Dopaminergic Modulation of Spinal Circuits for Walking in the Adult Mouse

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2018-09-06 Dopaminergic modulation of spinal circuits for walking in the adult mouse

Mayr, Kyle Andrew

Mayr, K. A. (2018). Dopaminergic modulation of spinal circuits for walking in the adult mouse (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/32925 http://hdl.handle.net/1880/107749 master thesis

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Dopaminergic modulation of spinal circuits for walking in the adult mouse

Kyle Andrew Mayr

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

GRADUATE PROGRAM IN NEUROSCIENCE

CALGARY, ALBERTA

SEPTEMBER, 2018

© Kyle Mayr 2018 Abstract Walking is a stereotyped rhythmic behavior that consists of alternating contractions of flexor and extensor muscles, as well as the left and right hindlimbs. The basic rhythmic pattern underlying locomotion is generated by a central pattern generator network within the lumbar spinal cord. The role of many neurotransmitters and modulators have been studied extensively, but dopamine’s (DA) role in modulating movement at the level of the lumbar spinal cord, is still not fully understood, especially in adult mice. The decerebrate mouse preparation allows us to examine modulation of stepping behaviour in adult mice with reduced descending inputs to the spinal cord, while intrathecally manipulating the lumbar spinal cord. Locomotor activity was measured by recording weight bearing during locomotion and electromyograms (EMG) from the flexor (tibialis anterior) and extensor (gastrocnemius) muscles of the hindlimbs. Our results show that intrathecal application of DA at the lumbar spinal cord increased the duration of locomotor bouts. Intrathecal DA led to an increase in weight bearing, suggesting that DA may promote extensor biased walking.

Furthermore, addition of D1-like agonists augmented weight bearing in the decerebrate animal, but not as much as DA alone. Using intact animals intrathecally injected with either DA, or DA antagonists we selectively activated or blocked DA receptor subtypes of the spinal cord. Using the open field test and ladder rung, we quantified the amount of activity, type of activity and scored the skilled steps during locomotion. Our findings show that DA decreased the amount of locomotor activity in the open field but did not have a significant effect on the ladder rung step score. Furthermore, we found that D1-like antagonists reduced locomotor activity (distance, velocity and bouts) while D2-like antagonists did not have a significant impact on open field activity, though there was an increase in errors while crossing the ladder rung. This thesis provides insight into the DAergic contribution to the modulation in adult mouse locomotion and bridges the gap from previous neonatal animal work. It shows that DA can have differential effects dependant on the state of the animal and provides a foundation for future work on DA neuromodulation.

ii Preface Chapter 1. Review of the literature and the relevant information pertaining to this thesis.

Chapter 2. Has been published as C. Meehan and K.A. Mayr, M. Manuel, S.T. Nakanishi, P.J. Whelan, “Decerebrate mouse model for studies of the spinal cord circuits.”. (2017) Nature protocols, vol. 12(4), issue: 4, pages: 732-747.

Chapter 3. In preparation for submission as K.A. Mayr and P.J. Whelan, “Dopaminergic modulation of stepping locomotion in adult mice”. Journal of Neuroscience.

Chapter 4: Is a general discussion of the material presented throughout this thesis and how it integrates with current knowledge on spinal network function.

iii Acknowledgements I would like to thank Dr. Patrick Whelan for his continuous support and mentorship throughout my master’s program. He has been an invaluable resource and mentor always giving me the freedom to explore new ideas but encouraging me to see the big picture in science. Words will never be enough to explain the gratitude I have for these past few years.

I would like to extend a sincere thank you to Dr. Simon Sharples for his friendship, expertise and help throughout this project and degree. You have been an invaluable resource to discuss and explore science with. Your intellect and persistence will make you an amazing PI one day.

This work would not have been possible without the funding and support of multiple agencies. I extend my deepest gratitude to the HBI for the funding support and the countless programs that are offered from this institution. HBI’s countless outreach, mentorship and involvement has allowed me to develop both professionally and personally. I would like to thank the Branch out Neurological Foundation for the funding support and for the numerous outreach events that I have been involved with.

I would like to thank my supportive lab and all the people that have made it so enjoyable to work in for the past few years including Adam Lognon, Shane Eaton and Dr. Charlie Kwok. You have always been an entertaining group of people, but were still there to lend an ear or a hand when needed. To Dr. Celine Jean-Xavier and Dr. Sandeep Sharma, you were great resources throughout my degree, you have both always been willing to teach me new things and provide honest feedback when necessary.

To my committee, Dr. Zelma Kiss and Dr. Dave Bennett. You have helped direct my studies and focus my work. You have both been instrumental in the creation of this thesis and making me a better scientist for it. To Dr. Shalina Ousman, thank you for taking the time to read this thesis and provide comments.

To Katie, you have been my rock over the past years. I can never express my gratitude for everything you do, thanks for being there for me.

iv Lastly, I would like to thank my parents, Wendy and Andy Mayr, and my brother Tylor. No one has been more important and supportive than my family. You have guided me throughout my life and for this I am eternally grateful. You listened even though many times the words that escaped my mouth seemed like gibberish. Your unconditional love and support will always drive me to succeed.

v Table of Contents Abstract ...... ii Preface...... iii Acknowledgements ...... iv Table of Contents ...... vi List of Tables ...... x List of Figures and Illustrations ...... xi List of Symbols, Abbreviations and Nomenclature ...... xiii

CHAPTER 1 ...... 1 1.1 Introduction ...... 1 1.1.1 The descending control of locomotion ...... 2 1.1.2 Central pattern generator for hindlimb locomotion ...... 2 1.1.3 Neurotransmitters and Dopamine ...... 4 1.1.4 DA receptor subtypes ...... 6 1.1.5 Previous work on DA and locomotion ...... 7 1.1.6 Decerebrate preparations ...... 8 1.1.7 Motor diseases ...... 9 1.2 Importance of this research ...... 9 1.3 Hypothesis ...... 10 1.4 Aims ...... 10 1.4.1 Aim 1: Development of a decerebrate preparation capable of producing sustained locomotor activity...... 10 1.4.2 Aim 2: Does intrathecal DA have an effect on locomotion in decerebrate animals on a treadmill...... 11 1.4.3 Aim 3: Does intrathecal DA have an effect on locomotion in the freely moving mouse ...... 11 1.5 Figures ...... 13

CHAPTER 2 ...... 17 2.0 General introduction ...... 17 2.0.1 CITATION ...... 17 2.1 ABSTRACT ...... 18 2.2 INTRODUCTION ...... 18 2.2.1 Development of the technique ...... 19 2.2.2 Overview of the procedure ...... 19 2.2.3 Advantages and limitations ...... 20 2.2.4 Applications ...... 21 2.2.5 Alternative methods ...... 21 2.2.6 Experimental design ...... 22 2.3 MATERIALS ...... 23 2.3.1 REAGENTS ...... 23 2.3.2 EQUIPMENT ...... 25 2.3.2.1 Surgical tools ...... 25 2.3.2.2 Other equipment ...... 25 2.3.2.3 Locomotor treadmill for experiments with stepping locomotion ...... 26

vi 2.4 PROCEDURE ...... 27 2.4.1 Induction of general anesthesia Timing: 10 min ...... 27 2.4.2 Carotid artery ligation and intubation ● TIMING 30 min ...... 27 2.4.3 Laminectomy or intrathecal application ● TIMING 30 min ...... 29 2.4.3.1 (A) Drug application pool in lumbar laminectomy ...... 29 2.4.3.2 (B) Intrathecal catheter subdural insertion ...... 30 2.4.4 Craniotomy and decerebration ● TIMING 30 min ...... 30 2.4.5 Box 1 | Nerve dissection for intracellular recordings and fictive locomotion ● TIMING 15 min ...... 31 2.4.6 Data acquisition ...... 33 2.4.6.1 (A) Fictive locomotion experiments ● TIMING 10 min ...... 33 2.4.6.2 (B) Stepping experiments ● TIMING up to 2 h 20 min ...... 35 2.4.7 BOX 2: Timing ...... 36 2.5 ANTICIPATED RESULTS ...... 36 2.5.1 Intracellular recordings ...... 36 2.5.2 Fictive stepping preparation ...... 38 2.5.3 Actual stepping preparation ...... 40 2.5.4 Optogenetics ...... 40 2.6 CONCLUSION ...... 41 2.6.1 Conclusion ...... 41 2.6.2 ACKNOWLEDGEMENTS ...... 41 2.6.3 AUTHOR CONTRIBUTIONS ...... 41 2.7 Figures ...... 42 2.8 REFERENCES ...... 64

CHAPTER 3 ...... 70 3.1 Abstract ...... 70 3.2 Introduction ...... 71 3.3 Methodology and materials ...... 72 3.3.1 Experiment 1: intrathecally applied DA and agonists post decerebration in a walking procedure ...... 72 3.3.1.1 Decerebration and laminectomy ...... 72 3.3.1.2 EMG insertion ...... 72 3.3.1.3 EMG data acquisition ...... 73 3.3.1.4 Treadmill experimental procedure...... 73 3.3.1.5 Post hoc verification of decerebration...... 73 3.3.1.6 Force transducer acquisition ...... 74 3.3.1.7 Exclusion criteria ...... 74 3.3.1.8 Data acquisition and analysis ...... 74 3.3.2 Experiment 2: intrathecal application of DA and DA antagonists in freely behaving animals ...... 75 3.3.2.1 Intrathecal injection ...... 75 3.3.2.2 Open field ...... 75 3.3.3 Data acquisition and analysis ...... 76 3.3.3.1 Ladder rung ...... 76 3.3.3.2 Verification of intrathecal injection ...... 76 3.3.3.3 Fluid extraction of spinal cord ...... 77 vii 3.3.4 Pharmacology ...... 77 3.3.4.1 For decerebrate preparations ...... 77 3.3.4.2 For intact animal preparations ...... 77 3.3.5 Statistics: ...... 78 3.3.5.1 Decerebrate ...... 78 3.3.5.2 Intact intrathecal injections ...... 78 3.3.5.3 Significance ...... 79 3.4 Results ...... 79 3.4.1 Intrathecal addition of pharmaceuticals in adult decerebrate mice ...... 79 3.4.1.1 Dopamine increases weight bearing during locomotion ...... 79 3.4.1.2 Dopamine increases bout duration in the adult decerebrate mouse ...... 79 3.4.1.3 Dopamine did not change EMG activity in decerebrate mice ...... 79 3.4.1.4 D1-like agonist augments weight bearing in adult decerebrate mice .....80 3.4.1.5 D2-like agonist do not have an effect on weight bearing in adult decerebrate mice ...... 80 3.4.2 Intrathecal injections in freely moving animals ...... 80 3.4.2.1 DA reduces number of locomotor bouts in the open field ...... 80 3.4.2.2 DA does not change ladder rung scores ...... 81 3.4.2.3 D1-like antagonists reduce overall locomotor activity and increase errors in skilled locomotor tasks...... 81 3.4.2.4 D2-like antagonists have no effect on locomotor activity in the open field but increase errors in skilled locomotor tasks ...... 81 3.5 Discussion ...... 82 3.5.1 Summary of major findings ...... 82 3.5.2 DA increases weight bearing but does not alter muscle EMG ...... 82 3.5.3 Rationale for differences between intact and decerebrate animals ...... 83 3.5.3.1 Cortical effects ...... 83 3.5.3.2 Off target activation ...... 84 3.5.4 D2-like receptor contribution to locomotion ...... 84 3.5.5 Sources of dopamine in the lumbar spinal cord ...... 85 3.5.6 Pharmacology and future experiments ...... 86 3.5.7 Next steps ...... 86 3.6 Conclusions ...... 87 3.7 Figures ...... 88

CHAPTER 4 ...... 103 4.1 General discussion ...... 103 4.1.1 Development of the decerebrate preparation ...... 103 4.1.2 Opposing results may be attributed to different recording parameters ...... 105 4.1.3 State may be responsible for the differential effects in modulating networks105 4.1.4 Cranial control may cause differential effects in intact vs decerebrate animals106 4.1.5 Differences in injection location ...... 106 4.1.6 Redundant systems of the CNS ...... 107 4.2 Therapeutics for Pathologies ...... 108 4.3 Contribution to technology development ...... 109 4.4 References ...... 111

viii APPENDIX ...... 134 Copyright permissions ...... 134

ix List of Tables Table 2.1: troubleshooting ...... 62

Table 3.1: Ladder rung step scoring ...... 101

x List of Figures and Illustrations Figure 1.1: Schematic model of a CPG network ...... 13

Figure 1.2: Monoamine metabolism ...... 14

Figure 1.3: Descending dopaminergic fibres within the spinal cord originate in the A11 ...... 15

Figure 2.1: Procedural steps for creating a decerebrate preparation...... 42

Figure 2.2: Surgical preparation of carotid to artery ligation and tracheotomy...... 43

Figure 2.3: Laminectomy with durotomy...... 44

Figure 2.4: Intracellular recording and antidromic stimulation...... 45

Figure 2.5: Sciatic nerve isolation of the hind limb...... 46

Figure 2.6: Hind-limb securing and antidromic stimulation of nerves...... 47

Figure 2.7: Craniotomy and decerebration cut location...... 48

Figure 2.8: Comparison of intracellular recordings from a motoneuron and a candidate interneuron...... 49

