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Single-Cell Electrophysiology and 2-Photon Imaging in Awake Mice with 2D-Locomotion Tracking

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In this webinar sponsored by Neurotar, experts present their research utilizing the Mobile HomeCage®, an experimental tool which ensures the stability required for high-precision neurophysiological techniques while allowing mice to navigate and explore their environment.

Case Study #1:
Dr. Sarah Stuart and Dr. Jon Palacios-Filardo of the University of Bristol present their studies combining analysis of goal-directed behavior with whole-cell recordings from the hippocampus of awake mice. The researchers share useful tips for the surgery protocol and for adjusting the head fixation angle in order to facilitate mouse motility and exploratory behavior.

Case Study #2:
Dr. Alexander Dityatev and Weilun Sun from the German Center for Neurodegenerative Diseases (DZNE) discuss 2-photon imaging of fluorescently labeled microglia in vivo in the context of neurodegenerative disease. They also present their recent data on the effects of different anesthetics on the microglial response to localized laser injury.

Case Study #3:
Dr. Norbert Hájos from the Hungarian Academy of Sciences presents his lab’s research into the amygdala’s role in reward-driven behavior. He shares the challenges of making single-unit recordings using silicon probes during mouse locomotion and subsequent morphological identification of active neurons in the amygdala.

Key topics covered during this webinar include:
- Requirements for stable single-cell recordings and 2-photon imaging in behaving mice
- Challenges of combining high-precision techniques with behavioral research
- Methodological considerations for improving exploratory behavior in head-fixed mice
- Quantitative analysis of microglial function using 2-photon microscopy in awake mice
- Recording neuronal activity in the amygdala of awake mice followed by morphological identification of recorded neurons

Published in: Science
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Single-Cell Electrophysiology and 2-Photon Imaging in Awake Mice with 2D-Locomotion Tracking

  1. 1. Single-Cell Electrophysiology and 2-Photon Imaging in Awake Mice with 2D-Locomotion Tracking Scientists present case studies focused on combining electrophysiology with 2D tracking, analyzing microglial function using 2-photon imaging and recording neuronal activity during reward-driven behavior.
  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. Sarah Stuart Research Associate University of Bristol Jon Palacios-Filardo Research Associate University of Bristol Alexander Dityatev Group Leader DZNE Magdeburg Weilun Sun PhD Student DZNE Magdeburg Single-Cell Electrophysiology and 2-Photon Imaging in Awake Mice with 2D-Locomotion Tracking Norbert Hájos Group Leader Hungarian Academy of Sciences
  5. 5. Click Here to Learn More About the Mobile HomeCage
  6. 6. In Vivo Electrophysiological Recording of Hippocampal Cells in Head-Fixed but Freely Exploring Mice Sarah Stuart, PhD Research Associate Universeity of Bristol Jon Palacios-Filardo, PhD Research Associate University of Bristol Copyright 2019 S. Stuart, J. Palacios-Filardo and InsideScientific. All Rights Reserved. Click Here to Watch the Webinar
  7. 7. Aim of the Study: Characterise normal reward-seeking and learning behaviours in head-fixed mice in the Mobile HomeCage Obtain intracellular CA1 pyramidal neuron recordings to visualize synaptic inputs and outputs Observe input/output plasticity during spatial navigation
