Epilepsy research employs sophisticated research methods such as fluorescence optical imaging and optogenetics, as well as novel electrophysiological techniques, to address unresolved questions about seizure generation and propagation on the cellular and circuitry levels. Since epilepsy research is most relevant when performed in non-anesthetized mice, it requires specialized tools that ensure stable head fixation during high-precision imaging and recordings. In this webinar, Dr. Anthony Umpierre (Prof. LongJun Wu group, Mayo Clinic, USA) and Prof. Rob Wykes (UCL, UK) present their research on microglial calcium signaling and epileptic networks carried out in awake head-fixed mice. In addition to sharing exciting new findings, the presenters address the challenges of working with awake mice. Key topics will include… - Mesoscopic investigations of seizure dynamics and propagation using widefield calcium imaging - Generating full-bandwidth electrophysiological recordings enabled by graphene micro-transistors to detect spreading depolarizations and seizures - On-demand optogenetic induction of spreading depolarizations to investigate pharmacological suppression in the awake brain - The impact of acute versus chronic window preparations on microglial calcium activity - The use of genetically encoded calcium indicators to study calcium dynamics in microglia - The effects of bi-directional shifts in neuronal activity caused by kainate-triggered status epilepticus and isoflurane anesthesia on microglial calcium

1. Rob C. Wykes, PhD
Senior Research Fellow,
UCL Queen Square Institute of Neurology;
Senior Lecturer,
University of Manchester, Dept. of Nanomedicine
Anthony Umpierre, PhD
Postdoctoral Researcher,
Mayo Clinic, Dept. of Neurology
Studying Epilepsy in Awake Head-
Fixed Mice Using Microscopy,
Electrophysiology, and Optogenetics

2. Experts discuss how microscopy,
electrophysiology, and optogenetics are used
to study microglial calcium signaling and
epileptic networks in awake head-fixed mice.
Studying Epilepsy in Awake Head-
Fixed Mice Using Microscopy,
Electrophysiology, and Optogenetics

3. Copyright 2020 R. Wykes and InsideScientific. All Rights Reserved.
Recording Seizures and
Spreading Depolarisations
in Awake, Head-Fixed Mice
Rob C. Wykes, PhD
Senior Research Fellow,
UCL Queen Square Institute of Neurology;
Senior Lecturer,
University of Manchester, Dept. of Nanomedicine

4. • Anaesthetics affect brain functions
• Imaging and electrophysiological
data obtained in non-anesthetized
animals are more relevant.
• We have developed an in vivo
head-fixed preparation that permits
optogenetic stimulation, with
mesoscopic calcium imaging and/or
electrographic recordings using
either epidural or penetrating arrays
of graphene micro-transistors.
Awake head-fixed experiments in mice
Graphene
micro-transistor
arrays

5. • The Mobile HomeCage is a combination
of a head-fixation system and an air table.
• The carbon fiber tray floats allowing the
animal to move it with its paws.
• This gives the mouse an illusion of freely
walking around the cage reducing stress.
• Habituation sessions of increasing length
(15 mins, 30 mins, 45 mins, 60 mins)
allow the animals to acclimatize to the
equipment prior to experimentation.
Awake head-fixed experiments in mice using a Neurotar Mobile HomeCage

6. Placeholder slide for video:
neuron_recording.mp4

7. Placeholder slide for video:
neuron_recording.mp4

8. Fluorescence imaging
of epileptic activity at
multiple scales
• Epilepsy research is rapidly adopting
novel fluorescence optical imaging
methods to tackle unresolved
questions on the cellular and circuit
mechanisms of seizure generation
and evolution.
• Two-photon microscopy can record
single neuron dynamics during
interictal and ictal discharges.
• Wide-field fluorescence imaging
provides mesoscopic mapping of
functional networks and can be used
to investigate seizure propagation.
Rossi LF, Kullmann DM & Wykes RC. (2018). “The Enlightened Brain: Novel Imaging Methods Focus on Epileptic
Networks at Multiple Scales”. Front Cell Neuroscience PMID: 29632475

9. Induction of epileptiform activity and seizures in the visual cortex
PTX, 10 mM
Interictal events occurred on average 13.4 ± 3.7 times per minute much more frequently
than ictal events, which occurred about once per minute (1.0 ± 0.2 events/min).
Brain expressing the genetically encoded
calcium indicator GCAMP6.
Rossi LF, Wykes RC, et al (2017) Nature Communications. PMID: 28794407.