Figure 2.9: Evoking fictive locomotion in a decerebrate mouse after L-DOPA treatment...... 52

Figure 2.10: Effect of spinalization on fictive locomotion...... 53

Figure 2.11: Assisted and spontaneous locomotion evoked on clutch-driven treadmill...... 55

Figure 2.12: Photostimulation of the dorsal L4/L5 spinal cord reduces amplitude of monosynaptic reflex...... 58

Figure 3.1: Data collection methods, parameters and timeline of decerebrate preparation ...... 88

Figure 3.2: Dopamine increases weight support during bouts of locomotion ...... 89

Figure 3.3: Dopamine increases EMG activity and locomotor bout duration ...... 91

Figure 3.4: D1-like agonist augments postural support during locomotion ...... 92

Figure 3.5: D2-like agonist injection does not change on postural support ...... 93

Figure 3.6: Intrathecal injection protocol...... 94

xi Figure 3.7: Intrathecal DA addition decreases bouts of locomotion in the open field and reduces center activity and crosses...... 96

Figure 3.8: Intrathecal D1-like addition abolishes locomotor activity but does not affect step score ...... 97

Figure 3.9: Intrathecal addition of D2-like antagonist does not change motor activity in the open field but increases errors per cross over the ladder rung test...... 99

xii List of Symbols, Abbreviations and Nomenclature 5-HT 5-hydroxy-tryptamine or serotonin A10 Aminergic nucleus of the VTA A11 Aminergic nucleus in the thalamus aCSF Artificial cerebral spinal fluid AADC aromatic amino acid decarboxylase ANOVA Analysis of variance ChR2 Channelrhodopsin 2 CPG Central pattern generator DβH Dopamine-β-hydroxylase DA Dopamine IHC Immunohistochemistry NA Noradrenaline NMDA N-methyl-D-aspartate PD Parkinson’s disease PFA Paraformaldehyde RLS Restless leg syndrome SCI Spinal Cord Injury TH Tyrosine hydroxylase VTA Ventral tegmental area

xiii

CHAPTER 1 1.1 Introduction Locomotion is, above all, a necessity of most animals for living, as no terrestrial animal on earth can survive if it cannot move. Prof. Daniel Wolpert suggests that the nervous system has evolved solely to produce movement (Wolpert, 2011). In his words, all functions of the brain, are ultimately in the service of a movement-orientated nervous system. Indeed, without the ability to move animals would be limited to the resources within a given area. Locomotion occurs in various forms, such as inchworm like movements, hopping in kangaroos, the coordinated movements of a millipede, and the graceful movement of a giraffe.

The brain is the control center of the body and sends specific signals for initiation and modifications of stepping to the spinal cord. Obviously, this is an oversimplification of a very complex system, but it is the premise around cranially modulated networks projecting to the spinal cord. This complex interplay between the brain and spinal cord creates a unique feedback system that provides for constantly adapting locomotor gait. The spinal cord has sufficient circuitry to create stereotypical locomotor patterns, and sensory input from the limbs can produce remarkable corrections to the gait pattern.

Movement, and locomotion in particular, needs to be carefully tuned to the task at hand. Neuromodulators, which can act across multiple synapses can alter multiple aspects of network function. To start to understand the impact of these neuromodulators more work needs to be completed at the level of the spinal cord, particularly in adult animals. My thesis is focused on DA, a neuromodulator associated with motor control. Studies starting with the lamprey have shown that DA plays an integral role in the coordination of a rhythmic sinusoidal locomotion (Grillner et al., 1998), while other studies have focused on a terrestrial locomotion model in mice. Previous work in mice has been invaluable in alluding to the importance of DA in the mammalian spinal cord in control of locomotion but has been limited to neonatal models looking at fictive locomotor patterns (Humphreys and Whelan, 2012; Sharples et al., , 2014, 2015). In this thesis, I used an adult stepping mouse model to elucidate the physiological effects that DA has when acting on spinal networks. Based on the previous work in this field, we know that there are high

concentrations of DA receptors within the spinal cord (Zhu et al., 2007; Sharples et al., 2014) and in dopamine receptor knockout mice, locomotion is abolished, and animals die within one week following birth (Kobayashi et al., 2004). Previous studies have demonstrated that DA can modulate locomotor network activity in neonatal rodents (Humphreys and Whelan, 2012; Delaloye et al., 2015), but it is still unknown the extent and role DA plays within the adult spinal cord. My thesis explores the contribution of DA to the control of locomotion in adults using a reduced preparation to examine effects on stereotypical locomotion. I then follow this with an examination of DA modulatory effects on locomotion in behaving freely moving mice.

1.1.1 The descending control of locomotion Planning and execution of locomotion are controlled by cortical structures, such as the motor and sensory cortices, which project to specific brain regions such as the mesencephalic locomotor region (MLR), subthalamic locomotor region (SLR), as well as the medullary reticular formation (MRF). These brain regions are located medio-caudally in the hindbrain and medio-rostrally in the brainstem. These projecting brain regions have been well studied in terms of both their locomotor function as well as the tracing of the projections (Kiehn, 2016a; Fluri et al., 2017; Kim et al., 2017; Noga et al., 2017). Previously, these regions of the brain were identified by electrical stimulation of these regions and recording the locomotor responses (Kiehn, 2016a). Through these studies, it was found that when these centers are activated, they are sufficient to promote and modulate locomotion (Shik et al., 1969; Whelan, 1996; Kiehn, 2006). With the advancement of genetic models and techniques, scientists have been able to better define and target specific cellular populations to further characterize the role and contribution these brain centers play in the cortical control of locomotion (Kiehn, 2016a; Arber, 2017). Motor control stemming from the brain is only one piece of this puzzle and any discussion of locomotion must consider the spinal cord networks.

1.1.2 Central pattern generator for hindlimb locomotion One key integration center is the lumbar spinal cord which is composed of a complex network of interneurons and motor neurons, known as the locomotor central pattern generator (CPG) (Kjaerulff and Kiehn, 1996; Branchereau et al., 2000; Rybak et al., 2015; Kiehn, 2016a). CPGs are defined as networks that are able to create rhythmic outputs without the need for rhythmic

inputs. Historically most work has focused on the hindlimb since it is most directly related to the bipedal human. However, cervical CPGs also exist in both quadrupeds and humans, but in my work we focused on the hindlimb. The distributed network within the thoraco-lumbo-sacral spinal cord is responsible for the complex coordination between muscles on the left and right limbs and alternation between antagonistic muscles within the same limb. The gastrocnemius and tibialis anterior of the lower leg are antagonistic to one another as one is an extensor and one is a flexor respectively. Extensor groups function to increase the angle of two bones that form a joint whereas flexor muscles act to decrease the angle of a joint. It is critical in locomotion that antagonistic muscles contract out of phase to efficiently and effectively control the joint for terrestrial locomotion. This is also true bilaterally; the same muscle groups need to alternate between the right and left sides of our body. It is worth noting that this definition is greatly simplified especially for biarticular muscles such as the semitendinosis which can have both a flexor and extensor role. Nevertheless, this mix of alternation bilaterally between limbs and unilaterally between muscle groups allows for the coordinated motor output we define as stepping. When many of these steps happen concurrently, this creates what we define throughout this thesis as locomotion, in reference to quadrupedal terrestrial locomotion in mice. For clarity and readability throughout the remainder of this thesis, walking locomotion in quadrupedal vertebrate mice will be referred to as locomotion, even though this term applies to diverse forms of movement.

This complex interplay between descending control and a localized pattern generator creates stereotypical locomotor patterns that contain left/right alternation coupled with antagonistic muscle activation. The first substantial contribution regarding CPG function came from Graham Brown (1911). This first model was described as a half center model where antagonistic muscles act through reciprocal inhibitory circuits to create an alternating flexor/extensor pattern. This reciprocal inhibitory model works on the principle that only one class of neurons are active at one time, allowing for antagonist muscle groups to activate out of phase from one another.

Since the proposal of this half center model, there have been many other network models proposed. For example, Dr. Sten Grillner discussed the concept of a unit burst generator that placed multiple symmetrical units bilaterally across the spinal cord segments contributing to the CPG function.

The key concept illustrated by Grillner is that individual oscillators could flexibly couple to generate diverse rhythmic patterns. These ideas were a fundamental foundation for CPGs and how they work, but over the decades since Grillner’s proposal, the general concept has evolved into a much more complex model. The previous models were a great start but lacked the complexity and ability to account for many locomotor patterns and the ability for differential speeds. The current best explanation comes from contributions of many research groups and is summarized by Kiehn 2016. The fundamental concept of this model is that there is a multi layered network with both pattern generating components and rhythm generating components to generate the many diverse patterns of locomotion (Figure 1.1). This model proposes that descending control terminates both at the pattern generating network and the rhythm generating network, but it is the rhythm generating network that sets the frequency of oscillations (Kwan et al., 2009; Harris-Warrick, 2011). This rhythm generating network projects to the pattern generating network to complete the overall pattern of locomotion. Ultimately, it is the pattern generator that projects to motoneurons to activate specific neuronal pools to create fluid locomotor output that is adaptable by both afferent input into the CPG circuit as well as supraspinal inputs to actively detect and modify movements across non-uniform terrains. This harmonious and complex production of adaptable locomotion is based on feedback loops to continuously monitor the environment of the animal and is driven by neuromodulation from the brain and brainstem.

1.1.3 Neurotransmitters and Dopamine Neurotransmission is the act of a neuron releasing neurotransmitters to regulate, activate or inhibit a variety of neurons directly at the projection site. Of the many neurotransmitters, some act as neuromodulators; to simplify the differences, neurotransmission is fast communication between neurons through ionotropic channels in contrast to neuromodulation, which is a slower and sustained process acting on whole networks through metabotropic receptors. Neuromodulation allows groups of cells to be modulated and thus, networks may react by modifying network properties as a whole. A specific class of neuromodulators, monoamines, act on multiple systems throughout the brain and spinal cord influencing mood, hunger, peristalsis of the gastrointestinal tract (Song and Avery, 2013), and motor control (Barbeau and Rossignol, 1991; Kiehn, 2006). Serotonin is one of the most well described monoamines in terms of descending motor control and,

along with other monoamines such as noradrenaline and DA, aids in stabilization and generation of locomotion (Grillner and Jessell, 2009; Humphreys and Whelan, 2012; Delaloye et al., 2015). Serotonin produces changes by acting at all levels of the network (Branchereau et al., 2000), including motoneurons (Harvey et al., 2006). Intrinsic and synaptic properties are altered in ways that differ depending on the class of cell identified (Kwan et al., 2009; Fouad et al., 2010; Harris- Warrick, 2011).

One specific class of monoamines are the catecholamines, which includes adrenaline, noradrenaline and dopamine. Catecholamines are classified as such due to the presence of a catechol base structure with varying side chains to differentiate between the different neurotransmitters. The major precursor of catecholamine neuromodulators within the CNS is L- DOPA which can be converted to dopamine, norepinephrine, and epinephrine. L-DOPA is synthesised from L-Tyrosine by Tyrosine hydroxylase (TH). DA is synthesised from L-DOPA via aromatic L-amino decarboxylase (AADC), while norepinephrine is synthesised from DA via dopamine β-hydroxylase (DβH), and epinephrine from NE via phenylethanol N-methyltransferase in this same pathway (summarized in figure 1.2). From the brain, there are 2 main regions of DAergic nuclei that project directly to the spinal cord, A10 and A11 (Björklund and Skagerberg, 1979; Pappas et al., 2008; Sharples et al., 2014), though there is growing evidence demonstrating that there are neurons within the spinal cord that contain all necessary machinery to produce DA locally (Ren et al., 2017).

DA plays a major role throughout the central nervous system (CNS) and is known for its role in mediation of reward, mood, hunger and is primarily known for its contribution in the nigrostriatal pathway to motor control. Pathologies like Parkinson's disease precedes with the death of DAergic nuclei within the striatum. This presents with various symptoms including tremor, rigidity, freezing of gait and general motor deficits. Studies in Parkinson's have been instrumental in demonstrating the importance of DA within these motor circuits. Current treatments of this disease are limited due to the irreversible death of neurons, but DA precursors, such as L-DOPA, are used to reduce symptoms.

