Sleep phenotyping of the animal models of neurodegenerative diseases ...€¦ · models of neurodegenerative diseases: Focus on the REM sleep behavior disorder (RBD) Pierre-Hervé

Sleep phenotyping of the animal models of neurodegenerative

diseases: Focus on the REM sleep behavior disorder (RBD)

Pierre-Hervé Luppi, Team "Physiopathology of the neuronal network

of the sleep-waking cycle” Centre of Neuroscience of Lyon

UMR 5292 CNRS/U1028 INSERM , Lyon, France

Michel Jouvet: the father of paradoxical (REM) sleep and RBD!

16 November 1925- 3 October 2017

CNRS Research Director (full time researcher)

2011- Head of the Team “Physiopathology of the neuronal network of the sleep-waking cycle" of the Center of Neuroscience of Lyon, UMR 5292 CNRS/U1028 INSERM, Université Claude Bernard Lyon 1, France.

2002-2010: Director of the CNRS laboratory UMR5167, University Claude Bernard Lyon I. 1999 – 2001 Director of Research (DR2), CNRS, INSERM U52, University Claude Bernard Lyon I. 1989 – 1999 CR1 CNRS, INSERM U52, University Claude Bernard Lyon I. 1984 – 1989 PhD in Neurosciences, University Claude Bernard Lyon I. Michel Jouvet, Advisor.1983 – 1984 DEA (Master) Neuroscience, University Claude Bernard Lyon I. Michel Jouvet, Advisor.

1990-91 Visiting scientist (6 months in total) at the Department of Mental Health Sciences, Hahnemann University, Philadelphia, USA (Pr. ASTON-JONES), Study of the afferents to the locus coeruleus.1992 Invited scientist (6 months), at the Brain Research Institute, UCLA, Los Angeles, USA. (Dr. MICHAEL CHASE), Study of the glycinergic neurons responsible for muscle atonia of paradoxical sleep.

What is paradoxical (REM) sleep (PS)?

Waking SWS or nonREM sleep

Paradoxical (REM) sleep

Hypnogram of 24h

PS

SWS

W

07h00 19h00 07h00

EEG

EEG

EEG

EMG

EMG

EMG

EOG

EOG

EOG

total sleep 12h50 (SWS 10h50 min - PS2h00 min)

A paradoxical (REM) sleep (PS)like state with muscle atonia is still present in “pontine cats”: muscle atonia of PS is generated by a brainstem circuit

Jouvet, 1962, Arch. Ital. Biol.

A: intact animalB: total decortication (pontine cat)C: mesencephalic section

SLD

The SLD generates PS

SLD: sublaterodorsal tegmental nucleus ±SubC: subcoeruleus nucleusPeri-LC: nucleus peri-LC alphaPIA: pontine inhibitory areaNPO: nucleus pontis oralis

The lesion of SLD in cats induces PS without atonia (RWA)and REM sleep behavior disorder (RBD) like behavior:

Sastre et coll., 1979, Physiology & Behavior

These results indicate that motoneurones1) are tonically inhibited during PS by a pathway arising from the SLD2) are phasically excited by a pathway (originating from the cortex?)

Courtesy of Dario Arnaldi

University of Genoa, IT

Rem Behavior Disorder

Mark Mahowald and Carlos Schenck 1985

W-on neurons

Excitatory pathways PS-off neurons SWS-on neurons

PS-on neurons Inhibitory pathways

MUSCLEATONIA

CORTICAL ACTIVATION

Identification of the neuronal circuit responsible for muscle atonia and movements during PS is required

MotorcortexMotorcortex

SLD

REM sleep behaviour disorder (RBD):Is the motor cortex inducing the movements?Which type of neurons degenerate? SLD?

What is the Neurotransmitter of the PSon neurons of the SLD ?Glutamate, acetylcholine or GABA?

« In situ » hybridization of vesicular glutamate transporter 2 (vGlut2), GAD67 or ChAt immunohistochemistry combined with CFos immunohistochemistry after PS hypersomnia

Clement et al., 2011

SLD section stained with vGlut2 and CFos

Conclusion: SLD PS-on neurons express only vGlut 2 and are glutamatergic

CFos/vGlut2 neurons

Control

Deprivation

Rebound

W

W

W

SWS

SWS

SWS

PS

PS

PS

13 h 16 h

Mean PS quantities

Control : 14,6%

Deprivation : 0%

Rebound : 50,4%

PS deprivation

Ascending and descending projections of the SLD PS-on neurons

The SLD neurons activated during PS project caudally to the ventral gigantocellular nucleus (GiV) but not rostrally to the thalamic intralaminar nuclei (Itha)

GiV

ITha

W-on neurons



Spinalmotoneurons

MUSCLEATONIA

CORTICAL ACTIVATION

Neuronal circuit responsible for muscle atonia during PS

Intralaminar thalamic nuclei


GiVGly/GABA?