  8. 8. Royers et al., 2012 The Mobile HomeCage Pressure air Physical restraint Linear treadmill Spherical treadmill Keller et al., 2012 Guo et al., 2014 Intracellular Recordings in Awake, Behaving Mice
  9. 9. T ra in in g S e s s io n #Laps 0 1 2 3 4 5 6 7 0 2 0 4 0 6 0 Overnight water deprivation (~16h) Activity in MHC stabilises over training sessions 10% sucrose reward (4ul) Trials/min L ig h t O ff L ig h t O n L ig h t O ff 0 2 4 6 - V is o r + V is o r Faecalcount - V is o r + V is o r 0 2 4 6 8 1 0 35° 37° 35° Naturalistic body posture Reducing light aversion Methodological Considerations B o d y w e ig h t %Startingweight 1 2 3 4 5 6 7 8 5 9 0 9 5 1 0 0 1 0 5 B o d y w e ig h t %Startingweight 1 2 3 4 5 6 7 8 5 9 0 9 5 1 0 0 1 0 5
  10. 10. Left and right LeftLeft Right Right Novel maze Reversal • Day 1: Animals freely explore a novel environment (T maze) and receive reward in both left and right arms • Day 2-3: Reward is delivered in one location only • Day 4 and 6: Reward location is switched to the previously unrewarded arm Left 2nd Reversal Characterising Normal ‘Foraging’ Behavior in the Mobile HomeCage
  11. 11. • Mice navigate around maze to find a ‘target zone’ (sandpaper) • Must wait in target zone for 2s to receive reward • Required to complete at least half a lap before returning to target zone to receive reward S e s s io n %Correcttrials 0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 3 0 4 0 5 0 6 0 7 0 8 0 9 0 S e s s io n %incorrectstops 0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 2 0 3 0 4 0 5 0 6 0 7 0 8 0 T m a ze C irc le S e s s io n #Targetcrosses 0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 Cued Goal-Localisation Task
  12. 12. 50 ms 50 pA 2 mV step In Vivo Hippocampal CA1 Recordings
  13. 13. Intracellular Recording from CA1 Pyramidal Neuron from Freely Exploring Mouse
  14. 14. Hippocampal CA1 Pyramidal Neurons Spatial Information Content
  15. 15. 100 ms 10 mV Super Burst Burst Single 100 ms 10 mV -60 mV Super Burst BurstSingle Single AP ≥ 5 AP = 1 AP [2-4] Group mean Single Super Burst [≥5 AP] Burst [2-4 AP] Exploration spikes Repose spikes R E Hippocampal CA1 Action Potential Pattern
  16. 16. CA1 Pyramidal Neuron Submembrane Dynamics Analysis
  17. 17. Acknowledgements Jack Mellor Jon Palacios Sarah Stuart Rachel Humphries Matt Udakis Pratap Tomar Mascia Amici Sonam Gurung Travis Bacon Matt Wilkinson Simon Griesius Heng Wei Zhu Leonard Khiroug Dmytro Toptunov
  18. 18. In Vivo Two-Photon Imaging of Microglial Surveillance and Photodamage-Directed Motility in the Mouse Cortex Alexander Dityatev, PhD Group Leader DZNE Magdeburg Weilun Sun PhD Student DZNE Magdeburg Copyright 2019 A, Dityatev, W. Sun and InsideScientific. All Rights Reserved. Click Here to Watch the Webinar
  19. 19. Microglia: Resident Innate Immune Cells of the CNS • CNS tissue macrophages, cells of mesodermal origin and myeloid lineage • Distributed throughout CNS tissues (including spinal cord and retina) • Have characteristic ‘ramified‘ appearance in the normal mature CNS tissue • Do constant monitoring of the tissue environment and scanning of synapses • Have a capacity to rapidly transform to alerted, activated and fully reactive states in response to stress, infection, tissue damage • Aiming at tissue maintenance, protection, and restoration • Guiding and assisting engagement of adaptive immune responses Alexander Dityatev, DZNE Magdeburg Hanisch & Kettenmann, Nat Neurosci 2007
