Ethology and Enrichment
The ABC of Animal Experiments
Vootele Voikar
Helsinki 09.01.2012
• Increased use of mice in biomedical research,
including behaviour
Different approaches to studying
behaviour
• Psychology – focus on questions about
proximate causation of behaviour (’how’
question), general processes of behaviour in a
few species under laboratory conditions
• Ethology – the biological study of behaviour,
how behaviour is controlled, but also what
behaviour is for and how it evolved (’why’
question) in natural conditions
Levels of Organization of the Nervous System
and Measuring Behaviour
Each successive level of organisation
has properties that cannot be
predicted from knowing the lower
levels of organisation.
Perhaps most satisfying of all,
therefore, are the studies in which an
analysis of an organism’s behaviour is
closely integrated with an analysis of
the neural, physiological and
molecular mechanisms that underlie
its actions. Knowledge of mechanism
can greatly inform understanding of
behaviour – and vice versa.
(Paul Martin and Patrick Bateson,
Measuring Behaviour, 3rd edition)
Ethology + Neurobiology = Neuroethology
evolutionary and comparative approach to the study of animal behavior and its underlying
mechanistic control by the nervous system
The four problems of behavioural biology
(from Tinbergen, 1963)
•
Proximate causation or control – ’How does it work?’ How do internal and
external causal factors elicit and control behaviour in the short term?
•
Development or ontogeny – ’How did it develop?’ How did the behaviour
arise during the lifetime of the individual; that is, how is behaviour
assembled?
•
Function – ’What is it for?’ What is the current use or survival value of the
behaviour? How does behaving in particular way help the individual to
survive?
•
Evolution or phylogeny – ’How did it evolve?’ How did the behaviour arise
during the evolutionary history of the species?
Tecott, Nestler 2004
How is behaviour studied?
Standard tests and test batteries
Distance travelled and time
spent in different zones of
various novel arenas
during limited time
(minutes – hours)
ETHOGRAM
An ethogram is a catalogue of the discrete behaviours typically employed by a
species. These behaviours are sufficiently stereotyped that an observer may record
the number of such acts, or the amount of time engaged in the behaviours in a time
budget.
Experimental ethograms are usually constructed to be exclusive and exhaustive:
- An exclusive ethogram is one where each behavior performed by the animal can
only be categorized as one behavior in the ethogram - that is, the animal can only
be recorded as doing one thing at a time.
- An exhaustive ethogram is one where every behavior performed by the animal
has a category in the ethogram, this is normally achieved in an experimental
ethogram by lumping all the behaviors of no interest to the hypothesis being tested
in an 'other' category. This greatly speeds the recording of behavior, as behaviors
irrelevant to the hypothesis can simply be ignored.
MOUSE ETHOGRAM
A Wiki Ethogram for the Laboratory Mouse -
http://www.ag.purdue.edu/ansc/mousebehavior/Wiki%20Pages/Home.aspx
Van Abeelen, J.H.F. (1963). Mouse mutants studied by means of ethological methods.
Genetica, 34, 79-94.
Van Oortmerssen, G. A. (1971). Biological Significance, Genetics and Evolutionary
Origin of Variability in Behaviour within and between Inbred Strains of mice (Mus
musculus): A Behaviour Genetic Study. Behaviour, 38(1/2), 1-92.
Mackintosh, J. H. (1981). Symposium of the Zoological Society of London. In Biology of
the House Mouse (Vol. 47, pp. 337-365).
A.
ACTIVE BEHAVIOURS
1. General activity
a) Exploratory behaviour
- search (locomotion, rearing)
- attend
- approach
- investigate (sniffing, whisking)
b) Affiliative interactions
- group sleeping
- allogrooming
c) Agonistic interactions
- threat behaviour
- aggressive behaviour
- flight and submissive behaviour
d) Sexual behaviour
e) Maternal behaviour
f) Abnormal behaviour (e.g. stereotypic behaviours,
barbering)
g) Miscellaneous
2. Maintenance behaviours
a) Drinking
b) Feeding
c) Grooming
d) Nesting
B. INACTIVE BEHAVIOURS
1. Sleeping
2. Still and alert
C. UNKNOWN BEHAVIOURS
Van Abeelen 1963
Ethological, species-specific behavioural tests
Nesting test (Deacon 2006)
5
35
30
4
25
3
CON
HIPP
2
20
15
10
1
5
0
0
nestscore
nestlet untorn, %
Burrowing test (Deacon 2006)
100
80
60
CON
HIPP
40
20
0
graved after 4h, %
graved after 24h, %
A robust automated system elucidates mouse home cage behavioral structure.
Goulding EH, Schenk AK, Juneja P, MacKay AW, Wade JM, Tecott LH.
Proc Natl Acad Sci U S A. 2008 Dec 30;105(52):20575-82.