GiVGly/GABA?


SLDGlutamate

REM sleep behaviour disorder (RBD):SLD glutamatergic descending neurons degenerate?

The new genetic method used induces a permanent inactivation of glutamatergic transmission in neurons of the SLD in rats

Expression of vGluT2 mRNAs within SLD Note the lack of cell body labeling in the SLD of vGluT2- versus ctrl mice.

Location with mCherry of AAVs injection sites centered on the SLD of control (n=5) and vGluT2- rats (n=5)

Collaboration with Michael Lazarus, Tsukuba, Valencia Garcia et al., Brain, 2017

Control shRNA vglut2

Inactivation of SLD glutamatergic neurons with adeno-associated virus (AAVs) carrying short-hairpin RNA (shRNA) targeting vesicular glutamate transporter 2

The removal of vGluT2 from glutamatergic SLD neurons induces only a 30% decrease in PS quantities in vGluT2- rats

Glutamatergic neurons of the SLD are not the only neurons generating PS?

W, PS and SWS quantities (24h) Number of SWS and PS episodes

Daily percentage of vigilance states and number of sleep bouts in vGluT2- vs control rats 30 days post shRNA injection.


The inactivation of glutamate transmission of SLD neurons in rats using short hairpin RNA for vglut2 induces REM sleep without atonia (RWA) and RBD like behaviors


How SLD glutamatergic neurons generate muscle atonia?

ControlStrychnine

SWSPS

A. Control

B. Strychnine

EEGEOGPGOEMG

Membranepotential

EEGEOGPGOEMG

Paradoxical sleep

30 s

-60

-70

-60

-70(Soja et al., 1991)

Membranepotential

Glycinergic premotoneurons are hyperpolarizing motoneurones during PS

Effect of strychnine (glycine antagonist) application during PS on spinal motoneurons in cats

Localisation of the glycine neurons specifically active during PS hypersomnia?

Only the glycine neurons of the ventral gigantocellular reticular nucleus (GiV) are specifically active during PS rebound

Expression of GlyT2 mRNAs in GiV and spinal cord neurons

Glycine transporter 2 (GlyT2) is a specific marker of glycine neurons

Localisation of the neurons active during PS hypersomnia and projecting to the lumbar motoneurons

Neurons of the GiV specifically activated during PS send direct projections to lumbar motoneurons

Résultats

PSC PSD PSR

GiV

The glycinergic neurons responsible for PS atonia are likely also GABAergic

Fos/GAD « in situ »

Sapin et al., 2009

GiV

W-on neurons



Spinalmotoneurons

MUSCLEATONIA

CORTICAL ACTIVATION

GiVGly/GABA

GiVGly/GABA


Neuronal circuit responsible for muscle atonia during PS: role of the GABA/glycinergic neurons of the Giv?

REM sleep behaviour disorder (RBD):GiV GABA/glycinergic descending neurons degenerate?

SLDGlutamate

Inactivation of the vesicular GABA/glycine transporter (vGAT) in GiV neurons using transfection with adenoviral vector of short hairpin RNA targeting the vGAT mRNA

Neurons lack expression of vGAT mRNA in the RMg, GiA and GiV in shRNA injected rats

GiVGiV

Valencia Garcia et al., Nat. Com., 2018

Second model of RBD:VGAT knockout in the GiV induces PS without atonia and RBD.

Collaboration with Michael Lazarus, Tsukuba, Valencia Garcia et al., Nat. Com., 2018

W-on neurons



Spinalmotoneurons

MUSCLEATONIA

CORTICAL ACTIVATION



GiVGly/GABA

GiVGly/GABA


SLDGlutamate

In summary:The glutamatergic neurons of the SLD and the GABA/glycinergic neurons of the

GiV are responsible for muscle atonia during PSTheir degeneration can be responsible for RBD

What is generating the movements? Motor cortex?

XX

Review ARticle published: 01 November 2010 doi: 10.3389/fneur.2010.00140

Beyond dreams: do sleep-related movements contribute to brain development? Mark S. Blumberg* Department of Psychology and Delta Center, The University of Iowa, Iowa City, IA, USA

In newborn rats, motor cortex is not mature and the animals still show muscle twitches after mesencephalic sectionAdult cats with mesencephalic section still show REMs and muscle twitching

Hypothesis: A brainstem system generate the movement and the sensory feedback from the vigorous movements trigger the violent dreams of RBD without intervention of the motor cortex

Alternative hypothesis

6h

72h 6h

72h 6h

72h

Control 10% of PS

PS deprivation 0%

PS hypersomnia 32%

Waking 100%

3h

Study of the distribution and number of neurons activated in the cortex during PS hypersomnia and waking