In addition to being implemented as a therapeutic to treat motor dysfunction, L-DOPA has also been used as a tool to study motor control and initiation of movement (Jankowska et al., 1967). Previous studies have demonstrated that systemic application of L-DOPA can promote stepping in the neonatal rodent (Barbeau and Rossignol, 1991; McEwen et al., 1997), a role that is at least partly due to actions at the spinal cord (Grillner and Zangger, 1979). L-DOPA is metabolized to DA and to NE if the appropriate enzymes are present. DA has the ability to evoke a variety of motor outputs in different species. DA contributes to the activation and modulation of locomotion in many invertebrate and vertebrate organisms such as the pond snail (Tsyganov and Sakharov 2000), leeches (Puhl and Mesce, 2008), newts (Matsunaga et al., 2004), lampreys (Svensson et al., 2003), and zebrafish (Boehmler et al., 2007). For example, in the leech when DA is applied to the CNS it induces a crawling motion (Puhl and Mesce, 2008). In the lamprey, DA is contained in plexuses within the spinal cord where it acts to prolong bursts and slow the classic S-shaped swimming pattern. It has been demonstrated that monoamines, including DA, are able to promote rhythmic motor output in cats, rats, and mice (Barbeau and Rossignol, 1991; Pearson and Rossignol, 1991). However, the data on DA function within the spinal cord in adult mice is sparse compared to serotonin and noradrenaline (Harris-Warrick and Cohen, 1985; Schmidt and Jordan, 2000; Madriaga et al., 2004; Liu and Jordan, 2005; Jordan et al., 2008; Gabriel et al., 2009).

1.1.4 DA receptor subtypes

DA has 5 main receptors that can be classified into two subfamilies; the D1-like receptors,

containing D1 and D5 receptors, are known to be excitatory, whereas the D2-like receptors,

including D2, D3, and D4, are inhibitory (Missale et al., 1998; Girault and Greengard, 2004; Zhu et al., 2007, 2008; Sharples et al., 2014). These receptors are all G-protein coupled receptors with

D1-like being Gs/q and D2-like being Gi/o. DA at high concentrations acts on other receptors such as adrenergic receptors but with much lower affinity. These receptor subtypes have varying affinity

for the DA ligand and specifically, the D2-like receptors have the strongest affinity for DA followed by D1-like (Marcellino et al., 2012) then followed by adrenergic receptors.

These receptor subtypes are distributed throughout the brain and spinal cord (Zhu et al., 2007, 2008; Sharples et al., 2014), but the roles of some of these receptors in synaptic transmission and

signaling pathways within spinal networks are not well understood compared to the brain (Kishore and McLean, 2015). That said, DA is thought to decrease sensory afferent transmission, affecting nociception (Kawamoto et al., 2012) and autonomic network function, in addition to modulating spinal motor circuits. In vertebrate animals, these receptors are expressed differentially across the dorsoventral axis (Zhu et al., 2007) (Figure 1.3). Most notable is the concentration of DAergic receptors within CPG networks in developing systems (Zhu et al., 2007), and adult systems (Atlas, 2006; Keeler et al., 2016) which along with DA’s role in modulation, suggests a clear role in adult spinal networks.

1.1.5 Previous work on DA and locomotion Early work using L-DOPA showed that systemic injection of the DA precursor was sufficient to evoke motoneuronal spiking in decerebrate and acutely transected cats (Jankowska et al., 1967; Rossignol and Dubuc, 1994). It was postulated that this was due to the descending NA input that disinhibits the system, though they did not consider DAergic contributions. Other work looking at postnatal stepping patterns demonstrated that L-DOPA increases postural tone for early stages of walking (Navarrete et al., 2002). Previous work from our lab and others demonstrated that application of DA onto an isolated neonatal spinal cord can modulate fictive locomotion (Barrière et al., 2004; Humphreys and Whelan, 2012; Delaloye et al., 2015; Sharples et al., 2015). These fictive locomotor-like bursts are recorded from the ventral roots that contain axons from motor neurons that innervate the muscles of the hindlimbs. These results demonstrate that after bath application of DA, spinal cord networks decrease the frequency of bursting and stabilize the rhythm (Humphreys and Whelan, 2012). Other work demonstrates that DAergic neurons project caudally down to the spinal cord from a region in the brain known as the A11 (Björklund and Skagerberg, 1979; Pappas et al., 2008; Koblinger et al., 2014). By activating the A11 neurons via optogenetic techniques, adult mice exhibit behavioral and locomotor changes (Koblinger et al., unpublished) including increased locomotor activity. Other groups have looked at the role of DA when exogenously applied following injury and showed that when the A11 nucleus was ablated using 6-OHDA, DA concentrations decreased in the lumbar spinal cord (Zhao et al., 2007), indicating a direct DAergic connection from the brain and the presence of DAergic connections.

Furthermore, studies in adult rats demonstrate that intrathecal injections of L-DOPA or DA agonists following transection of the thoracic spinal cord elicit stepping behaviour (Jankowska et al., 1967; McEwen et al., 1997; Lapointe et al., 2009). Conversely, animals treated with D1 and D2 antagonists abolished air stepping (McCrea et al., 1997). In humans, there is supporting evidence of a lumbar locomotor CPG; specifically, when humans with SCI were spinally stimulated with non-rhythmic electrical stimulation, they presented with rhythmic outputs that were alternating and rhythmic, resembling locomotion (Dimitrijevic et al., 1998; Minassian et al., 2016). Furthermore, paraplegic humans given electric stimulation during rehabilitation were able to perform more complex fine movements with one participant able to maintain a standing position with no external assistance, but arms resting on a support (Rejc et al., 2017). These studies were fundamental in showing that the spinal cord can be activated to elicit locomotor and locomotor like behaviours. We expanded on these findings using both a preparation with reduced cortical input as well as freely behaving animals to delineate the effects of direct DA addition to the lumbar spinal cord on locomotor activity.

1.1.6 Decerebrate preparations With previous work looking at descending control and connectivity along with the presence of DA receptors throughout the spinal cord, we first targeted all DA receptors in the spinal cord. We examined the contribution of DA receptors on locomotion at the level of the lumbar spinal cord in a preparation with reduced cortical descending control. We adopted and refined the adult decerebrate mouse preparation (Nakanishi and Whelan, 2012; Meehan et al., 2017) to help us understand lumbar network output in mature systems with reduced descending input. With the cerebrum removed, these animals are able to locomote freely on a treadmill while allowing allow access and relatively easy intrathecal delivery of drugs to the spinal cord. They also allow experiments to be designed in the future allowing fictive locomotion to be used in concert with intracellular recordings.

The decerebrate preparation has been widely used previously in other species such as the cat (Whelan, 1996; Kiehn, 2006; Meehan et al., 2012). This procedure utilizes many types of animals ranging from mice to rats but has been used primarily on cats (Drew and Rossignol, 1984; Noga

et al., 1988; Whelan, 1996; Kiehn, 2006; Oka et al., 1993; Takakusaki et al., 1993). The neonatal model is a strong model for research; it is a valuable tool in dissecting the mechanistic properties of spinal networks. However, to fully understand the function of dopamine, we needed to examine adult animals. Therefore, the adult decerebrate model was adopted and adapted for my thesis (Whelan, 1996; Whelan, 2003; Nakanishi and Whelan, 2012) to look at intact spinal networks with reduced descending connectivity, to invasively perturb the system with the goal of delineating the specific roles of neuromodulatory function. Adopting the decerebrate mouse model allows us to bridge the gap between these two experimental models in an attempt to describe the process of the descending dopaminergic pathways and how this can modulate locomotion within adult mice. Adaption of this model to the mouse required careful refinement of procedures, mainly because of the impact of blood loss. This preparation is powerful when considering the possibilities with transgenic mouse strains (Goulding, 2009) when looking at locomotion, which is makes our refinement of the preparation impactful.

Specifically, we aimed to describe whether direct addition of DA onto an adult mouse spinal cord in the lumbar region would modulate locomotion and if so, what parameters of locomotion would be affected.

1.1.7 Motor diseases With many motor diseases still lacking a treatment or cure, it is necessary to research the systems which we still do not completely understand. In the case of dopaminergic spinal modulation of locomotion, we may one day be able to use this as a therapeutic following injuries and diseases like SCI, ALS and PD. Indeed, of interest is that DA agonists can recover function in injured adult mice (Lapointe et al., 2009). SCI is of specific importance as it is an injury that disrupts descending control of locomotion. Since most humans with SCI incur contusion injuries it creates a viable target for downstream exogeneous neuromodulation.

1.2 Importance of this research My work is important as it provides insight into an understudied area in the field of research; the spinal cord and DAergic modulation of locomotion. We know relatively little regarding

dopamine’s effects on intact and brainstem evoked locomotor activity. Secondly, my thesis aims to identify new therapeutics. Movement disorders such as multiple sclerosis, restless leg syndrome (Clemens et al., 2006) and Parkinson's disease (Girault and Greengard, 2004) in addition to trauma such as traumatic brain injury and spinal cord injury may be addressed using intrathecal modulation of spinal cord networks through implanted catheters routinely used to control spasticity. I have focused here on DA since it has potential as a therapeutic and has been shown to be important in the activation and modulation of locomotor networks in diverse animal models. Since the basic spinal cord network architecture appears to be evolutionarily conserved, if we start with understanding simple systems in model organisms like the lamprey and mouse we will be better able to generate preclinical models that use combinatorial approaches to treat motor ailments.

1.3 Hypothesis Spinal dopamine modulates stepping locomotion in adult mice by acting on locomotor circuits in the lumbar enlargement.

1.4 Aims The objective of this thesis is to better understand the role DA plays at the level of the spinal cord in modulating locomotor output in adult mice. This study aims to bridge the current breadth of knowledge that was produced using isolated neonatal mouse spinal cords demonstrating a critical role for DA in modulating fictive locomotor-like rhythms.

1.4.1 Aim 1: Development of a decerebrate preparation capable of producing sustained locomotor activity. As previously stated (Chapter 1.2.6: decerebrate preparations), decerebrated animals have been used for many decades. The two main animal models used for this preparation were cats and rats. Given the widespread use of transgenic models of mice, we adapted and modified the previous protocols to use for mice. Mice present an increased difficulty in performing such a surgery as there are many considerations that must be accounted for including: blood loss, time in anesthesia, and the reduced size compared to the rat. We have successfully created a working protocol,

outlined in (Meehan et al., 2017), to guide current and future researchers into utilizing this preparation. The decerebrate mouse model is a valuable tool that will allow researchers to use transgenic mouse lines to specifically target descending inputs and localized cell populations at the level of the spinal cord. This preparation allows for further perturbations into localized networks, including but not limited to, the respiratory CPG of the brainstem-cervico-thoracic spinal cord, as well as networks involved in organ control such as bowel and bladder. More specifically, the development of this preparation will allow us to bridge the gap between neonatal preparations such as the isolated spinal cord and visualize stepping locomotion in adult preparations in the absence of the cerebrum; attempting to further understand the role of DAergic receptors in controlling lower limb locomotion in the developed spinal cord.

1.4.2 Aim 2: Does intrathecal DA have an effect on locomotion in decerebrate animals on a treadmill. Decerebrate animals confer several advantages as an animal model: (1) they allow for ease in manipulating the intrathecal environment, (2) locomotor bouts tend to be more consistent and, (3) they facilitate the use of intracellular recording to record fictive locomotion. By utilizing the decerebrate mouse preparation, we reduced descending dopamine inputs to the spinal network and could then systematically test the role of DA and specific DA receptor subtypes at the level of the spinal cord. Firstly, by adding DA subdurally to the lumbar spinal cord, we observed the changes to treadmill driven locomotor activity and quantified activity through EMG analysis and weight bearing. By using agonists applied intrathecally onto the lumbar spinal cord, we elucidated the contributions of DA-like receptors on locomotor output with reduced descending input. This provides crucial information about the role specific DA receptor classes play in modulating

stepping locomotion. Agonists used will be SKF81297 (D1-like) and quinpirole (D2-like). These specific agonists have been selected due to the high selectivity for their respective DA-like receptors and minimal off-target effects (Levant et al., 1992; Kim et al., 2000).

1.4.3 Aim 3: Does intrathecal DA have an effect on locomotion in the freely moving mouse By intrathecally injecting DA into the awake and freely behaving adult mice, we activated DA receptors in the spinal cord to observe the behavior and locomotor changes. We observed these

changes by monitoring the animals in a novel open field environment after DA injection. The open field tests the amount of locomotor activity via distance, number of bouts and velocity. Furthermore, mice were tested across a skilled locomotor task known as the ladder rung (Metz and Whishaw, 2009). This test looks at the paw placement across rungs, number of steps to cross and the time to cross. A modified scale was created and used to further classify stepping behaviours not accounted for in the previous scale. Details of these classifications and changes are outlined in table 3.1.

In separate experiments, we used antagonists intrathecally injected to inhibit specific DA receptor subtypes to identify their contribution to locomotor function using the same tests as above.

Antagonists used for this experiment are SCH23390 (D1-like) and sulpiride (D2-like). These antagonists have been selected due to their prevalence in the literature for other studies, use in in- vivo models, as well as the effective binding and minimal off target effects (Bowery et al., 1994; Bourne, 2001).

1.5 Figures

Figure 1.1: Schematic of model of a CPG network Model created by Rybak & McCrea adapted by Whelan 2010. Motor neurons are represented by squares while networks of interneurons are denoted with spheres. A three-layer model consisting of a rhythm generator, a pattern generator and an output layer. The proposed rhythm generator projects excitatory connections to both itself and the pattern forming layer. Populations in the rhythm generating layer project mutually inhibitory connections to both the rhythm generating layer and pattern forming layer to mold the pattern generation which is output to motor neurons. Used with permission from Whelan 2010.

