Predavanje 1

Boris [aki}

Psychology Building Rm. 303, 1280 Main St. West Hamilton, Ontario, Canada L8S 4K1  e‐mail: sakic@mcmaster.ca www.fhs.mcmaster.ca/psychiatryneuroscience/faculty/sakic www.brain‐body.ca/membership.html 

Associate Professor

Department of Psychiatry and Behavioural Neurosciences McMaster University

My field of expertise: Behavioural Neuroimmunology PsychoNeuroImmunology 1. Lupus of the nervous system and role of brain-reactive

antibodies in etiology of aberrant behavior

2. Role of autoimmunity in etiology of Alzheimer’s disease

Structure of the 2-h class: Presentation 50 min Break 10 min Presentation / Discussions 50 min

Please pay particular attention to slides with pinky brain in the upper corner. They reflect a topic or knowledge I would like you to keep during your career.

Topic-relevant slides from my PowerPoint presentations will be uploaded in PDF format to the University server or your e-mail.

What is behavior?

From forest to trees

From a big picture to details

What is life?

Uniquely organized dead matter.

The matter cannot be destroyed, it merely changes forms.

Definition of Life

Compared to dead matter, life expands on our planet.

THREE MANDATES OF LIFE?

1. TO ADAPT IN CONSTANTLY CHANGING EXTERNAL ENVIRONMENT

2. TO SURVIVE COMPETITON FOR RESOURCES (VS. OTHER FORMS OF LIFE)

3. TO PROCREATE AND EXPAND THE SPECIES

BEHAVIOR

CONSTELLATION OF ACTIONS AND TRAITS

THAT PROMOTE THREE MANDATES OF LIFE

HOMEOSTASIS

• physical stimuli (light, sound, heat etc.) • psychological stress

Nervous System

Endocrine System

• Viruses/bacteria • Toxins • Oncogens • Autoantigens

Immune System

Behavior

Homeostatic Metasystem

cytokines

Hormones (e.g., steroids)

External stressors

Internal stressors

Behavior: adaptive (normal) or non-adaptive (abnormal)

Psychology Ethology

Psychiatry Experimental Psychiatry

ETHOLOGY – THE BIOLOGICAL STUDY OF BEHAVIOR

(HOW AND WHY)

PSYCHOLOGY – THE STUDY OF HUMAN BEHAVIOR

(PROXIMATE CAUSATION – HOW)

Why study behavior?

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MEASURING BEHAVIOR DURING NEUROINFLAMMATION

Each successive level of organization has properties that cannot be predicted even from complete knowing the lower level of organization. Detailed knowledge about behavioral phenomenon at molecular, cellular or physiological level is not helpful in fully understanding what is happening at the behavioral level.

Miseries of the Human Kind

1. Natural disasters (force majeure)

2. Physical and mental diseases (including addiction)

3. Aggressive behavior, violence, and wars

4. To understand how to live a happier and more productive life

1. To better understand the nature of animals, including homo sapiens

2. To understand the causes of mental illness and brain damage

3. To find new treatments for brain diseases

Why measure behavior?

PROBLEM CLASSIFICATION

PROXIMATE CAUSATION – HOW DO INTERNAL AND EXTERNAL CAUSAL FACTORS ELICIT AND CONTROL BEHAVIOR?

DEVELOPMENT – HOW DID THE BEHAVIOR DEVELOP DURING THE LIFETIME OF THE INDIVIDUAL?

FUNCTION – WHAT IS THE CURRENT USE OR SURVIVAL VALUE OF THE BEHAVIOR?

EVOLUTION - HOW DID THE BEHAVIOR ARISE DURING THE EVOLUTIONARY HISTORY OF THE SPECIES?

Three phases of life (according to ancient East Indian book of Kama Sutra)

3. Acquiring spirituality

1. Acquiring knowledge

2. Acquiring material things

Adaptive behavior - a principal instrument in achieving success in private and professional life

Schooling Employment Selection of a life partner Marriage Child upbringing Retirement

Principles of experimental science

1. Measurability

2. Controllability

3. Reproducibility

The assessment of behavior depends on:

1. Time of occurrence

2. Context

4. Sequence of behavioral acts

3. Intensity (latency, duration, frequency)

Behavior: Observed / descriptive - qualitative analysis

Systematically measured - quantitative analysis

Distribution of vertebrate animal species used for research, testing, and education

V Baumans Gene Therapy (2004) 11, S64–S66. doi:10.1038/sj.gt.3302371 (mice + rats 77%)

In 2010 – 26,000,000 laboratory animals used for research (mice + rats 95%)

The discovery of anaesthetics and Darwin's publication on the Origin of Species in 1859, defending the biological similarities between man and animal.

