Video Tracking System Research Articles

Evidence of active aggregation behaviour in Lucilia sericata larvae and possible implication of a conspecific mark

Vectors of aggregation are well known for some arthropod species, but not for many others. We aimed to describe larval aggregation (experiment 1) in the carrion fly, Lucilia sericata (Diptera: Calliphoridae), and to investigate the effect of food and conspecifics on larval behaviour (experiment 2). In experiment 1, 40 larvae were placed in a petri dish with a homogeneous diet for 30 min, 1 h, 3 h, 5 h or 24 h. This experiment demonstrated for the first time under controlled conditions the active aggregation of L. sericata larvae. The results indicate that the aggregation took place quickly and was reinforced with time. After only 3 h, one main aggregate comprising a majority of individuals was observed. These results also highlight the likely use by necrophagous larvae of a signal left by conspecifics as an aggregation vector. In experiment 2, we used a video-tracking system to investigate whether such an aggregative signal exists. Fed and starved larvae were tracked for 5 min in a circular area with each half marked with a different signal combination. The time spent in the signal zones, the distance travelled, the velocity, the time at the stop and the number of stops in each zone were measured. The larvae were significantly retained by a signal (mark) left by conspecifics. Together, the results of this study demonstrate the existence of a contact and/or odour-mediated signal involved in the aggregative behaviour of necrophagous larvae.

Behavioural and toxicological responses of Blattella germanica (Dictyoptera: Blattellidae) to monoterpenes

Certain monoterpenes produced by aromatic plants are known to have lethal and sublethal effects on insects. As there is a need to replace conventional pesticides (organophosphates, carbamates, pyrethroids) with biorational pesticides, we evaluated the behavioural and toxicological responses of the German cockroach, Blattella germanica, exposed to the following monoterpenes, namely (+)-a-pinene, (-)-a-pinene, limonene, menthone, linalool, menthyl acetate and geraniol. Locomotor activity and repellency were quantified using a video tracking system. To study the effect on locomotor activity, larvae were exposed to filter papers treated with concentrations of monoterpenes ranging between 1.4 and 1,400 µg/cm2. Only menthyl acetate applied at a concentration of 14 µg/cm2 or higher increased the locomotor activity of the larvae. Repellency was evaluated by exposing larvae to circles of filter paper divided in two equal zones: one zone was treated with a solution of a monoterpene in acetone (14 or 140 µg/cm2) and the other with only acetone. Larvae were repelled by all the monoterpenes tested. The two concentrations of geraniol and the highest concentration of menthyl acetate and linalool were as repellent as DEET (positive control). The fumigant effect was evaluated by exposing larvae to vapour from 100 µl of pure monoterpene in a sealed container. The values of the 50% Knock-down Times (KT50), expressed in minutes, were: (+)-a-pinene: 11.8, (-)-a-pinene: 14.6, limonene: 81.0, menthone: 141.0, and linalool: 238.6. (+)-a-Pinene was as good a fumigant as dichlorvos (positive control, KT50 = 9.7 min). The least effective compounds were menthyl acetate and geraniol (both affected less than 50% of larvae after 490 min of exposure). In conclusion, (+)-a-pinene (for its fumigant effect), menthyl acetate (for its hyperactivant effect) and menthone and geraniol (for their repellent properties) could potentially be used for controlling B. germanica.

IPhone® applications as versatile video tracking tools to analyze behavior in zebrafish (Danio rerio)

