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.     

XPairIt Docking Protocol for peptide docking and analysis

The mechanics of peptide–protein docking has long been an area of intense interest to the computational community. Here we discuss an improved docking protocol named XPairIt which uses a multitier approach, combining the PyRosetta docking software with the NAMD molecular dynamics package through a biomolecular simulation programming interface written in Python. This protocol is designed for systems where no a priori information of ligand structure (beyond sequence) or binding location is known. It provides for efficient incorporation of both ligand and target flexibility, is HPC-ready and is easily extensible for use of custom code. We apply this protocol to a set of 11 test cases drawn from benchmarking databases and from previously published studies for direct comparison with existing protocols. Strengths, weaknesses and areas of improvement are discussed.     

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.     

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.     

Improvement and impairment of visually guided behavior through LTP- and LTD-like exposure-based visual learning

Cellular studies have focused on long-term potentiation (LTP) and long-term depression (LTD) to understand requirements for persistent changes in synaptic connections. Whereas LTP is induced through high-frequency intermittent stimulation, low-frequency stimulation evokes LTD. Because of the ubiquitous efficacy of these protocols, they are considered fundamental mechanisms underlying learning. Here we adapted LTP/LTD-like protocols to visual stimulation to alter human visually guided behavior. In a change-detection task, participants reported luminance changes against distracting orientation changes. Subsequently, they were exposed to passive visual high- or low-frequency stimulation of either the relevant luminance or irrelevant orientation feature. LTP-like high-frequency protocols using luminance improved ability to detect luminance changes, whereas low-frequency LTD-like stimulation impaired performance. In contrast, LTP-like exposure of the irrelevant orientation feature impaired performance, whereas LTD-like orientation stimulation improved it. LTP-like effects were present for 10 days, whereas LTD-like effects lasted for a shorter period of time. Our data demonstrate that instead of electrically stimulating synapses, selective behavioral changes are evoked in humans by using equivalently timed visual stimulation, suggesting that both LTD- and LTP-like protocols control human behavior but that the direction of changes is determined by the feature incorporated into the stimulation protocol.     

Differences in spatio-temporal behavior of zebrafish in the open tank paradigm after a short-period confinement into dark and bright environments

The open tank paradigm, also known as novel tank diving test, is a protocol used to evaluate the zebrafish behavior. Several characteristics have been described for this species, including scototaxis, which is the natural preference for dark environments in detriment of bright ones. However, there is no evidence regarding the influence of "natural stimuli" in zebrafish subjected to novelty-based paradigms. In this report, we evaluated the spatio-temporal exploratory activity of the short-fin zebrafish phenotype in the open tank after a short-period confinement into dark/bright environments. A total of 44 animals were individually confined during a 10-min single session into one of three environments: black-painted, white-painted, and transparent cylinders (dark, bright, and transparent groups). Fish were further subjected to the novel tank test and their exploratory profile was recorded during a 15-min trial. The results demonstrated that zebrafish increased their vertical exploratory activity during the first 6-min, where the bright group spent more time and travelled a higher distance in the top area. Interestingly, all behavioral parameters measured for the dark group were similar to the transparent one. These data were confirmed by automated analysis of track and occupancy plots and also demonstrated that zebrafish display a classical homebase formation in the bottom area of the tank. A detailed spatio-temporal study of zebrafish exploratory behavior and the construction of representative ethograms showed that the experimental groups presented significant differences in the first 3-min vs. last 3-min of test. Although the main factors involved in these behavioral responses still remain ambiguous and require further investigation, the current report describes an alternative methodological approach for assessing the zebrafish behavior after a forced exposure to different environments. Additionally, the analysis of ethologically-relevant patterns across time could be a potential phenotyping tool to evaluate the zebrafish exploratory profile in the open tank task.     