Claude Bernard published his book 'Introduction à l'étude de la médecine expérimentale' in 1865, introducing methodology as a tool for the design of physiological experiments.

The development of microbiology caused an increase in the use of animals, due to Koch's 'Postulates' where it is stated that pathogenicity of a microorganism can be proven after successfully infecting healthy, susceptible animals.

The development of pharmacology, toxicology and immunology.

Factors that led to increased use of animals in experimental research

Ethical standpoints

1. Absolutist (extremist) views: always justified, or never justified to use animals in biomedical research

1985 the Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS 123) of the Council of Europe 1986 the Directive for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes of the EU, based on ETS 123, but more stringent.

http://www.apa.org/science/leadership/care/guidelines.aspx

2. Relativist view: certain uses are justified, but not all

Guidelines for Ethical Conduct in the Care and Use of Nonhuman Animals in Research

Benefits of animal research: medical advances that required animal research for their development Benefits to Humans Immunization against polio, diphtheria, mumps, measles, rubella, smallpox Antibiotics Anesthetics and other painkillers Blood transfusions Radiation and chemotherapy for cancer treatment Open-heart surgery for coronary bypass and correction of birth defects Insulin treatment for diabetes Asthma medication Medication for epileptic seizures Organ transplantation and drugs to prevent organ rejection Medications to treat depression, anxiety, and psychosis Microsurgery to reattach severed limbs Rehabilitation of stroke and brain-damaged accident victims Benefits to Animals Vaccination against rabies, distemper, tetanus, and other infections Corrective surgery for hip dysplasia in dogs Orthopedic surgery and rehabilitation for horses Treatment for leukemia and other cancers in pets Improved nutrition for pets Source: Foundation for Biomedical Research, 1988

Guiding principles of three R's: Replacement - substitution of living animals by in vitro techniques (e.g., cells, tissues), computerized models, etc. Reduction - decrease of the number of animals using power analysis, reduce experimental procedures and number of housed animals for improved well being Refinement - environmental enrichment, adequate anaesthesia, analgesia and care, by guaranteeing skills of the researcher /animal staff, by improving experimental procedures and by determining a humane end point, where the animal can be euthanized to prevent unnecessary suffering (CCAC, AUP)

Russell & Burch in 1959: “The Principles of Humane Experimental Technique”

1. Better controlled 2. More consistently measured 3. Shorter than clinical studies 4. Can test cause-effect relationships

Advantages of animal studies:

Development of animal use in the 20th century

V Baumans Gene Therapy (2004) 11, S64–S66. doi:10.1038/sj.gt.3302371

Drop due to: - public awareness - strict legislation on animal use - the development of animal ethics committees

Increase due to development of genetically modified animals

75–100 million vertebrates per year are used in research and testing

Distribution of the purposes for animal use

V Baumans Gene Therapy (2004) 11, S64–S66. doi:10.1038/sj.gt.3302371

1. How worthwhile is a proposed piece of research in terms of its likely contribution to scientific understanding?

2. How likely is the research to bring benefits for humans, animals, economy or environment?

3. How much suffering to animals is likely to result from the research?

Types of judgment to be made before doing an experiment

The Bateson decision cube

The Canadian Council on Animal Care: http://www.ccac.ca/en_ McMaster's Animal Research Ethics Board (AREB) http://fhs.mcmaster.ca/healthresearch/areb_introduction.html

Institutional Animal Care and Use Committee (IACUC)

The subjective experiences of an animal may be totally different from humans, reflecting its different way of life and the different ways in which its body works.

Human interpretation of what is observed in an animal should not be based only on what is known from humans, but also on a good knowledge of its natural history and behavior.

= /

Animal behavior = human behavior ?

Using human emotions and intentions as explanations for animals’ actions can impede further attempts to understand the behaviour.

Absence of evidence is not evidence for absence.

Anthropomorphism Anthropomorphism or personification is any attribution of human characteristics to animals, objects, and natural phenomena.