•
Patterns of behavior exhibited by mice in their home cages reflect the function and
interaction of numerous behavioral and physiological systems. Detailed
assessment of these patterns thus has the potential to provide a powerful tool for
understanding basic aspects of behavioral regulation and their perturbation by
disease processes. However, the capacity to identify and examine these patterns in
terms of their discrete levels of organization across diverse behaviors has been
difficult to achieve and automate. Here, we describe an automated approach for
the quantitative characterization of fundamental behavioral elements and their
patterns in the freely behaving mouse. We demonstrate the utility of this approach
by identifying unique features of home cage behavioral structure and changes in
distinct levels of behavioral organization in mice with single gene mutations
altering energy balance. The robust, automated, reproducible quantification of
mouse home cage behavioral structure detailed here should have wide
applicability for the study of mammalian physiology, behavior, and disease.
A robust automated system elucidates mouse home cage behavioral structure.
Goulding EH, Schenk AK, Juneja P, MacKay AW, Wade JM, Tecott LH.
Proc Natl Acad Sci U S A. 2008 Dec 30;105(52):20575-82.
To quantitatively examine behavioral organization in
the freely acting mouse, daily movement, feeding,
and drinking are monitored in a simple stable home
cage environment. Mice are individually housed in
home cage behavioral monitoring (HCM) cages
consisting of 45x24x17 cm plexiglass enclosures with
feeders and water bottles mounted at one end. The
feeder consists of a wire ramp allowing entry into an
enclosure where mice access food via an aperture in
the ramp. Feeding is detected by interruptions of a
photobeam located below the opening in the ramp.
Drinking is detected by measuring changes in
capacitance resulting from lick contacts with the
metal spout of the water bottle. To monitor the
position of an animal's center of gravity, the home
cage enclosures are placed activity-monitoring
platforms, each of which has a central pivot point
and two load beams. Intake event onsets and offsets
(photobeam breaks and lick contacts) are sampled
every millisecond. Movement is sampled every 20
milliseconds, and a movement event onset and
distance moved in x and y is recorded if an animal's
center of gravity moves beyond a radius of 1 cm
from the previous sample.
State classification
Bout classification
Goulding E. H. et.al. PNAS 2008;105:20575-20582
Daily time budgets in WT (C57BL/6J), OB (leptin mutant),
and 2C (5-HT2C mutant) mice
Black – inactive; orange – feeding; blue – drinking; green – locomotion; red –
”other” behaviours during AS
Goulding E. H. et.al. PNAS 2008;105:20575-20582
Automated high-resolution behavior analysis
HomeCageScan
hanging upside
down
grooming
eating
drinking
The power of automated high-resolution behavior analysis revealed by its application to mouse
models of Huntington's and prion diseases
Steele, Jackson, King, and Lindquist, PNAS 2007
•
Automated analysis of mouse behavior will be vital for elucidating the genetic
determinants of behavior, for comprehensive analysis of human disease models,
and for assessing the efficacy of various therapeutic strategies and their
unexpected side effects. We describe a video-based behavior-recognition
technology to analyze home-cage behaviors and demonstrate its power by
discovering previously unrecognized features of two already extensively
characterized mouse models of neurodegenerative disease. The severe motor
abnormalities in Huntington's disease mice manifested in our analysis by
decreased hanging, jumping, stretching, and rearing. Surprisingly, behaviors such
as resting and grooming were also affected. Unexpectedly, mice with infectious
prion disease showed profound increases in activity at disease onset: rearing
increased 2.5-fold, walking 10-fold and jumping 30-fold. Strikingly, distinct
behaviors were altered specifically during day or night hours. We devised a
systems approach for multiple-parameter phenotypic characterization and applied
it to defining disease onset robustly and at early time points.
Steele et al. PNAS 2007
Steele et al. PNAS 2007
LABORAS (Metris b.v.)
Van de Weerd et al. 2001
Three R’s
(Russell and Burch, 1959)
Replacement refers to the preferred use of non-animal methods over animal methods
whenever it is possible to achieve the same scientific aim
Reduction refers to methods that enable researchers to obtain comparable levels of
information from fewer animals, or to obtain more information from the same number of
animals
Refinement refers to methods that alleviate or minimize potential pain, suffering or distress,
and enhance animal welfare for the animals still used
CAGE ENVIRONMENT
Breed and house animals in highly standardized conditions
reduced individual differences (intra-experiment variation) -> detection of
treatment effects
reduced differences between studies (inter-experiment variation) ->
increased reproducibility
Solution: Small, barren and monotonous housing
Science 284:1670, 1999
CAGE ENVIRONMENT
H. Wurbel ”Ideal homes? Housing effects on rodent brain and behaviour” Trends in Neurosci 24:
207-211 (2001)
Outbred ICR (left cage) and ICR nu mice (an athymic, nude mutation; right cage) develop two distinct forms of stereotypic
behaviour, bar-gnawing (mouse to the right in the left cage) and jumping, which originate from two different appetitive
behaviours to escape from the home cage. Performance of these patterns is highly repetitive, invariant and fixed in
orientation (see scratch marks in the left hand corner of the cage to the right). Although energetically costly, time consuming,
and apparently ineffective, mice persist in gnawing the bars of the cage lid or jumping up-and-down along the cage wall,
which might account for up to 50% of their total daily activity. Behavioural and pharmacological evidence relates cage
stereotypies in rodents to housing-induced basal ganglia dysfunction.
