Behavioral neuroendocrinology involves the study of the interactive effects of the steroid and peptide hormones, neuropeptides, cytokines and neurotransmitters on behavior. Previous chapters have mentioned the role of the hypothalamic nucleii in behavior (Section 4.1), the behavioral effects of neurotransmitter agonists and antagonists (Section 5.8), the neuroendocrine correlates of psychiatric disorders (Section 6.8), the behavioral functions of the steroid hormones (Section 9.9), the cognitive and behavioral effects of neuropeptides (Section 12.5), and the effects of the cytokines on the brain and behavior (Section 13.5). This chapter discusses the behavioral methods used for the study of neuroendocrinology, the neural and genetic mechanisms mediating the effects of hormones on behavior, and some of the special problems involved in conducting behavioral neuroendocrinology research.

Neuroendocrine research involves a number of specific methods such as radioimmunoassays (Chapter 8), autoradiography (Chapter 9), receptor binding assays (Chapter 10) and immunohistochemical techniques (Chapters 11 and 13). The study of behavioral neuroendocrinology relies on specific behavioral methodologies or behavioral bioassays. As discussed in Section 8.1, a bioassay measures physiological changes in an animal or cell culture to determine the concentration or potency of a hormone in the circulation. Thus, the size of a cock's comb is a bioassay for testosterone level and the size and weight of the adrenal glands are bioassays for the level of ACTH. A behavioral bioassay measures behavioral changes to determine the concentration or potency of a hormone.

BEHAVIORAL BIOASSAYS

A behavioral bioassay requires precise qualitative (verbal) descriptions of the behaviors of interest and accurate quantitative (mathematical) measures of the latency, frequency and duration of these behaviors.

This book is an introduction to neuroendocrinology from the point of view of the behavioral neurosciences. It is intended for students in Psychology, Biology, Nursing, Health Education, and other fields of Arts and Science and for more advanced students in physiology, anatomy and medicine who have not had a course on neuroendocrinology. It is based on the first half of my Hormones and Behavior lectures at Dalhousie University. While my lectures, and thus the book, focus primarily on mammalian research, the principles outlined apply to all vertebrates.

This book provides an outline of the neuroendocrine system and will give you the vocabulary necessary to understand the interaction between hormones and the brain. This information is essential to the understanding of the effects of hormones on behavior, but contains little reference to behavior until Chapter 14. In fact, it contains mainly endocrinology, physiology and a bit of cell biology, immunology and biochemistry. Do not despair. Once you master this material, the study of how hormones influence developmental processes and behavior will be easier to understand. This book focuses primarily on the neural actions of hormones, so many of the peripheral physiological actions of hormones, such as regulation of metabolism, water balance, growth, and the regulation of calcium, sodium and potassium levels, which are the focus of traditional endocrinology texts, are referred to only in reference to their importance in the neuroendocrine system.

THE AIM OF THIS BOOK

The aim of this book is to teach students the language and concepts of neuroendocrinology with an emphasis on how the neuroendocrine system influences behavior. It began with a classification of chemical messengers in the body as ‘true’ hormones, neurohormones, neurotransmitters, pheromones, parahormones, prohormones, growth factors and neuroregulators. As more became known about the neuroendocrine system, it was seen that these classifications are not clear cut and a single chemical could fit into two or more classes of messenger. While the classification of chemical messengers is useful to begin the study of neuroendocrinology, by the end it provides little help in understanding the different actions of peptides, steroids and neurotransmitters on different target cells.

The endocrine glands and the pituitary gland are generally thought to be the basis of the neuroendocrine system, but the traditional endocrine function of hormones acting on peripheral target cells provides only a small part of the neuroendocrine activity of a hormone such as testosterone, cholecystokinin or prolactin. These hormones also have significant effects on neural receptors and alter neural regulation of autonomic reflexes, behavior and emotional states. The hypothalamus provides the link between the traditional endocrine system and the brain and provides the mechanism for external factors to regulate the endocrine system. Thus, while the endocrine system appears to consist of a number of closed-loop feedback systems, with highly regulated physiological control over the synthesis, storage, release and deactivation of hormones, external stimuli can alter these systems. Environmental stimuli, social interactions and cognitive factors can greatly alter the functioning of the endocrine system by altering the ‘classical’ neurotransmitter pathways which regulate the release of hypothalamic hormones.

