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

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

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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. • 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. 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. 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. 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. 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. • 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. • 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. 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. 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. 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. 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. • 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. 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. 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. Results II: Locomotion data

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. 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. 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. • 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. • 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. 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. • 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. 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. 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. http://www.nobelprizemedicine.org
What are Place Cells?

30. Kislin M et al., J Vis Exp 2014
2-Photon Imaging in the Hippocampus
Pilz GA et al., J Neurosci 2016

31. 31
Neurotar Mobile
HomeCage:
Open Field
Experimental
Setup

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. 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. 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. 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. 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. rewardrewardrewardrewardrewardreward
An Example…
Top viewBehaviour-triggered reward Mouse trajectory
reward

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. Place Cell Mapping

40. Place Cell Re-mapping

41. Summary
• First demonstration of place cell imaging in
mice navigating an air-lifted homecage
• Place cells remapping in novel environments

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. 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
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