NEUR30002

Neurophysiology
Respiratory system: Rhythm of life
Foundations of respiratory system

Dr Angelina Fong
angelina.fong@unimelb.edu.au
Department of Physiology
School of Biomedical Sciences
 WARNING

This material has been reproduced and communicated to you

by or on behalf of the University of Melbourne in accordance
with section 113P of the Copyright Act 1968 (Act).

The material in this communication of this material by you

may be the subject of copyright protection under the Act.
 Learning objectives
By the end of this video, you should be able to:
1.Describe and explain the mechanics of breathing
2.Describe the 3 phases of respiratory rhythm

Department of Physiology, School of Biomedical Sciences

 Breathing:
“the act of taking air into your lungs and releasing it” Cambridge Advanced Learner's Dictionary

Department of Physiology, School of Biomedical Sciences

 Why do you need to breath?
Getting O2 …
Ventilation
Gas exchange:
Pulmonary diffusion • Get Oxygen
• Remove waste (CO2)

Maintain Blood pH
Circulation
Oxygen
cascade Match metabolism

Tissue Temperature regulation

diffusion
Tissue
metabolism
Department of Physiology, School of Biomedical Sciences
 Why do you need to breath?
Getting O2 …
Ventilation

Pulmonary diffusion
Carefully coordinated
interaction between the
respiratory and
Circulation cardiovascular system
Oxygen (autonomic nervous
cascade system)
Tissue
diffusion
Tissue
metabolism
Department of Physiology, School of Biomedical Sciences
 Breathing is continuous and automatic
Inspiration

Inspiration
VT

… must be a simple event

Respiration is not a simple event

But influenced and modulated continuously

Department of Physiology, School of Biomedical Sciences
 Breathing is continuous and automatic
Inspiration

Inspiration
VT

Breathing is continuously modulated

http://123ery.deviantart.com/

Conscious control
(feed forward) Swallowing Vocalisation

Department of Physiology, School of Biomedical Sciences

 Muscles of respiration
Muscle Innervation
Inspiration
Phrenic nerve
Diaphragm
Thoracic Intercostal
External intercostals
nerves
Genioglossus Hypoglossal nerve
Laryngeal abductor Recurrent laryngeal
muscles nerve

Expiration
Laryngeal adductor Recurrent laryngeal
muscles nerve
Thoracic Intercostal
Internal intercostals
nerves
Abdominal muscles Abdominal nerves

Department of Physiology, School of Biomedical Sciences Del Negro, et al., 2018. Nature Rev. Neurosci. 19:351–367
 Respiration is a 3 phase event In mammals
airflow
constriction
in
Subglottal
Pressure
pressure (mmHg) pressure (mmHg) dilation
Intra-alveolar

Recurrent
Laryngeal Branch of the vagus nerve
Intra-plural

Nerve

Phrenic
Volume of air

Nerve
moved (mL)

n
tio
ira
Diaphragm

sp
Insp

In
Department of Physiology, School of Biomedical Sciences
 Respiration is a 3 phase event In mammals
airflow
constriction
in
Subglottal
Pressure
pressure (mmHg) pressure (mmHg) dilation
Intra-alveolar

Recurrent
Laryngeal
Intra-plural

Nerve

Phrenic
Volume of air

Nerve
moved (mL)

n
tio

n
ira

tio
Diaphragm

sp

in st-
ira
Insp Post

In

Po
-Insp

sp
Department of Physiology, School of Biomedical Sciences
 Respiration is a 3 phase event In mammals
airflow
constriction
in
Subglottal
Pressure
pressure (mmHg) pressure (mmHg) dilation
Intra-alveolar

Recurrent
Laryngeal
Intra-plural

Nerve

Phrenic
Volume of air

Nerve
moved (mL)

n
tio

tio
n
ira

ra
tio
Diaphragm

pi
sp

in st-
ira
Insp Post Late

ex
In

Po
-Insp E

sp

te
La
Department of Physiology, School of Biomedical Sciences
 What do you need to know
1. The pressure and airways changes in the three phases of respiratory rhythm
2. The nerves and muscles involved in the three phases of respiratory rhythm (as much
detail as was described in the lecture)