Immunohistochemistry of FOS, Arc, Cox-2

« in situ » Hybridation of Bdnf

Renouard et al., 2015

*

Vigilance states quantities (%)

**###

#

W PSSWS

PSCPSDPSR

Distribution of FOS+ neurons in the hippocampus during PS hypersomnia

PS Hypersomnia Waking

Dentate gyrus CA3 CA10

50

100

150

200

250 PSCPSDPSRW

Number of FOS+neurones +

+

**##

*

****

**##

##

##

++

PS hypersomnia induces an increase in the number of Fos+ neurons specifically in the dentate gyrus Renouard et al., 2015

Distribution of FOS+ neurons in the somatosensory cortex during PS hypersomnia

cortex somatosensoriel0

500

1000

1500

2000

2500

3000

3500

4000 PSCPSDPSREV

Nombre de neurones FOS+

*##

++**##

PS deprivation PS hypersomniaWaking

SS

Number of FOS+

neurons

Somatosensory

cortex

Cortical activity is suppressed globally during sleep, being lowest during REM sleep

RBD-associated behaviors are reported to occur

more frequently during phasic REM sleep

Manni et al Sleep 2009; Frauscher et al 2009

De Carli et al Annals of Neurology 2016

Activation of the motor cortex during movement and in phasic REM sleep

rest mov

Tonic Phasic• During tonic REM sleep the

human Motor cortex exhibits an EEG pattern similar to the one observed during the resting state

• During phasic REM sleep the human Motor cortex exhibits an EEG pattern similar to the one observed when the Motor cortex is activated

mean frequency spectral values

Dauvilliers et al Sleep Medicine 2011; Mayer et al Brain 2015

Ictal SPECT in patients with RBD

Activation of the supplementary motor region

the neural activity generating movement during episodes of rapid eye movement sleep behaviour disorder by-passes the basal ganglia

Activation of the premotor-regions

Restoration of normal motor control in Parkinson’s disease during REM sleep

the movements during the RBD would be generated by the motor cortex and would follow the pyramidal tract bypassing the

extrapyramidal system.

Cochen De Cock et al Brain 2007

cortex cingulé antérieur0

100

200

300

400

500

600

700

800

PSC

Number of FOS+neurons

** **

**##

++

Distribution of FOS+ neurones in the anterior cingulate cortex during PS hypersomnia

ACA


Anteriorcingulate

cortexPSD

PSR

BDNF Arc

PSD

PSR

Distribution of FOS+ neurons in the retrosplenial and medial entorhinal cortex during PS hypersomnia

Cortex rétrosplénial0

500

1000

1500

2000

2500

3000

3500PSCPSD


**

****

##

++RS


PSD

PSR

BDNF Arc

PSD

PSR

Retrosplenial

cortex

Multiunit recordings in the rat retrosplenial cortex during the sleep-waking cycle

Pourcentages [ % ] Structure

State comparison RSC

aWk > SWS > REM 14,3

aWk > REM > SWS 14,3

SWS > aWk > REM 2,9

SWS > REM > aWk 0,0

REM > aWk > SWS 57,1

REM > SWS > aWk 11,4

total 97,2

Number of neurons Structure

State comparison RSC

aWk > SWS > REM 5

aWk > REM > SWS 5

SWS > aWk > REM 1

SWS > REM > aWk 0

REM > aWk > SWS 20

REM > SWS > aWk 4

sum 35

total 36

68% of the retrosplenial neurons recorded are more active during PS than W and SWS in agreement with functional neuroanatomical data

Correlation with theta activity

W SWS PS

In collaboration with Brain institute of Natal, Brazil, Koike et al., J. Neuro, 2017

TRAP method enables us to label neurons activated during two different successive periods of waking or paradoxical sleep and to study their function

What is the state of the cortex during PS? Use of TRAP mice (Targeted recombination in active population)

Few neurons are activated during PS in the cortex and they are different than those activated during waking

They are localized only in a few limbic cortical structure such as the dentate gyrus, retrosplenial, medial entorhinal and anterior cingulate cortices and claustrum

Paradoxical (REM) sleep and Waking are two completely different states

CLA

CLA

DG

RSP

Hippocampus

PAG

VIS

ACCA

Dentate gyrus

Retrosplenial cortex

Anterior cingulate

Supramammillary nucleus (Sum)

Localisation of the neurons activated during PS hypersomnia (FOS+) projecting to the dentate gyrus, anterior cingulate and retrosplenial cortices

Claustrum (CLA)

Iontophoretic Injections of CTb or Fluorogold retrograde tracers

Renouard et al., 2015

SLDGlutamate

SLDGlutamate

GiVGly/GABA

GiVGly/GABA

Excitatory pathways


Spinalmotoneurons

MUSCLEATONIA

CORTICAL ACTIVATION

ClaustrumGLU

ClaustrumGLU SumL

GABA/GLUSumL

GABA/GLU

DG

RS

ACAMotorcortexMotorcortex

What is the function of the activation of limbic cortical neurons during PS?Hypothesis:

In adults, they could generate dreams sceneries and activate downstream motor cortex

In Newborns, the cortical limbic structures would not be yet active and muscles twitches generated by a brainstem generator would help to set up the cortices

• Nearly all individuals with idiopathic REM sleep behaviour disorder will eventually develop Parkinson’s disease.

