Determining the role of the paratrigeminal (Pa5) in airway defence.

Author: Alexandria K. Driessen Affiliation: Department of Anatomy and Neuroscience, School of Biomedical Sciences, University of Melbourne Degree: Thesis to be submitted in total fulfilment of a Doctor of Philosophy – Medicine, Dentistry and Health Sciences Submitted: 24 November 2018

ORCID ID: https://orcid.org/0000-0002-7057-6852

Advisors A/Prof. Stuart Mazzone (Department of Anatomy and Neuroscience, School of Biomedical Sciences, University of Melbourne) Dr. Michael Farrell (Department of Medical Imaging and Radiation Sciences, Monash University) Dr. Alice McGovern (Department of Anatomy and Neuroscience, School of Biomedical Sciences, University of Melbourne)

Abstract

Respiratory sensations conveyed by airway sensory nerve fibres are poorly understood, yet they contribute significantly to morbidity in pulmonary disease. Our viral tracing studies identified a previously unknown airway sensory circuit in the brain projecting through an obscure medullary site known as the paratrigeminal nucleus (Pa5). The Pa5 is located in the dorsal lateral medulla and is commonly defined as a collection of interstitial cells in the dorsal tip of the spinal trigeminal tract. The Pa5 has been previously implicated in baroreceptor function and somatosensory processing. Astonishingly, very little investigations have been made to understand its role in airway afferent processing.

We therefore investigated the anatomical connectivity of the Pa5, its involvement in initiating and controlling respiratory reflexes as well as in more complex respiratory behaviours including evoked cough.

Conventional neuroanatomical tracing was conducted to determine the input and output connectivity of the Pa5. Firstly, retrograde tracing confirmed, in the guinea pig, that vagal afferents from the jugular versus the nodose ganglia project specifically into the primary afferent termination sites. That is the nodose vagal ganglia project almost exclusively to the nucleus of the (nTS), while the jugular vagal ganglia project predominately to the Pa5. Furthermore, anterograde tracing was employed from the guinea pig Pa5, showing that this nucleus has extensive projections throughout the cardiorespiratory column, including the nTS, reticular nuclei, pre-bötzinger nucleus and in particular the parabrachial and Kölliker-Fuse nuclei in the . We have shown functional significance of these projections by implicating the Pa5 in the initiation and control of laryngeal-evoked respiratory reflexes. Initially we found that laryngeal-evoked

I | P a g e respiratory slowing was significantly attenuated by blocking the Pa5 using the GABAA agonist muscimol (stimulus evoked change in breaths/min pre-muscimol was 40.9±3.5 breaths/min pre-muscimol vs. 21.4±2.9 post-muscimol, p=0.001), while this pharmacological modification in the nTS had no effect on the laryngeal-evoked respiratory slowing. Importantly, this novel finding showed that the Pa5 has a role in respiratory reflexes that is independent of the nTS. Furthermore, injection of glutamate receptor antagonists into the Pa5 were able to prevent respiratory slowing evoked by laryngeal stimulation (change in at the maximum response 40.3±4.1 breaths/min vs. 17.3±3.1 breaths/min; p=0.0002), while peptide receptor antagonists resulted in significantly smaller effects.

Intriguingly, while injection of glutamate into the Pa5 resulted in respiratory slowing and in some cases apnoea, mimicking the laryngeal-evoked reflex, the direct release of by capsaicin microinjection into the Pa5 resulted in the paradoxical increase in respiratory rate. This unique response could be blocked by Substance P receptor antagonists but not by glutamate receptor antagonists, suggestive of two types of

Pa5 postsynaptic neurons. Additional anatomical characterisation of the Pa5 revealed the existence of two phenotypically distinct subtypes of Pa5 neurons. That is, one expressing the neurokinin 1 receptor and the other expressing calbindin. Furthermore, dual retrograde tracing from nuclei important for respiratory rhythm and modulation and were shown to receive direct projections from the Pa5 (i.e. the ventrolateral medulla and ) revealed that less than 7% of Pa5 neurons were dual labelled and therefore revealed that Pa5 neurons project differentially throughout the cardiorespiratory column.

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Given the complex processing capability of the Pa5, it seemed plausible that this nociceptive region may be important in complex respiratory behaviours, such as evoked cough and the perception of airway irritations. We developed an assay in which we can study the conscious perception of an airway irritation using behaviours (enhanced grooming, moving and chewing), in addition to the standard assessment of evoked cough.

Conscious unrestrained guinea pigs were aerosolised bradykinin (a jugular and nodose C- fibre stimulant) and adenosine 5’-triphosphate (ATP, a nodose selective stimulant). While bradykinin evoked both dose dependent increases in cough (8.9±2.7 at 1mg/ml and

25.7±3.7 at 3mg/ml) and behaviours (20.7±45.8 behaviour duration during saline vs.,

350.4±46.7 total behaviour duration, p=0.04), ATP only evoked responses at the highest dose (22.7±5.6 coughs and 81.3 behaviour duration during saline vs. 404±109.5 total behaviour duration). These distinct cough and behavioural profiles are suggestive that jugular and nodose airway afferents differentially process respiratory sensations. This was further supported by showing that targeted toxin lesions (substance P-Saporin,

10ng/100nl) of the Pa5 resulted in decreased bradykinin evoked cough compared to controsl (i.e. 7.2±2.8 coughs control vs. 0±0 coughs lesion at 1mg/ml and 19.2±4.1 coughs control vs. 10.4±3 coughs lesion at 3mg/ml, p=0.02 and p=0.009 respectively).

Alternately, lesioning NK1 receptor expressing neurons in the Pa5 had no effect on ATP evoked cough, in turn implicating NK1 receptor expressing Pa5 neurons specifically process jugular C-fibre evoked cough. Together these data are the first to reveal a complexity of airway afferent processing in the Pa5. Better understanding this putative airway somatosensory system may help identify therapeutic targets to alleviate respiratory discomfort in disease.

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Declaration

I Alexandria Kathryn Driessen declare that this thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in text. I have clearly stated the contribution by others to jointly authored works that I have included in this thesis. In addition, due acknowledgement has been made throughout the text for all other material used.

The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree in any other university. Together this thesis contains fewer words than the maximum allowable limit, exclusive of tables, the reference list and appendices.

Alexandria K. Driessen

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Preface

Publications by candidate

McGovern AE, Driessen AK, Simmons DG, Powell J, Davis-Poynter N, Farrell MJ,

Mazzone SB (2015) Distinct Brainstem and circuits receiving tracheal sensory neurons inputs revealed using a novel conditional anterograde transynaptic viral tracing system. J Neurosci 35(18):7041-7055

Driessen AK, Farrell MJ, Mazzone SB, McGovern AE (2015) The role of the paratrigeminal nucleus in vagal afferent evoked respiratory reflexes: a neuroanatomical and functional study in guinea pigs. Front Physiol 21(6):378-389

Publication included in thesis – Chapter 1 Author Contribution Author 1 (Candidate) Performed experiments: 55%

Wrote and edited manuscript: 20%

Author 2 Wrote and edited manuscript: 20%

Author 3 Designed experiments: 50%

Wrote and edited manuscript: 30%

Author 4 Designed experiments: 50%

Performed experiments: 45%

Wrote and edited manuscript: 30%

Driessen AK, Farrell MJ, Mazzone SB, McGovern AE (2016) Multiple neural circuits mediating airway sensations: recent advances in the neurobiology of the urge-to-cough.

Respir Physiol Neurobiol S1 1569-9048(15): 30054-30059

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Driessen AK, McGovern AE, Narula M, Yang S, Keller JA, Farrell MJ, Mazzone SB

(2017) Central mechanisms of airway sensation and cough hypersensitivity. Pulm

Pharmacol Ther 226:115-120

Ajayi IE, McGovern AE, Driessen AK, Kerr NF, Mills PC, Mazzone SB (2018)

Hippocampal modulation of cardiorespiratory function. Respir Physiol Neurobiol 252-

253:18-27

Driessen AK, Farrell MJ, Dutschmann M, Stanic D, McGovern AE, Mazzone SB (2018)

Reflex regulation of breathing by the paratrigeminal nucleus via multiple circuits. Brain

Struct Funct (epub ahead of print)

Publication included in thesis – Chapter 2 Author Contribution Author 1 (Candidate) Designed experiments: 10%

Performed experiments: 95%

Wrote and edited manuscript: 60%

Author 2 Wrote and edited manuscript: 5%

Author 3 Wrote and edited manuscript: 5%

Author 4 Wrote and edited manuscript: 5%

Author 5 Designed experiments: 5%

Performed experiments: 5%

Wrote and edited manuscript: 5%

Author 6 Designed experiments: 85%

Wrote and edited manuscript: 0%

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Conference abstracts by candidate

2014 (Poster): The Australian Health and Medical Research (AHMR) Congress –

Melbourne, Victoria

Title: A comparison of the neuroanatomical connectivity of the paratrigeminal

nucleus and the nucleus of the solitary tract in the rat.

Authors: Driessen AK, Miller CJ, Mazzone SB and McGovern AE

2015 (Poster): Australian Neuroscience Society (ANS) – Cairns, Queensland

Title: Distinct organisation of upper airway nodose and jugular vagal circuits in

the brain: evidence from conditional anterograde transneuronal tracing and

physiology.

Authors: McGovern AE, Driessen AK, Simmons DG, Farrell MJ, Davis-Poynter

N and Mazzone SB

2015 (Poster): International Postgraduate Symposium in Biomedical Science –

Brisbane, Queensland

Title: Evidence for a somatosensory-like vagal afferent neural circuit arising from

the airways.

Authors: Driessen AK, McGovern AE, Farrell MJ and Mazzone SB

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2016 (Poster discussion): London Cough Symposium – Kensington, London

Title: Investigating the role of the paratrigeminal nucleus in respiratory sensation

and cough.

Authors: Driessen AK, McGovern AE, Farrell MJ and Mazzone SB

2016 (Oral): International Postgraduate Symposium in Biomedical Science –

Brisbane, Queensland

Title: An anatomical and functional investigation of the paratrigeminal nucleus

in the regulation of respiration and cough.

Authors: Driessen AK, McGovern AE, Farrell MJ and Mazzone SB

2016 (Oral): Australian Neuroscience Society – Hobart, Tasmania

Title: Anatomical and functional evidence that the paratrigeminal nucleus is

involved in laryngeal sensory neuron regulation of respiration.

Authors: Driessen AK, McGovern AE, Farrell MJ and Mazzone SB

2017 (Oral): Australian Society for Medical Research (ASMR) – Melbourne,

Victoria

Title: The role of the Pa5 in evoked cough and sensorimotor behaviours to

perceived noxious airway irritation.

Authors: Driessen AK, McGovern AE, Farrell MJ and Mazzone SB

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2017 (Oral): Australian Neuroscience Society – Sydney, NSW

Title: Airway sensory inputs to the paratrigeminal nucleus regulate cough and the

perception of noxious airway irritations.

Authors: Driessen AK, McGovern AE, Farrell MJ and Mazzone SB

2018 (Poster): Federation of European Neuroscience Societies (FENS) – Berlin,

Germany

Title: Airway afferent processing in the paratrigeminal nucleus is important for

respiratory reflexes and the perception of noxious airway irritations.

Authors: Driessen AK, McGovern AE, Farrell MJ and Mazzone SB

2018 (Oral): Central Cardiovascular and Respiratory Control: Future Directions

(CCRC:FD) – Melbourne, Victoria

Title: The role of the paratrigeminal nucleus in airway defence

Authors: Driessen AK, McGovern AE, Farrell MJ and Mazzone SB

2018 (Oral): Melbourne Brain Symposium – Melbourne, Victoria

Title: Revealing and unappreciated complexity and importance of the

paratrigeminal nucleus in airway afferent processing

Authors: Driessen AK, McGovern AE, Farrell MJ and Mazzone SB

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2018 (Poster): Australian Neuroscience Society (ANS) – Brisbane, Queensland

Title: Paratrigeminal processing of jugular airway afferents in the conscious

perception of airways irritation

Authors: Driessen AK, McGovern AE, Farrell MJ and Mazzone SB

Funding acknowledgements

This work was supported by the National Health and Medical Research Council

(NHMRC) on grant GNT1078943.

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Acknowledgments

My PhD. has been an incredible journey! One that started as a naïve undergraduate student who frequently was heard saying that she never wanted to do research. Now here

I am five years on, absolutely loving what I do and proud of everything I have achieved.

But I didn’t and couldn’t have made it to this point without a number fantastic people.

Firstly, thank-you to my family and friends. Taylor, I am incredibly grateful for our friendship you truly are a special person, whom without I would have definitely gone insane. To my family, thank you all for being there to push me through the hard times and cheer me on during the wins. I would especially like to thank my mum and dad who have encouraged and supported me through all of my endeavours. Also I must thank my new

Melbourne friends who have been amazing in making this new state feel like home, despite having to deal with my loudness, craziness and strong opinions.

To my laboratory colleagues (aka those forced to be my friends) it has been awesome to share my journey with you all. You are my second family and I will never forget the

Mazzone Crew! I especially would like to acknowledge the hard work of my supervisors.

Michael, thank-you for your support and always keeping my statistics in check. Alice, I admire the scientist that you are and cannot speak highly enough of your abilities. I am very fortunate to have been trained by you, of which required you to give me a lot of your time. I know this would have taken extreme patience and so thank-you for persisting.

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The final and most important acknowledgement is for my supervisor Stuart. When I think about what I have achieved, it literally would not have been have been possible without you. You have opened my world to countless opportunities and have been there every step of the way - from simple maths equations to helping me find the right path for my future and absolutely everything in between. You have always believed in me and over time have taught me the greatest lesson of all, which is to believe in myself. You are an amazing supervisor, mentor, leader and scientist. Truly an inspiration. It is hard to express how much you have done for me and how appreciative I am for it all. No words will ever be able to properly thank-you.

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Table of contents Abstract ...... I Declaration ...... IV Preface ...... V Acknowledgments ...... XI List of figures ...... 1 List of tables ...... 3 Abbreviations ...... 4 Glossary of terms ...... 6 1. Chapter 1: Introduction1 ...... 8 1.1 Overview of the airway afferent neural circuitry ...... 8 1.2 Nodose vagal ganglia derived airway afferents ...... 9 1.2.1 Airway afferent subtypes – mechanoreceptors ...... 10 1.2.2 Airway afferent subtypes – nociceptors...... 13 1.3 Jugular vagal ganglia derived airway afferents ...... 14 1.3.1 Airway afferent subtypes - nociceptors ...... 16 1.4 The paratrigeminal nucleus (Pa5) ...... 18 1.5 Respiratory sensations and reflexes initiated from the larynx...... 23 1.5.1 Apnoea ...... 24 1.5.2 Glottal closure and swallow ...... 25 1.5.3 Laryngeal cough ...... 26 1.5.4 Higher order vagal and the perception of laryngeal irritation... 28 1.6 Aims and hypotheses ...... 32 2. Chapter 2: The role of the paratrigeminal nucleus in vagal afferent evoked respiratory reflexes: A neuroanatomical and functional study in guinea pigs (reproduced exactly from publication Driessen et al., 2015) ...... 33 2.1 Introduction ...... 33 2.2 Methods ...... 36 2.2.1 Animals ...... 36 2.2.2 Conventional neuroanatomical tracing ...... 36 2.2.3 Tissue harvest and immunohistochemical processing ...... 37 2.2.4 Histology and tracing data analysis ...... 39 2.2.5 Physiology ...... 41 2.3 Results ...... 46 2.3.1 Retrograde tracing and immunohistochemical characterisation of vagal sensory neurons ...... 46 2.3.2 Functional studies ...... 51

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2.4 Discussion ...... 54 2.4.1 Evidence that vagal ganglia afferents have multiple distinct termination sites in the brainstem ...... 55 2.4.2 CGRP expression distinguishes jugular and nodose sensory neurons in the vagal ganglia and brainstem ...... 57 2.4.3 A jugular vagal ganglia-Pa5 neural circuit contributes to laryngeal evoked respiratory reflexes ...... 59 2.4.4 Conclusions ...... 62 3. Chapter 3: Reflex regulation of breathing by the paratrigeminal nucleus via multiple bulbar circuits (reproduced exactly from publication Driessen et al., 2018) ...... 64 3.1 Introduction ...... 64 3.2 Methods ...... 67 3.2.1 Animals ...... 67 3.2.2 Neuroanatomical studies ...... 67 3.2.3 Tissue harvest, immunohistochemical processing and microscopy ...... 70 3.2.4 Analysis of neuroanatomical studies ...... 72 3.2.5 Physiological studies ...... 74 3.2.6 Analysis of physiological studies ...... 78 3.2.7 Drugs and reagents ...... 79 3.3 Results ...... 81 3.3.1 Characterisation of jugular vagal ganglia laryngeal sensory neurons ...... 81 3.3.2 Anatomical characterisation of the rostro-caudal organisation of the Pa5 ...... 81 3.3.3 Respiratory effects of electrical and chemical stimulation of the larynx………………85 3.3.4 Respiratory effects of glutamate and capsaicin microinjections into the Pa5 ...... 89 3.3.5 Neuroanatomical connectivity of the Pa5…………………………………………………91 3.3.6 Dual retrograde labelling of Pa5 neurons from the dorsolateral pons and ventral lateral medulla ...... 94 3.4 Discussion ...... 97 3.4.1 A neuroanatomical framework for laryngeal sensory-evoked respiratory responses via the Pa5……………………………………………………………………………………………97 3.4.2 Functional and anatomical evidence for multiple Pa5 processes regulating respiration ...... 99 3.4.3 Physiological significance and conclusion ...... 102 4. Chapter 4: The role of neurokinin 1 receptor expressing neurons in the medullary paratrigeminal nucleus in cough and associated airway irritant behaviours in conscious guinea pigs...... 105 4.1 Introduction ...... 105 4.2 Methods ...... 108 XV | P a g e

4.2.1 Animals ...... 108 4.2.2 Conscious cough studies ...... 108 4.2.3 Chemical lesions of the Pa5 ...... 112 4.4.4 Analysis of lesion characterisation and conscious cough studies……………………114 4.3 Results ...... 117 4.3.1 Characterising an assay to study animal behaviours as a measure of the conscious perception of an airway irritation ...... 117 4.3.2 The effect of targeted toxin lesions of the Pa5 on cough and behavioural responses associated with nebulised airway irritants……………………………………………………..129 4.4 Discussion ...... 144 4.4.1 Studying behaviours of irritation is a sensitive and informative measure of airway irritation ...... 145 4.4.2 Cough and accompanying behavioural responses to nebulised irritant challenges in guinea pigs: the role of nodose versus jugular afferent pathways ...... 146 4.4.3 Evidence for NK1 receptor expressing Pa5 neurons regulating jugular C-fibre evoked cough...... 148 4.4.4 Physiological significance and conclusion ...... 149 5. Chapter 5: General discussion ...... 151 5.1 Jugular vagal ganglia-derived airway afferents project specifically to the Pa5 ...... 152 5.2 Distinct Pa5 neurons differentially influence respiratory behaviour ...... 156 5.3 Clinical significance and final conclusions ...... 161 References ...... 138 Appendix……………………………………………………………………………………...205

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List of figures

Figure 1.1……………………………………………………………………………17 Figure 1.2……………………………………………………………………………19 Figure 1.3……………………………………………………………………………29 Figure 2.1……………………………………………………………………………43 Figure 2.2……………………………………………………………………………47 Figure 2.3……………………………………………………………………………49 Figure 2.4……………………………………………………………………………50 Figure 2.5……………………………………………………………………………52 Figure 3.1……………………………………………………………………………83 Figure 3.2……………………………………………………………………………87 Figure 3.3……………………………………………………………………………90 Figure 3.4……………………………………………………………………………92 Figure 3.5……………………………………………………………………………95 Figure 4.1……………………………………………………………………………110 Figure 4.2……………………………………………………………………………118 Figure 4.3……………………………………………………………………………120 Figure 4.4……………………………………………………………………………121 Figure 4.5……………………………………………………………………………125 Figure 4.6……………………………………………………………………………126 Figure 4.7……………………………………………………………………………127 Figure 4.8……………………………………………………………………………130 Figure 4.9……………………………………………………………………………132 Figure 4.10…………………………………………………………………………..134 Figure 4.11…………………………………………………………………………..135 Figure 4.12…………………………………………………………………………..139 Figure 4.13…………………………………………………………………………..140 Figure 4.14…………………………………………………………………………..141 Figure 5.1……………………………………………………………………………156

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Figure 5.2………………………………………………………………………….158 Figure 5.3………………………………………………………………………….163

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List of tables

Table 1.1…………………………………………………………………………31 Table 2.1…………………………………………………………………………39 Table 2.2…………………………………………………………………………53 Table 3.1…………………………………………………………………………70 Table 3.2…………………………………………………………………………72 Table 3.3…………………………………………………………………………87 Table 4.1…………………………………………………………………………113 Table 4.2…………………………………………………………………………114 Table 4.3…………………………………………………………………………123 Table 4.4…………………………………………………………………………129 Table 4.5…………………………………………………………………………137 Table 4.6…………………………………………………………………………143

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Abbreviations

AAV Aden-Associated Virus ABP Arterial Blood Pressure AP Anterior-Posterior BDA Biotinylated Dextran Amine BDNF Brain Derived Neurotrophic Factor BOLD Blood Oxygen Level Dependent CGRP Calcitonin Gene Related Peptide ChAT Choline-acetyltransferase CRLR Calcitonin receptor-like receptor CT-B Cholera Toxin Subunit B DLH DL’-homocysteic acid DNIC Diffuse Noxious Inhibitory Control DRG Dorsal root ganglia DV Dorsal-Ventral eGFP Enhanced green fluorescent protein EF50 50% of the effective maximum response (related to frequency) Emax Effective maximum EV50 50% of the effective maximum (related to voltage) fMRI Functional Magnetic Resonance Imaging FoxP2 Forkhead Box Protein 2 HR Hear rate HSV1 H129 Herpes Simplex Virus 1 strain H129 ML Medial-Lateral NF-160kD Intermediate neurofilament protein NGF Nerve growth factor NK Neurokinin receptor nTS Nucleus of the Solitary Tract Pa5 Paratrigeminal nucleus

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PBS Phosphate Buffered Saline PFA Paraformaldehyde Phox2A/B Paired like homeobox 2A/B RAMP1 Receptor activity modifying protein 1 RAR Rapidly Adapting Receptor RLN Recurrent laryngeal Nerve RUNX1 Runt related transcription factor 1 SAR Slowly Adapting Receptor SLN Superior Laryngeal Nerve SSP-SAP Substance P Saporin

TP Tracheal pressure TRKA/B Tropomyosin Kinase Receptor A/B TRPV1 Transient Receptor Potential Vanilloid 1 vGlut1/2 Vesicular Glutamate Transporter 1/2 Wnt1 Wingless-type MMTV Integration Site Family Member 1

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Glossary of terms

Apnoea: The cessation of breathing that is important in

preventing inhalation of foreign material into the

lungs

Bronchoconstriction: Airway constriction due to smooth muscle

contraction

Cough: Deep rapid inspiration immediately followed by a

rapid and forced expiratory effort

Epibranchial placode: Embryological derive the sensory ganglia of the

facial, glossopharyngeal and vagal nerves

Extrapulmonary airways: Larynx, trachea and main stem bronchi (large

airways)

Intrapulmonary airways: Lungs that is the bronchioles and alveoli

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Laryngeal hypersensitivity: Characterised by increased perception of airways

irritation, irritation by innocuous stimuli and the

overproduction of cough

Neural crest: The ectoderm at the margins of the neural tube, of

which the body region embryologically derives the

sensory, sympathetic and parasympathetic ganglia

Tachypnoea: Increased respiratory frequency.

Urge-to-cough: The conscious perception of an airway irritation

that precedes the motor act of coughing

Ventrolateral medulla: Nuclei located in the ventral lateral aspect of the

brainstem that include the nucleus ambiguus,

ventral aspects of the reticular nuclei and the

ventral respiratory groups.

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1. Chapter 1: Introduction1

1.1 Overview of the airway afferent neural circuitry

The airway is vital as it performs constant surveillance of the airway environment to ensure adequate ventilation is maintained and that the airways themselves are protected from damage by inhaled material (Karlsson et al., 1988;

Widdicombe and Tatar, 1988). In turn, the respiratory system, consisting of the extrapulmonary airways (larynx, trachea and mainstem bronchi) and the intrapulmonary airways (small bronchi, bronchioles and lung parenchyma), are densely innervated by heterogenous populations of sensory receptors that respond to a wide variety of chemical and/or mechanical stimuli. Although more complex classification systems have been proposed, airway afferent neuron subtypes can be broadly differentiated into mechanoreceptors and nociceptors based on their physiological and pharmacological properties, bronchial or pulmonary based on their peripheral termination sites and vagal

(nodose and jugular) or spinal (dorsal root) based on the sensory ganglia from which they are derived (Widdicombe, 1982; Widdicombe and Tatar, 1988; Riccio et al., 1996;

Mazzone, 2005; Canning and Spina, 2009; Mazzone et al., 2009). Given that the airway afferent neural circuitry is heterogenous in nature, it is important to investigate this to aid in better understanding of the physiological mechanisms of evoked respiratory reflexes and complex respiratory behaviours to perceived sensations, which become dysfunctional in respiratory disease.

8 | P a g e 1 Text has been adapted and reproduced from published review originally written by the candidate: Driessen et al., 2016 (Appendix 1) and Driessen et al., 2017 (Appendix 2). Primarily, airway afferents are vagal in origin with their cell bodies located either in the jugular (superior) or nodose (inferior) vagal ganglia (Canning et al., 2004; Undem et al.,

2004; McGovern et al., 2012a). Conventional and viral neuroanatomical tracing studies of airway afferents have identified projections into the brainstem that in turn give rise to higher order brain circuits, presumably important for reflex and discriminative processing of airway sensations. For example, McGovern and colleagues inoculated the tracheal lumen of rats with the anterograde transynatpic viral tracer, Herpes Simplex Virus 1 strain

H129 (HSV1 H129), identifying detailed maps of the central networks likely involved in airway sensory processing (McGovern et al., 2012a; McGovern et al., 2012b; McGovern et al., 2015a; McGovern et al., 2015b). These studies confirmed the widely held notion that bronchial and pulmonary afferent neurons synapse with second order neurons in the caudal (predominately dorsolateral) nucleus of the solitary tract (nTS; McGovern et al.,

2012a; McGovern et al., 2012b; McGovern et al., 2015b). However, a second termination site for airway afferents within the medullary paratrigeminal nucleus (Pa5) was also identified, providing evidence for multiple bulbar and higher brain circuits arising from the airways and lungs (McGovern et al., 2012a; McGovern et al., 2012b; McGovern et al., 2015b). Collectively, these findings raise intriguing questions about how respiratory sensory processing occurs within the central nervous system and point towards a level of complexity not previously recognised. This represents the central rationale for the present doctoral studies.

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1.2 Nodose vagal ganglia derived airway afferents

The nodose vagal ganglia are derived from the epibranchial placode and as a result develop under the control of transcriptional elements such as Paired like Homeobox 2A and 2B (Phox2A and Phox2B), which are important for the genesis of parts of the autonomic nervous system (Figure 1.1A; Pattyn et al., 1997; Pattyn et al., 1999;

D’Autréaux et al., 2011). Airway sensory neurons derived from the nodose vagal ganglia have been shown to predominately innervate the intrapulmonary airways (McGovern et al., 2015a). They preferentially express the neurotrophin receptor Tropomyosin Kinase

Receptor B (TRKB) and in turn are physiologically sensitive to Brain Derived

Neurotrophic Factor (BDNF) and neurotrophin-3 (Lieu et al., 2011; McGovern et al.,

2015a). In addition, nodose vagal ganglia derived sensory neurons can be selectively activated by serotonin 5-HT3 receptor agonists, as well as by adenosine 5’-triphosphate

(ATP) as they uniquely express the purinergic receptors P2X2 and P2X3 that form a heteromeric complex required to confer sensitivity to ATP (Kwong et al., 2008;

Nassenstein et al., 2010; Potenzieri et al., 2012).

