Mouse Behavioral Testing: How to Use Mice in Behavioral Neuroscience

book.

Introduction to the Research Process

Chapter Outline

The Research Process: Scientific Aspects1

Adapting the approach to the stage of an investigation 2

Mice, mice, and more mice 4

Measuring instruments: Tests 4

The formal research design 4

Sample size 4

Ethics approval 5

Logistics 5

Obtaining subjects 5

The test day 6

Prelude to data analysis 6

Data analysis 7

Publishing results 7

The Institutional Context of Research7

People 8

Facilities 9

Policies 11

Testing mouse behavior is done as part of a scientific investigation of some larger question, such as the genetic bases for neuropsychiatric disorders or mechanisms of memory formation. Those who begin their career in science with behavioral testing naturally focus on the fine details of the task set for them by a more experienced person. Only gradually does a larger picture form of how their work relates to other parts of the research process. This chapter sketches some of those parts and puts the tests into a context.

The Research Process: Scientific Aspects

Whereas the research environment varies greatly in different countries and institutions, the actual process of doing research with mice is or should be the same everywhere. The main steps are illustrated in Figure 1.1, emphasizing things that are addressed in depth in this book. Conceptualization and literature review are governed by the existing knowledge and theories in a specific field of study. These are usually introduced to the student in an entire course such as behavioral neuroscience, behavioral genetics, psychopharmacology, or animal behavior. Through that kind of advanced study and extensive reading of the literature, the investigator becomes familiar with specialized methods of experimentation and learns what kinds of factors need to be controlled and measured in a good study. Little is said in this book about the first two steps in the process because they are thoroughly dealt with in many graduate training programs and postdoctoral settings. Several key reference works are listed at the end of this chapter for the benefit of those new to the field.

Figure 1.1 Broad outline of the research process followed by studies of both human and animal subjects. Shading of the box indicates the depth of discussion each step receives in this book. The darker the shading, the deeper the discussion.

Most researchers working with mice have taken at least an introductory course in statistical data analysis. Because not everyone will have a strong background in these procedures, there is some coverage of statistical methods in Chapter 5 but no attempt is made to condense a one-term statistics course into one chapter. Statistical principles will not be grasped firmly until the investigator needs to apply them to real data, at which time a review of the basics can be helpful. This is especially true for the planning phase of a project. Many texts on statistics devote chapter after chapter to what should be done with data that have already been collected, while little is said about critical matters such as choosing the proper sample size before the experiment begins. When analysis of data is discussed here, attention is focused on issues that are common in work with mouse behavior.

Adapting the approach to the stage of an investigation

Most long-term research programs involve two stages: an exploratory stage followed by a formal test of well-specified hypotheses. The latter methodology is elaborated in many courses, whereas the former receives little attention. The exploratory stage is perhaps the most important because it can be the time when a discovery is first made or a decisive clue is found. More often than not, an important discovery occurs by accident during the course of some other experiment. Students and technicians are supposed to follow a protocol rigidly and everything is supposed to happen according to well-laid plans, but nobody tells the mice about this, and some of the mice behave unexpectedly. Good researchers take note of the exceptions and surprises. An experienced researcher may know when a surprising result is potentially very important, but this same experience can also make the investigator jaded and too willing to attribute every exception to sampling error. There is considerable benefit in having people new to the field take a fresh look at the procedures used in a behavioral test and also at the mice as they are run through their paces.

It is good practice to conduct the exploratory stage of a project without rigid protocols and controls. The pilot study allows different test parameters and procedures to be evaluated informally using just a few animals in any one condition or perhaps using repeated testing of the same animals in practice runs. This kind of study scans a wide range of possibilities in a way that will detect those with very large effects. Later, when the most important variables have been brought under control and optimized for that lab, factors with more subtle effects can be studied in a formal experiment with the stricter controls and larger samples that are needed to detect them. The principal investigator (PI) faces a special challenge in convincing an animal ethics committee to approve a pilot study. Suggestions about how to do this are offered in Chapter 6. The purpose of a pilot study is not to circumvent the ethical principles of research with animals. Those principles should be respected in any kind of study with mice, which imposes limits on what range of test parameters can be tried in a pilot study.

