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

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.

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Sarah Stuart
Research Associate
University of Bristol
Jon Palacios-Filardo
Research Associate
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

Click Here to Learn More
About the Mobile HomeCage

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
Copyright 2019 S. Stuart, J. Palacios-Filardo and InsideScientific. All Rights Reserved.
Click Here to
Watch the Webinar

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

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

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

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

• 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

50 ms
50
pA
2 mV step
In Vivo Hippocampal CA1 Recordings

Intracellular Recording from CA1 Pyramidal Neuron from Freely Exploring Mouse

Hippocampal CA1 Pyramidal Neurons Spatial Information Content

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

CA1 Pyramidal Neuron Submembrane Dynamics Analysis

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

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

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

Microglia Modulate Synaptic Pruning and Spinogenesis
Kettenmann et al.,
2013, Neuron Parkhurst et al., 2013, Cell

Tetrapartite
Model of
Chemical
Synapses
Dityatev and
Rusakov, 2011, Curr
Opin Neurobiol

A Pentapartite State of Synaptic Dynamics
Modified
from
Kettenmann
et al., 2013,
Neuron
ECM

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

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

Procedures of Cranial Window and Head Holder Implantation
Weilun Sun,
DZNE Magdeburg

Imaging Procedures
Day 0
Surgery
1 2
3 4
Awake
Isoflurane
Resting Damage 1 Damage 2
2hImaging
Awake
Ketamine
2hImaging
Awake
Isoflurane
2hImaging
~4 mo
Awake
Ketamine
2hImaging
1 d
acute
chronic
1 d
7 d
AwakeIsofluraneKetamine
RSC

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

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
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
0
10
20
30
Day 1 Day 2
*
n.s.
n.s.
All processes: total terminal #
0
100
200
300
400
Day 1 Day 2
*
n.s. n.s.
All processes: total length (mm)

0
2
4
6
8
10
12
Day 1 Day 8
n.s.n.s.
n.s.
Primary processes: total process #
0
10
20
30
40
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
Day 1 Day 8
n.s.n.s.
n.s.
All processes: total terminal #
0
100
200
300
400
Day 1 Day 8
n.s.
n.s.
n.s.
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
Isoflurane significantly
increased the length of
microglial primary
processes while
ketamine had no effect
on the length and
number of processes in
chronic experiments.

All processes: change of category
Day 1 Day 2
0
20
40
60
80
100
120
Disappear
Disappear
no change
Increase
Appear
Primary processes: change of category
0
20
40
60
80
100
120
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
Disappear
Decrease
no change
Increase
Appear
Primary processes: change of category
Day 1 Day 8
0
20
40
60
80
100
120
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

0
100
200
300
400
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

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

0
1
2
3
4
5
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
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
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

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

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

• 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

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

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

Reference atlas, Allen Brain Institute
BLA
CEA
Amygdala is a complex neural structure affecting various brain functions
VGAT-Cre x LSL_ZsGreen1

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

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

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

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

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
presented/day

Habituation in MHC
for 7 days
Fear conditioning
Pairing CS+US in
context B
presented/day
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

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

Unit Recordings in the Amygdala of Awake Head-Fixed Mice in MHC

Juxtacellular Recordings in the Amygala of Awake Head-Fixed Mice in MHC

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.

Sarah Stuart
Research Associate
Jon Palacios-Filardo
Research Associate
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/

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

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