1.2.1 Airway afferent subtypes – mechanoreceptors

Airway low threshold mechanoreceptors are derived exclusively from the nodose vagal ganglia and they are exquisitely responsive to stretch, touch or other airway mechanical forces, but display limited sensitivities to a variety of chemical mediators, perhaps with the exception of ATP and protons (Canning et al., 2004; Undem et al., 2004; Gu and Lee,

2006; Nassenstein et al., 2010; Mazzone and Undem, 2016). Characteristically, these airway mechanoreceptors, are myelinated (that is they express the intermediate neurofilament protein NF-160kD) non-peptidergic neurons that conduct action potentials

10 | P a g e in the Aδ or Aβ range (Riccio et al., 1996; Undem et al., 2004; Chuaychoo et al., 2005).

Furthermore, they can be classified into three unique subtypes, based on their activation profiles; slowly adapting receptors (SARs), rapidly adapting receptors (RARs) and touch sensitive receptors (Yu, 2000; Canning et al., 2004).

SARs are the most well understood airway sensory neuron subtype. They have a complex structure with many receptive fields innervating the airway smooth muscle of the lungs

(particularly the alveoli and bronchioles; Widdicombe, 2001; Yu et al., 2003; Canning et al., 2006) and project centrally to a number of subnuclei of the nTS important for autonomic regulation including the intermediate, interstitial, ventral and ventrolateral nTS (Kubin et al., 1985). In the ventrolateral nTS, SARs terminate on the specialised respiratory modulating neurons, pump cells and inspiratory β-neurons, to directly influence respiration (Donoghue et al., 1982; Kubin et al., 1985). In addition, they play an integral role in the Hering-Breuer reflex (i.e. inhibition of inspiration to allow expiration and prevent over-inflation of the lungs) and respond tonically to sustained lung inflation with regular discharges and slow adaption rates (Knowlton and Larrabee, 1946;

Backman et al., 1984; Kubin et al., 1985; Widdicombe, 2001).

On the other hand, RAR terminals largely exist in the submucosa and airway epithelium and instead project to the caudal nTS, primarily in the commissural subnucleus (Kubin et al., 1985; Ezure et al., 1999; Widdicombe, 2001). Lung inflation and deflation stimulates these airway afferents in which they present a phasic response due to their rapid adaption rates (Kubin et al., 1985; Ezure et al., 1999). That is, RARs become active during the dynamic phase of lung deflation and then adapt during the sustained phase (Ezure et al.,

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1999; Canning et al., 2006). Together with SARs they work in concert to facilitate the cyclical transition between inspiratory and expiratory phases of breathing. However, it is important to note that, unlike SARs, RARs are more polymodal in that they are somewhat chemosensitive responding to a number of endogenous (inflammatory mediators released from the lungs) and exogenous (aerosols and fumes) stimulants (Mills et al., 1969;

Widdicombe, 1982; Canning et al., 2006). In turn, RARs have been implicated in a number of respiratory reflexes including bronchoconstriction, cough and the deflation reflex (Widdicombe, 1954; Mills et al., 1969; Widdicombe, 1982; Canning et al., 2006).

Interestingly, the evidence for RARs mediating cough in the guinea pig, a species that is used extensively to study cough, is relatively weak (Canning et al., 2006) and for many years the identity of the nodose cough-evoking mechanoreceptor went undiscovered.

Indeed, this gap in knowledge lead to the discovery of a specialised ‘cough receptor’ in the guinea pig (Canning et al., 2004; Mazzone et al., 2009), which is morphologically similar to RARs in that they are myelinated, non-peptidergic rapidly adapting mechanoreceptors derived exclusively from the nodose vagal ganglia (Mazzone and

McGovern, 2008), but are functionally distinct in that they do not respond to tissue stretch, but instead are exquisitely sensitive to punctate (touch-like) stimuli (Canning et al., 2004). The terminals of the nodose cough receptor are complex structures that innervate adjacent to the cartilage rings in the submucosal space of the extrapulmonary airways (Mazzone et al., 2009). This positioning of the cough receptor terminals is thought to be optimal for evoking reflex cough in response to punctate mechanical stimuli induced by inhaled foreign material, aspirate or airway mucous (Canning et al., 2004;

Mazzone et al., 2009). Whether other species have a comparable airway afferent subtype

12 | P a g e is unclear, although morphological studies in humans would suggest they are present and similarly positioned (West et al., 2015).

1.2.2 Airway afferent subtypes – nociceptors

Nociceptors represent the majority of airway sensory neurons (Coleridge and Coleridge,

1984), yet they remain the least well understood because of their neurochemical and functional heterogeneity. Nodose vagal ganglia derived pulmonary C-fibres innervate the superficial mucosal layers of the lungs (Lee and Pisarri, 2001; Undem et al., 2004) and primarily project to the intermediate and caudal subnuclei of the nTS, albeit innervating distinct subsets of nTS neurons compared to the mechanoreceptors (Kubin et al., 1985;

Mutoh et al., 2000; Peters et al., 2011; McDougall and Andresen, 2013). Nodose vagal ganglia derived nociceptors are activated by a range of endogenously produced mediators and/or exogenously produced chemicals (Riccio et al., 1996; Chung et al., 2002; Undem et al., 2004; Chou et al., 2008; Mazzone and Undem, 2016). These nociceptors are typically unmyelinated (small diameter) non-peptidergic C-fibres that express the

Transient Receptor Potential Vanilloid 1 (TRPV1; Riccio et al., 1996; Undem et al.,

2004), which confers sensitivity to capsaicin (the pungent chemical from hot chilli peppers; Caterina et al., 1997). While TRPV1 expression is characteristic of many visceral nociceptive populations, it is not absolute in defining nociceptors, as exemplified in the mouse that have a subset of C-fibres that are capsaicin insensitive (Kollarik et al.,

2003).

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1.3 Jugular vagal ganglia derived airway afferents

The jugular vagal ganglia are derived from the neural crest, which is a distinct embryological origin to that of the nodose vagal ganglia (D’Autréaux et al., 2011;

Nomaksteinsky et al., 2013). Recognising this distinction is not simply a matter of semantics and instead has important consequences for the anatomical, molecular and functional profiles of the sensory neurons derived from each of these vagal ganglia

(Figure 1.1A; Chuaychoo et al., 2006; Kwong et al., 2008; D’Autréaux et al., 2011;

McGovern et al., 2015a; McGovern et al., 2015b). However, the functional significance of these distinctions remains unclear and requires further investigation to understand the purpose of these different airway afferent lineages existing in parallel.

During development of the jugular vagal ganglia neurons express the neural crest marker

Runt-Related Transcription Factor 1 (Runx1), which promotes the expression of the neural crest migratory gene, Wingless-type MMTV Integration Site Family member 1

(Wnt1). In turn, jugular vagal ganglia neurons have a molecular fingerprint similar to that of somatosensory neurons derived from the dorsal root ganglia (DRG) and trigeminal ganglia (Nassenstein et al., 2010; D’Autréaux et al., 2011; Nomaksteinsky et al., 2013).

Unlike, those derived from the nodose vagal ganglia, jugular vagal ganglia neurons project predominately to the extrapulmonary airways and instead preferentially express

TRKA and thus are most sensitive instead to nerve growth factor (NGF) (Kwong et al.,

2008; Nassenstein et al., 2010), which induces a characteristically high expression of the neuropeptides, Substance P and calcitonin-gene related peptide (CGRP; Riccio et al.,

1996; Chung et al., 2002; Undem et al., 2004; Hayakawa et al., 2014). This is in striking

14 | P a g e comparison to sensory neurons derived from the nodose vagal ganglia that are largely devoid of neuropeptides.

Both Substance P and CGRP are known to influence bronchial and vascular smooth muscle tone, secretory cells and the cellular components of inflammation (Lundberg et al., 1983; Rosenfeld et al., 1983; Palmer et al., 1987; Martling et al., 1989; Riccio et al.,

1996; Undem et al., 2004; Hayakawa et al., 2014). Substance P is a tachykinin formed from the precursor protein pre-protachykinin A (Nakanishi, 1987). Tachykinins exert their effects by binding to cell surface neurokinin (NK) receptors; NK1, NK2 and NK3, which are differentially expressed on many target cell types (Mazzone and Geraghty,

1999; Boot et al., 2007; Naline et al., 2007). The CGRP is a 37-amino acid formed from the precursor hormone calcitonin (Rosenfeld et al., 1983) and signals through a heteromeric receptor consisting of a calcitonin receptor-like receptor (CRLR) and a receptor activity modifying protein 1 (RAMP1) that are commonly expressed on the vasculature (Joshi et al., 2014; Cauchi and Robertson, 2016). These neuropeptides are synthesised in the jugular vagal ganglia sensory neurons cell bodies and then transported along the axon to both the peripheral and central nerve terminals (Mazzone and Geraghty,

2000; Undem et al., 2004). Accordingly, many afferent fibres in the brainstem and extrapulmonary airways express Substance P and/or CGRP (Rosenfeld et al., 1983;

Palmer et al., 1987; Hayakawa et al., 2014).

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1.3.1 Airway afferent subtypes - nociceptors

The jugular vagal ganglia exclusively give rise to nociceptors that predominately innervate the extrapulmonary airway mucosa (Hunter and Undem, 1999; Canning et al.,

2004; Undem et al., 2004; McGovern et al., 2015a). Jugular vagal ganglia nociceptors, can be phenotypically distinguished into two subtypes. The first are the classical nociceptors that are TRPV1 positive, unmyelinated peptidergic C-fibres (Riccio et al.,

1996; Canning et al., 2004; Undem et al., 2004). The second, and less well studied, are the Aδ-nociceptors that are TRPV1 positive, myelinated and non-peptidergic (Canning et al., 2004). Both C- and Aδ-nociceptors derived from the jugular vagal ganglia are sensitive to an array of chemical irritants including many inflammatory mediators (Riccio et al., 1996; Chung et al., 2002; Undem et al., 2004; Chou et al., 2008; Mazzone and

Undem, 2016). Indeed, these subtypes have similar neurochemical profiles making them difficult to study in isolation. Therefore, it remains unknown if they influence airway afferent processing differentially. However, in this regard it is intriguing that the central projections of jugular vagal ganglia nociceptors differ substantially to the nodose vagal ganglia nociceptors. For example, our laboratory used conventional retrograde tracing in the rat to show that while the nodose vagal ganglia neurons display specific projections to the nTS (as described in section 1.2), jugular vagal ganglia neurons distinctly project to the Pa5 (Figure 1.1B; McGovern et al., 2015b). The physiological relevance of this distinction is unknown.

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17 | P a g e

Figure 1.1: Schematic depiction of the peripheral and central projections of nodose and jugular vagal ganglia airway afferents. A) Schematic depicting the vagal sensory innervation of the airways

(adapted Driessen et al., 2016). The vagal ganglia represent the primary source of afferent neural supply to the airways and lungs. These vagal ganglia contain heterogenous populations of neurons, which in part reflect their embryological origins. The superior jugular vagal ganglia are derived from the neural crest and develop under the control of the Runt Related Transcription Factor (RUNX1).

The resultant neurons (displayed in red) display a phenotype similar to the somatic spinal dorsal root ganglia. Their peripheral terminals dominate the afferent innervation of the extrapulmonary airways.

By contrast, the inferior nodose vagal ganglia are derived from the epibranchial placodes and develop under the regulation of the transcription factor Paired like Homeobox 2b (Phox2b), which is important for the development of much of the autonomic nervous system. The peripheral terminals of nodose neurons (displayed in green) represent the majority of afferents terminating in the intrapulmonary airways. Developmental distinctions between the nodose and jugular vagal ganglia determines differences in the molecular and functional profiles of the sensory neurons derived from each. B)

Diagram depicting the distinct innervation of nodose and jugular vagal ganglia neurons in the primary afferent termination sites of the medulla – the nucleus of the solitary tract (nTS - green) and the paratrigeminal nucleus (Pa5 – red; adapted Mazzone and Undem, 2016). Additional abbreviations: 5-

HT3; Serotonin receptor, CGRP; Calcitonin Gene Related Peptide, P2X2/P2X3; purinergic receptors,

RLN; Recurrent laryngeal nerve, SLN; superior laryngeal nerve, SP; Substance P, TRKA/TRKB;

Tropomyosin Kinase Receptor A/B.

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1.4 The paratrigeminal nucleus (Pa5)

In the early 1900’s, Ramón y Cajal first described vagal afferent terminations in the Pa5.

In fact, Cajal suggested two primary intramedullary pathways of vagal afferents and they are the principle vagal bundle that traverses the solitary tract terminating in the nTS and a smaller external bundle named Cajal’s trigeminal fascicle that travels through the spinal trigeminal tract (Cajal, 1909). Further investigations of Cajal’s trigeminal fascicle highlighted terminations primarily in the spinal trigeminal nuclei, which in the 1950’s were cytologically broken into three subnuclei that are now known as the subnucleus oralis, interpolaris and caudalis (Olszewski, 1950). Each of these subnuclei have been implicated in sensory processing of the orofacial structures and the best described are those subserving nociceptive processing (Nash et al., 2009; Jacquin et al., 2015; Nakaya et al., 2016; Mustafa et al., 2017). However, it was not until the mid-1960’s that Albert

Rhoton first identified the Pa5, in the monkey, as a distinct region within the spinal trigeminal interstitial system (Rhoton et al., 1966). Indeed, it is an obscure nucleus located in the dorsal lateral aspect of the medulla and has been defined more specifically as a collection of interstitial neurons situated within the dorsal segment of the spinal trigeminal tract (Figure 1.2; Chan-Palay, 1978; Phelan and Falls, 1989).

The spinal trigeminal interstitial system can be subdivided into five morphologically and functionally distinct components in the rat (Figure 1.2A); 1) interstitial cells extending from the medullary dorsal horn 2) dorsal paramarginal nucleus 3) trigeminal extension of the parvicellular 4) Pa5 and 5) insular trigeminal-cunealis lateralis nucleus (Phelan and Falls, 1989). The Pa5 has the largest rostro-caudal spread of these subdivisions, extending 1.5 to 2mm in the rat and this expands as large as 3.5 and 6mm

18 | P a g e in the monkey and human, respectively (Chan-Palay, 1978; Lapa and Watanabe, 2005;

Pinto et al., 2006). Morphological characterisation of the Pa5 neurons suggest they can be divided into two distinct subtypes based on their dendritic morphology; 1) those with long but few primary dendrites and 2) those with short dendrites and many branch points

(Phelan and Falls, 1989). Presynaptic terminations in the Pa5 synapse axo-dendritically

(>90%) with both types of postsynaptic neurons in the Pa5 (Chan-Palay, 1978; Saxon and

Hopkins, 2006). Furthermore, the Pa5 is an interesting region in that is thought to exclude interneuron processing but instead appears to contain a pronounced number of glia and astrocytes particularly in its most superficial layers (Chan-Palay, 1978; Saxon and

Hopkins, 2006; Caous et al., 2012).

Figure legend on the next page 19 | P a g e

Figure 1.2: Representative photomicrographs and adapted schematics to identify the

paratrigeminal nucleus (Pa5) and its location within the interstitial system of the spinal

trigeminal tract. A) Line diagrams of the interstitial system of the trigeminal tract as described by

Phelan and Falls, 1989. Each of the five components can be observed in the series; 1) the interstitial

cells that extend from the medullary dorsal horn (MDH; i-iii), 2) the dorsal paramarginal nucleus

(PaMd; i-iii), 3) trigeminal extension of the parvicellular reticular formation (V-RPc; iV-V), 4) the

Pa5 (PaV; iV-Vi) and 5) insular trigeminal-cunealis lateralis nucleus (iV-Cul; iii-Viii) (adapted from

Phelan and Falls, 1989). B) Photomicrographs of the rat medulla stained with Cresyl violet luxol

fast blue staining to identify the Pa5 (PTN) in the dorsal segment of the spinal trigeminal tract

(SpVt/SVT; adapted Saxon and Hopkins, 1998). C) Photomicrographs of the guinea pig medulla

(calamus scriptorius +0.5mm) depicting the Pa5 in the dorsal lateral region of the medulla. The left

brainstem hemisection shows Cresyl violet staining, while the right depicts the pan neuronal marker,

NeuN. A high magnification image from within the white square shows NeuN positive neurons

within the spinal trigeminal tract (Sp5) that is characteristic of the Pa5. Additional abbreviations:

AP; area postrema, LI/LII; lamina 1 and 2, NTS/nTS; nucleus of the solitary tract, IO; inferior olive

complex, SpVi/Vi; spinal trigeminal nucleus interpolaris, X; dorsal motor nucleus of the vagus, XII;

hypoglossal nucleus.

Conventional neuroanatomical tracing studies in the rat have shown that the Pa5 receives afferent input from trigeminal, glossopharyngeal and vagus nerves, as well as spinal afferents (Ciriello et al., 1981; Panneton and Burton, 1981; Altschuler et al., 1989;

McGovern et al., 2012a; McGovern et al., 2015b). It also projects extensively to bulbar and subcortical regions important for cardiorespiratory control and somatosensory processing including the nTS, spinal trigeminal nuclei, rostral ventrolateral medulla, ambiguus nucleus, reticular nuclei, , Kölliker Fuse nucleus, principal sensory trigeminal nucleus and the ventroposterior medial (Feil and Herbert,

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1995; Saxon and Hopkins, 1998; Caous et al., 2001; de Sousa Buck et al., 2001; Pinto et al., 2006; McGovern et al., 2015b). Importantly, Pa5 neuron output connectivity is not homogenous, but displays differences that are indicative of topographical organisation within this nucleus. In this regard, neuroanatomical tracing studies in the rat have identified distinctions between Pa5 projections to the nTS, parabrachial nuclei and ventroposterior medial thalamus (Saxon and Hopkins, 1998; Pinto et al., 2006). Initial dual retrograde tracing studies from these regions showed that while a number of neurons were dual labelled from the nTS and parabrachial nuclei, a distinct subset was observed projecting to the ventroposterior medial thalamus (Saxon and Hopkins, 1998).

Furthermore, retrograde tracing from different regions of the parabrachial nucleus revealed topographical projections rostro-caudally throughout the Pa5 (Pinto et al., 2006).

That is, Pinto and colleagues found that the caudal Pa5 (below obex at the level of the central canal and area postrema) projects to the ventral parabrachial nuclei, the intermediate (at the level of obex) to the external parabrachial nuclei and the rostral Pa5

(at the level of the inferior ) to the medial parabrachial nucleus (Pinto et al., 2006).

The Pa5 output projections feed into the key brainstem nuclei (as aforementioned) that constitute the cardiorespiratory control network particularly the ventrolateral medulla that is integral in the baroreceptor reflex given it directly influences sympathetic outflow

(Bonham and Jeske, 1989; Ruggiero et al., 1996; Koganezawa and Paton, 2014;

Tallapragada et al., 2016; Ghali, 2017). However, the first functional evidence for its involvement in the baroreceptor reflex came from microinjection of bradykinin directly into this nucleus, which resulted in an increase in arterial pressure (Lindsey et al., 1997).

In addition, lesions of the Pa5 result in increased resting arterial pressure and decreased

21 | P a g e resting heart rate, while also impairing baroreceptor reflex sensitivity (Lindsey et al.,

1997; Sousa and Lindsey, 2009). Electrophysiological recordings subsequently identified that 72% of the Pa5 neurons are barosensitive and of these almost all show rhythmic activity with the cardiac cycle (Junior et al., 2004; Sousa and Lindsey, 2009).

Further to a role in autonomic processing, the Pa5 is a key region involved in processing noxious stimuli. Indeed, stimulation of peripheral nociceptors results in the upregulation of activation markers (e.g. cFos and pERK) in Pa5 neurons (Bon et al., 1997; Yamazaki et al., 2008; Tsujimura et al., 2011). In particular, Pa5 neurons expressing calbindin are specifically activated by formalin induced orofacial pain (Ma et al., 2005). Chemical lesion studies have also implicated the Pa5 in altering nociceptor behaviour to noxious

(mechanical and chemical but not thermal) stimulation of the rat hindlimbs (Koepp et al.,

2006). Unilateral Pa5 lesions were sufficient to decrease paw withdrawal thresholds to

Von Frey tests, indicating that inhibition of nociceptive processing in the Pa5 increases sensitivity to mechanical noxious stimulation (Koepp et al., 2006). Interestingly, the opposite effect on sensitivity was seen for chemical noxious stimulation. Lesions of the

Pa5 instead decreased total nocifensive behaviours to formalin injections into the hind- paw, which suggests decreased sensitivity to chemical noxious stimulation when Pa5 processing is ablated (Koepp et al., 2006). Therefore, the Pa5 is involved in complex nociceptive processing, in that it can differentiate between types of nociceptive sensations. A processing complexity of this nucleus that has gone unrecognised in the airway sensory nervous system.

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1.5 Respiratory sensations and reflexes initiated from the larynx

The larynx represents an important reflexogenic site within the airways, which is essential to limit the accidental aspiration of foodstuffs and salivary secretions that ordinarily enter the oesophagus via the pharynx. In healthy individuals, activation of the sensory terminals in the larynx evoke a number of respiratory reflexes, that include apnoea, swallow, glottal closure, the expiration reflex and cough, all of which serve to limit the penetrance of foreign material beyond the glottis (Jafari et al., 2003; Ku et al., 2010). Indeed, six laryngeal receptor subtypes have been identified and characterised based on their activation profiles (Sant’Ambrogio et al., 1983; Widdicombe, 2001). These include the

C-fibres and irritant/RAR receptors (described in section 1.2 and 1.3) but also include flow, pressure, drive and cold receptors (Sant’Ambrogio et al., 1983; Widdicombe, 2001).

Flow receptors respond only to breathing of the extrapulmonary airways and instead are silent to tracheal breathing and/or occlusion (Sant’Ambrogio et al., 1983). Pressure receptors densely innervate the larynx and respond to inspiratory or expiratory discharge related to upper airway breathing, with most responding to pressures due to airway collapse (Sant’Ambrogio et al., 1983). Drive receptors are laryngeal mechanoreceptors that are stimulated by laryngeal contraction (Sant’Ambrogio et al., 1983). In the guinea pig, all laryngeal low threshold mechanoreceptors originate in the nodose vagal ganglia, while all laryngeal nociceptive-like afferents are jugular vagal ganglia derived (Canning et al., 2004). However, given the diversity in laryngeal receptor subtypes it has been difficult to interpret the individual contributions to respiratory reflexes. More focused studies on particular subsets of laryngeal afferents, such as those derived from the jugular vagal ganglia, will help to determine specificity of function within this airway structure.

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In humans, the larynx is recognised as the site in which individuals can perceive and localise sensations and in turn initiate behavioural coughing to satiate unpleasant and/or irritating sensory experiences (Ando et al., 2014; Hilton et al., 2015). In patients with respiratory disease these sensations are perturbed (often referred to as laryngeal hypersensitivity) resulting in an increased perception of irritation and in turn the overproduction of cough that is non-productive and potentially harmful (Polley et al.,

2008; Morice, 2013; Ando et al., 2014; Hilton et al., 2015). In patients with chronic cough hypersensitivity, eating dry crumbly food, inhaling perfumes, laughing and other normally innocuous stimuli can be sufficient to initiate reflexes from the larynx (Chung,

2011; Morice, 2013). These clinical observations suggest that the vagal sensory pathways innervating the larynx become sensitised. Given the preponderance of jugular vagal ganglia neurons terminating in this airway structure, it is conceivable that neural circuits involving the Pa5 may be involved in this enhanced reflexivity. Indeed, the previous implications of this nucleus in is perhaps consistent with a role for the Pa5 in laryngeal sensation, particularly those of a noxious nature. This represents a key research question to be addressed in this thesis.

1.5.1 Apnoea

Stimulation of nociceptive laryngeal afferents evokes apnoea (cessation of breathing) that is associated with a bradycardia (Marchal et al., 1982). Collectively this response is referred to as the laryngeal chemoreflex and is one of the most primitive respiratory reflexes studied. The laryngeal chemoreflex is most prevalent in infants (especially those that are premature) but becomes much harder to evoke in older species (Lucier et al.,

1979; Sasaki, 1979; Marchal et al., 1982). This is due to maturation of the afferents

24 | P a g e themselves (i.e. changes in conduction properties) as well as of the central networks involved in processing the laryngeal chemoreflex (Sasaki, 1979; Sun et al., 2011;

Donnelly et al., 2016). Electrophysiological studies have shown that nTS neurons receive direct input from the larynx (Dawid-Milner et al., 1995) and that serotoninergic mechanisms are particularly important in mediating nTS control of the laryngeal chemoreflex (Donnelly et al., 2016). Furthermore, inhibition of either the nTS or

Bötzinger complex are sufficient to abolish apnoea evoked by electrical stimulation of the larynx (Sun et al., 2011). In addition, many other brainstem regions have been shown to be activated by stimulation of the larynx and these include the loose formation of the nucleus ambiguus, rostral ventral respiratory group, spinal trigeminal nuclei and importantly the Pa5 (Wang et al., 2015), albeit involvement of these nuclei in the laryngeal chemoreflex are not well understood.

1.5.2 Glottal closure and swallow

Stimulation of the larynx also effectively evokes the laryngeal adductor reflex (glottal closure reflex) and swallow (Suzuki and Sasaki, 1976; Domer et al., 2013; Bautista and

Dutschmann, 2014). The laryngeal adductor reflex is essential in preventing unwanted material from entering and causing harm to the airways. It is characterised by stimulation of laryngeal afferents, particularly in the superior laryngeal nerve (SLN), causing contraction of adductor muscles of the larynx that include the thyroarytenoid muscle

(Suzuki and Sasaki, 1976; Bhabu et al., 2003; Kim et al., 2009). Working in coordination with glottal closure is laryngeal evoked swallow (Barkmeier et al., 2000), which serves to remove substances from the area of the glottis. Both of these reflexes rely on central networks that are common to those for laryngeal evoked apnoea, including the nTS,

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Bötzinger complex and the Kölliker Fuse nucleus in the pons (Oku et al, 1994; Bauman et al., 2000; Gestreau et al., 2000; Sang and Goyal, 2001; Bautista and Dutschmann,

2014). These regions are thought to directly influence laryngeal pre-motor neurons in the nucleus ambiguus that mediate laryngeal muscle activity necessary for both glottal closure and swallow (Oku et al, 1994; Bauman et al., 2000; Gestreau et al.,2000).

1.5.3 Laryngeal cough

Cough is characterised by a rapid deep inspiration followed by an active expiratory effort, initially against a closed glottis (the compression phase) and continuing upon glottal opening (Tsubone et al., 1991; Tatar et al., 1994). This patterned respiratory response can be initiated reflexively, without any involvement of conscious processing, or behaviourally with varying levels of higher brain sensorimotor control (see section 1.5.4)

(Magni et al., 2011; Mazzone et al., 2011). Importantly, coughing is associated with activity in both inspiratory and expiratory muscles, which distinguishes the cough reflex from other similar reflexes such as the aspiration reflex (inspiration only) evoked primarily from the epiglottis and the expiration reflex (expiration only) evoked from the vocal folds (Jakus et al., 2000).

Reflex cough has been extensively studied in anaesthetised and decerebrate animal models. Under these conditions both punctate mechanical stimuli and some chemical perturbations (such as rapid mucosal acidification) of the larynx and extrapulmonary airways can evoke coughing. Studies in guinea pigs suggest that a single

(mechanosensitive) afferent subtype innervating the larynx is responsible for cough evoked by airway mucosal probing and bolus application of citric acid (Tsubone et al.,

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1991; Widdicombe, 1996; Bolser and Reier, 1998; Canning et al., 2004). These mechanoreceptors likely represent the nodose vagal ganglia derived Aδ cough receptor, as described above (see section 1.2.1; Widdicombe, 1954; Mills et al., 1969;

Widdicombe, 1996; Canning et al., 2004). Cough-evoking stimuli result in the upregulation of the neuronal activation marker cFos throughout the cardiorespiratory regions of the brainstem (Gestreau et al., 1997; Jakus et al., 2008). These include the nucleus ambiguus, lateral reticular nucleus, nTS, spinal trigeminal nuclei, parabrachial nuclei, Kölliker Fuse nucleus and rostral (Gestreau et al., 1997; Jakus et al.,

2008). This medullary network is central to initiating reflex cough and importantly allowing for the appropriate modulation of the respiratory rhythm and pattern generators.

