What is normal animal behavior? Rat & · PDF fileWhat is normal animal behavior? Mouse & Rat Fiona Harrison, PhD Department of Medicine Vanderbilt University Medical Center . [email protected]
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What is normal animal behavior? Mouse & Rat Fiona Harrison, PhD Department of Medicine Vanderbilt University Medical Center [email protected]An Organ Systems Approach to Experimental Targeting of the Metabolic Syndrome
An Organ Systems Approach to Experimental Targeting of the Metabolic Syndrome
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This lecture should give you an idea of what to expect when working with live rodents, and by knowing a little more about them you will be able to get better data. The focus will mostly be general, and behavioral, because you will be learning a lot more of the physiology in the other lectures. Hopefully even those of you who have some experience working with rats or mice will learn something new that you haven’t considered before. A lot of what I go through today will be picked up in the two workshops this afternoon and tomorrow.
• Mouse (Mus musculus) • Rat (Rattus norvegicus) • For each species there are a number of different
strains with particular characteristics that may be more or less suitable for different types of research
C57Bl/6J 129/SVJ Sprague Dawley Lister Hooded
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rats of the genus Rattus and mice of the genus Mus bred for use in research are not currently considered USDA covered (or regulated) animals. Those not purpose-bred for research and all other genera of mice and rats may be considered USDA covered animals. They are still monitored by IACUC and AALAC so you do need proper housing conditions and approved protocols Note that C57s good for behavior, 129s much more docile, Hoodies also good for cognition. We will talk more about differences among the strains throughout the talk. Usually the mouse workshop has these two strains but Jax didn’t have the 129s so we have a similar strain from Charles River CD-1 which I haven’t worked with before but I believe to be similarly docile .. Be aware of differences in nomenclature and strain type between breeders (e.g. a Charles River 129 isn’t a white mouse)
Lab mice - brief history
• 1664 England: Robert Hooke used mice to study the effects of changing air pressure
• Gregor Mendel started his genetic inheritance studies in mice until one of the more senior Bishops requested he change to a less odorous and messy subject
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We have been collecting data with mice for a long time.Although Mendel did then have to switch to peas.
• Current lab strains derived from amateur breeders of “fancy mice”, an early 20th Century fad.
• Miss Abbie Lathrop of Granby, MA bred together the black-coated offspring from her female #57 and these became the widely-used C57Bl/6J line
• She also noticed some mice were more prone to tumors - early cancer research
• These groups and their mice led to the founding of Jackson Labs in 1929 under C.C. Little
• “In bred strains” are from 20+ consecutive brother X sister matings.
• Notation and records are important: A C57Bl/6J mouse direct from Jax labs ≠a C57Bl/6 mouse maintained in a university colony for several years/generations due to genetic drift
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Jax labs is a not for profit, research center as well as a supplier. There are slight differences among the strains so don’t switch suppliers half way through an experiment (even if you are offered discounts for example). Jax goes back to frozen embryos every 5 generations in order to avoid genetic drift.
Mice as experimental subjects
• Easy and (relatively) cheap to maintain • They eat a lot for their size, but their specific nutrition
requirements are inexpensive and simple. • Rodents eat cereals, grass, seeds, roots and stems of
various plants, insects, (cardboard and electrical wiring ..) • Rats also eat small mammals and birds
Remember that their teeth grow constantly so gnawing keeps them down. Genetic (heritable) trait for over growth but you can cut them with scissors. Wild rats kill more mice than lab rats but the two strains should always be kept totally separate.
Mice as experimental subjects
• Easy to breed – year-round breeding – large litters (6-12 pups) – tolerate in-breeding (genetic homogeneity) – complete mouse genome is available (2002) - genes
conserved >99% between mice and humans – easy to manipulate genetically
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Litter size will change with age, the first litter is often killed and eaten. Maternal abilities vary with strain but mice will accept foster pups. The complete genome has now been sequenced and there are thousands of transgenic and knockout animals available. In 2004 the count was >5000 knockout varieties but now it is much greater. There are different options including “humanized” proteins can be knocked in instead. Knockouts aren’t always the best model for disease because most diseases don’t involve a total loss of function. Conditional knockouts are a bit more flexible.
