A growing number of researchers are moving from reduced preparations such as dissociated cultured neurons or brain slices, to experimentation in live animals - in vivo - using advanced methods such as two-photon microscopy or combined optogenetics and patch-clamp recordings. In order to immobilize the animal during these challenging applications general anesthesia is often administered; however, the use of anesthetics greatly distorts brain function.
Is there a better way?
In this exclusive webinar sponsored by Neurotar Ltd, leading experts in the technology will discuss methodology, best-practices and show attendees how to immobilize the rodent’s head without restraining its body using the Mobile HomeCage™. The result is a controlled research environment for studying brain function in awake, freely-moving subjects with no stress to the animal. Discussion around how this technique can be applied to the study of neuronal plasticity, neurodegeneration, addiction, brain trauma and other pathophysiological conditions in longitudinal experiments will be included. Furthermore, presenters will demonstrate how this methodology is best combined with microscopy and electrophysiology techniques – all in vivo.
2. Making Optical and Electrophysiological Measurements in
the Brain of Head-Fixed, Freely-Moving Rodents
1. A brief history of The Mobile HomeCage (L. Khiroug, PhD)
2. Awake Plasticity (E. Castrén, MD, PhD)
3. Effects of drugs of abuse on dendritic spine plasticity:
intravitial microscopy in awake mice (P. Hyytiä, PhD)
4. Electrophysiological recordings of cortical activity in head-restrained
rodents in vivo (R. Khazipov, MD, PhD & M. Minlebaev, MD. PhD)
Sponsored by:
3. 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.
4. A brief history of
The Mobile HomeCage
Leonard Khiroug
Chief Scientific Officer (CSO),
Neurotar Oy Ltd
5. • 10 years as a PI at Univ.
Helsinki (after postdoc at
Duke and NIH); Adj. prof
• In vivo two-photon
imaging as a service to
global Pharma industry
• Going awake: solving our
own problem => Product
for labs world-wide
Evolution to
Revolution
Academic
group
(2003-2013)
Service
business
(2009 - )
Device
business
(2014 - )
7. • Mitochondrial
fragmentation in Ischemic
Stroke models
Example:
Transgenic “reporter” mice expressing
mitoCFP on Thy1 promoter, visualizing
mitochondrial of cortical neurons.
Laser-induced stroke, reversible and
drug-targetable fragmentation of
mitochondria
In vivo microscopy
as a service
8. • Mitochondrial
fragmentation in Ischemic
Stroke models
Example:
Transgenic “reporter” mice expressing
mitoCFP on Thy1 promoter, visualizing
mitochondrial of cortical neurons.
Laser-induced stroke, reversible and
drug-targetable fragmentation of
mitochondria
In vivo microscopy
as a service
Note the network-like organization of elongated
mitochondria prior to the stroke induction
9. • Mitochondrial
fragmentation in Ischemic
Stroke models
Example:
Transgenic “reporter” mice expressing
mitoCFP on Thy1 promoter, visualizing
mitochondrial of cortical neurons.
Laser-induced stroke, reversible and
drug-targetable fragmentation of
mitochondria
In vivo microscopy
as a service
Note the fragmented mitochondria 30 minutes
after stroke induction (blood flow obstructed)
15. Sleep research in
head-fixed mice
Chronic recording (wire electrodes)
Acute recording (multi-shank high-density silicon probes)
Locomotion
Sleep
Run
Dr. Nikolaos Karalis and
Prof. Anton Sirota
(LMU, Munich, Germany)
18. Head-fixed
in Mobile
HomeCage
Head-fixed
on treadmills
(linear, spherical)
+ VR
Carrying
head-mounted
miniaturized
devices
Short habituation and training,
minimized stress
+ - -
Cost-efficiency + - -
Natural environment
(flat floor, walls, obstacles)
+ - +
Allows high quality optics, multiple
precision electrodes
+ + -
Compactness and compatibility with
other equipment
+ - +
Allows place cell research or locomotion
over long distances
- + +
Summary
20. Neuronal Plasticity:
• The capacity of neurons and neural circuits to change structurally and
functionally in response to experience
• Plasticity is regulated by neuronal activity
• Plasticity is bidirectional: adding and removing
• Neurogenesis => selection by programmed cell death
• Axon growth => selection by retraction
• Synaptogenesis => selection by synaptic pruning
• Synaptic potentiation and depression
• Active neurons and synapses are selected and stabilized
• NOT THE QUANTITY, BUT THE QUALITY
21. CTRL ANESTH
CTRL ANESTH
Question &
Experimental
Procedure
AN
30’
M M MM
24 h 1h 1h 24 h
Does anesthesia
influence dendritic
spine plasticity?
