This research was supported by Grants-in-Aid for Young Scientists (A) (#16680015), Grant-in-Aid for Exploratory Research (#16653065), Grant-in-Aid for Scientific Research on Priority Areas (#18023022, #18016012 and #18053015) from the Ministry of Education, Culture, Sports, Science, and Technology in Japan, Grant-in-Aid from Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), Grant-in-Aid from Neuroinformatics Japan Center (NIJC), RIKEN

<ol><li>The apparatus used for the light/dark transition test consisted of a cage (21x42x25 cm) divided into two sections of equal size by a partition with door (Ohara &amp; Co., Tokyo).</li><li>Mice are housed three to four per cage in a room with a 12 hr light/dark cycle (lights on at 7:00 A.M.) with ad libitum access to food and water. Ideally, two controls and two mutants are housed together, by re-organizing the mice as soon as they are genotyped at the time of weaning. Behavioral testing is performed between 9:00 A.M. and 6:00 P.M. All the cages containing mice are transferred to the behavior testing room 30 min before the first trial begins.</li><li>One chamber is brightly illuminated by white diodes (390 lux), whereas the other chamber is dark (2 lux). Mice are placed into the dark side and the door is opened automatically 3 seconds after the mouse is detected by the infrared camera. The door is used so that the mice do not enter the light chamber immediately after the release with their motivation to escape from experimenter, since the latency to enter the light chamber may serve as an index of anxiety-like behavior. </li><li>Mice are allowed to move freely between the two chambers with door open for 10 min. The application used for acquiring and analyzing the behavioral data (Image LD4) is based on the public domain Image J program (developed byWayne Rasband at the National Institute of Mental Health and available at http://rsb.info.nih.gov/ij/), which was modified by Tsuyoshi Miyakawa (available through O'Hara &amp; Co., Tokyo, Japan). </li><li>The distance traveled in each chamber, the total number of transitions, the time spent in the each chamber, and the latency to enter the light chamberare recorded by Image LD4 program. </li><li>After each trial, all chambers are cleaned with super hypochlorous water to prevent a bias based on olfactory cues.</li></ol>

<ol>
	<li>Austin, C. P., Aiba, A., Takao, K., Miyakawa, T., Takao, K., Yamasaki, N., Miyakawa, T., Crawley, J. N., Crawley, J. N., Crawley, J., Goodwin, F. K., Miyakawa, T., Crabbe, J. C., Wahlsten, D., Dudek, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+knockout+mouse+project.">The knockout mouse project.</a> Nat Genet. 36, 921-924 (2004).</li></ol>

All procedures were approved by the Animal Use and Care Committee of Kyoto University.
Tsuyoshi Miyakawa is associated with BIRD, Japan Science and Technology Agency, Saitama, Japan.