Strain-specific problems
• C3H - retinal degeneration gene (early blindness) • 129 & C57 - early hearing loss • DBA/2J - audiogenic seizures and early hearing loss • Differing levels of aggression, anxiety, maternal
behaviors etc It is important to be aware of strain-specific characteristics when choosing strains for experiments
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It is important to be aware of differences among the strains, when choosing a background to cross your mouse with for behavior and for physiological features (glucose). Also important if you are trying to interpret literature, why did the same manipulation lead to the opposite effect in two studies.
Additional strain information • Mice
http://www.jax.org/phenome – Learn the baseline for your strain – Strain comparisons; data for grants, project planning – Detailed protocols including diagrams not available in literature
e.g. C57Bl6J is active, good at learning but has higher blood glucose than a lot of strains, response to dietary manipulations Mention Webinars for the Jax phenome project which has a huge wealth of information
For example here is blood glucose levels in different in-bred strains and their response to high fat diet. C57s naturally have high blood glucose and their response to the high fat diet is different to other strains.
Gender
Distance Fur
Male Female
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One of the more basic but very important things you need to be able to do is sex the mice. First you can all hopefully tell me which is the male. These mice are nice and big, around 6 months which makes it easier to tell. It is also possible to tell by size if the mice are the same age, and to my eye, males start to look a bit older a bit faster, so the females remain sleeker for longer. Look for the main features, Penis and testes in the male, the absence of these, but presence of nipples in the female. But in young mice it is actually quite tricky so the cheaters way is to look for the ano-genital distance, and the presence of fur along the line between them. Note the scruff hold, we will teach this in the workshop later for anyone who is not familiar
X
Typical behaviors Home-cage behavior
• Active and responsive to experimenter intervention
You also need to be able to recognise off the bat if all of your mice are healthy. Sick mice will confound your experiments, particularly behavior. We will discuss a number of different ways to assess your mice (Irwin screen) but you don’t perform this every time on every mouse so you need to be able to recognise signs and symptoms. Abnormal activity can include twitching, seizures, circling, jumping
Typical behaviors Home-cage
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He did not respond when I poked him Explain that he is actually not sick (no physical signs of fighting or weight loss) but he is extremely unhappy due to bullying by the SLOS mouse. This is a Wild-type mouse. When we have handled him he is very jumpy. Note the nesting material in the far corner (darkest) of the cage. His ears are back too.
Coding of facial expressions of pain in the laboratory mouse
Langford et al. Nature Methods (2010)
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Mouse Grimace scale (MGS) this was a great paper that Owen found and the differences are fine, but you can notice them, and you will hopefully be able to just tell when something is a little bit wrong
when planning your experiments you will also want to take age into account. 3-6 months will give you a good homogenous group. The older they get, the more heterogenous the data
Typical behaviors Social behaviors
• Mice are not small rats! This is especially noticeable in social behavior
• Mice have signaling postures to communicate intent, anxiety, dominance, excitement
• These can be quantified according to frequency, number/type & duration
Grant and Mackintosh (1973) Behaviour
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In the wild, rats and mice hold group territories. Mice typically have one male and one or two females and the pups leave and form their own social groups as adults. Rats have more adults in a group in the burrow. In each case a dominant male protects the group against intruders. In cages the animals cannot leave to set up their new territory so we see fighting. This is important not because you are going to become experts in social behaviors but because changes to the cage environment will affect your mice
• Social elements - visible when multiple animals in cage
- Social attention: detection of & orienting toward other mice - Social investigation: olfactory, tactile, visual exchanges - Sexual activity • Response spectrum
Attack -- Ambivalence/Maintain distance -- Escape Or defensive postures if escape is blocked
Social behaviors
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(e.g. mouse can’t be dominant/submissive if it’s the only mouse there)
• If escape is not possible - Submission Roll on back hiding rump and back which is commonly attacked/ provokes aggression
• Rat response can be used to predict hierarchy within the cage
RAT
From: The Laboratory Mouse Ed. Hans Hedrich
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Male and females have different attack patterns, The hierarchy is stable and ordered
• Lacks the same response patterns as rat
• Where escape is not possible mice freeze in “Defensive Upright Posture”. Circling around other mouse keeps rump away from attack
• Mice do not typically have the same clearly-defined hierarchy system as rats. Within cage find “Despot”, “Co-dominant”, and “Subordinate” mice
• Dominance can be assessed using tube test
MOUSE
Fig. 18.2 From: The Laboratory Mouse Ed. Hans Hedrich
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Male and females have different attack patterns, females will attack everywhere whereas males are focused on rump and also penis
Social defeat
Movie provided by Chris Olsen, Winder Lab
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First you see him assume the defensive upright posture, then he tries ‘avoidance’ but gets pushed to the ground
Social dominance
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Mice like to be in small places so retaining possession of the tube is desirable. They arent fighting, they are just exchanging olfactory information
• Aggression is useful in the wild to spread out demand for mates, food, territory
• In a confined setting (lab cage) it can lead to injury • From ≈7 weeks single housing will increase
territorialism and therefore aggression • Strain differences • Aggression is displayed less in females, but is
greater in nursing mice (defense of litter)
Social behaviors
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Why is this important? Even transporting mice to the lab - e.g. by throwing all in the same cage could have a huge effect. Or combining cages.