• Parkinson’s disease is a progressive neurodegenerative disorders characterised by abnormal aggregation and accumulation of the protein a-synuclein in neurons and dopamine deficiency in the nigrostriatal system

• Accumulating evidence suggests that α-synuclein aggregation is hypothesised to start in autonomic nerve terminals years before the appearance of motor symptoms, and subsequently spread via autonomic nerves to the spinal cord and brainstem

Rem Behaviour Disorder physiopathology

Risk and predictors of dementia and parkinsonism in idiopathic REM sleep behaviour disorder

Postuma et al. Brain 2019

• Mean interval between baseline evaluation and phenoconversion was 4.6 +/- 3.5 years.

• Overall phenoconversion rate of 6.25% per year

• 199 (56.5%) developed parkinsonism first

• 153 (43.5%) developed dementia first

• The average age was 66.3 +/- 8.4 and 82.5% were male.

uptake of ¹¹C-donepezil in the colon is lower

in patients with idiopathic RBD and PD than in healthy controls

indicating decreased cholinergic (including parasympathetic)

innervation of the gut

¹²³I- metaiodobenzylguanidine scintigraphy of the heart (arrow)

showing no uptake in patients with idiopathic RBD and PD measure

indicating decreased sympathetic cardiac innervation

In-vivo staging of pathology in REM sleep behaviour disorder

Knudsen et al. Lancet Neurol. 2018



Neuromelanin sensitive MRI of locus coeruleus The signal in the LC is lower in

patients with idiopathic RBD and PD indicating neurodegeneration of LC

neurons

Putamen ¹⁸F-DOPA signal (arrows) is decreased in some idiopathic RBD, but markedly decreased in PD indicating

decreased dopamine transmission

Thalamic MeNER binding potential (BPND; arrows)

is decreased in patients with idiopathic RBD and PD indicating decreased

noradrenergic innervation



Patients with idiopathic REM sleep behaviour disorder had fully developed pathology in the peripheral autonomic nervous system and the locus coeruleus, equal to that in diagnosed Parkinson’s disease. These patients also showed noradrenergic thalamic denervation, but most had normal putaminal dopaminergic storage capacity.

These findings indicate that the disease first target the periphery and the brainstem. They could help developing therapeutic strategies to halt or delay disease progression in its earliest stages

To this aim, there is a need to identify the specific populations of neurons involved using animal research.

Team "Physiopathology of the neuronal network of the sleep-waking cycle” Centre of Neuroscience of Lyon UMR 5292 CNRS/U1028 INSERM , Lyon, France

Olivier Clément, Sara Garcia Valencia, Patrice FortFrancesca Billwiller, Risa Yamazaki, Frédéric Brischoux, Christelle Peyron,and Patrice Fort

Michael Lazarus, University of Tsukuba, International Institute for Integrative Sleep Medicine, Japan

Second model of RBD:VGAT knockout in the GiV induces PS without atonia and RBD.

Collaboration with Michael Lazarus, Tsukuba, Valencia Garcia et al., Nat. Com., 2018

Muscle tone and muscle phasic movements are present during PS in vGluT2- compared to control rats.

Polysomnographic recordings illustrating SWS and PS in representative control vs vGluT2- rats


PS deprivation PS hypersomnia

Bdnf

ARC

COX2

Localisation of ARC+, Cox2+ and Bdnf+ neurons in the hippocampus during PS hypersomnia using immunohistochemistry and « in situ » hybridization

Granule cells of the dentate gyrus overexpress genes implicated in synaptic plasticity during PS hypersomniaRenouard et al., 2015

Claustrum0

20

40

60

80

100

120PSCPSDPSR


*

**

**##

##

Distribution of FOS+ neurones in the claustrum during PS hypersomnia

CLA


PSD

PSR

BDNF Arc

PSD

PSR

PSD

PSR

Dreamed Movement Elicits Activationin the Sensorimotor Cortex

(a study in lucid dreamers)

Han

d m

ov.

wake

Imag

. H

an

d m

ov

Dre

am

ed

Han

d m

ov

Dresler et al Current Biology 2011

Hand mov. wakeImag. Hand movDreamed Hand mov

MIMDM

Rapid eye movements (REMs) in sleep are associated with visual-like activity,

as during wakefulness

Andrillon et al Nature Communication 2015

visual-mnemonic responses

The Sum neurons activating dentate granule cells during PS are both GABA and glutamatergic

