On the Role of Pontine Cholinergic Neurons

in the Modulation of REM sleep and its

Respiratory Phenotype

KP Grace, H Liu, RL HornerDepartments of Medicine and Physiology,

University of Toronto

REM sleepNon-REM sleep

Genioglossus EMG

EEG

Diaphragm EMG

Introduction

Respiratory control network

REM sleep control network

Respiratory phenotype of REM

sleep

Sleep results in fundamental changes in respiratory muscle activity and control mechanisms, changes that can predispose individuals to disordered breathing during sleep. Genioglossus

muscle

Diaphragm muscle

adapted from ref. 4

adapted from ref. 1

Genioglossus muscleMaintains open airway for effective ventilation

ACh

ACh

To diaphragmPrimary respiratory pump

muscle

Pedunculopntine Tegmental (PPT) Nucleus

ACh

Sublaterodorsal region of the pons

Hypoglossal Motor Nucleus (HMN)Rostral ventral lateral

medulla

The cholinergic cell population of the pedunculopontine tegmental (PPT) nucleus fulfills these criteria by having:

(i) A population of neurons maximally active during REM sleep

(ii) Afferent and efferent connections to the site of REM sleep generation in the pons

(iii) Direct cholinergic projections to regions of the medulla containing respiratory neurons and motor neurons

IntroductionM

ean

dis

char

ge/s

ec

Active

Wake

Quiet

Wake

Non-

REMREM

REM with

twitching

adapted from ref. 2

Objective & Hypotheses

To determine whether the cholinergic REM sleep-active cell population of the PPT

nucleus is a necessary component of the circuitry responsible for the

generation of REM sleep and its respiratory phenotype

(i) ↑ endogenous acetylcholine at the hypoglossal motor nucleus ought to suppress genioglossus muscle activity

(ii) Targeted inhibition of REM sleep-active cholinergic neurons in the PPT nucleus ought to change:

(a) respiratory rate, diaphragm, and genioglossus muscle activities

consistent with the PPT having a necessary role in producing the respiratory phenotype of REM sleep.

(b)The normal temporal expression of REM sleep

Consistent with the PPT having a necessary role in modulating the state of REM sleep

EEG

DiaphragmNeck muscle

EEG Neck EMG

Genioglossus EMG

Diaphragm EMG

Hypoglossal Motor nucleus

Genioglossus muscle

adapted from ref. 3

PPT nucleus

(i) The hypoglossal motor nucleus (unilateral) & perfused with:

(A)Artificial cerebral spinal fuild (ACSF)

(B)Eserine dissolved in ACSF (100µM)Increases endogenous acetylcholine levels

(ii) The PPT nuclei (bilateral)& perfused with:

(A)Artificial cerebral spinal fuild (ACSF)

(B)8-OH-DPAT dissolved in ACSF (10µM)Produces targeted inhibition of cholinergic REM sleep-active cells 2

or

On the day of the experiment, microdialysis probes were inserted into either the:

MethodsM

ean

dis

char

ge/s

ec

Active

Wake

Quiet

Wake

Non-

REMREM

REM with

twitching

PPT Wake/REM sleep-active cell population

Me

an d

isch

arge

/se

c

Active

Wake

Quiet

Wake

Non-

REMREM

REM with

twitching

PPT REM sleep-active cell population

adapted from ref. 2

Genioglossus EMG

EEG

Neck EMG

5 sec

ACSF Eserine ACSF EserineACSF Eserine

Non-REM sleep REM sleepAwake

Diaphragm EMG

Amplitude(Au)

0

2

4

6

8

10

12

14

Wake Non-REM REM

Sleep-wake States

Respiratory-Related Genioglossus Activity

ACSFEserine

* Denotes significant effect of drug independent of state

*

* *

Amplitude (Au)

0

10

20

30

40

50

60

REM Sleep Specific Genioglossus Muscle Twitching

ACSFEserine

*

Enhanced Cholinergic activity at the Hypoglossal Motor Nucleus (n=10)

500µm

Microdialysis probe site

Genioglossus EMG

EEG

Neck EMG

Diaphragm EMG

ACSF 8-OH-DPAT ACSF 8-OH-DPATACSF 8-OH-DPAT

Non-REM sleep REM sleepAwake

Targeted inhibition of Cholinergic REM sleep-active cells at the PPT nuclei

5 sec500µm

Microdialysis probe site

0

5

10

15

20

25

70

75

80

85

90

95

100

Am

plit

ud

e(A

u)

Wake Non-REM REM

Sleep-wake States

Respiratory-Related Genioglossus Activity

Am

plit

ud

e (

Au

)

REM Sleep Specific Genioglossus Muscle Twitching

Respiratory Rate

Rat

e (

Bre

ath

s/m

in)

Wake Non-REM REM

Sleep-wake States

ACSF8-OH-DPAT

ACSF8-OH -DPAT

*

* Denotes significant effect of drug independent of state

* **

*

*

*

* Denotes significant effect of drug independent of state

0

20

40

60

80

100

120

140 ACSF8-OH -DPAT

500µm

PPT Cholinergic cells(NADPH diaphorase +)

(n=11)

ACSF

8-OH-DPAT

Targeted inhibition of Cholinergic REM sleep-active cells at the PPT nuclei (n=20)

W

N

R

W

N

R

10min

0

10

20

30

40

50

60

30

35

40

45

50

55

25

30

35

40

45

50

10

15

20

35

30

35

0

5

10

15

20

25

ACSF 8-OH-DPAT

ACSF 8-OH-DPAT

65

70

75

80

85

90

ACSF 8-OH-DPATACSF 8-OH-DPAT

% of TotalRecording

Time

% of TotalSleep Time

(tra

nsi

tio

ns/

hr)

Sleep-Wake State Transitions

Awake Non-REM sleep REM sleep

*

*

*

*

* *

Denotes significant difference relative to control

NOTE: Data from sham group not shown. Changes due

to time do not account for the changes resulting from

8-OH-DPAT treatment

*

Accounts for more than 50% of the

total increase in sleep time

Conclusions

• Increased endogenous acetylcholine at the hypoglossal motor nucleus suppresses genioglossus muscle activity in wakefulness and sleep

• This effect of increased acetylcholine may contribute to the major suppression of genioglossus muscle activity in REM sleep, since the hypoglossal motor nucleus is innervated by cholinergic cells of the PPT nucleus, a subset of which are maximally active during REM sleep.

• Selective inhibition of cholinergic REM sleep-active neurons in the PPT region alleviated suppression of genioglossus activity and respiratory rate across sleep-wake states.

• Therefore PPT REM sleep-active neurons are a necessary component of the circuitry responsible for shaping the phenotype of breathing in wakefulness and sleep.

Conclusions

•Fragmentation of sleep-wake state architecture, reduced wakefulness, and increased REM sleep following inhibition of REM sleep-active neurons in the PPT region shows that this cell population performs essential functions within the neural network controlling vigilance state.

• PPT REM sleep-active neurons act to restrain entry into REM sleep, contribute to the generation of wakefulness, and stabilize activity in state switching circuitry thereby limiting the frequency of state transitioning.

• These results are in opposition with long-standing models of REM sleep generation which regard the PPT REM sleep-active cholinergic cell population to be an integral part of that which generates REM sleep.

•This study provides the most definitive evidence to date regarding the role of this specific cell population within the REM sleep control network and therefore current models ought to be amended to reflect these findings.