Aggression in a single-housed mouse
Movie provided by Chris Olsen, Winder Lab
Social housing • Critical considerations for rodent housing
– 2 or more mice per cage
– Try to avoid constantly changing cage-mates
– Environmental enrichment (unless it conflicts with experimental design), running wheels, cardboard tubes, nesting materials
• Green group required 2 attempts to get a good blood sample
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Another example of why this is important - it is not just for the mouses wellbeing. Armario et al. 1996 measured these three increasing in medical students before tests. This example experiment has two groups. We know that 150 is about average for a C57 male BSL level. You can hazard a guess that perhaps the green group was treated with strep but in fact none were treated, the stress of bad handling increased glucose.
Typical behaviors Exploration v. avoidance
• Approach:Avoidance conflict - Exploration of environment for food/mate/potential escape routes - versus - - Open unprotected areas and risk of predator
• Conflict useful in several measures of anxiety: Open field, Light-Dark exploration, Elevated plus/zero maze
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In general we try to harness natural behaviors for our experiments. This is why mice hide in the corner of the room or under your fridge if you have them in your house. This is how you would measure but also something you need to be aware of when testing with them
Small rodents prefer to be in corners rather than open spaces to avoid potential predators
B6/C3 mouse following exploration of locomotor activity chamber
Wild mole trying to escape from a house cat
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Mouse had been in the box for a couple of hours. In both you can see the fecal boli around the edges of the chamber
Small rodents prefer to be in corners rather than open spaces to avoid potential predators
Reduce anxiety while working with the mice by holding them in small, dark boxes rather than large open containers
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For example during a glucose test. Transport the cages covered etc.
Response to handling
• Rats v. mice Rats can become comfortable with handling Mice become less anxious but still show signs of escape
• Biting - Defense or aggression (this will hurt) - To acquire information (taste - you will feel a nibble)
• A secure hold makes the animal feel safer and reduces stress and potential aggression
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Rats make excellent pets, mice not so much. Aggression is usually toward other rodents, defensive is towards you. Often students get bitten the first time they try to handle a mouse because they are nervous, so make jerky movements and scare the mouse. We will go over this in the workshops Rabbits go under your arm pit, guinea pigs hold close to body
Basal parameters - differences Human Rat Mouse
Heart rate 60-80 320-480 580-680
Respiratory rate 12-20 85-110 84-230
Core temperature (C)
37 38-39 37
Adult weight Varies widely 300-650 g 30-50 g
Estrous cycle (days)
~28 4-6 4-5
Sexual maturity 12-15 years ~ 5 weeks ~ 5 weeks
Nipples (pairs) 1 6 5
Nocturnal Personal preference
Yes Yes
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Difference in metabolic rate affects the metabolism of drugs, and so doses and treatment times. All animals can become very fat with age - bear in mind that standard lab chow is actually a high fat diet anyway compared to their wild diets. Pregnant or very old mice can get even bigger than this.