A computerized system for scoring primate behavior using standard keyboards

A scoring system has been developed for primate behavior which uses standard keyboards and minicomputers or microcomputers. The mnemonic, alphanumeric code used is easily learned, highly flexible, and can be recorded in longhand for later entry into a computer if a keyboard is not immediately available. The software consists of two programs, both of which can be written in BASIC. SCORE is used for data acquisition and appends the test time to each behavioral sequence. DATSUM decodes and summarizes the test data using table-driven logic. The minimum hardware required is a 16K microcomputer, an alphanumeric keyboard, a display, and cassette storage.     

msatcommander: detection of microsatellite repeat arrays and automated, locus-specific primer design

msatcommander is a platform-independent program designed to search for microsatellite arrays, design primers, and tag primers using an automated routine. msatcommander accepts as input DNA sequence data in single-sequence or concatenated, fasta-formatted files. Search data and locus-specific primers are written to comma-separated value files for subsequent use in spreadsheet or database programs. Binary versions of the graphical interface for msatcommander are available for Apple OS X and Windows XP. Users of other operating systems may run the graphical interface version using the available source code, provided their environment supports at least Python 2.4, Biopython 1.43, and wxPython 2.8. msatcommander is available from http://code.google.com/p/msatcommander/.     

The Modified Hole Board - Measuring Behavior,Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal’s behavioral profile can be investigated in one single test.The apparatus consists of a box, representing the ‘protected’ area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the ‘unprotected’ area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals’ adaptive capacities.Due to testing order effects in a behavioral test battery, naïve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.     

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals’ positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.     

How does variation in the environment and individual cognition explain the existence of consistent behavioral differences?

According to recent studies on animal personalities, the level of behavioral plasticity, which can be viewed as the slope of the behavioral reaction norm, varies among individuals, populations, and species. Still, it is conceptually unclear how the interaction between environmental variation and variation in animal cognition affect the evolution of behavioral plasticity and expression of animal personalities. Here, we (1) use literature to review how environmental variation and individual variation in cognition explain population and individual level expression of behavioral plasticity and (2) draw together empirically yet nontested, conceptual framework to clarify how these factors affect the evolution and expression of individually consistent behavior in nature. The framework is based on simple principles: first, information acquisition requires cognition that is inherently costly to build and maintain. Second, individual differences in animal cognition affect the differences in behavioral flexibility, i.e. the variance around the mean of the behavioral reaction norm, which defines plasticity. Third, along the lines of the evolution of cognition, we predict that environments with moderate variation favor behavioral flexibility. This occurs since in those environments costs of cognition are covered by being able to recognize and use information effectively. Similarly, nonflexible, stereotypic behaviors may be favored in environments that are either invariable or highly variable, since in those environments cognition does not give any benefits to cover the costs or cognition is not able to keep up with environmental change, respectively. If behavioral plasticity develops in response to increasing environmental variability, plasticity should dominate in environments that are moderately variable, and expression of animal personalities and behavioral syndromes may differ between environments. We give suggestions how to test our hypothesis and propose improvements to current behavioral testing protocols in the field of animal personality.     

“水生动物行为”专辑责编简介

行为学研究越来越受到动物科研工作者的重视, 尤其是动物个性研究。然而, 目前在个性的概念、实验设计和统计方法上仍存在不少问题, 因而规范行为学研究方法有着十分重要的意义。鉴于以上目的, 文章对动物个性和行为集概念的发展过程进行了简要回顾, 并归纳了动物个性和行为集研究的实验设计和目前最佳的数据统计分析方法。这些标准的建立, 可以帮助科研人员避免动物个性研究中常见的错误, 使个性研究的设计更加缜密、实验结果更加可靠, 从而推动行为生态学的蓬勃发展。     

Reliability-oriented bioinformatic networks visualization

SUMMARY: We present our protein-protein interaction (PPI) network visualization system RobinViz (reliability-oriented bioinformatic networks visualization). Clustering the PPI network based on gene ontology (GO) annotations or biclustered gene expression data, providing a clustered visualization model based on a central/peripheral duality, computing layouts with algorithms specialized for interaction reliabilities represented as weights, completely automated data acquisition, processing are notable features of the system. AVAILABILITY: RobinViz is a free, open-source software protected under GPL. It is written in C++ and Python, and consists of almost 30 000 lines of code, excluding the employed libraries. Source code, user manual and other Supplementary Material are available for download at http://code.google.com/p/robinviz/.     

