This document summarizes two techniques for collecting data from animals - traditional tethering methods using liquid swivels and commutators, and the Raturn cage system. The Raturn system avoids issues with traditional tethering by allowing the cage to rotate freely in response to animal movement without interfering with fluid or electrical connections. The document then discusses several studies comparing these techniques and how stress levels, drug administration accuracy, and data quality can be impacted by the method used. It also summarizes work combining microdialysis and electrophysiological recordings to simultaneously measure extracellular proteins and neuronal activity in awake, behaving animals.

1. John Cirrito, PhD Candace Rohde-Johnson
Director of in Vivo Products &
Services
BASi
Turn Away from Traditional Tethering
and Towards a Better Method for
Data Collection
Associate Professor &
Microdialysis Core Director
Washington University, St. Louis

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4. Candace Rohde-Johnson
Director of in Vivo Products and Services
BASi
Copyright 2019 C. Rohde-Johnson and InsideScientific. All Rights Reserved.
Turn Away from Traditional Tethering
and Towards a Better Method for
Data Collection

5. Turn Away From Traditional Tethering
• No liquid swivel
• No commutator
• Cage moves in response to the
animal
• Creates an extremely flexible
system for small (and some large)
animal models

6. What is the RaturnTM?

8. Why is it better? - Liquid Swivel
• What- a simple way to connect
fluid lines
• How- internal seals to
maintain connection
• Pros & Cons

9. Why is it better? - Commutator
• What- an electrical signal
relay (aka rotary joint, signal
transducer)
• How- multiple contact points
to relay
• Pros & Cons

10. The RaturnTM
• What- a movement
responsive caging system
• How- responds to animal
movement and turns the
cage
• Pros & Cons

11. Acclimation to the RaturnTM
Heart Rate
1 2 3 4 5 6
300
350
400
450
500
550
9 10
Days
Beatsperminute(min)
Body Temperature
1 2 3 4 5 6
34
35
36
37
38
39
40
41
42
9 10
Days
Temp(0
C)
Systolic Blood Pressure
1 2 3 4 5 6
100
110
120
130
140
150
9 10
Days
SystolicBP(mmHg)
Diastolic Blood Pressure
1 2 3 4 5 6
80
90
100
110
120
9 10
Days
DiastolicBP(mmHg)
Mean Arterial Blood Pressure
1 2 3 4 5 6
90
100
110
120
130
9 10
Days
MAP(mmHg)
Diastolic Blood Pressure
0 1 2 3 4 5 6 7 8
80
90
100
110
120
ABST
Home cage Telemetry
Days
DiastolicBP(mmHg)
H. Kamendi et al., 2010 J. Pharmacol. Toxicol. Methods
doi: 10.1016/j.vascn.2010.04.014

12. Sampling Method Influences Stress Hormone Release
H. Kamendi et al., 2010 J. Pharmacol. Toxicol. Methods
doi: 10.1016/j.vascn.2010.04.014
Stress hormones in ABST
vs tail bled and home cage rats
ACTH
(pg/m
l)
Corticosterone
(ng/m
l)
Insulin
(uIU/m
l)Prolactin
(ng/m
l)
0
25
50
75
100
125
150
175
200
225
Tail bleed
ABST
Home cage
**
* * *
** p< .0001
* p< .05
**
LevelsofHormones

13. Method of Human Intervention Can Impact Results
0
20
40
60
80
100
120
140
0 10 20 30 40 50 60
Nicotine(ng/ml)
Time (Min)
Automated Compared to Manual Intragastric Dosing - Nicotine
Automated Dosing Manual Dosing
*
*
*
* *
*
*

14. Possibilities with the Raturn™

15. Why Does That Matter?
Capture Data that isn’t possible
with other methods
Use Fewer Animals
Collect More Data

16. Copyright 2019 J. Cirrito and InsideScientific. All Rights Reserved.
Simultaneously Measuring
Extracellular Peptides and Neuronal
Activity In Vivo
John Cirrito, PhD
Associate Professor and Microdialysis Core Director
Department of Neurology
Washington University, St. Louis

17. Pools of Brain Aβ
Aβ is produced in neurons
then secreted into the brain
extracellular fluid, or
interstitial fluid (ISF)
Conversion from normal,
soluble ISF Aβ into
aggregated, toxic species is
concentration-dependent
Human AD brain

18. Both of which are performed in awake, behaving mice (or rats) either
separately or together
1. In vivo microdialysis
Measures extracellular proteins in interstitial fluid (ISF) over time in awake
animals
2. In vivo recording of neuronal activity
a. Depth EEG
(extracellular field potential recordings for gross measure of neuronal activity)
b. Single evoked potentials (field EPSPs) in the dentate gyrus
Two Technologies

