Neurosteroid dehydroepiandrosterone improves active avoidance retrieval and induces antidepressant-like behavior in rats.

Various studies reported beneficial effects of dehydroepiandrosterone (DHEA) and its sulphate (DHEAS), the neurosteroids involved in various brain functions, on synaptic plasticity, neuronal survival, memory, learning and behavior. This study aimed to investigate the behavioral profile of acute DHEA treatment by using active avoidance (AA) task, primarily predictive of the effects on the retrieval-based learning, and by applying forced swim test (FST), for assessment of antidepressant-like potential. Adult male Wistar rats received intraperitoneal injections of either DHEA (2, 10, 20mg/kg) or solvent, 30min prior to testing. DHEA, in a manner resembling an inverted U shape, influenced the retrieval imposed to rats in AA paradigm. The significant improvement of the performance in the retention session was observed following 10mg/kg DHEA treatment and it was not due to the changes in the motor activity, as indicated by unaltered locomotor parameters (inter-trial crossing). Moreover, 10mg/kg of DHEA significantly decreased the duration of immobility in FST, demonstrating antidepressant-like effects. The capability of bicuculline (2mg/kg) to antagonize the effects of DHEA has been evaluated simultaneously. The retrieval-facilitating as well as antidepressant-like effects of 10mg/kg DHEA were counteracted by bicuculline, a competitive antagonist of GABAA receptors, suggesting involvement of GABAergic system. These results support administration of DHEA as potential therapeutic strategy for treating depression and related cognitive impairments, but emphasized the importance of adequate dosing, as DHEA levels that are too high or too low may not be beneficial.

[The effects of resveratrol on rat behaviour in the forced swim test].

INTRODUCTION: The trans-isomer of resveratrol is the active ingredient of Poligonum cuspidatum, known for its medicinal properties and traditionally used in the treatment of neuropsychiatric disorders. It is also found abundantly in the skin of red grapes and red wine. Previous studies have suggested that trans-resveratrol demonstrates a variety of pharmacological activities including antioxidant, anti-inflammatory, as well as neuroprotective properties and procognitive effects. OBJECTIVE: The goal of the present study was to examine the influence of trans-resveratrol on behavior in rats and its antidepressant properties. METHODS: Male Wistar rats were treated intraperitoneally (i.p.) with the increasing doses of trans-resveratrol (5, 10 and 20 mg/kg) or vehicle (dimethyl sulfoxide--DMSO), 30 minutes before testing of the spontaneous locomotor activity or forced swimming. For the experiments, the behavior of the animals was recorded by a digital camera, and the data were analyzed by one-way ANOVA, followed by Tukey post-hoc test. RESULTS: Testing of spontaneous locomotor activity, after the application of vehicle or increasing doses of trans-resveratrol, showed no statistically significant difference between groups (p > 0.05). In the forced swim test, one-way ANOVA indicated statistically significant effects of trans-resveratrol (p < 0.001).Tukey post-hoc test showed that resveratrol significantly decreased immobility time at the doses of 10 mg/kg and 20 mg/kg, manifesting the acute antidepressant-like effects. There were no statistically significant differences between the resveratrol treatment of 5 mg/kg and vehicle (p > 0.05). CONCLUSION: The results from our study suggest that transresveratrol produces significant effects in the central nervous system. After single application, it has acute antidepressant effects, but without influence on locomotor activity.

DMCM, a benzodiazepine site inverse agonist, improves active avoidance and motivation in the rat.

