White Test Box Research Articles

NEUROPROTECTIVE EFFECTS OF POMEGRANATE JUICE ON LEAD INDUCED NEUROTOXICITY IN MICE

<p><strong>Objective: </strong>This study evaluates the potential neuroprotective of the pomegranate juice against chronic intoxication with lead acetate for 3<strong> </strong>months.</p><p><strong>Methods: </strong>Twenty-one female Swiss mice divided into 3 groups were employed in the present investigation. Control group: received drinking water for 90 days, neurotoxic group were exposed to 1000 ppm of lead acetate in the drinking water for 12 weeks, and neurotoxic treated group represents the mice received treatment with juice pomegranate diluted with distilled water (v/v) orally for 4 h / day followed by lead acetate at a dose of 1000 ppm orally for 20 h / day for 90 days. After cessation of treatment, neurobehavioral studies using the open field test, black and white test box and swimming test were made. In the next phase, brain injury was assessed histologically with hematoxylin-eosin staining.</p><p><strong>Results:</strong> Chronic exposure to lead led to significant increase in the level of anxiety, depression and the locomotor activity (P < 0.05). It was confirmed by histopathological alterations in many areas of the cerebral cortex and hippocampus including neuronal degeneration and decrease cell density. Treatment with the juice significantly improve the level of depression, locomotor function (P < 005) and anxiety (P > 0.05) in mice exposed to lead as well as restored the histological structure in cerebral cortex and hippocampus of mice. The total phenolic and flavonoids content in juice of pomegranate was found to be 3809. 8±29.404 mg GAE/l; 2109. 57±18.936 mg QE /l of juice.</p><p><strong>Conclusion: </strong>This finding suggests that phenolic compounds found in pomegranate juice provide a neuroprotective effect on behavioural impairments and histopathological change induced by lead.</p>

Zacopride: anxiolytic profile in rodent and primate models of anxiety.

Zacopride, a substituted benzamide derivative, was compared with diazepam in three models of experimental or provoked anxiety. The drug's action (i) in reducing aversion to a brightly lit environment was assessed in mice using a two compartment black and white test box system, (ii) in disinhibiting a suppressed behaviour was measured in the rat social interaction test under high light/unfamiliar conditions and (iii) in antagonizing a defensive response in the marmoset was assessed using the threat of a human presence. Both zacopride and diazepam enhanced exploratory behaviour and social interaction in the mouse and rat models and antagonized the defensive response in the marmoset, zacopride being 100 times more potent than diazepam. It is concluded that the 5-HT3 receptor antagonist, zacopride, alters rodent and primate behaviour in a manner consistent with that of an anxiolytic agent.

Interactions of buprenorphine and dipotassium clorazepate on anxiety and memory functions in the mouse

Buprenorphine, a partial mu-receptor agonist widely substituted for heroin in the treatment of addiction, is often misused in combination with benzodiazepines. Improved hedonic properties may result, but only at the cost of increased buprenorphine toxicity. In order to elucidate the appeal of the benzodiazepine-buprenorphine combination, the present study looked at its neuropsycho-pharmacological effects on various emotional and cognitive parameters in the mouse. On the basis of previous dose–response studies, the regimen used was buprenorphine 0.3 mg/kg, s.c. plus dipotassium clorazepate 1, 4 and 16 mg/kg, i.p. Anxiety-like behaviour was assessed using the black and white test box, and memory processes were examined via the spontaneous alternation paradigm in the Y-maze, and passive avoidance tests. Spontaneous locomotor activity was also evaluated. High doses of clorazepate impaired buprenorphine-induced hyperactivity and anxiogenic-like effects. They also increased buprenorphine-induced spontaneous alternation impairment, but did not modify its impact on long-term memory processes. These results suggest that the positive reinforcement experienced with the buprenorphine–benzodiazepine combination may be attributable, at least in part, to an increase in buprenorphine's sedative effect associated with a decrease in anxiogenicity.