Although all of the mouse genome sequences have been determined, the functions of most of these genes are not known. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice(Aiba et al., 2007; Austin et al., 2004). The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice(Takao and Miyakawa, 2006; Takao et al., 2007). As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice(Crawley, 2000). The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments(Crawley, 1985). The test is sensitive to anxiolytic drug treatment(Crawley, 1985). The apparatus consists of a dark chamber and a brightly illuminated chamber. A restricted opening, 3 cm high by 5 cm wide, connects the two chambers. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are used as indices of bright-space anxiety in mice.The light/dark transition test was originally developed by Crawley and colleagues (Crawley and Goodwin, 1980). There are two differences between their original version and our test. First, the light chamber is larger than the dark chamber in the original version, whereas the size of the two chambers is the same in our version of the test. Second, in the original version, the light chamber had no ceiling and the walls of the light chamber were transparent(Crawley and Goodwin, 1980), while we use opaque white plastic for the ceiling and walls of the light chamber. These differences, namely the size and openness of the light chamber, allow for the simultaneous detection of bright-space anxiety as well as open-space anxiety in the original version of the test. In our laboratory, however, open-space anxiety-like behavior in mice is tested in an elevated plus maze. The opaque walls and ceiling, size of the light chamber, and our specific light/dark transition test protocol is more specialized for detecting bright-space anxiety compared to the original version.Although the light/dark transition test and elevated plus maze are both used for assessing anxiety-like behavior, the results are not always consistent. For example, forebrain-specific calcineurin knockout mice spend an decreased amount of time in the light chamber in the light/dark transition test, but a increased amount of time in the open arms in the elevated plus maze(Miyakawa et al., 2003). We hypothesized that our light/dark transition test and elevated plus maze tests assess different aspects of anxiety-like behavior, such as bright-space anxiety and open-space anxiety-like behavior. To test this hypothesis, we used a large data set collected using the same mice in both tests. In our laboratory, we conduct behavioral test batteries covering many domains to screen for the behavioral significance of genes. The test battery requires several weeks to complete. During the testing period, mouse cages are kept on a rack in our facility. If mice are transported to our laboratory from other institutions, they are habituated to the environment of our facility for at least 1 week. After the habituation period, mice are subjected to a general health check and neurologic screening (Miyakawa et al., 2003), and after these simple examinations, mice are subjected to the light/dark transition test. The test is conducted in a sound proof experimental room after 30 min of habituation to the experimental room, as shown in the movie.We have assessed more than 60 strains of genetically engineered mutant mice using the protocol shown in the movie and have a large set of raw data for more than 3000 mice (including wild-type and mutant mice). We conducted factor analyses to take advantage of the large set of raw data. Indeed, we detected different factors from each test (unpublished data), although there were some significant correlations between indices (unpublished data).. Those data will be disclosed as part of a public database that we are now constructing.In our test battery, wild-type littermates are usually used as a control. As a background strain, C57BL/6J mice are commonly used. We collected the data of C57BL/6J mice used as a control of mutant mice in the test battery. The indices obtained from C57BL/6J mice in our light/dark transition test were as follows (n=795; mean SEM); distance traveled: 846.5 8.88 cm in the light chamber, 1478.5 9.53 cm in the dark chamber ; duration of time spent:215.8 1.85 s in the light, 395.8 1.86 s in the dark; number of transitions between compartments: 30.52 0.43; latency to enter the light chamber: 64.94 2.40 s. Movement of mice around the entrances to the chambers is detected by the camera and is counted as time spent in both chambers. Because of this, the sum of the mean duration of time spent in the light and that in dark compartments was greater than that of the entire duration of the test (600 s).In our protocol, a mouse is first placed in the dark chamber. Three seconds after placing the mouse in the dark chamber, the door between the chambers automatically opens and the mouse can move freely between the two chambers. C57BL/6J mice spend less time in the light chamber than in the dark chamber (p&lt;0.0001, duration if time spent in light: 215.8 1.85 s, duration of time spent in dark: 316.3 2.11 s [the time spent in the dark does not include the initial time spent in the dark prior to entering the light chamber for the first time], n=795, paired-t test). This indicates that C57BL/6J mice tend to avoid the light chamber, and that the time spent in the light is a good index of anxiety-like behavior.The results obtained from various strains of mutant mice are easily compared, because all data are obtained using the same protocol. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with data of other mutants, the behavioral test methods should be as identical as possible between laboratories. Crabbe and colleagues tested the interaction between mouse behavior and laboratory environment using exactly the same inbred strains and one null mutant strain. In their study, the apparatus, test protocols, and many environmental variables were rigorously standardized. Despite the standardization, they found systematic differences in behavior across laboratories, although they also detected strain differences in all behaviors(Crabbe et al., 1999). They reported that uncontrollable variables and experiments characterizing mutants may yield results that are idiosyncratic to a particular laboratory. The procedural differences that exist between laboratories make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated.Establishing visual documentation of the protocol will facilitate better understanding of the details of experimental procedures, allowing for standardization of the protocols used across laboratories and for comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. Movies of other protocols, such as for the elevated plus maze, open field test, porsolt forced swim test, fear conditioning, that we use in our behavioral test battery are currently being made for publication as future video journal articles.

light/dark transition test for mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

Watch this Scientific Journal Video about Light/dark Transition Test for Mice at JoVE.com

Light/dark Transition Test for Mice

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Research

JoVE Journal

Biology

52.4K Views.  Graduate School of Medicine, Kyoto University. He did. So what?