There are several modulatory sites at GABA(A) receptors, which mediate the actions of many drugs, among them benzodiazepine. Three kinds of allosteric modulators act through the benzodiazepine binding site: positive (agonist), neutral (antagonist), and negative (inverse agonist). The goal of the present study was to examine the influence of the inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) acting on α GABA(A) receptor and compare its dose-response effects on memory and depression-like behavior. We independently studied the effects of DMCM (0.05-1.0 mg/kg) on retention versus acquisition of active avoidance and depression-like behavior in the forced swim test. Throughout the study, drugs were given intraperitoneally, 30 min before testing. ANOVA has showed that treatment with DMCM significantly affected retrieval of avoidance response (p<0.05), exerted promnesic effects in inverted U-shape manner. Dunnett's test indicated that the DMCM avoidance-facilitatory dose was 0.1mg/kg. At the dose facilitating retrieval of avoidance memory, DMCM significantly (p<0.05, comparison of regression coefficients by Student's t-test) and progressively increased acquisition rate during 5 days training, compared to the saline group. In forced swim test, ANOVA indicated statistically significant effects of DMCM (p<0.05). Dunnett's analysis showed that DMCM significantly decreased immobility time at the dose of 0.1mg/kg, exerted acute antidepressant-like effects. Our results experimentally support the findings that under certain circumstances, nonselective benzodiazepine site inverse agonists, produce memory-enhancing and antidepressant-like effects. The molecular and neuronal substrates linking the actions of specific GABA-benzodiazepine receptor complex subunits remains to be further elucidated.

Are GABAA receptors containing alpha5 subunits contributing to the sedative properties of benzodiazepine site agonists?

Classical benzodiazepines (BZs) exert anxiolytic, sedative, hypnotic, muscle relaxant, anticonvulsive, and amnesic effects through potentiation of neurotransmission at GABA(A) receptors containing alpha(1), alpha(2), alpha(3) or alpha(5) subunits. Genetic studies suggest that modulation at the alpha(1) subunit contributes to much of the adverse effects of BZs, most notably sedation, ataxia, and amnesia. Hence, BZ site ligands functionally inactive at GABA(A) receptors containing the alpha(1) subunit are considered to be promising leads for novel, anxioselective anxiolytics devoid of sedative properties. In pursuing this approach, we used two-electrode voltage clamp experiments in Xenopus oocytes expressing recombinant GABA(A) receptor subtypes to investigate functional selectivity of three newly synthesized BZ site ligands and also compared their in vivo behavioral profiles. The compounds were functionally selective for alpha(2)-, alpha(3)-, and alpha(5)-containing subtypes of GABA(A) receptors (SH-053-S-CH3 and SH-053-S-CH3-2'F) or essentially selective for alpha(5) subtypes (SH-053-R-CH3). Possible influences on behavioral measures were tested in the elevated plus maze, spontaneous locomotor activity, and rotarod test, which are considered primarily predictive of the anxiolytic, sedative, and ataxic influence of BZs, respectively. The results confirmed the substantially diminished ataxic potential of BZ site agonists devoid of alpha(1) subunit-mediated effects, with preserved anti-anxiety effects at 30 mg/kg of SH-053-S-CH3 and SH-053-S-CH3-2'F. However, all three ligands, dosed at 30 mg/kg, decreased spontaneous locomotor activity, suggesting that sedation may be partly dependent on activity mediated by alpha(5)-containing GABA(A) receptors. Hence, it could be of importance to avoid substantial agonist activity at alpha(5) receptors by candidate anxioselective anxiolytics, if clinical sedation is to be avoided.

Benzodiazepine site inverse agonists and locomotor activity in rats: bimodal and biphasic influence.

Benzodiazepine site inverse agonists may increase or decrease locomotor activity in rodents, depending on the experimental settings. We have compared the behavioral responses to environmental novelty of rats treated with the non-selective inverse agonist DMCM (2 mg/kg) and the alpha1-subunit affinity-selective inverse agonist 3-EBC (15 mg/kg). The behavior in spontaneous locomotor assay (during 45 min) and elevated plus maze (EPM) was automatically recorded. In the EPM, general activity-related parameters were similarly decreased, whereas only DMCM inhibited open-arm activity. In the locomotor assay, both compounds depressed locomotion in the first 15 min and activity in the central zone of the chamber. However, the influence of 3-EBC was less pronounced. The alpha1-subunit selective antagonist beta-CCt (15 mg/kg) attenuated locomotor depression, but not the central-zone avoidance elicited by DMCM. When habituated to the chamber, DMCM-treated animals emitted a plateau phase of activity, which disappeared by adding beta-CCt. Hence, inhibition of activity in exposed areas may be mediated by non-alpha1-subunits, whereas both alpha1 and non-alpha1-subunits may participate in suppression of activity in more protective areas of an apparatus. Hyperlocomotion in habituated animals may depend primarily on the alpha1-subunit. Moreover, the bimodal influence of inverse agonists on locomotion can be biphasic, observable in the same experiment.

