Confusion and countermeasures of the non-conditioned anxiety test in rodent laboratory animals / 中华行为医学与脑科学杂志

Anxiety is a major mood disorder, and the high morbidity, co-morbidity and disability of anxiety disorders seriously affect people's quality of life, so the importance and urgency of research on anxiety cannot be overstated. Animal models are the main carriers for studying the mechanism of disease occurrence and development, drug efficacy evaluation and drug development.Unconditioned anxiety model is a common anxiety model.Elevated plus maze test, open field test and light-dark box test are widely accepted paradigms for the detection of unconditioned anxiety.This kind of behavioral paradigm based on environmental exposure takes advantage of the conflict between curiosity and fear of the unfamiliar environment to simulate and detect the anxiety of animals.However, the validity of these behavioral paradigms for evaluating anxiety in animals is questionable.In this paper, we discuss the concept of anxiety, the definition of anxiety behavior in the behavioral test of unconditioned anxiety, and the factors to be considered in the test of unconditioned anxiety behavior.On this basis, new solutions were proposed to the contradictions and blind spots in order to improve the test paradigm of anxiety behavior and provide a more reliable animal model for the evaluation of anxiety.This paper presents a new approach to address the contradictions and blind spots of this paradigm.

Light/dark box as an animal model of state anxiety in Kunming mouse: Conditions and factors / 中国药理学通报

Aim To explore the factors on behavior patterns of light/dark box(LDB)as an animal model of state anxiety in Kunming mice.Methods The behavior of adult,male,Kunming mice in LDB was recorded for five minutes,respectively.The following parameters were evaluated:percentage of time in the light area(Ltime%),percentage of squares crossing in the light area(Lcross%),percentage of rears in the light area(Lrear%),total number of squares crossing in the whole apparatus(Cross),total number of rears in the whole apparatus(Rear),total(Cross plus Rear),transitions between two areas(Transition),and number of fecal bolis in light box and dark box(Fbs).Subsequently,the factors,such as day-night rhythm,illumination area(L3/5 or L2/5 for ratio between Light box and Dark box:3:2 or 2:3,respectively),illumination color(in Dark box)and illumination intensity(in Light box),were investigated to screen the best experimental conditions.Results t-test showed that compared with night cycle,there was no significant difference in all LDB parameters during day cycle(P>0.05),while compared with 3/5 and 2/5,LDB parameters during day cycle such as Lcross%(t=5.363,P0.05),but a statistical influence of illumination area on Ltime%(F(1,20)=18.361,P0.05).Conclusions LDB as an animal model of state anxiety in Kunming mice can evaluate anxiety-,locomotion-exploration and emotionality- related behaviors,which cannot be affected by day-night rhythm,but illumination area(Ltime%,Lcross%,Lrear%),illumination color(Rear)and illumination intensity(Cross,Total).So it is recommended to adopt the uniform and fixed conditions,such as illumination area,color and intensity(less than 100 W).

A comparative study on depression and anxiety of mice (Mus musculus) on a prebiotic-supplemented diet versus a standard diet during Unpredictable Chronic Mild Stress (UCMS)

@#<p style="text-align: justify;"><strong>Background.</strong> Anxiety and depression are becoming increasingly prevalent today and are often aggravated by day-to-day stresses. Because current management strategies are usually accompanied by unpleasant side effects, there is a need to look into alternative treatment regimens - such as prebiotics - that may provide equally effective anxiolytic and antidepressant effects.</p><p style="text-align: justify;"><strong>Objective.</strong> Therefore, the study aims to determine the effect of a combined fructooligosaccharide (FOS) and galactooligosaccharide (GOS) supplemented diet on anxiety and depression levels in mice subjected to Unpredictable Chronic Mild Stress (UCMS).</p><p style="text-align: justify;"><strong>Methods.</strong> Forty male BALB/C mice were subjected to UCMS under a pretest-posttest control group design where the treatment group received prebiotic supplementation throughout the study. Repeated measures ANOVA was run to evaluate between, within, and time interactions of the measured anxiety parameters using the light-dark box test, and depression parameter using the fur coat state assessment.</p><p style="text-align: justify;"><strong>Results.</strong> Results show that (1) the FOS + GOS treatment did not give the treatment group an advantage over the control group during UCMS, (2) both groups grew more anxious and depressed over time, and (3) the treatment group grew more anxious with time in relation to control in terms of the total time spent in the light side.</p><p style="text-align: justify;"><strong>Conclusion.</strong> These imply that the UCMS protocol was successful in inducing stress in mice, but the FOS + GOS regimen failed to provide anxiolytic and antidepressant effects on male BALB/C mice exposed to UCMS.</p>