FOS/vGLUT2 + FOS/vGAT +

Neurochemical nature of Sum neurons active during PS

FOS/vGLUT2= 83,5% of FOS+ SumL neurons

FOS/vGAT= 80,5% ofFOS+ SumL neurons

Numberof neurons

SumMSumL

Billwiller et al., 2017

DG

RSP

ACA

CLA

0.76

6.36

11.9

5

17.5

5

23.1

4

28.7

4

34.3

3

39.9

3

45.5

20,6

0,8

1

1,2

1,4

1,6

Subcortical pathways responsible for cortical activation during PS

Sum

Powerspectrum analysis

Lesion/sham

Distribution of Fos+ cells in the DG

Effect of the Neurotoxic lesion of the Sum on DG

activation during PSSUMGLU

Macaque

Motor cortexREM sleep

parietal cortex

Mice

Activation of the sensorimotor cortex in animals

Electrophysiological studies

Jackson et al J Neurophysiol 2007; Brankack et al JSR 2012

Blumberg et al Sleep Medicine Reviews 2016

BOTTOM UP MODEL

Blumberg et al Sleep Medicine Reviews 2016

TOP DOWN MODEL

Andrillon et al Nature Communication 2015

Rapid eye movements (REMs) in sleep are associated with visual-like activity,

as during wakefulness

contacts pairs localized within the Mc and the dlPFc, as confirmed by postimplantation MRI, motor evoked potentials,

intracerebral electrical stimulation

Intracerebral Low Frequency Electrical

Stimulation

Motor Evoked potentials (MEP)

Paracentral Lobule(leg)

MC

dlPFC

Fz-Cz

EOG

EMG

Tonic REM sleep

MC

dlPFC

Fz-Cz

EOG

EMG

Tonic REM sleep

Hz

MC

0 5 10 15 20 25 30

MC

dlPFC

Fz-Cz

EOG

EMG

Phasic REM sleep

MC

dlPFC

Fz-Cz

EOG

EMG

Phasic REM sleep

Hz

MC

MC

dlPFC

Fz-Cz

EOG

EMG

Phasic REM sleep

Tonic and Phasic REM sleep


Tonic and Phasic REM sleep


the activation of the Mc during phasic REM sleep seems to reflect a widespread

involvement of the motor system

t test 1.58, df 6; NS

Mc pre REMS: 19.98 +/- 0.86 Hz Mc post REMS: 20.89 +/- 1.08 Hz


8-sec intervals preceding and following the onset of REM sequences

(Sequences of REMs, preceded by at least 10 seconds of silent EOG activity)

1 sec1 sec

REST Movement

Activation of the motor cortex during movement in wakefulness



(at least 2 leg movements; range 2–10)




ANOVA





Miller et al PNAS 2010; Pfurtscheller et al EEG Journal 1997


REM tonic REM phasic

Fz-Cz

EOG

EMG

Fz-Cz

EOG

EMG

Fz-Cz

EOG

EMG

RBD

Two different levels of Motor cortex activation during REM sleep?

SLDGlutamate

SLDGlutamate

GiVGly/GABA

GiVGly/GABA

LCNALCNA

TMNHIS

TMNHIS

Thalamus Thalamus vlPAG

dDPMeGABA GABA

W-on neurons

Excitatory pathways PS-off neurons PS-on cortical system


Hcrt PH

MCH/GABA

DRN5 HT

DRN5 HT

Spinalmotoneurons

MUSCLEATONIA

DPGiGABA

DPGiGABA

CORTICAL ACTIVATION

What is the function of the activation of limbic cortical system during PS? What is the link between the cortical system and the hypothalamic and brainstem network generating PS?

ClaustrumGLU

ClaustrumGLU Sum

GABA/GLUSum

GABA/GLU

DG

RS

ACA

MSGABAMS

GABA

Strong Expression of ChR2-EYFP in SumL fibers localized in the DG

G

GD

optrode

OPTRODE

SumM

GD

SumL

FERULE

Optic fiber

Catheter

LFP electrode

2

0.3

m

m6

mm

m

m

Optimal transfection of SumL neurons (n=10 mice)

AAV5 (ChR2)

Patch clamp recordings:Co-transmission GABA/Glutamate

Study of the SumL-DG pathway using optogenetic

Collaboration with Monique Esclapez (Marseille)

Transfection of ChR2 or NpHR3.0 in glutamatergic neurons of the Sum in

knock-in vGLUT2-Cre mice:

- Electrophysiological effect on DG- Effect on learning and memory

EPSCAMPA/NMDA

IPSCGABA-A

Blue Light

0 5 10 15 20 25 30 35 400

0,05

0,1

0,15

0,2

0,25

0,3

theta

delta thêta lent gamma 85-95 Hz0

0,5

1

1,5

2

delta thêta lent gamma 85-95 Hz0

0,20,40,60,8

11,21,41,61,8

2DG LFP EEG

Delta(2-5 Hz)

Theta (6-9 Hz)

*

Control

ChR2

Gamma(65-115 Hz)

Delta(2-5 Hz)

Theta (6-9 Hz)

Gamma(65-115 Hz)

Stimulation of the SumL/DG pathway during PS induces an increase in the power and frequency of theta

Effect on spectral composition of stimulation of the SumL-DG pathway during PS

6,8 Hz

8,3 Hz

Prestim

Stim

Billwiller et al., unpublished

EYFP/FOS double staining after optogenetic stimulation

Stimulation: 20 Hz during 15 minPerfusion after 90 min

ChR2

60 70 80 90 100 110 1200

0

0

0

0

0

0

0 5 10 15 20 25 30 35 400

0,05

0,1

0,15

0,2

Prestim

Stim

Stimulation of the Sum/DG pathway during W induces behavioral activation and an increase in the power and frequency of theta

Effect of stimulation of the Sum-DG pathway during W

CA3CA3

CA1CA1

SumLSumL

GABA

GLU

GABAa

NMDA

GD

PS

PSCPSDPSR Excitation

Synapticplasticity

Inhibition

- Glutamate receptors on dendrites?- GABAergic receptors on somas ?

Function of the GABA/GLU Sum-DG pathway activated during PS?

GD is implicated in pattern separation, fear conditioning (Deng et al., 2013)

PS deprivation: fear conditioning (Ravassard ↘ et al., 2015)

Hypothesis: activation of DG granule cells by the SumL during PS plays a role in consolidation of contextual learning and memory

Fig. 1. ArchT-mediated inhibition of medial septum (MS)

GABA neurons during REMS reduces theta rhythm

Fig. 3. Inhibiting MS GABA neurons selectively

during REMS impairs memory consolidation in NOPR

novel object place recognition (NOPR)

During development, the number of new dendritic spines in the motor cortex decreases during REM sleep while the size of the remaining spines increases

• Only a few cortical limbic structures are activated by the claustrum and the lateral supramammillary nucleus during paradoxical (REM) sleep

• The somatosensory cortices are deactivated• The medial septum GABAergic neurons drive theta during PS and play crucial in memory consolidation

• In the motor cortex, a large number of new dendritic spines prune and the remaining ones grow during REM sleep during development and learning

Summary on what’s happening in the cortex during paradoxical (REM) sleep

GABAHcrt

VLPOMCH

EveilSommeil lent

Thalamus

Cortex

TMNSCN

BF

Boissard et al., 2002

SLD

What turn on SLD neurons at the onset of PS? : pharmacological studies

Kynurenic acid

PSlikeSWSW

SLD PSon neurons are activated during PS by the removal of a GABAergic input present during W and SWS

Iontophoresis of bicuculline, kynurenic acid and atropine in head restrained rats

PSlike

12.3 Hz 12.4 Hz

67.1 Hz

Luppi et al., 2004

Kainic acidKainic acid

(GABAa antagonist)

What turns on GLU SLD neurons at the onset of PS? : pharmacological studies

PS induction by long iontophoretic application of bicuculline in the SLD

500 m

LDTscp

SLD

Localization of the neurons immunoreactive to CFos in the SLD after 90 min of bicuculline (GABAa antagonist) injection


PS-likeSWSW

SLD PSon neurons are activated during PS by the removal of a GABAergic input present during W and SWS

(GABAa antagonist)

What turns on the glutamatergic SLD neurons at the onset of PS? : pharmacological studies

Where are located the PSoff GABAergic neurones inhibiting the SLD PSon neurons during waking and slow wave sleep?


dDpMe

PS deprivation

Fos/GAD neurons

CTb/GAD neurons

Sapin et al., 2009

vlPAG

Conclusion: PS-off GABAergic neurons inhibiting SLD PS-on neurons during W and SWS are localized in the vlPAG/dDPMe

vlPAG: ventrolateral periaqueductal graydDpMe: dorsal deep mesencephalic nucleus

Delta(1.54Hz)

Theta(4.58.5Hz)

Sigma(914Hz)

Gamma(3050Hz)

Muscle

Hypnogram

30 60 90 120 150 180 210 minW

SWSPS

Mus 90min

Aq

4

DRN

1000µm

dDPMe

Hayashi et al., 2015 (vGAT-CRE mice)

Sapin et al., 2009 (rats)

Inhibition with GABAa agonist (muscimol) injection or DREADD of the vlPAG/ dDpMe GABAergic neurons: induction of PS

%

vlPAG

PSC PSD PSR PSD

Injection of the retrograde tracer CTb in the vlPAG/dDpMe GABAergic PS-off neurons in

PSC, PSD and PSR animals

Aq

4N

vlPAG

dDpMe DR

What is turning off the PSoff GABAergic neurons of the vlPAG/dDpMe at the onset of PS?

