophysiological responses in the brain, heart, and respiratory rate in Wistar rats. 8 experimental groups were determined: control, one alcohol group (20% at a dose of 1 ml/100 g VO), three Flunitrazepam groups (doses 0.1; 0.2 and 0.3 mg/kg) and three alcohol-Flunitrazepam groups (20% at a dose of 1 ml/100 g VO of alcohol, combined with 0.1; 0.2 and 0.3 mg/kg of Flunitrazepam, respectively). The results showed that there was a more pronounced reduction in alpha and theta wave power in the alcohol-Flunitrazepam groups, a decrease in the power of beta oscillations and greater sedation. There was a progressive decrease in respiratory rate linked to the increase of Flunitrazepam dose in the alcohol-Flunitrazepam associated administration. It was observed alteration in heart rate and Q-T interval in high doses of Flunitrazepam. Therefore, we conclude that the association alcohol-Flunitrazepam presented deepening of depressant synergistic effects according to the increase in the dose of the benzodiazepine, and this could cause alterations in low frequency brain oscillations, breathing, and hemodynamics of the patient.

Quantitative analyses of antagonism: combinations of midazolam and either flunitrazepam or pregnanolone in rhesus monkeys discriminating midazolam.

Adverse effects of benzodiazepines limit their clinical use; these effects might be reduced without altering therapeutic effects by administering other positive GABA(A) modulators (i.e., neuroactive steroids) with benzodiazepines. One concern with this strategy involves reversing these combined effects in case of overdose. The current study examined whether flumazenil can attenuate the combined effects of two benzodiazepines, midazolam and flunitrazepam, and the combined effects of midazolam and the neuroactive steroid pregnanolone, in four monkeys discriminating midazolam. Each positive modulator produced ≥80% midazolam-lever responding. Interactions between midazolam and either flunitrazepam or pregnanolone were additive. Flumazenil antagonized the benzodiazepines when they were administered alone or in combination. Schild analyses yielded slopes that did not deviate from unity, regardless of whether benzodiazepines were administered alone or together; the pA(2) value for flumazenil was 7.58. In contrast, flumazenil enhanced the effects of pregnanolone with 0.32 mg/kg flumazenil shifting the pregnanolone dose-effect curve 2-fold leftward. Flumazenil attenuated the combined effects of midazolam and pregnanolone, although antagonism was not dose-dependent. Thus, the interaction between two benzodiazepines was similar to that of a benzodiazepine and a neuroactive steroid; however, flumazenil more efficiently attenuated a combination of two benzodiazepines compared with a combination of a benzodiazepine and a neuroactive steroid. Although the magnitude of antagonism of a benzodiazepine combined with a neuroactive steroid was reduced, these results support continued exploration of the use of combinations of positive modulators to enhance therapeutic effects while reducing adverse effects.

Differential effects of short- and long-term zolpidem treatment on recombinant α1ß2γ2s subtype of GABA(A) receptors in vitro.

AIM: Zolpidem is a non-benzodiazepine agonist at benzodiazepine binding site in GABA(A) receptors, which is increasingly prescribed. Recent studies suggest that prolonged zolpidem treatment induces tolerance. The aim of this study was to explore the adaptive changes in GABA(A) receptors following short and long-term exposure to zolpidem in vitro. METHODS: Human embryonic kidney (HEK) 293 cells stably expressing recombinant α1ß2γ2s GABA(A) receptors were exposed to zolpidem (1 and 10 µmol/L) for short-term (2 h daily for 1, 2, or 3 consecutive days) or long-term (continuously for 48 h). Radioligand binding studies were used to determine the parameters of [(3)H]flunitrazepam binding sites. RESULTS: A single (2 h) or repeated (2 h daily for 2 or 3 d) short-term exposure to zolpidem affected neither the maximum number of [(3)H]flunitrazepam binding sites nor the affinity. In both control and short-term zolpidem treated groups, addition of GABA (1 nmol/L-1 mmol/L) enhanced [(3)H]flunitrazepam binding in a concentration-dependent manner. The maximum enhancement of [(3)H]flunitrazepam binding in short-term zolpidem treated group was not significantly different from that in the control group. In contrast, long-term exposure to zolpidem resulted in significantly increase in the maximum number of [(3)H]flunitrazepam binding sites without changing the affinity. Furthermore, long-term exposure to zolpidem significantly decreased the ability of GABA to stimulate [(3)H]flunitrazepam binding. CONCLUSION: The results suggest that continuous, but not intermittent and short-term, zolpidem-exposure is able to induce adaptive changes in GABA(A) receptors that could be related to the development of tolerance and dependence.