The lack of bicuculline and picrotoxin influence on midazolam depressant action on brain oxygen consumption.

In the previous study of the rat frontal cortex slices oxygen consumption (QO2), polarographically determined using the biological oxygen monitor, a moderate respiratory depressant action of midazolam ex vivo (1.0 mg/kg) has been observed. Antagonist of the benzodiazepine binding site, flumazenil, blocked the effect of the agonist. However, midazolam-gamma-aminobutyric acid (GABA) interactions pointed to the possibility that a part of midazolam action is independent of the classical GABA potentiation. To test this presumption, GABAA receptor antagonists bicuculline and picrotoxin were administered. Both blockers antagonized the QO2 reducing effect of the combination of per se effective doses of midazolam (1.0 mg/kg) and GABA (5 x 10(-4) mol/l), as well as of GABA (5 x 10(-4) mol/l) itself. However, neither effects of midazolam (1.0 mg/kg) on its own, nor those of midazolam in presence of the physiological, per se ineffective, concentration of GABA (10(-6) mol/l), were susceptible to antagonism. These results show that ex vivo influence of midazolam on cerebral metabolic activity should be partly ascribed to some of its cellular mechanisms probably associated to the GABA modulation, but distinct from the standard GABA-potentiating effects of benzodiazepines.

Bidirectional effects of benzodiazepine binding site ligands in the passive avoidance task: differential antagonism by flumazenil and beta-CCt.

Recent research on genetically modified mice has attributed the amnesic effect of benzodiazepines mainly to the alpha1-containing GABA(A) receptor subtypes. The pharmacological approach, using subtype selective ligands, is needed to complement genetic studies. We tested the effects of the non-selective antagonist flumazenil (0-20.0 mg/kg), the preferential alpha1-subunit selective antagonist beta-carboline-3-carboxylate-t-butyl ester (beta-CCt) (0-30.0 mg/kg), the non-selective agonist midazolam (0-2.0 mg/kg), the preferential alpha1-subunit selective agonist zolpidem (0-3.0 mg/kg), and the non-selective inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) (0-2.0 mg/kg) in the one-trial step-through passive avoidance task in rats. The compounds were administered intraperitoneally, before the acquisition test. Flumazenil and beta-CCt did not affect retention performance. Midazolam and zolpidem induced amnesia in a dose-dependent manner. The complete reversal of amnesia was unattainable. The effects of zolpidem were significantly attenuated by the both, flumazenil (10.0 mg/kg) and beta-CCt (30.0 mg/kg); by contrast, only flumazenil was considerably effective when combined with midazolam. DMCM exerted promnesic effects at 0.2mg/kg, in an inverted U-shape manner. Both antagonists tended to abolish this action. The results indicate that some other alpha-subunit(s), in addition to the alpha1-subunit, contribute to the amnesic actions of non-selective benzodiazepine site agonists in the passive avoidance task. On the other hand, a significant part of the DMCM-induced promnesic effect could involve the alpha1-subunit and/or other putative beta-CCt-sensitive binding site(s).

Bidirectional effects of benzodiazepine binding site ligands on active avoidance acquisition and retention: differential antagonism by flumazenil and beta-CCt.