Development of a computer-aided-controlling and image analysis system for light/dark box test in mice and rats / 中国比较医学杂志

Objective To develop a computer-aided-controlling and analysis system for light/dark box in mice and rats with a high degree of automation and intelligence.Methods Video recording and image processing were applied to develop the computer-aided-controlling and image analysis system for light/dark box test in mice and rats. The artificial environment was developed. The stability and reliability of the system was validated by male rats. Results The percentage of time spent in the lit chamber in total time was above 79.40%. The data showed that the artificial environment was successful. When the threshold was set at 18 cm/s, the data showed a high correlation coefficient of movement time between the computer and manual recordings(r > 0.99). Classical indexes including transition and time spent in both the lit and dark chambers also showed a high correlation. The model group showed a significantly decrease in the transitions and time spent in the lit chamber compared with the control group, indicating a high stability and reliability of the light/dark box test. Conclusions A stable and highly intelligent computer-aided-controlling and image analysis system for light/dark box test of mice and rats has been developed,and it could be used for pathological mechanism studies of anxiolytics.

Activation of locus coeruleus heme oxygenase-carbon monoxide pathway promoted an anxiolytic-like effect in rats

The heme oxygenase-carbon monoxide pathway has been shown to play an important role in many physiological processes and is capable of altering nociception modulation in the nervous system by stimulating soluble guanylate cyclase (sGC). In the central nervous system, the locus coeruleus (LC) is known to be a region that expresses the heme oxygenase enzyme (HO), which catalyzes the metabolism of heme to carbon monoxide (CO). Additionally, several lines of evidence have suggested that the LC can be involved in the modulation of emotional states such as fear and anxiety. The purpose of this investigation was to evaluate the activation of the heme oxygenase-carbon monoxide pathway in the LC in the modulation of anxiety by using the elevated plus maze test (EPM) and light-dark box test (LDB) in rats. Experiments were performed on adult male Wistar rats weighing 250-300 g (n=182). The results showed that the intra-LC microinjection of heme-lysinate (600 nmol), a substrate for the enzyme HO, increased the number of entries into the open arms and the percentage of time spent in open arms in the elevated plus maze test, indicating a decrease in anxiety. Additionally, in the LDB test, intra-LC administration of heme-lysinate promoted an increase on time spent in the light compartment of the box. The intracerebroventricular microinjection of guanylate cyclase, an sGC inhibitor followed by the intra-LC microinjection of the heme-lysinate blocked the anxiolytic-like reaction on the EPM test and LDB test. It can therefore be concluded that CO in the LC produced by the HO pathway and acting via cGMP plays an anxiolytic-like role in the LC of rats.

Locomotor Behaviour and Anxiety in the Open Field and Light/Dark Box in CD1 Mice Treated with Aspirin, Cataflam and Ethanolic Extract of Cannabis sativa.

Aims: To study the effects of cataflam, aspirin and the ethanolic extract of Cannabis sativa on nociception in CD1 albino mice of both sexes. Methods: Twenty (20) albino mice were divided into four (4) groups of five (5) each. The control group (group 1) received normal saline orally. Meanwhile groups 2-4 received p.o. cataflam (1.5 mg/kg), aspirin (13.5mg/kg) and Cannabis sativa (10 mg/kg) respectively. All four (4) groups were given access to normal mice chow and water ad libitum. The Open field apparatus and the light/dark box were used to measure locomotor/exploratory behaviour and anxiety. Results: There was a significant (p=0.05) reduction in the frequencies of rearing, walling and line crossing in the Open field test and a reduction in frequency of transition, rearing and line crossing in the light/dark box. There was no significant difference in the chamber durations in the light/dark box test. Conclusion: Cataflam, Aspirin and the ethanolic extract of Cannabis sativa all have anxiogenic effect and reduced locomotor behaviour. The ethanolic extract of Cannabis sativa seems to have the greatest anxiogenic effect, followed closely by Cataflam and Aspirin.

