Abstracts ·Joop S de Graaf, PhD ·David Whittaker, DVM&S DLAS MRCVS ·Bart A Ellenbroek, PhD ·Alma Gower, PhD ·Simon Bate, PhD ·Paul Moser, PhD ·Rob Voskuyl, PhD ·Jo Neill, PhD ·Pim Drinkenburg , PhD ·Arjan Blokland, PhD ·John L Waddington, PhD DSc ·Berend Olivier, PhD ·Frank Sams-Dodd, PhD, DSc ·Sietse F de Boer, PhD ·Martien J H Kas, PhD ·Workshop & Student Presentations ·Clare Stanford PhD ·Case Studies Joop S de Graaf, PhD Introductory Remarks The younger generation of pharmacologists, highly proficient in genomics-based and combinatorial methods, is facing the challenge to investigate an imminent wave of transgenic animals. This course offers an introduction to observing and adequately interpreting animal behaviour along with the necessary skills to set up behavioural experiments. << David Whittaker, DVM&S DLAS MRCVS Can we have Happy Animals and still deliver Good Science? Three key questions will be addressed in the context of laboratory animal welfare: • Can animals feel happy? • How can we make our animals happy? • Does happiness hinder or prevent good science? For the purpose of assessing animal welfcare we will consider “happiness” as a stae of freedom from pain, suffering, distress or lasting harm. The Five Freedoms developed by the Farm Animal Welfare Council (1993) best encapsulate the assessment of an animal’s welfare even for laboratory animals. Having developed and explored the conept of happiness for animals and what it might “look like” we will go on to explore how the Five Freedoms translate for our charges, the laboratory animal. Specific examples will illustrate how this concept is easily deployed in practice to evaluate and objectively assess an animal’s state of welfare (or happiness?). Finally, we will consider how limitless happiness may hinder or indeed prevent delivery of good science. Therefore by inversion we will explore why limiting or “damaging” the happiness may be necessary for experimjental (or other) reasons. In doing this we will close a circle by introducing the concept of Refinement which will be a principle feature of the day’s considerations. << Bart A Ellenbroek, PhD Issues of Animal Modelling Discovering novel compounds for human diseases requires the development of animal models for the specific disease under study. With respect to brain disorders, such models have proven to be notoriously difficult and, hence, many different models exist for each specific disease. Broadly, such models can be differentiated into screening models (where the emphasis lies on pharmacology) and simulation models (where the emphasis is on symptoms). The aim of the presentation is to discuss the general principles behind such animal models and to present practical guidelines for developing and validation animal models for psychiatric and neurological diseases. Most neurobiological research is performed with either rats or mice. Although the rat is the preferred species, especially when higher cognitive functions are involved, the mouse has gained more and more acceptance on the grounds of the relative ease with which the genetic material can be altered to produce, for instance knock-out mice. However, there are quite a lot of fundamental differences between rats and mice, both in terms of behaviour, as well as in the structure and function of the nervous system. In addition to differences between species, it is important to realise that there are also fundamental differences between strains of rats or strains of mice. Moreover, even within the same strain of animals, there can be differences between different breeders and suppliers, especially in so-called outbred strains of rats. This is not surprising since the make-up of the brain and behaviour is determined by both genetic and (early) environmental factors. Therefore, differences in breeding facilities may have far reaching consequences for the development of an animal. Finally, we will discuss the importance of individual differences within a strain from the same supplier. Although in the past such individual differences were often regarded as a nuisance they are now often used to selectively breed animals for a specific behavioural trait. << Alma Gower, PhD Experimental Design and Data Interpretation By definition, Behavioural Pharmacology involves living animals which imposes specific challenges to the scientist. Notwithstanding, it has proved to be a powerful tool for demonstrating specific and systematic effects in the integrated organism. The aim of this seminar is to highlight factors which impact on experimental design and study conduct and to discuss issues of data interpretation. Behavioural research may be prompted by various objectives which range from behavioural pharmacology to characterise CNS activity in order to predict and identify potential new CNS acting drugs, through to applications for CNS safety evaluation, both for new non-CNS drugs as well as for industrial and agro-chemicals. A more recent and increasing application is the evaluation of the impact of genetic modifications and general profiling of transgenic animals. Clearly the objective determines the choice of experimental paradigm. However, whatever the paradigm, living