Zebrafish (Danio rerio) are emerging as a promising model organism for experimental studies relevant to biological psychiatry. The objective of this study was to develop a novel video-based movement tracking and analysis system to quantify behavioral changes following psychoactive drug exposure in zebrafish. We assessed the effects of withdrawal from chronic ethanol exposure, and subsequent administration of fluoxetine (Prozac®), buspirone (Buspar®), and diazepam (Valium) using two behavioral paradigms; the Novel Tank Diving Test and the Light/Dark Choice Assay. A video tracking system was developed using two Apple® applications (Apps) to quantify these behaviors. Data from zebrafish exposed to the above treatments are presented in this paper not only to exemplify behavioral alterations associated with chronic exposure, but also more importantly, to validate the video tracking system. Following withdrawal from chronic ethanol exposure, zebrafish exhibited dose/time-dependent anxiogenic effects; including reduced exploration and freezing behavior in the Novel Tank Diving Test, and preference for the dark area for the Light/Dark Choice Assay. In contrast, the above drug treatments had significant anxiolytic effects. We have developed a simple and cost-effective method of measuring zebrafish behavioral responses. The iPhone® Apps outlined in this study offer numerous flexible methods of data acquisition; namely, ease of identification and tracking of multiple animals, tools for visualization of the tracks, and calculation of a range of analysis parameters. Furthermore, the limited amount of time required for interpretation of the video data makes this a powerful high-throughput tool with potential applications for pre-clinical drug development.

Selective Anesthesia-induced Neuroinflammation in Developing Mouse Brain and Cognitive Impairment

: Recent population studies have suggested that children with multiple exposures to anesthesia and surgery at an early age are at an increased risk of cognitive impairment. The authors therefore have established an animal model with single versus multiple exposures of anesthetic(s) in young versus adult mice, aiming to distinguish the role of different types of anesthesia in cognitive impairment. : Six- and 60-day-old mice were exposed to various anesthesia regimens. The authors then determined the effects of the anesthesia on learning and memory function, levels of proinflammatory cytokine interleukin-6 and tumor necrosis factor-α in brain tissues, and the amount of ionized calcium-binding adaptor molecule 1-positive cells, the marker of microglia activation, in the hippocampus. : In this article, the authors show that anesthesia with 3% sevoflurane for 2 h daily for 3 days induced cognitive impairment and neuroinflammation (e.g., increased interleukin-6 levels, 151 ± 2.3% [mean ± SD] vs. 100 ± 9.0%, P = 0.035, n = 6) in young but not in adult mice. Anesthesia with 3% sevoflurane for 2 h daily for 1 day and 9% desflurane for 2 h daily for 3 days induced neither cognitive impairment nor neuroinflammation. Finally, an enriched environment and antiinflammatory treatment (ketorolac) ameliorated the sevoflurane-induced cognitive impairment. : Anesthesia-induced cognitive impairment may depend on developmental stage, anesthetic agent, and number of exposures. These findings also suggest the cellular basis and the potential prevention and treatment strategies for anesthesia-induced cognitive impairment, which may ultimately lead to safer anesthesia care and better postoperative outcomes for children.

Simultaneous recordings of ocular microtremor and microsaccades with a piezoelectric sensor and a video-oculography system

Our eyes are in continuous motion. Even when we attempt to fix our gaze, we produce so called “fixational eye movements”, which include microsaccades, drift, and ocular microtremor (OMT). Microsaccades, the largest and fastest type of fixational eye movement, shift the retinal image from several dozen to several hundred photoreceptors and have equivalent physical characteristics to saccades, only on a smaller scale (Martinez-Conde, Otero-Millan & Macknik, 2013). OMT occurs simultaneously with drift and is the smallest of the fixational eye movements (∼1 photoreceptor width, >0.5 arcmin), with dominant frequencies ranging from 70 Hz to 103 Hz (Martinez-Conde, Macknik & Hubel, 2004). Due to OMT’s small amplitude and high frequency, the most accurate and stringent way to record it is the piezoelectric transduction method. Thus, OMT studies are far rarer than those focusing on microsaccades or drift. Here we conducted simultaneous recordings of OMT and microsaccades with a piezoelectric device and a commercial infrared video tracking system. We set out to determine whether OMT could help to restore perceptually faded targets during attempted fixation, and we also wondered whether the piezoelectric sensor could affect the characteristics of microsaccades. Our results showed that microsaccades, but not OMT, counteracted perceptual fading. We moreover found that the piezoelectric sensor affected microsaccades in a complex way, and that the oculomotor system adjusted to the stress brought on by the sensor by adjusting the magnitudes of microsaccades.

