How to measure rodent behavior and perform a neurological ... · • Stretch-attend postures • EZM has same exploration conflicts minus the central square . EPM is most widely used
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How to measure rodent behavior and perform a neurological screen. Fiona Harrison, PhD Department of Medicine Vanderbilt University Medical Center [email protected]An Organ Systems Approach to Experimental Targeting of the Metabolic Syndrome
• Mouse-centric lecture • Apparatus for mice are typically the
same as for rats but scaled down • However, mice are not just little rats.
Behavioral profiles differ
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Behavior isn’t like biochemistry where you follow a recipe. I would always recommend getting a behaviorist into your group, or at least to educate the group and get the tests set up and validated. The focus of behavior used to be in rats but with genetic manipulations being available in mice the switch was made, protocols and equipment were re-worked and now it would be too expensive to switch back. You will see this later in the 2 behavior cores we have the mouse space is much bigger than the rat space. Should have at least one task that is the same in both workshops.
Why study behavior?
• Psychology - the basics of learning and memory. Fundamental rules apply to animals and humans
• Genetic manipulations - to assess phenotypes caused by genetic manipulations
• Experimental manipulations - to assess phenotypic changes caused by pharmacological, dietary or environmental manipulations
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Brain areas and connections that govern each behavior. But behavior isn’t just complex learning, as we already discussed it is how they interact, how they respond to handling, whether they can walk in a straight line etc.
Colony Health
• Good practice • Eliminate artefactual effects in your
experiments (blindness, deafness, anosmic) • e.g. Irwin screen or SHIRPA • Appearance (grooming, fighting, coloration),
weight, temperature, posture and gait, activity, neurological reflexes
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Again - as we discussed earlier you can only get good data from healthy animals (unless your manipulation is what is making them sick in which case you need to know that your controls are healthy).Anosmia is lack of smell Handouts in the workshop Notice if your mouse has cataracts, or has been fighting
Feeding behaviors
• Feeding = Survival • Specialized circuits and mechanisms exist for
hunger, thirst and satiation and all associated behaviors (e.g. activity and foraging) Insulin and glucose - gastrointestinal feedback - hindbrain - hypothalamus
• Many injected peptides can influence feeding
Liao et al. 2012 Nature Medicine
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Most important for most metabolic type studies are not the behavior that a behaviorist might be thinking about, but are the things that mice do during the normal course of their day Probably mechanisms are covered in other lectures so just mention some basics here by way of introduction BDNF mutant
Feeding behaviors • 24 hour consumption of standard
chow and water (weight and volume) - Easy but not sensitive - Spillage, meal size, meal frequency, diurnal rhythm
• Restricted feeding (4 hours per day) - Easier to detect effects of short acting drugs
• Short access to highly palatable foods
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Some types of food are more crumbly than others, palatability Mice are pretty quick to stabilise their eating habits to this restricted time period. But dietary restriction can upregulate a whole number of other systems Remember to measure body weight too Mice love sucrose, sweetened condensed milk, peanut butter, cereals, traet pellets (including bacon) . Pellets dont crumble as much - but you can lose them in the bedding. Mouse and rat sized.
Feeding behaviors
• Continuous monitoring can be assessed in automated chambers
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Promethion from Sable Systems Ltd
Control diets Purina Standard lab chow
Research Diets Inc. Basic control diet
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So if you are changing diets for a particular supplement or deprivation, you will need to switch to the appropriate control diet. E.g. The Research Diets formula has double the amount of vitamin E so you have increased antioxidant potential. Also remember it can be a bit of a zero sum game – so if you increase the percentage of fat, you will also be decreasing something else (carbs/protein etc.). You need to maintain the nutrition per amount of calories, and not always per weight of diet because rodents are much better at consuming a calorie amount rather than a weight of food.
Feeding behaviors
Feeding behaviors/
Energy intake
BRAIN
Digestive tract - Stomach; intestines; pancreas
(ghrelin; GLP-1, PYY, CCK;
insulin)
Adipose tissue (leptin,
adinopectin)
Blood glucose
Hedonic control Psychosocial factors Environment
Homeostatic feedback Hunger/satiety
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Presentation Notes
Feeding is controlled by hormones, other substrates (glucose, lipids, amino acids), physiological (stomach distention), and psychological and social components (emotions, palatability). Remember to consider all of these. While we talk about other behavioral testing methods, mostly related to learning and memory, anxiety and other emotional function, the same concepts can affect your testing. Most of your experiments will be physiological tests and involve the light blue coloured circles but it is important to consider the Brain and psychological factors too because they all feed into each other
Feeding behaviors
• Novel taste neophobia • Social transmission of food preference
> Preference for novel food
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Because rats and mice cant vomit they won’t eat a lot of a food the first time the encounter it. They will if they have smelt it on another rat though. Remember this when introducing a new diet to them.