RATIONALE: The pharmacological approach, using subtype selective ligands, complements genetic studies on the specific contribution of individual receptor subtypes to the various effects of benzodiazepines. OBJECTIVE: The aim of this study was to examine the relative significance of alpha1-containing GABA(A) receptors in the effects of modulators at the benzodiazepine site on anxiety and memory processes. METHODS: We tested the effects of the nonselective antagonist flumazenil, the preferential alpha1-subunit selective antagonist beta-carboline-3-carboxylate-t-butyl ester (beta-CCt), the nonselective agonist midazolam, the preferential alpha1-subunit selective agonist zolpidem, and the nonselective inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) in a two-way active avoidance task in rats. The influence of flumazenil (10.0 mg/kg) and beta-CCt (30.0 mg/kg) on the effects of the two agonists were also examined. In the schedule 2 x 30 trials, drugs were administered i.p. 20 min before the training session. Avoidance responses in the training session are an anxiety-mediated behavior, whereas performance in the retention session relates to the effects on memory. RESULTS: Flumazenil and beta-CCt did not affect behavior. Midazolam (2.0 mg/kg) facilitated acquisition performance, while DMCM (1.0 and 2.0 mg/kg) induced the opposite effect. Flumazenil antagonized both effects. Beta-CCt potentiated the effect of midazolam, and partly antagonized the effect of DMCM. Midazolam (0.5 and 1.0 mg/kg) and zolpidem (1.0-3.0 mg/kg) impaired, while DMCM (0.1 mg/kg) facilitated the subjects' performance in the retention test. The amnesic effects were attenuated but not fully reversed, while the effect of DMCM was counteracted by both antagonists. CONCLUSION: The results indicate the alpha1-subunit interferes with the anxiolytic effect of a benzodiazepine site agonist and may contribute to the DMCM-induced anxiogenic effect. It is also substantially involved in the bidirectional memory processing in the active avoidance paradigm.

Memory effects of benzodiazepines: memory stages and types versus binding-site subtypes.

Benzodiazepines are well established as inhibitory modulators of memory processing. This effect is especially prominent when applied before the acquisition phase of a memory task. This minireview concentrates on the putative subtype selectivity of the acquisition-impairing action of benzodiazepines. Namely, recent genetic studies and standard behavioral tests employing subtype-selective ligands pointed to the predominant involvement of two subtypes of benzodiazepine binding sites in memory modulation. Explicit memory learning seems to be affected through the GABAA receptors containing the alpha1 and alpha5 subunits, whereas the effects on procedural memory can be mainly mediated by the alpha1 subunit. The pervading involvement of the alpha1 subunit in memory modulation is not at all unexpected because this subunit is the major subtype, present in 60% of all GABAA receptors. On the other hand, the role of alpha5 subunits, mainly expressed in the hippocampus, in modulating distinct forms of memory gives promise of selective pharmacological coping with certain memory deficit states.

Bidirectional effects of benzodiazepine binding site ligands in the elevated plus-maze: differential antagonism by flumazenil and beta-CCt.

Recent research using genetically modified mice has pointed to the specific contribution of individual receptor subtypes to the various effects of benzodiazepines. The aim of this study was to examine the relative significance of alpha(1)-containing GABA(A) receptors in the effects of modulators at the benzodiazepine site in the elevated plus-maze (EPM) under dim red light in rats. We tested the effects of the non-selective antagonist flumazenil (0-20.0 mg/kg), the preferential alpha(1)-subunit selective antagonist beta-carboline-3-carboxylate-t-butyl ester (beta-CCt, 0-30.0 mg/kg), the non-selective agonist midazolam (0-2.0 mg/kg), the preferential alpha(1)-subunit selective agonist zolpidem (0-2.0 mg/kg) and the non-selective inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM, 0-2.0 mg/kg). The influence of flumazenil (10.0 mg/kg) and beta-CCt (30.0 mg/kg) on the effects of both kinds of agonists were also examined. The standard spatio-temporal parameters reflecting anxiety (percentage of open arm entries and time) and locomotion (closed and total arm entries) were analyzed. beta-CCt did not affect behavior, while flumazenil at the highest dose (20.0 mg/kg) decreased indices of open arm activity and total arm entries. Midazolam at the dose of 1.0 mg/kg significantly increased the percentage of open arm time, whereas at 2.0 mg/kg both anxiety-related parameters were increased. In contrast to the open arm entries, the open arm time was independent of the decreased closed arm entries, observed at 2.0 mg/kg. Flumazenil abolished these effects, whereas beta-CCt partially potentiated the anxiolytic actions of midazolam. Zolpidem significantly increased both open-arm indices at 1.0 mg/kg, but the effect was dependent on the decreased closed arm entries. The selectivity of the anxiolytic-like effects of zolpidem was further checked under brighter white illumination. In these settings, the influence on anxiety-related, but not activity-related parameters, was absent. All of the activity-related effects of midazolam and zolpidem were mainly counteracted by both antagonists. DMCM produced significant anxiogenic effects at 1.0 mg/kg (open arm time) and 2.0 mg/kg (both parameters). beta-CCt (30.0 mg/kg) and flumazenil at higher dose (20.0 mg/kg) antagonized the effects of DMCM. The results indicate the anxiolytic effects of a non-selective benzodiazepine site agonist involve a predominant role of subunits other than alpha(1), whereas the behavioral indices of the anxiolytic-like properties of an alpha(1)-selective ligand, if observed, depend on the experimental settings and the changes in locomotor activity, and hence were behaviorally non-specific. The present results generally correspond well to the behavioral findings with the genetically modified mice. On the other hand, the relative significance of the alpha(1)-subunit in the anxiogenic effects of DMCM could not be clearly deduced.