Reduced anxious behavior in mice lacking the CCK2 receptor gene

Cholecystokinin 2 (CCK2) receptors have been implicated as mediators of anxiety in standard mouse models such as exploratory behavior both in black and white test boxes and in elevated plus-mazes. We investigated the role of the CCK2 receptor in anxiety by evaluating the behavior of mice lacking the gene for this receptor in these standard anxiety models (i.e., exploratory behavior in a black and white test box and exploratory behavior in an elevated plus-maze). In the black and white test box, mice lacking the CCK2 receptor gene showed significantly increased numbers of transitions between the boxes compared to control mice. In the elevated plus-maze, mice lacking the CCK2 receptor gene displayed significantly more head dips than control mice. These results suggest that mice lacking the CCK2 receptor gene are less anxious than normal mice.

Reduced anxiety-like and cognitive performance in mice lacking the corticotropin-releasing factor receptor 1

Corticotropin-releasing factor (CRF) has been hypothesized to be involved in the pathophysiology of anxiety, depression, cognitive and feeding disorders. Two distinct CRF receptor subtypes, CRFR1 and CRFR2, are thought to mediate CRF actions in the CNS. However, the role for each receptor subtype in animal models of neuropsychiatric disorders remains to be determined. Using CRFR1 deficient mice, the present study investigated the functional significance of this CRF receptor subtype in anxiety-like and memory processes. CRFR1 knockout mice displayed an increased exploratory behavior in both the Elevated Plus-maze (EPM) and the Black and White (B–W) test box models of anxiety, indicating an anxiolytic-like effect of the CRFR1 gene deletion. In contrast, during the retrieval trial of a two-trial spatial memory task wild type mice made more visits to and spent more time in the novel arm as opposed to the two familiar ones of a Y-maze apparatus. No increase in the level of exploration of the novel arm by the CRFR1 deficient mice was observed. This indicates that CRFR1 knockout mice are impaired in spatial recognition memory. These results demonstrate that genetic deletion of the CRFR1 receptor can lead to impairments in anxiety-like and cognitive behaviors, supporting a critical role for this receptor in anxiety and cognitive biological processes.

Acute stress enhances anxiolytic-like drug responses of mice tested in a black and white test box

Drug-induced facilitation of exploration of a two-compartment black and white test box was studied in mice applying different stressors before test. Tail suspension suppressed exploration of both compartments and enhanced diazepam- or eltoprazine-induced increase of exploration of the white compartment. (−)-Penbutolol reversed the effects of eltoprazine suggesting involvement of 5-HT 1A/1B receptors. Buspirone had weak effects both under standard conditions, and after tail suspension. Ritanserin suppressed exploratory behavior, and this effect was potentiated by tail suspension. In conclusion, application of acute tail suspension stress immediately before test in the black and white box enhanced the sensitivity to drugs. In contrast to this, forced swimming suppressed general behavioral activity, but in this case diazepam facilitated the behavior in both compartments. Foot shocks immediately before test reduced activity in the black compartment and did not affect the behavior in the white compartment. Thus different acute stressors modified behavior in the black and white box differently. Chronic stress (crowded housing) produced an anxiolytic-like response.

Behavioral profiles of SSRIs in animal models of depression, anxiety and aggression. Are they all alike?