On the other hand, nociceptive-selective stimuli have also long been known to evoke cough in conscious animals and humans. However, the role of nociceptive afferents in cough appears more complicated than originally thought. Firstly, not all airway projecting nociceptive afferents are cough-evoking. As such, C-fibres derived from the nodose vagal ganglia seem to be inhibitory to cough (Canning and Mori, 2011; Chou et al., 2018), whereas the activation of the extrapulmonary (jugular) C-fibres readily induce coughing.

Unlike mechanoreceptor-evoked cough, relatively little is known about the nociceptor pathways involved in evoking cough, which is in part due to the difficulty to mechanistically study this cough given that it does not persist under anaesthetics

(Mazzone et al., 2005). Therefore, nociceptor-evoked cough may involve significantly more complex neural processing, in that this type of cough may require intact higher order brain processing. Indeed, it has been suggested that nociceptor cough is not purely reflexive but includes significant behavioural contributions secondary to the perception of airway irritations (Mazzone et al., 2011). Together one could predict that the central

27 | P a g e representation of nociceptor-evoked cough may be notably different to that of mechanoreceptor-evoked cough and given that laryngeal nociceptors are exclusively derived from the jugular vagal ganglia, processing of these afferents in the Pa5 likely has important implications for mediating laryngeal nociceptor cough.

1.5.4 Higher order vagal sensory processing and the perception of laryngeal irritation

Accompanying motor responses evoked by airway afferent activation are perceivable sensations that can be discriminated with greater or lesser detail depending on the airway level involved. Often, laryngeal sensations are unpleasant and highly localisable sensations, described as an annoying tickle in the back of the throat, which in turn induce what is commonly referred to as the urge-to-cough (Ando et al., 2014; Hilton et al., 2015).

With the exception of purely reflex cough, this perceived irritation characteristically precedes the motor act of coughing (Davenport et al., 2007; Farrell et al., 2012) and is therefore thought to be an important ‘motivator’ for the behavioural regulation of coughing (Davenport et al., 2007). The urge-to-cough is a complex sensation requiring higher order processing, which has been well investigated in humans using functional magnetic resonance imaging (fMRI) that assesses blood oxygen level dependent (BOLD) intensity signal changes to measure neural activation (Mazzone et al., 2007; Mazzone et al., 2011). Following inhalation of an airway irritant, in healthy individuals, BOLD signal changes can be observed across a distributed brain network, including in the primary motor and somatosensory cortices, prefrontal cortex (particularly the orbitofrontal cortex), anterior insula cortex as well as limbic and cerebellar regions (Mazzone et al.,

2007). Perhaps of most interest is that regional activation responses in the insula cortex show a dose dependency relative to the magnitude of the irritant stimulus, while those in

28 | P a g e the primary sensory cortex correlate closely with the perceived (subjective) urge-to-cough intensity (Figure 1.3; Farrell et al., 2012).

Figure 1.3: regions encoding airway sensation (Driessen et al., 2016). A) During

functional magnetic resonance imaging (fMRI) scans participants inhaled nebulised capsaicin at two

dose levels; high and low. (I) Analysis of the fMRI scans involved a contrast between the two levels of

capsaicin to identify brain regions with stimulus dependent variation of blood oxygen level-dependent

(BOLD) signals. (II) The insula cortex in both hemispheres, shown here in the left hemisphere (red),

has levels of capsaicin-inhalation activation that are related to the dose. BOLD signal changes extracted

from the left insula clearly show increased levels during high versus low doses of capsaicin. B) Ratings

of urge-to-cough were obtained from participants after inhalation of a low or high dose of capsaicin

during fMRI scanning. (I) Ratings of urge-to-cough after inhalation of capsaicin show some variability

that is not explained by the dose of capsaicin stimulus. Analyses were performed to identify brain

regions where changes in BOLD signal correlated with the variance in urge-to-cough ratings. (II) Urge-

to-cough (perception) dependent activation occurs in the somatosensory cortex bilaterally and is

rendered here in the left hemisphere (blue). The region showing perception-related activation is at the

ventral aspect of the central sulcus and extends into the neighbouring operculum. The graph shows the

correlation between BOLD signal changes from the left somatosensory cortex and corresponding urge-

to-cough ratings.

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In animal studies, inputs to the primary sensory cortex from the airways have been reported (McGovern et al., 2012a) and our laboratory has provided anatomical evidence that the jugular vagal ganglia-Pa5 pathway may represent an important circuit contributing to primary sensory cortical projections (McGovern et al., 2015b). Thus, using a novel cre-conditional HSV1 H129 transynaptic anterograde tracer, our laboratory described differences between the ascending projections of nodose-nTS and jugular-Pa5 afferent pathways in that airway projecting nodose vagal ganglia neurons terminate in the nTS and then input into circuits that are largely involved in autonomic and limbic/paralimbic processing (McGovern et al., 2015b). By contrast, Pa5 neurons in receipt of airway jugular vagal ganglia afferents give rise to ascending circuits that resemble those involved in somatic nociceptive processing (McGovern et al., 2015b).

These observations, made in human and animal studies, collectively led to the hypothesis that the Pa5 is a likely candidate region important for airway afferent processing involved in the urge-to-cough. However, the identity of the specific airway afferents that contribute to the processing of the urge-to-cough remains unknown, as does how this afferent input is synaptically integrated at the level of the Pa5.

Inhalation of airway irritants (refer to Table 1.1) is used to study evoked cough in both humans and animals (Canning et al., 2004; Mazzone et al., 2005; Chou et al., 2008).

However, there are currently no animal assays to study the subjective experience of an urge-to-cough and instead cough has been the sole measure of airway irritation in species other than humans. In other behavioural studies, such as during nociceptive processing leading to the sensation of pain quantifying behaviours of irritation (e.g. the face grimace scale - Akintola et al., 2017), have been useful for understanding discriminative processing. An analogous behavioural approach for investigating airway sensations has

30 | P a g e not been reported but may be a more sensitive and informative measure for understanding the higher brain contribution to airway sensory-evoked behaviours. Perhaps it can relate to an animal’s conscious perception of airway irritation and therefore provide a model for detailed mechanistic studies of airway discriminative processing.

Table 1.1: Commonly used airway irritants to study evoked cough in conscious guinea pigs.

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1.6 Aims and hypotheses

Aim 1: To determine the organisation of the vagal ganglia central termination sites in the brainstem of the guinea pig.

Hypothesis 1: Neurons originating from the nodose vagal ganglia will predominately terminate within subnuclei of the nTS, whereas neurons originating within the jugular vagal ganglia will predominately innervate the Pa5 in the guinea pig.

Aim 2: To characterise the molecular profile of neurotransmission between jugular vagal ganglia sensory neurons and Pa5 second order neurons by investigating the central pharmacology of laryngeal evoked respiratory reflexes.

Hypothesis 2: Pharmacological blockade of the Pa5 will inhibit laryngeal jugular sensory neuron-evoked respiratory reflexes. Due to the high expression of neuropeptides in the jugular vagal ganglia derived airway afferents, compared to nodose airway afferents, it is hypothesised that peptidergic neurotransmission will play a prominent role in mediating airway afferent processing in the Pa5.

Aim 3: To develop a guinea pig model to study behaviours of irritation as a surrogate measure of the urge-to-cough and to implement this model to determine the role of the

Pa5 in behavioural responses to airway irritants in conscious unrestrained guinea pigs.

Hypothesis 3: Guinea pigs will display a range of behavioural responses during inhalation of airway irritants, that can be quantified to provide a useful predictive tool for studying the perception of airway irritations. Lesions of the Pa5 will inhibit evoked cough and associated behavioural responses to inhaled airway irritants that activate jugular vagal ganglia pathways but not those that activate nodose vagal ganglia pathways.

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2. Chapter 2: The role of the paratrigeminal nucleus in vagal afferent evoked respiratory reflexes: A neuroanatomical and functional study in guinea pigs (reproduced exactly from publication Driessen et al., 2015)

2.1 Introduction

The airways and lungs are innervated by heterogeneous populations of vagally-derived sensory neurons, which elicit reflexes and behaviours that contribute to the physiological control of respiration and protect the airways from potentially harmful stimuli (Riccio et al., 1996; Mazzone and Canning, 2002a; Mazzone and Canning, 2002b; Undem et al., 2004). There are two main types of airway sensory neurons that can be readily distinguished based on physiological and morphological properties; myelinated large diameter neurons that are exquisitely sensitive to mechanical stimuli and unmyelinated small diameter nociceptor (chemically sensitive) neurons that are activated by a range of irritant or pro-inflammatory chemicals. Low threshold mechanosensitive neurons are derived exclusively from the nodose vagal ganglia, while nociceptive neurons originate in both the nodose and jugular vagal ganglia (Riccio et al., 1996; Undem et al., 2004).

Indeed, the ganglionic origin of vagal sensory neurons imparts another source of heterogeneity as the nodose and jugular vagal ganglia have distinct embryological origins and in turn are under the control of distinct transcriptional elements (Nassenstein et al., 2010; D'Autréaux et al., 2011). Thus, the nodose and jugular vagal ganglia are derived from the epibranchial placode and neural crest, respectively (Nassenstein et al., 2010;

D'Autréaux et al., 2011).

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Consequently, the neurons comprising these vagal ganglia have differing phenotypes.

That is while the nodose vagal ganglia neurons are typically devoid of neuropeptides they instead express functional purinergic P2X receptors and display a visceral phenotype, while many jugular vagal ganglia neurons instead contain the neuropeptides Substance P and calcitonin gene-related peptide (CGRP) and exhibit a somatic-like phenotype similar to the dorsal root ganglia (Riccio et al., 1996; Undem et al., 2004; Nassenstein et al., 2010; D'Autréaux et al., 2011).

The embryological derivation of vagal sensory neurons not only imparts molecular and functional distinctions to the primary sensory neurons themselves, but also underpins differences in the organisation of the primary afferent projections to the brainstem and their resultant higher order central neural circuitry. Thus, we have recently reported in the rat that nodose vagal ganglia neurons almost exclusively project to the nucleus of the solitary tract (nTS), while the jugular vagal ganglia projects predominately to the paratrigeminal nucleus (Pa5) in the caudal medulla (McGovern et al., 2015b).

Furthermore, in the same study we employed novel anterograde transynaptic tracing using a conditional herpes simplex virus 1 strain H129 (HSV1 H129) to show differential higher order airway vagal projections arising from the nTS and Pa5 (McGovern et al., 2015b).

This previously unappreciated segregation of jugular and nodose neuronal circuits in the brain raises many questions about the functionality of nodose and jugular vagal ganglia sensory pathways and their respective roles in regulating respiratory reflexes and behaviours.

34 | P a g e

In the present study we first set out to determine whether nodose and jugular afferent neurons in guinea pigs display differential terminations in the nTS and Pa5, analogous with the observations we have made in the rat. Subsequently, we sought to assess whether the Pa5 plays any role in respiratory reflexes evoked by activation of jugular vagal ganglia derived laryngeal afferent neurons.

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2.2 Methods

2.2.1 Animals

Animal experiments were approved by an institutional Animal Ethics Committee and conducted on adult Dunkin Hartley guinea pigs of either sex (250–350g, n = 25). Animals were housed in a standard environment and given ad libitum access to food and water.

All efforts were made to ensure that a minimal number of animals were used and that they experienced little discomfort. No notable differences were observed between male and female animals, and as such this comparison did not form a component of the study.

2.2.2 Conventional neuroanatomical tracing

Dual retrograde tracing, with fluorescently conjugated cholera toxin subunit B (CT-B), was conducted to label the vagal ganglia neurons terminating in the guinea pig nTS and

Pa5 using techniques previously reported for rats (McGovern et al., 2015b). In each animal, CT-B594 and CT-B488 (Molecular Probes, Thermo Fisher Scientific) were microinjected into the nTS and Pa5, respectively. Guinea pigs (n =6) were anaesthetised with isoflurane (2.5% in medical oxygen) via a nose cone and their heads were placed into a stereotaxic frame and flexed at a 45° angle. A midline incision was made through the animal's , posterior neck muscles and dura mater to expose the medulla at the level between the occipital bone and C1 vertebra. Using Obex as a reference point (~0.5 mm rostral to the calamus scriptorius), unilateral (n =3 left and n =3 right) microinjections of 2% CT-B (250nl per injection) were made into the nTS (anterior- posterior (AP): +0.2 mm, medial-lateral (ML): +0.2 mm, dorsal-ventral (DV): +0.2 mm) and Pa5 (AP: +0.2 mm, ML: +2.8 mm, DV: +0.2 mm) using a calibrated glass

36 | P a g e micropipette (tip diameter =30μm) connected to a pneumatic pressure injector (Pneumatic

PicoPump; WPI, Sarasota FL). The glass micropipette was left in the injection site for five minutes after injecting the desired volume to prevent leakage of the tracer onto surrounding tissues. Following microinjections, the midline incision was sutured and animals were allowed to recover for seven days.

2.2.3 Tissue harvest and immunohistochemical processing

After seven days animals were overdosed with sodium pentobarbital (100mg/kg i.p.) and transcardially perfused with 200ml of 5% sucrose in 0.1M phosphate buffered solution

(PBS; pH 7.4) and 200ml of 4% paraformaldehyde (PFA) in 0.1M PBS. and nodose and jugular vagal ganglia (which are anatomically distinct in guinea pigs) were removed and postfixed overnight in 4% PFA, then cryoprotected in 20% sucrose at 4°C.

Before further processing, the vagal ganglia were cleaned of excess connective tissue and viewed as wholemounts to confirm the presence of retrogradely traced neurons, following which brainstems and vagal ganglia were frozen in OCT embedding compound and sectioned on a Leica CM1850 UV cryostat. The nodose and jugular vagal ganglia were sectioned at 12μm and thaw mounted onto gelatin-coated slides across six sets while the brainstems were sectioned at 50μm and collected serially in 0.1M PBS.

Sections of brainstem (free floating) and vagal ganglia (slide-mounted) were blocked with

10% goat serum in 0.1M PBS for one hour at room temperature, following which they were incubated overnight in the primary antibody of interest (diluted in 2% goat serum and 0.3% Triton-X 100 in 0.1 M PBS). For the vagal ganglia, vesicular glutamate

37 | P a g e transporter 1 (vGlut1), Neurofilament-160kD, α3- Na+/K+ ATPase, CGRP, and

Substance P immunohistochemistry was performed each on separate sets of slides (see

Table 2.1 for details) as our previous studies have shown that these markers help differentiate distinct subsets of vagal afferents (Mazzone and McGovern, 2008). For brainstems, every alternate section (i.e. every 100μm) was immunostained for the calcium binding protein calbindin to provide morphological detail. After several washes, tissues were incubated in the relevant secondary antibody (Table 2.1) for an hour at room temperature. Sections labelled with biotinylated secondary antibodies were further incubated with streptavidin conjugated to Alexa Fluor 350, (1:200 dilution; Molecular

Probes, Thermo Fisher Scientific). After immunostaining brainstem sections were mounted onto gelatin-coated slides and all slides were then coverslipped with an antifade mounting media (Fluroshield, Sigma Aldrich). Brainstems and vagal ganglia were also removed from four separate perfused fixed animals (i.e. containing no CT-B tracing) and sectioned as described above. These tissues were subsequently immunostained for both

CGRP and Substance P and used for quantifying the relative neuropeptide expression in the two ganglia (nodose vs. jugular) and two brainstem (nTS vs. Pa5) regions of interest.

All tissues were viewed using an Olympus BX51 fluorescent microscope equipped with appropriate filters, and images were captured using an Olympus DP72 camera.

Representative images were assembled in Adobe Photoshop CS6.

*All antibodies were optimised for concentration and specificity before use. This process was run in parallel with negative controls (i.e. omission of the primary antibody). 38 | P a g e

Table 2.1: Primary and secondary antibody specifications

2.2.4 Histology and tracing data analysis

Retrogradely traced CT-B neurons were quantified in the nodose and jugular vagal ganglia sections (ipsilateral to the injection site) by counting CT-B594 and CT-

B488 labelled cells with clearly identifiable nuclei in each vagal ganglia section collected.

Total CT-B positive cell counts for the nodose or jugular vagal ganglia were calculated by summing the cell counts obtained for each animal and then averaging these sums across animals. CT-B traced cells were further classified based on their co-expression of the selected immunomarkers and the percentages of CT-B and immunomarker double

39 | P a g e positive cells per tissue were calculated. For somal size analysis, stored digital images of

CT-B labelled neurons were imported into Image-J software (National Institutes of

Health, Bethesda, Maryland, USA) and cell perimeters were manually traced on screen using a calibrated drawing tool. Only cells with an identifiable nucleus were measured in order to increase the likelihood that perimeters were a reflection of true somal size. A minimum of 100 neurons were measured from both the nodose and jugular vagal ganglia.

There were no notable differences between experiments conducted with CT-B injections into the left vs. right brainstem (n =3 for each) and as such the vagal ganglia cell count data were pooled (n =6).

The brainstem injection sites of CT-B594 and CT-B488 in the nTS and Pa5, respectively were viewed in serial sections 100μm apart to calculate the rostro-caudal spread of injectate both relative to the identified injection site and the anatomical landmark Obex.

Photomicrographs of brainstem sections immunostained for Substance P and CGRP were collected at the same magnification and identical exposure for quantification of immunostaining intensity. Images were imported unmodified into Image-J and pixel density analyses was performed over a standardised area in each image of the nTS and

Pa5 (the area dimensions of the density tool equalled 428μm in the mediolateral direction and 284μm in the dorsoventral direction). A region of no immunostaining within the same image was always quantified to further normalise the data to the level of background pixel density. Total pixel density was defined as the sum of density measurements for a given region obtained from serial sections that approximately spanned the region of CT-B injectate spread, calculated above.

40 | P a g e

Such measurements were calculated for CGRP and Substance P in the nTS and Pa5 from an equal number of sections and data were background corrected and averaged across animals. All histological data are presented as the mean ±SEM and statistical comparisons were made using an unpaired Students t-test with significance set at p ≤ 0.05.

2.2.5 Physiology

Guinea pigs (n =17) were anaesthetised with urethane (1.5 g/kg i.p.), the level of which was tested by assessing the animal’s withdrawal reflex. Animals were placed in a supine position on a thermostatically controlled heating pad. An incision was made along the ventral surface of the neck and the muscles overlaying the trachea were retracted to expose the larynx, trachea and underlying nerves and blood vessels. The left carotid artery was cannulated using polyethylene tubing (internal diameter =0.5mm, outer diameter

=0.9mm) attached to a pressure transducer filled with heparinised saline (50 U/ml) to measure arterial blood pressure (ABP) and heart rate (HR). In addition, the distal extrathoracic trachea was cannulated. The cannula was connected via a sideport to a pressure transducer to measure the tracheal pressure (TP) changes associated with spontaneous respiration. Output from the pressure transducers were filtered and amplified

(NeuroLog Systems, Digitimer, Hertfordshire, UK), digitised (Micro1401 A-D converter,

CED, Cambridge, UK) and recorded using Spike II software (CED, Cambridge, UK) for offline analysis.

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In the guinea pig, the larynx is innervated by nodose-derived afferents principally traveling in the recurrent laryngeal nerves (RLNs) and jugular-derived afferents traveling principally in the superior laryngeal nerves (SLNs; Canning et al., 2004). Accordingly, both RLNs were gently blunt dissected from the larynx and trachea and subsequently cut in order to selectively denervate the nodose afferent innervation of the larynx, leaving intact the jugular-superior laryngeal nerve pathway (see Figure 2.1 for more details). A midline incision through the laryngeal thyroid cartilage was then made to expose the mucosal surface for the placement of a custom in-house built platinum bipolar stimulating electrode.

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Figure 2.1: Schematic representation of the vagal innervation of the guinea pig airways and preparation used in this study. In the guinea pig, the larynx is innervated by nodose (NG) derived afferents (green) principally travelling in the recurrent laryngeal nerves (RLN) and jugular (JG) derived afferents (red) travelling primarily in the superior laryngeal nerves (SLN). In spontaneously breathing anaesthetised guinea pigs, the distal-most portion of the trachea was cannulated and connected to a pressure transducer in order to measure tracheal pressure. In addition, the left carotid artery (CA) was cannulated and attached to a pressure transducer to measure arterial blood pressure and heart rate. The

RLNs were cut bilaterally (red crosses) to remove the nodose afferent innervation to the larynx, leaving intact the jugular-superior laryngeal nerve pathway. A midline incision through the laryngeal thyroid cartilage (dashed line) was then made to expose the mucosal surface for the placement of a custom in- house built platinum bipolar stimulating electrode (represented by +/-). Electrical stimulation of the larynx was used to evoked reflex changes in respiration. Additional abbreviations: VN; .

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After a 20-minute stabilisation period, electrical stimulation of the larynx was performed

(model s48, Grass Instruments) by delivering increasing voltages (0.1–10V) to the larynx at a constant stimulating frequency (32Hz), pulse durations (1ms) and train duration (10s), expected to activate mucosal and deeper laryngeal tissue afferents. The voltage that evoked a reproducible maximum respiratory response (defined as the optimum voltage, which on average =7.6 ± 0.4V) was then used to assess the frequency (1–32Hz) dependency of the evoked response under the same conditions. At the completion of the electrical stimulations, animals were placed into a stereotaxic frame and the brainstem exposed. The GABAA agonist, muscimol (20ng in 150nl) or vehicle (saline, 150nl) was then microinjected bilaterally into either the nTS (n =4 muscimol, n =3 vehicle) or Pa5

(n =4 muscimol, n =4 vehicle) following the same protocol as described above. Animals were immediately returned to a supine position and the stimulating electrode was replaced onto the laryngeal surface in order to repeat the frequency response stimulations at the predefined optimal stimulating voltage. Electrical stimulation following microinjection of either muscimol or vehicle was completed within a period of 30 minutes. In additional animals, the SLNs and the RLNs were cut bilaterally to completely denervate the vagal innervation to the larynx.

Physiological data at each stimulation frequency were calculated from the chart recordings and compared to baseline data (defined as the 60 second period prior to the commencement of the frequency response curve). Respiratory frequency was calculated over the 10 second period of the stimulation and multiplied by six to obtain the equivalent breaths/minute. Mean ABP responses were calculated at the peak effect during stimulation and compared to baseline data.

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All physiological data are represented as the mean ± SEM and differences between groups were tested using a two-way ANOVA with significance set at p ≤ 0.05. In addition, the evoked the maximum physiological effect (Emax) for each experiment was determined prior to vehicle or muscimol injection (defined as the maximum response evoked regardless of the stimulation parameters) and compared to the same stimulation parameters after microinjection of vehicle or muscimol into the nTS or Pa5. These data were compared using a paired t-test with significance set at p ≤ 0.05.

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2.3 Results

2.3.1 Retrograde tracing and immunohistochemical characterisation of vagal sensory neurons

Dual microinjection of fluorescent CT-B488 and CT-B594 conjugates successfully encompassed the target regions in all animals (Figure 2.2A,A′). The rostro-caudal spread of injectate within each nucleus was similar (nTS=0.94 ± 0.11mm and Pa5=1.68 ±

0.25mm) with the centre of the injection located just rostral to Obex (nTS=0.62 ± 0.16mm and Pa5=0.48 ± 0.19mm). The mediolateral spread never exceeded 0.2mm (nTS) and

0.3mm (Pa5) in any animal and as such there was never any overlap between the different injectates delivered to the two brainstem nuclei. Importantly, retrogradely labelled neurons were clearly visible within the nodose and jugular vagal ganglia, albeit with a distinct topographical arrangement, as shown in a representative wholemount image

(Figure 2.2B). Thus, there was a distinct separation of neurons retrogradely labelled from the nTS versus the Pa5 in the nodose and jugular vagal ganglia, respectively. Quantitative cell counts on tissue sections, confirmed that the jugular vagal ganglion had significantly more neurons retrogradely labelled from the Pa5 compared to the nTS (p =0.004; Figure

2.2C). On the other hand, the nodose vagal ganglia had significantly more retrogradely labelled neurons from the nTS compared to the Pa5 (p=0.0001; Figure 2.2C′). There were no systematic differences between vagal ganglia counts collected from animals receiving right versus left brainstem injections (data not shown).

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Figure 2.2: Differential terminations of nodose and jugular vagal ganglia neurons in the nucleus of the solitary tract (nTS) and paratrigeminal nucleus (Pa5). A,A′) Example photomicrograph and corresponding Nissl staining of a caudal brainstem section showing the nTS and Pa5 locations for microinjection of cholera toxin subunit B (CT-B) tracers conjugated with 488 (green) or 594 (red) fluorophores, respectively. B) Fluorescent photomicrograph of a wholemount preparation of the guinea pig vagal ganglia showing neuronal soma retrogradely labelled with CT-B from the nTS (red) and Pa5

(green). C) Quantitative cell counts performed on serial ganglia sections, demonstrating that Pa5 projecting neurons reside in the jugular ganglia (JG) whereas C′) nTS projecting neurons are located within the nodose ganglia (NG). Data represent the mean ± SEM number of neurons quantified from a minimum of 10 tissue sections of vagal ganglia obtained from n=6 separate dual tracing experiments.

**P≤ 0.01 and ***P≤0.001, significantly different pairwise comparison. Additional abbreviations:

10n; vagus nerve, 12; hypoglossal nucleus, IO; inferior olives, SLN; superior laryngeal nerve, Sp5; spinal trigeminal nucleus, Sp5; spinal trigeminal tract.

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Further analyses of the retrogradely traced vagal neurons revealed differences in their immunohistochemical profiles. Thus, there were significantly more peptidergic (CGRP and Substance P expressing) CT-B labelled neurons (presumably nociceptors) in the jugular vagal ganglia compared to the nodose vagal ganglia (p=0.002 and p=0.004 respectively; Figures 2.3D,E). Conversely, the number of CT-B traced neurons expressing neurofilament and α3 Na+/K+ ATPase, markers of myelinated neurons including low threshold mechanoreceptors, was higher in the nodose compared to the jugular vagal ganglia (p=0.012 and p=0.038 respectively; Figures 2.3B,C). However, vGlut1 also expressed by myelinated nodose neurons (Mazzone and McGovern, 2008) was expressed by a similar number of CT-B labelled nodose and jugular vagal ganglia neurons (Figure 2.3A). Quantification of traced cell perimeters confirmed that CT-B labelled a wide range of cell types from the nTS and Pa5 (encompassing the known distribution of somal sizes in the guinea pig vagal ganglia; Mazzone and

McGovern, 2008) and indeed there was no difference in the size distribution of traced cells between the two ganglia (Figure 2.3F), suggesting that our tracing experiments were not skewed toward a certain afferent subtype. Across the total population of vagal ganglia neurons there were significantly more neurons within the jugular compared to the nodose vagal ganglia that expressed the neuropeptides CGRP and Substance P (p<0.0001 and p =

0.03 respectively; Figures 2.4A,A′,A″). Quantification of immunohistochemical staining intensity within the brainstem revealed significantly higher levels of CGRP, but not

Substance P, present in the Pa5 compared to the nTS (p =0.002; Figures 2.4B,B′,B″).

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Figure 2.3: Characterisation of the vagal sensory neurons projecting to the nucleus of the solitary tract and paratrigeminal nucleus. Neurons retrogradely traced from either the nucleus of the solitary tract (red) of the paratrigeminal nucleus (green) were characterised based on their expression of several immunomarkers. Bar graphs demonstrate that A) VGlut1 is common to both nTS and Pa5 projecting vagal sensory neurons. More nTS projecting vagal neurons express B) neurofilament and C) α3 Na+/K+ ATPase. More paratrigeminal projecting neurons express the neuropeptides D) Calcitonin Gene Related Peptide (CGRP) and E) Substance P. Data represent the mean±SEM percentage of traced neurons expressing each immunomarker quantified from a minimum of 10 tissue sections of vagal ganglia obtained from n=6 separate dual tracing experiments. *P≤0.05 and **P≤0.001 significantly different pairwise comparison. F) The histogram shows that the distributions of traced neuron somal sizes are comparable between both nTS and Pa5 projecting vagal sensory neurons. These data represent pooled somal sizes of at least 100 neurons obtained from n=6 separate experiments. The relative frequency is the proportion of the total pooled population with a given somal size.