Locomotor activity
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Now a switch to measuring some basic behaviors - Mice want to explore a novel environment. Locomotor activity. 30 cm x 30 cm. You will be testing with these in the workshop later.
Locomotor activity
• Automated measurement by beam breaks
• Computer scored from top-view video • Hand-scored by square crossings
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You don’t even need specialized equipment, you just need a box and a video camera - don’t score this live, it is always better to video because then you can double check and have different people score it. The size and shape of the arena are important.
Locomotor activity
• Habituation - reduced exploration with repeated exposure
Within-trial and between-trials • Activity response to drug e.g. scopolamine
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Scopolamine is a muscarinic antagonist but affects cholinergic and also dopaminergic system. We will be using this in the lab later. Habituation over many weeks and months and can be used as a memory task for that reason
Anxiety
Open field
Light-dark box
• Using locomotor activity chambers
• Preferential exploration of dark, enclosed, wall/corner areas
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You can also count fecal boli and urination as a measure of anxiety So if you put a food reward in the middle of a big arena and see how much gets eaten you may be detecting anxiety rather than food preference.
• Elevated Plus/Zero Maze (EPM/EZM) • Time spent in Open versus closed arms
Should show preference for closed (dark)arms • Entries into each arm • Distance traveled • Stretch-attend postures • EZM has same exploration conflicts minus the central
square
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EPM is most widely used and so we will do that later because that is the vast majority of data you will come across. Zero maze is preferable because there is no dead space in the center.
In the EPM mice can choose between exploration in open and closed areas
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You see that he is happy to run in the closed area but more cautious about entering the light. This would change if the behavioral inhibition was altered. Note the STRETCH-ATTEND posture. They will usually spend between 70 and 90 % of their time in the closed arms.
Depressive-like behaviors • Porsolt or forced-swim - “Behavioral despair” indicated by time spent swimming - Be aware: Mice float, but rats sink
• Tail suspension test - “Behavioral despair” indicated by time spent immobile
• Behaviors improved by anxiolytic drugs
Abnormal behavior in db/db mouse
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Why are these things even relevant to your testing? Because they are!! Note weight, insulin, glucose and food and water intake. 5 weeks is juvenile, 10 weeks is pretty much adult. GO through the physiological differences in the table What else might you want to measure
Abnormal behavior in db/db mouse
a) Elevated Plus Maze
b) Y-maze
c) Porsolt forced swim
c)
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Choose the right age to test your mice Also deficits in Morris water maze Go through what each task is for Beware of confounding factors – ie if the mice are too fat their legs wont be long enough to swim properly and they will float more
Motor skills • Rotorod • Motor ability and also motor/procedural
learning across trials (cerebellum)
This image cannot currently be displayed.
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You will do a lot of these tests this afternoon so I won’t spend too much time now. These tasks are interesting so that you know about the neuromuscular abilities of the animals but they are also necessary as controls to make sure that your animals can be tested on other apparatus (no movement could be extreme anxiety or could be because they are paralysed).
• Gait Measured by specialized equipment or with (non-toxic) ink and paper
• Grip strength Measured by specialized equipment or by a rating scale
Hill et al. 2004 Nutritional Neuroscience
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These will be demonstrated in the mouse lab. Easy way and expensive way to do each.
Motor skills • Inverted screen
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Quatitative and qualitative - watch if they are moving around or just hanging on. Move to end of the video to demonstrate this
Motor skills • Horizontal beam • Wire hang
Score according to performance - grip, posture correction
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Scoring is based on performance, from FALL to maintains stability or escapes. Also latency measures. Experimenter must place appropriately Fatness of mouse will affect whether they can do this
Learning and memory
• There are a lot of different tasks • There are a lot of versions of each tasks • There are a lot of bad behavioral
experiments in the literature • Explicit/declarative v Implicit/procedural
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In case anyone is interested in doing these I will go over some of the more common tasks. You have to read the methods as well as the results and think about what they have and haven’t reported (ie control tasks for motor ability, vision etc)
Y-maze - alternation
Working memory Mice prefer novelty
To explore a new area, you must remember where you have already been
A
B
C
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Animal working memory is for 1 trial, human is 7+-2 info bits Different types of equipment, eg open arms, dark versus see through Number of Entries and Alternations in 5 min. trial Alternation = Entering 3 different arms within 3 consecutive arm choices�i.e. CBA but not ABA Discuss alternate short-term memory version
T-maze • Hippocampal-dependent, spatial learning • Rule: Non-match to sample • Forced-choice trial followed by rewarded free-choice trial • Advantage inclusion of time delays
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Food deprivation versus sucrose. Mice can learn these sorts of tasks quite quickly but it is labour intensive. But you can impose delays, and test repeatedly, eg different doses of drug
Morris water maze • First establish that your animal is physically
capable of performing the task Rats are naturally better swimmers than mice
• Visible/Cued platform Above surface Flag/marker
• Then Hidden Platform Use extra-maze cues to locate the platform
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Probably the most common spatial learning task
Data interpretation A B C
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Remember to consider other behaviors such as thigmotaxis and vision and physical ability when you choose and run this task/ 1 is clearly the best but 2 also knows what he is doing. How you report the data matters. So get as much information as possible when you are testing and look at it in everyway possible because you might be missing something subtle. Automation is great but it is no substitute for human observation. Whatever the behavior is, don’t accept the authors word for it, or assume that the interpretation is the normal one – that is why it is good to run your own data, and to keep videos a lot of the time.