The behavioral profiles of five clinically used selective serotonin reuptake inhibitors (SSRIs) citalopram, paroxetine, sertraline, fluvoxamine and fluoxetine, have been compared in animal models of antidepressant (mouse forced swim test), anxiolytic (exploration of black and white test box and foot-shock-induced ultrasonic vocalization in the rat) and antiaggressive (isolation-induced aggressive behavior in the mouse) activity. the results are discussed in relation to receptor binding data from the literature. Furthermore, affinities for the sigma 1 and sigma 2 binding sites are presented. Citalopram reversed the immobility induced by forced swimming with a potency similar to that of imipramine. Paroxetine, fluvoxamine and fluoxetine reversed swim-induced immobility less potently and with a maximum of 40-50% reversal. Citalopram produced a mixed anxiogenic-/anxiolytic-like response in rats tested in the two-compartment black and white box. Paroxetine induced an anxiogenic-like response at low doses and the other SSRIs were without major effects. Citalopram and paroxetine inhibited footshock-induced ultrasonic vocalization with high potencies. The dose-response curve was biphasic for citalopram with a maximum of 64% inhibition. Sertraline and fluvoxamine inhibited the vocalization less potently, and fluoxetine induced a weak inhibitory effect corresponding to a maximum of 32%. Sertraline, fluvoxamine and fluoxetine inhibited isolation-induced aggressive behavior, whereas citalopram and paroxetine were inactive. Both 5-HT1 and 5-HT2 receptors are involved, and there was a functional interaction between 5-HT1A and 5-HT2A or 5-HT2C receptors, as ritanserin potentiated the antiaggressive effect of 1,5-HTP as well as that of 8-OH-DPAT.

Serotonergic mechanisms involved in the exploratory behaviour of mice in a fully automated two-compartment black and white text box.

A fully automated version of the black and white two-compartment box for mice is presented. The anxiolytic-like effects of the benzodiazepines, diazepam and chlordiazepoxide, were confirmed, and the involvement of serotonergic mechanisms was studied in this animal model of anxiety. The partial 5-HT1A receptor agonists, buspirone and ipsapirone showed anxiolytic-like effects in a limited dose interval. The full agonist hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT) was inactive. The non-selective 5-HT1 receptor agonist, eltoprazine, induced marked increases of exploratory behaviour in the white compartment over a broad range of doses. Also pindolol a mixed 5-HT1A/1B and beta-adrenergic receptor antagonist showed anxiolytic-like effects, whereas another compound with a similar profile (-)-, penbutolol and the beta-adrenoceptor antagonist ICI 118,551, was inactive. The 5-HT2A/2C receptor antagonist, ritanserin, showed anxiogenic-like, and the 5-HT3 receptor antagonists, zacopride and ondansetron, showed anixiolytic-like effects. An overall increase of serotonergic activity by means of 5-HT uptake inhibition (citalopram), 5-HT release (fenfluramine) or administration of a 5-HT precursor (1-5-HTP) facilitated exploratory activity in the white compartment. Reduction of serotonergic activity by treatment with the 5-HT depletor p-chloro-phenylalanine methyl ester (PCPA) did not change the exploratory behaviour, but attenuated the response to fenfluramine significantly.

Effects of quinpirole on the behaviour shown by mice in the light-dark box and during social interactions

Quinpirole (0.25, 0.5 and 1.0 mg/kg) was administered by intraperitoneal injection to pair-housed adult DBA/2 mice. Controls received injections of physiological saline. Effects on behaviour during 5 min social encounters with untreated partners were examined by ethological procedures, commencing at 30 min after injection. Behaviour was examined in an aversive and less aversive situation, an unfamiliar neutral cage and the home cage. Behavioural effects were then assessed in a two-compartment black and white test box. Quinpirole dose-dependently increased the frequency and duration of flight, including the specific element “retreat”. At 0.5 mg/kg, the element, “freeze”, was also increased during encounters in the neutral cage. Immobility (a flaccid sitting posture) and sniffing of the substrate were increased by quinpirole to a similar extent at all dose levels, while non-social activity and social investigation were reduced. The significance of the effects of quinpirole in the home cage and neutral cage were qualitatively similar; the only quantitative differences were a greater enhancement of the duration of immobility and the frequency of substrate sniffing in the home cage. In the light-dark box, quinpirole reduced the number of transitions between light and dark compartments and decreased line crossings and scans/unit time in the light compartment, although it increased the amount of time in the light compartment into which mice had been originally placed. The induction of immobility and decrease of several active behavioural responses may arise from a D 2 autoreceptor inhibition of locomotor activity. The dose-related enhancement of flight by quinpirole may represent a useful model of fear-related behaviour which differs from behavioural effects induced by anxiogenic benzodiazepine inverse agonists.