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Figure 2.4: Expression of neuropeptides calcitonin gene related peptide (CGRP) and Substance

P (SP) in the vagal sensory ganglia and brainstem. Representative photomicrographs showing differential expression of CGRP in the A) nodose ganglia, A′) jugular ganglia, B) nucleus of the solitary tract (nTS) and B′) paratrigeminal nucleus (Pa5). Bar charts show quantitative analysis of

CGRP and SP expression in A′′) vagal ganglia and B′′) brainstem. Neuropeptide expression is more abundant in the jugular than the nodose ganglia. SP expression in the brainstem is similar in the nTS and Pa5, whereas expression of CGRP is significantly higher in the Pa5 compared to the nTS. Data represent the mean±SEM number of neurons A′′) or intensity of immunostaining B′′) quantified from a minimum of 10 tissue sections of vagal ganglia obtained from n=6 separate experiments. *P≤0.05 and **P≤0.01 significantly different pairwise comparison. Additional abbreviations: dmnX; dorsal motor nucleus of the vagus nerve, sp5; spinal trigeminal tract.

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2.3.2 Functional studies

Electrical stimulation of the larynx, in animals with RLN transections, evoked respiratory slowing resulting in complete apnoea for the stimulus duration at higher stimulus voltages

(above 8V). Rarely (less than 5% of occasions) animals coughed in response to electrical stimulation, consistent with the notion that the RLN transection removes most of the nodose afferent innervation of the larynx (Canning et al., 2004). At optimum stimulus intensities, increasing stimulus frequency evoked a frequency dependent reduction in respiratory rate as well as a modest decrease in ABP, while no effect was seen in heart rate (Figures 2.5A,B; Table 2.2). Microinjection of saline into the nTS or the Pa5 did not alter the frequency dependent fall in respiratory rate (Figure 2.5 B) or ABP (Table 2.2).

Bilateral microinjection of muscimol into the nTS did not alter basal respiration

(Figure 2.5B), but caused mean ABP to significantly decrease on average from 61.2± 4.18 to 44.8±4.66 mmHg (p =0.005). However, muscimol in the nTS did not alter the reduction in respiratory rate evoked by laryngeal stimulation in RLN transected rats (Figure 2.5 and

Table 2.2). The same dose of muscimol microinjected into the Pa5 neither altered basal respiratory rate (Figure 2.5B) nor ABP (63.6 ± 4.0 vs. 56.8 ± 2.8 mmHg before and after muscimol injection) but almost abolished the respiratory and ABP responses associated with laryngeal stimulation (Figure 2.5 and Table 2.2). Thus, the Emax for the respiratory slowing did not differ between pre (mean =10.5 ± 2.87 breaths/minute) and post (mean

=9.0 ± 1.73 breaths/minute) muscimol administration to the nTS (Table 2.2) but was significantly different between pre (mean =6.0 ± 3.46 breaths/minute) and post (mean

=25.5 ± 2.87 breaths/minute) muscimol administration to the Pa5 (p =0.001; Table 2.2).

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In two animals, bilateral denervation of both RLNs and SLNs completely abolished changes in respiration evoked by electrical stimulation of the larynx, confirming the vagal dependency of the evoked response (data not shown).

Figure 2.5: Laryngeal evoked respiratory reflexes are dependent upon a jugular-

paratrigeminal neural circuit. A) Representative physiological recording showing the slowing of

respiration and reduction in arterial blood pressure (ABP), without any change in heart rate (HR),

during electrical stimulation of the larynx in RLN transected animals. B) Mean frequency-dependent

effects on respiratory (Resp) rate before and after bilateral microinjection of vehicle or muscimol

into the nucleus of the solitary tract (nTS) or paratrigeminal (Pa5) nucleus. Each graph is the

mean±SEM of four animals *P≤0.05 significantly different responses at 4-32Hz compared to the

corresponding response before injection. Additional abbreviations: Au; arbitrary units, BPM; beats

per minute, TP; tracheal pressure.

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Table 2.2: Maximum laryngeal stimulation-evoked respiratory and cardiovascular responses (Emax).

Baseline (prior to any treatments of stimulation) respiratory rate (breaths/minute) = 46.93±1.69; blood pressure

(mmHg) = 60.34±2.19; heart rate (beats/minute) = 301.93±8.06. ** Significantly different compared to before

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2.4 Discussion

We have previously reported our investigations into the organisation of airway sensory neural circuitry in the rat brain, showing that anatomically distinct central circuits arise from nodose and jugular vagal ganglia airway afferents. Thus, in rats, nodose and jugular vagal afferent neurons specifically innervate second order neurons in the nTS and Pa5, respectively reflecting a previously unrecognised anatomical divergence of vagal afferent inputs to the brainstem (McGovern et al., 2015b). Importantly, while the role of vagal afferent processing in the nTS has been investigated in significant detail, the functionality of the jugular-Pa5 pathway with respect to responses evoked by airway irritation has not been previously explored. In the present study, we first confirmed that this anatomical segregation of vagal afferent circuitry in the brainstem is similarly expressed in the guinea pig and is therefore not peculiar to the rodent vagal system. Indeed, whilst heterogeneous nodose and jugular afferent neurons project to both brainstem integration sites, jugular vagal ganglia afferents can be readily differentiated from nodose vagal ganglia afferents both in the sensory ganglia and in the brainstem by the expression of the neuropeptide

CGRP. Secondly, we demonstrate that jugular vagal ganglia afferent mediated respiratory responses following laryngeal stimulation are abolished by inhibition of the Pa5 and not the nTS. Collectively, these data provide support for the existence of two parallel yet distinct airway vagal sensory processing pathways that likely differentially contribute to respiratory responses evoked by airway irritant stimuli.

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2.4.1 Evidence that vagal ganglia afferents have multiple distinct termination sites in the brainstem

A variety of studies confirm that airway sensory neurons originate in both the nodose and jugular vagal ganglia and that the distinct embryological origins of these ganglia confer the constituent neurons with meaningful differences in their functional, neurochemical, neuroanatomical and molecular expression characteristics (Riccio et al., 1996; Kollarik and Undem, 2002; Undem et al., 2004; Kwong et al., 2008; Nassenstein et al., 2010;

D'Autréaux et al., 2011; McGovern et al., 2015a). Indeed, investigations of the development of the cranial ganglia have identified transcriptional elements such as

Phox2b and Runx1 that determine a visceral versus somatic fate of ganglia neurons.

Specifically, nodose neurons represent visceral afferents inasmuch as they develop from an epibranchial placode lineage that is dependent upon transient developmental Phox2b expression, whereas jugular neurons, like the spinal dorsal root ganglia, reflect somatic neurons derived from neural crest cells under the control of Runx1 (Nassenstein et al., 2010; D'Autréaux et al., 2011). This fundamental source of afferent heterogeneity may in turn underpin differences in the organisation of nodose and jugular afferent terminals in the brainstem, which show preferential innervation of the nTS and trigeminal nuclei, respectively. Thus, our group recently showed in the rat that CT-B microinjections into the region of the Pa5 resulted in the retrograde labelling of jugular, but not nodose vagal ganglia neurons, whereas a comparable injection into a caudal lateral site in the nTS resulted in retrograde labelling of the nodose, but not jugular vagal ganglia neurons

(McGovern et al., 2015b). Furthermore, in several studies we have shown that both extra- and intrapulmonary injections of the anterograde transynaptic neurotropic virus HSV1

H129 results in time-dependent dorsolateral nTS and Pa5 infections that develop with

55 | P a g e comparable kinetics indicative of two distinct vagal pathways terminating in the brainstem (McGovern et al., 2015a).

In the present study, CT-B injections into the caudal lateral nTS and Pa5 of the guinea pig resulted in the selective retrograde labelling of nodose and jugular afferent neurons, respectively. Indeed, the anatomical specificity of the CT-B labelling from the two injection sites was extraordinary, with few nTS-projecting jugular or Pa5-projecting nodose neurons identified. Although our pathway tracing was not airway afferent specific

(i.e. the peripheral terminals of the retrogradely labelled sensory neurons were not identified) the striking segregation of nodose and jugular afferent terminations in brainstem regions that receive airway vagal afferent input (McGovern et al., 2015a;

McGovern et al., 2015b) would argue that the data can be confidently extrapolated to conclude that airway vagal afferents share this termination pattern. Thus, these results along with our previous studies, argue for parallel airway sensory processing pathways involving the jugular vagal ganglia projecting to the Pa5 and the nodose vagal ganglia projecting to the nTS. Furthermore, the data suggest that this anatomical organisation is conserved across species, at least for the specific brainstem loci targeted by the current microinjection strategy. However, we cannot rule out if other nTS sites receive jugular afferent inputs, such as the commissural nTS from which some retrogradely traced jugular neurons have been identified (Mazzone and Canning, 2002a). Even in our own studies we see a small but consistent population of jugular projections to the nTS in both the rat

(McGovern et al., 2015b) and guinea pig (present study). The reason why some jugular neurons may differ to others with respect to their central projections is presently unclear.

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2.4.2 CGRP expression distinguishes jugular and nodose sensory neurons in the vagal ganglia and brainstem

The present neuroanatomical studies were not intended to differentiate between vagal ganglia neurons terminating in different peripheral tissues. Nevertheless, important insight into the heterogeneity of vagal afferent neurons can be gained from such studies.

Thus, a heterogeneous population of sensory neurons innervates the airways, delineated by their vagal ganglia origin and neurochemical profile. For example, in the extrapulmonary airways (larynx, trachea, and main bronchi) the majority of nodose derived neurons express medium or large molecular weight neurofilament proteins, the

α3 subunit of Na+/K+ ATPase and vGlut1, while many of the large airway projecting neurons derived from the jugular vagal ganglia are characterised by the expression of the neuropeptides Substance P and CGRP (Undem et al., 2004; Mazzone and

McGovern, 2008). Consistent with this, we found that more of the nodose neurons that were retrogradely labelled from the nTS expressed neurofilament and α3 Na+/K+ ATPase

(but not vGlut1) compared to jugular neurons labelled from the Pa5. Whereas, retrogradely traced jugular neurons were more often immunoreactive for CGRP and

Substance P than those in the nodose vagal ganglia. This differential expression is unlikely due to selectivity of the CT-B neural tracer for different subsets of nodose and jugular neurons, as the profile of somal sizes of traced neurons was not different between ganglia.

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We further reasoned that the generalised differential molecular expression noted in the ganglia may be similarly conserved in the central terminals of the vagal afferents innervating the nTS and Pa5. Given that neurofilaments and α3 Na+/K+ ATPase are widely expressed in central neural tissue (Trojanowski et al., 1986; McGrail et al., 1991), we restricted this investigation to the neuropeptides that readily differentiate nodose from jugular neurons. Consistent with previous studies in other species (Franco-Cereceda et al., 1987; Sugimoto et al., 1997) and with the notion that jugular vagal ganglia neurons specifically project to the Pa5 (present study), we noted dense CGRP staining in the Pa5 that was significantly greater than that in the nTS. However, it is unlikely that vagal ganglia neurons represent the only afferent source of CGRP to the Pa5 as trigeminal ganglia neurons also express the neuropeptide (Eberhardt et al., 2008). By contrast, the expression of Substance P was not significantly different between the two brainstem nuclei. Indeed, Substance P staining intensity in the nTS appeared disproportionately high relative to the number of Substance P-positive neurons identified in the nodose vagal ganglia, perhaps reflecting alternative origins of Substance P terminals in the nTS.

Intrinsic neurons or the terminals of nTS inputs that arise from other peripheral or central neuronal populations can express Substance P (South and Ritter, 1986; Gallagher et al., 1992), which may explain why vagal denervation does not abolish Substance P immunoreactivity in the nTS (Gillis et al., 1980; Kawai et al., 1989). Given these findings it is tempting to speculate that CGRP may play an important role in the integration and/or sensitisation of jugular vagal ganglia afferent responses in the Pa5. Indeed, CGRP has been implicated in trigeminal nociception, orofacial pain, analgesic sensitisation and migraines (Devesa et al., 2014; Bigal et al., 2015; Romero-Reyes et al., 2015), and a comparable pro-nociceptive role for the neuropeptide in vagal-evoked responses seems

58 | P a g e plausible given the somatic nature of the juglar-Pa5 pathway. Nevertheless, this putative role for CGRP awaits further exploration.

2.4.3 A jugular vagal ganglia-Pa5 neural circuit contributes to laryngeal evoked respiratory reflexes

Electrical, mechanical or chemical stimulation of the guinea pig larynx has been shown to evoke a variety of respiratory and cardiovascular responses, including cough, apnoea/respiratory slowing, bronchoconstriction, and hypotension (Mazzone and

Canning, 2002a; Mazzone and Canning, 2002b; Canning et al., 2004; Chou et al., 2008).

Electrophysiological and neuroanatomical tracing studies have confirmed that the larynx receives both nodose and jugular afferent innervation, although the route via which nodose and jugular axons reach the larynx is not uniform (Canning et al., 2004; Mazzone et al., 2009). In electrophysiological mapping studies 88% of nodose vagal ganglia neurons (almost all are low threshold mechanoreceptors) innervate the rostral guinea pig trachea and larynx via the RLNs, while 84% of jugular vagal ganglia neurons (all C- and

Aδ-fibre nociceptors) innervate the same airway region via the SLNs (Canning et al., 2004). This is mimicked in retrograde tracing studies of the same airway segment in which prior unilateral sectioning of an RLN or SLN significantly and specifically reduces the number of retrogradely labelled neurons in the nodose and jugular vagal ganglia, respectively (Mazzone et al., 2009).

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In the present study, we exploited this differential anatomical arrangement of the afferent innervation to the larynx and bilaterally cut the RLNs in order to generate a physiological preparation in which the majority of the nodose afferent innervation was removed while the majority of the jugular innervation remained intact. In this preparation, almost none of the animals coughed in response to electrical stimulation of the larynx, which should not be interpreted as jugular vagal ganglia neurons not evoking cough, given the well described suppressive effect of anaesthesia on cough mediated by nociceptor pathways

(Canning et al., 2004). Rather the findings are consistent with the notion that nodose cough receptor innervation (which is not as sensitive to the suppressive effects of anaesthesia) was removed (Tsubone et al., 1991; Canning et al., 2004; Mazzone et al., 2005; Mazzone et al., 2009; Chou et al., 2008). In our preparation, electrical stimulation resulted in intensity and frequency dependent reduction in respiratory output, leading to apnoea at higher stimulus parameters suggesting that activation of laryngeal jugular afferents may be inhibitory to respiratory drive. This was accompanied by a modest but consistent fall in ABP, but no change in HR, perhaps reflective of a diminished respiratory dependent venous return during the apnoeic response. Compatible with these observations, Chou et al. (2008) previously reported in anaesthetised guinea pigs that stimuli selective for jugular vagal afferents evoked respiratory slowing and apnoea, while stimuli selective for nodose (mechanoreceptor) vagal afferents evoked cough and/or tachypnoea. Of particular interest, blocking the nTS did not alter laryngeal stimulation evoked reductions in respiration, consistent with the notion that nodose afferent pathways were denervated and unlikely involved in this reflex. Indeed, a significant fall in baseline

ABP following microinjection of muscimol into the nTS confirms the adequacy of the dosing regime for producing neuronal inhibition. By contrast, laryngeal-evoked

60 | P a g e respiratory slowing in RLN transected animals was abolished and the hypotension absent when the Pa5 was selectively inhibited by an equivalent dose of muscimol.

We don't know the identity of the vagal afferents mediating the evoked responses in the present study, other than they are of jugular vagal ganglia origin. In guinea pigs, most if not all jugular vagal ganglia afferents are chemically sensitive nociceptors, although both unmyelinated and myelinated subsets exist (Riccio et al., 1996). Consistent with this capsaicin application to the larynx evokes apnoea. However, other types of laryngeal afferents also exist, which are perhaps best characterised in larger species such as dogs where subtypes responding to temperature, pressure, “tracheal tug” during breathing or irritants (e.g. hypo-osmolar solutions) are definable in addition to those responding to classic nociceptor stimuli (reviewed in Sant'Ambrogio et al., 1995). Many of these afferents are nodose-derived mechanoreceptors yet they reach the larynx via the SLNs, suggesting that neither the anatomical pathway for innervation nor the physiological complexity of laryngeal afferent endings in the guinea pig mirrors that of the dog.

Accordingly, this makes drawing conclusions about the role of these other subsets in evoked responses difficult. Regardless of the identity of the primary afferents involved, we interpret our current data as strong evidence for a jugular vagal ganglia reflex circuit that can modify respiratory control via synaptic integration in the Pa5.

The present data may also point toward a role for the Pa5 in the regulation of autonomic outflow to the airways and other organs. Thus, chemical stimulation of the laryngeal mucosa can evoke profound bronchoconstrictor responses, which may in part reflect activation of the jugular vagal ganglia-Pa5 circuit. This was not assessed in the present

61 | P a g e study, but in previous studies in anaesthetised guinea pigs we have shown that laryngeal application of capsaicin evokes a bronchoconstrictor response which differs strikingly compared to that when capsaicin is inhaled into lower regions of the airways (Mazzone and Canning, 2002a; Mazzone and Canning, 2002b). Indeed, brief (bolus) application of capsaicin onto laryngeal mucosa in anaesthetised guinea pigs evokes an initial transient bronchodilation lasting one to five minutes that is replaced by a slowly developing increase in airway smooth muscle tone peaking at 75–100% of the maximum attainable response at 30–60 minutes after the initial bolus capsaicin challenge. When inhaled (in preparations bypassing the larynx) only a bronchoconstriction is observed and the response develops and decays quickly, typically lasting for the duration of the challenge only. Whether these differential responses relate to the nodose and jugular afferent circuits described herein is not known, but conceivably possible. Furthermore, studies from other groups have already demonstrated vagal afferent inputs from some baroreceptor afferents terminate in the Pa5 playing a role in cardiovascular control (Junior et al., 2004) and it is intriguing to speculate that these may also be of jugular vagal ganglia origin.

2.4.4 Conclusions

Our past and present data supports a previously unrecognised role of the Pa5 in mediating laryngeal evoked respiratory reflexes, specifically, those reflexes driven by jugular vagal ganglia derived afferent neurons. Given the somatic nature of the jugular vagal ganglia and the Pa5, it is possible however that this pathway is important for more than just bulbar mediated respiratory reflexes. Strong connectivity of the Pa5 to the somatosensory thalamus (Saxon and Hopkins, 1998; McGovern et al., 2015b) is indicative of jugular

62 | P a g e vagal ganglia neurons providing input to an ascending circuit that may underpin more complex airway sensations. Nevertheless, how these higher order circuits contribute to behaviours associated with airway irritation remain to be elucidated.

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3. Chapter 3: Reflex regulation of breathing by the paratrigeminal nucleus via multiple bulbar circuits (reproduced exactly from publication Driessen et al., 2018)

3.1 Introduction

The respiratory tract is innervated by heterogeneous populations of sensory neurons that respond to a diverse range of chemical and mechanical stimuli, and in turn initiate reflexes and behaviours that optimise breathing and protect the airways. Most of these airway sensory neurons originate from the nodose and jugular vagal ganglia, two embryologically distinct tissues derived from the epibranchial placode and neural crest respectively (Kummer et al., 1992; Nassenstein et al., 2010; D'Autréaux et al., 2011). This embryological distinction appears to drive anatomical, molecular and physiological differences between the airway sensory neurons that comprise the two vagal ganglia

(Riccio et al., 1996; Undem et al., 2004; Kwong et al., 2008; Nassenstein et al., 2010;

Lieu et al., 2011). Anatomically, for example, the extrapulmonary airways (larynx, trachea and mainstem bronchi) are predominately in receipt of afferents from the jugular vagal ganglia, while the intrapulmonary airways and lungs receive substantially more nodose vagal ganglia projections (Undem et al., 2004; McGovern et al., 2015a). In addition, we have shown in both rat (McGovern et al., 2015b) and guinea pig (Driessen et al., 2015) that the central terminal projections of nodose and jugular vagal ganglia neurons are different. Thus, nodose vagal ganglia neurons almost exclusively terminate centrally in the medullary nucleus of the solitary tract (nTS) and integrate with higher brain networks important for limbic and pre-autonomic control, while those from the jugular vagal ganglia project to the medullary paratrigeminal nucleus (Pa5) and are relayed to thalamocortical regions important for somatosensory processing (Driessen et al., 2015; McGovern et al., 2015b). Although the integration of nodose respiratory vagal

64 | P a g e afferents in the nTS has been studied extensively, respiratory vagal sensory processing in the Pa5 has escaped significant attention.

The Pa5 comprises the dorsal component of an interstitial system of neurons embedded in the medullary spinal trigeminal tract (Chan-Paley 1978; Phelan and Falls, 1989; Saxon and Hopkins, 1998; Koepp et al., 2006; Panneton et al., 2017), and has been shown to contribute to the processing of nociceptive and baroreceptive inputs (Carstens et al., 1995;

Lindsey et al., 1997; Zhou et al., 1999; Yu et al., 2002; Junior et al., 2004; Koepp et al.,

2006). Conventional anterograde and retrograde tracing studies in the rat and cat have identified inputs to the Pa5 from the airways, including the larynx, as well as direct projections of Pa5 neurons to the nucleus ambiguus, reticular nuclei, nTS, facial nucleus,

Kölliker-Fuse nucleus and the parabrachial nuclei (Panneton and Burton, 1985; Saxon and Hopkins, 1998; de Sousa Buck et al., 2001; Pinto et al., 2006), which are implicated in respiratory pattern formation and modulation, and associated orofacial behaviours

(Dick et al., 1994; Alheid et al., 2004; Dutschmann and Herbert, 2006; Bianchi and

Gestreau, 2009; Dutschmann and Dick, 2012; Bautista and Dutschmann, 2014).

Stimulation of the larynx evokes a diverse range of nodose and jugular vagal ganglia- dependent airway defence responses that include swallow, apnoea and cough (Canning et al., 2004; Ishikawa et al., 2005; Tsujimura et al., 2013) and we previously reported that laryngeal-induced apnoea in guinea pigs is absent following pharmacological inhibition of the Pa5 (Driessen et al., 2015).

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In the present study we set out to expand our understanding of vagal afferent processing in the Pa5 by carefully defining the anatomical connectivity of Pa5 neurons and characterising in detail the pharmacology of the laryngeal afferent inputs to this nucleus.

The findings identify novel complexity to sensory processing within the Pa5 and support the assertion that this nucleus is a functionally important regulator of laryngeal vagal afferent control.

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3.2 Methods

3.2.1 Animals

Animal experiments, approved by an accredited institutional Animal Ethics Committee, were conducted on adult Dunkin Hartley guinea pigs of either sex (n=82, weight range=

300-1000g). Anatomical and physiological data collected from male and female guinea pigs were not different and as such data reflects pooled outcomes from both sexes.

Animals were housed in a standard environment and given ad libitum access to water and food.

3.2.2 Neuroanatomical studies

Conventional and viral tracing of vagal afferents: Guinea pigs were anaesthetised with isoflurane (2.5% in medical oxygen) via a nose cone and were placed in a supine position on a thermostatically controlled heating pad. Retrograde tracing with fluorescently conjugated cholera toxin subunit B (CT-B594, Molecular Probes, C34777) was employed to identify the subpopulations of vagal ganglia neurons that innervate the laryngeal mucosa, following methods described for tracheal afferents (Springall et al.,

1987; Riccio et al., 1996; Hunter and Undem, 1999). A small incision was made along the ventral surface of the neck and the muscles overlaying the larynx were retracted. A

Hamilton syringe containing 5µl of CT-B594 (2mg/ml in 0.9% saline) was inserted into the laryngeal lumen, distal to the cricotracheal ligament, ensuring that the syringe bevel was facing towards the mucosal surface (n=5; Figure 1A). Tracer was injected over the course of 30 seconds and after injection the syringe was left in place for five minutes (to prevent backward leakage). Incisions were sutured and animals allowed to recover for 12 days.

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In an additional set of experiments, vagal ganglia neuron termination patterns in the guinea pig brainstem were qualitatively assessed by ganglionic neuron transduction of enhanced Green Fluorescent Protein (eGFP) using an adeno-associated viral vector

(AAV8 TRUFr eGFP, UNC Vector Core, Lot #AV5734B, Viral titre 6.3x10e12). AAV8 serotypes display modest retrograde mobility (Kollarik et al., 2010) and as such can be used to transduce both jugular and nodose neurons by injecting into the nodose vagal ganglia. The animals (n=4) were surgically prepared as outlined above but instead the vagus nerve was identified and blunt dissected from the surrounding tissues, carotid artery and hypoglossal nerve to allow access to the nodose vagal ganglia. Using a micromanipulator, a calibrated glass micropipette (30µm tip diameter) connected to a pneumatic pressure injector (Pneumatic PicoPump; WPL, Sarasota FL), was positioned parallel to the nerve for clean insertion into the nodose vagal ganglia. 2µl of AAV8

TRUFr-eGFP (AAV-eGFP) was slowly injected unilaterally and swelling of the ganglia was used as an indicator of adequate injection (Figure 3.1A schematic). Following injection, the micropipette was left in place for an additional five minutes before the incision was sutured and the animal allowed to recover for four weeks.

Anterograde and retrograde tracing of the Pa5: Guinea pigs were anaesthetised with isoflurane (2.5% in medical oxygen) via a nose cone and their heads were placed into a stereotaxic frame at a 45º angle. A midline incision was made through the skin, posterior neck muscles and dura mater to expose the medulla at the level between the occipital bone and C1 vertebra. Anterograde tracing with Biotinylated Dextran Amine (BDA) was employed to visualise the terminal projections of Pa5 neurons. Using calamus scriptorius as a reference point (~0.5mm caudal to Obex and at Bregma -17.3mm), unilateral microinjections of 250nl of 10% BDA (Sigma Aldrich, B9139) were made into

68 | P a g e the Pa5 (n=12) using a calibrated glass micropipette (30µm tip diameter) connected to a pneumatic pressure injector (Pneumatic PicoPump; WPL, Sarasota FL). After injection, the glass micropipette was left in place for five minutes to prevent leakage of the tracer before suturing the incision and allowing the animals to recover for 12 days. It is important to note that microinjections of BDA were made separately into the caudal, central or rostral Pa5 (see Table 3.1 for stereotaxic coordinates), based on previous analyses in the rat (Pinto et al., 2006). This rostro-caudal distinction was made for operationalisation purposes to best facilitate anatomical and functional studies of the Pa5.

In a series of separate experiments, we performed dual CT-B retrograde tracing from brainstem regions defined as having both projections from the Pa5 (results from BDA tracing above) and known roles in respiratory control. CT-B594 and CT-B488 were unilaterally microinjected (each 250nl of 2mg/ml CT-B), one into the Kölliker-Fuse nucleus and the other into the ventral lateral medulla (n=4), or one into the Kölliker-Fuse nucleus and the other into the lateral parabrachial nucleus (n=3). Injections into the ventral lateral medulla were performed as described above, using calamus scriptorius as a reference landmark (refer to Table 3.1 for stereotaxic co-ordinates). For pontine injections, the head was positioned flat in a stereotaxic frame and holes were drilled in the skull for microinjections at the corresponding coordinates relative to Bregma (Table

3.1). After each microinjection of CT-B, the micropipette was left in place for five- minutes to limit leakage into the surrounding sites. Wounds were sutured and animals allowed to recover for 12 days.

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Table 3.1: Stereotaxic co-ordinates for brainstem microinjection

* The Pa5 was divided into three subregions encompassing the rostro-caudal extent of this nucleus

to facilitate more precise anatomical and physiological investigations (de Sousa Buck et al., 2001;

Pinto et al., 2006; Caous et al., 2001). Co-ordinates relative to calamus scriptorius. #Co-ordinates

relative to Bregma.

3.2.3 Tissue harvest, immunohistochemical processing and microscopy

After the required recovery periods, animals were overdosed with sodium pentobarbital

(100 mg/kg i.p.) followed by transcardial perfusion with 150ml of 5% sucrose in 0.1M phosphate buffered saline (PBS; pH 7.4) and 200ml of 4% paraformaldehyde (PFA) in

0.1M PBS. Brains, vagal ganglia and the larynx were dissected. Brains were postfixed overnight in 4% PFA and then cryoprotected in 20% sucrose at 4⁰ Celsius, while the sensory ganglia and larynx were stored immediately in 20% sucrose at 4⁰ Celsius. In the case of tracing from the larynx, prior to further processing, both the larynx and vagal ganglia were cleaned and initially viewed as wholemounts to confirm the presence of the injection site as well as to identify retrogradely traced neurons in the vagal ganglia. In

70 | P a g e addition, the vagal ganglia were also viewed as wholemounts following ganglionic injection of AAV-eGFP to confirm successful injections. All tissues were frozen in OCT embedding compound and cryostat cut sections were either thaw mounted onto gelatin- coated slides across six sets (vagal ganglia, 14µm section thickness), or collected serially in 0.1M PBS (brain, 50µm section thickness). An additional four animals were perfused for tissue harvest and immunohistochemical processing of the rostro-caudal extent of the

Pa5 to identify the neuronal content in this region.

