Unconventional components of inhibition that contribute to the regulation of sensitization in the spinal cord dorsal horn

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Unconventional Components of Inhibition that

Contribute to the Regulation of Sensitization in the

Spinal Cord Dorsal Horn

Thèse

Jimena Pérez Sánchez

Doctorat en Neuroscience

Philosophiae Doctor (Ph. D.)

Québec, Canada

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Unconventional Components of Inhibition that

Contribute to the Regulation of Sensitization in the

Spinal Cord Dorsal Horn

Thèse

Jimena Pérez Sánchez

Sous la direction de:

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Abstract

The dorsal horn of the spinal cord contains a diverse population of neurons that are responsible for the integration and transmission of pain-related information into the brain. The excitability of these neurons is largely controlled by γ-aminobutyric acid (GABA) and glycine, the two main inhibitory neurotransmitters in the central nervous system. Conventional synaptic inhibition is mediated by chloride-permeable GABAA and glycine

receptors, located directly opposite to presynaptic terminals. However, a different subset of these receptors is located outside the synapse and provides further functional diversity of inhibition. The studies presented in this thesis employed a wide variety of approaches to explore two non-conventional components of inhibition in the regulation of pain-related transmission in the dorsal horn of the spinal cord. The first is the role of non-synaptic GABAergic receptors that produce tonic inhibition; the second is the regulation of intracellular chloride gradients that determine the strength of GABAergic inhibition.

The results show that there is a gradient of inhibition across the dorsal horn, with weaker inhibition in superficial levels, and increasingly stronger in deeper levels. This is evidenced by a differential contribution of α5 subunit-containing GABAA receptors (α5GABAARs) to

tonic inhibition across the dorsal horn of the spinal cord. Importantly, the basal activity of these receptors is not strong enough to counter sensitization of pain-related information but contributes to the recovery from these sensitized states. Enhancing the activity of α5GABAARs also does not constrain sensitization. In contrast, the activity of other

tonic-inhibition-producing GABAA receptors, namely those that contain δ subunits (δGABAARs),

can be enhanced to decrease sensitization in the dorsal horn. On the other hand, there is a also gradient in chloride homeostasis, which affects how different neurons integrate synaptic inputs. In fact, I found that chloride homeostasis modulates the propensity and stability of synaptic plasticity. As such, higher activity of the neuronal chloride extruder (KCC2), such as that present in deeper levels of the dorsal horn, is directly linked to a stable, or constrained, long-term potentiation (LTP). Conversely, low chloride extrusion capacity in superficial levels leads to enhanced and unstable LTP in the dorsal horn.

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In conclusion, I uncovered a gradient of inhibition across the dorsal horn that shapes the sensitization of nociceptive information. Weak inhibition at superficial levels leads to enhanced and unconstrained synaptic plasticity, whereas stronger inhibition stabilizes plasticity at deeper levels of the dorsal horn.

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Resumé

La corne dorsale de la moelle épinière contient une population diversifiée de neurones qui sont responsables de l'intégration et de la transmission de l'information liée à la douleur vers le cerveau. L'excitabilité de ces neurones est largement contrôlée par l'acide γ-aminobutyrique (GABA) et la glycine, les deux principaux neurotransmetteurs inhibiteurs du système nerveux central. L'inhibition synaptique conventionnelle est médiée par des récepteurs GABAA et glycine perméables aux chlorures, situés à l’opposé des terminaux

présynaptiques. Cependant, un sous-ensemble différent de ces récepteurs est situé à l'extérieur de la synapse et offre une diversité fonctionnelle supplémentaire d'inhibition. Les études présentées dans cette thèse ont utilisé une grande variété d'approches pour explorer deux composantes non conventionnelles de l'inhibition dans la régulation de la transmission liée à la douleur dans la corne dorsale de la moelle épinière. Le premier est le rôle des récepteurs GABAergiques non synaptiques qui produisent une inhibition tonique; le seconde est la régulation des gradients de chlorure intracellulaire qui déterminent la force de l'inhibition GABAergique.

Les résultats montrent qu'il existe un gradient d'inhibition à travers la corne dorsale, avec une inhibition plus faible dans les niveaux superficiels, et de plus en plus forte en profondeur. Ceci est démontré par une contribution différentielle des récepteurs GABAA contenant des

sous-unités α5 (α5GABAAR) à l'inhibition tonique à travers la corne dorsale de la moelle

épinière. L'activité basale de ces récepteurs n'est pas assez forte pour contraindre la sensibilisation à l'information liée à la douleur, mais contribue à la récupération de ces états sensibilisés. Le renforcement de l'activité de αGABAARs ne limite pas non plus la sensibilisation. En revanche, l'activité d'autres récepteurs GABAA producteurs d'inhibition

tonique, à savoir ceux contenant des sous-unités δ (δGABAAR), peut être améliorée pour

diminuer la sensibilisation dans la corne dorsale. D'autre part, il y a aussi un gradient dans l'homéostasie du chlorure, qui affecte la façon dont les différents neurones intègrent les entrées synaptiques. En fait, j'ai decouvert que l'homéostasie du chlorure module la propension et la stabilité de la plasticité synaptique. Une activité plus élevée de l'extrudeur neuronal du chlorure (KCC2), telle que celle présente dans les niveaux plus profonds de la

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Inversement, une faible capacité d'extrusion de chlorure dans les niveaux superficiels conduit à une LTP accentué dans la corne dorsale.

En conclusion, j'ai découvert un gradient d'inhibition à travers la corne dorsale qui détermine la sensibilisation de l'information nociceptive. Une inhibition faible aux niveaux superficiels conduit à une plasticité synaptique améliorée et sans contrainte, alors qu'une inhibition plus forte contraint la plasticité aux niveaux plus profonds de la corne dorsale.

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List of Figures

Figure 1.1 Anatomy of the spinal cord ... 6

Figure 1.2 Primary sensory neurons are pseudo-unipolar ... 7

Figure 1.3 Characteristics of primary afferent fibers ... 8

Figure 1.4 Laminar organization of primary afferent inputs in the dorsal horn. ... 9

Figure 1.5 Supraspinal projection sites ... 11

Figure 1.6 Neurons in lamina I ... 12

Figure 1.7 Neurons in lamina II ... 14

Figure 1.8 Physiology and connectivity of superficial dorsal horn neurons ... 15

Figure 1.9 Gate Control Theory ... 18

Figure 1.10 Descending pathways for the modulation of nociceptive information ... 20

Figure 1.11 Sensitization of spinal nociceptive circuits ... 22

Figure. 1.12 Synthesis and transport of GABA at synapses ... 27

Figure 1.13 GABAA receptor structure ... 28

Figure 1.14 Phasic and tonic inhibition ... 29

Figure 1.15 Gephyrin hexagonal lattice. ... 32

Figure 1.16 GABAA receptor scaffold ... 33

Figure 1.17 Action of benzodiazepines ... 37

Figure 1.18 Selectivity for anesthetic action ... 39

Figure 1.19 Regulation of Cl− by NKCC1 and KCC2 in neurons. ... 42

Figure 1.20 Regulation of KCC2. ... 45

Figure 1.21 Cell-type specific plasticity in the dorsal horn. ... 52

Figure 2.1 Cl−_{electrochemical gradient ... 81}

Figure 2.2 Shunting inhibition ... 82

Figure 2.3 Activity-dependent collapse of inhibition ... 83

Figure 2.4 Depolarizing GABA ... 84

Figure 2.5 Catastrophic collapse of inhibition ... 85

Figure 3.1 Measurement of field postsynaptic potentials ... 91

Figure 4.1 GFE3 specifically ablates gephyrin. ... 106

Figure 4.2 Regulation of GABAAR distribution by gephyrin. ... 107

Figure 4.3 Selective ablation of gephyrin reduces mIPSC amplitude and frequency. ... 108