•Water maze is more stressful to mice - elicits greater levels of serum corticosterone
•High corticosterone correlates with poorer performance in MWM but not BM
Harrison et al. (2009) Behavioural Brain Research 198, 247-251
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The reason I show this is to demonstrate a very clear effect of task demands and anxiety and hormonal response. These measures were taken 30 minutes post-test so think about leaving your animals in the test room for a while before you start work with them after moving them across campus Inverted U – cortisol/corticosterone improves performance up to a point at which it is detrimental
Fear conditioning • Learn and remember an association between an
aversive experience and an environmental cue • Memory of shock inferred from level of freezing on
subsequent trials when exposed to the same arena
Fear Conditioning
Test conditioned stimulus
Test context
8 weeks treatment with leptin improved performance in APP/PSEN1 mice Greco et al. (2010) J Alz. Dis.
24 hours
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Rat work shop. Test that make use of the rodents excellent memory for fear. This is a good demonstration that if you scare a mouse one day, it will remember and be scared of you next time it sees you. Other versions involve step down latency
Fear conditioning
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Freezing response to testing context and to the cue (auditory tone) that happened yesterday – a really obvious response, so don’t scare your mouse and expect it to be happy with you and relaxed the next day. Pre handling Don’t do an aversive test (blood draw) and then do a measurement the next day in the same room if you need them to be relaxed.
Now we are going to talk about some interventions that affect behavior. Activity is preparing for food.
Pharmacological interventions
• Must be able to cross blood brain barrier • Routes of administration
subcutaneous (s.c.) and intraperitoneal (i.p.) most common
• Dose required • Therapeutic response window • Side effects
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Only PSYCHOACTIVE if they cross the BBB. The BBB should be very tight junctions between cells but is impaired in diabetes. Other routes are tail vein and gavage, retro-orbital not a good option. This will be discussed in a separate pharm lecture.
Pharmacological interventions
• Leptin • Ghrelin • Neuropeptide Y
• Effects on activity • Effects on consumption
Other considerations
• Noise • Smells • Experimenter • Room size • Room cleanliness • Single housing • Time of day • Time of year
“Hey Ted, I would rather work with Tiffany”
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There are a lot of things that you wont be aware of but your mouse is. So cleaning equipment, and ways to transport mice etc should be a specific part of your protocol. EG Never have rats and mice in the same rooms.
Labs I & II - Aims
• Gain confidence with handling of animals and restraint techniques
• Learn injection techniques • See examples of behavioral tests • See responses to pharmacological
treatments
Lab I: Working with mice
• Location: Murine neurobehavioral lab • 1:30-4:30 pm • All groups: Mouse handling • Group 1: Neuromuscular testing • Group 2: Elevated plus maze • Group 3: Locomotor activity • If time allows - a tour of the rest of the behavior
facility
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Ale, Elizabeth and Marquicia Give out worksheets and tell them to bring them, same as in handbook but these will get dirty
Lab II: Working with rats
• Location: Murine neurobehavioral lab • 1:30-5 pm • Analisa Thompson-Gray & team • Rat handling & injections (s.c., i.p.) • Irwin screen with oxotremorin • Rat rotorod
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Oxotremorine is a cholinomimetic that acts as a non-selective muscarinic acetylcholine receptor agonist Oxotremorine produces ataxia, tremor and spasticity, similar to those symptoms seen in Parkinsonism, and has thus become a research tool in experimental studies aimed at determining more effective anti-Parkinsonian drugs We will use demonstration rats at the highest dose; you need to understand a sick animal to understand a normal animal. Rats will be euthanized at the end of the session. You will have lower dose animals.
Recommended reading
• Crawley JN (2000) What's wrong with my mouse? :
behavioral phenotyping of transgenic and knockout mice, Wiley-Liss, New York.