Department of Physiology, School of Biomedical Sciences

NEUR30002
Neurophysiology
Respiratory system: Rhythm generation
The hunt for the source of the rhythm

Dr Angelina Fong
angelina.fong@unimelb.edu.au
Department of Physiology
School of Biomedical Sciences
 WARNING

This material has been reproduced and communicated to you

by or on behalf of the University of Melbourne in accordance
with section 113P of the Copyright Act 1968 (Act).

The material in this communication of this material by you

may be the subject of copyright protection under the Act.
 Learning objectives
By the end of this video you should be able to
1. Describe the initial identification of the pre-Bötzinger complex as a potential site of
respiratory rhythm generation

Department of Physiology, School of Biomedical Sciences

 Muscles of respiration
Muscle Innervation
Inspiration
Phrenic nerve
Diaphragm
Thoracic Intercostal
External intercostals
nerves
Genioglossus Hypoglossal nerve
Laryngeal abductor Recurrent laryngeal
muscles nerve

Expiration
Laryngeal adductor Recurrent laryngeal
muscles nerve
Thoracic Intercostal
Internal intercostals
nerves
Abdominal muscles Abdominal nerves

Department of Physiology, School of Biomedical Sciences Del Negro, et al., 2018. Nature Rev. Neurosci. 19:351–367
 Ventral respiratory group (VRG)
Source of respiratory rhythm?
Neonatal rodent brainstem-spinal cord preparation

Brainstem regions produce synchronised

respiratory output to the spinal cord
Pons
XII
Hypoglossal nerve

VRG
VRG

Medulla
Hypoglossal C4
nerve
(CN XII)
Phrenic nerve
Cervical
spinal
cord PMN
External Intercostals
Phrenic Muscles (inspiratory)
nerve
Abdominal
PMN = Phrenic motor neurons Muscles
Smith et al., 1990. J. Neurophys. 64: 1149 - 1169
(expiratory)
Department of Physiology, School of Biomedical Sciences
 Ventral respiratory group (VRG)
Source of respiratory rhythm?
Neonatal rodent brainstem-spinal cord preparation

Which brainstem region is the source of

respiratory rhythm?
Pons
XII
Hypoglossal nerve

VRG
VRG

Medulla
Hypoglossal C4
nerve
(CN XII)
Phrenic nerve
Cervical
spinal
cord PMN
External Intercostals
Phrenic Muscles (inspiratory)
nerve
Abdominal
PMN = Phrenic motor neurons Muscles
Smith et al., 1990. J. Neurophys. 64: 1149 - 1169
(expiratory)
Department of Physiology, School of Biomedical Sciences
 pre-Bötzinger complex – the source of respiratory rhythm?
Modified from Alheid et al., Resp Physiol. Neurobiol., 2004
Smith et al., 1991,Science, 254: 726 - 729

Whole cell recording

DRG of Pre-BötC neurone
NTS

CVLM
RVLM

BötC rVRG
cVRG
Pre-BötC

rostral caudal

Hypoglossal nerve

Pre-BötC is sufficient to generate

respiratory rhythm
Department of Physiology, School of Biomedical Sciences
 Hypothesis:
Pre-BötC neurones are essential to respiratory rhythmogenesis.

1. If this hypothesis is true:

2. then what would happen if you destroy Pre-BötC neurones?

Department of Physiology, School of Biomedical Sciences

 Click here to access Poll Everywhere. (Does not require live presentation)
Department of Physiology, School of Biomedical Sciences
 What you need to know
1. Process involved in the identification of the pre-Botzinger Complex as the
potential respiratory rhythm generator

Department of Physiology, School of Biomedical Sciences

NEUR30002
Neurophysiology
Respiratory system: Rhythm generation
The Pre-Bötzinger Complex

Dr Angelina Fong
angelina.fong@unimelb.edu.au
Department of Physiology
School of Biomedical Sciences
 WARNING

This material has been reproduced and communicated to you

by or on behalf of the University of Melbourne in accordance
with section 113P of the Copyright Act 1968 (Act).