Copied from Animal Behaviour, 43, 185–188 (1992).

The use of animals in research raises important ethical issues. Studies in laboratory settings necessarily involve keeping animals in cages. Manipulative procedures and surgery may be necessary to achieve the aims of the research. Observation of free-living animals in their natural habitats may involve disruption, particularly if feeding, capture or marking is involved. While the furthering of scientific knowledge is a proper aim, and may itself advance an awareness of human responsibility towards animal life, the investigator should always weigh any potential gain in knowledge against the adverse consequences for the animals used as subjects, and also for other animals in the case of field studies.

In order to help their members make what are sometimes difficult ethical judgements, the Association for the Study of Animal Behaviour and the Animal Behavior Society have formed Ethical and Animal Care committees, respectively. These committees jointly produced the following guidelines for the use of all those who are planning and conducting studies of animal behaviour. These guidelines will be used by the Editors of Animal Behaviour. Submitted papers that appear to violate the spirit of the guidelines will be referred to one of the committees, and the evaluation of the committee will be used by the Editor in deciding whether to accept the manuscript.

Preliminary observations

Quantitative recording of behaviour should be preceded by a period of informal observation, aimed at understanding and describing both the subjects and the behaviour it is intended to measure. Preliminary observation is important for two reasons: first, because it provides the raw material for formulating questions and hypotheses; and second, because choosing the right measures and recording methods requires familiarity with the subjects and their behaviour. Preliminary observation is especially important if the problems or animals are new to the investigator.

The hypotheses that direct a study can seldom be formulated in isolation; rather, they reflect existing knowledge and theories, as well as the investigator's own interests and hunches. Effective research therefore requires the investigator to be familiar with the subjects, both by direct experience of watching them and by reading the literature about their biology and behaviour. A period of preliminary observation also provides a valuable opportunity for sharpening up questions and hypotheses, practising recording methods and generating additional or supplementary hypotheses. Most biologists would argue that an initial phase of description is an essential precursor to quantification, since before the right questions can be asked it is essential to know what there is to ask questions about.

We cannot over-state the importance of simply watching before starting to measure systematically. Beginners often falter early in a study because they rush to obtain ‘hard data’ and do not allow sufficient time to watch, think and frame interesting questions.

The response to the first edition of this book was gratifying. Reviewers seemed to agree that the need for such a source existed and that Measuring Behaviour satisfied that need. More than six years have slipped past since Measuring Behaviour first appeared, and the desirability of some changes became apparent. With the passage of time some sections of the first edition – notably those dealing with the technology for recording behaviour – had inevitably begun to show their age. In revising the book, we have taken the opportunity to make a number of changes to the order in which various topics are dealt with, thereby (we hope) making the ideas flow in a more cohesive manner. Some of the sections have been expanded and some entirely new ones added.

Despite our disclaimers in the introduction to the first edition, some reviewers said we should include detailed examples, both to illustrate the general points and to show in particular cases how research methods are selected and used. We have once again ignored their advice, because others – ourselves included – were pleased that we had kept the book concise. We remain convinced that a text book like this benefits from being as succinct as possible, and have therefore resisted the temptation to allow the new edition to burgeon in the cause of comprehensiveness.

We hope Measuring Behaviour will continue to be useful and that this new edition will help a new generation of scientists and students who wish to observe and measure the behaviour of animals and humans, both in the laboratory and in the field.

Why measure behaviour?