19. Raturn Caging System
The Raturn enables us to have a direct line from the
equipment to the animal’s head without the use of:
For us, the Raturn is a more costly, but
often the “simpler” route for long-term
continuous measures
Both techniques require tubing or wires to be connected to the head for 3-5 days
during each experiment while the mouse has freedom of movement and ad lib food
and water
1. Liquid swivels
Particularly prone to clogging and contamination, especially
when using protein in the perfusion buffer
2. Electrical commutators
Often made for specific equipment and not
interchangeable/flexible

20. 1. In vivo Microdialysis
Samples brain extracellular proteins every
hour for 3-5 days
Microdialysis is based on simple
diffusion across a semi-permeable
membrane
**Molecules smaller than the
membrane pore size will enter the
probe and be sampled over time
Microdialysis probes between 2-4 mm long
350um outer diameter
(from Bioanalytical Systems, SciPro or Eicom)
Microdialysis Probe
38-1,000kDa MWCO)

21. Selective serotonin reuptake inhibitor (SSRI) antidepressants reduce
ISF Aβ levels acutely in mice

22. Serotonin signaling requires extracellular regulated kinase (ERK) to
suppress Aβ generation
* Inhibition of MEK or ERK blocks the effect of
SSRIs on ISF Aβ
* Inhibition of JNK, a another MAPK, has no effect
on Aβ

23. 2a. Electroencephalography (EEG) - extracellular field potentials
Dual wires placed within the hippocampus or
cortex to record electrical activity
EEG generally measures the sum neuronal
activity within 1 cubic millimeter or so (gross
estimate)
Fig: Basal EEG activity and activity during
treatment with picrotoxin (GABAA receptor
antagonist)
Basal EEG
25uM Picrotoxin
Constructed of stainless steel or
platinum-iridium wires that are
teflon-coated for insulation
(wire 0.14mm outer diameter)

24. Electro-Microdialysis Probes
Wires glued to the shaft of the guide cannula so the
tips extend to the middle of the microdialysis probe
Electrodes
0.14mm
coated diameter
Microdialysis

25. Inhibiting neuronal activity with tetrodotoxin (TTX) reduces ISF Aβ levels
Tetrodotoxin (TTX)
a sodium channel blocker
1uM delivered via reverse
microdialysis
** TTX lowers ISF Aβ
levels which is reversible
when TTX is removed
from the microdialysis
perfusion buffer
Cirrito et al (2005) Neuron
EEG ISF Aβ

26. 2b. Evoked Potentials:
Simulation and recording
Stimulate perforant pathway and record
in the dentate gyrus
• Also possible to stimulate cortex and
measuring individual fEPSPs in
contralateral cortex (commissural
projections across corpus collosum)
• In rats, it is possible to stimulate
Schaffer collaterals from CA3 and
record in CA1

27. Combine microdialysis with stimulation and recording
High frequency electrical stimulation
of perforant pathway induces
hippocampal seizures which rapidly
increases ISF Aβ levels

28. “Mega Electrode”
For protein levels and sleep/wake measures
Components:
1. Microdialysis guide cannula
2. Depth EEG for hippocampal
activity around microd probe
3. EMG electrodes (loops of wire)
4. 2 cortical electrodes (screws)
5. Ground screw
• EMG with cortical EEG enables us to distinguish between
sleep/wake states of a mouse (awake, non-REM, REM)
• Aβ microdialysis
• EEG

29. ISF Aβ levels fluctuate with a diurnal rhythm in mice.
Aβ is high during wakefulness and low during sleep.
Sleep is good!
Important: mice habituate for 5-7 days in cage before behavioral studies.

30. Acknowledgements
Cirrito Lab:
Carla Yuede, Ph.D.
Rachel Hendrix, Ph.D.
Clare Wallace
Todd Davis
Woody Gardiner
Brooke Doherty
Kate Reardon
Kevin McBrearty
Derrick Ogola
Collaborators:
David Holtzman
Steve Mennerick
Chuck Zorumski, Yuki Izumi
Jin-Moo Lee, Ping Yan, Qingli Xaio
Former members:
Jon Fisher, Ph.D.
Jane Hettinger, Ph.D.
Hannah Edwards
Hollie Ridenbark
Kayla Yuede
Hyo Lee
Jessica Restivo
Jack Burchett
Funding:
R01 NIH/NIA, P01 NIH/NINDS, P50
NIH/NIA,
R21 NIH/NIA, Alzheimer’s Association,
CART Rotary Club International
Diana King
Katherine Young
Dorothy Schuler
Danielle Tripoli
Renee Ehrenstrom
Kaitlin Mallinson

31. John Cirrito, PhD Candace Rohde-Johnson
Director of in Vivo Products &
Services
BASi
Associate Professor &
Microdialysis Core Director
Washington University, St. Louis
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