1. Basic Mouse Neurobehavioral Exam (or how to give a mouse a physical) Julie Watson Vet MB DACLAM

2. Resources Jacqueline Crawley –Crawley,J and Paylor,R. Hormones & Behavior 197-211 (1997) –“What’s Wrong with my Mouse?” Wiley-Liss 2000. Samuel Irwin - First Phenotyping Screen –Psychopharmacologia (Berl.) 13 222-257 (1968) SHIRPA stage I –Mamm Genome. 1997 Oct;8(10):711- Rogers DC, Fisher EM, Brown SD, Peters J, Hunter AJ, Martin JE Websites: –Eumorphia http://www.eumorphia.org/EMPReSS/servlet/EMPReSS.Frameset –MRC Mutagenesis Program –http://www.mgu.har.mrc.ac.uk/facilities/mutagenesis/mutabase/http://www.mgu.har.mrc.ac.uk/facilities/mutagenesis/mutabase/ Clinical approach

3. Goals 1.Detect abnormalities likely to affect future behavioral tests –Blindness –Physical defects –Deafness

4. Background Strain Age-related hearing loss & vestibular defects (<3m) Many 129 strains A/J C57BR, C57L DBA I, LP, NOD, ALR, ALS C57BL/6J @12-18m Blindness (rd1 gene) FVB C3H BUB CBA SJL SWR NON P, PL

5. Goals (2) 2. Suggest further testing –Presence of abnormal behaviors –Absence of normal behaviors –Motor or neurological deficits

6. Pitfalls Variations due to: –Husbandry methods (see John Crabbe, Science 1999) –Age/sex/background strain –Noise/temperature/humidity/location etc. –Time of day Detect only sizeable deficits –Background strain not inbred. F1’s, N2’s –Are controls comparable ? –Insufficient group size Limited statistical analysis available - ordinal data Check genotyping reliability

8. BIOSAFETY CABINET

9. SIMPLE EQUIPMENT

10. WEIGHT PERTINENT INFORMATION TESTER BLINDED TO GENOTYPE

11. Tests Behavior in Empty Cage: –Gait, posture, general appearance –Are normal behaviors present? Exploring, thigmotaxis, digging, grooming, rearing –Are abnormal behaviors present? freezing, wild running, stereotypies, seizures, pruritus

12. REARING/ESCAPE

13. DIGGING

14. Knockout Mouse LIMITED REARING

15. Tests Pick up, record abnormal physical features –Whisker loss –Bald patches or unkempt haircoat, piloerection –Eyes –Legs and tail

16. NORMAL WHISKERS

17. WHISKER LOSS

18. General Reactivity Response to approach Body tone Petting escape Passivity

19. Normal Response to Approach

20. NORMAL BODY TONE

21. HIGH BODY TONE

22. PETTING ESCAPE

23. Unusual Escape Attempts

24. PASSIVITY

25. Postural Reactions and Reflexes Trunk curl Righting reflex Forelimb proprioceptive positioning Rearlimb withdrawal

26. TRUNK CURL

27. RIGHTING REFLEX

28. PROPRIOCEPTIVE POSITIONING

29. WITHDRAWAL - FAST

30. WITHDRAWAL - SLOW

31. Tests –V & VII Nerve Facial sensory and motor Whisker response Ear twitch Palpebral reflex (V, VII)

32. EAR TWITCH

33. EAR TWITCH – WHOLE HEAD RESPONSE

34. EAR TWITCH – UNRESPONSIVE

35. WHISKER RESPONSE (?)

36. PALPEBRAL RESPONSE

37. Sight Placing – visual II Placing – tactile

38. VISUAL PLACING

39. Tactile Placing

40. Tests Clicker / hearing Pupil / response to light Biting / aggression

41. CLICKER

42. Provoked Aggression

43. Grip Strength Normal

44. Grip Strength Abnormal

45. Impact of Physical Deficits on Behavioral Tests Impact of Blindness Learning and memory tests Water maze, contextual fear conditioning –animal cannot learn? –has limited spatial abilities? –Consider alternatives –2 lever or hole-poke test in operant chamber Anxiety tests Visual cliff, elevated T maze, light- dark box –Low anxiety?

46. Whisker Deficits, Itchy Skin Lack of Whiskers –Anxiety tests e.g. visual cliff, elevated T maze – Skew response compared with mice with whiskers ? Remove whiskers Itchy skin (B6 dermatitis) –open field, aggression hyperirritability, increased activity increased aggression

47. Hearing Deficits Cued fear conditioning –Use contextual fear conditioning Prepulse inhibition –Nb DBA still works

48. Examples of Further Testing Open Field Light Dark Box Elevated Plus Maze Accelerating Rotarod Cued and Contextual Fear Conditioning Morris Water Maze

49. Open Field total activity, discrete movements, distance traveled rearing

50. Light Dark Box Conflict between novelty exploration and avoidance of light open spaces Strain variations Responsive to anxiolytics

51. Elevated Plus Maze PICTURE CREDITS: EUROPEAN MOLECULAR BIOLOGY LABORATORY Exploration vs. aversion to open space - prefers dark enclosed arms Score entries into arms over 5 min trial Anxiolytics increase entries into open arms - widely used in drug discovery

52. Accelerating Rotarod

53. Cued and Contextual Conditioning Learning and memory test Pair footshock with auditory cue – see freezing behavior Day 2 return to same chamber - Freezing is a measure of contextual fear Move to different chamber, test freezing (control for context) Give auditory cue in different chamber – freezing is measure of cued fear

54. Computer image Morris Water Maze Visual cues Opaque water bath Containing hidden platform

55. Probe Trial

56. Visual Cliff Test of sight – plexiglass cover to apparent cliff Place on border 10 trials – see which way steps off Relies on ability to see cliff Original research showed blind strain 50/50 Vs. sighted strains 80% on solid side Confounded by other senses (whiskers)

57. Acknowledgements Jacqueline Crawley Christian Newcomer Michela Gallagher Nadine Forbes Questions?