Lecture 1

Kompjuterizovano merenje pona{anja

Human vs. computerized scoring of behavior

Isolated tests vs. home-cage behavior

Category Factors Ontogeny intrauterine position, litter size, maternal behavior Housing transport to animal facility, size of housing cage, environmental enrichment, social vs. individual housing, light/dark schedule, vibrations / ultrasonic sounds, temperature, humidity, diet Test situation size, shape, and cleanness of test apparatus, protocol, time of testing, light intensity

Factors affecting reliability and reproducibility of behavioural data

Human handling during cage cleaning and transportation to a testing room, observer’s presence, scoring experience, fatigue, and awareness of group origin

Sampling (which subjects and behavioral categories are observed)

Ad libitum Focal Scan Behavior

Ad libitum = no systematic constraints are placed on what is recorded and when. Observer simply notes down whatever is visible and seems relevant at the time. Can be useful during preliminary observations.

Focal = observing one individual or a social unit (e.g., dyad, litter) and recording all instances of its behavior.

Scan = a whole group of subjects is rapidly scanned at regular intervals and the behavior of each individual at that instant is recorded. Often restricts the observer to recording only one or a few simple categories of behavior.

Behavior = watching the whole group and recording each occurrence of a particular type of behavior, together with details of which individuals are involved.

Recording (for how long the behavior is observed)

Continuous Time sampling

Instantaneous One-zero

Continuous = provides an exact and faithful record of behavioral patterns, measuring true frequencies, times, and durations.

Time sampling = sampling of behavior in intervals. Less information is preserved and an exact record of the behavior is not necessarily obtained.

Sample points 

Sample intervals 

Time 

Start 

occurrences / sample points 

How good / right are your dependent measures? Reliability (“good”) - the extent to which the measurement is repeatable and consistent: that is, free from random errors Precision – how free is the measurements from random errors? Sensitivity – are small changes in the true value reflected by changes in the measured value? Resolution – what is the smallest change in the true value that can be detected? Consistency – do the repeated measurements of the same behavioural act produce the same results?

Validity (“right”) - the extent to which a measurement actually measures what the investigator wishes to measure and provides information that is relevant to the questions being asked. Accuracy – how free is the measurements from systematic errors? Specificity - to what extent does the measure describe what it is supposed to describe, and nothing else? 1. 3.

2. 4.

Human vs. computerized scoring of behavior

Company Country Website AccuScan Instruments, Inc. (1) U.S.A http://www.accuscan-usa.com

BioSignal Group Corp. (2) U.S.A http://www.biosignalgroup.com

Biobserve (1) Germany http://www.biobserve.com/experiments/fst-tst.html

Campden Instruments Ltd (1) UK http://www.campdeninstruments.com

Cleversys, Inc (1) U.S.A http://www.cleversysinc.com

Columbus Instruments (1) U.S.A http://www.colinst.com

Coulborn Inc (1) U.S.A http://www.coulbourn.com

HVS Image (2) UK http://www.hvsimage.com/index.htm

IITC Inc (3) U.S.A http://www.iitcinc.com/whatsnew.html

Kinder Scientific (3) U.S.A http://www.kinderscientific.com

Lafayette Instruments Co. Inc (1) U.S.A http://www.lafayetteneuroscience.com

MED Associates Inc. (1) U.S.A http://www.med-associates.com

NewBehaviour (1) Switzerland http://www.newbehavior.com

Noldus Information Technology (1) The Netherlands http://www.noldus.com

Panlab, S. L. (1) Spain http://www. panlab.com

San Diego Instruments Inc (1) U.S.A http://www.sandiegoinstruments.com

Stoelting Inc (1) U.S.A https://www.stoeltingco.com

TSE Tech Sci. Equipment GMBH (1) Germany http://www.tse-systems.com

(1): Limited video-tracking / event recording in home-cage environment; (2): Video-tracking only; (3): Limited hardware-based tracking out of home-cage environment.

Major software / hardware manufacturers of behavioral equipment

Reliable continuous measurements became feasible due to: 1. Increased speed of computer processors 2. Development of digital video formats 3. Increased capacity of storage media 4. Development of real-time video tracking

Computer > Human observer

From time sampling to continuous recording

1. Counting of behavioral responses (e.g., after drinking, eating, wheel running)

2. Position tracking of experimental animals (distribution of locomotion)

3. Classification of behavioral acts (e.g., grooming, sleeping, yawning, etc.) Human-based vs. Software-based applications)

Human observer > Computer

Computer > Human observer

Domains of computerized data acquisition

The list of behaviors in Ethogram Giant Panda

Manual counting of behavioral acts

Computerized counting of behavioral outputs

MedPC IV from Med Associates Inc.