Brain (free floating) and vagal ganglia (slide-mounted) sections were blocked in 10% goat or donkey serum in 0.1M PBS for one hour at room temperature before incubating in the primary antibody of interest (diluted in 2% goat or donkey serum and 0.3% Triton-

X 100 in 0.1M PBS) for 24-48 hours (see Table 3.2 for specific antibody details). Tissue sections were then washed three times with 0.1M PBS and incubated in the corresponding secondary antibody for one hour at room temperature. Immunostaining for Forkhead box protein P2 (FoxP2) to identify the Kölliker Nucleus (Stanic et al., 2018), choline acetyl transferase (ChAT) to identify the nucleus ambiguus (McGovern and Mazzone, 2010) and neurokinin 1 (NK1) receptor on brain sections was amplified using a tyramide signal amplification kit (Tyramide SuperBoost Kit Alexa Fluor 488-Streptavidin, Thermo

Fisher, B40932), following the manufacturer’s instructions (see Table 3.2 for specific antibody details). Tissues containing BDA were further incubated with streptavidin conjugated to Alexa Fluor 594 (strep-594; 1:300) to reveal BDA filled terminal projections. Brain sections were mounted onto gelatin-coated slides and coverslipped with an antifade mounting media (Fluroshield, Sigma Aldrich, F6182). All tissues were visualised under appropriate filter cubes using an Olympus BX51 fluorescent microscope and/or a Leica DM6B LED microscope and images were captured using an Olympus

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DP72 and/or a Leica DFC7000T camera. Representative images were assembled in

Adobe Photoshop CS6.

Table 3.2: Primary antibody specifications

3.2.4 Analysis of neuroanatomical studies

Immunohistochemical labelling of retrogradely traced laryngeal neurons in the jugular vagal ganglia were quantified by counting immunomarker positive and negative CT-B594 positive neurons with clearly identifiable nuclei in each section. Neuronal somal size analyses were conducted by capturing images of CT-B594 traced neurons and measuring their perimeter using a calibrated measuring tool on the Olympus BX51 fluorescent microscope software. These data are presented as frequency distribution histograms for each immunohistochemical marker. The laryngeal traced sensory neurons were characterised by immunostaining for sensory neuron markers including the neuropeptides

*All antibodies were optimised for concentration and specificity before use. This process was run in parallel with negative controls (i.e. omission of the primary antibody). 72 | P a g e calcitonin gene-related peptide (CGRP) and Substance P, that are characteristic of unmyelinated C-fibre afferents, as well as by the expression of markers of A-fibre afferents (Hermes et al., 2015), which include neurofilament-160kD (NF-160kD) or vesicular glutamate transporter 1 (vGlut1). These distinctions were confirmed by quantitatively assessing the co-expression of Substance P or NF-160kD with vGlut1 on a separate set of (untraced) jugular vagal ganglia sections. Counts of neurons expressing the pan neuronal marker NeuN, the calcium binding protein calbindin D-28k (calbindin) or NK1 receptor (n=5) were conducted on representative brainstem slices for three separate levels (caudal, central and rostral) of the Pa5, and where appropriate statistically compared using an unpaired student’s t-test. AAV-eGFP transduction efficiency was determined by quantifying the proportion of eGFP positive neurons in the nodose and jugular vagal ganglia. The central projections of vagal afferents revealed by AAV-eGFP transduction and subsequent anti-GFP immunohistochemistry are presented as representative photomicrographs only.

BDA and CT-B injection sites in brainstem target nuclei were assessed in serial sections

(100µm intervals) to identify the central injection locus and the rostro-caudal spread of injectate. 3 of the 12 experiments were excluded due to evidence of injectate outside of the Pa5. BDA-labelled Pa5 projections were subsequently analysed throughout the brainstem using a three-point weighted density scale, similar to that previously employed to analyse anterograde tracing in the rat (McGovern et al., 2015b). Regions of interest across multiple serial tissue sections per animal were assessed at 20x magnification to assign a score that approximates the number of traced terminals within the region 0=0 fibres, 1=1-50 fibres (sparse labelling), 2=50-300 (intermediate labelling), 3 = >300 fibres

(dense labelling). An overall peak density score was determined for each region for each

73 | P a g e animal to allow for semi-quantitative comparisons of Pa5 projections across brain regions and to identify those with the most robust labelling. Data are presented graphically in a density connectome diagram in which the weight of the interconnecting line between nuclei reflects the semi-quantitative density of traced terminals, as defined above. In dual retrograde tracing studies, single and double labelled neurons in the Pa5 were quantified by counting CT-B594 and CT-B488 positive cells with a clearly identifiable nucleus in each section of the Pa5, across its rostro-caudal extent. Traced neuron counts were summed across tissue sections and animals to allow a percentage of dual traced neurons relative to the total number of traced neurons to be calculated for each pair of injected regions.

3.2.5 Physiological studies

In the guinea pig, the laryngeal mucosa is innervated by jugular afferents travelling mostly within the superior laryngeal nerves (SLNs) and nodose vagal ganglia afferents travelling mostly within the (RLNs; Canning et al., 2004). Thus, surgical transection of the recurrent laryngeal nerves conveniently generates a laryngeal airway preparation that is almost exclusively innervated by jugular afferent fibres (Driessen et al., 2015). All physiological experiments described below were conducted in animals following bilateral resection of the recurrent laryngeal nerves.

Laryngeal stimulation-evoked respiratory responses in anaesthetised guinea pigs:

Guinea pigs were anaesthetised with urethane (1.5g/kg i.p.), the level of which was determined to be adequate by assessing the palpebral and toe withdrawal reflexes.

Animals were placed supine on a thermostatically controlled heating pad. A midline incision was made along the ventral surface of the neck and the muscles were retracted to

74 | P a g e expose the larynx, trachea and underlying nerves and blood vessels. RLN transection was performed by first blunt dissection of the nerves away from the trachea at the level of the third tracheal ring and then cutting and removing a 1cm segment of each nerve to ensure complete denervation. The left carotid artery was cannulated using polyethylene tubing

(internal diameter=0.5mm and outer diameter=0.9mm) attached to a pressure transducer filled with heparinised saline (50 U/ml, Sigma Aldrich) to measure arterial blood pressure

(ABP) and heart rate (HR). The distal extrathoracic trachea was cannulated and connected via a side-port to a pressure transducer to measure the tracheal pressure (TP) and changes associated with spontaneous respiration. Output from pressure transducers were filtered and amplified (Neurolog Systems, Digitimer, Hertfordshire, UK), digitised (Micro1401

A-D converter, CED, Cambridge, UK) and recorded using Spike II software (CED,

Cambridge, UK) for offline analysis. A midline incision was made through the larynx to expose the mucosal surface allowing placement of a platinum bipolar stimulating electrode (Driessen et al., 2015). Less than 7% of animals (2 out of 30) coughed in response to mechanical stimulation of the larynx after RLN transection, consistent with successful denervation of nodose-derived mechanoreceptors (Canning et al., 2004).

After a 20-minute stabilisation period, electrical stimulation (model s48, Grass

Instruments) of the larynx was performed. A voltage response curve was conducted by delivering increasing voltages (0.1-16V) to the larynx, at a constant maximum stimulating frequency (32Hz), pulse duration (1ms) and train duration (10s). On average the voltage required to elicit 50% of the maximum response (EV50) was 5.1±0.4V and the optimum voltage (i.e. the voltage that evoked a reproducible maximum respiratory response) was

10.9±0.4V. In each preparation, the optimum voltage was then used to conduct a frequency response analysis by sequentially increasing the stimulation frequency

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(1-32Hz). Following baseline electrical stimulation of the larynx, guinea pigs were placed onto a stereotaxic frame and the brainstem exposed to allow for bilateral microinjection of the following pharmacological agents into the Pa5 as previously described (Driessen et al., 2015); vehicle (0.9% saline, n=7), 5mM (of each in combination) of CNQX/AP-5

(ionotropic glutamate receptor agonists; n=8), 5mM (of each in combination) of

CP99994/ SB222200 (neurokinin 1 and 3 receptor antagonists; n=10) and 2mM

BIBN4096 (CGRP receptor antagonist; n=5). The appropriateness of the antagonist concentrations employed were determined from previous studies (Bongianni et al., 2005;

Mutolo et al., 2008; Hildreth and Goodchild, 2010; Hether et al., 2013). The volume of injection was uniformly 250nl per injection site and each animal received three microinjections bilaterally into the Pa5 (to cover the rostro-caudal extent; see Table 3.1).

Animals were immediately returned to a supine position and the frequency response of electrical stimulation of the larynx reassessed in a paired experimental design. In all instances, the second series of laryngeal stimuli was completed within a period of 30 minutes from the first microinjection. In animals where the respiratory response to laryngeal stimulation appeared unaltered by treatment, the SLNs were subsequently bilaterally transected and the optimum stimulation voltage and frequency reassessed to confirm that all responses were dependent upon laryngeal innervation.

In a separate series of experiments, animals received chemical, rather than electrical, stimulation of the larynx (n=10) by superfusing the laryngeal mucosa with capsaicin

(TRPV1 expressing neurons). A hooked 25-gauge needle was inserted into the intact rostral laryngeal lumen and connected to a 1mm syringe via polyethylene tubing

(internal diameter=0.5mm and outer diameter=0.9mm). A second 18-gauge cannula was positioned within a small incision at the caudal end of the laryngeal lumen, immediately

76 | P a g e below the cricotracheal ligament, and connected to gentle vacuum to allow for collection of superfused solutions. Stimuli consisted of 0.5ml of vehicle or capsaicin

(10µM) warmed to 37⁰ Celsius and slowly injected over a 40 second period. 10 minute rest periods between each perfusion ensured any evoked respiratory responses had returned to baseline. The same pharmacological modifications were made in the Pa5, as described above for the electrical stimulation of the larynx. Due to marked tachyphylaxis of capsaicin-evoked responses in this protocol, the study design was necessarily unpaired.

Pa5 stimulation-evoked respiratory responses in anaesthetised guinea pigs: To further assess the role of glutamate in respiratory responses mediated via the Pa5, we performed microinjection of DL-homocysteic acid (DLH, a glutamate analogue) into the caudal, central or rostral Pa5. We additionally reasoned that microinjections of capsaicin into the Pa5 should activate the central terminals of nearby afferents (Mazzone and

Geraghty, 1999; Geraghty and Mazzone, 2002) and thereby evoke responses sensitive to antagonism of glutamate and/or neurokinin receptors. Urethane anaesthetised guinea pigs were prepared for physiological monitoring of respiration, ABP and HR as described above. DLH (50mM; n=26 injection sites) or capsaicin (400nM; n=16 injection sites) were microinjected unilaterally across the rostral-caudal extent of the Pa5 (see stereotaxic coordinates in Table 3.1) with a five minute period between each to allow for evoked physiological responses to return to baseline. In separate animals, capsaicin responses were assessed in the central Pa5 (the most responsive site) following prior injection of the

Pa5 with either CNQX/AP-5 (5mM each in combination; n=4) or CP99994/SB222200

(5mM each in combination; n=5).

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3.2.6 Analysis of physiological studies

Respiratory rate, ABP and HR were calculated from the chart recordings at baseline and following each stimulation frequency or dose of capsaicin. For electrical stimulation, respiratory rates were calculated over 10 second periods immediately before (baseline) and during the stimulus train (evoked response) and multiplied by six to determine the equivalent respiratory rate per minute values. For chemical stimulation experiments and capsaicin microinjection into the Pa5, respiratory rates were similarly calculated over a

10 second period directly before each challenge/injection and again at the time of the peak effect. Alternately, responses evoked by DLH microinjection across the rostro-caudal extent of the Pa5 were assessed over five second periods because this more accurately characterised the highly transient responses observed for this stimulus. Changes in mean

ABP and HR were similarly calculated in all cases before and after the stimulation periods.

Physiological data were normalised to baseline values (before any stimulation) for each animal and then plotted as the mean±SEM within each cohort. This removed confounding influences of between animal variations in baseline parameters but importantly preserved the specific stimulus-evoked effects. The normality of each data set was assessed using the Maulchy’s test of sphericity and/or Brown-Forsythe test of variances. Each cohort was subsequently assessed for interaction effects of frequency and group using Pillai’s trace multivariate test. Post-hoc analyses of the electrical stimulus evoking the maximum reduction in respiratory rate (Emax), the electrical frequency required to elicit 50% of the maximum response (EF50) and the peak changes in respiratory rate for each intervention following superfusion of the larynx with capsaicin were conducted using a one-way

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ANOVA with a least significant differences multiple comparisons test. Respiratory effects evoked by DLH or capsaicin microinjected into the Pa5 were compared to vehicle microinjections using an unpaired Student’s t-test for each rostro-caudal level of the Pa5

(caudal, central and rostral). Microinjection of the ionotropic glutamate receptors or neurokinin receptors prior to capsaicin injection into the Pa5 were statistically compared using a one-way ANOVA with a least significant differences multiple comparisons test.

The level for statistical significance was set at P<0.05.

3.2.7 Drugs and reagents

Ionotropic glutamate receptor antagonists CNQX (#1045 - AMPA receptor antagonist) and AP-5 (#0105 - NMDA receptor antagonist), Substance P receptor antagonists

CP99994 (#3417 - NK1 receptor antagonist) and SB222200 (#1393 - NK3 receptor antagonist) and CGRP receptor antagonist BIBN4096 (#4561) were purchased from

Tocris. CNQX and AP-5 were made up in distilled water at stock concentrations of

10mM. CP99994 and SB222200 were made up individually at a stock concentration of

10mM in a 25% solution of 100% ethanol in 0.9% saline. BIBN4096 was made up at a stock concentration of 50mM in 1M hydrochloric acid. These stocks were diluted to a

2mM working concentration with distilled water (and pH adjusted to 7.4). Capsaicin and

DLH were purchased from Sigma Aldrich. Capsaicin was made up as a 400nM or 10mM working concentration in 10% ethanol and 10% tween in 0.9% saline, for Pa5 microinjection or laryngeal perfusion respectively. For laryngeal perfusion, the 10mM stock was diluted using 0.9% saline to create a 10µM working solution, used previously for laryngeal stimulation experiments (Mazzone and Canning, 2002). DLH was made up as 50mM stock solutions in distilled water (and pH adjusted to 7.4). In preliminary

79 | P a g e experiments, all vehicles were tested by microinjection into the Pa5 and shown to have no effect on physiological parameters measured, and as a result saline was used as the standard control for comparison purposes.

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3.3 Results

3.3.1 Characterisation of jugular vagal ganglia laryngeal sensory neurons

CT-B594 injections into the larynx labelled 224±73.7 neurons per jugular vagal ganglia

(n=5; Figure 3.1A). Traced neurons displayed a wide range of somal sizes (Figure 3.1B) consistent with labelling of both C and A-fibre laryngeal afferent subtypes.

Immunohistochemical analysis of the traced jugular vagal ganglia neurons also confirmed this (Figure 3.1B). Thus, a little over half were small-to-medium sized peptidergic laryngeal traced neurons that immunostained for the neuropeptides CGRP (58% positive; average perimeter=65.7±2.0µm) or Substance P (54% positive; average perimeter=

67.1±1.9µm), that is representative of the peptidergic C-fibre population (Riccio et al.,

1996; Undem et al., 2004). On the other hand, one third of the laryngeal traced neurons were larger in size and immunostained for NF-160kD (21% positive; average perimeter=

90.5±3.5µm) or vGlut1 (31% positive; average perimeter=79.1±2.8µm), which are representative of the A-fibre population. Consistent with this, we observed very minimal colocalization of vGlut1 with the neuropeptide Substance P (~7%), but substantial overlap of vGlut1 with NF-160kd (~55%) in untraced vagal ganglia tissues (n=4).

3.3.2 Anatomical characterisation of the rostro-caudal organisation of the Pa5

To our knowledge, the local architecture and rostro-caudal extent of the Pa5 has not been previously described in the guinea pig. NeuN immunohistochemistry revealed an interstitial neuronal group embedded in the dorsal tip of the trigeminal tract that extended from 0.1 to 1.4mm rostral to calamus scriptorius (defined herein as the Pa5). The density of neurons was highest in the caudal and central regions of the Pa5 and diminished appreciably in the rostral-most extent. Given that the jugular vagal ganglia contain both

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C- and A-fibre laryngeal afferents (Figure 3.1B), we subsequently assessed the distribution of Substance P (C-fibres) and vGlut1 (A-fibres) immunolabelling, notably observing apparent topographical distinctions between the location of these two markers in and around the Pa5 (Figure 3.1C).

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Figure 3.1: Identification and characterisation of guinea pig laryngeal jugular vagal ganglia sensory neurons and their terminations throughout the rostral-caudal extent of the paratrigeminal nucleus (Pa5). (A) A schematic depicting the experimental design of tracing from the larynx with CT-B594 (in red) and from the vagal ganglia with AAV-eGFP (in green). Example photomicrographs of a whole-mount preparation of the larynx showing the deposition of the retrograde neuronal tracer CT-B594 in the dorsal laryngeal mucosa (top), and a vagal ganglia whole-mount preparation displaying the distribution of CT-B594 retrogradely traced neurons predominately in the jugular (JG) rather than the nodose (NG) vagal ganglia (bottom). (B) Immunohistochemical characterisation of CT-B594 traced jugular ganglia neurons showing populations of both C-fibre (CGRP and SP-expressing) and A-fibre (NF 160KD and vGlut1-expressing) laryngeal neurons. Quantitative cell counts (pie charts) show the proportion of the total traced neuron population positive for each immuno-marker assessed. Subsequently somal size analyses are presented as histograms (10µm bins ) whereby the frequency is expressed relative to the total population of neurons that are both traced and immunopositive. Data were collated from n=5 jugular ganglia. (C) Representative photomicrographs of the rostro-caudal extent of the Pa5 showing brainstem hemisections immunostained for SP and vGlut1 on the left and the distribution of vagal afferent nerve terminals transduced by nodose vagal ganglia injections of AAV8 TRUFr-eGFP (AAV-eGFP) in hemisections on the right. The higher magnification photomicrographs of the Pa5 (signified by red boxes) show the organisation and relative density of the corresponding afferent terminals in the Pa5. The top photomicrograph demonstrates

AAV-eGFP transduced vagal afferents in close apposition to Pa5 neurons as stained by NeuN. Note the small number of NeuN positive Pa5 neurons and sparse afferent terminal labelling in the most rostral extent of this nucleus. The bottom photomicrograph displays the topographically distinct populations of SP and vGlut1 afferents in the Pa5. All scale bars represent 200µm, unless stated otherwise.

Additional abbreviations: AP; area postrema, CGRP; Calcitonin Gene Related Peptide, CS; calamus scriptorious, IO; inferior olives, NF-160kD; Neurofilament 160kD, nTS; nucleus of the solitary tract,

SP; Substance P, Sp5; spinal trigeminal nucleus, vGlut1; vesicular glutamate transporter 1.

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AAV-eGFP injections into the nodose vagal ganglia transduced approximately 40% of nodose neurons and 30% of jugular neurons, resulting in distributed eGFP labelling of afferent terminals in the brainstem. Not surprisingly these terminations were distributed bilaterally (ipsilateral predominance) in the nTS from 0.2mm caudal to 1.8mm rostral of the calamus scriptorius and importantly, terminal labelling was also evident ipsilaterally in and around the dorsal tip of the spinal trigeminal tract (Ciriello et al., 1981; Altschuler et a., 1989; McGovern et al., 2012a). Furthermore, labelling was in close apposition to

Pa5 neurons (NeuN positive neurons) at the caudal and central levels of the Pa5 but almost absent in the rostral-most extension of the nucleus (Figure 3.1C). Vagal afferent terminal labelling was absent from the Pa5 in one experiment where AAV injections into the nodose vagal ganglia efficiently transduced nodose neurons but failed to transduce any jugular vagal ganglia neurons, consistent with our previous data showing that vagal projections to the Pa5 originate from jugular neurons exclusively (Driessen et al., 2015).

Sparse ipsilateral labelling of terminals was also seen in the ventral geniculate trigeminal nucleus, interpolar part of the spinal trigeminal nucleus and the lateral edge of the caudal part of the spinal trigeminal nucleus, as well as in the medullary reticular formation, including in the region of the nucleus ambiguus, indicative of widely distributed vagal afferent terminations in the brainstem. No contralateral labelling was observed in any brainstem regions outside of the nTS.

3.3.3 Respiratory effects of electrical and chemical stimulation of the larynx

Prior to any interventions of the Pa5, baseline respiratory rate averaged 42.4±1.1 breaths/minute (n=30). In vehicle treated animals, electrical stimulation of the larynx (at an optimal voltage) produced a frequency dependent slowing of respiration (typically accompanied by visible swallows; not quantified) progressing to complete apnoea at

85 | P a g e higher stimulation frequencies (Figure 3.2A). The maximum reduction in respiratory rate

(Emax) following bilateral injection of vehicle into the Pa5 was 40.3±4.1 breaths/minute and the effective electrical frequency required to elicit 50% of the maximum response

(EF50) was 4.3±2.1Hz (Figure 3.2B and Table 3.3). A statistically significant interaction was detected between pharmacological treatment and electrical frequency dependent slowing in respiration (F (14,126) = 4.02; p<0.0001), indicative of one or more of the drug interventions modifying the magnitude of the evoked reflex. Post-hoc evaluations revealed that treatment with the neurokinin receptor antagonists CP99994/ SB222200 significantly reduced the Emax (p=0.007), but not the EF50, compared to vehicle (Figure

3.2C and Table 3.3), although this effect was significantly smaller than that induced by the ionotropic glutamate receptor antagonists CNQX/AP-5 which inhibited both the

Emax and EF50 compared to vehicle (Emax p=0.0002; EF50 p=0.01) and the neurokinin receptor antagonists (Emax p=0.032; EF50 p=0.02; Figure 3.2C and Table 3.3).

Microinjection of the CGRP receptor antagonist BIBN4096 into the Pa5 alone (Table 3.3 and see Appendix 3) or in combination with the neurokinin receptor antagonists (see

Appendix 3) failed to further inhibit electrically evoked respiratory responses. HR and

ABP effects evoked by electrical stimulation of the larynx were not significantly altered between pharmacological modifications of the Pa5 (see Appendix 3).

Chemical stimulation of the larynx using capsaicin superfusion evoked respiratory effects comparable to that seen with electrical stimulation. Following bilateral microinjection of vehicle into the Pa5, capsaicin superfusion on the larynx consistently produced visible swallowing (data not quantified) and decreased respiratory rate on average by 22±2.0 breaths/minute from baseline (often occurring with a brief period of apnoea; Figure 3.2C).

Comparable to the observations with electrical stimulation, capsaicin-induced respiratory

86 | P a g e slowing was not significantly altered by prior injections of neurokinin receptor antagonists into the Pa5 but was abolished (p=0.02) by glutamate receptor antagonists

(Figure 3.2C).

Table 3.3: The Emax and EF50 following pharmacological modification of the Pa5

* statistically compared to vehicle # statistically compared to CP99994/SB222200

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Figure 3.2: Glutamatergic and peptidergic neurotransmission in the paratrigeminal nucleus

(Pa5) mediate laryngeal-evoked respiratory reflexes. (A) Schematic representation of the vagal innervation of the guinea pig airways and the preparation used in the present physiological experiments. In the guinea pig the larynx is innervated by jugular ganglia (JG) derived afferents that travel primarily within the superior laryngeal nerves (SLN) and nodose ganglia (NG) derived afferents that travel primarily via the recurrent laryngeal nerves (RLN). Transection of the RLN’s (represented with an X on the schematic) allows for jugular-specific reflexes to be evoked by electrical or chemical stimulation of the larynx. The representative physiological recording demonstrates the slowing of respiration with a small reduction in blood pressure and no change in heart rate, in response to electrical stimulation of the larynx at optimal stimulation parameters. (B) Electrical stimulation of the larynx, following vehicle (VEH) microinjection into the Pa5, evokes frequency-dependent changes in respiratory rate. A significant interaction effect (p<0.0001) was observed between pharmacological modification in the Pa5 and frequency dependent respiratory slowing evoked by electrical stimulation of the larynx, indicating that bilateral microinjection of the ionotropic glutamate receptor antagonists

CNQX/AP-5 (αGluR) or the neurokinin 1 and 3 receptor antagonists CP99994/ SB222200 (αNKR) into the Pa5 is effective in altering laryngeal-evoked reflexes. Data represent the normalised mean±SEM for n=30 experiments, see Table 3.3 for further analyses. (C) Capsaicin superfusion across the laryngeal mucosal surface, after bilateral microinjection of VEH into the Pa5, evoked a reduction in respiratory rate. This respiratory slowing was significantly inhibited with bilateral microinjection of

+αGluR in the rostro-caudal extent of the Pa5 (*, p=0.02), but was unaffected following +αNKR bilateral microinjection. Data represent the mean±SEM change in respiration at the peak effect compared to immediately prior to stimulation, n=10 experiments. Additional abbreviations: ABP: arterial blood pressure, AU; arbitrary units, HR; heart rate, TP; tracheal pressure, VN; vagus nerve.

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3.3.4 Respiratory effects of glutamate and capsaicin microinjections into the Pa5

Microinjections of the glutamate analogue DLH or capsaicin into the Pa5 evoked opposing effects on respiration. That is, while DLH resulted in an abrupt reduction in respiratory drive (leading to brief arrest of breathing in three cases; Figure 3.3A,B), capsaicin initiated a slowly developing and persistent increase in respiratory rate

(tachypnoea; Figure 3.3C,D). Although respiratory slowing was induced by DLH across the entire rostro-caudal extent of the Pa5, the response was only statistically significant for the caudal level of the nucleus compared to vehicle injections (DLH decreased respiratory rate by 9.0±5.4 breaths/minute versus vehicle that increased respiratory rate by 4.5±3.2 breaths/minute p=0.049; Figure 3.3B). On the other hand, capsaicin-induced tachypnoea was significantly different compared to vehicle when injections were made into the central Pa5 (capsaicin increased respiratory rate by 22.5±5.1 versus vehicle that increased respiratory rate by 0±3.8 breaths/minute respectively p=0.009; Figure 3.3D).

A comparable response was only observed in two out of the seven cases following microinjection of capsaicin into the caudal Pa5, whereas capsaicin was without respiratory effect at the level of the rostral Pa5 (Figure 3.3D). At the level of the central

Pa5, capsaicin-evoked tachypnoea was not significantly altered by prior treatment with the glutamate receptor antagonists (CNQX/AP-5 increased respiratory rate by 18±4.9 breaths/minute, p=0.39; Figure 3.3E), but was abolished following pre-treatment with neurokinin receptor antagonists (p=0.001; Figure 3.3E). These data are consistent with a role for neuropeptides, and not glutamate, in centrally administered capsaicin-evoked tachypnoea. DLH and capsaicin produced small and inconsistent effects on ABP and HR parameters that were not significantly different between any groups.

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Figure 3.3: Glutamate and capsaicin microinjection into the paratrigeminal nucleus (Pa5) resulted in differential respiratory responses. (A) Example trace of tracheal pressure (TP) during microinjection of 50mM DL-homocysteic acid (DLH, glutamate analogue, red arrow) into the caudal

Pa5, evoking rapid but transient respiratory slowing. (B) Quantification of the peak change in respiratory rate following microinjection of vehicle (VEH) or DLH into either the caudal, central or rostral Pa5. *, p=0.049, significantly different to VEH in the caudal Pa5. (C) Representative trace of TP during microinjection of 400nM capsaicin (CAP, TRPV1 agonist, red arrow) into the Pa5, evoking a delayed, but persistent tachypnoea. (D) Quantification of the peak change in respiratory rate following microinjection of VEH or CAP into either the caudal, central or rostral Pa5. **, p=0.009, significantly different to VEH in the central Pa5. (E) Quantification of the effects of ionotropic glutamate receptor antagonists CNQX/AP-5 (αGluR) or neurokinin 1 and 3 receptor antagonists CP99994/SB222200

(αNKR) on peak changes in respiratory rate evoked by CAP microinjection into the central Pa5. **, p=0.001 significant attenuation of the response compared to CAP alone. Additional abbreviations: Au; arbitrary units, TRPV1; transient receptor potential vanilloid 1.