In addition to its intrinsic interest, the study of behaviour is both intellectually challenging and practically important. Animals use their freedom to move and interact, both with their environment and with one another, as one of the most important ways in which they adapt themselves to the conditions in which they live. These adaptations take many different forms such as finding food, avoiding being eaten, finding a suitable place to live, attracting a mate and caring for young. Each species has special requirements, and the same problem is often solved in different ways by different species.

Even though much is already known about such adaptations and the ways in which they are refined as individuals gather experience, a great deal remains to be discovered about the diversity and functions of behaviour. The principles involved in the evolution of increasingly complex behaviour and the role that behaviour itself has played in shaping the direction of evolution are still not well understood. Explanations of how behaviour patterns have arisen and what they are for will only come from the comparative study of different species and by relating behaviour to the social and ecological conditions in which an animal lives.

Animals are studied for many reasons (Driscoll & Bateson, 1988). Medical research has as its goals outcomes that are of direct benefit to humans. Such work is readily justified in the eyes of the public because its usefulness is obvious.

The options available

Having looked at the various forms of behavioural measure, we now move on to consider the mechanical processes involved in recording them. The choice of the medium, or physical means, used to record behavioural observations has important consequences for the sorts of data that can be collected and the sampling techniques that can be used. Five basic methods of recording behaviour are available: film or video recording; written or dictated verbal descriptions; automatic recording devices; check sheets; and computer event recorders. The most flexible and commonly used methods are check sheets and computer event recorders (described separately in sections 7.B and 7.C). Using check sheets, an event recorder or any other method obviously presupposes that the observer has formulated a set of discrete behavioural categories.

1 Film or video recordings give an exact visual record of the behaviour which can subsequently be slowed down for analysis. This is useful for studying behaviour that is too fast or too complex to analyse in real time. Similarly, exact records of vocalisations can be made with an audio tape-recorder and the sound patterns analysed later using instruments such as a sound spectrogram.

The major advantage of recording behaviour in this way is that the record can be analysed repeatedly and in different ways. However, filming or videotaping is rarely a complete replacement for observation. At some stage it is always necessary to code the behaviour – that is, to transcribe the record into quantitative measurements relating to specific behavioural categories.

Types of measure: latency, frequency, duration and intensity

Behavioural observations most commonly yield four basic types of measure.

1 Latency (measured in units of time; e.g., s, min or h) is the time from some specified event (for example, the beginning of the recording session or the presentation of a stimulus) to the onset of the first occurrence of the behaviour. For example, if a rat presses a lever for the first time 6 min after it is placed in a Skinner box, the latency to press the lever is 6 min. (See Fig. 5.1.)

If, as is normally the case, the period of observation is limited and each individual is tested more than once, then the behaviour pattern may not occur at all during some tests. This makes it impossible to calculate a simple average latency for each individual, although there are methods of combining repeated latency measures into an overall score for each subject (e.g., Theobald & Goupillot, 1990).

2 Frequency (measured in reciprocal units of time; e.g., s-1, min-1 or h-1) is the number of occurrences of the behaviour pattern per unit time. Frequency is a measure of the rate of occurrence. For example, if a rat presses a lever 60 times during a 30-min recording session, the frequency of lever pressing is 2 min-1. (See Fig. 5.1.)

An alternative usage, which is perhaps more common in the behavioural literature and in statistics, is when ‘frequency’ refers to the total number of occurrences.

This book came about almost without our realising it. We needed something to give to our undergraduate students who were about to embark on projects that involved measuring behaviour. No single source was concise enough or easily accessible to them, so we prepared some notes. These grew longer and longer, they generated more and more comments and, before long, we found ourselves doing something that neither of us had planned.

As the shape of the book started to emerge, we felt that its recommendations ought to represent, as far as possible, the collective view of the practitioners of the subject, rather than merely our own opinions. Therefore, a draft version was read by a large number of friends and colleagues. The eventual book was immeasurably improved by their advice, comments, criticisms and suggestions. Inevitably, some issues still provoke substantial disagreement; we have attempted to indicate where this is the case and where we have departed from common practice.