10. Simultaneous wide-field GCaMP imaging, LFP recordings
and behavioral measurements
Seizures were typically accompanied by pronounced
increases in pupil dilation and by bouts of running.
Rossi LF, Wykes RC, et al (2017) Nature Communications. PMID: 28794407.

11. Retinotopic
mapping of the
visual cortex
• The functional connectivity
within and across visual
areas can be easily
mapped.
• Higher visual areas inherit
their retinotopic
representation from V1.
• Do seizures preferential
follow contiguous or
homotopic propagation
pathways?
Rossi LF, Wykes RC, et al (2017) Nature Communications. PMID: 28794407.

12. Placeholder slide for video:
neuron_recording.mp4
Rossi LF, Wykes RC, et al (2017) Nature Communications. PMID: 28794407.

13. Seizure propagation
recruits homotopic
regions of cortex
6 to 8 s into the event, the ictal
activity shows a bimodal profile,
being stronger in the homotopic
regions in LM than in regions of
V1 that are closer to the focus.
average propagation speed of
0.48 ± 0.03 mm/s.
(underestimates the delays of
proximal territories and
overestimates those of distal
territories).
These results indicate
that delay to ictal
invasion depends
approximately linearly
on two factors: one
lateral, which grows with
distance in cortex, and
one homotopic, which
grows with distance in
visual preference.
Rossi LF, Wykes RC, et al (2017) Nature
Communications. PMID: 28794407.

14. Once the seizure spread to the entire visual cortex and beyond, we
observed more complicated patterns of propagation and oscillation.

15. Placeholder slide for video:
neuron_recording.mp4

16. Post-ictal Cortical
Spreading Depression
(CSD) recorded in an
awake mouse
• The CSD-like wave
started at a site distinct
from the seizure focus.
• The depolarisation
spread radially, with no
apparent relation to the
functional organisation
of the cortex.
CSD propagation ~3-5 mm/min.

17. • A major limiting factor is the absence of
experimental tools that would allow one to
perform concurrent recordings of both LFP
frequencies and ISA (CSD) in an awake brain
with high temporal and spatial resolution.
• Recording ultraslow signals (frequencies <0.1
Hz) with microelectrodes is severely
hampered by current electrode materials and
technology, primarily due to limitations
resulting from high electrode impedance and
voltage drift.
• Graphene Solution-gated field effect
transistors can overcome these limitations
Full bandwidth electrographic recordings using graphene micro-transistors
No signal attenuation and no voltage driftMasvidal-Codina E et al (2019) Nature Materials. PMID: 30598536

18. Recording spreading
depolarisations in
awake head-fixed mice
using graphene micro-
transistor arrays
• KCL injected into motor
cortex.
• Spreading depolarisation
detected using a gSGFET
array placed epidurally over
ipsilateral somatosensory /
visual cortex.
• Note the spatiotemporal
resolution capable using this
approach
400 mm

19. Full bandwidth
recording of a seizure
and spreading
depolarisation
• 14 channel penetrating probe
inserted through visual cortex to
the upper part of the
hippocampus.
• 2 channels displayed (one upper,
one lower) recording a seizure
evoked by focal injection of
picrotoxin into the cortex.
• Note DC shift at onset of ictal-like
activity in the top channel and a
post-ictal spreading depolarisation
recorded from the deeper channel.
400 mm

20. The transition
from inter-ictal to
ictal event
• Transient fluorescent
signals seen during
interictal events were
localized in V1. They
involved a region that was
markedly smaller than the
region that could be
activated by visual stimuli.
• Interictal and ictal events
had similar origins: 94±4%
of events started in V1,
within a radius of
0.32±0.12 mm).
Are there slow excitatory
processes accumulating
which allow/promote the
subsequent inter-ictal spike
to evolve into a seizure?