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3.3.5 Neuroanatomical connectivity of the Pa5

Conventional anterograde tracing using microinjections of BDA into the Pa5 was successful in nine cases, in which the average spherical spread of tracer was 0.8±0.2mm in diameter (Figure 3.4A). Anterograde labelling of terminals was broadly distributed across many bulbar nuclei, although they were always confined exclusively ipsilateral to the injection site. Suprabulbar projections from the Pa5, which have been described in rats (Saxon and Hopkins, 1998; Caous et al., 2001; McGovern et al., 2015b), were not observed in the guinea pig (data not shown). A similar pattern of BDA labelled terminal projections were observed regardless of the rostro-caudal location of the BDA injection site (Figure 3.4B). Common termination sites included the medullary spinal trigeminal nucleus, the nTS, the ventrolateral medulla in the region of the nucleus ambiguus and pre-

Bötzinger complex (Figure 3.4C), the principal sensory trigeminal nucleus and dense projections to the medial and lateral parabrachial nuclei and Kölliker-Fuse nucleus

(Figure 3.4D). However, the strength of connectivity to these common bulbar termination sites was not equal for caudal, central and rostral regions of the Pa5. Indeed, neurons in the caudal Pa5 displayed relatively strong connectivity with both medullary and pontine targets whereas neurons in the central and rostral Pa5 appear to preferentially project to pontine nuclei (Figure 3.4B).

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Figure 3.4: The guinea pig paratrigeminal nucleus (Pa5) connectome. (A) Schematic representation of the anterograde tracing experimental design. 10% Biotin Dextran Amine (BDA) injections were made unilaterally into the Pa5 and traced terminals were assessed in 100µm intervals of the brainstem. The brainstem photomicrograph shows an example of BDA injection into the central Pa5 of the guinea pig. The central Pa5 injection site is also presented at a higher magnification to highlight BDA filled paratrigeminal neurons within the injection site. (B) A density weighted connectome diagram graphically presenting the quantification of ipsilaterally traced BDA terminals from the caudal, central and rostral subregions of the Pa5 to medullary and pontine nuclei.

The weight of the interconnecting line between two regions corresponds to the extent of BDA labelling (sparse =dotted line, intermediate =solid line, dense =thick solid line). No labelled terminals were observed above the level of the pons. (C) Left hand side hemisection: choline acetyltransferase (ChAT) immunostaining of the ambiguus nucleus (Amb) as an anatomical landmark for distinguishing the ventrolateral medulla (VLM). Right hand side hemisection: representative photomicrograph of BDA traced terminals in the VLM. A high magnification photomicrograph of the area of the VLM confirming traced terminals in and around this region. (D)

Left hand side hemisection: Forkhead box protein P2 (FoxP2) staining of the Kölliker-Fuse (KF) and parabrachial nuclei. Right hand side hemisection: representative photomicrograph of BDA traced terminals in the pontine nuclei. A high magnification representative photomicrograph of the

KF, lateral parabrachial (LPB) nucleus and medial parabrachial (MPB) nucleus confirming dense terminal BDA labelling in these regions. The data represent the outcomes of n=9 successful BDA anterograde tracing experiments. Scale bars represent 1mm in all images. Additional abbreviations:

AP; area postrema, Cu; Cuneate nuclei, CS; calamus scriptorious, I5; intertrigeminal nucleus, IO; inferior olives, ml; , Mo5; , nTS; nucleus of the solitary tract, PR5; principal sensory trigeminal nucleus, PCR; parvicellular reticular nucleus, Rt; reticular nuclei, scp; spinal cerebral peduncle, Sp5; spinal trigeminal nuclei, VII; facial nucleus.

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3.3.6 Dual retrograde labelling of Pa5 neurons from the dorsolateral pons and ventral lateral medulla

Dual microinjections of CT-B into the Kölliker-Fuse and ventrolateral medulla or

Kölliker-Fuse and lateral parabrachial nucleus were successful in 7 out of 16 animals.

The central locus of the injection sites and diameter of injectate spread were as follows;

Kölliker-Fuse nucleus 9.4±0.1mm caudal to Bregma and a rostro-caudal spread of

0.3±0.1mm, lateral parabrachial nucleus 9.5±0.2mm caudal to Bregma and a 0.3±0.1mm rostro-caudal spread for and the ventrolateral medulla 0.6±0.1mm rostral to calamus scriptorius and a rostro-caudal spread of 1.6±0.4mm. Dual CT-B injections into the

Kölliker-Fuse and lateral parabrachial nucleus individually labelled a total of 305 and 315 neurons, respectively in the paratrigeminal nucleus (summed across all animals; Figure

3.5A). Similarly, dual tracing from the Kölliker-Fuse and ventrolateral medulla individually labelled 553 and 535 neurons, respectively in the Pa5 (Figure 3.5A). In both experimental groups we observed a small proportion of dual labelled neurons within the

Pa5. Only 8% of neurons (81 out of a total of 1007 individually traced neurons) were dual labelled from the Kölliker-Fuse and ventrolateral medulla and 17% of neurons (91 out of a total of 529 individually traced neurons) were dual labelled from the Kölliker-Fuse and lateral parabrachial nucleus (Figure 3.5A).

In separate experiments using immunohistochemistry we identified calbindin positive neurons in the Pa5, as described previously in the rat (Ma et al., 2005). However, across the rostro-caudal extent of the Pa5 we noted that calbindin positive neurons constituted a significantly smaller population (p=0.009) of Pa5 neurons compared to those labelled with the pan neuronal marker NeuN (compare Figure 3.5B with Figure 3.1C for NeuN

94 | P a g e staining). Double immunostaining for calbindin and the NK1 receptor revealed two distinct populations of neurons in the Pa5. Indeed, while calbindin and the NK1 receptor were expressed in a total of 264 and 398 individual neurons in the same tissue sections, only 5.7% (40 out of 702 counted neurons) were co-labelled with both calbindin and NK1 receptors immunoreactivity (Figure 3.5B).

Figure legend on the next page 95 | P a g e

Figure 3.5: The paratrigeminal nucleus (Pa5) consists of a heterogeneous population of neurons.

(A) Example photomicrograph of a coronal section through the central paratrigeminal nucleus showing

CT-B488 and CT-B594 retrograde labelled Pa5 neurons simultaneously traced from the ventral lateral medulla (VLM, arrows) and Kölliker-Fuse (KF, arrow heads) respectively. The higher magnification insert demonstrates clearly traced Pa5 neurons. Brainstem schematics depict the dual tracing experimental designs from the KF and VLM on the left and KF and lateral parabrachial nucleus (LPB) on the right. These correspond to quantified neuron counts of CT-B488 and CT-B594 single (green and red pie wedges) and dual (yellow pie wedge) labelled neurons in the Pa5. Only 8% (81 out of 1007 neurons, n=4 experiments) of Pa5 neurons were dual labelled from the KF and VLM and approximately 17% (91 out of 529 neurons, n=3 experiments) of Pa5 neurons were dual labelled from the KF and LPB. (B) Bar chart showing that calbindin (Calb) positive Pa5 neurons represent a significantly different population of neurons (** p=0.009) than the entire population represented by

NeuN positive Pa5 neurons (data represents the mean±SEM; n=3). Photomicrograph of neurokinin 1 receptor (NK1R, arrows) and calbindin (Calb, arrow heads) immunoreactivity in the medulla 0.5mm rostral to calamus scriptorius (CS), whereby the area corresponding to the Pa5 is highlighted by the red box. Quantified neuron counts were summed across n=3 experiments and are displayed using pie charts to show single labelled NK1R or Calb positive neurons (green and red wedges) and dual labelled neurons (yellow wedge). These data demonstrate that approximately 5.7% (40 out of 702) of Pa5 neurons express both markers. In addition, representative photomicrographs are also presented to show evidence of both NK1R and Calb positive neurons at a high magnification within the Pa5. Additional abbreviations: AP; area postrema, IO; inferior olives, nTS; nucleus of the solitary tract, Sp5; spinal trigeminal nucleus.

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3.4 Discussion

We have previously reported the existence of a novel respiratory sensory processing pathway that arises from jugular vagal ganglia afferents and principally innervates the larger airways and projects to the medullary Pa5 (McGovern et al., 2012a; Driessen et al.,

2015; McGovern et al., 2015a; McGovern et al., 2015b). Ascending projections from Pa5 neurons in receipt of airway jugular vagal ganglia inputs terminate in somatosensory processing regions of the brain (McGovern et al., 2015b), which has led us to propose that this sensory circuit may be important for discriminative processing of sensations arising from the large airways. In the present study, we investigated the reflexive regulation of respiration mediated by the Pa5 and demonstrate a level of complexity in

Pa5 sensory integration that has not been previously recognised. Notably, we report functional and anatomical evidence for the existence of at least two distinct postsynaptic targets for afferent inputs in the Pa5, which likely mediate opposing effects to respiratory drive. Collectively, these data advance our understanding of viscerosensory processing in the brainstem by highlighting a novel vagal afferent neural circuit with features that are consistent with an important role in respiratory sensation, breathing control and pulmonary defence.

3.4.1 A neuroanatomical framework for laryngeal sensory-evoked respiratory responses via the Pa5

The laryngeal mucosa is innervated by vagal sensory neurons, the activation of which induces the modulation of respiration and induction of protective expiratory responses that collectively serve to limit the effects of foreign material passing the glottis. However,

97 | P a g e the neural circuits underpinning afferent processing from this site have not been fully elucidated. In the guinea pig, the laryngeal mucosa is in receipt of sensory fibres arising from both the nodose and jugular vagal ganglia (Canning et al., 2004; Undem et al., 2004;

McGovern et al., 2015a). Nodose vagal ganglia neurons give rise to low threshold laryngeal mechanoreceptors that are important for defensive coughing and expiratory responses evoked by particulate matter and other mechanical stimuli. These neurons project exclusively to the nTS and were purposefully excluded from investigations in the present study but have been previously interrogated in detail (Canning et al., 2004; Chou et al., 2008; Mazzone and McGovern, 2008; Canning and Mori, 2011). Conversely, jugular vagal ganglia neurons innervating the laryngeal mucosa are not particularly mechanically sensitive but instead they respond to an array of chemical mediators of which the most notable is capsaicin (Riccio et al., 1996; Canning et al., 2004; Undem et al., 2004; Lieu et al., 2011). Our present data confirms previous studies in guinea pigs identifying a larger population of C-fibre and smaller population of Aδ-fibre laryngeal jugular vagal ganglia neurons, differentiated in retrograde tracing experiments by neuron somal size, peptidergic expression markers, myelination and vGlut1 (Riccio et al., 1996;

Pedersen et al., 1998; Canning et al., 2004; Mazzone and McGovern, 2008; Hermes et al., 2015).

We have previously shown in both rats and guinea pigs that jugular vagal ganglia neurons project exclusively to the Pa5 and not the nTS (Driessen et al., 2015; McGovern et al.,

2015b). Perhaps best studied in the rat, the Pa5 is part of an extensive interstitial system of the trigeminal nucleus (Chan-Paley, 1978, Phelan and Falls, 1989; Panneton et al.,

2017) and receives additional inputs from trigeminal and spinal nociceptive afferents (Ma et al., 2007; Alioto et al., 2008). Neuronal tracing studies in rats have shown output

98 | P a g e projections from Pa5 neurons to bulbar nuclei involved in autonomic and nociceptive processing, including the ventral lateral medulla and pontine nuclei (Feil and Herbert,

1995; de Sousa Buck et al., 2001; Pinto et al., 2006; McGovern et al., 2015b). Our data in the guinea pig is largely consistent with the rat in that we noted caudal to rostral differences in the organisation of the Pa5 and its vagal inputs, as well as comparable Pa5 pontomedullary connectivity. However, unlike the rat (McGovern et al., 2015b), guinea pig Pa5 neurons did not project to contralateral medullary nuclei or directly to the ventrobasal thalamus. Additionally, although the rat Pa5 projects heavily to the caudal sub-nucleus of the nTS (de Sousa Buck et al., 2001; McGovern et al., 2015b), this was not evident in the guinea pig and instead we observed that such connectivity was confined to rostral subnuclei. This argues that the Pa5 in the guinea pig likely acts independently of the nTS in the initial processing of cardiorespiratory-related primary afferent inputs to the brainstem.

3.4.2 Functional and anatomical evidence for multiple Pa5 processes regulating respiration

Our present data confirms our previous findings showing that laryngeal afferents arising from the jugular vagal ganglia evoke respiratory slowing when activated, which is a reflex mediated by afferent inputs to the Pa5 (Driessen et al., 2015). We have extended our previous findings by demonstrating a primary role for glutamatergic neurotransmission in the Pa5 in laryngeal jugular afferent evoked respiratory responses, regardless of whether reflexes were evoked by electrical or chemical stimulation of the laryngeal mucosa. However, we also consistently noted a relatively smaller but significant effect of blocking neurokinin receptors, but not CGRP receptors, on this reflex evoked respiratory slowing. The principle role of glutamatergic neurotransmission is also supported by the

99 | P a g e stimulus parameters and kinetics of the evoked responses, especially during electrical stimulation. Thus, large respiratory effects were evoked at low stimulation intensities, and proceeded with a rapid (almost immediate) onset and terminated equally rapidly after cessation of stimulation, consistent with ionotropic rather than metabotropic neurotransmission. However, although our studies are consistent with glutamate being the primary excitatory transmitter of laryngeal afferents in the Pa5, with neurokinins acting as neuromodulators, our data do not allow for the relative contribution of Aδ-fibre versus C-fibre laryngeal afferents to be determined. Indeed, almost all vagal ganglia neurons express vGlut2, suggesting that all are likely capable of releasing glutamate when activated (Juranek and Lembeck, 1997; Corbett et al., 2005; Mazzone and McGovern,

2008; Fenwick et al., 2014; Rogoz et al., 2015), and as yet a selective stimulus of either

A- or C-fibre jugular afferents has not been identified. Nevertheless, the low threshold voltages required to elicit reflex respiratory slowing might suggest a prominent role for jugular A-fibres, which to our knowledge, is the first report of a reflex specifically involving this airway afferent subtype.

Several lines of evidence suggest that the processing of afferent inputs within the Pa5 may be more complex than the simple notion that multiple afferent subtypes converge onto common postsynaptic Pa5 relay neurons. In this regard, the organisation of afferent inputs to the Pa5 may resemble the non-convergent specificity of A- and C-fibre vagal afferent synapses that has previously been reported in the nTS (Bailey et al., 2002;

McDougall and Andresen, 2013). Indeed, whilst microinjection of a glutamate analogue predictably mimicked the effect of laryngeal stimulation by evoking a transient interruption of respiration (presumably secondary to the activation of postsynaptic targets), capsaicin microinjection into the Pa5 paradoxically enhanced respiratory drive.

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Capsaicin is expected to activate the central presynaptic terminals of afferents in the Pa5, releasing neurotransmitter that subsequently activates Pa5 second order neurons

(Mazzone and Geraghty, 1999; Fenwick et al., 2014; Hofmann and Andresen, 2016).

Although we cannot discount a role for glutamate our data suggest that, under the circumstances employed, neuropeptides are the primary neurotransmitter mediating the centrally administered capsaicin-evoked response given that the tachypnoea was inhibited by neurokinin receptor antagonists but not by ionotropic glutamate receptor antagonists.

Of course, centrally injected capsaicin is expected to activate more than laryngeal afferent terminals in the Pa5. Indeed, the Pa5 is also in receipt of spinal, trigeminal and glossopharyngeal afferents (Cirello et al., 1981; Panneton and Burton, 1981; Panneton and Burton, 1985; Altschuler et al, 1989) and these could conceivably evoke changes in cardiorespiratory behaviours given the extensive projections of the Pa5 to brainstem nuclei important in modulating these effects. However, the opposing respiratory responses evoked by peripheral (laryngeal) versus central afferent activation as well as the rostro-caudal differences noted in functional responsive sites within the Pa5 suggest that several subsets of respiratory modulating Pa5 neurons may exist. It remains unclear as to why centrally administered capsaicin evokes respiratory responses that do not rely on glutamatergic neurotransmission in the Pa5, as it is known that both Aδ- and C-fibre laryngeal afferents are capsaicin sensitive (Riccio et al., 1996; Canning et al., 2004) and action potential dependent afferent responses are largely glutamatergic. Conceivably, the local calcium events in the afferent terminal differ between the activation of voltage gated

(action potential dependent) and TRP (capsaicin dependent) channels such that different pools of small and large dense core neurotransmitter vesicles are mobilized (Fawley et al., 2016; Hofmann and Andresen, 2016). However, such speculation requires further

101 | P a g e investigation. Regardless, transmitter release under these circumstances is presumably at different synapses given that we observed opposing effects on respiration.

Our dual retrograde neuronal tracing studies and immunohistochemical staining provide further evidence to support the existence of multiple postsynaptic neuron subtypes within the Pa5. Very few Pa5 neurons were dual labelled following retrograde tracing from the

Kölliker-Fuse and ventrolateral medulla, or the Kölliker-Fuse and lateral parabrachial nucleus. This contrasts with some previous finding in rats, which failed to show distinct

Pa5 neuron populations following dual retrograde tracing from the nTS and parabrachial nuclei, ventrobasal thalamus and nTS and the ventrobasal thalamus and the parabrachial nuclei (Saxon and Hopkins, 1998). However, we acknowledge that the discrete microinjections employed for this experiment would reduce tracing efficiency and thus our data may over-estimate apparent segregation. Nevertheless, we also demonstrated that at least two different populations of Pa5 neurons could be identified based on their immunoreactivity for either calbindin or the NK1 receptor. Taken together, and considering our functional data, these findings collectively support the hypothesis that types of postsynaptic neurons process afferent inputs in the Pa5. Whether A- and C-fibre laryngeal afferent inputs are segregated into parallel processing lines onto distinct postsynaptic recipient neurons within the Pa5, as has been reported for vagal afferent inputs to the nTS (Alheid et al., 2011), remains to be resolved.

3.4.3 Physiological significance and conclusion

In the present study, we employed neuroanatomical tracing as well as physiological and pharmacological experiments to assess the organisation of laryngeal afferent dependent

102 | P a g e responses mediated by jugular vagal ganglia inputs to the Pa5. The reflex changes in breathing pattern evoked by laryngeal stimulation or by centrally administered agents into the Pa5 undoubtedly reflect the intimate connectivity of this nucleus with bulbar regions important for respiratory pattern regulation. Our data reveal an underappreciated connectivity of the Pa5 with pontomedullary nuclei of the respiratory rhythm and pattern generating circuit (Feldman et al., 2003; Dutschmann and Dick, 2012; Ramirez et al.,

2012; Smith et al., 2013) and in turn provide a novel perspective to this field. In particular, the pronounced ascending projections into pontine respiratory nuclei that are critical for the modulation of airway resistance, respiratory airflow and the mediation of airway reflexes (Dutschmann and Dick, 2012) underline the significance of this pathway in sensory-motor controls of the upper airways. However, given that jugular vagal ganglia afferents comprise part of the neural crest-derived cranial ganglia (Kwong et al., 2008;

Nassenstein et al., 2010) and recipient neurons in the Pa5 connect extensively with thalamocortical networks involved in somatosensation, the results of this study have important broader implications for understanding the discriminative aspects of respiration and other visceral sensations.

Finally, the possibility that different circumstances govern glutamatergic and peptidergic neurotransmission in the Pa5 may be important for understanding mechanisms for altered central nociceptive afferent processing in disease. For instance, given that peripheral inputs in healthy animals are predominately dependent upon non-peptidergic mechanisms

(present study), how and when peptidergic neurotransmission occurs may be an important determinant for sensory plasticity. This could be especially relevant in conditions of laryngeal hypersensitivity in which altered neurochemistry of jugular-Pa5 afferent processing may be an underlying feature of sensory disturbances associated with

103 | P a g e respiratory disease. Accordingly, afferent sensitisation, phenotypic changes in sensory neurons and/or central alterations in afferent neurotransmission (Undem and Nassenstein,

2009; Undem et al., 2015; Mazzone and Undem, 2016; Driessen et al., 2017) will likely have a major impact on the nature of the output from the Pa5 and the ensuing reflex and behavioural respiratory responses that contribute to the symptoms of pulmonary disease.

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4. Chapter 4: The role of neurokinin 1 receptor expressing neurons in the medullary paratrigeminal nucleus in cough and associated airway irritant behaviours in conscious guinea pigs

4.1 Introduction

Physiologically, cough is a vital respiratory behaviour that acts as a defensive mechanism to protect the airways and lungs from damage by inhaled irritants and aspirate. In addition to the motor component of coughing, the presence of cough evoking stimuli in the airways is consciously perceived as an airway irritation (Davenport et al., 2007; Farrell et al.,

2012; Mazzone et al., 2013), resulting in a sensory experience defined as the urge-to- cough. It is believed that the urge-to-cough serves to motivate and regulate respiratory behaviours, like coughing, and to provide awareness of the environmental impacts upon pulmonary function (Mazzone et al., 2007; Davenport, 2009; Ando et al., 2014).

However, in pathological conditions the neural processes subserving cough and the urge- to-cough become hypersensitive (Lundberg et al., 1991; Polley et al., 2008; Hilton et al.,

2015; Kanemitsu et al., 2016; Ando et al., 2016; Zaccone et al., 2016), leading to abnormal pulmonary sensations and excessive coughing that no longer serves a physiological purpose (Chung et al., 2013; Morice, 2013; Irwin et al., 2018). This defines a clinical condition known as cough hypersensitivity syndrome (Chung, 2011; Morice,

2013), which in many patients proves to be a very difficult to treat component of pulmonary disease (Koo et al., 2018; Mazzone et al., 2018). Accordingly, a better understanding of the sensorimotor neural processes governing cough and their altered function is warranted.

The stimuli that induce cough do so by activating the terminals of sensory neurons which densely innervate the airway mucosa. Heterogenous populations of airway projecting

105 | P a g e sensory neurons are derived from both the nodose and jugular vagal ganglia and in turn ascend the neuraxis terminating in two primary brainstem regions; the nucleus of the solitary tract (nTS) and the paratrigeminal nucleus (Pa5; Canning et al., 2004; Undem et al., 2004; McGovern et al., 2012a; McGovern et al., 2012b; McGovern et al., 2015b).

Nodose and jugular vagal sensory pathways are embryologically distinct (D’Autréaux et al., 2011; Nomaksteinsky et al., 2013) and this manifests differences in the peripheral and central organisation of their fibres (Undem et al., 2004; Kwong et al., 2008; McGovern et al., 2015a; McGovern et al., 2015b). Indeed, the nodose vagal ganglia, derived from the visceral epibranchial placodes, predominately innervates the lungs and projects almost entirely to the nTS (D’Autreaux et al., 2011; Driessen et al., 2015; McGovern et al., 2015a; McGovern et al., 2015b). Whereas the jugular vagal ganglia, derived from the somatic neural crest, predominately innervates the extrapulmonary airways (larynx, trachea and mainstem bronchi) and projects almost exclusively to the Pa5 (D’Autreaux et al., 2011; Nomaksteinsky et al., 2013; Driessen et al., 2015; McGovern et al., 2015a;

McGovern et al., 2015b). With respect to cough, evidence exists for mediation from both nodose mechanoreceptors and jugular nociceptors, and although the central processing of nodose mechanoreceptor inputs in the nTS has been studied (Tsubone et al., 1991;

Widdicombe, 1996; Bolser and Reier, 1998; Canning et al., 2004; Canning et al., 2006;

Mazzone et al., 2009), there has been very few attempts to investigate nociceptor inputs or evoked defensive behaviours processed by the Pa5.

Recent work from our laboratory has revealed an unappreciated complexity of airway afferent processing in the Pa5. We have shown, in anaesthetised guinea pigs, that the jugular-Pa5 airway afferent processing pathway is important in laryngeal-evoked apnoea

(Driessen et al., 2015; Driessen et al., 2018). These studies showed that this effect is

106 | P a g e predominately mediated by glutamatergic neurotransmission in the Pa5 (Driessen et al.,

2018). Paradoxically, neuropeptide transmission in the Pa5, specifically that of Substance

P, results in increased respiratory rate (Driessen et al., 2018). These differential responses could plausibly be explained by the existence of multiple subtypes of postsynaptic neurons in this nucleus. Dual retrograde tracing of Pa5 neurons from the regions in the ventrolateral medulla and pons show low numbers of dual labelled neurons, which suggests that Pa5 neurons project differentially throughout the cardiorespiratory column

(Driessen et al., 2018). Subsequent anatomical studies defined a distinct population of

Pa5 neurons that express the Substance P neurokinin 1 (NK1) receptor (Driessen et al.,

2018). Given the potential role of the Pa5 in sensorimotor processing of jugular-related airway sensory inputs to the brain and the reported anti-tussive properties of NK1 receptor antagonists in animals and humans (Amadesi et al., 2001; Chapman et al., 2004; El-

Hashim et al., 2004; Grobman and Reinero, 2016; Smith et al., 2017), we hypothesised that NK1 receptor-expressing Pa5 neurons may be a central component regulating airway irritant induced behaviours, including cough. Therefore, the aim of this study was to assess the impact of selective NK1 receptor neuron ablation in the Pa5 on cough and associated behaviours induced by inhaled airway irritants in conscious unrestrained guinea pigs.

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4.2 Methods

4.2.1 Animals

Experiments, approved by an accredited institutional Animal Ethic Committee, were conducted on adult Dunkin Hartley guinea pigs of either sex (n=57, weight range = 350-

1000g). Animals were housed in a standard environment and given ad libitum access to water and food. No statistical differences were observed between the measured responses of male and female guinea pigs and in turn the data was pooled for all subsequent statistical comparisons.

4.2.2 Conscious cough studies

Conscious freely moving guinea pigs were exposed (up to four at a time) to aerosolised irritants in a four site Buxco whole body plethysmography system, without any prior intervention (no-intervention cohort), to assess irritant-evoked cough and associated changes in animal behaviour. The experimental design consisted of a 11-day protocol

(Figure 4.1) whereby animals were first acclimatised to the plethysmography chambers without challenge (days 1-6). During the acclimatisation, animals were placed individually into the chambers for 20 minutes in the morning of each day under full experimental conditions, including turning on the nebuliser delivery system without any loaded solutions. The animals were then challenged on day seven with increasing doses of the nodose and jugular C-fibre stimulant bradykinin (bradykinin acetate salt, Sigma

Aldrich, B3259; Kaufman et al., 1980; Hewitt et al., 2016). This was followed by a rest day (day 8) and a further two days of acclimatisation (days 9 and 10) before a final

108 | P a g e challenge on day 11 with increasing doses of the nodose specific stimulant adenosine 5’- triphosphate (ATP, Sigma Aldrich, A7699; Kwong et al., 2008; Muroi et al., 2013).

On challenge days (Figure 4.1), animals were weighed to calibrate the chambers for accurate recording of respiratory measures (Buxco Finepointe Software Version 2.1.0.9) and digital video cameras (Axis communications network cameras model M1054) were assigned and positioned correctly for each chamber to record (via Media Recorder 3 operating on a PC) the animal’s behaviour during the experiment. Animals were placed in the chambers for 20 minutes before receiving an initial 0.9% saline challenge followed by either bradykinin (0.3 (0.3), 1 (0.9) and 3 (2.8) mg/ml (mM); n=20) or ATP (1.1 (2),

11 (20) and 110 (200) mg/ml (mM); n=9). Bradykinin doses were made comparable to previous studies in the guinea pig (Mazzone et al., 2005; Hewitt et al., 2016), while ATP doses were made similarly to the concentrations previously used in human studies

(Basoglu et al., 2015; Fowles et al., 2017). In addition, all doses were confirmed in preliminary experiments. Challenges were nebulised (Buxco Aerosol Distribution Unit

10LPM, Aerogen nebuliser unit AG-AL1000 with filler cap of 3.1µm particle size) as one millilitre solutions over five minutes, at an air flow velocity of five litres/min and at

60% duty cycle. Airway irritants were nebulised for five minutes and this was followed by a five minute response period. In addition, an extra five minutes was allowed between each challenge to allow respiratory parameters and behaviours to return to baseline.