Figure 1.2: Monoamine metabolism Detailed schematic of the monoaminergic pathways including enzymes and products. The major pathway for dopamine starts with L-Phenylalanine and uses multiple enzymes to create DA. Major components of this system include the creation of L-DOPA from L-tyrosine through the tyrosine hydroxylase enzyme. DA itself is a widely used neurotransmitter, though it is also the precursor for NE and epinephrine. Used with permission from Meiser et al., 2013.

Figure 1.3: Descending dopaminergic fibres within the spinal cord originate in the A11 Schematic representation adapted from Zhu et al 2007 and used as open access from Sharples et al. 2014. Schematic representation of the descending fibers from the brain to the spinal cord. Notably, most DAergic fibers project from the A11 and A10 (not shown here) represented by green circles. Red circles (A5, A6, A7) represent NA descending fibers projecting from the pons. Inset: representation of dopamine receptor concentrations within the lumbar spinal cord, where all 5 DA receptors are present and diffuse through the dorsoventral axis.

CHAPTER 2

2.0 General introduction With the majority of work in the field looking at fictive locomotor patterns from neonatal spinal cord, we needed a tool to compliment our current work using intrathecal injection in intact animals. The decerebrate model is a tool that does just that. By using adult mice, we can selectively activate spinally located receptors with reduced efferent and afferent inputs. The decerebrate model removes the cerebrum from the animal to reduce the descending input, removes all pain perception while allowing intrathecal application and activation of neurons in stepping animals. This allows for the direct modulation of locomotor centers contained within different regions of the spinal cord to be selectively targeted with direct observation of locomotor changes. This preparation has been used for decades in cats and rats but until recently was not able to be used in mice. This is due to the small size of the animal where blood loss, time in anesthesia and overall difficulty are quite high. In collaboration with 3 labs across the world, we were able to create a protocol that is easy to understand and can be performed in many labs across the world. The adaptation of the decerebrate procedure required much dedication and commitment from all the labs that contributed to this paper. The fortification of this model provides a clear advantage over rats and cats, in that, it allows the use of transgenic mouse models which are not currently available in rat and cat preparations. The development of this model is a significant contribution to work occurring here at the University of Calgary but will also be pivotal in research of all spinal circuitry, for targeting of specific cell populations throughout the spinal cord.

2.0.1 CITATION Decerebrate mouse model for studies of the spinal cord circuits Claire F Meehan1,5, Kyle A Mayr2,5, Marin Manuel3, Stan T Nakanishi4 & Patrick J Whelan2 1Centre for Neuroscience, University of Copenhagen, Copenhagen, Denmark. 2Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, Alberta, Canada. 3CNRS UMR 8119, Université Paris Descartes, Paris, France. 4Department of Biology, University of Hawaii at Hilo, Hilo, Hawaii, USA. 5These authors contributed equally to this work. Correspondence should be addressed to P.J.W. ([email protected]).

Published online 9 March 2017; doi:10.1038/nprot.2017.001

2.1 ABSTRACT The adult decerebrate mouse model (a mouse with the cerebrum removed) enables the study of sensory-motor integration and motor output from the spinal cord for several hours without compromising these functions with anesthesia. For example, the decerebrate mouse is ideal for examining locomotor behavior using intracellular recording approaches, which would not be possible using current anesthetized preparations. This protocol describes the steps required to achieve a low-blood-loss decerebration in the mouse and approaches for recording signals from spinal cord neurons with a focus on motoneurons. The protocol also describes an example application for the protocol: the evocation of spontaneous and actively driven stepping, including optimization of these behaviors in decerebrate mice. The time taken to prepare the animal and perform a decerebration takes ~2 h, and the mice are viable for up to 3–8 h, which is ample time to perform most short-term procedures. These protocols can be modified for those interested in cardiovascular or respiratory function in addition to motor function and can be performed by trainees with some previous experience in animal surgery.

2.2 INTRODUCTION Over the past decade, the development of new genetic tools has induced a seismic shift in our knowledge of spinal cord circuitry (Grossmann et al., 2010; Whelan, 2010; Kiehn, 2011). These tools use transcription factor markers, which allow for discrete identification of sensorimotor circuits (Lanuza et al., 2004; Bretzner and Brownstone, 2013; Dougherty et al., 2013; Mendelsohn et al., 2015). Indeed, when combined with optogenetic and pharmacogenetic approaches, they facilitate selective manipulation of populations of interneurons and motoneurons (Wyart et al., 2009; Hägglund et al., 2013). Several laboratories have demonstrated the ability to genetically dissect circuits using isolated neonatal spinal cord preparations (Grossmann et al., 2010; Goulding et al., 2014). Elegant examples of targeted manipulation of neuronal populations, including motoneurons, have been published using these useful preparations. Yet these studies also highlight a critical gap in our knowledge. Although studies of these isolated young spinal cord preparations

have produced enlightening results in perinatal motor control, there has been less work concentrating on the adult mouse spinal cord. There is a disconnect between perinatal mouse models used to examine circuits and the adult mouse, in which generally only behavior can be examined (Akay et al., 2014). Spinal circuits are still developing perinatally, and weight support occurs only after the first neonatal week. Indeed, receptor densities, descending tracts, proprioceptive feedback during weight support, and spinal circuits are examples in which changes occur during development (Schmidt and Jordan, 2000; Whelan, 2003; Mentis et al., 2006, 2010).

2.2.1 Development of the technique The development of the decerebrate mouse (a mouse with the cerebrum removed) follows logically from the use of in vivo-anesthetized mouse preparations (Manuel et al., 2009; Meehan et al., 2010a, 2010b). It was driven by a desire to record both fictive locomotion and stepping behavior, which were not possible using the anesthetized preparation. Although decerebrate animals have been studied in many species, the mouse posed particular challenges due to its small size. Size is important because of the greater surface area to volume ratio of smaller mammals and the subsequent risk of heat loss. Furthermore, as metabolic rate per kilogram is inversely proportional to the total weight of the animal, mice have higher ventilatory rates, heart rates, and heat loss (Kleiber, 1947). As the average blood volume of a mouse is ~75 ml/kg, and the range of weights for standard C57/B6 mice is between 10 g (3 weeks old) and 30 g, the blood volumes are limited to 750 μl–2.25 ml. Therefore, the maintenance of decerebrate mouse preparations requires careful attention to ventilation rates during anesthesia, episodes of blood loss, and temperature regulation. We outline the step-by-step procedure for surgically preparing a decerebrate mouse with minimal blood loss, allowing recordings to be taken for many hours. These adaptations to a traditional decerebrate preparation are necessary because of the small size of the mouse. This allows invasive experiments to be designed that could not be performed on conscious mice.

2.2.2 Overview of the procedure We describe methods to record neuronal signaling in vivo, which can be difficult because of the small size of the mouse. We bring together a coordinated set of protocols from three laboratories, in Denmark, France, and Canada, to apply a best-practices approach to describing this preparation.

The first set of experiments using these preparations and intracellular approaches have been published(Alstermark and Ogawa, 2004; Manuel et al., 2009; Meehan et al., 2010a, 2010b, 2012; Iglesias et al., 2011; Nakanishi and Whelan, 2012; Delestrée et al., 2014; Hedegaard et al., 2015), and therefore this is an opportune time to describe the protocol in detail so that it can be applied in other laboratories.

2.2.3 Advantages and limitations Current models for studying the spinal sensory-motor system in adults have been largely limited to anesthetized preparations. A substantial limitation of such models, however, is that anesthetics themselves alter the excitability of the neurons by suppressing various conductances, such as persistent inward currents. Not only can this confound results regarding intrinsic neuronal excitability, but the use of different anesthetics can make it difficult to compare results across laboratories. Our decerebrate model therefore provides a major advantage over existing models in that the intrinsic properties of motoneurons can be measured unaffected by anesthetics. The other major advantage of our decerebrate model is that it is possible to activate spinal central pattern generators (CPGs) such as that for locomotion(Meehan et al., 2012; Nakanishi and Whelan, 2012), which is not possible in anesthetized mice(Meehan et al., 2012). Previously, investigations of this spinal circuitry in mice have been limited to neonatal preparations. Being an adult model, our decerebrate preparation allows for investigations of the mature spinal circuitry of the CPGs using transgenic and optogenetic technology. Adult mouse decerebrate models also have advantages for investigating neuronal excitability and connectivity in adult-onset motoneuron diseases such as amyotrophic lateral sclerosis (ALS). The model can also be adapted for use in all segments of the spinal cord and in both the dorsal and ventral horns, extending the potential use of the model to many fields.

The main limitation is that it requires greater technical expertise than in vitro electrophysiological approaches. A higher premature mortality will initially be observed as compared with anesthetized in vivo preparations; however, this can be reduced substantially with practice. In addition, as compared with that of behaving awake animals, the behavioral repertoire of decerebrate mice is limited. Finally, as they are decerebrate, these preparations can be used only acutely and are not

suitable for chronic long-term procedures. These limitations should be kept in mind when considering the adoption of the decerebrate model.

2.2.4 Applications The biological application of the adult decerebrate in vivo mouse provides a preparation in which to study sensory-motor integration and motor output from the spinal cord in control and transgenic adult mice without compromising these functions with anesthesia. For example, the ability to study the modulation of chronic neuropathic pain at a cellular and systems level using electrophysiological methods in a transgenic in vivo model is now possible. We are also able to measure kinematic data about limb position and angles at various speeds of locomotion, and we are in the process of applying optogenetic methods to modulate the activity of identified descending neuromodulatory projections and their effects on locomotion. Furthermore, a number of transcription factors and genetic markers have been identified and associated with subsets of interneurons in the spinal cord. Using this information, it will be possible to experimentally manipulate various subsets of interneurons in vivo.

We use examples of locomotion as a spinally coordinated behavior to demonstrate the application of this model, and include an example of photostimulation of VGAT+ neurons to illustrate its use. Beyond sensory-motor functions, however, the decerebrate mouse preparation can be used in other areas of research, including studies of spinal cord injury, multiple sclerosis, respiratory physiology, cardiovascular function, and muscle physiology.

2.2.5 Alternative methods Examining adult spinal cord motor function with in vitro preparations is more difficult than doing so in neonates. The crux of the issue is the requirement to maintain the viability of adequate amounts of spinal cord tissue to allow distributed motor networks to function (Wilson et al., 2003). Adult spinal cord slice approaches provide a complementary method for studying intrinsic and synaptic properties of identified neurons (Husch et al., 2015), but only thin slices of spinal cord can be kept alive for recording interneuron and motoneuron spike activity (Husch et al., 2011; Mitra and Brownstone, 2012). An alternative approach is the use of in vitro adult sacral spinal cord

tissue with multiple intact segments (Long et al., 1988; Bennett et al., 2001; Jiang and Heckman, 2006). Anesthetized in vivo preparations are another alternative for labs that are initiating attempts at in vivo experiments. However, these approaches are not appropriate for studying locomotor behavior. By using in vivo decerebrate approaches, neurons can be examined in a more physiological relevant state, with descending projections and sensory input left intact. Correlations between behavior such as locomotion and neural activity in the same in vivo system are also possible. We believe that the decerebrate mouse model provides a complementary preparation that can be combined with the alternative methods listed here to build a comprehensive overview of motor function.

2.2.6 Experimental design As pointed out above, blood loss is an important concern for mouse preparations. For these reasons, we would highly advise researchers to consider using anesthetized mice (Manuel et al., 2009; Meehan et al., 2010b) to learn intracellular recording before adopting the decerebrate procedure. This allows trainees to practice their surgical skills and collect intracellular recording data in anesthetized preparations before moving on to the protocol outlined here.

Researchers adopting the decerebrate preparation need to ensure that the preparation is kept hydrated to compensate for blood loss. Once established, the preparation is quite stable; however, control experiments need to be run to ensure that bouts of locomotion or motor activity are stable over the proposed duration of the experiment. This will help ensure that any experimental manipulation is not occurring over a backdrop of preparation run-down.

The Axoclamp 2B (no longer sold by Molecular Devices—but available second-hand) is used by investigators here and is preferred due to its ease in manipulating current injection and other parameters during sharp recordings. We have listed the Multiclamp 900A as an alternative, but the responsiveness of the analog interface of the Axoclamp 2B is better for in vivo recordings for which adjustments need to be performed in the process of data collection.

This protocol describes steps to create a fictive or actual stepping preparation. A fictive preparation is ideal for making intra-axonal or intracellular recordings. One could use these preparations to examine changes in afferent transmission or changes in intrinsic firing properties of neurons in the spinal cord. On the other hand, stepping preparations are ideally suited to examining behavioral output of the network. Afferent inflow remains intact and can be manipulated to examine changes in locomotor function. Motor output can be measured by directly recording muscle electromyograms (EMGs), kinematics, or force. In some cases, both preparations can be used. For example, if the effects of afferent inflow were being manipulated, it would be advantageous to test this in a stepping preparation to examine changes in behavior. These could be compared and contrasted with fictive locomotion in which mechanisms of action could be addressed at a cellular level. Figure 2.1 presents a flowchart of the procedure.