Figure 4.4 Expression of GFE3 does not affect frequency or amplitude of mEPSCs. ... 109

Figure 4.5 The magnitude of GABAergic currents is not affected by GFE3 expression. .. 110

Figure 4.6 Noise analysis of GABA-evoked currents. ... 111

Figure 4.7 Gephyrin ablation by GFE3 is transient and reversible. ... 112

Figure 5.1 Laminar distribution of α5GABAARs in the mouse spinal cord. ... 132

Figure 5.2 Distribution of α5GABAARs in different compartments of spinal LI and LII. . 133

Figure 5.3 Contribution of α5GABAARs to tonic vs. synaptic inhibition in the dorsal horn. ... 135

Figure 5.4 α5GABAARs constrain late phase sensitization but not acute thermal and mechanical nociception, nor early phase sensitization. ... 136

Figure 5.5 Time-course of sensitization. ... 137

Figure 6.1 Etomidate prolongs decay time of inhibitory synaptic currents in lamina II neurons, without affecting the amplitude. ... 150

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Figure 6.3 Enhancing tonic inhibition with etomidate does not prevent the potentiation of

fPSP responses to LFS. ... 152

Figure 6.4 Enhancing δGABAAR activity with THIP abolishes spinal LTP. ... 153

Figure 6.5 Tonic inhibition enhancers decrease sensitization to capsaicin. ... 154

Figure 7.1 Cl− extrusion capacity in the superficial dorsal horn is mediated by KCC2. .... 173

Figure 7.2 Interlaminar heterogeneity of Cl−_{extrusion capacity in the superficial dorsal} horn. ... 174

Figure 7.3 Activity-dependent collapse of inhibition in the superficial dorsal horn. ... 175

Figure 7.4 GABAA receptors mediate collapsible inhibition ... 176

Figure 7.5 Distribution of KCC2 in the superficial dorsal horn. ... 177

Figure 7.6 Interlaminar gradient of KCC2 expression in the SDH depends on TrkB signalling. ... 179

Figure 7.7 Activity-dependent gradient in [Cl]i in LI and LII. ... 180

Figure 7.8 Differences in synaptic potentiation between superficial and deep fPSP recordings in the superficial dorsal horn. ... 181

Figure 7.9 Interlaminar difference in synaptic plasticity is determined by TrkB receptors. ... 182

Figure 8.1 Weaker inhibition in LI gives rise to increased and unstable synaptic plasticity. ... 188

Figure 8.2 Development of the modulation by α5GABAARs. ... 191

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List of Tables

Table 2-1 Proteins involved in neuronal Cl− homeostasis ... 64 Table 2-2 Cl− dysregulation in disease ... 76

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Abbreviations

[Cl]i intracellular concentration of chloride

ACSF artificial cerebrospinal fluid

AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid AP action potential

ATP adenosine triphosphate

BDNF brain-derived neurotrophic factor BIC bicuculline

Ca2+ calcium

CCC cation-chloride cotransporter CCI chronic-constriction injury CFA complete Freund’s adjuvant CGRP calcitonin-gene related protein

Cl− _chloride

CNQX 6-cyano-7-nitroquinoxaline-2,3-dione CNS central nervous system

CVLM caudal ventrolateral medulla

D-APV D(−)-2-amino-5-phosphonopentanoic acid DF driving force

DG dystroglycan

DH dorsal horn

DOX doxycycline

DRG dorsal root ganglion DRR dorsal-root reflex E equilibrium potential

eIPSC evoked inhibitory postsynaptic current EPSP excitatory postsynaptic potential

FingR fibronectin intrabody generated with mRNA display fPSP field postsynaptic potential

GABA γ-aminobutyric acid

GABAAR γ-aminobutyric acid receptor type A

GAD glutamic acid decarboxylase GAT γ-aminobutyric acid transporter GFE3 gephyrin-FingR E3-ligase construct

HCN hyperpolarization-activated cyclic nucleotide-gated HCO3 bicarbonate

HFS high-frequency stimulation

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I/O input-output

IASP International Association for the Study of Pain IB4 isolectin-binding 4

IPSC inhibitory postsynaptic current

K+ potassium KA kainate KCC2 potassium-chloride cotransporter 2 LC locus coeruleus LFS low-frequency stimulation LI lamina I LII lamina II LTD long-term depression LTMR low-threshold mechanoreceptor LTP long-term potentiation

mIPSC miniature inhibitory postsynaptic current

NA noradrenaline Na+ sodium NK1 neurokinin 1 NKCC1 sodium-potassium-chloride cotransporter 1 NL2 neuroligin 2 NMDA N-methyl-D-aspartate

NpHR Nathromonas pharaonis halorhodopsin

NPY neuropeptide Y NS nociceptive-specific

PAD primary afferent depolarization PAG periaqueductal grey

PAM positive allosteric modulator PB parabrachial nucleus

PDZ postsynaptic density zone

PG prostaglandin

PLCγ phospholipase C

PNS peripheral nervous system RVM rostral ventromedial medulla

S-ACSF sucrose-based artificial cerebrospinal fluid SDH superficial dorsal horn

SEM standard error of the mean

SP substance P

STN solitary tract nucleus STRY strychnine

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TBS theta-burst stimulation

THIP 4,5,6,7-tetrahydroisoxazolo[4,5c]pyridine-3-ol TNF-α tumor necrosis factor α

TrkB tyrosine kinase receptor B

TRPV1 transient receptor potential vanilloid 1 TTX tetrodotoxin

V voltage

VGLUT vesicular glutamate transporter Vm membrane potential

WDR wide-dynamic range

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Acknowledgements

First and foremost, I would like to express my sincere thanks to my supervisor, Pr. Yves De Koninck, for welcoming me in his lab for my PhD. In particular, I thank Yves for his trust and patience, for helping me develop my independence and allowing me to grow as a scientist. I will be forever in awe of his capacity to always think outside the box and integrate every piece of information to make sense of everything. I hope one day I can do the same. I want to thank Annie Castonguay, Karine Bachand and Jacqueline Turmel for their technical support. The lab life felt easy with your help. Thanks also to Sylvain Côté, for always pushing me toward a better attitude, and for his amazing figures which make clear even the most difficult concepts.

I want to thank my ‘patching’ team: Rob Bonin, Francesco Ferrini, Cyril Bories and Isabel Plasencia for being there in sometimes very frustrating occasions. I have learned a lot with you. My gratitude extends to the rest of my fellow lab-mates (YDK and all CERVO) for greatly improving the quality of the life during these years.