animals are used and the research is thus subject to the variability and special complexities which this entails. It is therefore extremely important to optimise testing conditions to achieve a reliable and reproducible experimental system. The variables controlling behaviour exert subtle effects but these may be sufficiently disrupting to interfere with behavioural consistency and study reproducibility and may be account for an inability to replicate published findings. In addition, the testing procedures often rely on subjective evaluation so the role of the experimenter is of particular relevance. The purpose of this seminar is to create an awareness of factors which impinge on the experimental design and on the study conduct, to optimise achievement of the objective. Consideration will be given to choice of experimental test system and where appropriate, how this extrapolates to the clinical situation. In this context, it is important to be sure that what is intended to be measured will actually be measured. The choice of automated versus non-automated approaches will also be considered. Having selected the test system, the factors influencing the experimental design will be reviewed, ranging from species and animal characteristics, pre-testing manipulations, through to determination of appropriate test-groups including relevant controls, etcetera. At this stage, the eventual statistical analysis should be taken into account (although this topic will be discussed separately by another speaker). Even the most carefully designed study can founder if the study is not carried out carefully. Although the experimental design should take into account and control for many factors which could affect study outcome, it is nevertheless very important to be aware of additional factors which directly affect the study conduct, such as location and environmental conditions, the role and consistency of experimenter as well as the actual manipulation of the animal to minimise stress. Finally, the seminar will consider problems related to data interpretation and what additional steps may be necessary to realise the objective. Over the course of the past 50 years, behavioural pharmacology has proved to be a powerful tool for demonstrating specific and systematic effects in the integrated organism. With careful and appropriate study design and execution, it will continue to play a major role in the evaluation of pharmaceutical and industrial agents. << Simon Bate, PhD Workshop Experimental Design and Statistics The purpose of this session is to apply the techniques learnt in the seminar on Experimental Design and Data interpretation and to experience the impact of experimental design on the eventual result. Delegates are invited to work with a dataset representing the outcome of an imaginary experiment (preferably on their own laptops, but extra computers are available on request). Additionally, participants are invited to bring along existing datasets they have generated in previous studies. Statistical computing software will be provided as well. Simon Bate and Alma Gower will be available to offer help and advice on the statistical analysis of their datasets. This will include tips on the appropriate graphical and statistical tools to use and the most powerful statistical approaches to get the most information out of the dataset. << Paul Moser , PhD Behavioural Receptor Typing Virtually all neurotransmitter receptors show a heterogeneous distribution in the CNS. It is therefore not surprising that stimulating a particular receptor with more-or-less specific agonists often leads to characteristic symptom patterns that can be used as an index of functional activity at that receptor system. Typical examples include the head-twitch response (5-HT2), orofacial stereotypies (D2) and the tetrad (CB1). These effects can then be used to investigate in vivo drug effects at these receptors to evaluate potency, duration of action etc. In addition, these signs can also be used to investigate interactions between neurotransmitter systems. Classification of agonist effects are usually more accurate when symptom clusters are used (eg the serotonin syndrome or the cannabinoid tetrad), as individual symptoms are rarely dependent on activity at a single receptor subtype. In contrast, antagonists can be evaluated using agonist effects against less specific endpoints such as hyper- or hypo-activity and hyper- or hypo-thermia. The advantage of this approach is that the endpoint can be a convenient and simple measure allowing relatively high throughput evaluation. Finally, the drug discrimination technique will be described which allows us to detect effects of CNS active substances that are not detectable by simple observation. << Rob Voskuyl , PhD Mechanism-based PK/PD: focus on animal to human extrapolation In recent years, important progress has been made in the development of chronically instrumented rat models in which the time course of drug effects can be determined in conjunction with the time course of drug concentrations in plasma. Studies in chronically instrumented rat models can advance the drug development process in many ways. Useful applications