Profiles of interaction of R(+)/S(-)-zacopride and anxiolytic agents in a mouse model

The mouse black and white test box was used to measure changes in behaviour in an aversive situation where the administration of R(+)-zacopride (but not S(-)-zacopride) alone decreased aversive responding to the white area. A similar anxiolytic profile of action was observed using parachlorophenylalanine (PCPA), whose effects were antagonised by a co-treatment with R(+)-zacopride and reversed by S(-)-zacopride to an exacerbation of the aversive response. An anxiolytic profile of action was also observed using ondansetron, granisetron, chlodiazepoxide, diazepam, ritanserin, 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), E4424 (2-[4-[4-(4-chloro-l-pyrazolyl)butyl]-l-piperazinyl]-pyrimidine), umepsirone, DuP753 (2-n-butyl-4-chloro-5-hydroxy-methyl-1-[2(1H-tetrazol-5-yl)biphenyl-4-yl)methyl)]-imidazole), SQ29,852 ((S)-1-[6-amino-2[hydroxy)(4-phenyl-butyl)phosphinyl]-oxy)-1-nexy]-2-proline), devazepide and guanfacine, and this was retained following co-treatment with PCPA. The anxiolytic profile of action of PCPA was also retained following co-treatment with renzapride which when administered alone failed to modify behaviour. However, the ability of chlordizepoxide, diazepam, ondansetron and E4424 (but not devazepide, DuP753 or SQ29,852) to reduce aversive responding was inhibited by co-treatment with R(+) and/or S(−)-zacopride. It is concluded that the reduction in aversive responding caused by pharmacological manipulation at the benzodiazepine, 5-HT receptor subtypes 5-HT 1A, 5-HT 1C/5-HT 2 and 5-HT 3 (but not at the cholecystokin CCK A or angiotensin receptors or inhibition of angiotensin converting enzyme) can be inhibited by R(+) and S(−)-zacopride. The data is discussed in terms of zacopride having an agonist or partial agonist effect at the 5-HT 3 receptor.

The interaction of R(+)- and S(-)-zacopride with PCPA to modify rodent aversive behaviour

The interaction of R( + )- and S( − )zacopride (0.00001–10 mg/kg i.p.) with parachlorophenylalanine (PCPA, 3 day treatment 100 mg/kg i.p.) to modify behaviour in an aversive situation was investigated in the mouse black and white test box and rat social interaction test. R( + )-Zacopride (but not S( − )zacopride) and PCPA had an anxiolytic profile of action to reduce aversive responding in both species. Their established anxiolytic profiles were abolished by a subsequent treatment with S( − )zacopride. In contrast, S( − )-zacopride was less or ineffective if administered simultaneously with R( + )-zacopride. A co-treatment of PCPA with R( + )-zacopride also inhibited the anxiolytic profiles observed to the individual treatments. It is concluded that there is a complex interaction between the isomers of zacopride to modify responding to an aversive situation that is dependent on the temporal sequence of drug administration, and which may be modified by changes in basal 5-hydroxytryptamine (5-HT) tone and agonist, partial agonist and antagonist effects at the 5-HT 3 receptor.

Effects of the benzodiazepine receptor inverse agonist, DMCM, on the behaviour of mice: An ethopharmacological study

Ethopharmacological procedures and a two-compartment black and white test box were used to examine behavioural effects produced by the benzodiazepine receptor inverse agonist, methyl-6,7- dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), when given by intraperitoneal injection (0.4 and 1.0 mg/kg) to pair-housed adult CD1 male mice. Control mice received injections of the solvent. Behaviour in the light-dark box was examined at 30 min after the injection and behaviour during encounters with an untreated group-housed male, in a neutral cage, was then assessed by ethological procedures. In dominant mice, aggressive behaviour was significantly reduced and the ratio of flight, relative to aggression received, was significantly increased by DMCM at 0.4 mg/kg. At 1 mg/kg but not 0.4 mg/kg, DMCM decreased time spent by dominant mice in the light compartment of the test box. In both dominant and subordinate mice, flight was increased by DMCM at 1 mg/kg to a level close to statistical significance. Treatment with DMCM had no other detectable effect on the behaviour of subordinate animals. It is suggested that anxiogenic activity of this compound might induce a shift of agonistic behaviour from aggression to “fear-induced flight”.