Thanks to my first « family » in Quebec: Farida El gaamouch, Bruce Mesnage, Liliana Fadul and Rene Alfonso. Thank you for your friendship and guidance.

And finally, my heartfelt thanks to my family, my mother and father; Marcela and Roman, and to my brother, Octavio. I would not have been able to do it without your support.

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Foreword

This thesis was written as completion for a PhD degree in Neuroscience accorded by Laval University. The first section, Chapters 1 and 2, are introductory chapters. Chapter 1 constitutes an overview of the organization of the spinal cord dorsal horn and inhibitory mechanisms that can be affected to produce an alteration in information processing. This chapter includes the rationale and the objectives addressed in subsequent chapters. Chapter 2 contains a submitted review article that reflects on the importance of chloride homeostasis for synaptic plasticity in neurons. Chapter 3 provides a description and justification of the methods employed in the thesis. The results section is distributed among several chapters (Chapters 4, 5, 6 and 7) that are composed of text and figures that have contributed to the publication of original research articles or are being prepared for submission. The final section (Chapter 8) contains the general discussion of the results obtained, as well as future perspectives and conclusions.

The particulars of the chapters that have been already submitted and/or accepted for publication go as follows:

Chapter 2. This chapter consists of a specialized review that has been submitted for publication:

« Regulation of chloride gradients and neural plasticity. » Jimena Perez-Sanchez and Yves De Koninck.

Submitted to Oxford Encyclopedia of Neuroscience on May 25th_{, 2018 and is currently being revised}

for publication.

Chapter 4. The results in this chapter contributed to the publication of a peer-reviewed paper: « An

E3-ligase-based method for ablating inhibitory synapses. »

Garrett G. Gross, Christoph Straub, Jimena Perez-Sanchez, William P. Dempsey, Jason A. Junge, Richard W. Roberts, Le A. Trinh, Scott E. Fraser, Yves De Koninck, Paul De Koninck, Bernardo L. Sabatini and Don B. Arnold

Accepted in Nature Methods (2016) 13: 673–678

For this publication I performed all the electrophysiological experiments in cultured neurons. The details of my contribution are addressed in Chapter 4.

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Chapter 5. The results of this chapter have been published and the article is reproduced in this thesis.

« α5GABAA receptors mediate tonic inhibition in the spinal cord dorsal horn and contribute to the

resolution of hyperalgesia. »

Jimena Perez-Sanchez, Louis-Etienne Lorenzo, Irene Lecker, Agnieszka A. Zurek, Charalampos Labrakakis, Erica M. Bridgwater, Beverley A. Orser, Yves De Koninck and Robert P. Bonin

Accepted in Journal of Neuroscience Research (2017) 65: 1307–1318

For this publication I performed all the electrophysiological experiments in mice slices as well as their statistical analysis. Charalampos Labrakakis contributed to the interpretation of the electrophysiological data. Immunohistochemical experiments were performed by Louis-Etienne Lorenzo. Behavioral assays were performed by Irene Lecker, Agnieska A. Zurek, Erica M. Bridgewater and Robert P. Bonin. Experimental procedures were supervised by Beverly A. Orser, Yves De Koninck and Robert P. Bonin. Robert P. Bonin and I wrote the paper.

On the other hand, Chapters 6 and 7 are being prepared for submission:

Chapter 6. The results of this chapter are being prepared for submission as « Etomidate enhances

tonic currents by α5GABAA receptors but fails to constrain synaptic plasticity in the spinal cord dorsal horn. »

Jimena Perez-Sanchez and Yves De Koninck

I performed all electrophysiological experiments in this section. Both in mice slices and whole spinal cord tissue explants. Behavioral analysis were performed with the help from Modesto R. Peralta. Yves De Koninck supervised the experiments.

Chapter 7. The results of this chapter are being prepared for submission to integrate an article entitled

« TrkB-driven differential chloride homeostasis in the superficial dorsal horn locally shapes synaptic metaplasticity. »

Francesco Ferrini, Jimena Perez-Sanchez, Louis-Etienne Lorenzo, Martin Cottet, Antoine Godin, and Yves De Koninck

For this article in preparation, Francesco Ferrini and I performed the electrophysiological experiments and their analysis. Immunohistochemical experiments were made by Louis-Etienne Lorenzo.

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Antoine Godin developed the tools for the analysis of immunohistochemical data. Martin Cottet executed the in vivo imaging trials and analysis. Yves De Koninck supervised the work.

_

Hitherto I have described the research publications which I considered most relevant to integrate my thesis. Nevertheless, during the course of my PhD I have contributed to the publication of other articles which are not described in subsequent chapters. These are:

Gagnon M, Bergeron MJ, Lavertu G, Castonguay A, Tripathy S, Bonin RP, Perez-Sanchez J, Boudreau D, Wang B, Dumas L, Valade I, Bachand K, Jacob-Wagner M, Tardif C, Kianicka I, Isenring P, Attardo G, Coull JAM, De Koninck Y (2013) Chloride extrusion enhancers as novel therapeutics for neurological diseases. Nature Medicine 19: 1524–1528.

Khoutorsky A, Bonin RP, Sorge RE, Gkogkas CG, Pawlowski SA, Jafarnejad SM, Pitcher MH, Alain T, Perez-Sanchez J, Salter EW, Martin L, Ribeiro-da-Silva A, De Koninck Y, Cervero F, Mogil JS, Sonenberg N (2015) Translational control of nociception via 4E-binding protein 1. eLife 4:e12002. Zhang J, Echeverry S, Shi XQ, Huang H, Wu YC, Lorenzo LE, Perez-Sanchez J, Bonin RP, De Koninck, Y, Yang M (2017) Spinal microglia are required for long term maintenance of neuropathic pain. PAIN 158(9): 1792–1801.

Gagnon M, Bergeron MJ, Perez-Sanchez J, Plasencia-Fernández I, Lorenzo LE, Godin AG, Castonguay A, Bonin RP, De Koninck Y (2017) Reply to Cardarelli et al. Nature Medicine 23(12): 1396–1398.

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Chapter 1 Introduction

1.1 Opening Remarks

It is highly probable that you have felt pain, as I and almost everyone has, although perhaps we have not felt it in the same way. Pain is always a subjective experience. It is defined by the International Association for the Study of Pain (IASP) as « an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage » (Merskey and Bogduk, 1994). It has therefore an individual connotation and is influenced by previous experiences (Baliki and Apkarian, 2015). On the other hand, pain is also an important sensory modality whose function is to alert and protect an organism of potential or ongoing damage. This function is clearly evident in acute manifestations of pain, such as a pinprick or sunburn, which are time delimited and disappear once the stimulus, or the associated lesion, are gone. In fact, people with congenital insensitivity to pain often present severe physiological problems that remain untreated because tissue damage that would normally produce pain is not detected nor avoided (Prescott et al., 2014). However, pain loses its protective significance when it persists over time, independent of a stimulus. In this case, pain may become a pathological condition by itself and is referred to as chronic pain.

