Making Optical and

Electrophysiological Measurements

in the Brain of Head-Fixed, Freely-

Moving Rodents

Sponsored by:

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:

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the sharing and distribution of scientific information regarding innovative technologies, protocols, research

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A brief history of The Mobile HomeCage

Leonard Khiroug

Chief Scientific Officer (CSO),

Neurotar Oy Ltd

• 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 - )

• Trans-BBB Pharmacokinetics and Biodistribution

Example:

Client’s proprietary antibody tagged with Alexa Fluor 647 (red), kinetics of penetration through BBB, colocalization with Abeta tangles labelled with Methoxy X04 (green)

In vivo microscopy as a service

• 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

• 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

• 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)

Full-body constraint

Previous solutions for studies in awake mice

Linear treadmill Head-mounted devices

Air-lifted ball (spherical treadmill)

Previous solutions for studies in awake mice

Our Solution: “Flat Ball With a Wall”

Front view Side view Air

Natural enclosed environment, short habituation, stable fixation, compact design

Our Solution: “Flat Ball With a Wall”

Our Solution: “Flat Ball With a Wall”

Two-photon microscopy

Optogenetics

Intrinsic optical imaging

Glutamate uncaging

Voltage-sensitive dyes

Whole-cell patch clamp

Multichannel electrodes

Voltammetry

Ion-sensitive electrodes

Microdialysis

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)

Sleep research in head-fixed mice

Dr. Nikolaos Karalis and Prof. Anton Sirota (LMU, Munich, Germany)

Sleep

Run

Sleep research in head-fixed mice

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

Awake Plasticity

Eero Castrén, MD, PhD Neuroscience Center University of Helsinki

Finland

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

CTRL ANESTH

CTRL ANESTH

Question & Experimental Procedure

AN

30’

M M M M

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

Generally about technique

• 2-photon imaging in awake, freely moving mice

Generally about technique

• Allows repeated imaging of an identified neuronal process

Isoflurane does not influence spine dynamics

Somatosensory cortex

• 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

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

• Introduction

• Why intravitial microscopy in awake mice?

• Our hypotheses

• Experimental design

• Preliminary data

• Summary of main points

What we are going to cover today?

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

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

• 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

Cocaine and morphine produce opposite effects in spine density and dendritic branching

Cocaine Morphine

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

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

• Other cortical areas: motor, perirhinal, orbital cortex

• Thalamocortical projections

• Corticostriatal projections

Primary somatosensory cortex (S1) connectivity

Primary somatosensory cortex activation by drugs of abuse

• Metabolic mapping

• Activation marker c-Fos expression

• fMRI

• 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

Effects of subchronic morphine - Spine turnover imaging protocol

Week 1

M 1d

M M M Week 3

M 1d

M Week 2

Morphine PBS

Effects of subchronic morphine - Spine turnover imaging protocol

Week 1

M 1d

M M M Week 3

M 1d

M Week 2

Morphine PBS

Effects of subchronic morphine - Spine turnover imaging protocol

Week 1

M 1d

M M M Week 3

M 1d

M Week 2

Morphine PBS

Effects of subchronic morphine - Spine turnover imaging protocol

Week 1

M 1d

M M M Week 3

M 1d

M Week 2

Morphine PBS

Effects of subchronic morphine - Spine turnover imaging protocol

Week 1

M 1d

M M M Week 3

M 1d

M Week 2

Morphine PBS

Effects of subchronic morphine - Spine turnover imaging protocol

Week 1

M 1d

M M M Week 3

M 1d

M Week 2

Morphine PBS

Effects of subchronic morphine - Spine turnover imaging protocol

100 mm

Baseline, before PBS and Morphine 2 weeks after Morphine injections

100 mm

Imaging stability (raw data comparison)

• 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

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

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:

Surface EEG

Intracortical recordings of the local field potentials

Multi-electrode arrays

Minlebaev & Khazipov, 2009

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

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

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

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

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:

Spontaneous hippocampal activity in CA1 region of awake mice

Theta oscillations in Stratum Radiatum

Cross-Frequency Coupling between Theta and Gamma frequency bands in Stratum Radiatum

PAC measure = cfc Dataset = Structures = -

Am

p f

req /

Hz -

Phase freq / Hz -

5 10 15 20 25 30 35 400

10

20

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60

70

80

90

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

0.05

Multi-unit activity (StrR and StrPyr) phase lock to StrRad Local Field Potential

1s

Current clamp whole cell in vivo recordings in the somatosensory cortex

2962029600295802956029540

Time (ms)

Sig

nal 00

(mV

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• 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

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/

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.