A novel fear conditioning memory model formation and erasing by foot-shock in tree shrew / 安徽医科大学学报

Objective To investigate the way of fear conditioning memory model evoked and erased by foot-shock in tree shrew. Methods First, detect the tree shrew activities regularly in light/dark box. Second, test a suitable voltage degree of foot shock on tree shrew. Third, investigate the memory formation and erasing of fear conditioning on tree shrew of trial group. Results The duration of tree shrew (n=4) stay in the dark-box was significantly lon-ger than that of in the light box (P<0. 01) in normal condition. In the same environment of two light boxes, given different voltage degrees, the durations of tree shrew (n=6) stay in the stimulating chamber gradually reduced and the durations of tree shrew stay had significant difference between stimulatus chamber and no stimulatus chamber when the stimulus voltage up to 12 V ( P<0. 05 ) , 16 V ( P<0. 01 ) and 20 V ( P<0. 01 ) . The animal of trial group ( n=4 ) could build up the fear conditioning memory of the dark box with the stimulus of 16 V foot-shock in the dark box ( P<0. 001 ) . After formation of the fear conditioning memory, the same stimulus in light box ap-peared for 4 days. The durations of tree shrew stay in trial group (n=4) decreased in light box, and there was no significant difference between the trial group and the control group. Conclusion Tree shrew prefers to stay in the dark box. The suitable voltage for foot-shock on tree shrew is 16 V. The fear conditioning memory can be evoked and erased by foot-shock.

Influence of 8-alkyl-berberine on ethology and neurotransmitter in brain tissue of anxiety model mice / 中草药

Objective: To investigate the anti-anxiety effect of 8-alkyl-berberine in anxiety model mice and explore the mechanism. Methods: The anti-anxiety effect of 8-alkyl-berberine was evaluated with light-dark box test and elevated plus-maze (EPM). The contents of norepinephrine (NE), dopamine (DA), and 5-hydroxytryptamine (5-HT) in brain tissue were detected by high performance liquid chromatography-fluorescence detector (HPLC-FLD). Results: The light-dark box test indicated that 8-butyl-, 8-octyl-, 8-dodecyl-, and 8-hexadecyl-berberine could obviously increase the time spent in light box (P < 0.05) compared with the control group. The number of transitions in 8-octyl-berberine group was significantly increased (P < 0.05). The results of EPM showed that 8-octyl-berberine significantly increased the percentage of the times entering the open arms (OE%) and staying duration in the total period (OT%) (P < 0.05). There was variation observed in the concentration of monoamine neurotransmitters of 8-octyl-berberine. Conclusion: 8-Alkyl-berberine has a certain anxiolytic effect. The mechanism may be related to the regulation of the contents of NE, DA and 5-HT in the brain tissues.

Correlation between two animal models of state anxiety in Kunming mice: evidence from open field test and light-dark box / 中华行为医学与脑科学杂志

ObjectiveTo explore the correlation between open field test(OFT) and light-dark box (LDB),as two animal models of state anxiety in Kunming mice.MethodsThe behavior of adult,male,Kunming mice in OFT and LDB was recorded by sequence,for five minutes,with a one-week inter-trial interval.The following parameters were evaluated:percentage of time exploring in the OFT central area( OFT-Ctime％ ) ; percentage of squares crossing in the OFT central area( OFT-Ccross％ ) ; total number of squares crossing in OFT(OFT-Cross) ;total number of rears in the OFT(OFT-Rear) ; number of fecal boli in OFT(OFT-FB) ; percentage of time exploring in the LDB light area( LDB-Ltime％ ) ; percentage of squares crossing in the LDB light area( LDB-Lcross％ ) ;and percentage of rears in the LDB light area( LDB-Lrear％ ) ; transitions between two areas in LDB( LDB-Transition) ; total number of squares crossing in LDB(LDB-Croas) ; total number of rears in LDB(LDB-Rear) ; number of fecal boli in LDB (LDB-FB).Subsequently,factor analysis,cluster analysis and correlation analysis were calculated for these parameters.ResultsFactor analysis and cluster analysis of variances from either OFT or LDB revealed a three behavioral dimensions:anxiety factor ( loaded by OFT-Ctime％,OFT-Ccross％ or LDB-Ltime％,LDB-Lcross％,LDB-Lrear％ ),activity factor ( loaded by OFT-Cross,OFT-Rear or LDB-Transition,LDB-Cross,LDB-Rear) and emotionality factor(loaded by OFT-FB or LDB-FB).While all the variances from both OFT and LDB in combination were analyzed,it could be seen as five components:' LDB-anxiety factor ( loaded by LDBLtime％,LDB-Lcross％ and LDB-Lrear％ ),' OFT-anxiety factor ( loaded by OFT-Ctime％ and OFT-Ccross％ ),'LDB-activity factor (loaded by LDB-Transition,LDB-Cross and LDB-Rear),'OFT-LDB-emotionality factor( loaded by OFT-FB and LDB-FB) and 'OFT-activity factor (loaded by OFT-Cross and OFT-Rear).Good correlation were found between OFT/LDB factors alone and in combination,such as OFT-emotionality factor and LDB-emotionality factor(Pearson =0.383,P＜0.05),LDB-anxiety factor and 'LDB-anxiety factor( Pearson =0.989,P＜0.01 ),OFT-anxiety factor and 'OFT-anxiety factor( Pearson =0.934,P ＜ 0.01 ),LDB-activity factor and 'LDB-activity factor ( Pearson =0.956,P ＜ 0.01 ),OFT/LDB-emotionality factor and ' OFT-LDB-emotionality factor ( Pearson =0.835,P＜0.01 ;Pearson =0.696,P＜0.01 ),OFT-activity factor and 'OFF-activity factor( Pearson =0.926,P＜0.01 ).ConclusionEither OFT or LDB comprised three behavioral dimensions:anxiety factor,activity factor and emotionality factor; however,it was difficult to establish face validity as a point-to-point concordance between OFT and LDB.Attention should be paid to heterogeneity of animal models when OFT and LDB were in combination as a behavionomics to evaluate anxiolytics in Kunming mice.