Chronic pain is a widespread condition, with an estimated 10–55% of the general population affected by it, and as many as 7–10% of adults experience chronic pain with neuropathic characteristics (Voscopoulos and Lema, 2010; van Hecke et al., 2014). Chronic pain can arise from a wide variety of causes, including nerve injury, trauma, diabetes, HIV infection, shingles, cancer, and surgery, as well as peripheral causes such as chronic inflammation. With this diverse etiology, chronic pain is often difficult to handle because its treatment remains only partially effective. Further understanding of its root physiological causes is essential to improve analgesic strategies and to generate novel alternative treatments. To study the mechanisms that produce pain, it is often useful to distinguish the sensory

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capable of activating a sensory pathway without producing outright pain. The term

nociception was introduced in 1906 by C. S. Sherrington to describe this sensory component.

Nociception can be set apart from cognitive pain as it does not involve an emotional or perceptual response. The contrary is also true, pain can occur in a spontaneous manner, in absence of a nociceptive stimulus, as is the case of phantom pain (Flor et al., 2013). Therefore, nociception refers to the neural processes of encoding and processing of a noxious stimulus (Merskey and Bogduk, 1994) — namely its detection, integration and transmission — including reflex responses that protect the organism. In contrast, pain is a conscious experience, it carries a necessary affective component, and requires the cortical interpretation of nociceptive information.

Altered nociceptive information processing can affect how a nociceptive signal is transmitted to higher brain structures and, therefore, how pain is perceived. Accordingly, hypersensitivity to sensory input can produce pain by normally innocuous stimuli, such as light touch; a phenomenon known as allodynia (Merskey and Bogduk, 1994). Similarly, the term

hyperalgesia describes an excessive pain response to a moderately noxious stimulation

(Merskey and Bogduk, 1994). Indeed, both allodynia and hyperalgesia are a consequence of aberrant processing in the nociceptive system, produced by increased responsiveness of nociceptive neurons to their normal afferent input (hyperalgesia), or by the possible recruitment of normally subthreshold input (allodynia). Often, the term sensitization is used to portray these enhanced responses (Merskey and Bogduk, 1994).

1.1.1 Types of Pain

Nociceptive pain

Despite the aforementioned distinction between nociception and pain, it must be acknowledged that activation of nociceptors and nociceptive pathways can certainly give rise to pain. As such, nociceptive pain is the « normal » acute pain sensation in response to noxious stimuli (Costigan et al., 2009). It is driven by strong mechanical, thermal, and/or chemical stimulation and persists for as long as the stimulus is present. Nociceptive pain consists of receptor activation in the periphery, transmission of nociceptive input to the central nervous system (CNS), spinal and cortical processing of this information to generate

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a pain perception, and engagement of descending circuits for its modulation (Basbaum et al., 2009; Das, 2015b). This system is responsible for the vital protective role that informs the body of danger. Therefore, altering or disabling this system can produce severe consequences such as the development of chronic pain and the loss of its protective quality (Woolf et al., 2004).

Inflammatory pain

As nociceptive pain, inflammatory pain is also a physiological, protective mechanism. It originates as a result of a tissue damage and the development of local inflammation around the injury (Das, 2015b). It is characterized by swelling, heat, redness and pain hypersensitivity. The typical features of this hypersensitivity is that normally innocuous stimuli become painful (allodynia) and/or there is an exaggerated response to noxious stimuli (hyperalgesia) (Woolf et al., 2004). This hypersensitivity provides protection to the damaged tissue to facilitate healing and recovery, and gradually disappears as inflammation goes away. There are cases, however, in which inflammatory pain becomes chronic (such as after severe trauma or inflammatory diseases) and requires proper management to maintain its protective role (Woolf et al., 2004; Das, 2015b).

Neuropathic pain

We refer to neuropathic pain when pain persists for long periods of time, thus losing its protective function and becomes harmful. It therefore contrasts with nociceptive and inflammatory pain described above, which are physiological ways to protect an organism. Neuropathic pain is caused by a lesion or a disease to the somatosensory system. Damage can occur in the peripheral nervous system (PNS) due to trauma, infections, tumors, or metabolic diseases such as diabetes mellitus or in the CNS after stroke, spinal cord injury or multiple sclerosis (Woolf et al., 2004; Costigan et al., 2009). This pain is characterized by sensory allodynia, hyperalgesia and spontaneous pain. In particular, spontaneous pain is generated by abnormal neuronal activity which yields the firing of action potentials without any evoking stimuli. This ectopic activity is the result of hyperexcitability in primary sensory neurons which transmit information into the spinal cord (Woolf et al., 2004; von Hehn et al.,

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close proximity to the injury can potentially also give rise to abnormal activity without an identified stimulus and thereby also contribute to enhanced pain sensations (von Hehn et al., 2012). Second order neurons in the spinal cord, which in some cases transmit nociceptive information directly to the brain, respond to this enhanced input and increase their excitability themselves to produce further sensitization that outlasts the intial stimulus.

Other types of pain

Other pain states can develop without any apparent damage to the CNS. The IASP has recently coined the term « nociplastic pain » to describe pain that arises from altered nociception when there is no clear indication of actual or threatened tissue damage which may cause the activation of peripheral nociceptors, nor evidence for a disease or a lesion of the somatosensory system that causes the pain (Kosek et al., 2016). This new term helps explain the pain in fibromyalgia and musculoskeletal pain, for whom activation of nociceptors cannot be confidently established.

In brief, nociceptive information is first relayed at the spinal cord before moving towards the brain for further processing and interpretation. Nevertheless, as a first relay point, significant processing already occurs in this region. Aberrant processing at the spinal cord level can evenually affect how pain is perceived. Therefore, it is of utmost importance to understand how sensory processing at the spinal cord can be altered or affected in order to come up with potential treatments for chronic pain and similar disorders.

1.2 Organization of the spinal dorsal horn

The spinal cord is a fundamental part of the CNS, even if it is often disregarded as a simple structure. It contributes to important bodily functions that include the provision of autonomic innervation, the execution of voluntary movements and the control of muscle tone. More importantly, as the subject of the present thesis, it is also the main harbor for the reception and integration of sensory information, including nociceptive information from most regions of the body.

Along its rostro-caudal axis, we often divide the spinal cord into segments, which are associated with each pair of spinal nerves that innervate it (Fig. 1.1A). When viewed in a

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cross-section, the spinal white and grey matter can be distinguished, as described by Rolando in 1824; with the grey matter classically separated into dorsal and ventral horns. The shape and size of the horns is not homogeneous from one segment to the next. The ventral horns are quite large at the cervial and lumbar enlargements and much narrower in upper cervical and thoracic levels (Fig. 1.1B). The most current description of the spinal grey matter was provided by Bror Rexed (1952). This description is based upon cyto-architectonic studies using Nissl-stained preparations in the cat spinal cord, and reveals that neurons are longitudinally organized into layers or laminae. However, a similar laminar pattern has been described in most species, including primates and humans. In this arrangement, the dorsal horn is formed by the ensemble of laminae I to VI (Fig. 1.1C). Lamina VII embodies mostly the intermediate grey matter, and laminae VIII–X form the ventral horn. In particular, lamina I (LI); the outermost layer (marginal zone) and lamina II (LII), which corresponds to the substantia gelatinosa (of Rolando), conform the superficial dorsal horn (SDH) (Light, 1992). This region contains an ensemble of different types of neurons, and is regarded as an important site of relay and modulation of nociceptive information as I will endeavour to describe in the following sections.

