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Place Cell Mapping and Stress Monitoring in Head-Fixed Mice Navigating an Air-Lifted Homecage

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In this webinar sponsored by Neurotar, experts present their research on 2-photon imaging of hippocampal place cells and on stress monitoring in head-fixed awake behaving mice. Dr. Konrad Juczewski from the National Institutes of Health (NIH)/National Institute on Alcohol Abuse and Alcoholism (NIAAA) discusses the impact of head fixation on animal’s stress, locomotion and performance in classical behavioral paradigms.

Dr. Mary Ann Go from the Laboratory of Neural Coding and Neurodegenerative Disease at Imperial College London led by Prof. Simon Schultz presents her research using 2-photon microscopy aimed at place cell mapping in the hippocampus during exploration and navigation of a circular linear track.

Key Discussion Topics Include:
- Stress reduction in head-fixed rodents
- Improving data reproducibility and translational value of the data acquired from head-fixed rodents
- Effects of head fixation on blood corticosterone concentration, locomotion patterns and performance in stress-associated behavioral tests
- Optimizing habituation protocol for head-fixed mice
- Monitoring neural activity and mapping of place cells using 2-photon microscopy during navigation and exploration behavior
- Automating the experiments using a closed-loop approach and behavior-triggered reward systems

Published in: Science
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Place Cell Mapping and Stress Monitoring in Head-Fixed Mice Navigating an Air-Lifted Homecage

  1. 1. Konrad Juczewski, PhD Post-Doctoral Fellow Laboratory for Integrative Neuroscience National Institute on Alcohol Abuse and Alcoholism National Institutes of Health Mary Ann Go, PhD Post-Doctoral Fellow Neural Coding and Neuro- degenerative Disease Lab Department of Bioengineering Imperial College London Place Cell Mapping and Stress Monitoring in Head-Fixed Mice Navigating an Air-Lifted Homecage
  2. 2. InsideScientific is an online educational environment designed for life science researchers. Our goal is to aid in the sharing and distribution of scientific information regarding innovative technologies, protocols, research tools and laboratory services
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  4. 4. Background Stress in the Head- Fixed Method: Can we Ignore it? Konrad Juczewski, PhD Copyright 2019 K. Juczewski and InsideScientific. All Rights Reserved. Post-doctoral Fellow with Dr. David M. Lovinger Laboratory for Integrative Neuroscience National Institute on Alcohol Abuse and Alcoholism National Institutes of Health
  5. 5. • Introduction: Why do we care? • Methods: Considerations • Results I: Blood sampling data • Results II: Locomotion data • Results III: Behavioral data • Discussion, summary & the future Outline
  6. 6. Why do we care? – Stress factor • Chronic stress – a serious confound - shown to affect behavior (e.g. elevated plus maze) and physiology (e.g. food and water intake) in mice - severe lack of information on stress in the head-fixed situation. • My interests + David Lovinger’s lab - mechanisms of synaptic plasticity - the function and roles of cortico-basal ganglia circuits in habit formation and addiction - also, their involvement in motor skill learning • Head-fixed method in the Lovinger lab - understanding changes related to methodology (head-fixation) to be able to interpret physiological and behavioral data correctly Harper & Austad, 2000 Chiba et al., 2012
  7. 7. Why do we care? – Popular methods • Head-fixed method = old technique gaining popularity in the rodent research world - new environments, e.g. treadmill, virtual reality + spherical ball - new methods, e.g. optogenetics, photometry, new types of microscopes • Head-fixed method advantages - no anesthesia that always affects brain function - stable environment for sensitive recordings (e.g. single- cell patch-clamp electrophysiology) - paired behavioral measurements (e.g. licking, locomotion) • Head-fixed method disadvantages - complex surgeries and experimental set up - limited movements = movement restriction, abnormal posture - potentially very stressful • Variety of head-fixed set ups
  8. 8. Why do we care? – Variety of head-fixed set ups Giovannucci et al., 2018 modified Type II: cylindrical (left) and flat (right) treadmill + less restriction & potentially less stressful – uncomfortable posture (in cylindrical) – limited planes for movement (in flat)
  9. 9. Why do we care? – Air-lifted platform & questions Type IV: air-lifted platform + reduction of disadvantages (optimal range of movement & optimal body posture) + additional benefits: compact, easily transported & modifiable – still limited range of movement Pursue the best experimental protocol - habituation to reduce stress - lack of a standardized protocol for any method (variety of set ups + variety of tasks) - 0-5 days habituation to head-fixation for 0-2 hours (e.g. Nashaat M. et al., 2016; Lee D., et al., 2017 Voigts J. et al., 2018) - additional flannel wrapping (Kislin M. et al., 2014) Habituation in the air-lifted platform - different ideas about habituation but is it justified in terms of stress? - what is the stress level in the head-fixed mouse with partial restraint (only head) versus other stressors? - is habituation procedure necessary or can we skip it? - what about locomotion – is it related to stress level? - and behavior? How is it affected by head-fixing?