The effects of umespirone as a potential anxiolytic and antipsychotic agent

Umespirone was compared to buspirone, diazepam and clozapine as a potential anxiolytic and antipsychotic agent. In the mouse black and white test box, umespirone was considerably more potent than diazepam or buspirone to reduce aversive responding, tolerance to its effects was not observed and sedation was absent, a chronic treatment and withdrawal was not associated with an anxiogenic profile, and umespirone prevented the behavioural consequences of withdrawal from diazepam. Umespirone also had an anxiolytic profile of action in the tests of rat social interaction and in the marmoset exposed to a human threat. Both umespirone and clozapine reduced the hyperactivity induced by the infusion of dopamine into the nucleus accumbens of rat. In radioligand binding assays umespirone demonstrated nanomolar affinity for the α 1-adrenoceptor and the 5-HT 1A and dopamine D 2 receptors. It is concluded that umespirone may present as a novel psychotropic agent with anxiolytic and antipsychotic potential.

The effect of ketotifen in rodent models of anxiety and on the behavioural consequences of withdrawing from treatment with drugs of abuse

Ketotifen was compared to diazepam to inhibit aversive responding of the mouse in a black and white test box and in the rat social interaction test. Both drugs reduced aversive responding in the mouse to the brightly illuminated area of the test box and facilitated social interaction in the rat; ketotifen was approximately 100 times more potent than diazepam. The chronic administration of diazepam, ethanol, nicotine and cocaine in the mouse also reduced aversive responding but their withdrawal was associated with an increased behavioural suppression. The administration of ketotifen during the period of withdrawal from diazepam, ethanol, nicotine and cocaine prevented the exacerbation in aversive responding. It is concluded that ketotifen, like diazepam and 5-HT3 receptor antagonists, can reduce behavioural suppression in rodent models of anxiety and attenuate the behavioural consequences of withdrawal from treatment with drugs of abuse.

Behavioural effects in gerbils of the 5-HT3 receptor antagonists, BRL 43694 and ICS 205-930, under circumstances of high and low light intensity

The 5-HT3 receptor antagonists, BRL 43694 and ICS 205-930, were each given for 21 days in the drinking fluid at 1.3 mg/l (120 micrograms/kg daily), to Mongolian gerbils, while the controls received tap water to drink. Effects of the treatments in reducing aversion to a brightly lit environment were assessed on behaviour during social encounters with an unfamiliar untreated resident, under bright white light and in a two-compartment black and white test box, after 12-16 days of treatment. Effects on behaviour under dim red illumination, when encountering unfamiliar untreated residents, were examined after 17-19 days. Behaviour during social encounters was recorded by ethological procedures. During encounters under bright white light, the frequency and duration of the social element "attend" were increased by BRL 43694 and ICS 205-930 and the frequency of "nose" was increased by BRL 43694. In the light-dark box, BRL 43694, though not ICS 205-930, reduced the time spent in the dark compartment. Under dim red light, BRL 43694 and ICS 205-930 increased the occurrence of the social elements, "sniff", "follow" and "sniff chin", suggesting increased sensitivity to olfactory stimuli. Increases of social investigation were associated with compensatory changes to non-social behaviour. It is suggested that 5-HT3 receptor antagonists may, on the one hand, increase sensitivity to social stimuli under dim red illumination and, on the other hand, show an apparent anxiolytic potential, associated with increase of other elements of social investigation under the more aversive test conditions of bright white light.