are: 1) development of mechanism-based PK/PD models, 2) selection of drug candidates and lead optimisation, 3) development, evaluation and validation of biomarkers and 4) prediction of exposure response in man. Application of PK-PD modelling for CNS disorders has been and still is a great challenge. Use of the EEG as a marker for the pharmacological effect has proven to be a powerful tool, because the EEG provides nearly continuous information on the state of the CNS in several ways. Analysis of the EEG has been particularly successful in the investigation of benzodiazepines, neurosteroids and synthetic opiates. Recently, behavioural analysis has been introduced as another technique to assess the efficacy of drugs. Epilepsy evidently lends itself to such an approach, because motor components of epileptic seizures can be suppressed or modified in character or intensity by drugs. However, the technique should be applicable as well in other CNS disorders that are characterized or accompanied by behavioural changes. A new element in PK-PD modelling is a growing interest in the influence of disease progression on the pharmacodynamics of drugs. This presentation will illustrate the application of PK-PD analysis in studies on CNS active drugs and include examples of the above-mentioned issues. << Jo Neill PhD The Psychopharmacology of Feeding Behaviour Obesity is an increasing problem worldwide and is associated with various disease states, including type 2 diabetes, hypertension, cardiovascular disease, stroke and osteoarthritis. The mechanisms controlling food intake are complex and multifactorial. One way to improve our understanding of these mechanisms is through the use of animal models. This presentation will outline the various paradigms to investigate ingestive behaviour in the laboratory, and the way in which pharmacological agents can alter feeding, with particular emphasis on models of antipsychotic-induced weight gain. << Pim Drinkenburg , PhD Bridging sleep-wake behaviour from animal to human Next to sexuality and intake behaviour, sleep constitutes one of the most fundamental behaviours in the animal kingdom. Although its function is still not fully understood, it is clear that sleep is essential for survival. Sleep has many behavioural and physiological correlates, which can be monitored and appear to be conserved across species. The same holds true for the way in which drugs modulate sleep and its correlates. Examples will be given of the manifestations of sleep and sleep dysfunction and how this can be used to bridge the understanding of drug effects and extrapolation from animal to human. << Arjan Blokland, PhD Preclinical models of cognition Human cognition is not a unitary process - it falls apart in many different sub-processes. Modeling human cognition in animals is therefore not a simple task and the effects of e.g. pharmacological manipulations can therefore best be studied using different models for different aspects of cognition. In this seminar, the main focus will be in memory. Memory research has a long-standing tradition in human and animal research and a vast amount of models are available. The huge list of tasks allows to measure different aspects of memory. However, it is not always easy to decide if a test is ‘good’ or ‘bad’. In this presentation I would like to explain more about the good and the bad and some ugly tasks. This is not only related to construct- and face validity but also related with test protocol and measuring behavior. I hope to demonstrate that memory can be assessed in animals provided that great care is taken to make valid definitions and to rule out confounding factors. << John L Waddington PhD DSc Behavioural phenotyping Mutant mice with targeted gene deletion [‘knockout’] or insertion [transgenic/‘knockin’] now constitute a primary tool in neuroscience research. Though molecular and cellular biology are important phenotypic domains, the ultimate level of phenotype is behaviour, for which there are two main sources of methodological variation: that attributable to the ‘knockout’ technique itself and that attributable to the manner in which the associated phenotype is assessed. It is widely accepted that careful behavioural evaluation is integral to phenotypic evaluation of any ‘knockout’. This can involve at least two approaches. One advocates a broad, hierarchically based strategy, to capture the breadth of phenotype across a diversity of general behaviours in the absence of a priori considerations. Another emphasises a priori roles in the regulation of behaviour that require a level of resolution and sensitivity commensurate with the specificity of the entity deleted. This presentation will discuss several of these issues, including the value of ethologically based vis-à-vis other behavioural approaches. << Berend Olivier, PhD Sexual Behaviour Sexual behaviour and the effects of (psychoactive) drugs on sexual performance and activities is an important topic in our human society. Fundamental research into brain and spinal cord mechanisms involved in all aspects of this important branch of social behaviour is strongly on the increase and various drugs are on the market already