Upregulation of Uric Acid Production and Caspase 3 Signalling Mediates Rohypnol-Induced Cardiorenal Damage.

The global prevalence of illicit drug use is on the increase with attendant complications like cardiorenal collapse. One such substance of abuse is rohypnol. Despite its ban in most countries, it remains a popular substance of abuse. Whether or not rohypnol induces cardiorenal injury and the associated mechanism is yet to be elucidated. Therefore, the present study investigated the effect of rohypnol on cardiorenal integrity and functions, and glucolipid metabolism. Forty-eight male Wistar rats randomized into six groups (n = 8/group) received (per os) vehicle, low-dose (2 mg/kg) and high-dose (4 mg/kg) rohypnol once daily for twenty eight days, with or without a cessation period. Data revealed that rohypnol exposure irreversibly caused insulin resistance, hyperglycaemia, and dyslipidaemia. This was accompanied by reduced cardiorenal mass and impaired cardiorenal cytoarchitecture and function. Furthermore, rohypnol treatment promoted oxidative stress, inflammation, genotoxicity, and decreased cardiorenal activities of Na+-K+-ATPase, Ca2+-ATPase, and Mg2+-ATPase. These alterations were associated with enhanced uric acid generation and caspase 3 activity in the cardiorenal complex. Thus, this study reveals that rohypnol exposure triggers cardiorenal toxicity with incident insulin resistance, glucolipid and cardiorenal proton pump dysregulation, altered redox state, and inflammation via enhancement of uric acid generation and caspase 3-dependent mechanism.

Sedative-Hypnotic Agents That Impact Gamma-Aminobutyric Acid Receptors: Focus on Flunitrazepam, Gamma-Hydroxybutyric Acid, Phenibut, and Selank.

There are many nonopioid central nervous system depressant substances that share a gamma-aminobutyric acid (GABA) receptor-related mechanism of action. These sedatives-hypnotics can be indicated to treat anxiety, seizures, depression, and insomnia but are also used as substances of abuse and used to facilitate sexual assault. Barbiturates, methaqualone, and glutethimide were among the first type A GABA receptor-mediated sedative-hypnotics. Their clinical use was limited for most indications by serious adverse events and strong abuse potential but continue to be used illicitly around the world. The benzodiazepines supplanted barbiturates for most indications because they were less likely to cause severe adverse events in monotherapy. Flunitrazepam is a newer benzodiazepine that is preferentially used recreationally and to facilitate sexual assault. Flunitrazepam has greater potency and higher affinity for the type A GABA receptor than most benzodiazepines. Gamma-hydroxybutyric acid is sought illicitly for its hypnotic, euphoric and anabolic effects as well as to facilitate sexual assault. When any of these GABAergic drugs are used in high doses or with other sedative hypnotic agents, respiratory depression, coma, and death have occurred. Chronic use of these GABAergic drugs can lead to significant withdrawal syndromes. Phenibut and selank are poorly studied Russian drugs with GABAergic mechanisms that are inexplicably sold to US consumers as dietary supplements. Poison control center calls regarding phenibut have increased substantially over the past 5 years. Desired euphoriant effects account for the recreational and illicit use of many GABA-modulating agents. However, illicit use can lead to significant toxicities related to abuse, dependence, and subsequent withdrawal syndromes. Significant evaluation of developing agents with GABA properties should be conducted to determine abuse potential before public access ensues.

Triton X-100 inhibits agonist-induced currents and suppresses benzodiazepine modulation of GABA(A) receptors in Xenopus oocytes.

