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 of Lyon UMR 5292 CNRS/U1028 INSERM , Lyon, 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 , University Claude Bernard Lyon I. Michel Jouvet, Advisor. 1990-911983 – 1984Visiting DEA (Master) scientist Neuroscience, (6 months in Universitytotal) at the Claude Department Bernard of Lyon I. MentalMichel Jouvet,Health Sciences,Advisor. 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

EEG EEG

EMG EMG

EOG EOG Paradoxical (REM) sleep

EEG

EMG

EOG

total sleep 12h50 (SWS 10h50 min - PS2h00 min) Hypnogram of 24h

PS

SWS

W

07h00 19h00 07h00 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.

SLD

The SLD generates PS

SLD: sublaterodorsal tegmental nucleus ± SubC: subcoeruleus nucleus Peri-LC: nucleus peri-LC alpha A: intact animal PIA: pontine inhibitory area B: total decortication (pontine cat) NPO: nucleus pontis oralis C: mesencephalic section 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 motoneurones 1) are tonically inhibited during PS by a pathway arising from the SLD 2) are phasically excited by a pathway (originating from the cortex?) Rem Behavior Disorder

Courtesy of Dario Arnaldi University of Genoa, IT

Mark Mahowald and Carlos Schenck 1985 REM sleep behaviour disorder (RBD): Is the motor cortex inducing the movements? Which type of neurons degenerate? SLD?

CORTICAL ACTIVATION

Motor cortex

SLD

W-on neurons PS-on neurons Inhibitory pathways

SWS-on neurons PS-off neurons Excitatory pathways MUSCLE ATONIA

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

SLD section stained with vGlut2 and CFos PS deprivation Control Mean PS quantities PS

SWS ­ Control : 14,6% W

PS Deprivation

SWS ­ Deprivation : 0%  W CFos/vGlut2 neurons Rebound PS

SWS

­ Rebound : 50,4% W

13 h 16 h

Conclusion: SLD PS-on neurons express only vGlut 2 and are glutamatergic Clement et al., 2011 Ascending and descending projections of the SLD PS-on neurons

GiV ITha

The SLD neurons activated during PS project caudally to the ventral gigantocellular nucleus (GiV) but not rostrally to the thalamic intralaminar nuclei (Itha) Neuronal circuit responsible for muscle atonia during PS

CORTICAL ACTIVATION

Motor cortex

IntralaminarIntralaminar thalamic nuclei

SLD Glutamate

GiV Gly/GABA? Spinal motoneurons

W-on neurons PS-on neurons Inhibitory pathways

SWS-on neurons PS-off neurons Excitatory pathways MUSCLE ATONIA

REM sleep behaviour disorder (RBD): SLD glutamatergic descending neurons degenerate? Inactivation of SLD glutamatergic neurons with adeno-associated virus (AAVs) carrying short-hairpin RNA (shRNA) targeting vesicular glutamate transporter 2

Control shRNA vglut2

Location with mCherry of AAVs injection sites centered on the SLD of control (n=5) and vGluT2- rats (n=5) Expression of vGluT2 mRNAs within SLD Note the lack of cell body labeling in the SLD of vGluT2- versus ctrl mice.

The new genetic method used induces a permanent inactivation of glutamatergic transmission in neurons of the SLD in ratsCollaboration with Michael Lazarus, Tsukuba, Valencia Garcia et al., Brain, 2017 Daily percentage of vigilance states and number of sleep bouts in vGluT2- vs control rats 30 days post shRNA injection.

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

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

Collaboration with Michael Lazarus, Tsukuba, Valencia Garcia et al., Brain, 2017 How SLD glutamatergic neurons generate muscle atonia? A. Control Paradoxical sleep

EEG EOG PGO EMG ControlStrychnine Membrane -60 potential B. Strychnine -70 30 s EEG EOG SWS PGO PS EMG Membrane -60 potential (Soja et al., 1991) -70 Effect of strychnine (glycine antagonist) application during PS on spinal motoneurons in cats

Glycinergic premotoneurons are hyperpolarizing motoneurones during PS Localisation of the glycine neurons specifically active during PS hypersomnia?