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Figure 1.1 Anatomy of the spinal cord

(A) Spinal segmentation along the rostro-caudal axis. Each spinal segment is associated with a pair of peripheral nerves. Broadly, they can be grouped into cervical, thoracic, lumbar and sacral segments. Cervical and lumbar enlargements are marked with an asterisk (*). (B) Cross-section at different spinal segments (dashed line in A), showing the grey and white matter divided in dorsal and ventral horns. (C) Grey matter divided into laminae. This laminar arrangement is conserved for most species. The most superficial laminae (LI-LII) are grouped as the superficial dorsal horn (SDH).

1.2.1 Nociceptive input through primary afferent fibers

The detection of a noxious stimulus occurs in the periphery by the activation of specific sensory receptors known as nociceptors. These terminals originate from pseudo-unipolar neurons located in dorsal root ganglia (DRG) on either side of the spinal cord. As such, these neurons have an axonal projection that terminates in the peripheral tissue and another directed toward the spinal cord (Fig. 1.2). The axons of these cells are known as primary afferent fibers and can be distinguished by their diameter, conduction velocity, degree of myelination and response to sensory stimuli (Light and Perl, 1979; Sugiura et al., 1986).

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Figure 1.2 Primary sensory neurons are pseudo-unipolar

Cell bodies of sensory neurons reside in the dorsal root ganglia (DRGs) along the spinal cord. They project peripherally to innervate skin, muscle and viscera and centrally to the spinal cord. Modified from Purves et al. (2004).

The primary afferent fibers associated with nociceptors are usually thin and slow conducting axons that are either lightly myelinated (Aδ fibers) or unmyelinated (C-fibers) (Fig. 1.3). Nociceptive Aδ fibers, with conduction velocities in the range 5–30 m/s, are responsible for an acute pain response, leading to immediate withdrawal action (Almeida et al., 2004). On the other hand, C-fibers, the slowest conducting axons (0.2–2 m/s), mediate a slower pain response with summation of the response over time (Almeida et al., 2004). Primary afferents are excitatory and use glutamate as the main neurotransmitter. However, they are known to also release other excitatory neuromodulators. In fact, C-fibers can be further subdivided into two major subpopulations according to the expression of specific neurochemical markers. Therefore, fibers that express neuropeptides like substance P (SP) or calcitonin gene related peptide (CGRP) are peptidergic C-fibers, whereas non-peptidergic C-fibers do not contain neuropeptides but exhibit binding to isolectin-B4 (IB4) (Julius and Basbaum, 2001).

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Figure 1.3 Characteristics of primary afferent fibers

(A) Peripheral nerves include large diameter Aβ fibers, medium diameter Aδ fibers, as well as small diameter C fibers. (B) The conduction velocity of each fiber is directly related to its diameter as evidenced from the compound action potential recording from a peripheral nerve. Most nociceptors are either Aδ or C fibers and their different conduction velocities are associated with the first (acute) and second (slow) pain responses to injury (Julius and Basbaum, 2001).

Axons classified as Aβ fibers are large, highly myelinated with fast conduction velocities (16–100 m/s) and usually transmit non-nociceptive tactile stimuli (Fig. 1.3). This characterization, however, is not conclusive; while most nociceptors are indeed associated with Aδ and C-fibers, the opposite is not the case. It is now generally accepted that not all cutaneous sensory receptors with Aδ or C-fiber characteristics relay only noxious stimuli. Low-threshold mechanoreceptors (LTMRs) conducting in the Aδ and C-fiber range respond to small-hair movements (Djouhri and Lawson, 2004) and gentle caressing touch (Bessou et al., 1971; Liu et al., 2007; Vrontou et al., 2013).

The centrally-directed terminals of nociceptive fibers enter the spinal cord and form synapses upon second-order neurons in the dorsal horn. Their termination pattern displays a regional distribution (Fig. 1.4) (Ribeiro-da-Silva and De Koninck, 2008). Cutaneous nociceptive input is mainly targeted to neurons in SDH (LI–LII) and to deeper LV, whereas LTMRs terminate in deeper laminae (LIII-V) (Abraira and Ginty, 2013). In particular, afferent input in LI stems mainly from Aδ- and peptidergic C-fibers, in a similar way as the outer part of LII (LIIo).

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The deeper part of LII (inner LII or LIIi) is mainly innervated by non-peptidergic C-fiber

afferents (Bráz et al., 2005). This segregation also explains the distribution of different sensory modalities in the SDH. Noxious thermal sensation is transmitted by a distinct population of afferents that express the heat-sensitive and capsaicin-activated cation channel, the so-called transient receptor potential vanilloid 1 (TRPV1) (Cavanaugh et al., 2009; McCoy et al., 2013). The expression of this channel is usually restricted to the peptidergic population of C-fiber afferents which terminate predominantly in LI (Cavanaugh et al., 2011). Instead, non-peptidergic afferents, which mainly terminate in LII, express the Mas-related G protein-coupled receptor, MrgD, and are required for the detection of noxious mechanical stimuli (Cavanaugh et al., 2009).

Figure 1.4 Laminar organization of primary afferent inputs in the dorsal horn.

(A) Cross-section of a mid-lumbar spinal cord immunostained with a neuron-specific antibody (NeuN). Lamina boundaries are shown with dashed lines. (B) Primary afferents target specific laminae of the dorsal horn. Aβ tactile and hair afferents arborize mainly in lamina III–V, with some extension into lamina IIi. Aδ afferents arborize on either side of the border between lamina II and

lamina III, whereas Aδ nociceptors end mainly in lamina I. Peptidergic primary afferents (which also include some Aδ nociceptors) arborize mainly in lamina I and lamina IIo, with some fibres penetrating

more deeply, whereas most non-peptidergic C fibres form a band that occupies the central part of lamina II (Todd, 2010).

The specialization of some afferents to a particular sensory modality supports a specificity theory to explain the representation of sensory stimuli. The specificity, or labeled line theory,

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neurons, that respond selectively to that stimulus (Prescott et al., 2014). However, there is growing evidence that a particular stimulus may activate more than a single neuronal type but in unique combination, which gives rise to a combinatorial theory (Prescott and Ratté, 2012; Prescott et al., 2014; Wang et al., 2018).

In contrast, deeper laminae (LIII–LV) receives input from both nociceptive and non-nociceptive afferents. Nociceptive input is transmitted monosynaptically by Aδ fibers and polysynaptically by C-fibers, while non-nociceptive signals are relayed by myelinated Aβ fibers (Almeida et al., 2004).

1.2.2 Cellular components of the superficial dorsal horn

Projection neurons

In order for a nociceptive stimulus to be interpreted as pain, the signal must be transmitted to supraspinal centers in the brain for its interpretation. LI and a smaller hub of neurons in deeper LV host the main nociceptive output pathways in the dorsal horn (Todd, 2002). Even then, projection neurons in LI account for only 5% of all cells (Spike et al., 2003).

