Imaging rodent behaviors

Transcription

1 Imaging rodent behaviors Anni-Maija Linden, PhD Institute of Biomedicine, Pharmacology Analysis of rodent behaviors? Other manipulation 2 1

2 Analysis of rodent behaviors Brains, other tissues, blood samples for analysis? 3 Detecting and imaging of rodent behaviors Operational devices Video recordings Automatic image analysis Infra-red beam breaks Conversion of raw data (digitized video, beam breaks ) to behavioral parameters Interpretation Piezoelectric-sensors Transponder based methods Observed behaviors 2

3 Testing different behaviors Basic behaviors Locomotor activity Motor functions, motor coordination Sensory functions Nociception, pain For analysis of in vivo effects of new chemical compounds, investigational drugs Adverse effects, toxicity To test expected beneficial effects requires spesific disease models For analysis of new transgenic mouse/rat strain Behavioral phenotyping 5 Testing behavioral responses Drug-induced behaviors to model components of diseases Morphine-induced place preference Addiction Amphetamine-induced hyperlocomotion Psychosis, schizophrenia Disrubtion of memory To test cognition enhancers Genetic models of diseases (transgenic animals) Mice overexpressing risk genes Alzheimer s disease Knockout mice lacking risk genes Schizophrenia, anxiety, depression Other methods to mimic diseases to be studied Experimentally-induced ischemia in brain Stroke models Deletion of brain regions/neuronal population s e.g. Parkinson s dis. Nerve injuries Neuropathic pain models 6 3

4 Behavioral animal models Modeling human disorders, used in research of, Psychiatric diseases Neurological diseases (stroke, neurodegenerative diseases) Other diseases affecting behavior (e.g. muscle strenght, weakness motor performance) Used to study mechanisms Used to test novel experimental drugs or other treatment options Usually one test can model only one component of the disorder, not the whole human disease Only models! 7 Validity of animal models Face validity comparable to symptoms How well the model mimics the symptoms? Construct validity similar mechanisms Theoretical rationale linking the cause of the human disease to an animal model. Requires that the etiology of the disease is known. Predictive validity clinically effective drugs effective Does this requirement prevent finding of drugs with novel targets and novel mechanisms of actions? 8 4

5 Preclinical rodent models of human disorders Requirements (Crawley JN, 2000, What s wrong with my mouse?) Replicates at least one symptom of the human disease Responds to treatments that are effective in the human disease Is unaffected by treatments that are ineffective in the human disease Conceptual analogy to the etiology of the human disease desirable Conceptual analogy to multiple components of the human disease desirable Endophenotypes Separate components, specific measurable behaviors or other physiological responses Examples of rodent models for human psychiatric/neurological diseases Unconditioned stress/fear (EPM,L/D-test) anxiety disorders Conditioned fear (conditioned frear freezing) anxiety disorders Prepulse inhibition (amount of startle) psychosis, schizophrenia Learned helplessness depression Cognitive tests (long term memory, working memory) neurodegenerative disorders, schizophrenia Conditioned place preference (reward) addiction Self-administration of addictive drugs - addiction 10 5

6 Analysis of motor coordination Basal motor coordination Motor learning during training Effects of pharmacological treatments (ataxia, sedation) Rotarod protocols How long the animal stays on the rod Rotarod Rotating rod Accelerating Constant speed Pretrained/no training Horizontal beams Beams Ø 0.8 cm and 1.2 cm round beams of 1.2 and 0.8 cm in diameter elevated approx. 50 cm latency to traverse the beam, number of falls, number of slips/errors, lenght of beam before 1st fall 11 Example: Effects of GABAergic drugs on rotarod performance Single dose of muscimol, testing at different time points, speed 15 rpm Latency to fall (s) TASK-1-KO Flurazepam Rotarod +/+ ** *** sal mg/kg Latency to fall (s) TASK-3-KO Flurazepam Rotarod +/+ -/- -/- sal mg/kg Cumulative doses of benzodiazepine (flurazepam) every 30 min, testing 15 min after each injection, speed 5-30 rpm A Latency to fall (s) C Latency to fall (s) E Latency to fall (s) Males 0.75 mg/kg 0 sal mg/kg 60 *** 0 *** sal mg/kg *** 0 sal Time points (min) B Females 0.75 mg/kg c57 thy 0 sal D * 1.0 mg/kg 0 *** *** *** *** sal F mg/kg 0 sal Time points (min) *** 12 6

