The phenomenon of "one-trial tolerance" to the anxiolytic effect of chlordiazepoxide in the elevated plus-maze is abolished by the introduction of a motivational conflict situation.

A single exposure to the elevated plus-maze test of anxiety reduces or abolishes the anxiolytic efficacy of benzodiazepines. The present study was designed to examine whether this phenomenon of "one-trial tolerance" resulted from a motivational deficit on trial 2. We hypothesized that whereas there is a motivational conflict on trial 1 in relation to the open arms (exploration drive X natural fear of open spaces), there is no "reason" for an animal to explore it on trial 2. A motivational conflict was introduced on trial 2 by rendering the enclosed arms of the apparatus aversive on trial 1. Thus, every time rats entered the enclosed arms, an aversive situation (light and hot air blow) was produced until they left the arm. On trial 2, rats did not receive this aversive stimulation. Chlordiazepoxide significantly enhanced the percent open arm time as well as the percent open arm entries on trial 2 in rats that had been submitted to the aversive stimulation in the enclosed arms on trial 1, but was not effective in rats which had been exposed to the apparatus in the absence of the aversive stimulation on trial 1. In addition, there was no difference in the percent open arm time and entries on trial 2 between saline-treated rats submitted to the aversive or non-aversive condition on trial 1. The aversive condition on trial 1 did not modify the number of total arm entries on trial 2, either. The results suggest that the anxiolytic effect of chlordiazepoxide in the elevated plus-maze depends on the presence of a motivational conflict situation.     

One-trial tolerance to the effects of chlordiazepoxide in the elevated plus-maze is not due to acquisition of a phobic avoidance of open arms during initial exposure

A single exposure to the elevated plus-maze (EPM) test of anxiety reduces or abolishes the anxiolytic-like efficacy of benzodiazepines. This phenomenon called one-trial tolerance has been suggested to represent the acquisition of a phobic-like response to the open arms during trial 1. The present study was designed to examine the effects of chlordiazepoxide (5 mg/kg, ip) on the behaviour of rats in a conventional EPM apparatus after previous exposure to a four-open-arm EPM, a four-enclosed arm EPM or a conventional EPM, as well as in naive rats. Chlordiazepoxide had clear-cut anxiolytic-like effects (increased percentage of time spent on the open arms) in a traditional EPM in naive rats and in animals previously exposed to a four-open-arm EPM. However, it was ineffective in rats previously exposed to a traditional or a four-closed-arm EPM. Thus, the phenomenon of one-trial tolerance does not depend upon initial open-arm experience.     

Role of learning of open arm avoidance in the phenomenon of one-trial tolerance to the anxiolytic effect of chlordiazepoxide in mice

A single exposure to the elevated plus-maze (EPM) test of anxiety reduces or abolishes the anxiolytic efficacy of benzodiazepines on a second trial. Some possible explanations to the occurrence of this phenomenon (one-trial tolerance-OTT) involve behavioral modifications thought to be consequence of some kind of learning in the first trial. In the present study, the influence of learning-impairing situations on the effects of the benzodiazepine chlordiazepoxide on mice re-tested in the EPM is investigated. The results showed that: (1) as expected, the administration of chlordiazepoxide to mice re-tested in the EPM- under the same conditions of the first trial- failed to induce anxiolysis; (2) a decreased percent time in the open arms was observed on the second trial of mice exposed to both trials under the same experimental conditions; (3) neither the increase in open arm avoidance by mice re-exposed to the EPM nor the OTT to chlordiazepoxide effect were modified by administration of the amnestic agent scopolamine; (4) the decrement of the duration of the first trial to 1 min or the change in light and noise conditions in both trials counteracted the increase in open arm avoidance on trial 2; (5) none of the later procedures modified the phenomenon of OTT. Although not discarding the modulation exerted by other memory processes in the OTT phenomenon, the results indicate that situations that impair the learned avoidance response to the open arms in the EPM do not modify the phenomenon of OTT.     