Different classes of projection neurons can be recognized based on the supraspinal target of their axons (Fig. 1.5). The spinothalamic tract (STT) represents the main output pathway from the spinal cord to the brain, including the transmission of nociceptive information, but also temperature and touch (Almeida et al., 2004). Nociceptive input to the STT arises mostly from LI and LV. Nevertheless, most LI neurons (~80%) project to the parabrachial (PB) nucleus instead (Todd et al., 2000)(Fig. 1.5). This projection is important for pain interpretation, since PB is closely associated with forebrain limbic structures such as the amygdala and hypothalamus, which contribute in giving the pain experience its emotional quality (Almeida et al., 2004).

As many as 80% of PB-projecting LI neurons express the neurokinin-1 receptor (NK1) (Todd et al., 1998). This receptor is the target of SP, which suggests that these neurons are innervated by a subset of peptidergic nociceptive afferents that express SP. As such, many of these neurons respond only to noxious stimulation and are thus considered as nociceptive-specific (NS) neurons (Bester et al., 2000). The proportion of NS cells in LI varies from 40–

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60%, depending on the species (Han et al., 1998; Seagrove et al., 2004), but at least 75% project to the PB (Bester et al., 2000). The remaining 25% of projecting neurons respond to both noxious and innocuous stimulation and are therefore considered as wide dynamic range (WDR) neurons (Bester et al., 2000).

Figure 1.5 Supraspinal projection sites

Axons of projection neurons in LI target several brain areas. (From top to bottom) Thalamus: several nuclei in the thalamus receive input from the superficial dorsal horn (SDH). These pathways are likely to contribute to both sensory-discriminative and affective-motivational aspects of pain. Periaqueductal grey (PAG): it is involved in organizing strategies to cope with stressors. It is therefore important to orchestrate the descending modulation of dorsal horn circuits. Lateral parabrachial nucleus (LPB): it is the major target site for LI projection neurons. These nuclei receive input from the superficial dorsal horn and project to other brain areas such as the amygdala and hypothalamus. They are likely involved in emotional and autonomic components of pain. Nucleus of the solitary tract (NTS): this region also receives cardio-respiratory inputs and has a role in the reflex tachycardia that results from noxious stimulation. Caudal ventrolateral medulla (CLVM): this region gives rise to a descending projection to the dorsal horn and may integrate nociceptive and cardiovascular responses. Many of these are interconnected and therefore also receive indirect nociceptive inputs from the SDH. Modified from Todd (2010).

Other postsynaptic targets include the periaqueductal grey matter (PAG) and various parts of the medullary reticular formation (Todd et al., 2000; Spike et al., 2003)(Fig. 1.5). Very few

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neurons that project to the PAG express NK1 (Spike et al., 2003). This area contributes to aversive behavior and in the modulation of descending systems (Almeida et al., 2004). From a morphological standpoint, LI projection neurons can be identified as multipolar, fusiform, flattened or pyramidal (Lima and Coimbra, 1986)(Fig. 1.6). Mostly, they display local dendritic arbours, which do not branch beyond LI (Cordero-Erausquin et al., 2009). The multipolar and fusiform neurons are largely NK1-receptor-expressing and thus considered to be nociceptive (Almarestani et al., 2007). As such, they largely project to the PB, but also to the caudal ventrolateral medulla (CVLM) (Lima and Coimbra, 1989). In contrast, pyramidal neurons are known to project to the PAG and also to the solitary tract nucleus (STN) (Gamboa-Esteves et al., 2001). These neurons do not express NK1-receptor and are considered to be non-nociceptive (Polgár et al., 2008). They respond to innocuous cold and probably receive input from TRPM8-afferents (Ribeiro-da-Silva and De Koninck, 2008).

Figure 1.6 Neurons in lamina I

Morphological classification of neurons that are characteristic of lamina I (LI): fusiform, flattened, multipolar and pyramidal. Modified from Lima and Coimbra (1986).

Often, the spiking pattern of neurons is used to classify different neuronal subpopulations. Different spiking patterns can arise from varying levels of ion channel expression in neurons. In particular, different combinations of two potassium (K+) channels, namely a low threshold non-inactivating (Kv1-type) K+ conductance and an inactivating (A-type) K+ conducance,

have been shown to account for this heterogeneity in the SDH (Balachandar and Prescott, 2018). In particular for LI, there is a good association between different firing patterns,

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measured in slices, with the morphology of these neurons (Prescott and De Koninck, 2002). Accordingly, the majority of fusiform neurons show tonic firing in response to a depolarizing pulse. Pyramidal cells typically show phasic firing and those with multipolar morphology show delayed or single spike firing (Fig. 1.6C).

Interneurons

Locally-projecting interneurons comprise most of all the cells in the SDH. This region is not a simple relay station for nociceptive information to be transmitted to the brain. It is clear that the incoming nociceptive information must be processed by a complex circuit of these local neurons before it is passed on (Fig. 1.6B). In this regard, virtually all cells in LII are interneurons and in LI they account for 90–95% of cells (Bice and Beal, 1997; Spike et al., 2003; Todd, 2017). Multiple efforts to address the complex circuitry of the SDH have amounted to an ever-growing description of different neuronal populations, whose function we are only just beginning to clarify (Todd, 2010; Duan et al., 2014; Smith et al., 2016; Abraira et al., 2017; Cheng et al., 2017).

The first morphological descriptions already distinguished different cell types in LII, such as stalked cells, large islet and small islet neurons (Todd and Spike, 1993). More recently, patch-clamp experiments have defined four distinct morphological types: central, islet, radial and vertical cells (Grudt and Perl, 2002)(Fig. 1.7). Islet cells have relatively large somata and display the largest dendritic arbor (~400 µm) with rostro-caudal orientation. Central cells also have dendrites extending along the rostro-caudal axis, but they are much shorter than those of islet neurons (150–200 µm). They are localized in the mid-zone of LII. Radial cells have a typical stellate form, with dendrites that radiate in all directions. Finally, vertical cells are located mostly in LIIo proper, sometimes near to the border with LIIi. They have a distinct

orientation of their dendrites, spreading significantly in the dorsoventral plane, but with a predominant ventral extension. Most neurons can be assigned to these four classes. However, there is still a rather large proportion of neurons that remain unclassified (~25%) (Grudt and Perl, 2002; Yasaka et al., 2007). In contrast, an unbiased genetic labelling approach revealed a broad range of morphological complexity in deeper lamina neurons (LIII–LV), implicated in the processing of low-threshold mechanoreceptive information (Abraira et al., 2017).

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Figure 1.7 Neurons in lamina II

Lamina II (LII) neurons can be morphologically classified as radial, central, vertical and islet neurons (Yasaka et al., 2007).

The association between the morphology and the electrophysiological properties of LII neurons is not as clear as we saw before for LI (Prescott and De Koninck, 2002). There are, however, some patterns that have been described (Fig. 1.8). Islet cells fire in tonic fashion when injected with a depolarizing current and they receive monosynaptic input from C-fibers. Vertical cells also give tonic discharges to maintained depolarization, but mostly show a delayed discharge of action potentials, and receive monosynaptic input from Aδ fibers. Radial cells also display a delayed firing pattern. On the other hand, central cells receive monosynaptic input from C-fibers. They show both tonic and phasic firing patterns and are therefore sub-classified into tonic central cells and transient central cells (Grudt and Perl, 2002; Lu and Perl, 2005; Yasaka et al., 2007). Monosynaptic input from Aβ fibers is targeted to neurons in deeper laminae. In these cells, depolarizing current steps have reveled distinct firing patterns including single spike, initial bursting, phasic, delayed, gap, regular spiking and tonic (Abraira et al., 2017).