Impact of psychosocial stress on airway inflammation and its mechanism in a murine model of allergic asthma

Background It has already been recognized that psychosocial stress evokes asthma exacerbation; however, the mechanism of how stress gets inside the body is not clear. This study aimed to observe the impact of psychosocial stress on airway inflammation and its mechanism in the ovalbumin-induced asthmatic mice combined with social disruption stress. Methods Thirty-six male BALB/c mice were randomly divided into: control group, asthma group (ovalbumin-induced), asthma plus social disruption stress group (SDR), and SDR group. The open field video tracking system was used to assess animal behaviors. The invasive pulmonary resistance (RL) and dynamic lung compliance (cdyn) test system from Buxco was applied to detect pulmonary function. The enzyme-linked immunosorbent assay (ELISA) was utilized to determine OVA-IgE, T-helper type 2 (Th2) cytokines (IL-4, IL-5, IL-13) and corticosterone in mouse serum, the Th2 cytokines (IL-4, IL-5, IL-13, IL-6, TNF-α) in bronchoalveolar lavage fluid (BALF), and IL-6 and TNF-α levels in the supernatant of splenocytes cultured in vitro. Hematoxylin-eosin (H&E) staining was used to assess airway inflammation in lung histology. The cell count kit-8 assay (CCK-8) was applied to evaluate the inhibitory effect of corticosterone on splenocyte proliferation induced by lipopolysaccharide (LPS). Real time-PCR and Western blotting were utilized to determine glucocorticoid receptor (GR) mRNA and GR protein expression in lungs. Results The open field test showed that combined allergen exposure and repeated stress significantly shortened the time the mice spent in the center of the open field (P <0.01), increased ambulatory activity (P <0.01) and the count of fecal boli (P <0.01), but deceased vertical activity (P <0.01). Results from pulmonary function demonstrated that airway hyperresponsiveness (AHR) was enhanced by psychosocial stress compared with allergy exposure alone. The ELISA results showed that cytokines in serum and BALF were significantly increased (P <0.05). Moreover, the lung histology showed that infiltrated inflammatory cells were significantly increased in the asthma-SDR group compared with the asthma group (P <0.05). Interestingly, serum corticosterone was remarkably raised by psychosocial stress (P <0.05). In addition, the inhibitory effect of corticosterone on IL-6 and TNF-α in LPS-stimulated splenocyte cultures in vitro was diminished in the asthma-SDR group compared to the asthma group. The CCK-8 test revealed that the inhibition effect of corticosterone on splenocyte proliferation induced by LPS was significantly impaired in the SDR and asthma-SDR groups, while no significant effect was observed in the control and asthma groups. Furthermore, expression of GR mRNA and GR protein were significantly reduced in the lung tissues of the asthma-SDR group (P <0.05). Conclusions Social disruption stress can promote anxiety behavior, activate the hypothalamic-pituitary-adrenal (HPA) axis, increase AHR and inflammation, and also impair glucocorticoid sensitivity and its function in a murine model of asthma. The down-regulation of GR expression induced by social disruption stress is in part associated with glucocorticoid insensitivity, which leads to asthma exacerbation.

Memory-enhancing effects of the leptin hormone in Wistar albino rats: sex and generation differences

Leptin is a hormone secreted by adipose tissue that informs the brain about the fat stores of the body. In this study, we examined the effects of timed leptin injections on spatial memory performances of adult and juvenile male and female Wistar rats with the Morris water maze test. We applied the injections and conducted the training trials for 4 days. On the fifth day after leptin injections, the experiments were performed. The activities were recorded and analyzed with the Noldus Ethovision Animal Video Tracking System. The results show that the spatial memory performance of the rats was improved by the leptin injections since they shortened the latency to find the platform and elongated the time spent in the correct quadrant. The age of the rats was found to be important since the adults found the platform faster than the juveniles. There was an interaction effect between sex and generation in all parameters examined except for the time spent in the correct quadrant. The results of the present study suggest that leptin administration via timed injections strengthens spatial memory in Wistar albino rats, but this effect depends upon the sex and the generation of the rats.

