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Turn Away from Traditional Tethering and Towards a Better Method for Data Collection

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Experts discuss the use of a novel movement responsive rodent caging system as a means to minimize animal stress and enable unique discovery in many research applications, namely neuroscience, animal behaviour, drug discovery and cardiometabolic disease.

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Turn Away from Traditional Tethering and Towards a Better Method for Data Collection

  1. 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
  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
  3. 3. To access webinar content, Q&A reports, FAQ documents, and information on lab workshops, subscribe to our mail list
  4. 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. 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. 6. What is the RaturnTM?
  7. 7. Why is it better? - Liquid Swivel • What- a simple way to connect fluid lines • How- internal seals to maintain connection • Pros & Cons
  8. 8. Why is it better? - Commutator • What- an electrical signal relay (aka rotary joint, signal transducer) • How- multiple contact points to relay • Pros & Cons
  9. 9. The RaturnTM • What- a movement responsive caging system • How- responds to animal movement and turns the cage • Pros & Cons
  10. 10. 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
  11. 11. 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
  12. 12. 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 * * * * * * *
  13. 13. Possibilities with the Raturn™
  14. 14. Why Does That Matter? Capture Data that isn’t possible with other methods Use Fewer Animals Collect More Data
  15. 15. 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
  16. 16. 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
  17. 17. 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
  18. 18. 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
  19. 19. 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)
  20. 20. Selective serotonin reuptake inhibitor (SSRI) antidepressants reduce ISF Aβ levels acutely in mice
  21. 21. 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
  22. 22. 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)
  23. 23. 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
  24. 24. 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
  25. 25. 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
  26. 26. Combine microdialysis with stimulation and recording High frequency electrical stimulation of perforant pathway induces hippocampal seizures which rapidly increases ISF Aβ levels
  27. 27. “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
  28. 28. 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.
  29. 29. 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
  30. 30. John Cirrito, PhD Candace Rohde-Johnson Director of in Vivo Products & Services BASi Associate Professor & Microdialysis Core Director Washington University, St. Louis For additional information on the products and applications presented during this webinar, please contact the speakers below. Thank you!

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