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Figure 1.8 Physiology and connectivity of superficial dorsal horn neurons

Summary of synaptic input and connectivity between different types of spinal neurons (Left). On the right are some examples of firing patterns elicited by a current injection into the cell body. Modified from (Prescott and Ratté, 2012).

There are also some generalities that can be made regarding the excitatory or inhibitory nature of LII interneurons (Figure 1.8). Commonly, two thirds of the entire interneuron subpopulation is considered to be excitatory, whereas the rest is considered to be inhibitory (Polgár et al., 2003). In particular, islet cells are mostly GABAergic, with a subset of these also expressing glycine, and are therefore inhibitory (Todd, 2010). Central cells can be both inhibitory and excitatory (Yasaka et al., 2007). In contrast, radial and vertical cells express mainly glutamate, and thus presumed to be excitatory (Maxwell et al., 2007). The expression of VGLUT2, the vescicular glutamate transporter, in many of these neurons confirms their excitatory profile (Punnakkal et al., 2014). In LIII–LV, seven interneuron subtypes are considered to be excitatory and comprise about 70% of neurons, whereas the remaining 30% are inhibitory and belong to four distinct interneuron subpopulations (Abraira et al., 2017). Furthermore, both excitatory and inhibitory neurons can express other neurochemical markers which define additional populations (Todd, 2010). Some of these, including neuropeptide Y (NPY), galanin, parvalbumin and nitric oxide synthase are present in

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inhibitory neurons in the SDH, whereas PKCγ, somatostatin, neurotensin, neurokinin-B and SP are found predominantly in excitatory interneurons. Others, like enkephalin and dynorphin are expressed in both inhibitory and excitatory interneurons (Todd, 2010). In the deep DH, 11 interneuron subtypes have been identified by the expression of uniquely expressed genes, each with a unique combination of morphological and physiological properties (Abraira et al., 2017).

Non-neuronal cells

Another population of cells which participates in the integration and modulation of nociceptive information is conformed by glial cells. Glial cells were originally believed to be the interstitial « glue » (hence, the name glia) surrounding neurons to keep them in place (Allen and Barres, 2009). However, glial cells are much more than a simple scaffold; they are important to maintain the ionic balance in the extracellular space, to insulate axons for information transmission and to provide nourishment to neurons (Magistretti, 2006; Allen and Barres, 2009). They are also important to clear away transmitter molecules, but also release their own, and are considered as a functional component of synapses (Araque et al., 1999; Perea et al., 2009). Therefore, despite the fact that they are not neurons, they deserve their place within the functional components of the dorsal horn and will be briefly described here. Of these, astrocytes and microglial cells are important allies in the modulation of nociceptive information (Milligan and Watkins, 2009; Beggs and Salter, 2010; Chiang et al., 2012; Old et al., 2015).

Astrocytes are thus named to describe the « star-shaped » cells that were observed in the supporting system of neurons. Due to their stellate aspect, their multiple processes envelop synapses made by neurons (Perea et al., 2009). Astrocytes respond to most neurotransmitters released into the synaptic cleft. Upon activation, they in turn release a multitude of neuroactive molecules such as glutamate, ATP, nitric oxide (NO), prostaglandins (PGs). These molecules are known to affect neuronal excitability and are important for the development of sensitizing phenomena, such as allodynia and hyperalgesia (Milligan and Watkins, 2009; Beggs and Salter, 2010; Chiang et al., 2012; Old et al., 2015).

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On the other hand, microglia in the CNS are the homologues of macrophages in the periphery. After a lesion or an infection, microglial cells activate and adopt a different morphology, which allows them to translocate toward the lesion as a protective mechanism (Beggs and Salter, 2007). The activation of microglia is important for the transmission of nociceptive information, as well as for the development of neuropathic pain (Zhang et al., 2007b; Echeverry et al., 2008; Echeverry et al., 2017). Activated microglia release several factors including cytokines and chemokines, reactive oxygen species (ROS), NO and brain-derived neurotrophic factor (BDNF) which are associated with the immune response after a lesion (Milligan and Watkins, 2009; Beggs and Salter, 2010; Chiang et al., 2012; Old et al., 2015).

1.2.3 Modulation of nocicieptive information

Segmental control

A prevailing model for the modulation of nociceptive signals in the SDH was postulated more than 50 years ago. The « gate control » theory was described in 1965 by Melzack and Wall (Fig. 1.9) (Melzack and Wall, 1965). This model has seen multiple revisions since then, but the main idea behind it is that pain is not simply the result of the activation of nociceptive-specific afferents and the ability of the brain to understand this input as pain (Craig, 2003; Braz et al., 2014; Todd, 2017). Instead, it has been proposed that the brain receives the product of the activity generated by a complex network of cells located in the SDH; a network that can be regulated by the activity of nociceptive and non-nociceptive afferents. This theory described a rather simple circuit, in which inhibitory interneurons in LII acts as a « gate control » unit, or key, for nociceptive signals entering the spinal cord. The gate is closed when activity of large afferent fibers (non-nociceptive) activate an inhibitory interneuron, which then inhibits the activity of projection neurons that transmit the message into the brain. On the other hand, noxious stimulation activates nociceptive afferents, which « open » the gate by inhibiting the LII interneuron. As the inhibitory interneuron no longer inhibits the projection neuron, this results in increased transmission of the nociceptive message and increased pain.

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Figure 1.9 Gate Control Theory

Large (L)- and small (S)-diameter afferent fibers project into the SDH and activate substantia gelatinosa (SG) and transmission (T) neurons. T neurons activate neural mechanisms including the action system responsible for response and perception. The inhibitory effect exerted by SG neurons on afferent fiber terminals is increased by activity in L-fibers and decreased by activity in S-fibers. The central control mechanisms represent descending efferent fibers which can influence afferent conduction. These mechanisms project into the SDH to the gate control system. + : excitation; – : inhibition (Melzack and Wall, 1965).

Since the gate theory was proposed, some of the synaptic arrangements in the original model have turned out to be inaccurate. In particular, we know that all primary afferents express glutamate and are thus excitatory. As such, the proposed inhibitory action of the nociceptors that opens the gate is presumed to arise from the activation of other interneurons, that in the end produce the LII interneuron inhibition (Braz et al., 2014; Todd, 2017). Indeed, when this theory was proposed, there was a quite limited grasp on the vast heterogeneity of the cells that conform the SDH, as well as the complex circuits in which they interact.

Descending modulation

Another feature of the gate-control theory that is often overlooked is the suggestion of descending systems (« central control ») from supraspinal regions that modulate nociceptive processing in the SDH (Melzack and Wall, 1965). There are several brain regions that project into the spinal cord and target second-order neurons in the SDH, but also central terminals of primary afferent fibers, to control nociceptive transmission (Fig. 1.10). The nature of this

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descending modulation can be GABAergic, serotonergic, noradrenergic and/or opioidergic depending on the origin of the descending system (Heinricher et al., 2009). Probably the best known orchestrator of descending modulation is the PAG nucleus. This region is ideally suited to control descending systems as it receives direct spinal cord input. The PAG projection to the spinal cord is mainly opioidergic, which attenuates nociceptive transmission (Budai and Fields, 1998). On the other hand, PAG neurons can also project to other supraspinal centers such as the rostral ventromedial medulla (RVM) in the brainstem. The PAG–RVM descending pathway is considered to be serotonergic because the RVM contains a subset of serotonergic neurons that project into the spinal cord. The release of serotonin in the SDH normally has antinociceptive effects, but its action can become facilitatory under chronic pain states (Heinricher et al., 2009).