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

Expression of GlyT2 mRNAs in GiV and spinal cord neurons

Only the glycine neurons of the ventral gigantocellular reticular nucleus (GiV) are specifically active during PS rebound 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 The glycinergic neurons responsible for PS atonia are likely also GABAergic

Gi V

Fos/GAD « in situ »

GiV

PSC PSD PSR Sapin et al., 2009 Neuronal circuit responsible for muscle atonia during PS: role of the GABA/glycinergic neurons of the Giv?

CORTICAL ACTIVATION

Motor cortex

SLD Glutamate

GiV Gly/GABA Spinal motoneurons

W-on neurons PS-on neurons Inhibitory pathways

SWS-on neurons PS-off neurons Excitatory pathways MUSCLE ATONIA

REM sleep behaviour disorder (RBD): GiV GABA/glycinergic descending neurons degenerate? Inactivation of the vesicular GABA/glycine transporter (vGAT) in GiV neurons using transfection with adenoviral vector of short hairpin RNA targeting the vGAT mRNA

GiV GiV

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

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 In summary: The glutamatergic neurons of the SLD and the GABA/glycinergic neurons of the GiV are responsible for muscle atonia during PS Their degeneration can be responsible for RBD What is generating the movements? Motor cortex?

CORTICAL ACTIVATION

Motor cortex

IntralaminarIntralaminar thalamic nuclei

SLD GlutamateX GiV Gly/GABAX Spinal motoneurons

W-on neurons PS-on neurons Inhibitory pathways

SWS-on neurons PS-off neurons Excitatory pathways MUSCLE ATONIA Review ARticle Alternative hypothesis 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 section Adult 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 Study of the distribution and number of neurons activated in the cortex during PS hypersomnia and waking Control 10% of PS

Vigilance states PSC quantities (%) PSD 72h 6h PSR PS deprivation 0% # # #** # PS72h hypersomnia6h 32% * W SWS PS

72h 6h Immunohistochemistry Waking 100% of FOS, Arc, Cox-2 « in situ » Hybridation of Bdnf

3h Renouard et al., 2015 Distribution of FOS+ neurons in the hippocampus during PS hypersomnia

PS Hypersomnia Waking

# Number of FOS+ PSC 250 ** # neurones PSD + + 200 #+ PSR # ** **+ 150 # W # # 100 #** 50 * 0 Dentate gyrus CA3 CA1

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

PS deprivation Waking PS hypersomnia NombreNumber de neurones of FOS+ 4000 neuronsPSC + ## PSD **+ SS 3500 PSR EV

3000

2500

2000

1500

1000 * 500 # # 0Somatosens cortex somatosensorielory 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

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

mean frequency spectral values rest mov

• During tonic REM sleep the Tonic Phasic 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 De Carli et al Annals of Neurology 2016 Ictal SPECT in patients with RBD

Activation of the supplementary motorActivation region of the premotor-regions

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

Dauvilliers et al 2011; Mayer et al Brain 2015 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 Distribution of FOS+ neurones in the anterior cingulate cortex during PS hypersomnia Number of FOS+ PS deprivation Waking PS hypersomnia neurons 800 + #+ PS **# ACA 700 C

600

500

400

300 BDNF Arc 200

100 ** **

0 Anterior PS PS PS PS cortex cingulécingulate antérieur D R D R cortex Distribution of FOS+ neurons in the retrosplenial and medial entorhinal cortex during PS hypersomnia PS deprivation Waking PS hypersomnia Number of FOS+ neurons 3500 PS + RS C +# **# 3000 PS D

2500

2000

1500

1000 BDNF Arc 500 ** ** 0 Cortex rétrosplénial Retrosplenia PS PS PS PS l D R D R cortex Multiunit recordings in the rat retrosplenial cortex during the sleep- waking cycle