VTS를 이용한 두류종실에 대한 노린재류의 선호성 평가

Preference of stink bugs to various leguminous seeds was evaluated by using VTS (Video Tracking System) in laboratory. Major soybean stink bugs such as bean bug, Riptortus pedestris (Fabricius), one-banded stink bug, Piezodorus hybneri (Gmelin), eastern green stink bug, Nezara antennata (Scott), and sole bug, Dolycoris baccarum (L.) were significantly most attracted to Cheongjakong, a soybean variety, baited fish-net trap, followed by soybean varieties Ilpumgeomjeongkong and Taekwangkong, a peanut variety Daekwangdangkong, a kidney bean variety Gangnangkong, and a adzuki bean variety Jungwonpat, respectively in a soybean field. VTS observation in laboratory showed that R. pedestris and D. baccarum had significantly higher frequency of visit on Cheongjakong, followed by Ilpumgeomjeongkong. However, P. hybneri, N. antennata and Halyomorpha halys (Stal) had significantly higher number of visits on Cheongjakong, Seonnogkong and Jinpumkong, followed by Ilpumgeomjeongkong. The sojourned time of stink bugs, however, was significantly longer on Cheongjakong regardless of species of the bugs. Accordingly, Cheongjakong was evaluated as the most preferred soybean seed by fish-net trap and VTS. Thus, VTS is found to be an effective means to evaluate the food preference of stink bugs.

Survival and behavioural response to acaricides of the coconut mite predator Neoseiulus baraki

The coconut mite, Aceria guerreronis Keifer, is a major pest of coconut palm in the world. The control of this pest species is done through acaricide applications at short time intervals. However, the predators of this pest may also be affected by acaricides. Among the predators of A. guerreronis, Neoseiulus baraki (Athias-Henriot) has potential for biological control. The objective of this study was to assess the effect of acaricides on the survival and behavior of N. baraki. The survivorship of N. baraki was recorded in surface-impregnated arenas. Choice and no-choice behavioral bioassays were carried out using a video tracking system to assess the walking behavior of the predator under acaricide exposure. Although all acaricides negatively affected the survival of N. baraki, chlorfenapyr and azadirachtin caused lower effect than the other acaricides. No significant differences in walking behavior were observed under exposure to fenpyroximate, chlorfenapyr and chlorpyrifos on fully-contaminated arenas. Azadirachtin and chlorpyrifos caused repellence. Irritability was observed for all acaricides, except for abamectin. Chlorfenapyr was the most suitable product for managing the coconut mite because of its low effect on survival and behavior of N. baraki.

<em>C. elegans</em> Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

<em>C. elegans</em> Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior, locomotion, and general mutational phenotyping. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

Early impairment in a water-finding test in a longitudinal study of the Tg2576 mouse model of Alzheimer's disease

Behavioral assessments of mouse models of neurodegenerative disorders are useful for investigating the molecular basis of the pathologies of the diseases. Here, we investigated the utility of a water-finding test using a video tracking system as a tool for evaluating cognitive deficits in Alzheimer's disease model mice. Transgenic mice expressing mutant amyloid precursor protein that incorporated the Swedish mutation (Tg2576 mice) were tested for behavioral alterations at 3, 5, 6, or 10 months of age. Tg2576 mice, which are widely used as a model of Alzheimer's disease, exhibited significant cognitive deficits in the water-finding test as early as 5 months of age. The impairments progressively worsened at 6 and 10 months of age. In addition, we analyzed spontaneous physical activities, such as locomotor activity, in the home-cage environment with an automated video analysis system (HomeCageScan). Our longitudinal study revealed that spontaneous behavior was altered in the Tg2576 mice, starting at the age of 10 months. Impairment in the Morris water maze (MWM) task was also first observed in the Tg2576 mice at the age of 10 months. These results indicated that the ability to perform the water-finding test was more susceptible to age-related cognitive deterioration in Tg2576 mice than the MWM test. We therefore propose that the water-finding test is a rapid and sensitive method that can be used to assess cognitive and/or behavioral deficits in mouse models of Alzheimer's disease.

