On the role of pontine cholinergic neurons in the modulation of rem sleep and its respiratory...
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Transcript of On the role of pontine cholinergic neurons in the modulation of rem sleep and its respiratory...
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.