  10. 10. • Preparation: - head-plate surgery (7 days before handling) - 2 days of handling (15 minutes each day; first day hands, second day flannel wrapping) • Head-fixed habituation: - 25 daily sessions of 120-minutes head-fixation - blood sample (BS) collection every 5 days (about 40 uL of blood = 20 uL of plasma) - corticosterone blood plasma concentration (ELISA kit) - locomotion recordings with video camera - locomotion analysis with EthoVision software (tracking the yellow dot) BS#1 BS#5 BS#10 BS#15 BS#20 BS#25 BS#30 Preparation BehaviorHead-fixed habituation Methods: Experimental protocol
  11. 11. • Choice of blood sampling method = tail vein bleeding (TVB): - moderately stressful - awake animal – avoiding anesthesia - partial restraint in the small container – avoiding full-body restraint Vein bleeding: RBB = retro-orbital TVB = tail SVB = saphenous FVB = facial JVB = jugular Tsai et al., 2015 Methods: Considerations
  12. 12. Gong at al., 2015Gong at al., 2015 Methods: Considerations • Choice of biomarker = corticosterone: - corticosterone – adaptation-related response (e.g. repeated restraint) - cortisol – acute stress response (e.g. unpredictable stress)
  13. 13. https://www.jax.org/ Methods: Considerations • Choice of animal’s age = mature adult (13-24 weeks): - youngsters (<13 weeks) have higher and less stable corticosterone level
  14. 14. Gong at al., 2015 Female mice Gong at al., 2015 Male mice Methods: Considerations • Choice of gender = males: - corticosterone fluctuations related to estrous cycle (females) - corticosterone fluctuations related to circadian rhythm (less in males)
  15. 15. Challenge I: tail cut off Solution: better control of cut depth and placement Challenge III: object missing/not found in the EthoVision analysis Solution: NEUROTAR tracking software Challenge II: loss of head-plate Solution: gentle fixing procedure with some movement flexibility different glue/dental acrylic more screws Methods: Challenges
  16. 16. • Paired animals = housed 2 per cage • Head-fixed group procedure: - weight checking - flannel wrapping and transferring to the frame - head-fixing to the frame for 2-hours - placing animal in the container and collecting blood samples (up to 5-minutes altogether) - placing animal back to the cage • Control group procedure: - weight checking - flannel wrapping for about 3-5 minutes (time corresponding to the head-fixing procedure) - placing animal back to the cage in the same room as the head-fixed apparatus with the air-pump on D a y 1 D a y 5 D a y 1 0 D a y 1 5 D a y 2 0 D a y 2 5 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 Cort.conc.(ng/ml) H e a d -fixe d C o n tro l BS#1 BS#5 BS#10 BS#15 BS#20 BS#25 BS#30 Preparation BehaviorHead-fixed habituation Results I: Blood sampling data
  17. 17. Blood sampling data in the context (Sadler et al., 2016) - corticosterone and behavioral response to restraint stress = similar to our studies - full body restraint for 2 hours each day = more severe restraint than our studies (head-fixing with full body movements allowed) - blood sampling from the lateral vein immediately after restraint (between 11:00 and 13:00) = similar to our studies but we had 4 times for head-fixing (8:00-10:00; 10:15-12:15; 12:30-14:30; 14:45-16:45 +/- 15 min) Results I: Blood sampling data
  18. 18. Blood sampling data in the context (Bowers et al., 2008) - corticosterone and immune response to various type of stressors - types of stressors: full body restraint, low temperature, forced swim test, social isolation, handling - stressor specific alterations in the corticosterone levels Results I: Blood sampling data
  19. 19. Results II: Locomotion data
  20. 20. Results II: Locomotion data Movement time in the context (Kislin et al., 2014) - 120 minutes sessions - absolute movement time (%) but calculated per second not per frame – seconds with detected movement versus non-movement
  21. 21. Results II: locomotion data Average distance - animals active throughout entire session - more activity at the beginning - detailed analysis – task engagement over 25-days Average velocity - general information about the movement - running on the air-lifted platform = motor skill learning - detailed analysis – movement quality (bouts of activity) 0 5 1 0 1 5 2 0 2 5 0 1 2 3 4 5 A v e ra g e v e lo c ity a ll a n im a ls c o m b in e d D a y Velocity(cm/sec) 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 A v e ra g e d is ta n c e p e r a n im a l T im e (m in ) Distancetraveled(cm) A 2 A 3 A 1 3 A 1 5 A 1 8 Results II: Locomotion data