  20. 20. Microglia Modulate Synaptic Pruning and Spinogenesis Kettenmann et al., 2013, Neuron Parkhurst et al., 2013, Cell
  21. 21. Tetrapartite Model of Chemical Synapses Dityatev and Rusakov, 2011, Curr Opin Neurobiol
  22. 22. A Pentapartite State of Synaptic Dynamics Modified from Kettenmann et al., 2013, Neuron ECM
  23. 23. How to Image Microglia In Vivo • In anaesthetized or awake mice? • At which time after implantation of transcranial window? Madry et al., 2018, Neuron Suppression of microglia responses to ATP and cell depolarization after application of isoflurane in vitro
  24. 24. Aim of the Study: To compare microglial surveillance and damage- directed motility in awake mice versus mice anaesthetized by ketamine or isoflurane in acute (1-2 days) versus chronic (> 1 month) preparations
  25. 25. Procedures of Cranial Window and Head Holder Implantation Weilun Sun, DZNE Magdeburg
  26. 26. Imaging Procedures Day 0 Surgery 1 2 3 4 Awake Isoflurane Resting Damage 1 Damage 2 Resting Damage 1 Damage 2 2hImaging Awake Ketamine Resting Damage 1 Damage 2 Resting Damage 1 Damage 2 2hImaging Awake Isoflurane Resting Damage 1 Damage 2 Resting Damage 1 Damage 2 2hImaging ~4 mo Awake Ketamine Resting Damage 1 Damage 2 Resting Damage 1 Damage 2 2hImaging 1 d acute chronic 1 d 7 d AwakeIsofluraneKetamine RSC
  27. 27. Imaging Examples 10 mm 50 mm Z = 6 (10 mm) t = 1 t = 100 (20 sec interval) 100 images, 33 min 0 min 30 min 0 min 30 min Resting Photodamage-directed 50 mm 10 mm Z = 6 (10 mm) t = 1 t = 100 (20 sec interval) 100 images, 33 min 0 min 30 min 0 min 30 min Resting Photodamage-directed 50 mm
  28. 28. Dynamics of Microglial Processes in Acute Experiments 0 2 4 6 8 10 Awake Iso Awake Keta Day 1 Day 2 n.s.n.s. n.s. Primary processes: total process # 0 10 20 30 Awake Iso Awake Keta Day 1 Day 2 * n.s.* Primary processes: avg. length (mm) Primary processes 1 2 3 4 5 acute T= 0-3 min T= 30-33 min All processes 12 3 4 5 6 7 8 9 10 11 12 acute T= 0-3 min T= 30-33 min Anesthesia as well as time interval after window implantation differentially affect microglia processes in acute preparation. Awake Isoflurane Awake Ketamine Awake Isoflurane Awake Ketamine Awake Isoflurane Awake Ketamine Awake Isoflurane Awake Ketamine 0 10 20 30 Awake Iso Awake Keta Day 1 Day 2 * n.s. n.s. All processes: total terminal # 0 100 200 300 400 Awake Iso Awake Keta Day 1 Day 2 * n.s. n.s. All processes: total length (mm) Awake Isoflurane Awake Ketamine Awake Isoflurane Awake Ketamine
  29. 29. 0 2 4 6 8 10 12 Awake Iso Awake Keta Day 1 Day 8 n.s.n.s. n.s. Primary processes: total process # 0 10 20 30 40 Awake Iso Awake Keta Day 1 Day 8 * n.s. n.s. Primary processes: avg. length (mm) Primary processes 1 2 3 4 5 T= 0-3 min T= 30-33 min chronic 0 10 20 30 Awake Iso Awake Keta Day 1 Day 8 n.s.n.s. n.s. All processes: total terminal # 0 100 200 300 400 Awake Iso Awake Keta Day 1 Day 8 n.s. n.s. n.s. All processes: total length (mm) All processes 12 3 4 5 6 7 8 9 10 11 12 T= 0-3 min T= 30-33 min chronic Dynamics of Microglial Processes in Chronic Experiments Awake Isoflurane Awake Ketamine Awake Isoflurane Awake Ketamine Awake Isoflurane Awake Ketamine Awake Isoflurane Awake Ketamine Isoflurane significantly increased the length of microglial primary processes while ketamine had no effect on the length and number of processes in chronic experiments.