An open-ended exploratory study can be done in some situations. Crowcroft (1973), in his charming book Mice All Over, described a study of mice free to run loose in an entire building. At the Institute for Behavioral Genetics in Colorado, Gerald McClearn had a device constructed that he dubbed the hypothesis generating apparatus. It was a large and complex environment populated with mice of both sexes from several inbred strains. The students and postdoctoral fellows sat in chairs and watched the antics of 5 to 10 mice for hours at a time at different phases of the light–dark cycle (dim light for the dark phase). This was an excellent way to become familiar with the range of behaviors that mice commonly express, and hypotheses flowed freely from this device (e.g., Yanai & McClearn, 1972). David Sternthal, a graduate student at the University of Waterloo, went one step further by filling a small lab room with topsoil covered by grass turf with places to build deep burrows and a hut where an outcast could find shelter away from the main group of mice. Those inbred mice that sometimes seem so dull and listless in a shoebox colony cage or boring behavioral tests came to life and utilized every portion of an environment that was vastly larger than the typical colony cage. A complex mouse society emerged quickly and soon the entire environment was transformed with tunnels, nests, and home bases. Hans-Peter Lipp and co-workers (Lipp, Amrein, Slomankia, Wolfer, 2007) at the University of Zürich and Moscow State University took inbred mice to the outer limits of practical research by releasing them into large outdoor pens in a rural research station north of Moscow, where they were exposed to the rigors of natural selection. To the amazement of almost everyone, inbred mice survived and some of them even thrived in the Russian winter . This was high-risk, exploratory research at its finest, because nobody knew if the study would provide any useful data.

Another source of ideas about testing mouse behavior is the Internet site YouTube. There are fascinating videos of the smartest mouse in the world that rapidly navigates an astoundingly complex sequence (probably aided by odor trails) over barriers and through a wide variety of devices that put anything we do in the lab to shame. There are mice on surf boards that exhibit the notorious aversion of mice to water, even at the beach. At the beginning of scientific work with mice early in the twentieth century, laboratory researchers went to mouse fanciers to obtain their subjects and learn about how to breed and maintain mice (Chapter 2). Today there is still a thriving community of mouse fanciers with fascinating Web sites who keep these animals as pets and have a greater interest in behavior than fanciers 100 years ago, who were interested mainly in coat color variants.

One perfectly legal approach to exploratory work is to buy mice at a local pet shop and keep them in a basement or garage. This approach needs to respect the needs of the mice and adhere to regulations that proscribe animal cruelty. Never should a pet be made to suffer just in case it might give a scientist some prize winning idea. At the same time, all the things that are done to train cats and dogs for the show ring are reasonable for training mice and testing the limits of what they can achieve. Animal trainers have been able to induce bears to play ice hockey and porpoises to serve cocktails on silver trays at poolside. There is no good reason why a professional scientist who studies mouse behavior in the lab cannot also be an animal trainer or observer after hours, provided friends and family can live with the mouse odors.

Mice, mice, and more mice

Pets are fun to watch, but research is done with special mice developed for use in laboratories around the world. Their surprising origins are now fairly well understood. Modern lab mice are a mixture of genes from four wild Mus subspecies. Mouse fanciers in Europe, the United States, China, and Japan kept mice as pets, and those animals were convenient sources of research subjects in the twentieth century when the scientific study of mouse behavior commenced. Before long, standard inbred strains were created and mouse genetics progressed rapidly. With the advent of molecular genetic technology to sequence the genome and introduce mutations into known genes, mice became more popular than ever. Today, mouse models of human genetic disorders have become prime subjects for neuroscience research. Chapter 2 reviews mouse history and the many kinds of mice that inhabit labs today.