• Steady state spine
number or shape?
• Spine dynamics?
C A C A C A
25. • Imaging, stimulation and electrophysiology in living brain
• Increasingly evidence that anesthesia influences
• Need for recording/ imaging in awake animals
• Imaging/recording in behaving and learning animals
• Behavior in familiar, non-stressed environment
• Imaging in home cage environment would be optimal
ERA of connectomics
26. Effects of drugs of abuse on
dendritic spine plasticity:
intravitial microscopy in awake mice
Petri Hyytiä
PhD, adjunct professor,
Department of Pharmacology
Biomedicum
University of Helsinki Finland
27. • Introduction
• Why intravitial microscopy in awake mice?
• Our hypotheses
• Experimental design
• Preliminary data
• Summary of main points
What we are going to cover today?
28. The “Addicted” Spine
• Drug addiction marked by long-
lasting changes in behavior
• Persistent structural changes in
neurons in limbic brain regions
• Changes in size of cell bodies,
dendritic arborization, spine
morphology and dynamics
The brain reward pathways
29. Spiga et al. 2014
The “Addicted” Spine
• Drug addiction marked by long-
lasting changes in behavior
• Persistent structural changes in
neurons in limbic brain regions
• Changes in size of cell bodies,
dendritic arborization, spine
morphology and dynamics
30. • Robinson & Kolb 1997
• Medium spiny neurons in
nucleus accumbens
• First demonstration of drug-
induced structural plasticity
• Persistent increase in total
spines per 10 µm of dendrite
and in the number of spines
with multiple heads
(branched spines)
Amphetamine-
induced structural
modifications
31. Cocaine and morphine produce opposite effects in spine
density and dendritic branching
Cocaine Morphine
32. Fraction of spines gained
between imaging sessions
Cocaine induced structural plasticity in frontal cortex –
2-photon imaging via cranial windows
Spine accumulation during
cocaine treatment
New persistent spines gained
during cocaine conditioning:
correlation w/ cocaine CPP
Munoz-Cuevas et al 2013
33. Why intravitial microscopy in awake mice?
Benefits:
• Long-term longitudinal imaging
• No interfering anesthesia
• No need for control group
Limitation:
• Depth: only superficial layers of neocortex
34. • Other cortical areas:
motor, perirhinal,
orbital cortex
• Thalamocortical
projections
• Corticostriatal
projections
Primary
somatosensory
cortex (S1)
connectivity
36. • Changes in spine dynamics correlate with the
synaptic activity at the spines and therefore
serve as indicators of changes in neural circuitry
produced by drugs of abuse
• Persistence of the changes in spines point to a
mechanism behind the long-term drug effects,
including susceptibility to relapse
• Changes in dendritic spine turnover are
correlated with behavioral alterations
Our hypotheses
Thy1-YFP mice
38. Week 1
M
1d
M M M
Week 3
M
1d
M
Week 2
MorphinePBS
Effects of subchronic morphine - Spine turnover imaging protocol
39. Week 1
M
1d
M M M
Week 3
M
1d
M
Week 2
MorphinePBS
Effects of subchronic morphine - Spine turnover imaging protocol
40. Week 1
M
1d
M M M
Week 3
M
1d
M
Week 2
MorphinePBS
Effects of subchronic morphine - Spine turnover imaging protocol
41. Week 1
M
1d
M M M
Week 3
M
1d
M
Week 2
MorphinePBS
Effects of subchronic morphine - Spine turnover imaging protocol
42. Week 1
M
1d
M M M
Week 3
M
1d
M
Week 2
MorphinePBS
Effects of subchronic morphine - Spine turnover imaging protocol
43. Week 1
M
1d
M M M
Week 3
M
1d
M
Week 2
MorphinePBS
Effects of subchronic morphine - Spine turnover imaging protocol
44. 100 mm
Baseline, before PBS and Morphine 2 weeks after Morphine injections
100 mm
Imaging stability (raw data comparison)
45. • In vivo two-photon microscopy in non-anesthetized Thy1-
YFP mice yields unbiased information of drug-induced
changes in spine dynamics in the somatosensory cortex
• Monitoring these changes over extended periods of time
gives insight into drug-induced structural plasticity in the
mammalian neocortex
• Our pilot project with morphine demonstrates the
feasibility of the procedures using the mobile home cage
in the context of two-photon microscopy
Summary of main points
46. Electrophysiological recordings
of cortical activity in head-
restrained rodents in vivo
Roustem Khazipov
Directeur de Recherche,
INSERM U901
Marat Minlebaev
Charge de Recherche,
INSERM U901
47. 1. Overview of the extracellular and patch-clamp
recording techniques (Khazipov)
2. Application of the Mobile HomeCage (Minlebaev)
3. Tips for stable electrophysiological recordings from the
head-fixed animals in Mobile HomeCage
What we are going to cover today:
50. Intracortical recordings of the local field potentials
Minlebaev & Khazipov, 2009
• Local field potentials and
multiple unit activity (MUA -
spikes of individual neurons) can
be recorded from different
depth
• Current source density (CSD)
profile shows sinks and sources
of the population events
• MUA and cross-correlation
analysis shows how neurons in
different layers are activated
during population events
51. Multiple and single neuron action potentials
Mitrukhina et al., 2014
• Extracellular recordings
of neurons spikes with
tetrodes enable to
isolate spikes of
individual neurons
through cluster analysis
• One tetrode may give a
description of activity of
several neurons
52. Whole-cell recordings from a single neuron
Minlebaev et al., 2011
• Whole-cell recordings from
individual neurons enable
to access synaptic
correlates of the network
activity
• GABA and glutamate
synaptic currents can be
studied in isolation
• In current clamp mode,
firing of recorded cell and
subthreshold conductances
53. Cell-attached recordings
• Cell-attached recordings
enable to record spikes from
individual neurons and
• Currents through single ion
channels
• Reversal potential of GABA
currents can be deduced
from single GABA channel
activity without altering
intracellular chloride
Tyzio et al., 2006
54. 1. Overview of the extracellular and patch-clamp
recording techniques (Khazipov)
2. Application of the Mobile HomeCage (Minlebaev)
3. Tips for stable electrophysiological recordings from the
head-fixed animals in Mobile HomeCage
What we are going to cover today:
59. Current clamp whole cell in vivo recordings
in the somatosensory cortex
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60. • Coordinates (atlases, papers)
• Cortical surface better to keep wet (agarose better)
• Ensure mechanical stability (make the hole in the skull
as small as possible. Put dental cement close around
the hole)
• Minimize pulsations (put agar, silicone oil if needed)
• Avoid multiple insertions (not more than 5-10 attempts
through the same hole)
• Vertical penetration (‘+’ extra cortical immobilization,
‘-‘ tissue damage; ~10 penetrations).
• Pipette length depends on the recorded structure
(shorter – better, otherwise extra capacitance; for
striatum, up to 3 mm in mice)
Patch-clamping in awake mice
61. Thank You!
For additional information on the Mobile HomeCage and
methods for using this device in awake rodents with optical
imaging or electrophysiological techniques please visit:
http://www.neurotar.com/
62. 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.