4


c-FOS/CTb

MCHc-FOS/CTb/MCH

The lateral hypothalamus area (LHA) contain PS-on neurons controlling the vlPAG/dDPMe PS-off GABAergic neurons?

The LHA contains 12.2% of the CTb/c-Fos double-labeled neurons in PSR animals after CTb injection in the vlPAG/dDPMe

44% of these neurons express melanin concentrating hormone (MCH)

MCH: melanin concentrating hormone

C-Fos MCH

CTb

Triple labeling c-FOS/CTb/MCH


LHA

mLH

PSC PSD PSR

PeF

PSC PSD PSR

PSD

PSR

rZI

PSC PSD PSR

The tuberal hypothalamus contains a huge number of Fos/GAD double-labeled neurons after PS hypersomnia

Numberof cells

Fos/GAD neurons

Sapin et al., , 2011

Approximately 1/3 of the GABAergic neurons active during PS located in the tuberal hypothalamus express MCH peptide

Melanin concentrating hormone (MCH) immunohistochemistry combined with GAD67 « in situ » double staining

Sapin et al., , 2011

Hypothalamic MCH neurons are active during PS

Hassani et al., 2009

MCH icv injection induces PS

Verret, Goutagny et al., 2003

MCH is an hypnogenic factor:Treatment of insomnia?

Optogenetic stimulation of MCH neurons induces PS

Jego et al., 2013, Konadhode et al. 2013

SLDGlutamate

SLDGlutamate

GiVGly/GABA

GiVGly/GABA

LCNALCNA

TMNHIS

TMNHIS

Thalamus Thalamus vlPAG

dDPMeGABA GABA

W-on neurons

Excitatory pathways PS-off neurons


Hcrt PH

MCH/GABA

DRN5 HT

DRN5 HT

Spinalmotoneurons

MUSCLEATONIA

DPGiGABA

DPGiGABA

CORTICAL ACTIVATION

Paradoxical (REM) sleepgenesis

SLDGlutamate

SLDGlutamate

GiVGly/GABA

GiVGly/GABA

LCNALCNA

TMNHIS

TMNHIS

ThalamusThalamusvlPAG

dDPMeGABA GABA

W-on neurons

Excitatory pathways PS-off neurons


Hcrt PH

MCH/GABA

DRN5 HT

DRN5 HT

Spinalmotoneurons

MUSCLEATONIA

DPGiGABA

DPGiGABA

CORTICAL ACTIVATION?

Paradoxical (REM) sleep (PS)generating network

ChR2% awakening

0,8 1 1,21,41,61,8 2 2,20

102030405060708090

100

ControlN=4

ChR2N=6

Effect of stimulation of the Sum-DG pathway using optogenetic

STIMULATION : 20 Hz 10 sec


SUMGABA/G

LU

DG

RSP

ACA

CLA

GABA/glutamate neurons of the Sum activate dentate granule cells during PS, the claustrum activate the other limbic cortical structures

These results provide evidence for the first time that selective populations of neurons

from the dentate gyrus and a subset of limbic cortices are activated and display

synaptic plasticity during REM sleep. They pave the way for the identification of the

function of REM sleep by means of behavioral and electrophysiological studies combined

with local inactivation of the subcortical pathways revealed.

Functional studies in Human: Cortical activity during PS is close but not identical to Waking

Hypoactive regions during PS: dorso-lateral prefrontal, posterior cingulate and inferior parietal cortices (Maquet et al., 1996)

Limbic region more active during PS : amygdala, hippocampus, anterior cingulate cortex … (Maquet et al., 1996)

Cortex

ThalamusBasal

Telencephalon

LDT/PPT

SLD

Steriade et al., 1982

W

SWS

PS

Hassani et al., 2009

Mechanisms of cortical activation during PS

ChR2-EYFPChR2-EYFP

Injection of AAV in the Sum

EF-1ITR WPRE ITRpolyAloxP

Cre

XNa+

K+

Cl-

H+

Channelorhodopsin (ChR2)

Halorhodopsin (eNpHR3.0)

470 nm 530 nm

loxP

Transfection of ChR2 and NpHR3.0 in glutamatergic neurons of the Sum in

knock-in vGLUT2-Cre mice:

- Electrophysiological effect on DG- Effect on learning and memory

X

Study of the Sum-DG pathway using optogenetic

Strong decrease in the number of FOS+ neurons in the dorsal DG

vGLUT2 +Optic density

ShamLesion

FOS +neurons

PSR- Sham PSR- Lésion

PSR- Sham PSR- Lésion

Disappearance of vGLUT2+ fibers in the dorsal DG

Effect of the Neurotoxic lesion of the Sum on the Dorsal DG

PS hypersomnia


No decrease in the number of Fos+ neurons

ShamLesion

FOS +neurons

vGLUT2 + optic density

PSR- Sham PSR- Lesion

No decrease of vGLUT2+ fibers

PSR- Sham PSR- Lesion

Effect of the Neurotoxic lesion of the Sum on the ventral DG

PS hypersomnia

The suprammammillary nucleus projection induces Fos expression in dorsal but not in ventral dentate

gyrus neurons

Origin of the activation of the ventral DG during PS

The medial enthorinal cortex projection induces Fos expression in the ventral dentate gyrus neurons

Optogenetic stimulation induces the expression of FOS in the dorsal GD with a distribution close to that seen after PS

hypersomnia

GD

HG

M

Control ChR2

Stimulation: 20 Hz during 15 min Perfusion90 min

Effect of stimulation of the Sum-DG pathway on Fos expression in the DG

IO

100µmGiV neurons immunoreactive to glycine contacted by PHAL fibers from SLD neurons

Localization of the glycinergic neurons responsible for muscle atonia

CFos and glycine neurons in the GiV after PS induction

CFos/Gly neurons


GiV

Glycinergic neurons hyperpolarizing motoneurons during PS are localized in the Giv?

CFos and glycine neurons in the medullary ventral gigantocellular reticular nucleus (GiV) after PS hypersomnia

Multiunit recordings in the rat dentate gyrus during the sleep waking cycle

Number of neurons Structure

State comparison DGY

aWk > SWS > REM 7

aWk > REM > SWS 15

SWS > aWk > REM 12

SWS > REM > aWk 50

REM > aWk > SWS 15

REM > SWS > aWk 13

sum 112

total 128

Pourcentages [ % ] Structure

State comparison DGY

aWk > SWS > REM 6,3

aWk > REM > SWS 13,4

SWS > aWk > REM 10,7

SWS > REM > aWk 44,6

REM > aWk > SWS 13,4

REM > SWS > aWk 11,6

total 87,5

55% of the DG neurons recorded are more active during SWS than PS and W while only 25% are more active during PS than W and SWS: granule cells?

W SWS PS

MCH/GABA PS-on neurons of the HLA control PS genesis?Muscimol (GABAa agonist) and clonidine (alpha2 adrenergic

agonist) injections in the LHA

DPGi1,9%

After muscimol injection in the LHA, the MCH and the Hrct neurons are inactivated (Fos-)

c-FOS/Hcrt or c-FOS/MCH neurons

Muscimol and clonidine injections in the LHA inhibit PS:


The activity of LHA neurons is necessary for PS to occur

By which mechanisms LHA MCH neurons control PS:C-Fos/GAD and C-FOS/CTb double labeling in the vlPAG/dDPMe

The lateral hypothalamic area control PS by means of inhibition of vlPAG/dDpMe PS-off GABAergic neurons projecting to the SLD

c-FOS/GAD or c-FOS/CTb double-labled neurons Clement et al., 2012



The rate of phenoconversion was significantly increased with:1) abnormal quantitative motor testing (HR = 3.16)2) objective motor examination (HR = 3.03)3) olfactory deficit (HR = 2.62)4) mild cognitive impairment (HR = 2.37)5) erectile dysfunction (HR = 2.13)6) motor symptoms (HR = 2.11), 7) abnormal DAT scan (HR = 1.98)8) colour vision abnormalities (HR = 1.69)9) constipation (HR = 1.67)10)REM atonia loss (HR = 1.54)11)Age (HR = 1.54).



No significant predictive value of:• Sex• Daytime somnolence• Insomnia• Restless legs syndrome• Sleep apnoea• Urinary dysfunction• Orthostatic symptoms• Depression• Anxiety• Hyperechogenicity on substantia nigra ultrasound


• Standardized motor examination• Standardized motor symptoms• Quantitative motor testing• Olfaction• Colour vision• Physician-documented insomnia• Excessive daytime somnolence• Restless legs syndrome• Sleep apnoea• REM sleep without atonia• Constipation

• Urinary symptoms• Erectile dysfunction• Orthostatic symptoms• Orthostatic blood pressure• Cognition, neuropsychological testing• Cognition, office-based diagnosis• Depression• Anxiety• DAT-SPECT• Substantia nigra pars compacta hyperechogenicity


Documents

Sleep phenotyping of the animal models of neurodegenerative diseases ...€¦ · models of neurodegenerative diseases: Focus on the REM sleep behavior disorder (RBD) Pierre-Hervé