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Figure legend on the next page 110 | P a g e

Figure 4.1: Schematic representation of the experimental timeline. Cough studies were conducted in conscious unrestrained guinea pigs using whole body plethysmography over an 11 day protocol. Before any challenge, animals were acclimatised in the plethysmography chambers under experimental conditions for a week (day 1 to day 6). On day seven the animals were challenged with increasing doses of nebulised bradykinin (0.3, 1 and 3mg/ml). Guinea pigs then had a rest day (day 8) before being re- acclimatised on day nine and ten prior to challenges with increasing doses of nebulised adenosine 5’- triphosphate (ATP, 1.1, 11 and 110mg/ml) on day 11. On challenge days, animals were acclimatised for

20 minutes (-15 – 5 minutes) with the respiratory and behavioural recording beginning at time zero to measure five minutes of baseline activity before the initial challenge of 0.9% Saline (Sal) followed by each dose of bradykinin. Each solution was preceded by a five minute baseline period (blue) and then nebulised for five minutes (green) with a further five minutes for the response time. During each of these time periods evoked cough and associated behaviours of irritation were recorded. An example of the corresponding changes in the respiratory chart trace during a coughing event to 3mg/ml of bradykinin, showing the well-defined cough pattern of a large inspiratory effort followed by a forced expiration. In addition, during cough animals also demonstrated a characteristic stance that consisted of raising on their forelimbs in a hunched posture, as can be seen in the still image. Finally, a range of behaviours were measured including oral (e.g. chewing), respiratory (e.g. cough and yawn), grooming and gross motor movements of which a few example photographs have been provided. These behaviours were video recorded and then imported into a behavioural analysis program (The Observer XT12) to measure the duration of behaviour during each phase of the challenge.

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4.2.3 Chemical lesions of the Pa5

Targeted toxin lesions of the Pa5 were conducted using Saporin conjugated to Substance

P (SSP-SAP, Advanced Targeting Systems, IT-11) following protocols described for other central nuclei (Wilkinson et al., 2011; Fu et al., 2017). SSP-SAP is a ribosomal inactivating protein that binds specifically to the NK1 receptor upon which it is internalised and results in cell death in neurons that express this receptor (Riley et al.,

2002; Abdala et al., 2006). Guinea pigs were prepared for recovery surgery with subcutaneous injections of the muscarininc antagonist, atropine (0.2mg/kg, atropine sulfate salt monohydrate, Sigma Aldrich, A0257) to decrease oral and airway secretions.

30 minutes post atropine treatment animals were anaesthetised with isoflurane (2.5% in medical oxygen) via a nose cone and their heads were placed in a stereotaxic frame at a

45⁰ degree angle. A midline incision was made through the skin, posterior neck muscle and dura mater to expose the medulla at the level between the occipital bone and C1 vertebra. Using calamus scriptorius as a reference point (~ 0.5mm caudal to obex at

Bregma -17.3mm) bilateral microinjections of 200nl of 10ng/100nl SSP-SAP (lesion cohort) or 0.9% sterile saline (control cohort) were made across three sites of the Pa5 (to cover the rostro-caudal extent of this nucleus as previously described in Driessen et al.,

2018; see Table 4.1 for stereotaxic coordinates). Incisions were sutured and animals received subcutaneous injections of 1ml/kg of 0.5mg/ml Meloxicam (Boehringer

Ingelheim), 1ml/kg of 5mg/ml Endofloxacin (Bayer Health Care) and 3ml of 0.9% sterile saline. Animals were allowed to recover for three weeks, and then were challenged with bradykinin (n=14 lesion and n=14 control) and ATP (n=10 lesion and n=12 control) following the 11 day protocol described above in section 4.2.2. Note the difference in sample size between bradykinin and ATP challenges is due to some animals not being challenged to ATP for the purposes of histological analyses.

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Table 4.1: Stereotaxic co-ordinates for the microinjection of Substance P-

Saporin into the paratrigeminal nucleus (Pa5)

Lesion characterisation: Following completion of the 11 day protocol, animals were overdosed with sodium pentobarbital (100mg/kg i.p.) and transcardially perfused with 150ml of 5% sucrose in 0.1M phosphate buffered saline (PBS; pH7.4) and 200ml of

4% paraformaldehyde (PFA) in 0.1M PBS. Brainstems were dissected and postfixed overnight in 4% PFA before being cryoprotected in 20% sucrose at 4º Celsius. Tissues were frozen in OCT embedding compound and 50µm cryostat cut sections were collected serially in 0.1M PBS. Sections were blocked in 10% goat serum in 0.1M PBS for one hour at room temperature. Tissue sections were then incubated for 48 hours in the primary antibody of interest (see Table 4.2 for antibody specifications) diluted in antibody diluent

(2% goat serum and 0.3% Triton X-100 in 0.1M PBS). After the required incubation, sections were washed three times with 0.1M PBS and then incubated for an hour in donkey anti-rabbit Alexa Fluor 488 (1:500, Thermo Fischer Scientific, A-21206).

Immunostained brainstems were mounted onto gelatin-coated slides and coverslipped with an antifade mounting media (Fluroshield, Sigma Aldrich, F6182). All tissues were visualised under the appropriate filter cubes using a Leica DM6B LED microscope and

113 | P a g e images were captured using a Leica DFC7000T camera. Representative photomicrographs were assembled in Adobe Photoshop CS6.

Table 4.2: Primary antibody specifications for lesion characterisation

4.2.4 Analysis of lesion characterisation and conscious cough studies

Lesion quantification: Pa5 neurons were quantified in brainstems from lesion and control animals by counting immunopositive neurons across the rostro-caudal extent of the Pa5 (-0.13–1.4 mm CS, four sections/animal/marker). These data are graphically presented as the region-pooled total count ± SEM for each marker (NK1 receptor, NeuN and calbindin). For each immunostain the total counts were statistically compared between lesion and control animals using an unpaired two-tailed Student’s t-test.

Respiratory responses and associated behaviours: Respiratory frequency

(breaths/minute), tidal volume (ml), inspiratory and expiratory time (seconds, see

Appendix 4) were automatically sampled every 0.02 seconds and have been presented as

*All antibodies were optimised for concentration and specificity before use. This process was run in parallel with negative controls (i.e. omission of the primary antibody). 114 | P a g e the mean per minute across the extent of the recording. Individual coughs were counted manually by assessing the Buxco chart traces and video recordings to confirm the characteristic airflow and behavioural ‘cough stance’ posture (Tsubone et al., 1991; Fox et al., 1996; Figure 4.1). For this study, a clinical definition of cough was adopted and herein defined as any large expiratory airflow with a preceding inspiration that commonly occurred in rapid succession (Figure 4.1). In addition, video files were imported into a behavioural analysis software (Observer XT12) to code for the duration of behaviours that accompanied nebulised challenges (Figure 4.1). Behaviours were categorised as oral

(i.e. chewing and licking that involved mastication of themselves or their environment), grooming (i.e. use of the paws to wipe the nose/face and scratch the body), movement

(i.e. notable movement around the enclosure and ‘dog’ shakes) or respiratory (i.e. time spent coughing, ‘cough stances’ and yawn).

Both cough and duration of behaviours are reported as total per minute values, the total duration for the baseline, nebulised and response periods individually (i.e. data not normalised) and the normalised cumulative total duration (sum of across doses) ± SEM.

Both evoked cough and associated duration of behaviours were statistically compared by a first level analyses using a Kruskal Wallis one-way ANOVA with a Dunn’s multiple comparisons test across doses for the no-intervention cohorts and using a two-way

ANOVA with a Tukey’s multiple comparisons test across dose and cohort for SSP-SAP lesion experiments. Additionally, post-hoc analyses, using non-parametric Mann

Whitney T-tests, were conducted to compare evoked effects between the doses of challenges in the no-intervention cohort and to compare differences between lesion and control animals at each dose. Finally, the relationship between cough and behaviour was explored using a χ2 test to assess the probability of behaviours predicting evoked cough

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(defined here as the behaviour probability index), and through regression analyses to investigate correlative relationships between measured endpoints. All analyses were conducted single blinded and for all comparisons statistical significance was set at p<0.05.

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4.3 Results

4.3.1 Characterising an assay to study animal behaviours as a measure of the conscious perception of an airway irritation

Mechanistic studies investigating the conscious perception of an airway irritation have been limited by the difficulty to study this subjective experience in animal models.

Although evoked cough is considered the gold standard measure of airway irritation, we hypothesised that a more sensitive measure may be to investigate the associated behaviours of irritation. Therefore, we exposed conscious un-restrained guinea pigs to nebulised bradykinin (nodose and jugular C-fibre activator; Kaufmann et al., 1980;

Hewitt et al., 2016) and ATP (nodose selective stimulant; Kwong et al., 2008; Muroi et al., 2013) and assessed cough, behaviours and their relationships.

Bradykinin: Aerosolised bradykinin evoked cough in the nebulised phase of the 1 and 3mg/ml doses that usually occurred within one to three separate coughing events

(Figure 4.2, Figure 4.4 and Table 4.3). Bradykinin evoked coughs were strongly dose dependent with significant increases in cough evoked by the 1mg/ml (9.2±2.8 coughs, 8 out of 20 responders) and 3mg/ml (15.5±2.2 coughs, 17 out of 20 responders) doses compared to no coughing under baseline and saline conditions (p=0.003 and p<0.0001 respectively; Figure 4.4A). These effects where observed in the absence of any preceding or proceeding changes in respiratory rate or tidal volume (Figure 4.2). Bradykinin also evoked notable changes in behaviour durations (Figure 4.3, Figure 4.4 and Table4.3).

Behaviour durations were significantly increased in the nebulised phase of the 3mg/ml bradykinin dose compared to baseline (112.7±12.4 vs. 37.7±12.7 seconds respectively, p=0.0009; Figure 4.4B). After normalisation, to account for inter-animal variability in

117 | P a g e baseline behavioural profiles, the cumulative total duration of behaviours at each dose of bradykinin was in fact significantly increased when compared to saline at all tested bradykinin challenge doses (p=0.03 for 0.3mg/ml, p=0.003 for 1mg/ml and p<0.0001 for 3mg/ml; Figure 4.4B).

Figure 4.2: Bradykinin evokes cough in the conscious guinea pig without altering basal

respiration. Cough number, respiratory frequency (breaths/minute) and tidal volume (ml) are presented

as per minute values over the course of the entire bradykinin challenge (0-65 minutes) in the no-

intervention cohort (n=20). Blue bars represent baseline recordings and the green are for periods of

nebulised solutions that is saline (Sal) and 0.3, 1 and 3 mg/ml of bradykinin. These traces show

bradykinin evoked cough predominately occurred in the nebulised phased to 1 and 3mg/ml of

bradykinin, while not significantly altering respiratory frequency or tidal volume.

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A range of individual behaviours were evoked by bradykinin challenges. For example, normalised chewing duration was significantly increased following nebulisation of

3mg/ml bradykinin compared to saline (p=0.02; Figure 4.3 and Table 4.3), while normalised grooming behaviours were significantly increased over the entire bradykinin challenge compared to saline (p=0.001; Table 4.3). Not surprisingly we also identified that in response to bradykinin challenges guinea pigs spent an increased time in the cough stance and performing respiratory behaviours (p=0.0004 for 1mg/ml and p<0.0001 for

3mg/ml; Figure 4.3 and Table 4.3). However, these behaviours were not statistically likely (64.5% probability) to predict cough (χ2 p=0.13; Figure 4.4C) and the cumulative total behaviour duration was not correlated to the total number of evoked coughs

(R2=0.02; Figure 4.4D).

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Figure 4.3: Bradykinin evokes behaviours associated with irritation in the conscious guinea- pig. Total durations (seconds) of cough stance, chewing and the sum total of all measured behaviours are shown as per minute values across the entire challenge. Blue bars represent periods of baseline recordings throughout the challenge, while green bars indicate the periods of nebulised solutions. These graphs show a clear dose-dependent increase in behaviour duration at each dose of bradykinin (0.3, 1 and 3mg/ml) compared to that observed during the saline (Sal) challenge. It can also be seen that increased behaviour appears to be driven predominately by chewing during bradykinin challenges.

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Figure legend on the next page 121 | P a g e

Figure 4.4: Bradykinin dose dependently evokes cough that is preceded by associated behaviours of irritation. (A) The total number of coughs evoked by Saline (Sal) and each dose of bradykinin individually for each of the baseline (B), nebulised (N) and response (R) periods. Evoked cough was significantly increased during the nebulisation of both 1 and 3mg/ml of bradykinin (** p=0.003 and **** p<0.0001 respectively) compared to their corresponding baselines. In addition, the cumulative total coughs, that is the sum across all dose, demonstrates a clear dose dependency in evoked cough. (B) The total behaviour duration (seconds) evoked by each of the individual periods of the challenge showing an increase in duration during the nebulisation of 3mg/ml of bradykinin compared to baseline (** p=0.009).

In addition, the cumulative behaviour duration, sum total across the challenges, also reveals a dose- dependent response in behaviour. Notably, at 0.3mg/ml there was a significant increase in evoked behaviour duration compared to saline (*p=0.03), despite no cough being evoked in any animals at this dose, which is indicative of behaviours preceding coughing. However, these behaviours were not found to predict cough as shown in panel (C) which investigates the difference between the time to first behave above baseline compared to the time for cough induction for individual coughing events (defined as the probability index) at 1mg/ml (circles) and 3mg/ml (squares) bradykinin challenge doses. A positive probability index reflects behavioural responses predicting evoked coughing, which was not found to be statistically likely (62.5% likelihood). (D) In addition, regression analysis revealed no significant relationship between the magnitude of bradykinin evoked behavioural and cough responses (R2=0.02).

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Table 4.3: List of behaviour durations and events associated with inhalation of

increasing doses of bradykinin in the no-intervention guinea pigs

* p=0.02; ** p=0.001; *** p=0.0004; **** p<0.0001

ATP: Aerosolised ATP evoked cough exclusively at the highest dose tested

(22.7±5.6 coughs, 8 out of 9 responders, p=0.0004) without influencing changes in respiratory frequency or tidal volume (Figure 4.5 and Figure 4.7B). Similar, to bradykinin, ATP evoked cough was generally confined to one to three coughing events.

However, in distinction to bradykinin, 110mg/ml of ATP significantly evoked cough in both the nebulised and response phase of the challenge compared to baseline (p=0.009;

Figure 4.7A). Also of note, changes in evoked behavioural responses were less robust compared to bradykinin and as such significant differences in cumulative scores compared to saline were only noted at the highest ATP challenge dose tested (404±109.5

123 | P a g e vs. 81.3±47.4 seconds, p=0.01; Figure 4.6 and 4.7B). Whereas chewing was the primary behavioural response to bradykinin, ATP evoked behaviours that largely reflected guinea pigs spending longer durations in the ‘cough stance’ (p=0.0003) and performing respiratory behaviours (p=0.0003; Figure 4.6 and Table 4.4). Finally, the duration of ATP evoked behaviours did not predict cough (37.5% likelihood; Figure 4.7C) and nor were they dependent on the level of cough (Figure 4.7D).

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Figure 4.5: Adenosine 5’-triphosphate (ATP) evokes cough in the conscious guinea pig without altering basal respiration. Cough number, respiratory frequency (breaths/minute) and tidal volume

(ml) are presented as per minute values over the course of the entire ATP challenge (0-65 minutes) in the no-intervention cohort (n=9). Blue bars represent baseline recordings and the green are for periods of nebulised solutions that is saline (Sal) and 1.1, 11 and 110 mg/ml of ATP. These traces show ATP evoked cough predominately occurring in the nebulised phase of 110mg/ml of ATP, while not significantly effecting respiratory frequency or tidal volume.

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Figure 4.6: Adenosine 5’-triphosphate (ATP) evokes behaviours associated with irritation in the conscious guinea-pig. Total durations (seconds) of cough stance, chewing and the sum total of all measured behaviours are shown as per minute values across the entire challenge. Blue bars represent periods of baseline recordings throughout the challenge, while green bars indicate the periods of nebulised solutions. These graphs show no dose-dependency of behavioural responses evoked by ATP (1.1, 11 and 110mg/ml), with increased durations only evident at the highest tested dose of ATP. Additional abbreviations: Sal; Saline.

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Figure legend on the next page 127 | P a g e

Figure 4.7: Adenosine 5’-triphosphate (ATP) evokes behaviours only at the cough evoking dose. (A)

The total number of coughs evoked by Saline (Sal) and each dose of ATP individually for each of the baseline (B), nebulised (N) and response (R) periods. Evoked cough was significantly increased during the nebulisation and recovery period of 110mg/ml of ATP (** p=0.009) compared to their corresponding baselines. In addition, the cumulative total coughs, that is the sum across all doses, demonstrates that the total evoked cough to ATP was significantly increased compared to Sal (*** p=0.0004). (B) The total behaviour duration (seconds) evoked by each of the individual periods of the challenge showing no significant increase in behaviour compared to corresponding baselines. In addition, the cumulative total behaviour duration, sum total across the challenges, revealed an appreciable increase in total behaviour duration compared to Sal (*p=0.01). (C) Behaviours evoked by ATP were not found to predict cough as shown in panel (C) which investigates the difference between the time to first behave above baseline compared to the time for cough induction for individual coughing events (defined as the probability index) at 110mg/ml (circles) ATP challenge doses. A positive probability index reflects behavioural responses predicting evoked coughing, which was not found to be statistically likely (37.5% likelihood). (D) In addition, regression analysis revealed no significant relationship between the magnitude of ATP evoked behavioural and cough responses (R2=0.06).

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Table 4.4: List of behaviour durations and events associated with inhalation of

increasing doses of adenosine 5’-triphosphate (ATP) in the no-intervention guinea

pigs

** p=0.002; **** p=0.009

4.3.2 The effect of targeted toxin lesions of the Pa5 on cough and behavioural responses associated with nebulised airway irritants

Microinjections of SSP-SAP into the Pa5 significantly reduced the number of NK1 receptor expressing Pa5 neurons (27.2±5.8 vs. 62.3±13.7 NK1 receptor-positive Pa5 neurons in lesioned animals compared to controls, p=0.04; Figure 4.8A). Neurons devoid of NK1-receptor expression were seemingly spared as SSP-SAP did not affect the number of Pa5 neurons expressing calbindin (30.8±4.6 in lesioned animals vs. 30.8±13.7 controls;

Figure 4.8B), previously shown to label a distinct population of neurons in the Pa5

(Driessen et al., 2018). Consequently, although the total number of Pa5 neurons, as

129 | P a g e labelled by the pan-neuronal marker NeuN, was reduced (44.2±20.1 in lesioned animals vs. 81.1±18.5 controls; Figure 4.8C), this did not reach statistical significance.

Figure 4.8: Characterisation of Substance P-Saporin (SSP-SAP) lesions of the paratrigeminal

nucleus (Pa5) in the guinea pig. Example photomicrographs of neurokinin 1 receptor (NK1, A),

Calbindin D-28k (Calb, B) and NeuN (C) in the Pa5 (+0.5mm Calamus Scriptorius) of control (left)

and lesion (right) animals. Total cell counts of immunoreactive Pa5 neurons in the control and

lesion animals are represented in the histograms. Quantification shows that NK1 receptor positive

Pa5 neurons are significantly decreased (* p=0.04) in lesion animals compared to control, while no

changes were seen in the total population of Pa5 neurons labelled by Calb or NeuN positive

populations.

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Bradykinin: SSP-SAP lesions of the Pa5 had no effect on tidal or stimulus-evoked respiratory frequency or volume, when compared to controls (Figure 4.9) or to animals without prior intervention (compare with Figure 4.2). In control animals, nebulised bradykinin reliably evoked cough (Figure 4.9), consistent with responses in guinea pigs without prior intervention. In lesioned animals, although the highest dose of bradykinin

(3mg/ml) was sufficient to evoke cough in the nebulising phase comparable to that in controls (Figure 4.11A), lesioned animals exhibited significantly less cough to 1mg/ml bradykinin compared to controls (0±0 coughs in lesioned animals vs. 7.2±2.8 coughs in controls, p=0.04; Figure 4.11A). Consequently, the cumulative total of bradykinin- evoked cough was significantly reduced by SSP-SAP lesions (9.9±2.9 coughs in lesioned animals vs. 20.7±3.9 coughs in controls cough, p=0.04; Figure 4.11A). Consistent with this, the proportion of animals that evoked cough to bradykinin was lower in lesioned

(50%) versus control (78.6%) animals, as was the number of coughing events (p=0.04;

Table 4.5). However, no significant relationship between lesion size and the degree of cough reduction was observed.

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Figure 4.9: Substance P-Saporin lesions of the paratrigeminal nucleus (Pa5) reduces bradykinin evoked cough in the conscious guinea pig without altering basal respiration. Cough number, respiratory frequency (breaths/minute) and tidal volume (ml) are presented as per minute values over the course of the entire bradykinin challenge (0-65 minutes) for control (black circles) and lesion (red squares) animals (n=14). Blue bars represent baseline recordings and the green are for periods of nebulised solutions that is saline (Sal) and 0.3, 1 and 3 mg/ml of bradykinin. These traces show bradykinin evoked cough was reduced in lesion animals compared to controls, while not significantly altering respiratory frequency or tidal volume.

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Despite the reduction in evoked cough, SSP-SAP lesions produced negligible changes in bradykinin evoked behaviours compared to controls (Figure 4.10 and Figure 4.11B).

Extensive analysis of all the recorded behaviours only identified a significant reduction in cough related behaviours following SSP-SAP lesions. For example, lesioned animals displayed a decrease in ‘cough stance’ duration during the 1mg/ml bradykinin dose compared to the controls (p=0.04; Table 4.5), consistent with the accompanying reduction in cough number observed. Furthermore, neither the lesion nor the control animals showed any correlation between cough and behaviour, consistent with the data obtained from animals without any prior intervention (Figure 4.11D and see section

4.3.1). However, interestingly in control animals evoked behaviours were statistically likely to predict cough (70% likelihood, p=0.0001), while this relationship did not exist in lesioned animals (Figure 4.11C).

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Figure 4.10: Substance P Saporin (SSP-SAP) lesions of the paratrigeminal nucleus (Pa5) do not alter behaviours evoked by nebulised bradykinin in the conscious guinea pig. Total durations (seconds, s) of cough stance, chewing and the sum total of all measured behaviours are shown as per minute values across the entire challenge. Blue bars represent periods of baseline recordings throughout the challenge, while green bars indicate the periods of nebulised solutions.

These graphs show both control (black circles) and lesion (red squares) evoke similar behaviour durations to saline (Sal) and increasing doses of bradykinin (0.3, 1 and 3mg/ml).

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Figure legend on the next page 135 | P a g e

Figure 4.11: Substance P-Saporin (SSP-SAP) lesions of the paratrigeminal nucleus (Pa5) significantly reduce bradykinin evoked cough but do not alter evoked behaviours. (A) The total number of coughs evoked by Saline (Sal) and each dose of bradykinin individually for each of the baseline (B), nebulised (N) and response (R) periods for control (black) compared to lesion (red) animals. In addition, the cumulative total coughs, that is the sum across all dose, demonstrates inhibition of cough to 1mg/ml in the lesion group and a significant reduction in total evoked cough to bradykinin (* p=0.04) compared to control animals. (B) The total behaviour duration (seconds) evoked by each of the individual periods of the challenge, as well as the cumulative behaviour duration both showed no differences in evoked behaviour between the control and lesioned guinea pigs.

However, interestingly in control animals these behaviours were found to predict cough, while this relationship was lost in lesion animals as shown in panel (C). which investigates the difference between the time to first behave above baseline compared to the time for cough induction for individual coughing events (defined as the probability index) at 3mg/ml bradykinin for control (black circles) and lesioned (red squares) animals. A positive probability index reflects behavioural responses predicting evoked coughing, which was statistically likely in the control group (70% likelihood, p=0.0001) but not the lesioned cohort. (D) In addition, regression analysis revealed no significant relationship between the magnitude of bradykinin evoked behavioural and cough responses for either cohort (R2=0.12 for controls and R2=0.03 for lesions).

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Table 4.5: List of behaviour durations and events associated with inhalation of

increasing doses of bradykinin in control (black) and lesioned1 (red) guinea pigs.

1Lesioned animals: Substance P-Saporin microinjections bilaterally into the paratrigeminal nucleus (Pa5)

* p=0.04

ATP: SSP-SAP lesions of the Pa5 similarly had no effect on respiratory rate and tidal volume responses associated with nebulised ATP (Figure 4.12). However, in contrast to that observed with bradykinin, lesioned animals showed no changes in ATP- evoked cough compared to control animals (Figure 4.12 and Figure 4.14A). In addition, behaviours of irritation associated with ATP challenges were also negligibly altered by

SSP-SAP lesions (Figure 4.13, Figure 4.14B and Table 4.6). Indeed, none of the behavioural measures analysed, including ‘cough stance’ duration, showed significant

137 | P a g e differences between the lesion and control cohorts (Figure 4.13 and Table 4.6). It is worth noting that while the normalised cumulative total behaviour duration appears to be trending downwards compared to controls, this trend was also evident during saline challenges (Figure 4.14B). Consequently, when the data is corrected for the relative response to saline, lesioned animals displayed an overall 96.2% increase in their behaviour duration during ATP challenges, which was slightly more than that observed in control animals (82.2% increase). Neither lesioned nor control animals showed any correlation between cough and behavioural responses, lesion and effect size or predictiveness of behaviours to cough (Figure 4.14C,D).

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Figure 4.12: Substance P-Saporin lesions of the paratrigeminal nucleus (Pa5) does not alter adenosine 5’-triphosphate (ATP) evoked cough or basal respiratory parameters. Cough number, respiratory frequency (breaths/minute) and tidal volume (ml) are presented as per minute values over the course of the entire ATP challenge (0-65 minutes) for control (black circles, n=12) and lesion (red squares, n=10) animals. Blue bars represent baseline recordings and the green are for periods of nebulised solutions that is saline (Sal) and 1.1, 11 and 110 mg/ml of ATP. These traces clearly show no difference in response of control and lesioned guinea pigs to nebulised ATP.

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Figure 4.13: Substance P Saporin (SSP-SAP) lesions of the paratrigeminal nucleus (Pa5) do not alter behaviours evoked by nebulised adenosine 5’-triphoshate (ATP) in the conscious guinea pig. Total durations (seconds) of cough stance, chewing and the sum total of all measured behaviours are shown as per minute values across the entire challenge. Blue bars represent periods of baseline recordings throughout the challenge, while green bars indicate the periods of nebulised solutions. These graphs show both control (black circles) and lesion (red squares) evoke similar behaviour durations to saline (Sal) and increasing doses of ATP (1.1, 11 and 110mg/ml).

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Figure legend on the next page 141 | P a g e

Figure 4.14: Substance P-Saporin (SSP-SAP) lesions of the paratrigeminal nucleus (Pa5) do not alter evoked cough or associated behaviours to aerosolised adenosine 5’-triphosphate (ATP).

(A) The total number of coughs evoked by Saline (Sal) and each dose of ATP individually for each of the baseline (B), nebulised (N) and response (R) periods for control (black) compared to lesion

(red) animals. Both cohort of animals similarly evoked cough to the highest tested dose of ATP

(110mg/ml). (B) The total behaviour duration (seconds) evoked by each of the individual periods of the challenge, as well as the cumulative behaviour duration both showed no differences in evoked behaviour between the control and lesioned guinea pigs. (C) This panel investigates the differences between the time to first behave above baseline compared to the time for cough induction for individual coughing events (defined as the probability index) at 110mg/ml ATP for control (black circles) and lesioned (red squares) animals. A positive probability index reflects behavioural responses predicting evoked coughing, which was not observed for either the control of lesioned animals. (D)

In addition, regression analysis revealed no significant relationship between the magnitude of bradykinin evoked behavioural and cough responses for either cohort (R2=0.03 for controls and

R2=0.3 for lesions).

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Table 4.6: List of behaviour durations and events associated with inhalation of increasing doses of adenosine 5’-triphosphate (ATP) in control (black) and

1 lesioned (red) guinea pigs.