Measuring instruments: Tests

Behavior is a measurable phenotype (Wahlsten & Crabbe, 2007). The process of measuring it is termed a test. The test involves a physical apparatus and a protocol instructing how to conduct the trials. Virtually every kind of behavior, with the possible exception of some simple reflexes, is complex and can be measured in several ways. The test chosen for a study must pertain to the kinds of behavioral or neural processes that are the subjects of the study. The researcher needs to decide on the specific behavioral tests to employ in a study quite early in the process before there can be sample size estimation or ethics approval. Chapter 3 introduces more than 50 kinds of tests. The entire second half of the book (Chapters 10 to 14) is devoted to an in-depth discussion of many kinds of tests. Chapters on general features of behavioral tests cover a wide range of topics from ways to construct a test battery to methods for assessing reliability. Specialized techniques to provide adequate motivation and obtain good results from computer-based video tracking are explored in considerable depth. Finally, the challenge of obtaining comparable results in different labs is described, and possibilities for standardizing the tests as well as the conditions in a lab are discussed.

The formal research design

Many brilliant discoveries in a pilot study, accidental findings in a project aimed at studying something else, or flashes of insight while watching pets at play arise from events that cannot be repeated. Many hunches turn out to be wrong. Good ideas need to survive a rigorous, formal test before the rest of the field needs to take notice. For this purpose, the investigator must adopt a research design that is capable of proving the truth of an idea to a skeptical audience of peers. Many elaborate designs with sophisticated control groups are used for genetic studies of mouse behavior. A few of the main ones are described in Chapter 4. Design of a study is critically important for sample size determination, ethics approval, and logistics of running the experiment. Once those hurdles have been crossed, the proper execution of an elegant design is essential for obtaining good data. It is fairly easy to design a study on a piece of paper so that logic appears to govern all else. Taking that design into a real laboratory, however, soon teaches the researcher to anticipate and respect the many pitfalls and complications that beset the practical implementation of well-laid plans. It is for this reason that most of the remaining chapters address the practical side of research on mouse behavior.

Sample size

Only after the design of the study and the specific behavioral tests are chosen can the researcher rationally decide the appropriate number of mice to be tested. This step is taken before the application for ethics approval and then grant funding, because the number of animals in the study is an important aspect of external review and consent. After the data are collected and the report is submitted for publication, the author may be asked by a skeptical reviewer to justify the sample size. If the number of mice is found to be too small at that stage of the process, the ship of science will break apart on the shoals of bad methodology. It is better by far to plan ahead and take great care to get this number right. Chapter 5 is devoted to this topic, and a series of statistical utilities is provided to make the process as quick and painless as possible.

Ethics approval

Having decided upon the design, the tests, and the number of mice, permission to conduct the study must be given. This is usually done through some kind of peer review by colleagues who also work with animals or have expertise pertinent to the study. The specific rules and procedures differ greatly in different countries, and within a single country an institution may have considerable latitude in implementing ethical principles. The journal to which an article is submitted for publication may also, through its peer review process, judge the ethical aspects of a study. Official bodies sanctioned by a government agency may be given powers to pass judgment on a research proposal, and those bodies provide instructions on how to pass over the hurdles and through the hoops of regulations. The discussion in Chapter 6 does not attempt to replace or reduce the need for an investigator to engage the official mechanism. Instead, it offers a researcher’s perspective on the process and advice on how to get the job done well and done quickly. Specific examples of research procedures that are considered good practice or in some cases unethical in work with mice are presented, and a scheme for rating the degree of discomfort or invasiveness for a wide range of behavioral tests is proposed.

Logistics

After funding and permissions are obtained, it is time to plan the details of execution of the experiment. This step could be taken earlier in the process, but everything could change after the sample size calculation or the ethics review. Planning means a list of which mouse is to be tested at what time on which day under what conditions, and it requires other aspects of the lab to be arranged so that the testing can progress smoothly according to protocol. This stage is complete when the researchers have produced a stack of data sheets, one per mouse, in the correct order for the entire experiment. The study should not begin until every detail of every trial for every mouse in the entire experiment has been specified.