2.3 MATERIALS 2.3.1 REAGENTS Mice: C57BL/6J (JAX Mice Strain; Charles River, cat. no. 000664) or Swiss Webster Crl:CFW(SW) (CFW; Charles River) ! CAUTIONAll experiments should be performed in accordance with relevant guidelines and regulations. The following local ethics committees approved all procedures described here: the University of Calgary Health Sciences Animal Care Committee (Canadian Council of Animal Care guidelines), the Paris Descartes Ethics Committee (CEEA34.MM.064.12), and the Danish Animal Experiments Inspectorate (Dyreforsøgtilsynet; permission nos. 2012-15-2934-00501 and 2015-15- 0201-005450. All procedures were performed in accordance with EU Directive 2010/63/EU. Guidance regarding decerebrate animals not perceiving pain should be provided by the relevant institutional animal care and use committee (US)(Silverman et al., 2014). Where applicable, refer to local guidelines.

CRITICAL: We have used two strains of mice in our decerebrate experiments: Swiss Webster (CFW) and C57BL/6. The C57BL/6 is the most popular strain of mouse used, as it forms the background strain for many transgenic lines. We did observe some differences in the quality of stepping produced, with the CFW strain producing more consistent actual stepping. For

intracellular recording, however, we were able to record consistent fictive steps with C57BL/6 animals. Isoflurane USP (Fresenius Kabi) !CAUTION: Halogenated gases can cause dizziness, nausea, eye irritation, and headache. Long- term effects are not fully known. Leakage of isoflurane usually occurs from nose cone and the induction box. The induction box should be used in a fume hood, and gases escaping the nose cone should be vented to a fume hood. Medical-grade oxygen—for vaporized anesthetic (100% medical grade, tank grade: 1072; Praxair) 95% (vol/vol) isopropyl alcohol/5% DiO2 (vol/vol) (MilliporeSigma, cat. no. PX1830-4) Ophthalmic gel (Optixcare Eye Lubricant; CLC Medica) Mouse intubation cannula (Harvard Apparatus, model no. VK32(73-2844)) Braided silk suture thread, needle unattached, 4-0 (Ethicon, cat. no. SA83H) Braided silk suture thread, needle unattached, 6-0 (Fine Science Tools, cat. no. 18020-60) Suture needles, nonthreaded (Fine Science Tools, cat. no. 12050-03) Rounded diamond-embedded burr bit (Hager & Meisinger, model no. IRF-009) Glass micropipettes (Harvard Apparatus, borosilicate glass electrodes with filament GC120F-10, cat. no. 30-0044 or GC150F-10, depending on your electrode holder) Absorbable hemostat strip (Surgicel; Ethicon, cat. no. 63713-0019-55) CRITICAL: Surgicel should be cut into strips that can be easily inserted into the skull during decerebration.

Bone wax (SMI, cat. no. Z046) Sterile lactated Ringer’s solution (Braun Medical, cat. no. L7501) 23-Gauge needle (PrecisionGlide IM; BD, cat. no. 305145) 30-Gauge half-inch needle (PrecisionGlide IM; BD, cat. no. 305106) 35-Gauge, 3-strand Teflon-coated stainless steel wire (A-M Systems, cat. no. 793400) Cotton-tipped applicator (VWR International, cat. no. 89031-270) Gauze sponge (Professional Preference, cat. no. 110808) No. 10 round stainless steel surgical blade (Integra Miltex, cat. no. 4-310)

aCSF (128 mM NaCl, 4 mM KCl, 1.5 mM CaCl2, 1 mM MgSO4, 0.5 mM Na2HPO4, 21 mM NaHCO3, 30 mM d-glucose) CRITICAL: Alternatively, Ringer’s solution (Sigma-Aldrich, cat. no. 96724) can be used. Intrathecal catheter (Alzet, cat. no. catheter MIT-02) Vetbond surgical glue (3M Canada, cat. no. 1469SB) Kwik-Cast Sealant (WPI, cat. no. Kwik-Cast) Pancuronium bromide (Sigma-Aldrich, cat. no. P1918) Monoamine oxidase inhibitor, nialamide CRITICAL: This monoamine oxidase inhibitor has been shown in cats to reduce dopamine metabolism and therefore potentiate the effect of L-DOPA (Carlin et al., 2000; Heckman and Lee, 2001; Silverman et al., 2014). This is best administered in two doses of 100 mg/kg with at least 1 h in between doses, at least 2 h before surgery commences.

2.3.2 EQUIPMENT 2.3.2.1 Surgical tools Extra-fine Graefe Forceps (Fine Science Tools, cat. no. 11152-10) Extra-fine Graefe toothed forceps (Fine Science Tools, cat. no. 11154-10) 5/45 Forceps (Fine Science Tools, cat. no. 11251-35) Needle driver with inset scissor (Fine Science Tools, cat. no. 12002-12) Microscissors (Fine Science Tools, cat. no. RS-5620) Rongeurs (Fine Science Tools, cat. no. 16221-14) Dumont no. 2 laminectomy forceps (Fine Science Tools, cat. no. 11223-20) Curved spatula, length: 6.25 inches (16 cm; Fisher, cat. no. 21-401-25A) This can also be custom- made from a simple small metal spatula (Supplementary Fig. 2.1a)

2.3.2.2 Other equipment CRITICAL Update equipment to newer versions as appropriate. Isoflurane vaporizer (SurgiVet Vaporizer; Smiths Medical, model no. 100) Mouse respirator (Minivent; Harvard Apparatus, model no. 845) Wireless hair trimmers (Wahl Industries, model no. 9960)

Hand-held cauterizer tool (Acu-Tip Portable; Practicon, model no. 7051711) High-speed micro-drill (Stoelting, model: Ideal micro 2953) Stereotaxic frame with ear bars ( Stoelting, model no. 51625 or Narashige, model no. STS-A) Compact spinal cord clamps (Narashige, model: Narashige STS-A) or, alternatively, Cunningham spinal stereotaxic adaptors (Harvard Apparatus, model no. 51690). The recordings shown in this article were made using the Narashige clamps. Narashige vertebral clamps limit the working space above the mouse but offer the flexibility of clamping larger regions, tilting the cord and stretching the vertebrae slightly to reduce movement and improve stability Custom-made leg holder (Supplementary Fig. 2.1b) Temperature-controlled variable heat lamp, 100W (Leviton Manufacturing) Temperature-controlled variable heat pad (K&H Manufacturing, model no. 1009) Rectal thermal probe (Raytek Thermalert, model no. TH-5 or CWE, model no. TC-1000) Extracellular recordings headstage ( Dagan Instruments, model no. 4001) Amplifier (Dagan Instruments, model no. ex4-400 or Digitimer Neurology System) Digitizer (Power 1401, CED, or Digidata 1550, Molecular Devices) Force transducer (UFI, model no. 1030) Carbon dioxide air measurement system (MicroCapstar End-tidal CO2 analyzer, CWE) Analysis software (Spike 2, CED or Clampex/Clampfit 10, Molecular Devices or MatLab, MathWorks) Intracellular amplifier (Molecular Devices, model: Multiclamp 900A or Axon Instruments, model: Axoclamp 2B) Micropipette puller (Sutter Instruments, model no. P-97) Stepper motor (Micropositioner, Kopf, model no. 2660)

2.3.2.3 Locomotor treadmill for experiments with stepping locomotion Half-inch Plywood (Home Depot Canada, cat. no. 894001020) 2-inch-thick polystyrene sheet (Rona, cat. no. 0510004) Abec 5 1/2 -inch ball bearing (Rona, cat. no. 0696094) 3/8-inch threaded stainless steel rod (Home Depot Canada, cat. no. 5091-318) Lego Mindstorms NXT Kit (LEGO, cat. no. 8547)

8-inch-circumference rubber bands (Staples Canada, cat. no. 39380) Stereotaxic facial holder (Stoelting, cat. no. 51629) Adjustable-height bar (Home Depot Canada, cat. no. 801637)

2.4 PROCEDURE 2.4.1 Induction of general anesthesia Timing: 10 min 1| Administer nialamide in two doses of 100 mg/kg with at least 1 h in between doses at least 2 h before surgery commences. This step is required for fictive locomotion only. 2| Place the mouse in a sealed induction chamber with 1 ml of isoflurane (1-chloro-2,2,2- trifloroethyldifluoro-methyl ether, PPC) added to a small cloth at the bottom. 3| Verify that the mouse is in an anesthetized state by checking the righting reflex by tilting the induction chamber. The righting reflex is defined as the mouse’s ability to turn right side up after being flipped over. Check the anesthetized state by testing withdrawal reflexes to toe and tail pinch. 4| After verification of complete anesthesia, put the mouth and nose into a nose cone delivering 2– 2.5% (vol/vol) isoflurane with oxygen (medical-grade oxygen) at 0.4 l/min. The amount of anesthesia required will vary per mouse, but normally it needs to be adjusted to between 1.5 and 2.0%. Throughout the procedure, withdrawal reflexes should be periodically tested. Ventilation rates can vary from a low of 72 breaths/min for fictive locomotion to a high of 168 breaths/min for actual walking. CRITICAL STEP: Look for irregularities in breathing; isoflurane at desired levels will slightly reduce the breathing rate. ?TROUBLESHOOTING

2.4.2 Carotid artery ligation and intubation ● TIMING 30 min 5| Place the mouse on its dorsal side on a covered, heated pad. Lightly tape the forelimbs to the table to allow for stability and better surgical access. 6| Shave the skin around the trachea with a pair of small hair clippers, and remove excess fur using 95% (vol/vol) isopropyl alcohol on a piece of gauze. 7| Using a round-tip 10-mm scalpel blade, make an incision on the skin midline from the sternum to the trachea, moving rostrally toward the bottom of the jaw (Figure 2.2a,b).

8| Isolate the left and right carotid arteries via blunt dissection using the extra-fine Graefe forceps along with the 5/45 forceps (Figure 2.2c). CRITICALSTEP: The vagus nerve runs parallel to the carotid artery; it must be separated before ligation, with extra care taken not to damage it via cutting or crushing. 9| Dissect the vagus nerve away from the carotid artery and exclusively tie off the artery using 6- 0 suture thread (Figure 2.2c–e). CRITICALSTEP: The carotid arteries are fragile; use caution when tying these off. Two ligation sutures are recommended to completely occlude blood flow. TROUBLESHOOTING 10| Expose the trachea with blunt dissection of the sternohyoid muscle, located ventrally to the trachea, to expose the cartilage rings of the trachea (Figure 2.2f,g). 11| Run two lengths of 4-0 suture thread underneath the trachea to the opposing side (Fig. 2.2h). 12| Using microscissors, perform a tracheotomy by cutting a small incision between two pieces of cartilage horizontal to the trachea; the incisions should be large enough to allow insertion of the stainless steel endotracheal tube (Figure 2.2i). !TROUBLESHOOTING 13| Insert the tube ~1–2 cm through the dissected tracheal incision. CRITICALSTEP: This process must be completed quickly to ensure that the mouse does not wake up during the procedure. It is important to increase the anesthetic level slightly beforehand to allow more time for the cannulation procedure. TROUBLESHOOTING 14| Secure the tracheal tube in place by using the two nylon 4-0 sutures previously run underneath the trachea and securing with square knots. Close the incision using nylon suture thread and standard interrupted suturing techniques (Figure 2.2j).

CRITICALSTEP: After intubation, reduce the anesthetic level while maintaining a lack of reflex response to tail pinch and whisker stimulation. ? TROUBLESHOOTING

2.4.3 Laminectomy or intrathecal application ● TIMING 30 min 15| Shave the head and lower back, as well as the left side of the mouse, completely to minimize fur entering the open incisions. Make a superficial cut ~3 cm in length along the midline of the spine around the end of the fat pad (T8–T9). 16| Blunt-dissect the connective tissue to expose the vertebra of interest completely (Figure 2.3a). 17| With the vertebra fully exposed, remove the top portion of the vertebra with rongeurs or laminectomy forceps to expose the spinal cord. If required, extend the laminectomy across several sections to create an elongated well for direct drug application (Figure 2.3a–d). ? TROUBLESHOOTING 18| Administer drugs by performing a lumbar laminectomy pool (option A) or intrathecal catheter subdural insertion (option B). Option A is preferred for intracellular recordings, and option B is preferred for stepping experiments. CRITICALSTEP: Some drugs do not pass through the blood–brain barrier; therefore, for many drugs intrathecal application is required. For these applications, it is important to fully remove the dura (obligatory for intracellular recording experiments).