Anxiolytic profile of girisopam and GYKI 52,322 (EGIS 6775). Comparison with chlordiazepoxide and buspirone.

The anxiolytic action of two 2,3-benzodiazepines: girisopam: GYKI 51,189 (EGIS 5810): (1-(3-chlorophenyl)-4-methyl-7,8-dimethoxy-5H-2,3-benzodiazepine), and GYKI 52,322 (EGIS 6775): (1-(4-aminophenyl)-4-methyl-7,8-dimethoxy-5H-2,3-benzodiazepine) was investigated in comparison to chlordiazepoxide and buspirone using three different animal models of anxiety: the lick conflict, the elevated plus maze and the open field methods in rats. Both 2,3-benzodiazepines exerted anxiolytic effect in all three tests used, however their pharmacological profile differs considerably from that of either chlordiazepoxide or buspirone. Using the animal models mentioned above the order of potency was GYKI 52,322 (EGIS 6775) > chlordiazepoxide > girisopam > buspirone.     

Tolerance in the anxiolytic profile following repeated administration of diazepam but not buspirone is associated with a decrease in the responsiveness of postsynaptic 5-HT-1A receptors

To understand the role of serotonin (5-hydroxytryptamine; 5-HT)-1A receptors in the treatment of anxiety and the development of tolerance to benzodiazepines the present study was designed to monitor the responsiveness of postsynaptic 5-HT-1A receptors following repeated administration of diazepam and buspirone. Results show that tolerance in the anxiolytic profile is produced following repeated administration (2 weeks) of diazepam (2 mg/kg) but not buspirone (0.5 mg/kg). The behavioral effects of 8-OH-DPAT at a dose of 0.25 mg/kg were monitored 3 days after repeated administration of saline or buspirone or diazepam. The results show that 8-OH-DPAT elicited forepaw treading was smaller in repeated diazepam but not repeated buspirone injected rats, while hyperlocomotive effects of 8-OH-DPAT were smaller in both repeated buspirone and repeated diazepam injected rats. The results suggest that postsynaptic 5-HT-1A receptor-dependent responses were attenuated following long-term administration of diazepam but not buspirone. Role of 5-HT-1A receptors in the development of tolerance to the anxiolytic effects of diazepam but not buspirone is discussed.     

Increases in plasma corticosterone and stretched-attend postures in rats naive and previously exposed to the elevated plus-maze are sensitive to the anxiolytic-like effects of midazolam

A single exposure to the elevated plus-maze test (EPM) reduces or abolishes the anxiolytic efficacy of benzodiazepines on a second trial. This phenomenon known as one-trial tolerance (OTT) is considered to be due to a shift in the emotional state of the animals across the test/retest sessions. Activation of the hypothalamic-pituitary-adrenocortical (HPA) axis has been considered to be an adaptive response to stressful or challenging situations such as height and openness of the EPM. This work looks at the phenomenon of OTT to the benzodiazepine compound midazolam through the conjoint examination of the novel ethological measures of the EPM and adrenocortical response of rats exposed to single and repeated sessions of the EPM. The results obtained confirmed that the approach/avoidance conflict on the first trial of the EPM is very sensitive to the anxiolytic effects of benzodiazepines. Moreover, stressful stimuli present upon initial exposure to the EPM render the standard measures of the EPM resistant to the anxiolytic effects of benzodiazepines on retest. However, the increases in plasma corticosterone and in risk assessment behavior observed in rats submitted to single or repeated sessions in the EPM were reversed by pretreatment with midazolam. The administration of metyrapone, a glucocorticoid synthesis blocker, decreased risk assessment but did not affect locomotion and anxiety-like behaviors. It is suggested that the detection of the dangerous environment through the stretched-attend postures in the second trial leads to the known low level of exploration and the consequent OTT upon retest. Moreover, in view of the similarity between the risk assessment and plasma corticosterone patterns in both naive and experienced rats, this hormone-behavior profile may be crucial for the expression of OTT to benzodiazepines in rodents exposed to the EPM.     