CAGE ENVIRONMENT
H. Wurbel ”Ideal homes? Housing effects on rodent brain and behaviour” Trends in Neurosci 24:
207-211 (2001)
ENVIRONMENTAL ENRICHMENT
BEHAVIOURAL NEUROSCIENCE
EE concerns how the brain is affected by the stimulation of its information processing
provided by its surroundings
Donald Hebb – 1947 The Organization of Behavior (1949)
Mark Rosenzweig - 1960’s Enriched and Impoverished Environments: Effects on Brain
and Behavior (1987)
’enrichment’ items are changed regularly to measure effects on neuronal plasticity (hippocampus – learning
& memory; amygdala – emotional reactivity)
Enrichment-induced changes in the behaviour of knockout mice
Tsien, Huerta and Tonegawa. The essential role of hippocampal CA1 NMDA
receptor-dependent synaptic plasticity in spatial memory (Cell, 1996, 87:132738)
Rampon, Tang, Goodhouse, Shimizu, Kyin and Tsien. Enrichment induces
structural changes and recovery from nonspatial memory deficits in CA1
NMDAR1-knockout mice (Nature Neurosci, 2000, 3:238-44)
ENVIRONMENTAL ENRICHMENT
LABORATORY ANIMAL SCIENCE / ANIMAL WELFARE
Any modification of a captive animal‘s environment by providing physical or social
stimuli
Any modification in the environment of the captive animals that seeks to
enhance its physical and psychological well-being by providing stimuli meeting
the animals’ species-specific needs
Standardized set-up of items validated for having a lasting positive effect on
parameters related to laboratory animal welfare
ENVIRONMENTAL ENRICHMENT
ANIMAL NEEDS –
Physiological needs:
eating
drinking
sleeping
Behavioural needs:
social behaviour, exploration, foraging, grooming, digging, nest building,
seeking shelter – essential innate behaviours
It is generally agreed that environmental enrichment is beneficial for the well-being
of laboratory animals and that it should be applied whenever appropriate or
practical
ENVIRONMENTAL ENRICHMENT
Goals of Environmental Enrichment (Baumans 2005):
• Improving the quality of the captive environment so that the animal has a greater
choice of activity and some control over its social and spatial environment;
• Increasing behavioral diversity;
• Reducing the frequency of abnormal behavior;
• Increasing positive utilization of the environment;
• Increasing the animal’s ability to cope with challenges
Bringing crucial features of the environment into the laboratory so that natural
behaviours may be expressed and reinforced (Blanchard and Blanchard, 2003)
ENVIRONMENTAL ENRICHMENT
Types of environmental enrichment (Baumans 2005):
1. Social enrichment
Social Contact and Non-Contact Enrichment
2. Physical Enrichment
Complexity – appropriate structuring of the cage (areas for feeding, resting, excretion)
shelters, nest boxes, nesting material, tubes, platforms, etc.
Sensory enrichment – visual, auditory, olfactory stimuli
Nutritional enrichment – scattering food in the bedding
ENVIRONMENTAL ENRICHMENT
ENVIRONMENTAL ENRICHMENT
ENVIRONMENTAL ENRICHMENT
Evaluation of Enrichment:
Species
Strain
Age
Sex
Type and Material of Enrichment
B6 and D2 male mice:
Gr – group-housed
Ind – single-housed
Voikar et al. 2005
B6 female mice:
GN – group-housed with nesting material
GNN – group-housed without nesting material
SN – single-housed with nesting material
SNN – single-housed without nesting material
Voikar et al. 2011
C57BL/6NCrl
DBA/2NCrl
FINAL COMMENTS:
We should be aiming to provide mice with the appropriate amount of space
containing the appropriate diversity of environment that takes into account their
species-specific characteristics and needs.
i.e. Quantity of space and Quality of space
(C.M. Sherwin 2002 Comfortable Quarters for Mice in Research Institutions)
http://www.animalethics.org.au/__data/assets/pdf_fi
le/0004/249898/draft-guidelines-housing-mice.pdf
(accessed on 2011-01-04)
Refers to 459 original publications
European Cooperation in the field of
Scientificand Technical Research (COST) is an
intergovernmental framework for European Cooperation in the field of Scientific and Technical
Research, allowing the co-ordination of
nationally funded research on a European level.
COST Actions cover basic and pre-competitive
research as well as activities of public utility.
Established in 1971, COST has developed into
one of the largest frameworks for research cooperation.
COST networks are called Actions. In December
2003, COST launched a new Action - B24. The
main objective of the Action B24 is to increase
knowledge necessary for both ethically
sustainable and scientifically valid use of
laboratory animals in research.
Baumans V, Augustsson H, Perretta G. 2011.
Animal Needs and Environmental Refinement.
In The COST Manual of Laboratory Animal Care
and Use: Refinement, Reduction, and Research,
(ed. B Howard, T Nevalainen, G Perretta), pp.
75-100. CRC Press.