  30. 30. All processes: change of category Day 1 Day 2 0 20 40 60 80 100 120 Awake Iso Awake Keta Disappear Disappear no change Increase Appear Primary processes: change of category 0 20 40 60 80 100 120 Awake Iso Awake Keta Disappear Decrease no change Increase Appear Day 1 Day 2 * * All processes: change of category Day 1 Day 8 0 20 40 60 80 100 120 Awake Iso Awake Keta Disappear Decrease no change Increase Appear Primary processes: change of category Day 1 Day 8 0 20 40 60 80 100 120 Awake Iso Awake Keta Disappear Decrease no change Increase Appear acute chronic Isoflurane anesthesia affected the turnover of microglia processes with fewer primary processes disappearing and more primary processes shortening in acute experiments. Morphological Changes of Microglial Processes Primary processes: change of category All processes: change of category Primary processes: change of category All processes: change of category 10 mm 0 min 30 min 10 mm Awake Isoflurane Awake Ketamine Awake Isoflurane Awake Ketamine Awake Isoflurane Awake Ketamine Awake Isoflurane Awake Ketamine
  31. 31. 0 100 200 300 400 Awake Iso Awake Keta All processes: total length (mm) Acute Chronic Animal 1 Animal 2 Animal 3 Animal 4 Animal 5 Animal 6 * * *n.s. Dynamics of Microglial Process Between Acute and Chronic Preparation Acute Total length of all processes increased, suggesting that in the chronic preparation microglia are more in the surveilling rather than activated state in contrast to the acute preparation. Chronic Awake Isoflurane Awake Ketamine
  32. 32. A3 Distance from the damage center (mm) (mm) Imaging time (min) Distancefromthedamagecenter y = -2.3787x + 39.222 R² = 0.9893 0 10 20 30 40 0 5 10 15 2’20” 5’40” 9’00” 12’20” 0 0.2 0.4 0.6 0.8 1 1.2 0 10 20 30 40 50 Relativeintensity 4’00” 12’20” 10’40” 9’00” 7’20” 5’40” 2’20” 50 mm Response of Microglia to Photo- Damage 50 mm
  33. 33. 0 1 2 3 4 5 Awake Iso Awake Keta MG process velocity (mm/min) Acute Chronic Animal 1 Animal 2 Animal 3 Animal 4 Animal 5 Animal 6 * 0 1 2 3 4 5 Awake Iso Awake Keta MG process velocity (mm/min) Day 1 Day 8 n.s. n.s. * ** chronic Day 1 Day 2 p* < 0.0001 *n.s. n.s. 0 1 2 3 4 5 Awake Iso Awake Keta MG process velocity (mm/min) Day 1 Day 2 * n.s. n.s. *** acute Motility of microglia to a photodamage is strongly affected by preparation type (acute vs. chronic) and isoflurane anesthesia. Quantification of MG Process Velocity in Acute and Chronic Experiments Awake Isoflurane Awake Ketamine Awake Isoflurane Awake Ketamine Awake Isoflurane Awake Ketamine
  34. 34. Injury size ( m2 ) 0 250 500 Velocity(m/min) 0 2 4 6 Acute Chronic Number of cells 0 5 10 Velocity(m/min) 0 2 4 6 Mean distance ( m) 40 60 80 Velocity(m/min) 0 2 4 6 Activation area ( m2 ) 0 1000 2000 Velocity(m/min) 0 2 4 6 Correlation Between Velocity and Other Factors Activation area Injury size
  35. 35. Summary of Results Motility type Acute cranial window Chronic cranial window Isoflurane Ketamine Isoflurane Ketamine Microglial surveilling Length ↑ ↑ Ramification ↑ Processes’ disappearance ↑ Damage- directed Velocity ↑ Activation area
  36. 36. • This study reveals potentiating effects of isoflurane on the length of surveilling microglial processes and microglial response to a damage. • We recommend to use awake mice with a chronically implanted transcranial window for the studies of microglia morphology and function in vivo. • When imaging in awake mice is not feasible, ketamine anesthesia in chronic preparation is preferable to isoflurane. Conclusions
  37. 37. FUNDING: 2nd Young Glia Japan- Germany collaboration program (coordinated by Frank Kirchhoff & Kazuhiro Ikenaka) Michisuke Yuzaki Leonard Khiroug Kunimichi Suzuki Janelle Pakan Stoyan Stoyanov Carla Cangalaya Acknowledgements Dmytro Toptunov
  38. 38. Recordings of Single Neuron Activities in the Amygdala of Behaving, Head-Fixed Mice Norbert Hájos Group Leader Laboratory of Network Neurophysiology Institute of Experimental Medicine Hungarian Academy of Sciences Budapest, Hungary Gergő A. Nagy, Bence Barabás, Richárd Kozma Copyright 2019 N. Hájos and InsideScientific. All Rights Reserved. Click Here to Watch the Webinar