for certain purposes, including the PDE5-inhibitors for erectile dysfunction (sildenafil-Viagra(r)) and the anti-premature ejaculation drugs (certain SSRIs). Furthermore, side effects of many drugs are considered a big nuisance in therapeutic use and many new drug appliances want to know potential influence on sexual capacities before going to clinical trials. Male and female sexual behaviour in rats offers many possibilities to model certain human sexual dysfunctions, including motivational (libido) and performance (orgasm, ejaculation, erection) aspects. Normal sexual interaction between a male rat and an oestrus female will be illustrated as starting point. This is highly interactive behaviour in which the male and female display highly motivated activities that are highly synchronized, and in which all kind of sensory and motor modalities play a role, including ultrasonic sounds. Males display approach, following, mounting, intromissions and ejaculations, followed by rest periods in which they display postejaculatory songs of approx. 22 kHz. Females hop, dart, and show ear wiggling and display lordosis behaviour upon mounting of the male. The female produces high-pitched ultrasonics during the courting behaviour and refrain from courtship behaviour during the 22 kHz song of the male. This cycle can be repeated many times till sexual exhaustion occurs. In large groups (we have tested approx. 500) of male rats, sexual endophenotypes are found, strongly reminiscent of the human male population. When male rats are tested weekly for 30-min/test,, each rat displays a very stable ejaculation pattern after 3-4 tests. The number of ejaculations is distributed in a Gaussian way; there are animals showing no or 1 ejaculation per test (the retarded or 'slow' ejaculation group), but also animals that perform 4-5 ejaculations/test, the premature or 'fast' ejaculators. The remaining animals fall in the 'normal' range of 2-3 ejaculations.Depending on the endophenotype of the rat, effects of drugs may vary. Several studies using this approach have been performed and relevant examples of prosexual and sexual inhibitory effects of drugs will be described. Acute and chronic dosing of antidepressants (and other drugs) may be of help to delineate putative side effects in humans. Various implications of drug effects on (aspects of) sexual behaviour and its dysfunctions will be outlined. << Frank Sams-Dodd, PhD, DSc Social Interaction In a number of psychiatric disorders the ability to have normal social relations is disturbed, a symptom that severely affects the lives of the patients and their families. Social behaviour can easily be studied and quantified in animals and studies have shown that these behaviours are sensitive to drugs in a manner that correlate well with clinical findings. The purpose of the seminar is to discuss the use of social behaviour as a parameter in pharmacological studies and in disease models, the design of appropriate behavioural tests and how confounding variables may affect the interpretation of observations. << Sietse F de Boer, PhD Ethology, neurobiology and psychopharmacology of aggressive behaviour in rats and mice. A considerable part of our current knowledge on the ethology, neurobiology and pharmacology of normal and deviant forms of human aggression is based on experimental laboratory models of aggressive behaviour in animals. Much of this pre-clinical aggression research is conducted in territorial male or maternal female resident rats/mice confronting an intruder conspecific (resident-intruder conflict paradigm). By recording the frequencies, durations, latencies and temporal and sequential patterns of all the observed behavioral acts and postures in the combatants during these confrontations, a detailed quantitative picture (ethogram) of offensive (resident) and defensive (intruder) aggression is obtained. Normally the display of aggressive behavior serves an important adaptive biological function and does not exceed species-typical levels or patterns. However, escalated excessive or maladaptive forms of aggressive behavior, that are more relevant to the clinical situation, can also be induced in this model by various neuropharmacological/genetic (alcohol, benzodiazepines, single gene mutations) and environmental/behavioral (social instigation, frustration, repeated winning experience) means. Furthermore, using this experimental approach, classical neuroanatomical tracing and lesion/stimulation studies have revealed the global neural substrates of aggression. In addition, recent studies using Immediate-Early Gene expression mapping have yielded a more detailed picture of the individual neurons in the brain that become activated during the expression of aggressive behavior. Accordingly, traditional pharmacological studies and new molecular genetic approaches have resulted in an impressive list of molecular substrates (i.e, neurotransmitters/hormones/cytokines and their respective enzymes, receptors and intraneuronal signalling molecules) that, within these neural circuits, may profoundly influence aggression. Among the most promising lines of inquiry are those that focused on targeting subtypes of