There are two major parts to the planning process. First, the throughput for the test, how much time on how many days is required to obtain measures on just one animal, must be determined. Second, the order in which animals from the various genotypic and treatment groups are to be tested must be decided. The order should be carefully balanced and randomized so there is no preponderance of mice from any one group at any particular period of testing. The effects of test order must always be considered. Some mouse must go first or last and early or late each day. A skillful execution of test order causes variance arising from time of day and sequential effects such as odor trails to end up in the within-groups variance, rather than creating spurious between-group effects. Chapter 7 explains how to achieve this and provides several elaborate examples from real experiments.

Obtaining subjects

After everything is ready for the experiment, it is time to bring the mice to the laboratory, if they are to be purchased from a commercial supplier. In labs where the mice are to be bred locally, as when unique genotypes are involved, the planning for obtaining research subjects is far more difficult and requires months of advance notice to animal care personnel. Mice are born in litters ranging in size from 2 to 13 pups, and not all mated females become pregnant, especially in some of the less fertile inbred strains. Commercial suppliers such as the Jackson Laboratory in Bar Harbor, Maine, breed so many mice of the more common strains that average litter size and pregnancy rates can be tuned to the anticipated demand for each genotype, with the happy consequence that the experimenter can simply ask for 10 mice of each strain and sex to be shipped at a narrow age range on a specific day. Obtaining the same numbers in the same age range at the same time locally can challenge the resources of most laboratories when more than two or three genotypes are involved.

Obtaining mice includes things done the day they arrive in the lab, such as unpacking, marking and assigning them to cages placed in specific locations on a rack in a colony room, and everything that happens to them until the first day of testing. The researcher must make some deliberate choices: how much cage enrichment to use, what kind of food and water to provide, the light–dark cycle, and so forth. Life in the colony room can have a significant impact on behavioral test results. Even when there is little the experimenter can do to control these phenomena, it is very important to be cognizant of their presence when planning the study and interpreting results. Chapter 8 discusses many of these issues, and Chapter 15 explores them in greater depth.

The test day

After all the planning is completed and everyone is ready for the first day of testing, the real action begins. Everything should go smoothly if the conditions have been properly prepared. In reality, however, something almost always goes wrong and adjustments need to be made on the fly. A major study simply cannot be put on hold while the researcher ponders what went wrong and what to do next. It can be terminated early, and this is a disaster. To avoid such disasters, it is wise to have contingency plans. This is best done by looking closely at exactly what is done during the test day and identifying vulnerable points in the protocol where a small mouse can run a large experiment right off the rails. This happened in the author’s lab when the roster of strains in a large study for the Mouse Phenome Project included four wild-derived inbred mouse species. Inbreeding had not bred the wildness out of them, and more than once the testing schedule was imperiled when the technician was led on a merry chase after an escapee around a cluttered testing room.

There are events too large to accommodate in any plan, such as major weather calamities or terrorist attacks. Sadly, the mouse lab can be a target for deliberate disruption. Security remains a concern at many institutions. Options are briefly considered here, but rare events are difficult to anticipate. In a long career, the author has never experienced a truly major disruption of an experiment, but others have. Gordon Harrington ( personal communication) at the University of Northern Iowa maintained eight unique inbred strains of rats, but the entire building housing his collection burned to the ground. His students responded quickly to the emergency and carried every one of the rats in their cages onto the lawn, saving a valuable genetic resource. Harrington had not organized them in advance into fire brigades with little rat resuscitation apparatuses; instead, the students exercised good judgment and acted spontaneously. What Harrington had done was teach his students the importance of unique strains of research animals. They knew there was something housed in that building that was really worth saving.