Another important modulatory system is noradrenergic (NA). This pathway originates from the locus coeruleus (LC) and subcoeruleus (Tsuruoka and Willis, 1996). NA modulation can be inhibitory or facilitatory depending on its target receptors. NA action is inhibitory through α2-NA receptors in presynaptic terminals, but excitatory when activating α1-NA receptors in inhibitory interneurons (Peng et al., 1996b). In both cases, NA modulation is mainly associated with antinociceptive effects.

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Figure 1.10 Descending pathways for the modulation of nociceptive information

Neurons located in the periaqueductal grey (PAG) matter of the mid-brain project to the serotonergic (5-HT) neurons in the nucleus raphe magnus (NRM) that is located in the rostral ventromedial medulla (RVM). Locus coeruleus (LC) noradrenergic (NA) neurons are located in the upper pons. The axons of serotonergic medullary and noradrenergic LC neurons descend to all levels of the spinal cord and synapse on neurons located in the SDH.

Descending inhibition also involves the activation of last-order inhibitory interneurons in the SDH. Most of the inhibitory effects produced by activation of supraspinal centers can be blocked by the local administration of GABAA and/or glycine receptor antagonists,

regardless of the nature of the descending system (Peng et al., 1996a; Kato et al., 2006). Therefore, inhibitory interneurons activated by central mechanisms release GABA and glycine in the SDH to modulate nociceptive transmission.

1.2.4 Alteration of spinal nocicieptive processing

Nowadays, we are still quite at large regarding how specific population of cells interact in the processing and modulation of nociceptive information, as well as in the development of

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chronic pain. One thing seems to be clear though, and that is the critical role of inhibitory neurotransmitters, GABA and glycine, in this modulation (Zeilhofer et al., 2012). Indeed, SDH circuits are normally under constant inhibitory control. When this inhibitory control is lost, such as in chronic pain conditions, there is cross-modal activation of primary afferents and aberrant spread of excitation. This can lead to the perception of innocuous stimuli as painful (allodynia) or to enhanced pain responses (hyperalgesia) (Koch et al., 2018).

Peripheral and central sensitization

Following tissue injury, nociceptors in the affected zone become sensitized due to the release of proinflammatory mediators, such as bradykinin, prostagalandins (PGs), chemokines (e.g. CCL-2, CX3CL1), cytokines, histamine, proteases and protons from damaged cells and blood vessels at the site of injury as well as from immune cells that invade the site of injury (Basbaum et al., 2009; Costigan et al., 2009; Das, 2015b) (Fig. 1.11). These mediators are detected by specialized receptors on the peripheral endings of sensory neurons, which trigger transcriptional and translational changes that eventually lead to their enhanced excitability (Stemkowski and Smith, 2012). The resultant aberrant activity of primary sensory neurons is generally known as peripheral sensitization (Basbaum et al., 2009). This process can lead to further release of pronociceptive transmitters and neurotrophins such as glutamate, SP, CGRP, adenosine triphosphate (ATP), PGs, dynorphin and BDNF from primary afferents both peripherally and centrally (Das, 2015b).

In the periphery, proinflammatory molecules released from injured afferents can recruit more immune cells to the injured area, leading to further sensitization of nociceptors (Das, 2015a). Centrally, these mediators stimulate further release of proinflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) in the spinal cord, which enhance excitatory input into second-order neurons and reduce inhibitory currents in these neurons, thereby contributing to central sensitization (Basbaum et al., 2009; Latremoliere and Woolf, 2009; Das, 2015b). The action of these mediators on microglia promotes the expression of the ionotropic purinergic receptor P2X4R on these cells (Tsuda et al., 2003; Trang et al., 2009). This activation of spinal microglia can produce further release of cytokines, chemokines and BDNF (Coull et al., 2005; Beggs and Salter, 2013; Old et al., 2015).

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Figure 1.11 Sensitization of spinal nociceptive circuits

Tissue damage leads to the release of inflammatory mediators by activated nociceptors or non-neuronal cells in the periphery. These mediators include bradykinin (BK), histamine (Hist), cytokines (e.g. IL-1β), prostaglandins (PG), adenosine triphosphate (ATP), chemokines (e.g. CX3CL1). These factors excite and sensitize nociceptor terminals by interacting with cell-surface receptors expressed in these neurons (peripheral sensitization). Intense activation of sensitized nociceptors increases the release of glutamate (in blue), and neuromodulators such as SP and CGRP from the terminals. Thus, normally silent NMDA glutamate receptors (NMDAR) located in the postsynaptic neuron now signal and increase the postsynaptic calcium (Ca2+_{) levels, thereby activating a host of Ca}2+_-dependent

signalling pathways. This cascade of events will increase the excitability of the output neuron and facilitate the transmission of pain messages to the brain (central sensitization). Under normal circumstances, inhibitory interneurons continuously release GABA and/or glycine to decrease the excitability postsynaptic neurons in the SDH and modulate pain transmission. However, in the setting of injury, this inhibition can be lost, resulting in hyperalgesia (disinhibition). Nerve injury also leads to a central reorganization of Aβ fibers, which sprout from their deep dorsal horn laminar location up into the SDH and make functional synaptic contacts, which may result in allodynia (sprouting of Aβ

fibers). Modified from Woolf and Salter (2000).

BDNF is a neurotrophin with important effects in neuronal survival and differentiation, as well as synapse formation and plasticity (Park and Poo, 2013; Smith, 2014). Despite these beneficial functions, especially during development, BDNF released from microglia is an essential trigger for central sensitization (Beggs et al., 2012; Beggs and Salter, 2013). BDNF signalling is mediated by both p75NTR and tropomiosin-related kinase B (TrkB) receptors

Unconventional components of inhibition that contribute to the regulation of sensitization in the spinal cord dorsal horn

Unconventional Components of Inhibition that

Contribute to the Regulation of Sensitization in the

Spinal Cord Dorsal Horn

Thèse

Jimena Pérez Sánchez

Doctorat en Neuroscience

Philosophiae Doctor (Ph. D.)

Québec, Canada

Unconventional Components of Inhibition that

Contribute to the Regulation of Sensitization in the

Spinal Cord Dorsal Horn

Thèse

Jimena Pérez Sánchez

Sous la direction de:

Abstract

Resumé

Table of Contents

List of Figures

List of Tables

Abbreviations

Acknowledgements

Foreword

Chapter 1

Introduction

1.1 Opening Remarks

1.1.1 Types of Pain

1.2 Organization of the spinal dorsal horn

1.2.1 Nociceptive input through primary afferent fibers

1.2.2 Cellular components of the superficial dorsal horn

1.2.3 Modulation of nocicieptive information

1.2.4 Alteration of spinal nocicieptive processing