serotonergic, GABAergic, glutaminergic, dopaminergic and several neuropeptidergic (CRH, opioids, vasopressin, oxytocin) receptors and their genes as potential targets for pharmacotherapeutic interventions. Additionally, by measuring aminergic activity during the initiation, execution and termination of aggressive bouts as well as in anticipation of such confrontations via in vivo microdialysis promises to be relevant to the fundamental neurobiological question of integrating serotonin deficiency that characterizes certain violent-prone individuals with the phasic changes in this amine that are triggered by aggressive behavior itself. Finally, an important concept in aggression research that relates to an understanding of the individual vulnerability for stress-related diseases concerns the enduring behavioral, physiological and neuroadaptive changes that result from brief aggressive experiences. << Martien J.H. Kas, PhD Exploration and Anxiety Survival of an organism in its ever-changing environment highly relies on proper behavioural responses to external cues. For example, exploratory behaviour for food or social interaction has been evolved to deal with variation in the availability of food resources or reproduction partners, on one hand, and with the presence of threat from natural predators or territorial attacks from con-specifics, on the other hand. The identification of biological substrates underlying basic behavioural survival mechanisms will greatly contribute to our understanding of normal and maladaptive behaviour in animals and humans, however, novel research approaches are necessary to tackle its complexity. Conventional laboratory tests for studies on exploratory behaviour reflect behavioural differences following pharmacological or genetic interventions, however, the nature of these traditional tests makes it difficult to interpret the data. Therefore, the aim of this seminar is to discuss short-comings of conventional behavioural tests for exploratory and anxiety-related behaviours and to introduce novel phenotyping approaches that address distinct behavioural domains within behavioural strategies. Furthermore, new mouse genetical genomic approaches will be discussed that will allow the identification of biological substrates underlying complex neurobehavioural traits. << Workshop & Student Presentations This afternoon session is intended for the presentation of the experimental results obtained in the morning session so that experience and problems can be shared with the other students. Each group will have approximately 40 minutes to present and discuss their experimental test system and data. Prepare group presentations using the following guidelines: • Assign one member of the group as co-ordinator. • Give a short Introduction to the topic. • Describe the Methodology. Include a video clip to show the actual behaviour which was observed and evaluated. • Present the data- on an individual basis and treated (e.g. means ± SEM) as appropriate. • Discussion/interpretation. • Comment on what you have learned/ problems encountered/ tips for future etc. • Allow 5 or 10 minutes for questions 16:30-18:30: Presentations and Discussion << Clare Stanford PhD Genes, Environment and Behaviour Walter Cannon (1911) was the first to propose that animals’ emotional (flight / fight) response to an environmental stimulus differed within a group of individuals.  He even suggested that this variability was coded chemically. In recent years, technological developments have enabled us to monitor gross physiological and neurochemical responses in freely-moving animals when they are exposed to non-noxious environmental stimuli. We can now study a wide range of animals’ responses to conditioned and unconditioned stimuli at the same time as recording their behaviour.  As a result of this work, we have learned that both animals’ genes and past experiences contribute to individual differences in neurochemistry and behaviour in later life. However, investigating the interactions between all these factors is not a straightforward process. This talk will discuss some of the procedural difficulties and how best to avoid them. A major difficulty is that the neurochemical and behavioural response to an experimental challenge are often assumed to be causally linked. Even worse, the neurochemical response is usually assumed to underlie the change in behaviour (rather than the other way around). Such assumptions need careful validation.  Finally, as the expanding list of mutant mice will testify, comparing responses in inbred strains of animals is helping to unravel the neurochemical coding of behaviour. Even so, it is important not to make invalid assumptions about what needs measuring.  This seminar will end with a discussion of some pros and cons of using genetically-modified mice in psychopharmacology research, particularly when trying to explain the neurobiology of mood and behaviour.  << Bart Ellenbroek, PhD; Joop S de Graaf, PhD Case Studies Examples from every-day practice will be analysed as an exercise in comprehensive whole-animal pharmacology. Participants are invited to contribute their ownexamples for discussion and/or clarification. <<