Prelude to data analysis

After reading current texts on research design and data analysis, it appears that analysis with sophisticated multivariate methods begins the day after data collection is complete. What a false impression this is. In reality, the study may appear to progress smoothly to a brilliant finale, but the data can later prove to be gravely flawed or even worthless. All behavioral test data need to be subjected to a rigorous process of quality control. This is usually done after data collection is completed because the tight testing schedule leaves little spare time. In recent studies in the author’s lab involving video tracking of mice in complex apparatus, almost as much time was spent checking for and correcting tracking errors as was spent doing the study in the first place. Those errors were obvious from the bizarre patterns on the computer screen (Bailoo, Bohlen, & Wahlsten, 2010); a mouse simply could not have jumped from one arm of a maze to another in 0.04 sec. More insidious are those occasional errors or oddities in the data that generate outlier data points and have a major impact on the statistical analysis. Hunting down exceptional events requires good tactics and a keen eye, and deciding when an outlier is a real event or an outright error requires wise judgment. This stage in the process is critically important. Chapter 9 suggests ways to detect and expunge bad data. The finest design and perfect balancing of test order can be defeated by poor quality control after data collection is finished. This stage includes the backing up of data in whatever form it was collected. Hard disks fail; it has happened to me more than once.

Data analysis

Almost everyone who conducts a thorough statistical analysis of behavioral test data will do so after taking at least one formal course on statistics. Nevertheless, it is seen time and again that lessons from a formal course are not permanently implanted into the mind until the researcher is faced with the task of making sense of his own real data and then trying to convince other scientists that the conclusions are justified by those data. It is suggested in Chapter 9 that the researcher should first compile descriptive statistics and graphs so that the major results of the study are evident, then inquire whether the apparent effects of treatments are indeed statistically significant. The first part of this process can be immediately rewarding, even fun, while the second generates severe pains in the frontal lobes.

Publishing results

An experiment does not become part of the body of scientific knowledge until it is published or somehow made available to other scholars. On occasion a student may conduct an experiment with mice to obtain a grade in a course or even an advanced degree but then fail to follow through with this last step in the process. The study then is not a part of science, and is rendered utterly worthless. This is not a happy outcome. The data must reach others interested in the same topic. Publishing is a difficult art usually learned from a master through close collaboration on a project.

Those are the major steps in research on mouse behavior from conceptualization through to publication. This book emphasizes aspects of the process that generally are not explained in-depth in texts on behavioral neuroscience or research design and statistical data analysis. This information will be most helpful to those entering the field and hopefully will provide some added insights for experienced researchers as well. The field is continually changing and there are always new things to learn.

The Institutional Context of Research

Scientific research is done in an institutional context consisting of people, facilities, and policies. The personnel involved in research can be divided between those in the academic or scientific world and those in the administrative or service realm (Figure 1.2). In a very small institution, the researcher may be in charge of almost every aspect of the enterprise, whereas at a large university he or she may feel remotely connected to officials in the upper administration and service sector. Whatever the case, there are essential roles in research and steps in the process that are the same almost everywhere. Here we focus on those parts that are important for testing mouse behavior.

Figure 1.2 Organization of a research laboratory operating in an institutional and regulatory context. The configuration can differ greatly among institutions. Arrows indicate the approximate frequency of interactions based mainly on the author’s experiences at four universities in Canada and the United States. In some institutions the technical services are based in a department and responsible to the PI, whereas in others they are centralized.

The research usually follows a series of steps that is almost the same for work with both human and animal subjects. These steps are introduced in this Chapter and become the basis for organizing most of this book. The principles of science are universal and apply to all domains of investigation, while the realization of the scientific study is achieved in a local context using a kind of organism that may require slight modifications in the approach.

People

Every laboratory exists in a larger setting governed by a director or head of a department or institute, but these individuals, who may be eminent scientists, are usually involved very little in a research project when a PI is in the lab. The PI is responsible for all aspects of the research, from conceptualization of the problem, gaining ethics approval, applying for a grant, hiring and training staff, analyzing data, and writing articles for publication. With sufficient grant funds, the PI can have a laboratory manager, possibly someone with a Ph.D. in neuroscience or psychology, to organize the daily work of the lab. The actual testing of the mice may be done by career technicians with a bachelor’s degree or a technical certificate in lab animal science. A postdoctoral fellow may do the work of both a PI and lab manager, whereas a graduate student may function as both lab manager and technician. Students sometimes believe they are doing the work of the PI too because the boss is traveling to conferences so much of the time. Whatever the titles and academic status of the people doing the research, the roles of PI, lab manager, and technician are essential. All of these individuals need to have a good understanding of the methodology of behavioral testing.

