Assessing mouse behaviors: Modeling pediatric traumatic brain injury Bridgette Semple Ph.D. Postdoctoral Fellow, Noble Laboratory Department of Neurological Surgery, UCSF
Pediatric Traumatic Brain Injury (TBI) TBI is a contributing factor to a 1/3 of all injury-related deaths in the US. Children aged 0-4 years, adolescents aged 15-19 years, and adults >65 years are most likely to sustain a TBI. In children, injury is super-imposed upon ongoing maturation. Children < 4 years show poorest long-term outcomes after TBI. Increased vulnerability of the immature brain Disrupted brain development? Deficits emerge over time
How do you model TBI in mice?
Modeling brain development Semple et al. (2013) Progress in Neurobiology
Human Mouse Developmental milestones Behavioral phenotype 24-28 wk gestation 36-40 wk gestation pnd 2-3 pnd 7-10 Peak in neurogenesis. Predominance of pre-oligodendrocytes. Peak brain growth spurt. Switch to predominance of immature oligodendrocytes. Consolidation of the immune system. Ongoing astrogenesis. 2-3 years pnd 17-21 Plateau of total brain weight. Peak in synaptic density. Peak in myelination rate. Neurotransmitter changes Weaning. Increased activity levels. Increased sociability. 4-11 years pnd 25-35 Fractionation/specialization of prefrontal cortex neural networks (structural maturation). 12-18 years pnd 35-49 Reduced synapse density, reaching a plateau at adult levels. Refinement of cognitive-dependent circuitry. Ongoing myelination resulting in increasing white matter volume. 20 years + pnd 60+ -Adult levels of neurotransmitters. -Adult levels of synaptic density. Ongoing region-specific myelination. Increase in social play. Development of working memory and inhibitory control. Adolescent-type behaviors (sociability, risk-taking, impulsivity). Onset of sexual maturity. Increased cognitive capacities (e.g. working memory). Adult-type behaviors (reduced risk-taking, reduced impulsivity, increased parental tendencies).
Pediatric TBI a mouse model Focal, unilateral controlled cortical impact to the parietal lobe at p21 Behavior of TBI mice compared to sham controls (littermates who receive anesthesia + craniotomy but no impact) Randomized group allocation and blinded assessments
Quantifying clinically-relevant functional outcomes Sensorimotor Cognitive Psychosocial
Open Field Laser beam detection of mouse movements in x, y and z Assessment of activity levels, exploration and locomotion Time in center versus periphery is an indicator of anxiety
TBI mice show hyperactivity
Elevated Plus and Zero Mazes Automated detection of mouse movement Mice normally prefer the closed, darkened arms compared to the open arms Quantify time in open versus closed arms as a measure of anxiety
Reduced anxiety at adolescence after ptbi Pullela et al (2006) Dev Neurosci 28:396-409
Motor function and coordination Locomotion is mediated by many different CNS systems, including the dopaminergic system and the cerebellum, as well as by peripheral abnormalities (e.g. muscle weakness or nerve degeneration). A change in locomotor activity could also result from general ill health of an animal, and may non-specifically alter performance in other behavioral tests. The rotarod is a simple task to measure motor function, particularly in mutant mice suspected of having impaired cerebellar function.
Accelerating Rotarod Gradual acceleration to constant maximal speed Motor function: coordination and balance Latency to fall = measure of maximal performance Repeated measures possible: - motor learning - relative to baseline
Normal motor function after parietal ptbi
Motor learning deficit after frontal ptbi
Detecting a unilateral forelimb deficit Shelton et al., 2008, J Neurosci Methods 168(2):431-442
Learning and memory Many aspects of learning that can be measured in rodents including attention, working memory, memory consolidation and retrieval, and long-term memory retention. Memory tasks may dependent on hippocampal function (e.g. Morris Water Maze spatial) or independent of hippocampal function (e.g. fear-conditioning).