Substance P enhancement of inhibitory avoidance learning: mediation by the N-terminal sequence.

Experiments were performed to investigate the effects of intraperitoneally administered undecapeptide substance P (SP), its N-terminal fragment SP(1-7) (SPN) and the C-terminal analog [pGlu6]-SP(6-11) (SPC) on inhibitory avoidance learning, using a one-trial up-hill avoidance task. In Experiment 1 rats were injected with either SP (50 micrograms/kg), SPN (3.3, 33, 167, 333 micrograms/kg) or SPC (2.7, 27, 134, 268 micrograms/kg) immediately after the training trial. Controls received the diluent vehicles. When tested 24 hr later, rats injected with 50 micrograms/kg SP (37 nmol/kg) and 167 micrograms/kg SPN (185 nmol/kg) exhibited longer step-up latencies than vehicle-treated controls. None of the other doses of SPN nor of the C-terminal fragment influenced performance. In Experiment 2, 167 micrograms/kg SPN or vehicle was injected posttrial either immediately or 5 hr after the training trial. Retention latencies 24 hr later were longer for rats treated with 167 micrograms/kg SPN immediately after the training trial. Performance of the SPN 5-hr delay group did not differ from that of the vehicle-injected controls, ruling out proactive effects of SPN on recall.     

Comparison of neurokinin SP with diazepam in effects on memory and fear parameters in the elevated T-maze free exploration paradigm.

The elevated T-maze was combined with a free exploration protocol, which, in contrast to the conventional procedure, dispenses with handling of the animals during the experimental sessions. This allows measurement of fear indexes derived from the elevated plus-maze as well as assessment of acquisition of open arm avoidance and open arm escape in one continuous session. Retention of the different fear-responses is measured 72 h later without drug treatment. In order to assess the effects of two known anxiolytics in this paradigm, rats received an IP injection of diazepam (1 to 4 mg/kg), substance P (5 to 500 microg/kg) or vehicle (1 ml/kg) and were tested on the T-maze for 5 min. Diazepam elevated open arm activity, indicative of an anxiolytic effect. The drug also increased the latency to escape from the open arms, but did not significantly affect acquisition of open arm avoidance. During the retention trial, diazepam in higher doses impaired the performance of both fear-responses, suggestive of an anterograde amnesic effect. Substance P did not influence acquisition and retention of open arm avoidance and escape. However, in high doses, the peptide increased the sojourn time in the central arena of the maze, indicating reduced fear and, hence, a dissociation between anxiolytic and amnesic effects. The present findings demonstrate that the elevated T-maze free exploration paradigm is sensitive to anxiolytic and memory-modulating effects of drugs.     

Naltrexone potentiates both amnestic and anxiolytic effects of chlordiazepoxide in mice

Studies have suggested that opioid antagonists potentiate the anxiolytic effect but not the amnestic action of chlordiazepoxide (CDZ). We investigated the effects of naltrexone (NAL) on the anxiolytic and amnestic effects of CDZ in mice tested in the plus-maze discriminative avoidance task (DAT). Mice are conditioned to choose between two enclosed arms (one of which aversive) while avoiding the two open arms of the apparatus. This task measures memory (time spent in the aversive vs. time in the non-aversive enclosed arms) and anxiety (time spent in the open arms). Mice treated with saline (SAL) or 5 mg/kg NAL, and SAL or 2.5 mg/kg CDZ were submitted to DAT training. The test was performed 24 h later, without aversive stimuli. In the training, NAL + CDZ group showed higher percent time spent in the open arms than all the other groups. In the test, NAL + CDZ (but not SAL + CDZ) group showed higher percent time spent in the aversive enclosed arm than SAL + SAL and NAL + SAL groups. The data suggest that NAL potentiates the small decreases in anxiety and retention induced by a subeffective dose of CDZ.     