P W SWS S

Number of neurons Structure Pourcentages [ % ] Structure State comparison RSC State comparison RSC aWk > SWS > REM 14,3 aWk > SWS > REM 5 aWk > REM > SWS 14,3 aWk > REM > SWS 5 SWS > aWk > REM 2,9 SWS > aWk > REM 1 SWS > REM > aWk 0,0 SWS > REM > aWk 0 REM > aWk > SWS 57,1 REM > aWk > SWS 20 REM > SWS > aWk 11,4 REM > SWS > aWk 4 total 97,2 sum 35 total 36 Correlation with theta activity 68% of the retrosplenial neurons recorded are more active during PS than W and SWS in agreement with functional neuroanatomicalIn collaboration data with Brain institute of Natal, Brazil, Koike et al., J. Neuro, 2017 What is the state of the cortex during PS? Use of TRAP mice (Targeted recombination in active population)

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

RSP CLA

DG

Hippocampus

CLA

Paradoxical (REM) sleep and Waking are two completely different states Localisation of the neurons activated during PS hypersomnia (FOS+) projecting to the dentate gyrus, anterior cingulate and retrosplenial cortices Iontophoretic Injections of CTb or Fluorogold retrograde tracers Dentate gyrus Supramammillary nucleus (Sum)

Anterior Claustrum cingulate (CLA)

CA AC

Retrosplenial cortex VIS

PAG

Renouard et al., 2015 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

CORTICAL RS ACTIVATION Motor cortex ACA cortex

DG

Claustrum GLU SumL GABA/GLU SLD Glutamate GiV Gly/GABA Spinal motoneurons

PS-on neurons Inhibitory pathways

Excitatory pathways MUSCLE ATONIA Rem Behaviour Disorder physiopathology

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

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

• 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

Postuma et al. Brain 2019 In-vivo staging of pathology in REM sleep behaviour disorder

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

Knudsen et al. Lancet Neurol. 2018 In-vivo staging of pathology in REM sleep behaviour disorder

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 Thalamic MeNER binding potential (BPND; arrows) is decreased in patients with idiopathic RBD and PD indicating decreased noradrenergic innervation Putamen ¹⁸F-DOPA signal (arrows) is decreased in some idiopathic RBD, but markedly decreased in PD indicating decreased dopamine transmission

Knudsen et al. Lancet Neurol. 2018 In-vivo staging of pathology in REM sleep behaviour disorder

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. Knudsen et al. Lancet Neurol. 2018

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

Team "Physiopathology of the neuronal network of the sleep-waking cycle” Centre of Neuroscience of Lyon UMR 5292 CNRS/U1028 INSERM , Lyon, France 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 Polysomnographic recordings illustrating SWS and PS in representative control vs vGluT2- rats

Muscle tone and muscle phasic movements are present during PS in vGluT2- compared to control rats. Collaboration with Michael Lazarus, Tsukuba, Valencia Garcia et al., Brain, 2017 Localisation of ARC+, Cox2+ and Bdnf+ neurons in the hippocampus during PS hypersomnia using immunohistochemistry and « in situ » hybridization PS deprivation PS hypersomnia

Bdn f

ARC

CO X2

Granule cells of the dentate gyrus overexpress genes implicated in synaptic plasticity during PS hypersomniaRenouard et al., 2015 Distribution of FOS+ neurones in the claustrum during PS hypersomnia Number of FOS+ PS deprivation Waking PS hypersomnia neurons

120 PSC PS # D **# 100 PSR CLA

80

60

# 40 **# PS PS D R BDNF Arc 20 *

0 Claustrum PS PS PS PS D R D R Dreamed Movement Elicits Activation in the Sensorimotor Cortex (a study in lucid dreamers) e k a Hand mov. wakeImag. Hand movDreamed Hand mov w