  22. 22. Behavior: - day #26 AM – open field test (10-minutes session) - day #26 PM – forced swim test (6-minutes session) - day #27 AM – elevated plus maze (7-minutes session) - day #27 PM/overnight – 2-bottle cages for sucrose preference test & nesting behavior test - day #28-29 – sucrose preference test - day #30 – final blood sampling BS#1 BS#5 BS#10 BS#15 BS#20 BS#25 BS#30 Preparation BehaviorHead-fixed habituation Methods: Experimental protocol continued
  23. 23. • Elevated plus maze (EPM): behavior day #2 AM (overall day #27) - 7-minute recordings (6-minutes analyzed beginning from minute 1) • Head-fixed group spend more time in the open arms but no change in total locomotion - floating container = closed space becomes a stressful environment? • Similar results at day 7 but not day 14 (Sadler et al., 2016) - studies with full-body restraint repeated every day for 2 hours C o n tro l H F 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 E P M d is ta n c e Totaldistancetraveled(cm) C o n tro l H F 0 1 0 0 2 0 0 3 0 0 4 0 0 E P M c u m u la tiv e d u ra tio n o p e n Time(sec) C o n tro l H F 0 1 0 0 2 0 0 3 0 0 4 0 0 E P M c u m u la tiv e d u ra tio n c e n te r Time(sec) C o n tro l H F 0 5 0 1 0 0 1 5 0 2 0 0 E P M la te n c y to firs t o p e n Time(sec) Results III: Behavioral data
  24. 24. • Sucrose preference test (SPT): behavior day #3 & day #4 (overall day #28 & day #29) - animals placed in new cages with 2 bottles with water overnight at day #27 - bottles changed in the morning at day #28 AM (one bottle with sucrose, one bottle with water) - bottles weighed and sides swapped at day #29 AM (side preference control) - bottles weighed at day #30 AM • No sucrose preference in the head-fixed group - sign of anhedonia developed over the course of 25 days • Similar results at day 14 but not day 7 (Sadler et al., 2016) - studies with full-body restraint repeated every day for 2 hours W a te r S u c ro s e 0 2 4 6 8 1 0 S P T c o n tro l Consumedvolume(mL) ** W a te r S u c ro s e 0 2 4 6 8 1 0 S P T h e a d -fix e d Consumedvolume(mL) C o n tro l H F 0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 S P T S c o re Ratiosucrosetototalvolume C o n tro l H F 0 2 4 6 8 1 0 1 2 S P T w a te r + s u c ro s e Totalconsummedvolume(mL) Results III: Behavioral data
  25. 25. www.jax.org Age: start to end 0 5 1 0 1 5 2 0 2 5 9 0 9 5 1 0 0 1 0 5 1 1 0 D a y Normalizedbodyweight(%) H e a d -fix e d C o n tro l 0 5 1 0 1 5 2 0 2 5 2 5 2 7 2 9 3 1 3 3 3 5 D ay Bodyweight(g) H e a d -fix e d C o n tro l • Body weight change - about 3-4% decrease in body weight of the head-fixed animals Results III: Other measurements
  26. 26. • The closest future: Juczewski et al. (late 2019 or early 2020) - Manuscript in preparation, submission planned before the end of 2019 - Presented data supplemented – more animals and complete behavior - Additionally – detailed analysis of locomotion pattern (bouts of activity) corticosterone blood concentration (CBC) versus time of the day; CBC versus locomotion pattern; CBC in single-housed animals; CBC in the head-fixed animals on the moving/non-moving platform (possibly); locomotion analyzed with Ethovision and Neurotar tracking system side by side (hopefully) http://members.madasafish.com/ D a y 1 D a y 5 D a y 1 0 D a y 1 5 D a y 2 0 D a y 2 5 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 Cort.conc.(ng/ml) H e a d -fixe d C o n tro l Discussion, summary & the future • Discussion & summary - Can we ignore background stress in the head-fixed method? – it depends - Corticosterone blood concentration at the level of social stress – but it is still possible to reduce - Context-dependent fear (elevated plus maze) + anhedonia (sucrose preference test) – but extended head-fixation and no entertainment apart from running - What is the optimal protocol? – again, it depends but most likely 5 to 10 days
  27. 27. Do not miss, contact & acknowledgements • Do not miss - SfN poster 433 / 3806 (Neuroscience, Chicago 2019) – certain Monday October 21, afternoon session (1:00 PM - 5:00 PM) *K. JUCZEWSKI, J. KOUSSA, A. KESNER, D. M. LOVINGER. - Publication in preparation (presented data + more on stress & locomotion) Juczewski et al. (early 2020) – almost certain ;) • Contact details konrad.juczewski@nih.gov konrad.juczewski@gmail.com • Thank you note - Members of the David Lovinger’s lab - Members of the Veronica Alvarez’s lab - Especially: Jonathan Koussa (running head-fixed experiments) - Andrew Kesner and Miriam Bocarsly (behavioral consultation) Daniel da Silva (teaching the blood sampling method)
  28. 28. Mary Ann Go, PhD Copyright 2019 M. A. Go and InsideScientific. All Rights Reserved. Post-Doctoral Fellow Neural Coding and Neurodegenerative Disease Lab Department of Bioengineering Imperial College London Place Cell Mapping and Stress Monitoring in Head-Fixed Mice Navigating an Air-Lifted Homecage
  29. 29. http://www.nobelprizemedicine.org What are Place Cells?