Absence of anxiolytic response to chlordiazepoxide in two common background strains exposed to the elevated plus-maze: importance and implications of behavioural baseline

Although genetic background is acknowledged as a potentially important determinant of mutant phenotypes, publications on genetically modified mice far outnumber those on progenitor strains. We have recently reported major differences in basal anxiety levels (elevated plus-maze & light/dark exploration) among three strains (C57BL/6JOlaHsd, 129/SvEv and 129S2/SvHsd) employed as progenitor stock in European laboratories (Rodgers et al. in press). Furthermore, the phenotypes of these inbred strains differed significantly from that of an outbred strain (Swiss-Webster) commonly used in behavioural pharmacology. In view of these findings, the present study assessed possible differences in the anxiolytic efficacy of chlordiazepoxide (0, 7.5 & 15.0 mg/kg, IP) in three of these strains (Swiss-Webster (SW), C57BL/6JOIaHsd (C57) & 129S2/SvHsd (129)). Experimentally naive mice were exposed to the elevated plus-maze, sessions were videotaped and behaviour analysed using ethological software. The performance of control subjects confirmed significant strain differences in basal levels of activity (SW > C57 > 129) and anxiety-related behaviours (129 = SW > C57), with hypolocomotion dominating the 129 profile. SW mice displayed an anxioselective response to both doses of chlordiazepoxide (CDP), with significant reductions in open arm avoidance and risk assessment observed in the absence of any change in general activity. In direct contrast, the lower dose of CDP (7.5 mg/kg) was without effect in either inbred strain, whereas treatment with 15.0 mg/kg induced a profile indicative of muscle relaxation/mild sedation in C57 mice and virtually abolished all behavioural activity in 129 mice. Although the absence of an anxiolytic response to CDP in C57 mice may be attributed to their low basal anxiety levels, the profile of 129 mice strongly suggests an abnormality in benzodiazepine/GABAA receptor function. The implications of these findings for research on mutant mice are discussed.     

Anxiolytic effects of lavender oil inhalation on open-field behaviour in rats

To establish a valid animal model of the effects of olfactory stimuli on anxiety, a series of experiments was conducted using rats in an open-field test. Throughout, effects of lavender oil were compared with the effects of chlordiazepoxide (CDP), as a reference anxiolytic with well-known effects on open-field behaviour. Rats were exposed to lavender oil (0.1-1.0 ml) for 30 min (Experiment 1) or 1h (Experiment 2) prior to open-field test and in the open field or injected with CDP (10 mg/kg i.p.). CDP had predicted effects on behaviour, and the higher doses of lavender oil had some effects on behaviour similar to those of CDP. In Experiment 3, various combinations of pre-exposure times and amounts of lavender oil were used. With sufficient exposure time and quantity of lavender the same effects were obtained as in Experiment 2. Experiment 4 demonstrated that these behavioural effects of lavender could be obtained following pre-exposure, even if no oil was present in the open-field test. In Experiments 2-4, lavender oil increased immobility. Together, these experiments suggest that lavender oil does have anxiolytic effects in the open field, but that a sedative effect can also occur at the highest doses.     

Effects of chronic treatment with ipsapirone and buspirone on the C57BL/6J strain mice under social stress

We experimented on inbred C57BL/6J strain mice who experienced social stress caused by defeat in inter-male confrontations for 20 days. From the fifth fight on, some mice were injected with ipsapirone (3 mg/kg), and some with buspirone (1 mg/kg) on a daily basis, for 14 days. Post-treatment behavior was examined in the plus-maze, partition, and Porsolt forced swim test (Porsolt's test). Each of these drugs had anxiolytic effects in the plus-maze, suggesting that they reduce state anxiety. Neither had any effect in the partition test, which provides further support to the hypothesis that normally the C57BL/6J strain mice have a high level of trait anxiety and for that reason they did not respond to the drugs. Chronic treatment with neither drug had any effect in the Porsolt's test. It is proposed that ipsapirone and buspirone fail to alleviate the depressive-like behaviors in the C57BL/6J mice because of a high level of trait anxiety, which might be inherent to this mouse strain.     