21. Transition of Epileptic Activity Across Brain Regions – I
Cortical injection of 4-AP

22. Transition of Epileptic Activity Across Brain Regions – II

23. Optogenetic induction of CSD

24. • The ability to combine
optogenetics, mesoscopic calcium
imaging and full bandwidth
electrophysiology in an awake
head-fixed mouse preparation
offers a powerful method to
study seizure dynamics.
• In particular, these approaches
will allow us to gain a better
understanding of the role of
spreading depolaristaions in the
epileptic brain.
Summary

25. Calcium Imaging
Federico Rossi (Matteo Carandini & Dimitri Kullmann)
Graphene Transistors
Martin Smith, Eduard Masvidal, Andrea Bonaccini, Daman Rathore, Yunan Gao, Monique Keane, Amirhossein
Jafarian
Design & Fabrication of Graphene Transistors
Instituto de Microelectronica de Barcelona- Eduard Masvidal
Instituto de Microelectronica de Barcelona - Anton Guimera Brunet
Instituto de Microelectronica de Barcelona - Xavi Illa
Catalan Institute of Nanoscience and Nanotechnology - ICN2 - Andrea Bonaccini
Catalan Institute of Nanoscience and Nanotechnology - ICN2 - Jose Garrido
Funding
Acknowledgements

26. Anthony Umpierre, PhD
Postdoctoral Researcher,
Mayo Clinic, Dept. of Neurology
Imaging Microglial Calcium
During Seizures and Epilepsy
Development in Awake Mice
Copyright 2020 A. Umpierre and InsideScientific. All Rights Reserved.

27. Microglia and their Unique Structural Responses
to the Brain Environment
Microglia
• Innate immune cells of the CNS
• Dynamic processes survey the local environment
• Respond to injury and molecular landscape
• Can be both reparative and pro-inflammatory

28. Microglial Processes Sense Brain Hyperactivity
CX3CR1 Microglia: GFP
Thy-1 Neurons: YFP
Upkong Eyo, Postdoc.
Asst. Prof., Univ. of Virginia
Eyo et al., (2014) J. Neurosci.

29. Microglial Processes Sense Brain Hypoactivity
Yong Liu, Postdoc.
Asst. Prof., S. China Univ. of Technology
Liu et al., (2019) Nat. Neurosci.

30. Complimenting Structural Studies:
Understanding Microglia from a Functional Lens
Structure (Process Dynamics) Function (Calcium Dynamics)
Liu et al., (2019) Nat. Neurosci. Umpierre et al., (2020) eLife

31. Methodology
Neurotar Mobile HomeCage
Window
GCaMP6s
R26-LSL-GCaMP6s Cx3Cr1CreER-IRES-eYFP

32. Key Point:
Acute Window
Higher Spontaneous Ca2+
Chronic Window
Low Spontaneous Ca2+
Acute Chronic
0
5
10
15
SignalArea(F/Fs)
****
Layer I Calcium
Microglial Processes
Umpierre et al., (2020) eLife
See also: Eichhoff et al., (2011)
Biochim. Biophys. Acta.; Pozner
et al., (2015) Front. Mol. Neurosci.
: Platform Training
D0: Window Surgery (P35+)

33. Studying Microglia through the Lens of Calcium Dynamics
Brawek et al. (2014) Acta. Neuropathol.
Eichhoff et al. (2011) Biochim. Biophys. Acta.
Pozner et al. (2015) Front. Mol. Neurosci.
Microglia
Calcium
Signaling
LPS
Laser Burn
Pozner et al.
mM ATP
Neuron
Damage
A-Beta
Plaques
Bicuculline
Pozner et al.Eichhoff et al.
Eichhoff et al.
Brawek et al.
Pathology
Neuronal
Activity
(Low at rest)

34. Baseline
(Awake)
Hypoactivity
• Anesthesia
• Gi DREADD
Hyperactivity
• Seizures
• Gq DREADD
NeuronalAlterationMicroglialResponse
Hypothesis and Approach

35. Hypothesis and Approach
Baseline
(Awake)
Hypoactivity
• Anesthesia
• Gi DREADD
Hyperactivity
• Seizures
• Gq DREADD
NeuronalAlterationMicroglialResponse

36. microglial calcium signaling
Kainic Acid
Kainate Receptor
AMPA Receptor
Increasing neuronal activity