Light-dark box as a model of state anxiety in Kunming mice:three behavioral dimensions by factor analysis / 中华行为医学与脑科学杂志

Objective To explore the behavioral dimensions and validity of the light-dark box(LDB),as an model of state anxiety in Kunming mice.Methods The behavior of adult,male,Kunming mice in LDB was recorded twice,for five minutes,with a one-week inter-trial interval.The following parameters were evaluated by factor analysis:latence for the first entry into the dark area(Latence);percentage of time exploring in the light area (Ltime%);percentage of squares crossing in the light area(Lcross%);percentage of rears in the light area (Lrear%);transitions between two areas(Transition);total number of squares crossing in the whole apparatus (Cross);total number of rears in the whole apparatus(Rear);and number of fecal boli(FB).Results There was a good intercorrelation both among Ltime%,Lcross%,Lrear%,and among Transition,Cross,Rear.A good correlation also was found between test and retest for Transition,Rear and FB.Ltime%,Lcress%and Lrear%had significant loadings on Anxiety factor(Loading%:39.54%,35.40%);Transition loaded on Locomotor factor(Loading%:23.78%,19.66%),combined with Cross;Rear loaded on Exploration factor(Loading%:13.75%,22.60%);and FB loaded on Emotionality factor(Loading%:14.42%,13.02%);Latence was not stable.Conclusion Light-dark box,as an animal model of state anxiety in Kunming mice,comprised three behavioral dimensions:anxiety factor,LDB-activity factor(locomotor factor and exploration factor)and emotionality factor.

Light-dark box as a model of state anxiety in Kunming mice: test-retest reliability and correlations between parameters / 中华行为医学与脑科学杂志

Objective To explore the test-retest reliability and correlations of the light-dark box (LDB).MethodsThe behavior of mice in LDB was recorded twice, for five minutes,with a one-week inter-trial interval.Following parameters were evaluated:latency time for the first entry into the dark area (Latence); percentage of time exploring in the light area ( Ltime％ ); percentage of squares crossing in the light area ( Lcross％ ); total number of squares crossing ( Cross ); percentage of rears in the light area ( Lrear％ ); total number of rears (Rear); total score (Total); transitions between two areas (Transition); and number of fecal boli (Fbi). Subsequently,the intraclass correlation coefficient (ICC), the Kappa agreement coefficient (Kappa) and the Pearson correlation coefficient (Pearson) were calculated for these parameters. Results ①ICC for Lrear％ ( ICC =0.5207, P＜0.01 ) showed a fair reliability. ②Kappa for all parameters indicated a poor reliability. ③Pearson displayed weak negative correlations between Transition and Ltime％ ( Pearson = - 0.4030, P ＜ 0.05; Pearson =- 0. 1490, P＞ 0. 05 ), Lcross％ ( Pearson = - 0. 4331, P ＞ 0. 05; Pearson = - 0.1893, P ＞ 0.05 ), Lrear％ ( Pearson = - 0. 4485, P ＜ 0. 05; Pearson = 0. 02845, P ＞ 0. 05 ), however, fair positive correlations between Transition and Cross (Pearson =0. 8603, P＜0.01; Pearson =0.6341, P＜0. 01 ) ,Rear(Pearson =0.4271, P＞0. 05;Pearson = 0. 7245, P ＜ 0. 001 ). Conclusion Rear is found to be stable over time, and indicated a fair reliability.There is a weak negative correlation between Transition and Ltime％, Lcross％, Lrear％, but fair positive correlations between Transition and Cross, Rear.