Assessment of scopolamine on learning and memory on PND 22–28 juvenile rats using an automated video tracking system

Assessment of scopolamine on learning and memory on PND 22–28 juvenile rats using an automated video tracking system

Effects of a sublethal pesticide exposure on locomotor behavior: A video-tracking analysis in larval amphibians

Organochlorine pesticides such as endosulfan have been shown to have both lethal and sublethal effects on amphibians. In this context, behavioral endpoints have proved their usefulness in evidencing impacts of such chemicals at environmental concentrations that do not necessarily cause mortality. The recent development of video-tracking technologies now offers the possibility of accurately quantifying locomotor behaviors. However, these techniques have not yet been applied to evaluating the toxicity of pesticides in amphibians. We therefore aimed at determining the potential toxicity of endosulfan on endpoints associated with locomotion after short-term environmental endosulfan exposure in Rana temporaria tadpoles and at using these data as warning systems for survival alterations after a longer exposure. To this end, we analyzed video-tracks of 64 tadpoles (two pesticide treatments: 5 and 50μgL−1, one control and one solvent-control) with Ethovision XT 7 software. The highest endosulfan concentration had a significant effect on all four behavioral endpoints. Contaminated tadpoles traveled shorter distances, swam less often, at a lower mean speed, and occupied a less peripherical position than control tadpoles. The lowest endosulfan concentration had similar but lower effects, and did not affect mean speed during swimming. Survival was reduced only after a long-term exposure to endosulfan and was associated with short-term behavioral dysfunctions. These results show that endosulfan strongly affects the behavioral repertory of amphibian tadpoles, but in different ways depending on concentration, thus suggesting that the pesticide has complex modes of action. Given the importance of locomotion and space use in tadpole success in their aquatic environment, these results confirm the toxic action of endosulfan. By highlighting effects before mortality markers, video-tracking systems also show their potential as sentinels of sublethal effects of pesticides.

Endogenous dynorphin protects against neurotoxin-elicited nigrostriatal dopaminergic neuron damage and motor deficits in mice

BackgroundThe striato-nigral projecting pathway contains the highest concentrations of dynorphin in the brain. The functional role of this opioid peptide in the regulation of mesencephalic dopaminergic (DAergic) neurons is not clear. We reported previously that exogenous dynorphin exerts potent neuroprotective effects against inflammation-induced dopaminergic neurodegeneration in vitro. The present study was performed to investigate whether endogenous dynorphin has neuroprotective roles in vivo.Methods1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (MA), two commonly used neurotoxins in rodent models of Parkinson’s disease, were administered to wild-type (Dyn+/+) and prodynorphin-deficient mice (Dyn−/−). We examined dopaminergic neurotoxicity by using an automated video tracking system, HPLC, immunocytochemistry, and reverse transcription and polymerase chain reaction (RT-PCR).ResultsTreatment with MPTP resulted in behavioral impairments in both strains. However, these impairments were more pronounced in Dyn-l- than in Dyn+/+. Dyn−/− showed more severe MPTP-induced dopaminergic neuronal loss in the substantia nigra and striatum than Dyn+/+. Similarly, the levels of dopamine and its metabolites in the striatum were depleted to a greater extent in Dyn−/− than in Dyn+/+. Additional mechanistic studies revealed that MPTP treatment caused a higher degree of microglial activation and M1 phenotype differentiation in Dyn−/− than in Dyn+/+. Consistent with these observations, prodynorphin deficiency also exacerbated neurotoxic effects induced by MA, although this effect was less pronounced than that of MPTP.ConclusionsThe in vivo results presented here extend our previous in vitro findings and further indicate that endogenous dynorphin plays a critical role in protecting dopaminergic neurons through its anti-inflammatory effects.