The influence of midazolam on active avoidance retrieval and acquisition rate in rats.

The purpose of the present study was to examine the influence of midazolam on the retrieval and acquisition rate of two-way active avoidance in rats. In the schedule 2 x 100 trials, the effects of midazolam (0.5-5.0 mg/kg), benzodiazepine binding site antagonist flumazenil (2.5-10.0 mg/kg), specific antagonist of GABA(A) receptor, bicuculline (0.5-4.0 mg/kg), and the blocker of GABA(A) receptor containing Cl(-) channels, picrotoxin (1.0-5.0 mg/kg), on the second day retrieval of avoidance performance were investigated, as well as the influence of the used blockers of GABA(A) receptor function on midazolam effects. Furthermore, the effect of midazolam (1.0 mg/kg) on acquisition rate in the 5 x 50 schedule, as well as the effects of third day treatment changing in that paradigm, was examined. Throughout the study, drugs were given intraperitoneally, 30 min before testing. Midazolam at the dose of 1.0 mg/kg facilitated avoidance retrieval, whereas flumazenil and bicuculline did not significantly change behavior. Picrotoxin (5.0 mg/kg) diminished performance. All three kinds of blockers used abolished facilitatory action of midazolam, confirming GABAergic mediation of the effect of benzodiazepine. Midazolam (1.0 mg/kg) increased acquisition rate during five consecutive days relative to saline, but without significant effect on the first day acquisition. In the case of third day changing of treatments, the intersection of regression rate lines was detected. Results from active avoidance paradigm experimentally support the findings from human studies that in certain circumstances, benzodiazepines, potentiating GABAergic neurotransmission, could produce retrieval-enhancing effects in memory tasks.

The influence of diazepam on atropine reversal of behavioural impairment in dichlorvos-treated rats.

Acute effects on the behaviour of the organophosphate insecticide dichlorvos and its standard antidotes possessing behavioural activity, atropine and diazepam, were studied separately and in combinations in male Wistar rats. In the spontaneous locomotor activity test, dichlorvos and diazepam decreased, whereas atropine increased performance. The effect of dichlorvos was obtained at a dose (5 mg/kg) that induced overt intoxication, and could not be reversed during first half hour-period after administration of any combination of drugs. In the other two tests, active avoidance learning and rotarod performance, the effective dose of dichlorvos (2 mg/kg) was devoid of somatic signs of intoxication. In these more sensitive tests, the effective atropine dose (40 mg/kg) completely reversed dichlorvos-induced incapacitation. In the rotarod test, diazepam (0.5 mg/kg) contributed to the incapacitating effect of dichlorvos, and impeded desirable influence of atropine as well. In the active avoidance test, diazepam (2.5 mg/kg) contributed to failure to escape; it did not influence the dichlorvos-induced decrease of avoidance performance, nor did it impair the completely reversing effects of atropine. The results point to the possible summation of acute incapacitating effects of organophosphates and diazepam on motor performance, which seems to be, at least partly, antagonized by sufficiently high doses of atropine. However, taking into account the long-term neuroprotective role of the anticonvulsant diazepam, and hence its delayed beneficial influences on behaviour, the immediate testing of atropine/diazepam treatment of organophosphate intoxication in active avoidance paradigm could possess beside sensitivity the predictive value as well.