An inexpensive,automation‐friendly protocol for recovering high‐quality DNA

Although commercial kits are available for automated DNA extraction, ‘artisanal’ protocols are not. In this study, we present a silica‐based method that is sensitive, inexpensive and compliant with automation. The effectiveness of this protocol has now been tested on more than 5000 animal specimens with highly positive results.     

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic,Biomedical, and Agricultural Research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.     

Efficient TPA-based auditing scheme for secure cloud storage

In recent years, how to design efficient auditing protocol to verify the integrity of users’ data, which is stored in cloud services provider (CSP), becomes a research focus. Homomorphic message authentication code (MAC) and homomorphic signature are two popular techniques to respectively design private and public auditing protocols. On the one hand, it is not suitable for the homomorphic-MAC-based auditing protocols to be outsourced to third-party auditor (TPA), who has more professional knowledge and computational abilities, although they have high efficiencies. On the other hand, the homomorphic-signature-based ones are very suitable for employing TPA without compromising user’s signing key but have very low efficiency (compared to the former case). In this paper, we propose a new auditing protocol, which perfectly combines the advantages of above two cases. In particular, it is almost as efficient as a homomorphic-MAC-based protocol proposed by Zhang et al. recently. Moreover, it is also suitable for outsourcing to TPA because it does not compromise the privacy of users’ signing key, which can be seen from our security analysis. Finally, numerical analysis and experimental results demonstrate the high-efficiency of our protocol.

     

Automated high-throughput process for site-directed mutagenesis, production, purification, and kinetic characterization of enzymes

Site-directed mutagenesis followed by functional characterization is a widely used approach to obtain information on the structure-function relationship of proteins. Due to time and cost considerations, the number of amino acids studied is frequently reduced. To address the need for convenient parallel production of numerous point mutants of a protein, we developed an automated method to perform classical site-directed mutagenesis, protein purification, and characterization in a high-throughput manner. The process consists of a succession of six fully automated protocols that can be adapted to any automated liquid handling systems. Our procedure allows construction, validation, and characterization of hundreds of site-directed mutants of a given protein in just 4 days. The method is especially adapted to projects aiming at the study of unique or multiple mutants without the need to construct and screen large libraries of random mutants. The usefulness of the technique is illustrated by the construction and characterization of tens of single mutants of the penicillin-binding protein 2x (PBP2x) from Streptococcus pneumoniae. Moreover, seven mutations of PBP2x were obtained simultaneously in a single experiment with efficiency close to 90%.     

The cow as an induced ovulator: Timed AI after synchronization of ovulation

Timed-AI after synchronization of ovulation has become one of the most used reproductive technologies developed during the past 40 years. Various adaptations of this technology are now extensively used worldwide, in the beef and dairy cattle industry. Our well-cited report, published in Theriogenology in 1995, presented a method termed Ovsynch, that used GnRH and PGF_2α to perform synchronization of ovulation and timed AI in lactating dairy cows. This report introduced Ovsynch, more as a concept of induced ovulation, and demonstrated the ovarian dynamics during the protocol. Validation and improvements on this method were subsequently performed in numerous university studies and on commercial dairies, worldwide. This review will provide a brief historical background, some personal recollections, and certain modifications that have been made in synchronization of ovulation protocols. Each section emphasizes the physiology that underlies the most widely-used synchronization of ovulation protocols and key modifications and some practical application of these protocols on commercial operations. Finally, the effect of timed AI in the US dairy industry and in the Brazilian beef cattle industry are compared. Although numerous studies have been done using these protocols, there is still substantial need for research to improve the synchronization, efficacy, simplicity, and practical application of these protocols.