The material in this communication of this material by you

may be the subject of copyright protection under the Act.
 Learning objectives
By the end of this video you should be able to
1. Describe the processes that resulted in the identification of the pre-Bötzonger
complex as a rhythmogenic site
2. Describe and explain the importance of the pre-Bötzinger complex in the generation
of respiratory rhythm

Department of Physiology, School of Biomedical Sciences

 Identity of pre-Bötzinger complex neurones

Pre-BötC are:
Functionally • Pre-inspiratory neurones
(electrophysiological) or early Inspiratory
• Little to no expiratory neurones
Extracellular
recording

Phrenic nerve
recording

NAm

Pre-BötC

RVLM /
CVLM
Biotin =
recorded neurone NK1R
Department of Physiology, School of Biomedical Sciences Guyenet & Wang, 2001. J. Neurophysiol. 86:438-446.
 Identity of pre-Bötzinger complex neurones Chemical marker
Pre-BötC are:
• No unique markers
NAm • Glutamatergic
Pre-BötC • Subset that is glycinergic
• NK1-receptor expressing
RVLM /
CVLM • Somatostatin (SST) expressing
• Dbx-1 (transcription factor) expressing

NK1-receptor expressing
Gray PA et al., 2001. Nature Neuroscience, 4: 927-930.
Fong & Potts, 2006. J. Physiol. 575: 869-885.

Biotin =
Stornetta et al., 2003. J. Comp. Neurol. 455:499-512
recorded neurone NK1R
Department of Physiology, School of Biomedical Sciences Guyenet & Wang, 2001. J. Neurophysiol. 86:438-446.
 Neurokinin 1 receptors (NK1-R)
1.Endogenous ligand = substance P
2.G-protein coupled receptor
3.NK1-R internalised following stimulation by ligand
(substance P)
Attached ligand also internalised

Mantyh et al.,1995. Science. 268:1629-32.

Department of Physiology, School of Biomedical Sciences

 Saporin (SAP)

• Ribosomal inactivating protein (RIP)

• Inhibits protein synthesis
• cell death

• Cells selectivity through TA

• Require chaperon to enter cell

ZAP = SAP
TA (Targeting agent) = Substance P
Department of Physiology, School of Biomedical Sciences
 pre-Bötzinger complex – necessary for respiratory rhythm
Selective lesion of > 80% of NK1 receptor expressing cells in Pre-BötC using Saporin-Substance P
sigh

Control eupnea

Gray PA et al., 2001. Nature Neuroscience, 4: 927-930.

apnea
SAP-SP

Ipeak 1.0

5ss
Gray PA et al., 2001. Nature Neuroscience, 4: 927-930.

NK1-R expressing cells in Pre-BötC is a required for

normal breathing rhythm generation

Department of Physiology, School of Biomedical Sciences Saporin = ribosome-inactivating protein (RIP)

 pre-Bötzinger complex – necessary for respiratory rhythm

Viral delivery of SST promoter driving

expression of EGFP and Allatostatin receptor.

SST-EGFP AAV2–infected pre-BötC neurons

(green)

Allatostatin = insect peptide

Department of Physiology, School of Biomedical Sciences Tan et al., 2008. Nature Neuroscience, 11: 538-540.
 pre-Bötzinger complex – necessary for respiratory rhythm

Allatostatin = insect peptide

AlstR = allatostatin receptor Tan et al., 2008. Nature Neuroscience, 11: 538-540.