Video: Light/dark Transition Test for Mice

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Behavior

Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the &#34;cloud&#34; and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in&#160;cabinet; two such cabinets (16 environments) may be controlled by one computer.

Fully automated system for measuring physiologically meaningful properties of the mechanisms mediating spatial localization, temporal localization, duration, rate&#160;and probability estimation, risk assessment, impulsivity, and the accuracy and precision of memory, in order to assess the effects of genetic and pharmacological manipulations on foundational mechanisms of cognition in mice.

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and ...

Measuring Circadian and Acute Light Responses in Mice using Wheel Running Activity

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2. They are responsible for timing our sleep/wake cycles and hormone secretion. Since this timing is not precisely 24-hours, it is synchronized to the solar day by light input. This is accomplished via photic input from the retina to the suprachiasmatic nucleus (SCN) which serves as the master pacemaker synchronizing peripheral clocks in other regions of the brain and peripheral tissues to the environmental light dark cycle3-7. The alignment of rhythms to this environmental light dark cycle organizes particular physiological events to the correct temporal niche, which is crucial for survival8. For example, mice sleep during the day and are active at night. This ability to consolidate activity to either the light or dark portion of the day is referred to as circadian photoentrainment and requires light input to the circadian clock9. Activity of mice at night is robust particularly in the presence of a running wheel. Measuring this behavior is a minimally invasive method that can be used to evaluate the functionality of the circadian system as well as light input to this system. Methods that will covered here are used to examine the circadian clock, light input to this system, as well as the direct influence of light on wheel running behavior.

This article will review methods that can be used to determine circadian function and light responsiveness in mice.

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2.  They ...

Neuroscience

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

Investigating Migraine-Like Behavior Using Light Aversion in Mice

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as photophobia. Whilst it is impossible to confirm that a mouse is experiencing migraine, light aversion can be used as a behavioral surrogate for the migraine symptom of photophobia. To test for light aversion, we utilize the light/dark assay to measure the time mice freely choose to spend in either a light or dark environment. The assay has been refined by introducing two critical modifications: pre-exposures to the chamber prior to running the test procedure and adjustable chamber lighting, permitting the use of a range of light intensities from 55 lux to 27,000 lux. Because the choice to spend more time in the dark is also indicative of anxiety, we also utilize a light-independent anxiety test, the open field assay, to distinguish anxiety from light-aversive behavior. Here, we describe a modified test paradigm for the light/dark and open field assays. The application of these assays is described for intraperitoneal injection of calcitonin gene-related peptide (CGRP) in two mouse strains and for optogenetic brain stimulation studies.

Rodents are not able to report migraine symptoms. Here, we describe a manageable test paradigm (light/dark and open field assays) to measure light aversion, one of the most common and bothersome symptoms in patients with migraines.

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as ...

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

Medicine

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

Bioengineering

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model to study animal avoidance behavior. This protocol introduces a light-spot assay to investigate larval phototactic behavior. The experimental set-up includes two main parts: a visual stimulation system that generates the light spot, and an infrared light-based imaging system that records the process of larval light avoidance. This assay allows tracking of the behavior of larva before entering, during encountering, and after leaving the light spot. Details of larval movement including deceleration, pause, head casting, and turning can be captured and analyzed using this method.

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

This protocol introduces a light-spot assay to investigate Drosophila larval phototactic behavior. In this assay, a light spot is generated as light stimulation, and the process of larval light avoidance is recorded by an infrared light-based imaging system.

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model ...

Light-Dark Preference Test: A Method to Study Anxiety-Related Behavior in Zebrafish

Source: Holcombe, A., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/51851/" target="_blank">A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal</a>. &#160;J. Vis. Exp.&#160;(2014)
The video describes the light-dark preference assay to study the anxiety-related behavior in zebrafish following the exposure to ethanol.

Source: Holcombe, A., et al. A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal. &#160;J. Vis. ...

Light/dark Transition Test for Mice

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Summary

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