7 Locomotor activity Locomotor activity in home-cage Exploration in novel environment Habituation Circadian rhythm Rearing events (standing up) Thigmotaxis (staying close to walls) Drug-induced changes (sedation, hyperstimulation) Muscimoleffect in GABA-A alpha6 overexpressing transgenic mice (Chandra et al. 2009). Novelty-induced hyperactivity of AMPA receptor GluA1-KOs. (Procaccini, Aitta-aho et al 2011). Imaging locomotor activity Detection based on infra-red beam frames (e.g. Med Associates) Animal location is tracked using I/R transmitters and receives (beam arrays), software calculates coordinates at each time points and few behavioral paramaters Horizontal activity (moving around) Vertical activity (standing up), Fine movements Zone analysis, time intervals Limitations: - not for long-term measurements (beddings, food, water bottles disturb detection) - analyze only robust behaviors Computer Software 7

8 Imaging locomotor activity Detection based on infra-red beam frames Modifications: - For analysis of anxiety (fear of brightly lid areas, preference for dark areas) - Dark compartment from material which do not disturb infra-red beam detection Infra-red beam arrays Imaging locomotor activity Video tracking systems (e.g. Ethovision system, Noldus) Digitized video of behaving animal (on-line or recorded and stored) Image analysis software detects the object (animal) from the background Data: A track of the detected object in the arena Data analysis: zone analysis, time intervals, various parameters Advanced versions: social contacts of two animals in the same arena Limitations: - the animal must be detectable (black on white ok, but white on white not) - analyze only rather robust behaviors reliably (not standing up, circling, stereotypy) vehicle 1.8 g/kg EtOH 8

9 Imaging locomotor activity Video tracking systems (e.g. Ethovision XT, Noldus) Activity detection, freezing Body position, detection of nose, tail Social interactions, at least some parameters (not as detailed as observation) atch?v=dq9ylop3pci ch?v=rmkwrbo0sea Imaging locomotor activity Voluntary running in home cage Circadian rhythms Small running wheels in home cages, wheels have wireless transponders, computeraided detection and collection of running data for days or weeks Home-cage detection of behaviors using systems with transponders under the skin Individual behaviors of mice living in social groups Microchip under the skin of each mouse 9

10 Morris water-maze spatial long-term memory test The animal needs to learn where the hidden platform is located Confounding factors: stress, defects in swimming performance, sensory functions, risk of hypothermia in mice Modifications: Different type of reversal learning tests Ethovision video camera Ethovision-detected tracks of the mouse swimming to the hidden platform. Water-maze A track to the hidden platform on the 1st training day. After 2-day training. Probe trial: A track when the platform was removed after 4-day training. 19 Working memory T-maze spontaneous alternation The mouse needs to remember which arm it visited previously Locomotor activity affects the results No need for food-restriction Start arm T-maze Remotely controlled doors to choice arms TASK-3 KO mice show reduced spontaneous alternation (Linden et al JPET) Chance level being 50% 20 10

11 Touchscreen based methods for cognitive functions Learning different rules, flexibility to adapt new rules, reversal learning Correct choice (touching the image) is rewarded Learning and memory Working memory Executive functions From Horner et al 2013, Nature Protocols 8 From Mar et al 2013, Nature Protocols 8 Anxiety models: tests of unconditioned stress Elevated plus-maze 5 min test Closed arm Track Zones Open arm Light-dark choice 5 min test Effect of gaboxadol (3 mg/kg, i.p.), Saarelainen et al. 2008, J Neurochem 22 11