2.4.3.1 (A) Drug application pool in lumbar laminectomy (i) Remove the dura by using a 30-gauge needle to create a cut horizontal to the spinal cord, creating a small incision. Through this hole, lift the dura with the 5/45 microforceps and use the microscissors to cut the dura and expose the spinal cord (Figure 2.3a). CRITICALSTEP: When the spinal cord is exposed, it is critical not to let it dry out. Always keep it well saturated with gauze soaked in aCSF or Ringer’s solution. (ii) Place the mouse within a stereotaxic frame by securing ear bars into ear canals and placing the mouth into a tooth holder for stability. (iii) For intracellular recordings, form a pool of mineral oil on top of the spinal cord. This is done in one of two ways, depending on the spinal clamps used. When using Narashige vertebral clamps, tie and extend the skin dorsally with silk threads (Figure 2.4a). Tie the threads to the frame to create a mineral oil pool. If you are using Cunningham spinal stereotaxic adaptors, clamp the spinal column from the lateral aspects, which has the advantage of more working space dorsal to the cord. For these adaptors, use a custom-made plastic pool and suture the skin flaps to it. Next, render the

plastic pool leak-proof by applying Kwik-Cast sealant (WPI). Once the sealant has cured (5 min), pour mineral oil over the spinal cord (Figure 2.4b).

2.4.3.2 (B) Intrathecal catheter subdural insertion (i) Create a small incision in the dura by using a 30-gauge needle to create a cut horizontal to the spinal cord. (ii) Fill and moisten the catheter with aCSF. (iii) Run the catheter s.c. through an opened skin incision from the midline between the left and right scapula down to the laminectomy site. (iv) Insert the catheter, subdurally and parallel to the spinal cord, into the previously opened dural incision and slide caudally toward the region of interest of the spinal cord. (v) Secure the catheter at the laminectomy site with a small drop of Vetbond on the skin, taking extra care not to contaminate the laminectomy site. Secure the rostrally facing end of the catheter to the skin by two interrupted sutures through the skin if desired. 19| If you wish to perform nerve dissection for intracellular recordings and fictive locomotion, follow the procedure described in Box 1 and illustrated in Figures 2.5 and 2.6. Otherwise proceed to the next step.

2.4.4 Craniotomy and decerebration ● TIMING 30 min 20| Shave and clean the head with 95% (vol/vol) isopropyl to remove excess hair. 21| Make a superficial cut across the midline of the skull, along its entire length, in the rostral-to- caudal direction. 22| Score the skull on the lambda and bregma fissures with a microdrill and rounded burr bit as in Figure 2.7. CRITICALSTEP: It is not necessary to penetrate the bone—rather, lightly score to create weak spots for complete and easy removal. 23| Remove the skull with rongeurs, exposing the brain and resulting square field ~1–2 mm caudal to the lambda and 1–3 mm rostral to the bregma and extending perpendicular to the midline roughly 1–2 cm to the left and right sides of the midline (Figure 2.7b).

? TROUBLESHOOTING 24| Perform the decerebration resection with a surgical scalpel (no. 10 blade; Fine Science Tools or the custom-made tool, Supplementary Figure 2.1a) at the previously existing lambda crest at a 45° angle. Make the incision from the dorsal section of the brain ventrally at a 45° angle, with the blade perpendicular to the midline, in a quick slicing motion across the horizontal plane (Figure 2.7b). The goal is to end with a premammillary decerebrate preparation. CRITICALSTEP: Try to keep the mammillary bodies as intact as possible, as this will aid in both viability and successful spontaneous stepping (in case of a stepping preparation). 25| Remove the rostrally separated section by using a small curve-bladed microspatula. Fill the hole with Surgicel strips to help keep bleeding to a minimum and fill the newly formed space. CRITICALSTEP: It is critical to curtail bleeding; make sure that the tools are ready for resection of the cerebrum and that hemostatic Surgicel is ready to be inserted into the skull. 26| Suture the skin on the skull using interrupted suturing techniques for stepping preparations. CRITICALSTEP: For experiments that use drugs that increase blood pressure, such as L-DOPA, it is useful to leave the area exposed to monitor for fresh bleeding. If this occurs, add fresh Surgicel to the skull cavity. 27| Once the bleeding has subsided, remove the Surgicel and confirm the completeness of the decerebration before removing the isoflurane anesthetic from the flow. CRITICALSTEP: For fictive locomotion experiments, it is necessary to allow at least 40 min for the isoflurane to be metabolized before administering L-DOPA. For intracellular recording of intrinsic properties of neurons, it is advised to wait for at least 1 h after removal of the anesthetic to ensure that reliable data are obtained.

2.4.5 Box 1 | Nerve dissection for intracellular recordings and fictive locomotion ● TIMING 15 min Additional materials required: • Blunt-tipped scissors (Fine Science Tools, cat. no. 14072-10) • Bipolar hook electrodes (custom made, see Figure 2.6b) • Constant-voltage isolated stimulator (AMPI ISO-Flex)

PROCEDURE 1. Completely shave one hind limb. 2. Using a no. 10 round-blade scalpel, cut the skin of the hind limb from the hip joint to the ankle, then expose the lateral muscles using blunt-tipped scissors. The edge of the biceps femoris muscle, where the gluteal muscles intersect, should be visible as a thin white line (Figure 2.5a). 3. Using fine scissors, cut along this white line from the knee to the hip. CRITICALSTEP: There are many superficial blood vessels in the hind limbs; use precautions to avoid cutting blood vessels close to the knee or close to the hip to avoid blood loss. Use a handheld cauterizer as needed, but never use it too close to the nerves. 4. Cut the biceps femoris along the tibia. 5. Pull the biceps femoris up to expose the sciatic nerve and, using blunt forceps, dissect the nerve from the surrounding connective tissue (Figure 2.5b–c). CRITICALSTEP: The biceps can be resected completely or simply dissected to the side at this point. CRITICALSTEP: The sciatic nerve has been used here as an example; the various branches of the sciatic nerve can be individually dissected, cut, and placed on bipolar stimulation electrodes for antidromic identification of pools of motoneurons. 6. Tie the leg to the custom-made leg holder with a thread attached to the ankle and place a pin through the knee joint and into the leg holder. This easily constructed leg holder allows the skin to be pinned to the holes in the side to create a leg pool, which can be filled with paraffin oil to keep the muscle and nerves moist and at the correct temperature (Figure 2.6a, Supplementary Figure 2.1b). CRITICALSTEP: It is important to regularly monitor the temperature of the leg oil pool independently of the core temperature, as even small drops in temperature can substantially affect force recordings and conduction velocity. 7. Using bipolar hook electrodes (custom made, see Figure 2.6b), hang the nerve into the hook (Figure 2.6b) and connect to a constant-voltage isolated stimulator (AMPI ISO-Flex). Publishers

2.4.6 Data acquisition 28| Acquisition of data. Follow option A for fictive locomotion experiments and option B for stepping experiments.

2.4.6.1 (A) Fictive locomotion experiments ● TIMING 10 min (i) Preparation for intracellular recordings. Administer the neuromuscular blocking agent pancuronium bromide to prevent movements (dilute 1:10 with saline and then give a 0.1-ml dose i.p. initially, followed by 0.05-ml doses every hour). (ii) Place a monopolar recording electrode on the surface of the spinal cord. Place the reference electrode on a muscle near the vertebral column. Connect this recording electrode to an AC amplifier (bandwidth: 10 Hz–5 kHz). This electrode is used to record the afferent volley reaching the spinal cord ~1 ms after electrical stimulation of the sciatic nerve (or one of its branches).? ?TROUBLESHOOTING (iii) Stimulate the hind limb nerve with a 50-μs pulse of supra-maximal intensity at a frequency of 1–5 Hz. (iv) By slowly moving the recording electrode along the spinal cord rostro-caudally, identify the region where the afferent volley is at a maximum. This corresponds to the area in which the density of motoneurons is the highest. Make a patch in the pia at this level with the extra-fine Graefe forceps. (v) Using a stepper motor, bring an intracellular electrode into contact with the spinal cord. Using an approach through the dorsal columns, angle the electrode 10–15° from the vertical and point it toward the lateral part of the spinal cord to reach the lateral motoneuron pools in the ventral horn. (vi) Drive the microelectrode through the dorsal part of the spinal cord. CRITICALSTEP: Care should be taken to avoid compressing the spinal cord at this point. (vii) Throughout the track, use a small pulse of current to monitor the resistance of the electrode (which should have been zeroed at the surface of the spinal cord using the bridge balance knob of the amplifier). The resistance of the electrode increases when in contact with a cell. CRITICALSTEP: The shape of the electrode and its impedance is critical for successful intracellular recordings. We routinely use KCl-filled glass micropipettes pulled to an ~1-μm tip

with a resistance of 10–15 MΩ or 2 M potassium acetate with a resistance of ~25 MΩ (or lower for voltage clamp). (viii) Once the electrode is ~800 μm deep, drive the electrode using 3- to 6-μm steps. (ix) Track for motoneurons up to a depth of ~1,500 μm. Observe the electrode potential recording after each electrical stimulation of the nerve. When close to the motoneuron pool innervating the stimulated nerve, this stimulation elicits a distinctive field potential (visible as a downward deflection on the electrode potential recording). If no field potential is observed in response to nerve stimulation, bring the electrode back out of the spinal cord, and move the electrode 50–100 μm in either the rostro-caudal direction or the mediolateral direction. Repeat Step 28A(ii–ix) until a field potential is observed. ? TROUBLESHOOTING (x) Identify motoneurons by the invasion of an antidromic action potential following the electrical stimulation of the nerve. Following impalement and identification, apply a series of protocols to characterize the electrical properties of the motoneuron (see ANTICIPATED RESULTS and Figure 2..8). Methods for intracellular recordings are described in detail in previous work 16,17. To measure the after-hyperpolarization (AHP), a short depolarizing current pulse of 1 ms can be applied through the microelectrode (using bridge mode on the Axoclamp amplifier) and successive trials can be averaged (using the Spike 2 software). Repetitive firing can be achieved in two ways. The first method uses short (500 ms) square current pulses of progressively stronger current intensities injected into the cell body through the microelectrode (with the Axoclamp amplifier in discontinuous current (DCC) mode). The steady-state firing frequency can be measured and plotted against the current intensity to obtain the I–f gain for the neuron. Alternatively, triangular depolarizing–repolarizing current ramps can be used to elicit repetitive firing (also in DCC mode) and the instantaneous firing frequencies (calculated by the Spike 2 software) are plotted against the current intensity. This second method allows for a faster acquisition of the data needed to calculate the I–f gain and allows for a fast comparison of recruitment versus de-recruitment current intensities to test for the presence of persistent inward currents. The disadvantage of using triangular current ramps is that persistent sodium currents are activated on the ascending ramp before reaching the threshold for the action potential. Measurements for voltage threshold and rheobase are therefore more accurately obtained using square current pulses of increasing intensity.

A series of small-amplitude (−3 to +3 nA), 500-ms square current pulses (in DCC mode) can be used to plot the I–V relationship. ? TROUBLESHOOTING

2.4.6.2 (B) Stepping experiments ● TIMING up to 2 h 20 min (i) Force transducer setup for estimating weight-support during walking. Make a small incision of 1 cm along the midline above the sacrum in the rostro-caudal direction. (ii) Thread a suture through the gluteal muscle or the skin perpendicular to the midline of the body with 4-0 suture thread. (iii) Tie a knot in the end of this thread, creating a loop. Attach the force transducer through the main force blade hole, securing it with three overhand knots. (iv) Calibrate the force transducer by adjusting the variable resistor until the baseline rests at zero. (v) EMG hook electrode implantation for actual stepping experiments. Thread 12–16 inches of 35- gauge, three-strand, Teflon-coated stainless steel wire through the lumen of the 23-gauge needle (Supplementary Figure 2..1c). (vi) Strip 2–3 mm of the Teflon coating from the end closest to the bevel of the needle (Supplementary Figure 2.1d). (vii) Fold the 2-mm end 180° (parallel to the existing wire) and pull it into the lumen (Supplementary Figure 2.1e,f). (viii) Repeat Step 28B(v–vii) as needed; two wires are required for each muscle group for recording. (ix) Insert two wires into each muscle for recording. Place one wire distally in the muscle and one located more proximally. (x) While withdrawing the needle, rotate slowly clockwise and counterclockwise, leaving the hooked wire implanted inside the muscle. (xi) Obtainment of locomotor recordings. Using these methods, recordings can be obtained for 1– 2 h. After this period, euthanize the mouse using sodium pentobarbital. ? TROUBLESHOOTING

2.4.7 BOX 2: Timing For surgery on a single decerebrated mouse: Steps 1–4, induction of general anesthesia: 10 min Steps 5–14, carotid artery ligation and intubation: 30 min Steps 15–19, laminectomy and intrathecal preparation: 30 min Steps 20–27, craniotomy and decerebration: 30 min Steps 28A(i–x), preparation for intracellular recordings: 10 min Steps 28B(i–iv), force transducer setup: 5 min Steps 28B(v–x), EMG hook electrode implantation: 15 min Step 28B(xi), locomotor recording and euthanization: up to 2 h Box 1, nerve dissection for intracellular recordings and fictive locomotion: 15 min

2.5 ANTICIPATED RESULTS 2.5.1 Intracellular recordings We can attest that following the described procedure decerebrate preparations can be maintained for up to 8 h after decerebration, during which time it is possible to obtain intracellular recordings of a quality similar to those we observe in anesthetized mouse preparations (Figures 2.8 and 2.9). Stable recordings can be obtained, allowing measurements of all the intrinsic properties of motoneurons, including features of single action potentials such as AHP, rheobase, and voltage threshold for the action potentials; input resistance; and repetitive firing behavior such as current- firing frequency slope. We have successfully used similar techniques in anesthetized mice (up to 750 d old) to investigate the intrinsic properties of spinal motoneurons both in normal mice (Manuel et al., 2009; Meehan et al., 2010a; Iglesias et al., 2011) and in transgenic models of the diseases affecting motoneurons, such as ALS (Meehan et al., 2010b; Delestrée et al., 2014; Hedegaard et al., 2015) and Charcot–Marie Tooth disease (Lehnhoff et al., 2014), and we anticipate that the decerebrate preparation will expand the questions that can be asked in aged and diseased mouse models. We have also applied these intracellular recording techniques in the decerebrate mouse model to study the firing activity of spinal neurons during fictive locomotion and fictive scratch activity (Meehan et al., 2012; Nakanishi and Whelan, 2012), as well as that of afferents using intra-axonal dorsal root recordings (Nakanishi and Whelan, 2012).