Every institution has some kind of administrative apparatus that governs all research operations and work with animals. This apparatus will be under the sway of some high institutional official (IO), perhaps a vice president of research or an associate provost, who is rarely involved directly with researchers. The domain of the IO includes three offices of great importance for work in the behavioral test lab: the animal ethics committee, the office of the IO, and animal care personnel. No research can be done until the project is approved by the animal ethics committee (Chapter 6). After it is approved, funds must be obtained to support the work, which usually requires applications to external granting agencies. The office of the IO includes people who know where and how to apply. Once funds are available, care of the animals will often be done by animal care staff that report to the IO, not the PI. This staff will include a professional veterinarian as well as workers who maintain, change, and wash the animal cages. These people are a critical part of the laboratory environment (Chapter 15) a component that is especially difficult for the PI to alter. Although it would be wonderful if the IO, animal ethics committee, and animal care personnel knew about behavioral testing principles, this is unlikely to happen. These people need to deal with a very wide range of research projects and cannot be expected to read an entire book on behavioral testing. They have power over crucial aspects of the research but lack the expertise to plan and conduct a program of behavioral research. The PI and everyone in the lab, therefore, need to develop educational and diplomatic skills when working with colleagues who report to the IO. If a good working relationship cannot be established among all the participants, the research process will not progress smoothly, and in extreme cases will not generate good data.

Technical services such as statistical consulting, computer and Internet technology, and machine and electronics shops are of great importance for the mouse lab. If the lab is very large or the department has many well-funded researchers, these people may be employed to serve one or a few PIs and their staff. Otherwise, the institution may have a central service used by people in all fields of research as well as classroom instruction or medical practice. In some settings, these services are obtained externally from private companies. These people are unlikely to know much about mice; therefore, the researchers need to be able to clearly explain the problems and available options related to the special features of the test animals.

Facilities

A behavioral testing facility usually consists of rooms devoted to the tests, colony rooms for housing the animals over several weeks, and rooms for cleaning the cages and storing supplies. Offices for personnel often are not part of the test facility per se and are not allowed in the animal facility in many institutions. The researcher should have considerable control over who enters the testing rooms and how things are arranged there, but the colony rooms and cleaning areas often are part of a central animal facility managed by an independent professional staff. This can create challenges if routine operations like cage changing, washing the floors and cage racks, or inspections are done without regard to the behavioral testing schedule.

Both federal government and granting agencies provide extensive recommendations on the design of animal facilities. For example, the Canadian Council on Animal Care (CCAC) likes to see all animal housing and procedure rooms contained in one large, centralized facility with a secure means to prevent entry by unauthorized people. These facilities are also supposed to prevent intrusions by wild mice, but mice can be ever so devious and find ways to breach all but the most solid barrier. Hollow walls and ceilings where there are unfilled gaps along heating ducts and water pipes sometimes provide ready access. For example, on one occasion in the author’s lab at the University of Alberta, a technician was surprised to find a wild deer mouse (Peromyscus maniculatus) sitting atop a mouse cage dining on the lab chow.

From the standpoint of the researcher, there are major advantages to having mice in a small satellite facility under the control of the PI. The author designed one such facility (Figure 1.3) at the University of Windsor that was housed in the Great Lakes Institute for Environmental Research where there were no other mammals.. The entire lab consisted of only four rooms in one suite having a single unmarked door to the hallway. All air was vented to the outdoors, so that nobody walking in the hallway even knew it was a mouse lab. Two side-by-side colony rooms could be maintained on opposite light–dark cycles. It was a very short distance from colony to test room, with transit through the cleaning area. Cage washing was scheduled when no behavioral testing was being done. We hired undergraduate students at a modest wage to do the cage changing and cleaning. We got the job done with a small commercial dishwasher (Hobart 7i) instead of the humongous, expensive, and temperamental cage washer often found in central facilities. The Hobart 7i could accommodate only three cages at a time but had a 2 minute cycle time with a bleach rinse at the end, yielding cages that were clean enough for people to eat from. The net result of these features was high quality data and healthy