The influence of midazolam and flumazenil on rat brain slices oxygen consumption.

This study investigated the impact of benzodiazepine receptor agonist, midazolam and antagonist, flumazenil, on the rat frontal cortex slices oxygen consumption (QO(2)), in presence and absence of gamma-aminobutyric acid (GABA). QO(2) was polarographically determined, using the biological oxygen monitor. As it was previously shown, GABA on its own decreases QO(2) moderately. Midazolam decreased QO(2) at 1.0mg/kg, whereas flumazenil had no effect. In combination with per se ineffective GABA (10(-6)mol/l), flumazenil showed respiratory depressant action, presumably revealing partial agonistic activity at some of GABA(A) receptor subtypes. However, it completely antagonized effects of midazolam on QO(2), on its own and in presence of GABA. Our results show that in vivo well-established effects of midazolam on cerebral metabolic activity could be reproduced in in vitro settings. Moreover, flumazenil antagonized this action, indicating the role of GABA(A)-benzodiazepine receptor complex activation in QO(2) regulation.

Gaba-benzodiazepine receptor complex in brain oxidative metabolism regulation.

This study investigated the impact of modulating the gamma-aminobutyric acid(A) (GABA)(A)-benzodiazepine receptor complex activity on the rat frontal cortex slices oxygen consumption (QO(2)), polarographically determined using the biological oxygen monitor. Throughout the study, diazepam, flumazenil and picrotoxin were administered i.p. 30 min before sacrificing animals and obtaining slice preparations, while GABA was added directly into the medium in the reaction chamber. GABA decreased QO(2) in concentrations of 5 x 10(-4), 10(-2) and 5 x 10(-2)mol l(-1), while 10(-5) and 10(-6)mol l(-1) GABA had no effect, as well as diazepam, flumazenil and picrotoxin. All diazepam doses (1, 2.5 and 5 mg kg(-1)) increased action of 5 x 10(-4)mol l(-1) GABA, whereas 2.5 mg kg(-1) dose amplified the effect of 10(-6)mol l(-1) GABA. Flumazenil and picrotoxin (5 mg kg(-1) both) blocked diazepam's effects. Flumazenil augmented 10(-6)mol l(-1) GABA effects, while picrotoxin and flumazenil abolished the effects of 5 x 10(-4)mol l(-1) GABA. To our knowledge, this is the first study to examine the influence of modulation of GABA(A)-benzodiazepine receptor function on cerebral metabolism of oxygen in in vitro settings. The results are in accordance with those obtained in numerous in vivo studies, pointing to the moderate level of influence of GABA(A)-benzodiazepine receptor complex on QO(2) regulation.

[Effect of uremia and peritoneal dialysis on peritoneal mesothelial cells]. / Uticaj uremije i peritoneurske dijalize na celije mezotela peritoneuma.

The aim of the study was to investigate the morphology of mesothelial cells of the peritoneum of patients with terminal renal failure (TRF), taken by the biopsy immediately before the onset of peritonal dialysis (PD), and to compare it with the findings in patients with PD. The samples were prepared in the way standard for light microscopy and transmission electron microscopy. In patients with TRF intracytoplasmatic inclusions could be observed, unusual protrusions of mesothelial apical surfaces, deformation of mesothelial cells and their detachment from the basal membrane, as well as the dilatated cisternae of granulated endoplasmatic reticulum with filamentous structures in some of them. In patients on PD cytoplasmic protrusions of different shapes and contents were observed at the surface of mesothelial cells, multiplication of basal membrane, occurrence of young forms of mesothelial cells as well as the detachment of those cells from the basal lamina.