. v o m d n a H v o m d n a H

. g a m I v o

m M

d IM n DM a H d e m a e r D

Dresler et al Current Biology 2011 Rapid eye movements (REMs) in sleep are associated with visual-like activity, as during wakefulness

visual-mnemonic responses

Andrillon et al Nature Communication 2015 Neurochemical nature of Sum neurons active during PS

FOS/vGLUT2 + FOS/vGAT + Number of neurons

SumM SumL

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

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

The Sum neurons activating dentate granule cells during PS are both GABA and glutamatergic Billwiller et al., 2017 Subcortical pathways responsible for cortical activation during PS

RSP

ACA DG

Effect of the Neurotoxic CLA lesion of the Sum on DG SUM activation during PS GLU

Sum

Powerspectrum analysis 1,6 Lesion/sham 1,4 1,2 1 0,8 Distribution of Fos+ cells 0,6 in the DG 6 6 5 5 4 4 3 3 2 .7 .3 .9 .5 .1 .7 .3 .9 .5 0 6 1 7 3 8 4 9 5 1 1 2 2 3 3 4 Activation of the sensorimotor cortex in animals Electrophysiological studies

Macaque Mice

REM sleep Motor cortex

parietal cortex

Jackson et al J Neurophysiol 2007; Brankack et al JSR 2012 BOTTOM UP MODEL

Blumberg et al Sleep Medicine Reviews 2016

TOP DOWN MODEL

Blumberg et al Sleep Medicine Reviews 2016

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

Andrillon et al Nature Communication 2015 contacts pairs localized within the Mc and the dlPFc, as confirmed by postimplantation MRI, motor evoked potentials, intracerebral electrical stimulation

Paracentral Lobule (leg)

Intracerebral Motor Evoked potentials Low Frequency Electrical (MEP) Stimulation Tonic REM sleep

MC

dlPFC

Fz-Cz

EOG

EMG Tonic REM sleep

MC

MC

dlPFC

Fz-Cz 0 5 10 15 20 25 30 Hz

EOG

EMG Phasic REM sleep

MC

dlPFC

Fz-Cz

EOG

EMG Phasic REM sleep

MC MC

dlPFC

Fz-Cz

Hz

EOG

EMG Phasic REM sleep

MC

dlPFC

Fz-Cz

EOG

EMG Tonic and Phasic REM sleep

De Carli et al Annals of Neurology 2016 Tonic and Phasic REM sleep

De Carli et al Annals of Neurology 2016 the activation of the Mc during phasic REM sleep seems to reflect a widespread involvement of the motor system

mean frequency spectral values Mc pre REMS: 19.98 +/- 0.86 Hz

Mc post tREMS: test 1.58, df 20.89 6; NS +/- 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) Activation of the motor cortex during movement in wakefulness

1 sec 1 sec

REST Movement Activation of the motor cortex during movement in wakefulness (at least 2 leg movements; range 2–10)

De Carli et al Annals of Neurology 2016 Activation of the motor cortex during movement in wakefulness (at least 2 leg movements; range 2–10)

ANOVA

De Carli et al Annals of Neurology 2016 Activation of the motor cortex during movement in wakefulness (at least 2 leg movements; range 2–10)

mean frequency spectral values

De Carli et al Annals of Neurology 2016 Activation of the motor cortex during movement in wakefulness

Miller et al PNAS 2010; Pfurtscheller et al EEG Journal 1997 Two different levels of Motor cortex activation during REM sleep?

Fz-Cz Fz-Cz

EOG EOG EMG EMG REM tonic REM phasic

Fz-Cz

EOG EMG RBD 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?