Changes in lipid bilayer elastic properties have been proposed to underlie the modulation of voltage-gated Na(+) and L-type Ca(2+) channels and GABA(A) receptors by amphiphiles. The amphiphile Triton X-100 increases the elasticity of lipid bilayers at micromolar concentrations, assessed from its effects on gramicidin channel A appearance rate and lifetime in artificial lipid bilayers. In the present study, the pharmacological action of Triton-X 100 on GABA(A) receptors expressed in Xenopus laevis oocytes was examined. Triton-X 100 inhibited GABA(A) alpha(1)beta(3)gamma(2S) receptor currents in a noncompetitive, time- and voltage-dependent manner and increased the apparent rate and extent of desensitization at 10 muM, which is 30 fold below the critical micelle concentration. In addition, Triton X-100 induced picrotoxin-sensitive GABA(A) receptor currents and suppressed allosteric modulation by flunitrazepam at alpha(1)beta(3)gamma(2S) receptors. All effects were independent of the presence of a gamma(2S) subunit in the GABA(A) receptor complex. The present study suggests that Triton X-100 may stabilize open and desensitized states of the GABA(A) receptor through changes in lipid bilayer elasticity.

GABA(A) receptors mediate the attenuating effects of a 5-HT(3) receptor antagonist on methamphetamine-induced behavioral sensitization in mice.

We previously showed that 5-HT(3) receptors are involved in the development and expression of methamphetamine (MAP)-induced locomotor sensitization in mice. Here, we examined whether the dopaminergic or the GABAergic systems are involved in the attenuating effects of the 5-HT(3) receptor antagonist MDL72222 on MAP-induced locomotor sensitization. Quantitative autoradiography of D1 ([(3)H]SCH23390), D2 ([(3)H]raclopride) receptor, and GABA(A) receptor benzodiazepine ([(3)H]flunitrazepam) binding was carried out in the brains of mice treated with chronic MAP and pretreatment with MDL72222. No significant differences were found in D(1) and D(2) binding between the two groups, suggesting that the attenuating effects of MDL72222 on MAP-induced locomotor sensitization is not medicated by D1 and D2 receptors. Postsynaptic dopamine (DA) receptor supersensitivity was measured by challenge with apomorphine, a dopamine D(1) and D(2) receptor agonist, after repeated MAP treatment or pretreatment with MDL72222 before MAP. Apomorphine induced an enhanced locomotor activity in both chronic MAP-treated mice and mice pretreated with MDL 72222, with no significant differences between the two groups. The binding of [(3)H]flunitrazepam was significantly decreased in the motor and cingulate cortex, caudate putamen, and nucleus accumbens of mice in the repeated MAP treatment group compared with the control group, and this effect was reversed by pretreatment with MDL72222. This suggested that GABA(A) benzodiazepine binding sites are involved in the attenuating effects of a 5-HT(3) receptor antagonist on MAP-induced locomotor sensitization.

Remarkable selectivity of the in vivo binding of [3H]Ro15-4513 to α5 subtype of benzodiazepine receptor in the living mouse brain.

To evaluate the binding characteristics of [(3)H]Ro15-4513 with the central benzodiazepine (BZ) receptor, inhibition experiments of [(3)H]Ro15-1788 and [(3)H]Ro15-4513 were performed both in vitro and in vivo, using two BZ ligands, flunitrazepam (FNP), and ethyl-ß-carboline-3-carboxylate (ß-CCE). FNP inhibited the binding of [(3)H]Ro15-1788 and [(3)H]Ro15-4513 in a dose-dependent manner in the mouse cerebral cortex, hippocampus, and cerebellum, both in vitro and in vivo. ß-CCE also inhibited the binding of [(3)H]Ro15-1788 and [(3)H]Ro15-4513 in all the aforementioned brain regions in vitro. However, in vivo, ß-CCE inhibited the binding of [(3)H]Ro15-4513 in the cerebral cortex and cerebellum, but not in the hippocampus, even at an injected dose of up to 1mg/kg. In contrast, more than 50% of the in vivo binding of [(3)H]Ro15-1788 was inhibited by 1 mg/kg of ß-CCE in all regions. The time-activity curve of [(3)H]Ro15-4513 in the hippocampus also showed no alteration of the peak uptake between the control group and 0.3 mg/kg of ß-CCE coinjected group. These results indicated that the binding characteristics of [(3)H]Ro15-4513 with the BZ receptor differed markedly between the in vitro and in vivo condition, and the selectivity of [(3)H]Ro15-4513 binding to α5 subtype of BZ receptor in the mouse brain seemed to be remarkable under the in vivo condition.