To demonstrate that the motoneurons in the decerebrate preparations are healthy, we compared the I–f gains (in the primary range) of motoneurons in decerebrate mice with those of mice anesthetized with different anesthetics: fentanyl citrate (Hypnorm) and midazolam, ketamine and xylazine, and sodium pentobarbital (while keeping all other parameters, such as ramp speed, constant). The results of this demonstrate that motoneuron health was not affected by the decerebration; in fact, successful intracellular penetrations of motoneurons displaying repetitive firing within these ranges could be obtained up to 8 h after the decerebration. The main difference with the I–f gain in the decerebrate occurs with respect to the secondary range, which reflects the onset of persistent inward currents, presumably originating in proximal dendrites from activation of L-type calcium channels (Carlin et al., 2000). Here we see that in the decerebrate preparations the transition from primary to secondary range occurs earlier than in anesthetized preparations. The fact that the secondary range is so pronounced in the decerebrate is consistent with descending monoaminergic innervation in the decerebrate preparation.

One of the remaining limitations of intracellular recording in mice is the technical difficulty of recording from interneuronal populations. Although difficult, it is possible, and over the years we have obtained a small sample of ventral horn candidate interneuron recordings (Figure 2.8b,d,e). As with larger animals, these can be identified by a combination of criteria, including location/depth, a lack of antidromic activation, a distinctive high-frequency injury discharge on initial penetration, high firing frequencies, steep I–f slopes, and short AHPs. The AHP durations and I–f slopes of interneurons such as that illustrated in Figure 2.8 are well outside of the range that we have recorded in antidromically identified motoneurons in both anesthetized and decerebrate preparations over the years. To demonstrate this, we have illustrated the I–f slope of the interneuron in Figure 2.8d along with an average I–f slope from a mouse motoneuron and the steepest I–f slope we have ever recorded from a mouse motoneuron. Because of the small size of the mouse spinal cord, however, antidromic identification of descending or ascending axons is complicated by stimulus spread even at low stimulation intensities.

One can not only record in current clamp; voltage-clamp recordings are also possible using sharp microelectrodes. We have achieved this in vivo by using a device developed by R. Lee (Emory University) and C.J. Heckman (Northwestern University)(Lee and Heckman, 1998; Heckman and Lee, 2001). The device is connected to an intracellular amplifier (e.g., Axoclamp 2B) and acts as an external feedback loop to enhance low-frequency gain, which allows for an increase in gain without the high-frequency oscillations or ringing that can occur in voltage-clamp, particularly those using sharp microelectrodes. Using this setup, we can record voltage-clamp synaptic events such as excitatory postsynaptic currents (EPSCs), as shown in Figure 2.8. By incorporating step protocols that increase voltage, one can obtain measurements of the I–V relationship or one can use gradually increasing voltage ramps that are helpful in detecting persistent inward currents (Figure 2.8). We can observe the onset of a negative region at −53 mV, showing the onset of a persistent inward current, which can be detected by measuring the∼ deviation of the negative slope region from the theoretical line expected without activation of a persistent inward current (Figure 2.8h)(Jordan et al., 2008).

2.5.2 Fictive stepping preparation Fictive locomotion can be elicited from mice, and reliable patterns can be recorded from muscle nerves innervating muscles of the hind limbs. After our decerebration, the appearance of spontaneous fictive locomotion is rare. We usually wait 40 min for the anesthetic to leave the system before we try to evoke locomotion with drugs. Our∼ previous work established that i.p. administration of L-DOPA facilitated these bouts of locomotion 21.

We have found that locomotion is evoked with dosages of L-DOPA as low as 2 mg for a 25-g mouse (80 mg/kg)(Meehan et al., 2012). Rhythmic activity usually starts as slow synchronous activity on all nerves, and then gradually the frequency of the rhythm increases and alternates between the left and right sides and flexor and extensor electroneurograms (ENGs) (Figure 2.9). In our current experiments, we have found that at this point the pattern is improved by the administration of 25 μg of 5-hydroxytryptophan (5-HTP). Eventually, the rhythmic activity starts to become less robust, the frequency decreases again and becomes synchronous. Here we have found that another 25-μg dose of 5-HTP restores the rhythm.

Rhythmic activity can also be activated by spinalization (Figure 2.10). This can improve the quality of ongoing L-DOPA-elicited locomotor activity—by speeding up and improving alternation—and also induce activity when none has been achieved by L-DOPA alone or when it has started to fail (Figure 2.10b). This effect is usually fairly short-lived, and thus to test whether it was just the release from inhibition or that we were stimulating descending pathways with the lesion, we performed 2 lesions. This is shown in Figure 2.10b. After the first cervical lesion, fictive locomotor activity is evoked. After the rhythm ceases, it can be reinitiated by a thoracic lesion. We have also shown that peripheral nerve stimulation can elicit rhythmic activity in L-DOPA-treated mice before a sustained episode of L-DOPA-evoked locomotion begins (Figure 2.9b). Furthermore, we confirmed that stimulation of the peripheral nerves can reset an ongoing L- DOPA-evoked rhythm (Figure 2.9b) as has been shown in cats (Conway et al., 1987).

Intracellular recording techniques can be combined with ENG recordings to observe the activity of single neurons during locomotor-like activity (Figure 2.9c). The neurons fire action potentials during the activity phase on the corresponding nerve and are usually silent when the corresponding ENG is silent. The firing frequency for the neuron varies throughout its active phase and invariably matches the ENG activity in the corresponding nerve. The firing patterns vary from cell to cell but generally show a decrementing firing pattern during the active phase (two-thirds of motoneurons, Figure 2.9d,g), starting with high firing frequencies and decreasing. Incrementing firing patterns (Figure 2.9e) and incrementing–decrementing ramps are occasionally observed but are rare. The maximum firing frequencies recorded during the active phase range from 86 Hz to 356 Hz (mean 178 Hz), and the minimum firing frequencies during the active phases range from 11 Hz to 83 Hz (mean 26 Hz). In just over half of cells, the onset of firing in the active phase occurred in the form of doublets or triplets (or even quadruplets, Figure 2.9g). These had an interspike interval of between 2.6 and 11 ms, followed by a prolonged interspike interval, as has been described for other species. In just over half of the motoneurons, clear periods of pronounced inhibition were observed during periods of activity on the antagonist nerve ENG (Figure 2.9d,e,f). The degree of inhibition matched the degree of activity in the antagonist nerve, suggesting a common drive to

both motoneurons and to interneurons inhibiting antagonist motoneurons (reciprocal inhibition) during L-DOPA-evoked locomotion in mice.

2.5.3 Actual stepping preparation Historically, decerebrate nonparalyzed cats walking on treadmills have been used to demonstrate the influence of afferent feedback on the locomotor pattern. This is also possible in nonparalyzed mice. After decerebration and discontinuation of anesthesia, we observed that within 10–30 min an increase in muscle tone and reflex responses occurred (Supplementary Figure 2.2). The increase in muscle tone, evident by an increase in EMG activity (Figure 2.11a), could be visualized as an increase in weight support. This increase in activity would prompt the experimenter to start the treadmill, and, with practice, stepping could be routinely elicited from decerebrate mice. This is in contrast to fictive locomotion, where bouts of non-drug-evoked locomotion were rare—although afferent nerve stimulation can evoke rhythmic alternating activity of flexor and extensor nerves. Although treadmills can be used, decerebrate mice can also locomote well over a wheel, which in our case was manufactured out of light-weight Styrofoam (Figure 2.11b). The advantage of this setup is that spontaneous bouts of locomotion can be recorded in which the mouse runs at its own speed or by motorizing the wheel with a simple clutch mechanism. Supplementary Video 1 shows an example of stepping using this apparatus. Figure 2.11c,d illustrates the various data types that can be recorded simultaneously from a decerebrate mouse preparation. The wheel can be adapted to fix the spinal cord for extracellular recordings. Compared with freely behaving mice, decerebrate mice allow for more invasive procedures to be used. For example, limbs can be partially fixed in place (Duysens and Pearson, 1980), optical recording and stimulation techniques can be more easily deployed, and the effects of acute transections of the spinal cord can be assessed (Whelan, 1996).

2.5.4 Optogenetics A clear potential for the use of the preparation is the use of optogenetic approaches to examine activation and inactivation of specific neurons within the spinal cord (Fig 2.12). We show here an example of photostimulation of VGAT neurons in the spinal cord to depress activation of the H- reflex recorded from the flexor digitorum brevis—which functions to measure the monosynaptic

stretch reflex; such photostimulation can be achieved using a custom-made micro-LED array implanted over the dorsal surface of the spinal cord. This device also potentially can be used in stepping animals.

2.6 CONCLUSION 2.6.1 Conclusion The decerebrate preparation can be used to both examine stepping behavior and record from motoneurons and candidate interneurons in the spinal cord. These procedures can be adapted to record from intra-axonal afferents and motor units. Although the examples are from the hind-limb motor system, the decerebrate model described here can be easily adapted for use in respiratory studies, cardiovascular function, and pain studies, for example.

2.6.2 ACKNOWLEDGEMENTS This work was supported by Natural Sciences and Engineering Research Council grants to P.J.W. M.M. received funds from NIH NINDS R01NS077863. C.F.M. received funds from an EU FP7 Marie Curie Fellowship and project grants from the Lundbeck Foundation. C.F.M. acknowledges the technical assistance of L. Grøhndahl of the Meehan laboratory, the assistance of A. Hedegaard of the Meehan laboratory for the voltage clamp experiments, and advice regarding the voltage clamp and the voltage clamp external gain instrument from C.J. Heckman (Northwestern University). K.A.M. received a studentship from the Branch Out Neurological Foundation and the Hotchkiss Brain Institute. P.J.W. and K.A.M. acknowledge the technical assistance of A. Krajacic of the Whelan laboratory.

2.6.3 AUTHOR CONTRIBUTIONS C.F.M., K.A.M., M.M., and S.T.N. performed the experiments, analyzed the data, and prepared figures. P.J.W. wrote the paper and edited figures. C.F.M., K.A.M., M.M., S.T.N., and P.J.W. conceived of the experiments. C.F.M., K.A.M., S.T.N., M.M., and P.J.W. edited the manuscript.

The authors declare no competing financial interests.

2.7 Figures

Figure 2.1: Procedural steps for creating a decerebrate preparation. Schematic block representation of decerebration procedure and steps to create a fictive or actual stepping preparation. The step numbers refer to the Procedure steps in the protocol.

Figure 2.2: Surgical preparation of carotid to artery ligation and tracheotomy. (a) Red dotted line indicates location of superficial cut along the midline of the neck. (b) Excavation of the superficial adipose tissue to expose the sternohyoid muscles. (c) Blunt dissection of the carotid artery and vagus nerve. (d) 6-0 sutures run underneath the carotid artery, avoiding the vagus nerve. (e) Unilateral tied sutures of the carotid artery. (f) Location of sternohyoid and sternomastoid muscles. (g) Separation of the sternohyoid muscle to expose the trachea. (h) 4-0 sutures run under the trachea for future use. (i) Tracheal cannula inserted into the trachea. (j) Tracheal cannula sutured into place for attaching to a ventilator. Local ethics committees approved the procedures shown here. This figure illustrates Steps 5–14 of the PROCEDURE.

Figure 2.3: Laminectomy with durotomy. (a) Representation of a three-vertebra laminectomy with dura mater attached. Rongeurs or laminectomy forceps are used to clip away the lamina. (b) Spinal cord with the dura mater in the process of being opened with small spring scissors. (c) The dura—held by forceps. (d)Close-up view of the image within the orange square in c. This laminectomy is suitable for application of substances and optogenetics, but smaller laminectomies can improve stability of the spinal cord, which is useful for intracellular recordings. This figure illustrates Steps 15–17 of the PROCEDURE. Local ethics committees approved the procedures shown here.

Figure 2.4: Intracellular recording and antidromic stimulation. (a) Intracellular recording setup as viewed from the right side of the animal, showing the use of Narashige clamps, which provides an excellent range of motion. (b) Alternative setup showing the use of Cunningham spinal clamps, which provides for greater working space on the dorsal surface due to the lateral placement of clamps. This figure illustrates to Step 18A of the PROCEDURE. Local ethics committees approved all procedures shown here.