Pharmacological Activity of Ruthenium Complexes trans-[RuCl2(L)4] (L = Nicotinic or i-Nicotinic acid) on Anxiety and Memory in Rats

Many biological properties have been attributed to ruthenium complex I (trans-[RuCl₂(nic)₄]) and ruthenium complex II (trans-[RuCl₂ (i-nic) ₄]) including nitric oxide synthase inhibition. In this study, we evaluated pharmacological effects of these complexes on anxiety and memory formation. Memory was evaluated with inhibitory avoidance and habituation to an open-field and anxiety was tested with elevated plus-maze. Adult male Wistar rats (250 to 350 g) received intraperitoneal injections of vehicle, ruthenium complex I (45.2, 90.4, or 180.7 μmol/kg), or ruthenium complex II (0.08, 4.5, or 13.6 μmol/kg) 30 min prior open-field training or elevated plus-maze test and 30 min or 0 h after training. No effects were observed in the anxiety parameters and habituation to an open-field. The ruthenium complexes impaired memory retention compared with vehicle group in the inhibitory avoidance, as when administrated 30 min prior as immediately after training. The memory impairment induced by ruthenium complexes may be due to their nitric oxide synthase inhibition capacity.     

Potentiation of the behavioral effects of pentobarbital, chlordiazepoxide and ethanol by thyrotropin-releasing hormone

Effects of pentobarbital, chlordiazepoxide and ethanol were studied alone and in combination with thyrotropin-releasing hormone (TRH), IM, on punished behavior. Key-peck responses of pigeons were maintained by food presentation under a fixed-interval 3-min schedule in which every 30th response produced shock. Moderate doses of pentobarbital, chlordiazepoxide and ethanol increased punished responding to 150-200% of control values while the higher doses of these drugs almost completely eliminated responding. TRH (0.01-1 mg/kg) had little effect on punished responding and 3 mg/kg produced 50% decreases. Although the lower doses of TRH were without effect when given alone, doses of 0.03 mg/kg and greater markedly potentiated the rate-increasing effects of pentobarbital, chlordiazepoxide and ethanol. Increases in punished responding of 350% were obtained with combinations of TRH and these drugs. The rate-decreasing effects of the sedative-hypnotic and anxiolytic compounds were not reversed by TRH. Potentiation of the behavioral effects of sedative-hypnotic and anxiolytic drugs by TRH suggests that TRH may play an important role in modulating the behavioral effects of these compounds and that combinations of neuroactive peptides with certain psychotherapeutic agents may be of some therapeutic value.     

Effects of chlordiazepoxide and beta-carboline 3-carboxylic acid ethyl ester on non-suppressed and minimally-suppressed responding in the squirrel monkey.

Lever-pressing of squirrel monkeys was maintained under a multiple fixed-interval (FI) 5-min schedule of food presentation. In one component, responding was suppressed to various degrees by the presentation of electric shock following each 30th response. When responding was either substantially or minimally suppressed, intermediate doses of chlordiazepoxide (CDAP, 1-30 mg/kg) increased both suppressed and non-suppressed responding. Beta-carboline 3-carboxylic acid ethyl ester (beta-CCE, 0.1-3 mg/kg) had little effect at low to intermediate doses (0.1-0.3 mg/kg) and decreased both minimally-suppressed and non-suppressed responding to a comparable extent at higher doses. Repeated daily dosing with beta-CCE (up to 10 mg/kg) resulted in rapid tolerance to its rate-decreasing effects. As agonists do not typically exhibit rapid tolerance for anxiolytic efficacy, the current results suggest that some behavioral effects of inverse agonists may not be strictly opposite those of benzodiazepines.     

Naloxone blocks conditioned place preference induced by substance P and [pGlu6]-SP(6-11).