  39. 39. Reference atlas, Allen Brain Institute BLA CEA Amygdala is a complex neural structure affecting various brain functions VGAT-Cre x LSL_ZsGreen1
  40. 40. Amygdala is involved in different processes including: i) memory ii) decision-making iii) emotional responses (fear, aggression, anxiety etc.) iv) social interactions v) food intake Amygdala is a complex neural structure affecting various brain functions VGAT-Cre x LSL_ZsGreen1
  41. 41. Aim of the Study: Reveal the information flow within the amygdala complex during aversive and appetitive behaviors that are known to be processed by this brain region Record spiking activity of individual neurons in different regions of the amygdala in head-fixed, behaving mice. Goal 1: Establish conditions that allow us to study the behavior of head-fixed mice Goal 2: Perform recordings of spiking activities of well isolated single neurons in the amygdala region in behaving mice
  42. 42. In line with our research goals we aim to use a setup that, in addition to providing the recording stability of head-fixed conditions, offer behavioural capacities comparable to freely moving recordings. To this end we perform behavioral experiments in awake head-fixed mice, we use a Mobile HomeCage (Neurotar Ltd). To conduct monitoring of neural activities in awake head-fixed mice, we obtain unit recordings by silicon probe and juxtacellular electrodes in mice placed in a Mobile HomeCage. Juxtacellular recording 10 s 0.5 mV 3% Neurobiotin in 0.5 M NaCl solution Methods 0.2 s 100µV Silicon probe recording
  43. 43. Cue-Dependent Fear Learning Using MHC 4x7x 30s + 2s Habituation in MHC for 7 days Fear conditioning Pairing CS+US in context B Testing for fear memory in MHC Day 1,2,3; 4xCS presented/day
  44. 44. Cue-Dependent Fear Learning Using MHC 0 200 400 600 800 0 20 40 60 80 100 Immobility(%) Time (s) mouse# 2 mouse# 3 4x7x 30s + 2s Habituation in MHC for 7 days Fear conditioning Pairing CS+US in context B Testing for fear memory in MHC Day 1,2,3; 4xCS presented/day
  45. 45. Cue-Dependent Fear Learning Using MHC 0 200 400 600 800 0 20 40 60 80 100 Immobility(%) Time (s) mouse# 2 mouse# 3 4x7x 30s + 2s Habituation in MHC for 7 days Fear conditioning Pairing CS+US in context B Testing for fear memory in MHC Day 1,2,3; 4xCS presented/day
  46. 46. Habituation in MHC for 7 days Fear conditioning Pairing CS+US in context B Testing for fear memory in MHC Day 1,2,3; 4xCS presented/day Cue-Dependent Fear Learning Using MHC 0 20 40 60 80 100 Immobility(%) 0 20 40 60 80 100 Immobility(%) 0 20 40 60 80 100 Immoblity(%) 4x 4x 4x Mouse# 1 Mouse# 2 Mouse# 3 Test day 1 Test day 2 Test day 3 baseline CS baseline CS baseline CS
  47. 47. Running in MHC Motivated by Reward Day 1 Day 4 Day 6 0 20 40 60 80 100 Mobility(%) Solution: 10 % sucrose Liquor delivery system
  48. 48. Unit Recordings in the Amygdala of Awake Head-Fixed Mice in MHC
  49. 49. Juxtacellular Recordings in the Amygala of Awake Head-Fixed Mice in MHC
  50. 50. Conclusions MHC is a platform for studying neural activity in a head-fixed configuration, while the mouse is engaged in affective behaviors. Both fear memory processes and the motivationally driven behavior can be studied in MHC. Using either silicon probes or juxtacellulary positioned glass electrodes, stable recordings of spiking activities of individual neurons can be reliably obtained in head-fixed, behaving mice using the MHC.
  51. 51. Sarah Stuart Research Associate University of Bristol Jon Palacios-Filardo Research Associate University of Bristol Alexander Dityatev Group Leader DZNE Magdeburg Weilun Sun PhD Student DZNE Magdeburg Norbert Hájos Group Leader Hungarian Academy of Sciences Thank You For additional information on the products and applications presented during this webinar please visit https://www.neurotar.com/research-instruments/

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