CORTICAL RS ACTIVATION

ACA

DG vlPAG Thalamus dDPMe GABA GABA DRN 5 HT LC 5 HT NA Hcrt NA PH Claustrum DPGi GLU Sum MCH/GABA GABA MS GABA MS GABA/GLU SLD GABA Glutamate TMN Glutamate HIS GiV Gly/GABA Spinal motoneurons

W-on neurons PS-on neurons Inhibitory pathways

PS-on cortical PS-off neurons Excitatory pathways MUSCLE system ATONIA Study of the SumL-DG pathway using optogenetic

Transfection of ChR2 or NpHR3.0 in AAV5 (ChR2) FERULE

OPTRODE m m

glutamatergic neurons of the Sum in 6 knock-in vGLUT2-Cre mice: GD

m Catheter 2 m

SumM Optic fiber

- Electrophysiological effect on DG SumL m 3 . m 0 - Effect on learning and memory LFP electrode

Optimal transfection of SumL Patch clamp recordings: neurons (n=10 mice) Co-transmission Strong Expression of ChR2- BlueGABA/Glutamate Light EYFP in SumL fibers localized in the DG

optrode

GD

G EPSC IPSC AMPA/NMDA GABA-A Collaboration with Monique Esclapez (Marseille) Effect on spectral composition of stimulation of the SumL-DG pathway during PS

0,3 Stimulation: 20 Hz during 15 min Perfusion after 90 min 0,25 8,3 Hz

0,2

Prestim 0,15 6,8 Hz Stim 0,1

0,05

0 EYFP/FOS double staining after 0 5 10 15 20 25 30 35 40 optogenetic stimulation theta Control 2 DG LFP 2 EEG ChR2 1,8 1,6 1,5 * 1,4 1,2 1 1 0,8 0,6 0,5 0,4 0,2 0 0 Delta Theta Gamma deltaDelta thêtaTheta lent gammaGamma 85-95 Hz (2-5delta Hz) (6-9thêta Hz) lent (65-115gamma 85-95 Hz) Hz (2-5 Hz) (6-9 Hz) (65-115 Hz) Stimulation of the SumL/DG pathway during PS induces an increase in the power and frequency of theta Billwiller et al., unpublished Effect of stimulation of the Sum-DG pathway during W

ChR2

0,2 Prestim 0,15 Stim

0,1

0,05

0 0 5 10 15 20 25 30 35 40 0 0 0 0 0 0 0 60 70 80 90 100 110 120

Stimulation of the Sum/DG pathway during W induces behavioral activation and an increase in the power and frequency of theta Function of the GABA/GLU Sum-DG pathway activated during PS?

PS CA1 PSC PSD PSR Excitation CA3 Synaptic plasticity GD GABAa

GABA Inhibition NMDA GLU

SumL

- Glutamate receptors on dendrites? - GD is implicated in pattern separation, fear conditioning GABAergic receptors on somas ? (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 novel object place recognition (NOPR)

Fig. 1. ArchT-mediated inhibition of medial septum (MS) Fig. 3. Inhibiting MS GABA neurons selectively GABA neurons during REMS reduces theta rhythm during REMS impairs memory consolidation in 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 Summary on what’s happening in the cortex during paradoxical (REM) sleep

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

Eveil Sommeil lent

Kynurenic acid Cortex

Thalamus

PS­like SWS W

GABA Hcrt Iontophoresis of bicuculline, BF kynurenic acid MCH SLD VLPO and atropine in SCN TMN head restrained rats Boissard et al., 2002 SLD PS­on neurons are activated during PS by the removal of a GABAergic input present during W and SWS What turns on GLU SLD neurons at the onset of PS? : pharmacological studies

(GABAa antagonist) PS­like Kainic acid Kainic acid 67.1 Hz

12.3 Hz 12.4 Hz

Luppi et al., 2004 What turns on the glutamatergic SLD neurons at the onset of PS? : pharmacological studies

(GABAa antagonist)

PS-like SWS W

PS induction by long iontophoretic application of bicuculline in the SLD

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

Boissard et al., 2002 500 m SLD PS­on neurons are activated during PS by the removal of a GABAergic input present during W and SWS Where are located the PS­off GABAergic neurones inhibiting the SLD PS­on neurons during waking and slow wave sleep?