The effect of prior treatment with the opioid receptor (opioceptor) antagonist naloxone on conditioned place preference produced by the neurotachykinin substance P (SP) and its C-terminal hexapeptide analog [pGlu6]-SP(6-11) (SPC) was investigated in rats. Place conditioning was assessed using a circular open field partitioned into four quadrants that were equally preferred by the rats prior to drug treatment. On three successive days, rats received an intraperitoneal (i.p.) injection of naloxone-HCl (1 mg/kg) or vehicle 15 min before an i.p. injection of either 37 nmol/kg SP, equimolar dosed SPC or corresponding diluent vehicle. After injection the rats were placed into their assigned treatment corral for 15 min. During the test for conditioned corral preference (CCP), when provided a choice between the four quadrants, rats injected with SP or SPC spent more time in the treatment corral compared to vehicle controls, indicative of a positive reinforcing action of these peptides. The pre-treatment with naloxone blocked the positive reinforcing effects of both SP and SPC; when injected alone, naloxone did not influence the preference behavior. Gross locomotor activity was affected by neither treatment. Thus, the positive reinforcing effects of SP and SPC may be mediated via interactions with the endogenous opioid system(s).     

Effects of chronic buspirone treatment on female mice exposed to the long-lasting psychoemotional influence

The effects of chronic treatment (30 days) with the 5-HT1A receptor partial agonist buspirone (0.05, 1 and 10 mg/kg i.p.) on the behaviour of C57BL/6J female mice exposed to long-lasting psychoemotional influence were studied. The influence involved forced living of each female with an aggressive male separated with a perforated transparent partition in the same cage and daily female's presence during 10-min intermale confrontations behind a partition caused by introducing of another male to the aggressive male. Chronic buspirone injection (in all used doses) did not affect the behaviour of females estimated in the "partitions" and "open field" tests at the end of the drug treatment. The anxiolytic effect of buspirone only at the dose of 1 mg/kg on the female's plus-maze behaviour was revealed. In the Porsolt, test buspirone in the dose of 1 mg/kg caused a slight increase in the duration of immobility indicating a slight pro-depressive effect. Thus, chronic buspirone treatment of females exposed to the long-lasting psychoemotional impact has a different effect on their behaviour depending on the dose and test conditions.     

Disruptive effects of naloxone on development of morphine tolerance

In rats the development of one-trial tolerance to the analgesic actions of morphine is disrupted by the post-administration of naloxone at 5 min, 3 hrs, or 24 hrs. Naloxone injections alone 24 or 48 hrs prior to the analgesic test failed to counteract morphine induced analgesia. It is suggested that naloxone initiates long term biochemical changes that oppose those produced by morphine.     

Effects of chlordiazepoxide on drinking compared in rats challenged with hypertonic saline, isoproterenol or polyethylene glycol

Several investigators have shown that anxiolytic benzodiazepines stimulate additional water consumption in rats made thirsty by water deprivation. The present report extends this work by showing that chlordiazepoxide (CDP) enhanced drinking in rats challenged with either cellular or extracellular dehydration, following hypertonic saline or polyethylene glycol injection respectively. Since CDP also increased drinking in control animals, it may have produced a direct dipsogenic effect which acted additively with respect to the physiological thirst challenges. In contrast, CDP did not enhance water intake during the dipsogenic action of the beta-adrenergic agonist, isoproterenol. The data provide new evidence that benzodiazepine mechanisms may be involved in thirst and the controls of drinking.     

Influence of semax on the emotional state of white rats in the norm and against the background of cholecystokinin-tetrapeptide action

The effects of the adrenocorticotropic hormone (ACTH(4–10)) analog, Semax (MEHFPGP), on the level of anxiety and depression in white rats have been studied in the normal state and against the back-ground of cholecystokinin-tetrapeptide (CCK-4) action. Semax was injected intranasally in doses of 50 and 500 μg/kg 15 min before the testing. CCK-4 was administered intraperitoneally in a dose of 400 μg/kg 40 min before the testing. The level of anxiety was estimated in the elevated plus-maze test, and the degree of depression, in the forced swimming test. Semax administration did not influence the emotional state of animals in the normal state. The CCK-4 injection led to an increase in anxiety and depression in rats. Semax normalized the animal behavior disturbed by the CCK-4 administration, which attests to its anxiolytic and antidepressant effects at elevated levels of anxiety and depression.