Circadian rhythm
• On a 12:12 Light:Dark cycle mice will be most active in the dark
• Most food and water intake, defecation and urination occurs in dark cycle
• Most ovulation, mating, births occur during dark cycle • Changes in body temperature and metabolism can
affect experiments
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Especially behavior, also glucose experiments should be run at the same time of day Check your housing facility for lighting - try to get your animals away from the noisy fans etc Hormonal variations can affect your experiments
Strain comparison in locomotor activity chambers
data from http://phenome.jax.org
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Here is another example of data form the Jax phenome project. This is 5 to 30 meters in a small box over about 10 or 15 minutes. But mice can actually be much more active than you would think if they are not in a strange testing box environment. Strains highlighted are the ones we will be working with (or a 129 equivalent)
Running wheel: distance traveled per day (km) 12:12 h light:dark cycle
0.4
C57Bl/6J 7.5
Lightfoot et al. J Appl Physiol (June 10, 2010)
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Depend on mice in cage, size and type of wheel, whether they have to share etc Mice on a 24 hour dark cycle will actually run on the wheel for 22-23 hours (messed up diurnal rhythms) They want to run around and we are restricting them in the cage
Sensory systems - Olfaction
• Best developed sense in most rodents • Required for food, territory, predators, social
cues, sexual cues • On meeting, mice orient toward each other Exchange of information - eyes/ears/nose They touch (nose) and sniff head, body and
ano-genital region
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And now for a slight change in pace we are going to think about mouse communication and information collection. Mice can respond to cues that we don’t realize are there (noise, sound, lighting differences). Need all of this information to decide on strategy or response
• Information from urine, breath, saliva, skin secretions, anal and preputial glands
• Urine contains MUPs - major urinary proteins. Especially in males these carry information about dominance etc. and can elicit a range of behaviors
• Useful for mouse-mouse intraspecies communication
• MUPs also present in ‘snake essence’ and cat saliva which act as a natural repellent to mice even if never exposed to the predator
• Exposure to rats or rat smell leads to increases in corticosterone and decreases in cognitive performance
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This is important because it matters if you do your tests taking the mice from a dirty cage (nasty for us but better for them), or if you throw 2 mice in together to transport them, or just put everyone in a clean cage. If you hold a cotton bud dipped in urine of an opposite sex they will investigate, but if it is from a predator they will avoid it like the plague. So don’t test rats and mice in the same room.
Sensory systems: Vision • Least important sense to rodents that are naturally
nocturnal • Vision is dichromatic - see from UV through to green • Generally poor visual acuity
Worse in albinos - retinal degeneration due to light flooding
• A number of common inbred strains are blind from 10 weeks
• Both rats and mice can see large, high contrast cues
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lower vc in the eye because less UV damage Other senses can adapt and take over from vision much more so than in humans. Sight worsens with age. Typical behavioral experiments have shapes like above.
• Ultrasonic signaling in response to cage intruders • Signaling between young pups and mothers • Complex signals
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They are talking to you behind your back - like bird song, noises are part of the social behaviors http://www.lsu.edu/deafness/HearingRange.html , www.ratbehavior.org Very well developed - much more sensitive than ours.
Sensory systems: Touch
• Excellent touch sensation particularly in the vibrissae • Specialized regions of the cortex encode information • Includes feeling pain, and sensitivity to handling
• Overall Size
• Relative size of specific areas
• Surface area of cortex ~2500 cm2 in human ~6 cm2 in rat
Key differences involve size, surface area and size of olfactory bulbs Structures remain the same, connections and basic functions Olfactory bulbs have connections to specific cortical areas for encoding information http://faculty.washington.edu/chudler/facts.html
Differences Human Rat
Cerebral cortex volume (% of whole brain)
77 % 31 %
Brain weight 1300-1400 (g) 2 g
Brain % total body weight 2 % (150 lb)
0.5 % (400 g)
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Brain specializations are what have helped us develop as a species
Similarities • Subdivision of cortex
• Specific structures
• Connections between structures
• Homologous brain regions, neurotransmitter systems, connections and responses to pharmacology offer validity
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Neocortex (because developmentally newest area) or cerebral cortex Specific structures are the same (hippocampus, cerebellum, striatum), the order and type of neurons that dominate in particular areas, the types of neurotransmitters and responses to pharmacology. Different functional areas
Which are the wild-type mice?
Lijam et al. 1997. Cell
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And example of NESTING, HUDDLING and ALLOGROOMING (grooming by a conspecific mouse or another) The knockout mice also tended to win more often at the tube test - failure to recognise social cues
Recommended reading
• Hedrich HJ, Bullock GR (2004) The laboratory mouse, Elsevier Academic Press, Amsterdam ; Boston.