Morris Water Maze Day 1 Day 2 Day 3 Day 4 Day 5 Visible (1) Visible (3) Hidden (5) Hidden (7) Hidden (9) Visible (2) Visible (4) Hidden (6) Hidden (8) Hidden (10) 3 trials per session (am or pm)
Brody et al (2006) Exp Neurol 197: 330-340
Spatial memory deficits after TBI visible hidden
Probe trials reveal memory retention deficits
Radial Arm Water Maze (RAWM) A F B E C D visible hidden hidden hidden open pool Day 1: Spatial Learning 15 trials; alternating visible and hidden platforms for the first 12. Day 2: Spatial Memory 15 trials; all hidden platform. Day 2: Visual and swimming abilities Open pool (no inserts), visible platform
Novel Object Recognition (NOR) Familiarization (2 identical objects) Testing (familiar vs. novel object) time interval
Social behaviors in rodents Survivors of childhood TBI are at increased risk of social dysfunction, with a considerable impact on long-term QoL. Study of social behaviors in rodents is common in psychiatric disorder research (schizophrenia, autism). Several common mouse strains are highly social (C57Bl/6J, FVB/NJ) while others are not (Balb/C). Social recognition in mice is primarily dependent upon olfactory cues.
Partition test Time spent at partition quantified. Resident-Intruder Home cage of test mouse. Interactive and investigative behaviors quantified from video.
Social investigation is reduced by adulthood after ptbi * * Semple et al, (2012) J Neurotrauma
Three-chamber social approach task Stage 1: habituation Stage 2: sociability Stage 3: social novelty
Reduced sociability emerges by adulthood after ptbi * *** * adolescence adulthood Semple et al, (2012) J Neurotrauma
Communication: Ultrasonic Vocalizations USV s elicit social approach and explorative behaviors. Complex underlying genetics; call rates and characteristics differ depending on stimulus, sex, development age, strain Altered USV s reported in mouse models of neurodevelopmental and neuropsychiatric disorders. Hanson and Hurley (2012) PlosOne 7(7): e40782
Male to female call: Isolated pup call: Hammerschmidt K et al. Biol. Lett. 2009;5:589-592 Outcome measures: call rate, average duration, latency to call, max and median frequency, patterns (e.g. frequency of different call types)
Frequency (khz) Adult C57Bl/6 male exposed to female bedding Time (sec)
Communication scent marking Rodents deposit urinary pheromones traces in a context-specific manner to mark territories, attract mates and communicate information about health and dominance status. Reduced scent marking is observed in strains with low sociability. Arakawa et al (2007, 2008); Wohr et al (2011); Roullet et al (2011)
Scent marking is reduced by brain-injured mice sham TBI
Study design, considerations and limitations
Study Design how many mice? Typical mouse behavior studies require 10-15 subjects per experimental group for adequate statistical power. Separate cohorts (tested at different times) may be required to obtain adequate numbers. In this case, each cohort should include mice of each genotype/experimental treatment. Randomization Blinding Control groups
Appropriate Controls Matched for age, gender and background strain. Use of littermate controls is strongly recommended for genetically-altered/mutant strains. Behavioral results are highly sensitive to environmental factors such as home cage conditions and parental care. Ensure matched housing conditions prior to and during testing procedures. Strains can differ dramatically in behavioral phenotype.
Picciotto and Wickman (1998) Physiology Reviews 78(4): 1131-1163. Mouse Phenome Database: http://phenome.jax.org/
Additional Considerations Measures of activity are sensitive to the circadian clock. o Mice are most active just after lights off and just before lights on. o Ensure experiments are conducted at the same time of day for all subjects under consistent lighting. Consider distractions: noises (ultrasonic and audible) olfactory cues (perfumes, other mice) Personnel: Adequate handling training, consistency Task order/sequence: Least to most stressful; limit tasks per day
Some Limitations Behavioral experiments can be timeconsuming! Lack of standardized protocols can limit cross-validation of findings between laboratories. Individual behavioral assays may be misinterpreted when they are performed in isolation; o E.g. a motor deficit may mask cognitive deficits in the MWM. o Evaluation of a array of behavioral domains improves the extent to which each individual assay may be correctly interpreted.
In Summary Behavioral evaluations can help you understand how your gene/molecule/physiology/manipulation manifests. o Consider clinically-relevant phenotypes Multiple facets of behavior: o Somatosensory o Cognitive o Psychosocial Study design appropriate controls, blinding, randomization, experimental conditions, ethics approval (IACUC).