37. Key Point:
Umpierre et al., (2020) eLife
0
5
10
15
20
SignalArea(F/Fs)
****
Layer I Microglial Calcium
Soma Processes
n.s.
Details:
• Time series (15 min, 1Hz)
• Layer I Imaging (50-70 µm)
• Awake
• Within-subject comparison to baseline
• Soma and Process domains

38. microglial calcium signaling
Decreasing neuronal activity
Isoflurane
General Anesthesia

39. Baseline 0-10 10-20 20-30
0
5
10
15
SignalArea
(F/Fs)
Isoflurane (1.5-2.0%)
**** ****
Soma
Processes
Key Point:
Umpierre et al., (2020) eLife

40. DREADD-based alterations in neuronal activity
Local CaMKIIa neurons
Gq DREADD
CNO
Gi DREADD
Hyperpolarization*
Less NT
Release
Decreases local excitatory
neuron activity
AAV5.CaMKIIa.Gi-DREADD
Gi DREADD
CNO
Gq DREADD
Calcium
NT Release
Increases local excitatory
neuron activity
AAV5.CaMKIIa.Gq-DREADD
*K channels and additional mechanisms

41. ***
5.0 1.25 0 0 2.5 5
0
100
200
300
SignalArea
(%ofBaseline)
***
*
*
Microglia Process Ca2+
Gi DREADD Gq DREADD
CNO dose:
Not Shown:
No change in Soma Ca2+
Neuronal Peak Response
Microglial Peak Response
Key Point:
Umpierre et al., (2020) eLife

42. Longitudinal Changes in Microglia Calcium
1. Tse et al (2014), PLOS One; Umpierre et al (2016), Exp. Neurol.
2. Vezzani et al (2013), Exp. Neurol.; Hauser et al (2017), Neuroscientist
Epilepsy
Injury
Event
Epilepsy Development
“Epileptogenesis”
Status Epilepticus (SE)
• prolonged seizure event
• systemic kainate injection (i.p.)1

43. Increased Ca2+
Transients
B
aseline
D
ay
1
D
ay
2
D
ay
3
D
ay
7
D
ay
10
D
ay
14
2
4
8
16
32
64
128
256
SignalArea(F/Fs)
Soma
Processes
Day Post-Kainate
***
**
***
**
**
***
*
**
*
**
ns
ns
Umpierre et al., (2020) eLife

44. Spreading Ca2+
Waves
Observed across all animals
Not confined to early or late response
See also:
Pozner et al. 2015
LPS + Bicuc. waves
Umpierre et al., (2020) eLife

45. Umpierre et al., (2020) eLife
%ofProcesses
Timing of Nearest Ca2+ Event
<30s <120s<60s
Extension
Retraction
10%
18%
23%
35%
51%
55%
**
** **
Motility/Ca2+
Correlations

46. Motility/Ca2+ Correlations
Umpierre et al., (2020) eLife
Isoflurane
Gi DREADD
Kainate (24 hrs)
Gq DREADD

47. Summary
Baseline Day 1 Day 2 Day 3 Day 7 Day 10 Day 14
1
2
4
8
16
32
64
128
256
512
SignalArea(F/Fs)
Somata
Processes
Day Post-Kainate
ACUTE Studies LONGITUDINAL Study
***
**
***
**
**
***
*
**
*
**
ns
ns

48. Acknowledgements
Yong Liu Yujia Wei Dale Bosco [me] Qian Wang Tingjun Chen
Lauren Bystrom Christina Vasquez Yanlu Ying Long-Jun Wu
Fiona Zheng Sidra Jabeen Manling Xie Colleen Allen Min-Hee Yi
Mentor
Dr. Long-Jun Wu
Funding
Mayo Foundation (LJW)
NIH NINDS F32 NS114040 (ADU)
NIH NINDS R01 NS112144 (LJW)
NIH NINDS R01 NS088627 (LJW)
NIH NINDS R01 NS110825 (LJW)
NIH NINDS R01 NS110949 (LJW)

49. Rob C. Wykes, PhD
Senior Research Fellow,
UCL Queen Square Institute of Neurology;
Senior Lecturer,
University of Manchester, Dept. of Nanomedicine
Anthony Umpierre, PhD
Postdoctoral Researcher,
Mayo Clinic, Dept. of Neurology
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