vlPAG dDpMe  CTb/GAD neurons

Boissard et al., 2003

PS deprivation vlPAG: ventrolateral periaqueductal gray dDpMe: dorsal deep mesencephalic nucleus Fos/GAD neurons

Sapin et al., 2009

Conclusion: PS-off GABAergic neurons inhibiting SLD PS-on neurons during W and SWS are localized in the vlPAG/dDPMe Inhibition with GABAa agonist (muscimol) injection or DREADD of the vlPAG/ dDpMe GABAergic neurons: induction of PS

Delta (1.5­4Hz)

Theta (4.5­8.5Hz)

Sigma (9­14Hz)

Gamma (30­50Hz)

Muscle

Hypnogram Mus 90min PS SWS W 30 60 90 120 150 180 210 min

%

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

vlPAG

DRN dDPMe 4 1000µm Sapin et al., 2009 (rats) What is turning off the PS­off GABAergic neurons of the vlPAG/dDpMe at the onset of PS? PSC PSD PSR PSD

Aq

vlPAG

dDpMe 4 DR 4N

Injection of the retrograde tracer CTb in the vlPAG/dDpMe GABAergic PS-off neurons in PSC, PSD and PSR animals

Clement et al., 2012 The lateral hypothalamus area (LHA) contain PS-on neurons controlling the vlPAG/dDPMe PS-off GABAergic neurons?

Triple labeling c-FOS/CTb/MCH

LHA C-Fos MCH

CTb c-FOS/CTb c-FOS/CTb/MCH MCH: melanin concentrating hormone MCH

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) Clement et al., 2012 The tuberal hypothalamus contains a huge number of Fos/GAD double-labeled neurons after PS hypersomnia Number of cells PSD

rZI

PSC PSD PSR

mLH PSR

PSC PSD PSR

PeF

 Fos/GAD neurons PSC PSD PSR Sapin et al., , 2011 Melanin concentrating hormone (MCH) immunohistochemistry combined with GAD67 « in situ » double staining

Sapin et al., , 2011

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

Hassani et al., 2009 MCH icv injection induces PS

Verret, Goutagny et al., 2003

Optogenetic stimulation of MCH neurons induces PS

MCH is an hypnogenic factor: Treatment of insomnia?

Jego et al., 2013, Konadhode et al. 2013 Paradoxical (REM) sleep genesis

CORTICAL ACTIVATION

vlPAG Thalamus dDPMe GABA GABA DRN 5 HT LC 5 HT NA Hcrt NA PH DPGi MCH/GABA DPGi GABA SLD Glutamate TMN Glutamate HIS GiV Gly/GABA Spinal motoneurons

W-on neurons PS-on neurons Inhibitory pathways

PS-off neurons Excitatory pathways MUSCLE ATONIA Paradoxical (REM) sleep (PS) generating network

CORTICAL ACTIVATION?

vlPAG Thalamus dDPMe GABA GABA DRN 5 HT LC 5 HT NA Hcrt NA PH DPGi MCH/GABA DPGi GABA SLD Glutamate TMN Glutamate HIS GiV Gly/GABA Spinal motoneurons

W-on neurons PS-on neurons Inhibitory pathways

PS-off neurons Excitatory pathways MUSCLE ATONIA Effect of stimulation of the Sum-DG pathway using optogenetic

% awakening 100 ChR2 90 80 70 60 50 40 30 20 10 0 0,8 1 Control1,21,41,61,8ChR22 2,2 N=4 N=6

STIMULATION : 20 Hz 10 sec

Billwiller et al., unpublished GABA/glutamate neurons of the Sum activate dentate granule cells during PS, the claustrum activate the other limbic cortical structures

RSP

ACA DG

CLA

SUM GABA/G LU

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

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

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

Steriade et al., 1982

W x Corte SW S

P S Thalamu s LDT/P Basal PT Telencepha lon SLD

Hassani et al., 2009 Study of the Sum-DG pathway using optogenetic

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

Channelorhodopsin Halorhodopsin Injection of AAV in the Sum (ChR2) (eNpHR3.0) 470 nm 530 nm K+ H+ Cre