  30. 30. Kislin M et al., J Vis Exp 2014 2-Photon Imaging in the Hippocampus Pilz GA et al., J Neurosci 2016
  31. 31. 31 Neurotar Mobile HomeCage: Open Field Experimental Setup
  32. 32. • hSyn1-GCaMP6s-mRuby • hippocampus (1.6 mm ML; 2.0 AP; -1.5 DV) A B C 0 7 14Day 21 Circular linear track Open field Viral injection Hippocampal window surgery Imaging Behavioural training 0 7 14Day 21 Start H2 0 restriction 28 28 Viral injection Hippocampal window surgery Imaging Behavioural training Start H20 restriction 2-Photon Imaging Methodology https://www.addgene.org/50942/
  33. 33. A B C 0 7 14Day 21 Circular linear track Open field Viral injection Hippocampal window surgery Imaging Behavioural training 0 7 14Day 21 Start H2 0 restriction 28 28 Viral injection Hippocampal window surgery Imaging Behavioural training Start H20 restriction 2-Photon Imaging Methodology Pilz GA et al., J Neurosci 2016
  34. 34. 2-Photon Imaging Methodology A B C 0 7 14Day 21 Circular linear track Open field Viral injection Hippocampal window surgery Imaging Behavioural training 0 7 14Day 21 Start H2 0 restriction 28 28 Viral injection Hippocampal window surgery Imaging Behavioural training Start H20 restriction • 1-3 mL water • Maintain ≥80% starting weight
  35. 35. 2-Photon Imaging Methodology A B C 0 7 14Day 21 Circular linear track Open field Viral injection Hippocampal window surgery Imaging Behavioural training 0 7 14Day 21 Start H2 0 restriction 28 28 Viral injection Hippocampal window surgery Imaging Behavioural training Start H20 restriction
  36. 36. HPC cannula H2O 2-Photon Imaging Methodology A B C 0 7 14Day 21 Circular linear track Open field Viral injection Hippocampal window surgery Imaging Behavioural training 0 7 14Day 21 Start H2 0 restriction 28 28 Viral injection Hippocampal window surgery Imaging Behavioural training Start H20 restriction
  37. 37. rewardrewardrewardrewardrewardreward An Example… Top viewBehaviour-triggered reward Mouse trajectory reward
  38. 38. Data Processing Steps Raw Ca2+ imaging video Motion correction ROI segmentation Signal extraction and neuropil correction Spike estimation Place field mapping Raw Motion corrected
  39. 39. Place Cell Mapping
  40. 40. Place Cell Re-mapping
  41. 41. Summary • First demonstration of place cell imaging in mice navigating an air-lifted homecage • Place cells remapping in novel environments
  42. 42. Come see my dynamic poster in SfN! • SfN dynamic poster 333.13 • 10/21/2019, 8:00 am – 12:00 pm • *M. Go, J. Rogers, C. Davey, S. V. Prado, G. Gava, L. Yio, L. Khiroug, S. R. Schultz
  43. 43. Konrad Juczewski, PhD Post-Doctoral Fellow Laboratory for Integrative Neuroscience National Institute on Alcohol Abuse and Alcoholism National Institutes of Health Mary Ann Go, PhD Post-Doctoral Fellow Neural Coding and Neuro- degenerative Disease Lab Department of Bioengineering Imperial College London Thank You For additional information on the products and applications presented during this webinar please visit www.neurotar.com/research-instruments/

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