12 Tests of emotionality: helplessness Based on the assumption that animals will eventually stop trying to escape when an aversive, inescapable stimulus is continuously presented Forced swim test (Porsolt) Rats, 1st day training, 2nd day analysis of behavior Mice, one day test (6 min exposure, analysis of movements last 4 min) Antidepressants decrease immobility Tail suspension test only for mice (rats too heavy) 6 min exposure, analysis of movements, last 4 min Antidepressants decrease immobility Automated analysis (TST) or manually from video recordings. 23 Forced swim test (FST) and Tail-suspension test (TST) 25 C water Total 6 min (2 min + 4 min analysis) From the experiments of Teemu Aitta-aho and Chiara Procaccini 24 12

13 Acute escitalopram (5 mg/kg) reduces immobility in wildtype mice in the tail suspension test (TST) Mean time of immobility ( s ) GluR-A+/+ Sal ** ** ** GluR-A+/+ Esc GluR-A-/- Sal GluR-A-/- Esc Procaccini et al Models: Model of fear memory: Fear conditioning Anxiety disorders, PTSD Cognition: fear learning, extinction Anxiolytic drugs can be tested Conditioning phase: the animal is trained to pair a tone (sound) with a mild foot-shock ( ma, duration 1-2 s) Fear conditioning unit, infra-red video recordings (Med Associates) Motion track to analyze freezing Sound Fear experssion 24 h later: the amount of freezing is analyzed in the same context after the tone (cue) in a different context Elli Leppä 13

14 Testing sensory-motor integration Acoustic startle Startle intensity db sound TASK-1KOs Speaker Mouse holder Piezoelectric sensor to detect movements/ startle Linden et al JPET 27 Testing sensory filtering: Prepulse inhibition (PPI) Gating/filtering of sensory information Defective in many psychiatric disorders such as schizophrenia Speaker Mouse holder Piezoelectric sensor to detect startle Drugs reducing PPI may model some aspects of schizophrenia Dunnett SB et al, 2003 Mouse Behavioral Phenotyping (Crawley JN, ed) SFN course 28 14

15 Conditioned place preference Conditioning: The animal is trained to associate a floor material with a drug (e.g. morphine) or vehicle administration. Testing: The time spent on two different materials is measured during a 15-min test using Ethovison detection. Mor? Sal? Morphine-induced place preference in wildtype mice. 50% Teemu Aitta-aho Researcher is observing and rating behaviors manually Observational analysis Rogers et al 1997 Fast screening of robust behaviors Shirpa screening protocol Primary phenotypic characterization of transgenic mice Detects clear abnormalities or defects Viewing jar -body position, transfer arousal, repiratory rate Open field -locomotor activity, gait, touch escape 15

16 Observational analysis Researcher is observing and rating behaviors manually because automatic detection not available or not applicable Social behaviors Difficult to automate, although automatic analysis systems exist Stereotypic behaviors (drug-induced, transgenic animals) Circling, repetitive jumping, repetitive head movements Withdrawal symptoms (benzodiazepine, morphine, alcohol) Jumping, digging, stretching, wet-dog like shakes, forepaw tremor, paw treading, head twitching, writhing, circling behavior, and pelvic elevation Additional reading material Review articles Dennis C, 2005, Nature 438, : All in the mind of the mouse. Takao K, et al., 2007, Neuroscience Research 58, : Impact of brainbehavior phenotyping of genetically-engineered mice on research of neuropsychiatric disorders. Horner AE, et al., 2013, Nature Protocols 8, : The touchscreen operant platform for testing learning and memory in rats and mice. Books Jacqueline N. Crawley, 2007 (Wiley), What s Wrong With my Mouse: Behavioral Phenotyping of Transgenic and Knokcout mice