Position tracking

Manual

Video-tracking (center of gravity, 3-point detection)

Infra-red technology

Infra-red technology

Video-tracking

Video-tracking with EthoVision XT 8

EthoVision XT – Zone Setting / Calibration

EthoVision XT – Data Acquisition

EthoVision XT – Data Acquisition in Multiple Arenas

Software-based

Automated classification of behavioral acts

Human-based

Observer XT

CleverSys

Behavioral Instruments (BI)

Analytical technique that identifies the behavior of organisms based on positional and vibratory information

Behavioral Spectroscopy

Automated Seizure Detection in Mice

Automated Forced Swim Assay

Multiple tests vs. monitoring of home-cage behavior

1. Assume a single behavioral domain or category 2. Restricted to a novel environment 3. Short lasting

Disadvantages of “standard” behavioural tests:

Relevant dynamic processes, such as circadian, monthly, and annual rhythms, are not included.

Behavior is a process, not an event.

Important information on baseline conditions and interactions between motivational systems cannot be obtained.

Neuron, Vol. 41, 825–837, March 4, 2004

1. Allows continuous measurements of both baseline behaviour and experimental manipulations.

2. By introducing stimuli to elicit specific behavioural responses, multiple behavioural domains and their interactions can be addressed in a single test set-up.

3. Minimalized stress induced by handling and transportation (confounding factors).

4. Use an standardized testing environment, thereby reducing between-laboratory variance.

5. Fully automated home cage system is time and cost saving, minimizes the need for human intervention and biased analysis.

Advantages of home cage testing

1. Enables video recording independent of light conditions in the laboratory. 2. Enhances the standardization of behavioral experiments. 3. Extendable with specially designed home cage accessories and integrated visual and acoustic stimuli. 4. PhenoTyper offers the test set-up for activity and open field studies, interactive home cage testing, and circadian studies.

Phenotyper by Noldus

Phenotyper

Phenomaster by TSE

social interaction cage (placed when required)

Novel object (placed when required)

running wheel (CT-ENV-043A)

PC with EthoVision XT5,Observer XT, MedPC software packages

stimulus light (ENV-221M)

climbing mesh (CT)

removable tray

water (photobeam lickometer ENV-351W)

Food dispenser -20 mg pellets (ENV-203-20)

digital interface package (DG-716P2)

sucrose solution (photobeam lickometer ENV-351W)

Picolo Diligent BNC card (live tracking and/or MPG4 video recording)

Infra-red Panasonic WV-CP484 camcorder

Integrated Behavioral Station (InBeSt) designed by B. Sakic

Shelter (see through) (IR-010 Infrared Plastic)

Molex cable

BNC cable

INBEST = EthoVision + Med PC IV

Variables: MedPC IV Total Number of Licks on Lickometer 1 (water intake) Latency to First Lick on Lickometer 1 Total Number of Licks on Lickometer 2 (sucrose intake) and volume ingested (manually recorded) Latency to First Lick on Lickometer 2 Total Number of Food Pellets Taken Latency to First Food Pellet Taken Total Number of Counts on Running Wheel Total Number of Runs on Running Wheel Total Time Spent on Running Wheel Latency to Running Wheel Total Number of Climbs on Climbing Mesh Total Time Spent on Climbing Mesh Latency to Climbing Mesh from EthoVision XT 8 Number of entries into the shelter Time spent in the shelter Distance traversed, moving time, locomotor speed Number of visits to the running wheel Latency to approach a novel object Exploration of a novel object (frequency, duration) Sleeping episode (duration, frequency) Grooming (duration, frequency) Seizures (duration, frequency)

InBeSt

Variables used in behavioral assessments

MedPC + EthoVision

Summary Benefits of computerized analysis of behavior

Computerized methodology led to: 1. increased reliability of behavioral measures 2. improved standardization of behavioral protocols 3. minimized confounding factors associated with scoring by human

observer 4. high-throughput phenotyping Automated home cage monitoring allowed: 1. minimized exposure to stressors associated with multiple tests 2. measurement of behavior is conducted in ethologically relevant

context