Department of Physiology, School of Biomedical Sciences

 What you need to know
1. Pre-BötC neurones are
Can be identified and characterised functionally and chemically.
There are intrinsic bursting properties of pre-Bötzinger complex neurons
2. Destruction or transient silencing of pre-BötC neurons results in apnea or
altered breathing

Department of Physiology, School of Biomedical Sciences

NEUR30002
Neurophysiology
Respiratory system: Properties of the
pre-Bötzinger Complex

Dr Angelina Fong
angelina.fong@unimelb.edu.au
Department of Physiology
School of Biomedical Sciences
 WARNING

This material has been reproduced and communicated to you

by or on behalf of the University of Melbourne in accordance
with section 113P of the Copyright Act 1968 (Act).

The material in this communication of this material by you

may be the subject of copyright protection under the Act.
 Learning objectives
By the end of this video you should be able to
1. Describe the characteristics of the neurones in the pre-Bötzinger complex
2. Explain how these characteristics of neurones enable the rhythmogenic properties of
the pre-Bötzinger complex

Department of Physiology, School of Biomedical Sciences

 Properties of pre-Bötzinger neurones
CNQX, APV, BIC & STR =
synaptic blockade

Intrinsic bursting persists

CNQX, APV, BIC & STR =

synaptic blockade Ril = riluzole
(blocks persistent sodium current)

Intrinsic bursting lost

Persistent sodium currents

(INaP) important in generation of
Del Negro et al., 2005. J. Neurosci. 25: 446-453.

Department of Physiology, School of Biomedical Sciences

bursts in pre-BötC
 Properties of pre-Bötzinger neurones

Block ICaN = smaller intrinsic burst

Block ICaN + INaP all activity lost

Ca2+ activated nonselective

cationic current AND persistent
sodium currents (INaP) important
in generation of bursts in pre-
BötC

Question:
Can you explain why there addition
of SP (substance P) produces a small
return of bursts?
FFA = flufenamic acid blocks Ca2+ activated nonselective cationic current (ICaN)
Del Negro et al., 2005. J. Neurosci. 25: 446-453.

Department of Physiology, School of Biomedical Sciences

 Respiratory rhythm is an emergent property

Kam, Worrell et al., 2013, J Neurosci. 33: 3332-3338.

Department of Physiology, School of Biomedical Sciences
 Human pre-Bötzinger Complex
Human

SOM = somatostatin

Rat

NAm

Pre-BötC

RVLM /
CVLM
IO

Department of Physiology, School of Biomedical Sciences Schwarzacher et al., 2011. Brain. 134: 24-35.
 Human pre-Bötzinger Complex
MSA =
multiple system atrophy

SCA3 =
spinocerebellar ataxia type 3 brains

Schwarzacher et al., 2011. Brain. 134: 24-35.

https://academic.oup.com/brain/article/
134/1/24/294932
Department of Physiology, School of Biomedical Sciences
 What would you expect to happen here:
A subset of pre-Bötzinger neurones are found to express μ-opioid receptors
• μ-opioid receptors is associated with Gαi.
• You are recording from pre-Bötzinger pre-inspiratory neurones in the slice
• You add a μ-opioid receptor agonist
• What would you predict would 

happen to:
Q1: Membrane potential of the neurone?
Q2: Respiratory rhythm?

Department of Physiology, School of Biomedical Sciences

 Click here to access Poll Everywhere. (Does not require live presentation)
Department of Physiology, School of Biomedical Sciences
 1. https://PollEv.com/multiple_choice_polls/eLuvR3gRE3SYpYsyu9AFP/respond

Click here to access Poll Everywhere. (Does not require live presentation)
Department of Physiology, School of Biomedical Sciences
 Click here to access Poll Everywhere. (Does not require live presentation)
Department of Physiology, School of Biomedical Sciences
 What you need to know
1. Pre-BötC neurones have intrinsic bursting properties
2. Production of respiratory rhythm is an emergent property of the pre-Bötzinger
complex
3. Evidence from humans support the importance of the pre-Bötzinger complex in
respiratory function

Department of Physiology, School of Biomedical Sciences