Some of our reviewers asked us to put in detailed examples, to illustrate the general points and to show in particular cases how methods are selected and used. We decided not to do so to the extent they would have liked for several reasons. First, we wanted to keep the book as concise as possible, in order to make the information in it readily accessible. Second, we wished to draw attention to general principles of measurement and analysis.

Different approaches to studying behaviour

Not all scientists study behaviour in the same way or ask the same sorts of questions. Historically, psychology (which originally grew out of the study of the human mind) was distinguished from ethology (the biological study of behaviour) in terms of the methods, interests and origins of the two sciences. In this century, comparative and experimental psychologists have tended to focus mainly on questions about the proximate causation of behaviour (so-called ‘how’ questions), studying general processes of behaviour (notably learning) in a few species under laboratory conditions. In contrast, ethologists have their roots in biology and have asked questions not only about how behaviour is controlled but also about what behaviour is for and how it evolved (‘why’ questions).

Biologists are trained to compare and contrast diverse species. Impregnated as their thinking is with the Darwinian theory of evolution, they habitually speculate on the adaptive significance of the differences between and within species. Indeed, many ethologists are primarily interested in the biological functions of behaviour and are wary of proceeding far with laboratory experiments without first understanding the function of the behaviour in its natural context. Studies in unconstrained conditions of animals, and increasingly of humans, have been an important feature of ethology and have played a major role in developing the distinctive and powerful methods for observing and measuring behaviour. In contrast, psychologists have traditionally placed greater emphasis on experimental design and quantitative methods.

General advice

Our aim in this chapter is to consider briefly some of the main issues that arise in the statistical analysis and interpretation of behavioural data. This is not a statistics textbook, however, and for an account of statistical methodology readers should refer to one of the many excellent statistics books available.

Our general advice is not to become obsessed by statistical techniques, nor too cavalier in their use. Statistical analysis is merely a tool to help answer questions, and should be the servant rather than the master of science. The great physicist Lord Rutherford was perhaps over-stating this point when he wrote: ‘If your experiment needs statistics, you ought to have done a better experiment’; biological systems have much greater variability than is encountered in physics experiments and statistical analysis is often essential for understanding what is going on. Nonetheless, excessively complicated statistics are sometimes used as a poor substitute for clarity of thought or good research design. Statistical analysis, no matter how arcane or exquisite, can never replace real data.

Besides being sometimes over-used, statistical techniques are frequently misused in the behavioural literature. We have already outlined one common error – that of pooling data in the mistaken belief that they are independent of one another (section 3.F). Other familiar pitfalls, covered in this chapter, include the inappropriate use of one-tailed tests (section 9.C.5), the conflation of effect size with statistical significance (9.D), the application of parametric tests to data that violate their underlying assumptions (9.F), the misinterpretation of multivariate statistics (9.1) and the various misuses of correlation coefficients (9.G).

Reliability versus validity

Measuring behaviour, like measuring anything else, can be done well or badly. When assessing how well behaviour is measured, two basic issues must be considered: reliability and validity (sometimes expressed as the distinction between ‘good’ measures and ‘right’ measures).

1 Reliability concerns the extent to which measurement is repeatable and consistent; that is, free from random errors. An unbiased measurement consists of two parts: a systematic component, representing the true value of the variable, and a random component due to imperfections in the measurement process (see section 9.K and Martin & Kraemer, 1987). The smaller the error component, the more reliable the measurement. Reliable measures, sometimes referred to as good measures, are those which measure a variable precisely and consistently.

At least four related factors determine how ‘good’ a measure is:

(a) Precision: How free are the measurements from random errors? The degree of precision is represented by the number of significant figures in the measurement. Note that precision and accuracy are not synonymous: accuracy concerns systematic error (bias) and can therefore be regarded as an aspect of validity. A clock may tell the time with great precision (to within a millisecond), yet be inaccurate because it is set to the wrong time.

(b) Sensitivity: Do small changes in the true value invariably lead to changes in the measured value?

(c) Resolution: What is the smallest change in the true value that can be detected?