1 Lesioned animals: Substance P-Saporin microinjections bilaterally into the paratrigeminal nucleus

(Pa5)

No significant differences observed

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4.4 Discussion

The Pa5 is a poorly studied nucleus in the dorsal lateral medulla that is in receipt of inputs from vagal, glossopharyngeal and spinal sensory neurons (Ciriello et al., 1981; Panneton and Burton, 1981; Altschuler et al., 1989; McGovern et al., 2012a; McGovern et al.,

2015b). We have shown that the Pa5 receives primary vagal afferents in guinea pigs and rats, but only those originating from the jugular vagal ganglia (Driessen et al., 2015;

McGovern et al., 2015b). We have also demonstrated a role for this nucleus in the integration of laryngeal-evoked respiratory reflexes, a tissue heavily innervated by jugular vagal ganglia neurons (Driessen et al., 2015; Driessen et al., 2018). Interestingly,

Pa5 neurons appear to be important for somatic nociception and complex sensorimotor behaviours associated with nociceptive experiences (Bon et al., 1997; Ma et al., 2005;

Koepp et al., 2006), suggesting that this nucleus plays a broad role in integrating noxious sensory inputs to the central nervous system. In this regard, Pa5 neurons provide ascending inputs to thalamocortical circuits involved in sensory discrimination (Caous et al., 2001; Chang et al., 2012; McGovern et al., 2015b). Since the jugular vagal ganglia share embryological and functional similarities with somatic sensory neurons

(Nassenstein et al., 2010; D’Autréaux et al., 2011; Nomaksteinsky et al., 2013) and because evoked cough and the associated sensations experienced during airway irritations resemble somatic nociception (Ando et al., 2014; Hilton et al., 2015), we hypothesised that the Pa5 may be an important medullary integration site for airway sensory inputs that in turn induce cough and the behaviours associated with the perception of an airway irritation. Consistent with this, the findings of the present study suggest that selective lesioning of NK1 receptor expressing Pa5 neurons reduces cough evoked by stimuli of jugular vagal ganglia nociceptor neurons but does not influence cough evoked by nodose afferent stimulation or many of the behaviours that accompany nebulised irritant

144 | P a g e challenges. These data implicate the jugular-Pa5 airway afferent processing pathway as a central component of jugular nociceptor evoked cough and in turn further highlights its importance in airway defence.

4.4.1 Studying behaviours of irritation is a sensitive and informative measure of airway irritation

The inhalation of airway irritants evokes perceivable sensations in human subjects, consistent with the notion that sensory inputs from the respiratory tract are processed in sensory discriminative regions of the brain. One sensory experience that commonly accompanies inhaled challenges is referred to as the urge-to-cough and is characterised by the perception of an airway irritant, often at the level of the larynx, that precedes the motor act of coughing (Davenport et al., 2007; Farrell et al., 2012). Cortical regions involved in the sensory processing linked to the urge-to-cough have been reasonably well defined in humans using fMRI during inhalation of cough evoking chemicals such as capsaicin. In such studies, capsaicin-evoked urge-to-cough is associated with increased neural activation within a distributed network that encompasses the mid-cingulate cortex, somatosensory cortices, inferior parietal lobe, insula and the pre-central cortex (Mazzone et al., 2007; Farrell et al., 2012). Mechanistic studies have suggested that the primary sensory cortex is particularly important for encoding the level of an urge-to-cough, because regional responses correlate strongly with an individual’s subjective reports of their sensory experiences (Farrell et al., 2012). This suggests that ascending sensory pathways analogous to those involved in somatosensory processing may be involved in aspects of respiratory sensory discrimination.

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The precise organisation of these higher order circuits involved in subjective experiences have been difficult to study, in part because detailed mechanistic experiments are difficult to perform in animals given that a surrogate measure of the urge-to-cough has not been developed. Indeed, this is a challenge for any animal studies investigating subjective experiences. In the field of pain research, in addition to measuring the motor responses to painful stimuli (e.g. tail flick or withdrawal reflexes) behavioural correlates of pain perception, including assessing face grimace, have proved informative in rats and mice

(Langford et al., 2010; Sotocinal et al., 2011; Akintola et al., 2017; Tuttle et al., 2018).

This motivated our attempts to develop a model for assessing behavioural responses accompanying cough evoking airway irritations in guinea pigs.

4.4.2 Cough and accompanying behavioural responses to nebulised irritant challenges in guinea pigs: the role of nodose versus jugular afferent pathways

In guinea pigs, ATP is a selective stimulus of the nodose airway afferents (Kwong et al.,

2009; Nassenstein et al., 2010; Muroi et al., 2013), which we have shown to integrate within brainstem and subcortical regions that are important for autonomic and limbic processing (Driessen et al., 2015; McGovern et al., 2015b). Nodose nociceptive C-fibres do not readily evoke cough (Canning and Mori, 2011; Muroi et al., 2013; Chou et al.,

2018), while mechanically sensitive nodose A-fibre cough is primarily reflexively

(persisting under anaesthesia) driven (Tsubone et al., 1991; Widdicombe, 1996; Bolser and Reier, 1998; Canning et al., 2004; Canning et al., 2006). On the other hand, although bradykinin stimulates both nodose and jugular nociceptors, it presumably evokes coughing via its action on jugular afferents (Hewitt et al., 2016). Interestingly, however, bradykinin and other jugular afferent stimuli only evoke cough in conscious animals and

146 | P a g e humans (Canning et al., 2004; Mazzone et al., 2005), which might suggest and important role for higher brain processing and conscious perception in jugular evoked responses.

Consistent with prior studies, both bradykinin and ATP nebulised challenges evoked variable amounts of coughing in our conscious guinea pigs, dependent on the challenge dose employed. In addition to cough, nebulised challenges evoked a variety of behaviours consistent with the perception of an irritation associated with the challenge stimulus.

These included periods of increased locomotor activity, chewing and grooming during and after nebulised challenges. In humans, the urge-to-cough threshold for a given inhaled stimulus is typically lower than the stimulus threshold needed to evoke coughing (Farrell et al., 2012). In other words, the perception of irritation precedes the motor act of coughing on a stimulus response curve. In the present study, bradykinin-associated behavioural responses similarly displayed a lower threshold for induction than for evoked cough. Interestingly, while bradykinin showed a dose dependent increase in both cough and associated behaviours, ATP did not, and instead only evoked increased behaviours at the same dose as that for evoked coughing. These data suggest that sensations associated with jugular C-fibre activation evoke behaviours that are consistent with the urge-to- cough experience in humans. On the other hand, neither stimulus demonstrated behavioural correlates that were predictive of cough or any correlation between the magnitude of the behavioural measures and coughing, perhaps suggestive of independent sensory processing. Regardless, the observation of distinct cough and behavioural profiles associated with bradykinin compared to ATP nebulised challenges provide functional evidence for nodose and jugular afferents processing contributing differently to sensory experiences.

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4.4.3 Evidence for NK1 receptor expressing Pa5 neurons regulating jugular C-fibre evoked cough.

We previously identified two populations of Pa5 neurons in the guinea pig differentiated by the expression of NK1 receptors and calbindin (Driessen et al., 2018). In the present study, we were interested in the role of the NK1 receptor expressing population in evoked cough and the conscious perception of an airway irritation as neurons of a similar phenotype elsewhere in the nociceptive system are essential for processing painful sensations (Iadorala et al., 2017; Li et al., 2017). Additionally, NK1 receptor antagonists have been shown to be anti-tussive in both animals and humans and as such represents a targetable population (Armadesi et al., 2001; Chapman et al., 2004; El-Hashim et al.,

2004; Grobman and Reinero, 2016; Smith et al., 2017).

Consistent with our hypothesis, SSP-SAP toxin lesions of the NK1 receptor positive Pa5 neurons significantly reduced cough evoked by bradykinin but not cough evoked by ATP.

Furthermore, while control animals often responded with a number of distinct coughing events across different bradykinin dose challenges, this pattern was dramatically altered in the lesioned animals that typically only responded with a single bout of coughing at the highest dose of bradykinin tested. This finding is consistent with a putative role of jugular and nodose afferent pathways in bradykinin and ATP-evoked cough, respectively.

The reduction in bradykinin-evoked cough was accompanied by a reduction in cough- related respiratory behaviours (e.g. time spent in cough stance). However, contrary to our hypothesis, NK1 receptor neuron lesions did not alter any of the other behavioural responses associated with airway irritation during nebulised challenges with either bradykinin or ATP.

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This observation, and the lack of correlation between behaviours and cough, cast doubt on the nature of the behavioural responses to nebulised challenges. Indeed, it is conceivable that behavioural responses are not reflective of selective irritation of the airways but may also be driven by nebulised agents irritating ocular, nasal and orofacial tissues. However, it is also conceivable that while NK1 receptor expressing neurons play a specific role in the manifestation of cough, instead an alternative Pa5 neuron subtype

(e.g. those expressing calbindin) may be responsible for the associated behaviours.

Alternatively, the magnitude of the lesion (approximately 50% reduction in NK1 receptor expressing neurons) may not have been sufficient to reveal a behavioural phenotype given the sensitivity of the measure. Non-specific lesions of the Pa5 (for example using electrolytic approaches) to equally reduce all neuronal populations with greater efficiency may help to shed more light on these possibilities.

4.4.4 Physiological significance and conclusion

Chronic cough can be a difficult to treat symptom in many patients with respiratory disease. This is in part relates to an apparent heterogeneity in the mechanisms precipitating cough (Mazzone et al., 2018) as well as a poor understanding of the organisation of the neural circuits that are responsible for cough induction. It is further complicated by the fact that reflexive cough is an essential protective mechanism for airway clearance and therapeutics that diminish protective cough may negatively compromise respiratory function and leave patients vulnerable to aspiration pneumonia.

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The findings of the present study provide functional evidence to suggest that nodose and jugular airway afferent pathways regulate cough through different central neural circuits.

Our data support previous findings consistent with the notion that nodose airway afferents are important for reflex defensive coughing, which is dependent upon afferent integration within the nTS (Driessen et al., 2015; McGovern et al., 2015b). For example, mechanical or acidic stimuli applied to the glottis, larynx or large airways evokes rapid defensive cough, even under general anaesthesia and in decerebrated animals (Tsubone et al., 1991;

Widdicombe, 1996; Bolser and Reier, 1998; Canning et al., 2004), responses that are inhibited by glutamatergic antagonists applied to the nTS (Ezure et al., 1999; Canning and Mori, 2011). On the other hand, stimuli that activate jugular airway afferent pathways only evoke cough in conscious animals (Canning et al., 2004; Mazzone et al., 2005) and these responses are readily modified by voluntary and cognitive processes, including placebo manipulations and anxiety (Davenport et al., 2009; Leech et al., 2013; Janssens et al., 2014). Our current data may suggest that the jugular vagal ganglia pathway evokes cough via circuitry that is distinct to that involved in nodose coughing. As jugular afferents are thought to be important for the manifestation of chronic cough in disease, our findings reveal a novel opportunity for therapeutic intervention, potentially allowing for cough in disease to be targeted independent of reflex (nodose) protective cough.

Furthermore, we have identified a distinct subtype (NK1 receptor positive) of postsynaptic neuron in the Pa5 important for processing jugular afferent evoked cough and learning more about the phenotype of these neurons might identify novel pharmacological targets for cough suppression. Taken together, our findings implicate the jugular-Pa5 airway afferent processing pathway as a candidate target for treating cough without perturbing reflex function.

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5. Chapter 5: General discussion

Respiratory sensations conveyed by airway afferents are poorly understood, despite contributing significantly to morbidity associated with pulmonary disease (Polley et al.,

2008; Chung, 2011; Morice, 2013; Hilton et al., 2015). Physiologically, the perception of an airway irritation may be important for informing individuals about the environment and functioning of the respiratory system and for initiating top down modulation of respiratory behaviours to help protect the airways from damage and maintain adequate ventilation. While studies have identified the cortical networks activated by the conscious perception of an airway irritation and the associated urge-to-cough (Mazzone et al., 2007;

Mazzone et al., 2011; Farrell et al., 2012; Ando et al., 2016), we do not have the same level of understanding regarding the brainstem and subcortical brain regions that are involved in this process.

In the present doctoral studies, it was hypothesised that the paratrigeminal nucleus (Pa5) is a candidate brainstem region for processing airway irritant-related inputs. This hypothesis, in part, evolved because of previous studies implicating the Pa5 in baroreceptor reflex function and noxious somatosensory processing (Lindsey et al., 1997;

Koepp et al., 2006; Yamazaki et al., 2008; Sousa and Lindsey, 2009). In addition, using viral tracing techniques, we had shown that the Pa5 is in receipt of vagal airway primary afferents (McGovern et al., 2012a; McGovern et al., 2012b). However, astonishingly no prior studies had investigated the involvement of the Pa5 in airway afferent processing.

Therefore, the overarching aim of this thesis was to investigate the role of the Pa5 in airway defence. To address this aim I completed several neuroanatomical, neurophysiological and behavioural studies that were designed to understand the input

151 | P a g e and output connections of the Pa5 and its involvement in both respiratory reflexes and complex respiratory behaviours. In doing so, I was able to show for the first time that airway afferents projecting to the Pa5 are both anatomically and functionally distinct from those that project to the well-studied nucleus of the solitary tract (nTS; Driessen et al.,

2015). In addition, the present results revealed an unappreciated complexity of processing in the Pa5 that has important implications for both the initiation of laryngeal-evoked respiratory reflexes and complex respiratory behaviours, specifically in evoked cough.

5.1 Jugular vagal ganglia-derived airway afferents project specifically to the

Pa5

Our laboratory previously reported the results of studies in the rat using neuroanatomical tracing approaches showing airway afferents derived from the jugular vagal ganglia projecting almost exclusively to the Pa5 (McGovern et al., 2015b). In the studies presented in this dissertation, I used a comparable experimental strategy to demonstrate the same anatomical distinction in the guinea pig (Driessen et al., 2015). Furthermore, physiological experiments confirmed functionality of this anatomy. Together these data suggested the existence of a previously unrecognised organisation for airway sensory processing in the brainstem. Jugular vagal ganglia afferents have not been studied extensively but are thought to have a fairly restricted pattern of peripheral innervation.

Although, many terminal fields are associated with somatic tissues of the head and neck, afferents from the jugular vagal ganglia also terminate in tissues that would traditionally be considered visceral in nature, including the large airways and neighbouring oesophagus (Khurana and Petras, 1991; Undem et al., 2004; Yu et al., 2005; McGovern et al., 2015a; Driessen et al., 2018). The larynx, an important reflexogenic site for airway

152 | P a g e defence and is particularly heavily innervated by vagal afferents derived from the jugular vagal ganglia (Driessen et al., 2018), raising important questions about the role of these afferents in airway protective reflexes and associated sensations.

Jugular vagal ganglia-derived laryngeal neurons are classified as either Aδ- or C-fibre nociceptors, based on conduction velocity, degree of myelination and their peptidergic content (Riccio et al., 1996; Canning et al., 2004; Undem et al., 2004; Driessen et al.,

2015; Driessen et al., 2018). These nociceptors can be activated by chemical irritants and endogenous inflammatory mediators including capsaicin (TRPV1 agonist) and bradykinin (B2 receptors), that are also known to elicit responses from nodose C-fibres

(Undem et al., 2004; Chou et al., 2008). Alternately, more selective stimuli have been proposed in cell culture or ex-vivo conditions and they include low extracellular calcium concentrations that preferentially activate jugular C-fibres (Undem et al., 2003) and hyperosmolar solutions that more readily stimulate jugular Aδ-fibres (Pedersen et al.,

1998). However, these latter stimuli have not proven to be good cough stimuli in vivo, despite implications for jugular vagal ganglia-derived nociceptors in respiratory reflexes and evoked cough (Muroi et al., 2013; Hewitt et al., 2016).

Experiments in the anaesthetised guinea pig have shown that jugular and nodose vagal afferents effect respiration differentially (Chou et al., 2008; Chou et al., 2018). That is while selective stimulation of the nodose vagal ganglia-derived neurons results in tachypneic responses (i.e. increased respiratory rate) the activation of jugular vagal ganglia-derived neurons is associated with apnoea (i.e. cessation of breathing) and respiratory slowing (Chou et al., 2008). Aligning with these results, recent data has also

153 | P a g e suggested that while jugular C-fibres are pro-cough, the nodose C-fibres are inhibitory to this respiratory reflex (Chou et al., 2018). In addition, studies in the conscious guinea pigs and humans are suggestive that jugular C-fibre evoked cough is dependent on higher brain processing given that cough via this pathway is inhibited by anaesthesia (Tsubone et al.,

1991; Widdicombe, 1996; Bolser and Reier, 1998; Canning et al., 2004). However, we are yet to understand the individual contribution of the jugular Aδ- and C-fibre populations to cough genesis or how they integrate into higher ordering processing of sensory disturbances associated with airway irritation.

Investigating jugular vagal ganglia-derived afferent processing in more detail has been limited by our lack of understanding of this sensory neuron population. Our broad classifications of airway sensory neurons are likely a gross under-representation of the true diversity of the afferent populations. For example, single cell-RNA sequencing of dorsal root ganglia neurons has revealed at least eight functionally distinct types of nociceptors in the somatosensory nervous system (Hu et al., 2016; Li et al., 2016;

Jurcakova et al., 2018). Comparable studies have not been reported for vagal afferents, although investigating if this level of heterogeneity exists in the airway sensory nervous system will be important to better understand afferent physiology and to improve our ability to specifically study and target the jugular vagal ganglia afferent processing pathway.

Additionally, studies presented in this thesis suggest a level of complexity of how afferent fibres and postsynaptic partners might be arranged in the Pa5, although the fine synaptic organisation of these afferent subtypes remains to be investigated. One possibility is that

154 | P a g e there exists a convergent organisation, whereby each postsynaptic Pa5 neuron is innervated by both Aδ- and C-fibre jugular nociceptors (Figure 5.1A). Alternately, different afferent subpopulations could form synapses with specific populations of Pa5 neurons (Figure 5.1B) similar to the non-convergent organisation of A- and C-fibre terminations in the nTS (Bailey et al., 2002; McDougall and Andresen, 2013). In addition, it is well known that the Pa5 receives many more inputs than solely from the vagal ganglia, including from the trigeminal, glossopharyngeal and spinal nerves (Ciriello et al., 1981; Panneton and Burton, 1981; Altschuler et al., 1989; McGovern et al., 2012a;

McGovern et al., 2015b). In fact, anatomical investigations in the rat have identified convergence of the trigeminal and vagal or glossopharyngeal afferents in the Pa5 (Saxon and Hopkins, 2006; Ma et al., 2007), evidence that the Pa5 is a site for integration of visceral and somatic information. Understanding the specificity of this integration in regard to subtypes of jugular afferents and Pa5 neurons remains to be understood.

155 | P a g e

Figure 5.1: Diagrammatic representation of the potential synaptic organisation in the

paratrigeminal nucleus (Pa5). (A) Schematic depicts the possibility of non-specific

organisation within the Pa5. That is different subtypes of Pa5 postsynaptic neurons receive

convergent information from both the Aδ- and C-fibre jugular airway afferents, relying on

differential release mechanisms for functional specificity. (B) Alternately, the peripheral inputs

into the Pa5 could be organised specifically such that one type of postsynaptic neuron receives

one type of peripheral input to allow for specificity in the system. Additional abbreviations:

NK1 receptor +ve; neurokinin 1 receptor positive, Calb+ve; calbindin positive.

5.2 Distinct Pa5 neurons differentially influence respiratory behaviour

The present studies provide evidence against the notion that the Pa5 is simply a relay nucleus for diverse nociceptive inputs, but instead likely plays an important role in multimodal afferent integration, needed for both reflex regulation and higher order sensory discrimination. Investigations into the output connectivity of Pa5 neurons revealed that the Pa5 displays specificity in projections throughout the cardiorespiratory column (Driessen et al., 2018). This was further supported by the characterisation of two

156 | P a g e distinct populations of Pa5 neurons; one being defined by expression of the NK1 receptor and the other by expression of the calcium binding protein, calbindin (Driessen et al.,

2018). However, an obvious question, not addressed in the experimental chapters of this thesis, is do different phenotypes of Pa5 neurons display selective connectivity with recipient bulbar nuclei? Studies are currently under way to address this by investigating the phenotype of pontine versus ventrolateral medullary projecting Pa5 neurons.

Interestingly, with respect to these two recipient sites, the preliminary data suggests that

NK1 receptor expressing Pa5 neurons project exclusively to the pons, while calbindin expressing Pa5 neurons preferentially project to the ventrolateral medulla (Figure 5.2).

This is further consistent with notion that afferent integration through the Pa5 is organised such that it can conceivably drive selective physiological responses.

157 | P a g e

Figure 5.2: Characterisation of distinct projections of paratrigeminal (Pa5) neurons to the (A) ventrolateral medulla (VLM) and (B) pontine nuclei (preliminary data). (A) Pie charts representing the total population of Pa5 neurons retrogradely traced from the VLM (n=2) in green and the proportion of those that either stained for neurokinin 1 receptor (NK1; orange) or calbindin (Calb; red). The histograms show that while calbindin expressing Pa5 neurons project to the VLM, NK1 receptor expressing Pa5 neurons do not. (B) Pie charts show the total population of Pa5 neurons retrogradely labelled from the pons (n=6) in green and the proportion of those that either stained for NK1 receptor (orange) or calbindin (red). The histograms show that the pontine nuclei receive a mixed proportion of both NK1 receptor and calbindin expressing Pa5 neurons.

158 | P a g e

The neuroanatomical data presented is supported by functional evidence for two distinct subtypes of Pa5 neurons, capable of differentially controlling respiration. Glutamatergic neurotransmission in the Pa5 was shown to evoke apnoeic responses, while peptidergic neurotransmission was shown to evoke tachypnoea (Driessen et al., 2018). These contrasting responses may reflect specificity in peripheral terminations in the Pa5 (Figure

5.1B), but they may also reflect the capability of central terminals to differentially release neurotransmitters (i.e. glutamate and neuropeptides). In this regard, studies have shown that local synaptic calcium events are associated with specific types of channel activation that can differentially regulate the release of small (glutamatergic) or large (peptidergic) dense core neurotransmitter vesicles (Fawley et al., 2016; Hofmann and Andresen, 2016).

This is consistent with the effects of microinjected capsaicin reported in the present thesis, which would mobilise nerve terminal calcium via different ionic mechanisms compared to action potential (voltage)-dependent processes. While this needs to be tested directly in the Pa5, if correct it raises an important question about whether this system is perturbed in disease in a manner that potentiates peptidergic signalling. Given that we have demonstrated a role for the NK1 receptor expressing Pa5 neurons in the processing of jugular C-fibre evoked cough, it is conceivable that upregulated peptidergic signalling in the Pa5 could lead to exaggerated respiratory sensations and cough associated with disease.

159 | P a g e

The data presented herein are also suggestive that the heterogeneity of Pa5 neurons is not limited to the NK1 receptor and calbindin expressing populations, because collectively these neuronal subtypes do not account for all neurons in the Pa5 identified with a pan neuronal marker. Interestingly, both calbindin and the NK1 receptor expressing neurons have similarly been described in the trigeminal nucleus and dorsal horn, along with other neuronal subtypes defined by their expression of markers such as serotonin, nicotinic- acetylcholine and opioid receptors (Oliveras et al., 1981; Classey et al., 2010; Wang et al., 2018). Given the functional similarity between the Pa5 and these other areas, for primary afferent processing, it might be predicted that a similar diversity in Pa5 neuronal subtypes exists. If true, it will then be important to investigate how the afferent inputs are synaptically arranged with respect to each of these postsynaptic neuron types and explore specificity in the local bulbar connectivity arising from these neurons. In this regard, it is also important to acknowledge that the current body of work regarding the Pa5 connectome in the guinea pig has not attempted to assign airway afferent specificity to the circuitry. However, significant advancements have been made in circuit mapping technologies that would allow for this type of investigation, including our ability to deliver circuit tracing constructs to the Pa5 in the guinea pig, via viral (e.g retrograde adeno-associated viruses) or non-viral (e.g. CRISPR) technologies. Therefore, while the present data have advanced our current understanding of Pa5 processing it has arguably only scratched the surface and there is still much to be learnt about this important afferent processing region. Identifying the heterogeneity in postsynaptic neuronal populations, their connectivity with afferent inputs and recipient brainstem targets and their involvement in noxious sensory integration will increase our capability to functionally study this nucleus and modulate it for therapeutic benefit.

160 | P a g e

5.3 Clinical significance and final conclusions

This thesis has clearly shown that the Pa5 is an important functional region involved in airway afferent processing. The presented data builds upon our foundational evidence from the rat (McGovern et al., 2015b) showing anatomically and functionally that the nodose-nTS and jugular-Pa5 represent two parallel processing pathways for sensing the airway environment. Importantly, our investigations of the Pa5 in evoked cough and associated behaviours of irritation showed that these two pathways likely support different components of airway sensations. That is, selective stimulation of nodose airway afferents (by ATP) weakly evoked cough and behaviours of irritation in an all or none response that would align with a more reflexive airway neural circuit. Alternately, activation of jugular afferents readily evoked cough and behaviours of irritation in a dose dependent manner, perhaps highlighting the influence of higher order processing. As this is consistent with the known central connectivity of the Pa5 (McGovern et al., 2015b), it seems conceivable that this nucleus is therefore integral for directing airway sensory inputs to areas of the higher brain responsible for aspects of sensory discrimination associated with airway irritations.

In addition, we predict that the Pa5 may not only integrate primary sensory information across multiple ascending inputs but may also be an important site for the regulation of afferent processes by modulatory networks, including the descending analgesia system and diffuse noxious inhibitory control (DNIC). Simply DNIC is the phenomenon whereby one pain inhibits another at the level of the brainstem (Le Bars et al., 1979; Dickenson et al., 1981; de Resende et al., 2011; Youssef et al., 2016). This neural circuit is an important gating mechanism in pain and when disrupted results in conditions of chronic pain

161 | P a g e

(Dickenson et al., 1981; Yarnitsky, 2010; Ness et al., 2014; Bannister et al., 2015). It is yet to be determined if this circuit also modulates visceral nociception from the airways, but ultimately it could present as an alternate therapeutic target to better treat perturbed airway sensations that are associated with respiratory disease.

Finally, it is important to recognise that the present body of work has been conducted in healthy animals and therefore likely under-represents the role of the Pa5 in conditions of sensory hypersensitivity. As such, it will be intriguing to investigate the potential plasticity in afferent processing that occurs in this region in a disease context (Figure 5.3), as this may provide new insights in the manifestation of heightened sensitivity and exaggerated cough that is characteristic of many pulmonary conditions. This includes investigating changes in the distribution and phenotype of afferent terminal and synaptic organisation (neuronal plasticity), the induction of central neuroinflammation and neuronal hyper-excitability (including increased peptidergic signalling) and the possibility that therapeutic modulation at the level of the Pa5 leads to clinical benefit. It is a matter of interest because sensory hypersensitivities accompanying respiratory diseases have proven difficult to treat and consequently many patients continue to suffer from chronic cough and related sensory disturbances. To this end, the Pa5 may represent a novel alternate target to treat perturbed sensations of respiratory disease, potentially without impacting vital respiratory reflexes.

162 | P a g e

Figure 5.3: Flow diagram proposing anatomical and functional alterations in the paratrigeminal nucleus (Pa5) in a pathological context. In disease, processing in the Pa5 may be altered and contribute to chronic cough and persistent airways irritation. Three of the major contributing factors may include induction of neuronal plasticity that alters afferent organisation and function in the Pa5, such that innocuous stimuli may be processed by pro-nociceptive pathways. In addition, general increases in Pa5 neuronal excitability may be observed with increased peptidergic signalling. This could involve both increased presynaptic neuropeptide release or up-regulation of the peptide receptors on the postsynaptic neurons. Finally, neuroinflammation is a likely component that will allow maintenance of hyperexcitable and hypersensitive states by both microglial infiltration and communication with Pa5 neurons. Together these effects will increase nociceptive drive from the Pa5 and in turn potentiate evoked cough and sensations of airway irritations. Additional abbreviations:

Glu; Glutamate, SP; Substance P.

163 | P a g e

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Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Driessen, Alexandria

Title: Determining the role of the paratrigeminal nucleus (Pa5) in airway defence

Date: 2018

Persistent Link: http://hdl.handle.net/11343/221395

File Description: Alexandria Driessen 879290 Final Thesis

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