Immunomodulating properties of gamma-hydroxybutyrate (GHB), flunitrazepam and ethanol in 'club drugs' users.

Despite the increasing concern about gamma-hydroxybutyrate (GHB) toxicity in users, no studies have addressed GHB and other club drugs effects on the immune system under controlled administration. Lymphocyte subsets and functional responsiveness of lymphocytes to mitogenic stimulation were measured in 10 healthy male recreational users of GHB who participated in five experimental sessions within the framework of a clinical trial. The study was randomized, double blind, double dummy and cross-over. Drug conditions were: a single oral dose of GHB (40 mg/kg or 60 mg/kg), ethanol (0.7 g/kg), flunitrazepam (1.25 mg) and placebo. Acute GHB produced a time-dependent immune impairment in the first 4 hours after drug administration associated with an increase in cortisol secretion. Although total leukocyte count remained unchanged, there was a significant decrease in the CD4 T/CD8 T-cell ratio, as well as in the percentage of mature T lymphocytes, probably because of a decrease in both the percentage and absolute number of T helper cells. A significant decrease was also observed in natural killer cells and in functional responsiveness of lymphocytes to mitogenic stimulation. Flunitrazepam administration did not produce any change in the immune system, while ethanol intake produced a decrease in B lymphocytes and in lymphocyte proliferative response to mitogens. These results provide the first evidence that GHB intake under a controlled environmental setting impairs the immunological status and confirms the alterations in the immune function caused by ethanol.

Metabolic activation of benzodiazepines by CYP3A4.

Cytochrome P450 3A4 is the predominant isoform in liver, and it metabolizes more than 50% of the clinical drugs commonly used. However, CYP3A4 is also responsible for metabolic activation of drugs, leading to liver injury. Benzodiazepines are widely used as hypnotics and sedatives for anxiety, but some of them induce liver injury in humans. To clarify whether benzodiazepines are metabolically activated, 14 benzodiazepines were investigated for their cytotoxic effects on HepG2 cells treated with recombinant CYP3A4. By exposure to 100 microM flunitrazepam, nimetazepam, or nitrazepam, the cell viability in the presence of CYP3A4 decreased more than 25% compared with that of the control. In contrast, in the case of other benzodiazepines, the changes in the cell viability between CYP3A4 and control Supersomes were less than 10%. These results suggested that nitrobenzodiazepines such as flunitrazepam, nimetazepam, and nitrazepam were metabolically activated by CYP3A4, which resulted in cytotoxicity. To identify the reactive metabolite, the glutathione adducts of flunitrazepam and nimetazepam were investigated by liquid chromatography-tandem mass spectrometry. The structural analysis for the glutathione adducts of flunitrazepam indicated that a nitrogen atom in the side chain of flunitrazepam was conjugated with the thiol of glutathione. Therefore, the presence of a nitro group in the side chain of benzodiazepines may play a crucial role in the metabolic activation by CYP3A4. The present study suggested that metabolic activation by CYP3A4 was one of the mechanisms of liver injury by nitrobenzodiazepines.

Neonatal acute stress by novelty in the absence of social isolation decreases fearfulness in young chicks.

Two hours after hatching (Day 0), groups of chicks from both sexes were housed either individually (IND) or socially in pairs (SOC) for 24 h. On Day 1, for each of the two conditions, half of the chicks were individually exposed to early novelty for 10 min, which comprised being placed in a novel-cage with small pebbles glued to the floor. The other half (controls) remained in the home-cage (IND-C and SOC-C). Thus, the IND-N group was exposed to early novelty, and the SOC-N+I group was exposed to early novelty and social isolation. Subsequently, all groups were mixed and socially reared until reaching 15 days of age. At this time, chicks were exposed to open field (OF) and tonic immobility (TI) tests. The IND-N group showed a shorter latency to ambulate in the OF test, shorter immobility duration in the TI test, a reduced plasma corticosterone concentration and increased flunitrazepam sensitive-GABA(A) receptor basal forebrain density compared with other groups, indicating that a neonatal novelty induced lower fearfulness in young chicks. In contrast, the effect of neonatal novelty was abolished by a simultaneous effect of social isolation in the SOC-N+I group. Thus, early post-hatch life events such as early novelty could improve a bird's later ability to cope with new stressful events. In addition, it is possible that both novelty and social isolation act on different neurobiological processes.