Actions of ORG 5222 as a novel psychotropic agent

ORG 5222 is a tetracyclic compound with high affinity for dopamine and 5-HT 2 receptors. ORG 5222 was compared to fluphenazine in behavioural tests and was shown to be less potent to cause catalepsy on peripheral administration or to induce asymmetric body posturing following intrastriatal injection. On injection into the nucleus accumbens, ORG 5222 antagonised spontaneous and amphetamine-induced hyperactivity. The peripheral administration of ORG 5222 antagonised the hyperactivity induced by infusion of dopamine into the nucleus accumbens of rat or ventral striatum of the marmoset and, unlike the use of fluphenazine, there was no evidence of a ‘rebound’ hyperactivity after discontinuation of treatment. Furthermore, ORG 5222 prevented changes in responsiveness to dopamine agonist challenge following dopamine infusion. In a mouse black and white test box and the rat elevated plus maze ORG 5222 released exploratory behaviour suppressed by the aversive white or elevated environments. It is concluded that ORG 5222 is effective to antagonise mesolimbic dopamine function in the rodent and primate and an aversive behaviour in rodent tests. Such effects reveal a novel profile of action of ORG 5222 in behavioural paradigms predictive of antipsychotic and anxiolytic potential and may relate to a dopamine and 5-HT receptor antagonism.

Exploration of mice in a black and white test box: Validation as a model of anxiety

The validity of a black and white test box to measure changes in mouse exploratory behaviour relevant to assessment of anxiety was investigated by variation of the illumination within the test box, the use of different strains of mice, holding conditions and drug treatments. The suppression of exploratory activity in the white section caused by bright illumination was antagonised by anxiolytic agents from the benzodiazepine series, buspirone, 5-HT 3 receptor antagonists, alcohol, nicotine, morphine and SCH23390. The anxiogenic agent FG7142 exacerbated the behavioural suppression. Black C57/BL/6, brown DBA 2 and albino BKW mice were sensitive to the effects of drug treatments, whereas albino Tuck mice were less responsive. It is concluded that the characteristic change in mouse exploratory behaviour caused by anxiolytic agents is to preferentially increase exploratory behaviour in the white aversive section of the black and white test box. It is most consistently shown by (a) an increased time spent in the white section with proportional increases in (b) rearings and (c) ambulation and (d) a delay in the initial transition from the white to the black section.

Neuroanatomical sites of action of 5‐HT3 receptor agonist and antagonists for alteration of aversive behaviour in the mouse

1. The cerebral topography of the action of diazepam and the action of the 5-hydroxytryptamine 5-HT3 receptor antagonists GR38032F and ICS 205-930 in attenuating an aversive response was studied in the mouse. 2. Mice which had been cannulated to allow drug injection into the dorsal and median raphe nuclei, the amygdala, nucleus accumbens or caudate-putamen were placed in a two compartment black (dimly illuminated) and white (brightly illuminated) test box. Measurements were made of the time spent, rearing and line crossings in the two sections and the latency of initial movement from the white to the black area. 3. The injection of diazepam (0.1-10 ng), GR38032F (0.01-1.0 ng) and ICS 205-930 (1.0-10 ng) into the dorsal raphe nucleus and amygdala, and the injection of diazepam (0.1-10 ng) into the median raphe nucleus, reduced an aversive response to the brightly illuminated white area, delaying the initial movement into the black section and increasing the time spent, rearings and line crossings in the white area. Concomitantly such activities were decreased in the black section. 4. The injection of the 5-HT3 agonist 2-methyl-5-hydroxytryptamine (0.1-10 ng) into the dorsal raphe nucleus and amygdala caused the opposite response, decreasing the time taken to move into the black section and increasing the time spent, rearings and line crossings in the black section, decreasing such activities in the white area. 5. The 5-HT3 agonist and antagonists showed little or no effect following injection into the median raphe nucleus and there were no changes in exploratory behaviour following their injection, or injection of diazepam, into the nucleus accumbens or caudate-putamen. 6. It is concluded that in the mouse the cerebral topography of action of GR38032F and ICS 205-930 in attenuating an aversive response follows that of diazepam in the dorsal raphe nucleus and amygdala but that diazepam may have additional effects mediated via the median raphe nucleus.