Phosphine‐induced walking response of the lesser grain borer (Rhyzopertha dominica)

In spite of the intensive worldwide use of phosphine against stored-product insects, its potential sublethal effects on targeted insect species is seldom recognised and may compromise the efficacy of this fumigant, particularly against phosphine-resistant insects. Therefore, the behavioural response of three populations of the lesser grain borer Rhyzopertha dominica (Coleoptera: Bostrichidae) to sublethal phosphine exposure was assessed. Concentration-mortality bioassays with phosphine confirmed the resistance status of the insect populations studied, with levels of phosphine resistance of 40.8-fold and 85.7-fold compared with the susceptible population. Regarding walking behaviour, determined using a digital video-tracking system, the phosphine-resistant populations were less active than the susceptible population. In addition, sublethal phosphine exposure decreased the walking activity of all three populations. Phosphine exposure decreased walking activity. Such reduced walking activity is likely to lower the respiration rate, thereby minimising phosphine uptake. As higher phosphine resistance was also associated with reduced walking activity, phosphine-resistant populations may resist phosphine fumigation even more efficiently on account of this behavioural trait, further aiding their physiological resistance, and should be a matter of concern.

Using video‐tracking to assess sublethal effects of pesticides on honey bees (Apis mellifera L.)

Concern about the role of pesticides in honey bee decline has highlighted the need to examine the effects of sublethal exposure on bee behaviors. The video-tracking system EthoVisionXT (Noldus Information Technologies) was used to measure the effects of sublethal exposure to tau-fluvalinate and imidacloprid on honey bee locomotion, interactions, and time spent near a food source over a 24-h observation period. Bees were either treated topically with 0.3, 1.5, and 3 µg tau-fluvalinate or exposed to 0.05, 0.5, 5.0, 50, and 500 ppb imidacloprid in a sugar agar cube. Tau-fluvalinate caused a significant reduction in distance moved at all dose levels (p < 0.05), as did 50 and 500 ppb imidacloprid (p < 0.001). Bees exposed to 50 and 500 ppb spent significantly more time near the food source than control bees (p < 0.05). Interaction time decreased as time in the food zone increased for both chemicals. This study documents that video-tracking of bee behavior can enhance current protocols for measuring the effects of pesticides on honey bees at sublethal levels. It may provide a means of identifying problematic compounds for further testing.

Chemical analysis of three herbal extracts and observation of their activity against adults of Acanthoscelides obtectus and Leptinotarsa decemlineata using a video tracking system

Ethanol extracts of Rosmarinus officinalis, Lavandula angus-tifolia and Ruta graveolens were tested against adults of Acanthoscelides obtectus and Leptinotarsa decemlineata. Using a newly developed computer tracking system, a choice test revealed that all of the extracts have a repellent action. The highest repellent activity against the bean weevils adults was the ethanol extract of rue, whereas the highest repellent activity against the Colorado potato beetle adults was the lavender extract. We suggest that a cocktail of volatile components in the ethanol extracts was responsible for the observed repellent action. All three of the extracts have insecticidal effects on bean weevils, reducing F1 adult emergence, with no side effects on the germination of the bean plants. In contrast, the extracts did not demonstrate an insecticidal effect on Colorado potato beetles.

Locomotor activity assay in zebrafish larvae: Influence of age, strain and ethanol

Several characteristics warrant the zebrafish a refining animal model for toxicity testing in rodents, thereby contributing to the 3R principles (Replacement, Reduction, and Refinement) in animal testing, e.g. its small size, ease of obtaining a high number of progeny, external fertilization, transparency and rapid development of the embryo, and a basic understanding of its gene function and physiology. In this context we explored the motor activity pattern of zebrafish larvae, using a 96-well microtiter plate and a video-tracking system. Effects of induced light and darkness on locomotion of zebrafish larvae of different wild-type strains and ages (AB and TL, 5, 6 and 7 dpf; n=25/group) were studied. Locomotion was also measured in zebrafish larvae after exposure to different concentrations of ethanol (0; 0.5; 1; 2 and 4%) (AB and TL strain, 6 dpf; n=19/group). Zebrafish larvae showed a relatively high swimming activity in darkness when compared to the activity in light. Small differences were found between wild-type strains and/or age. Ethanol exposure resulted in hyperactivity (0.5–2%) and in hypo-activity (4%). In addition, the limitations and/or relevance of the parameters distance moved, duration of movements and velocity are exemplified and discussed. Together, the results support the suggestion that zebrafish may act as an animal refining alternative for toxicity testing in rodents provided internal and external environmental stimuli are controlled. As such, light, age and strain differences must be taken into account.

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of α-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that α-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that α-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.