Relative potency and effectiveness of flunitrazepam, ethanol, and beta-CCE for disrupting the acquisition and retention of response sequences in rats.

Despite the knowledge that gamma-aminobutyric acid(A) modulators can affect learning and memory, their capacity for disrupting each of these complex processes is rarely compared, and often mistakenly assumed to occur with identical potency. For these reasons, the effects of flunitrazepam (0.056-3.2 mg/kg), ethanol (0.25-1.5 g/kg), and ethyl-beta-carboline-3-carboxylate (beta-CCE; 1-17.8 mg/kg) were compared in groups of rats responding under baselines that assessed learning and memory separately. The first baseline was a multiple schedule of repeated acquisition and performance of tandem response sequences, whereas the second baseline was a retention or memory procedure where a tandem response sequence was acquired and then retested after a 30-min delay. Under both procedures, responding was maintained under a second-order fixed-ratio-2 schedule of food reinforcement, and incorrect responding (errors) produced a 5-s timeout. With regard to the effects of the three drugs on sequence acquisition (learning), all three drugs dose dependently decreased the overall response rate and increased the percentage of errors. Both flunitrazepam and beta-CCE affected accuracy more potently than response rate, whereas ethanol was equipotent in affecting these two dependent measures. With regard to the effects of these drugs on sequence retention (memory), both flunitrazepam and ethanol dose dependently decreased retention at doses that had little or no effect on sequence acquisition under the multiple schedule, whereas beta-CCE decreased retention and sequence acquisition similarly at the doses tested. Together, these data show that drugs with differing capacities for altering the function of gamma-aminobutyric acid(A) receptors differ in their capacity for disrupting the acquisition and retention of response sequences and that positive modulation of this receptor complex may be more predictive of disruptions in memory than disruptions in learning.

The effects of benzodiazepines on urotensin II-stimulated norepinephrine release from rat cerebrocortical slices.

BACKGROUND: Urotensin II (UII) and its receptor (UT) are implicated in mood disorders, such as stress and anxiety, and this may result, at least in part, from increased norepinephrine release from the cerebral cortex. Benzodiazepines have been widely used as hypnotics and anxiolytics, producing a decrease in cerebrocortical norepinephrine release. We hypothesized that there was some interaction between benzodiazepines and the UII system in the cerebral cortex. METHODS: In the present study, we have examined the effects of benzodiazepines on UII-increased norepinephrine release from rat cerebrocortical slices and intracellular Ca(2+) concentrations ([Ca(2+)]i) in HEK293 cells expressing rat UT receptor (HEK293-rUT cells). RESULTS: Midazolam, diazepam and flunitrazepam concentration-dependently inhibited UII-evoked norepinephrine release but did not affect [Ca(2+)]i. The IC(50) of midazolam for inhibition of UII-evoked norepinephrine release (0.32 microM, P < 0.01) was significantly lower than that of diazepam (187 microM) or flunitrazepam (40 microM). The inhibitory effects of midazolam on UII-evoked norepinephrine release were significantly attenuated by flumazenil, a benzodiazepine site antagonist. CONCLUSION: The present study suggests that midazolam, at clinically relevant concentration, significantly inhibited UII-evoked norepinephrine release. This inhibitory effect may be partially mediated via central benzodiazepine receptors.

Neurosteroids act on recombinant human GABAA receptors.

The endogenous steroid metabolites 3 alpha,21dihydroxy-5 alpha-pregnan-20-one and 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiate GABA-activated Cl- currents recorded from a human cell line transfected with the beta 1, alpha 1 beta 1, and alpha 1 beta 1 gamma 2 combinations of human GABAA receptor subunits. These steroids are active at nanomolar concentrations in potentiating GABA-activated Cl- currents and directly elicit bicuculline-sensitive Cl- currents when applied at micromolar concentrations. The potentiating and direct actions of both steroids were expressed with every combination of subunits tested. However, an examination of single-channel currents recorded from outside-out patches excised from these transfected cells suggests that despite the common minimal structural requirements for expressing steroid and barbiturate actions, the mechanism of GABAA receptor modulation by these pregnane steroids may differ from that of barbiturates.