ITR EF-1 XloxP ChR2-EYFPChR2-EYFP XloxP WPRE polyA ITR

Na+ Cl- Effect of the Neurotoxic lesion of the Sum on the Dorsal DG

PS hypersomnia PSR- Sham PSR- Lésion

Sham Lesion

Disappearance of vGLUT2+ fibers in the dorsal DG vGLUT2 + Optic density PSR- Sham PSR- Lésion

Strong decrease in the number of FOS+ neurons in FOS + neurons the dorsal DG Billwiller et al., unpublished Effect of the Neurotoxic lesion of the Sum on the ventral DG

PS hypersomnia PSR- Sham PSR- Lesion

Sham Lesion

vGLUT2 + optic density No decrease of vGLUT2+ fibers PSR- Sham PSR- Lesion

FOS + neurons No decrease in the number of Fos+ neurons

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

90 min Stimulation: 20 Hz during 15 min Perfusion

Control ChR2

M

H G

GD

Optogenetic stimulation induces the expression of FOS in the dorsal GD with a distribution close to that seen after PS hypersomnia Localization of the glycinergic neurons responsible for muscle atonia

Gi V  CFos/Gly neurons C­Fos and glycine neurons in the medullary ventral C­Fos and glycine neurons in the GiV after PS gigantocellular reticular nucleus (GiV) after PS induction hypersomnia

IO

GiV neurons immunoreactive to glycine 100µm contacted by PHAL fibers from SLD neurons

Glycinergic neurons hyperpolarizing motoneurons during PS are localized in the Giv? Boissard et al., 2002 Multiunit recordings in the rat dentate gyrus during the sleep waking cycle W SWS PS

Number of neurons Structure Pourcentages [ % ] Structure State comparison DGY State comparison DGY aWk > SWS > REM 7 aWk > SWS > REM 6,3 aWk > REM > SWS 15 aWk > REM > SWS 13,4 SWS > aWk > REM 12 SWS > aWk > REM 10,7 SWS > REM > aWk 50 SWS > REM > aWk 44,6 REM > aWk > SWS 15 REM > aWk > SWS 13,4 REM > SWS > aWk 13 REM > SWS > aWk 11,6 sum 112 total 87,5 total 128 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? MCH/GABA PS-on neurons of the HLA control PS genesis? Muscimol (GABAa agonist) and clonidine (alpha2 adrenergic agonist) injections in the LHA

DPGi 1,9%

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

After muscimol injection in the LHA, the MCH and the Hrct neurons are inactivated (Fos-) Muscimol and clonidine injections in the LHA inhibit PS:

Clement et al., 2012 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

c-FOS/GAD or c-FOS/CTb double-labled neurons Clement et al., 2012 The lateral hypothalamic area control PS by means of inhibition of vlPAG/dDpMe PS-off GABAergic neurons projecting to the SLD Risk and predictors of dementia and parkinsonism in idiopathic REM sleep behaviour disorder

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).

Postuma et al. Brain 2019 Risk and predictors of dementia and parkinsonism in idiopathic REM sleep behaviour disorder

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

Postuma et al. Brain 2019 Risk and predictors of dementia and parkinsonism in idiopathic REM sleep behaviour disorder

• Standardized motor examination• Urinary symptoms • Standardized motor symptoms• Erectile dysfunction • Quantitative motor testing • Orthostatic symptoms • Olfaction • Orthostatic blood pressure • Colour vision • Cognition, neuropsychological testing • Physician-documented insomnia• Cognition, office-based diagnosis • Excessive daytime somnolence• Depression • Restless legs syndrome • Anxiety • Sleep apnoea • DAT-SPECT • REM sleep without atonia • Substantia nigra pars compacta hyperechogenicity • Constipation

Postuma et al. Brain 2019