Figure 2.5: Sciatic nerve isolation of the hind limb. (a) Superficial cut starting at the hip joint, proceeding straight to the knee joint and ending at the ankle joint. The location of the intersection point between the gluteal muscles and the biceps femoris is indicated by a thin white line in the tissue. White arrows identify muscle facia, and yellow arrow indicates the rostro-caudal orientation of the mouse. (b) Dissection of the gluteal muscles and the biceps femoris after cutting along the connecting line. (c) Visualization of the (1) sciatic nerve and the nerve branches consisting of the (2) tibial, (3) common peroneal, and (4) sural nerves. (d) The various branches of sciatic nerve shown in schematic form. Local ethics committees approved all procedures shown here. See REAGENTS section for details. This figure to illustrates Box 1, steps 1–5. Local ethics committees approved the procedures shown here.

Figure 2.6: Hind-limb securing and antidromic stimulation of nerves. (a) Hind-limb stimulation. Left hind limb is secured in a custom-made holder with nerves dissected. Red dashed box indicates the placement of hook electrodes with oil bath. (b) Stimulating hook electrodes (close-up view of the area outlined by the dashed red box in a). Two sets of hook electrodes are placed under the tibial nerve and common peroneal nerve for evoking antidromic action potentials. See REAGENTS section for details. This figure illustrates Box 1, steps 6 and 7. Local ethics committees approved all procedures shown here.

Figure 2.7: Craniotomy and decerebration cut location. (a) Craniotomy and brain exposure are performed by first scoring a square outline (red dashed box) between the lambda and the bregma using an electric handheld drill. The mouse rongeur tool is used to remove the skull as one piece by lifting the scored area, exposing the brain and meninges. (b) Superior sagittal sinus vein and other major vasculature are cauterized using hand cauterization tool (Acu-Tip Portable or Bovie Change-A-Tip) at the most caudal end to reduce bleeding. (c) Schematic representation of the removal of the cerebral cortex (decerebration) and underlying structures. Decerebration is performed with a no. 10 round blade, cutting through the brain at a 45o angle; cut is represented by an asterisk and the red dotted line. (d) Rostral portion of brain is carefully removed from the cut (asterisk) using a microspatula and without damage to the remaining caudal section. In the absence of the rostral brain, the remaining cavity is filled with a hemostatic sponge and/or Surgicel. This figure illustrates Steps 20–27 of the PROCEDURE. Local ethics committees approved all procedures shown here. 48

Figure 2.8: Comparison of intracellular recordings from a motoneuron and a candidate interneuron. These intracellular recordings were performed in adult female C57BL/6J mice, and all recordings in decerebrates were performed at least 2 h after removal of isoflurane from the ventilation flow, to allow for metabolism of the isoflurane. (a) Average antidromic action potentials (n = 18) from stimulation of the sciatic nerve, recorded from a motoneuron in a decerebrate mouse. (b) An average of six spontaneous action potentials recorded in a candidate ventral horn interneuron in an anesthetized mouse. (c) Repetitive firing (middle trace) evoked in a motoneuron in a decerebrate mouse following intracellular ramp current injection (lower trace). Upper trace shows instantaneous firing frequency. (d) Repetitive firing evoked (middle trace) in the same interneuron as in b, following intracellular current injection (lower trace). Upper trace shows instantaneous firing frequency. (e) Graph showing an example I–f slope obtained for a ventral horn interneuron (green) in a mouse. This can be compared with an example of a typical I–f slope from a mouse motoneuron (light blue) and an example of one of the steepest I–f slopes that we have recorded over the years (from a presumptive fast motoneuron, darker blue). (f) Comparison of current–frequency (I–f) slopes recorded from individual motoneurons (single dots) in decerebrate mice and in mice under different anesthesia types (n = 99 motoneurons (decerebrate n = 21 cells from 3 mice, Hypnorm & midazolam n = 21 cells from 5 mice, ketamine & xylazine n = 24 cells from 4 mice, sodium pentobarbital n = 33 cells from 5 mice; a total of 18 mice were used)).

Horizontal lines and error bars represent mean ± s.d. Cells from individual mice within each group are color-coded. The data in this figure come from a series of experiments designed specifically to compare I–f slopes in decerebrate mice with those of anesthetized mice and so all parameters were kept constant (and the examples from e are not included). (g) Current clamp (upper two traces) and voltage clamp (lower two traces) of a series of intracellular recorded EPSPs in an adult mouse spinal motoneuron in vivo evoked by stimulation of the tibial nerve. (h) The I–V function recorded under voltage clamp during a voltage ramp command. A negative inflection is seen at –53 mV, consistent with the onset of persistent inward currents calculated by measuring the∼ deviation of the negative slope region from the theoretical line expected without their activation. Local ethics committees approved all procedures used to obtain these results. See REAGENTS section for details.

Figure 2.9: Evoking fictive locomotion in a decerebrate mouse after L-DOPA treatment. (a) Schematic illustrating the spinal cord and the dissected peripheral nerve branches of the sciatic nerve: the common peroneal (CP) and tibial (Tib) nerves. (b) The effects of peripheral nerve stimulation. Left: an example of rhythmic activity evoked by peripheral nerve stimulation (tested after L-DOPA administration and before spontaneous L-DOPA locomotion started). Right: once the L-DOPA-induced rhythm develops, the rhythm can be reset by stimulation of a peripheral nerve (in this case the nerve branches innervate posterior biceps and semitendinosus (PBST) muscles). In this example, the rhythm was enhanced by a rostral spinalization. (c) Intracellular recording from a CP motoneuron (upper trace) during a period of L-DOPA-evoked fictive locomotion. This is depicted as rhythmic ENG activity (lower traces) alternating between nerves innervating flexor muscles (CP) and nerves innervating extensor muscle (Tib) extensor on a single side. (d) An example of a recording from a Tib motoneuron showing a decrementing pattern of discharge during the active phase on the corresponding ENG recorded from the Tib nerve. Note the inhibition of the Vm of the motoneuron during the active phase of the antagonist nerve (CP). (e) An example of a recording from a CP motoneuron showing an incrementing pattern of discharge during the active phase on the corresponding CP ENG. Inhibition is also seen during the antagonist (Tib) ENG phase. (f) An example of a recording from a slightly more hyperpolarized Tib motoneuron, revealing a clear phase of inhibition and excitation during the active phases of the Tib and CP nerve ENG, respectively. (g) An example of a recording from a CP motoneuron showing an initial high-frequency burst of action potentials (a quadruplet) at spike onset, followed by a rapidly decrementing pattern. Local ethics committees approved all procedures used to obtain the results shown here. See REAGENTS section for details. ENG, electroneurogram.

Figure 2.10: Effect of spinalization on fictive locomotion. (a) Spontaneous locomotion recorded from common peroneal (CP) and tibial (Tib) ENGs after administration of L-DOPA. Lower left insert shows zoomed in section illustrating slow synchronous activity on all nerves. Lower right insert shows a time period later in the recording, at which the activity on all nerves is faster, with alternation between left and right

legs and flexor and extensors. (b) An example of the effect of spinalization on rhythmic activity. L-DOPA-induced locomotion had ceased. A spinalization at the cervical level resulted in rhythmic activity between the flexor nerve, CP, and the extensor nerve, gastrocnemius (Gast). Once locomotion ceased, it could be elicited again by a second thoracic spinal transection. Local ethics committees approved all procedures used to produce data shown here.

Figure 2.11: Assisted and spontaneous locomotion evoked on clutch-driven treadmill. (a) Schematic representation of the left side of the mouse, with EMG electrode insertion into the tibialis anterior and gastrocnemius muscles. The EMG cable is secured subcutaneously to reduce EMG motion artifacts. (b) Schematic of the free-wheel treadmill with clutch system allowing for spontaneous and assisted treadmill locomotion. (c) Example traces of spontaneous and assisted locomotion from CFW decerebrate mice—EMG traces were recorded from the left tibialis

anterior (LTa) and gastrocnemius (LGn). (d) Example trace of a 40-s bout of locomotion demonstrating the weight-bearing at baseline compared with weight-bearing during intrathecal administration of dopamine using a force transducer. Weight change in the force transducer is inversely proportional to the weight-bearing by the mouse. The black bars indicate the stance phase of the left and right limbs. Local ethics committees approved all procedures used to produce data shown here. See REAGENTS section for details. Figure 2.11 refers to Step 28B of the PROCEDURE.

Figure 2.12: Photostimulation of the dorsal L4/L5 spinal cord reduces amplitude of monosynaptic reflex. (a) Schematic representation of stimulation and recording setup. (b) Electrical stimulation of the sciatic nerve was performed on VGAT-ChR2 (H134R)-EYFP-BAC mice in the presence and absence of trains of photostimulation targeted toward VGAT neurons in the spinal cord. Photostimulation was accomplished using an array of six micro-LEDs in a flexible silicone bilayer, allowing stimulation of the surface of the spinal cord. (c) Example traces show a reduction in the H-reflex recorded from the flexor digitorum brevis (marked H in diagram) and expanded in right panels to show reduction in the H-reflex amplitude during light-on conditions. (d) Graph summarizing effects across conditions. Error bars refer to mean ± s.d. n = 20 for each bar. Local animal ethics committees approved these procedures. See REAGENTS section for details. Local ethics committees approved all procedures used to produce results shown here.

Supplementary Figure 2.1: Intramuscular hook electrode for recording electromyogram activity within muscles (a) Rounded and sharpened curved spatula used for decerebration. (b) Custom made leg holder used to easily make a mineral oil bath for the hindlimb muscles and nerves. (c) One completed intramuscular EMG hook electrode using 3 stranded Teflon coated wire (A-M systems, cat No.793400) run through the lumen of a 23-gauge needle (B-D precisionGlide IM, cat No.305145). (d) Stripped 2-3 mm of Teflon coating from the end of the stainless steel wire. (e) 180o bend backwards, creating a hook. (f) Stainless steel wire hook pulled backwards to rest in

the lowest part of the bevel of the lumen. a refers to Step 28 of procedure, b refers to Step 22. c-f refers to Step 32B.

Supplementary Figure 2.2: Increase in EMG tone indicating a bout of locomotion. Increase in the amplitude of flexor and extensor EMG (tibialis anterior and gastrocnemius respectively), in the decerebrate preparation, indicating that a locomotor bout was imminent and that the treadmill should be turned on. Tibialis Anterior (TA), Gastrocnemius (Gast). All experiments should be performed in accordance with relevant guidelines and regulations. Local ethics committees have approved all procedures.

Table 2.1: troubleshooting Step Problem Possible reason Solution

1 Breathing is irregular Isoflurane is too Adjust isoflurane as needed.

high

1 Sudden change in CO2 Temperature, Alter heat-lamp. Check tubing.

tracheal tube has

increased

altering dead-

space.

7 Breathing becomes slow Potential vagus Check to see if the vagus nerve was

and/or sporadic nerve injury or ligated with carotid artery. If yes,

ligation remove sutures and retie only

carotid artery.

Verify vagus nerve is not injured.

12 Ventilator is not providing Tracheal cannula Disconnect cannula from ventilator

flow is blocked. and check for blockages, verify air

flow, and reconnect.

17 Excess bleeding during Ruptured Use bone wax or haemostatic

laminectomy vertebral arteries sponges to minimize blood loss.

20/21 Excess blood loss during Vasculature of Use bone wax to seal the open

craniotomy skull is bleeding bone vessels

30 Spinal cord is compressed Various Adjust the shape of the electrode

by the intracellular taper

electrode.

34 Field potential not visible Incorrect Move the electrode in the rostro-

placement of the caudal or medio-lateral direction.

electrode

Change the angle of the electrode

with respect to the vertical. The

larger the angle, the more lateral

the motoneuron pools targeted.

35 Intracellular recordings are Movements of Decrease the inspiration volume

unstable the vertebral and adjust the other parameters to

column maintain adequate ventilation.

Improve fixation of the vertebral

column on each side of

laminectomy to prevent

movements

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CHAPTER 3 3.1 Abstract The role of many neurotransmitters and modulators have been studied extensively, but dopamine’s (DA) role in modulating movement at the level of the lumbar spinal cord is not fully understood, especially in adult mice. The decerebrate mouse preparation was used to examine modulation of stepping behaviour in adult mice with reduced descending inputs, while applying drugs intrathecally to the lumbar spinal cord. Locomotor activity was measured by recording electromyograms (EMG) from the flexor (tibialis anterior) and extensor (gastrocnemius) muscles of the hindlimbs. Our results show that intrathecal lumbar application of DA produced significantly longer bouts of locomotion. Furthermore, DA application significantly increased

weight bearing and suggests that DA may promote extensor biased walking. D1-like receptors also showed an increase in weight bearing although, not as significantly as when DA was applied. D2- like receptors did not seem to change weight bearing following administration.

Intrathecal application of DA in awake freely moving animals led to a decreased number of locomotor bouts, velocity and distance travelled in the open field while there was no deficit in stepping score, as compared to paired controls in the ladder rung test. Furthermore, DA led to the decrease in locomotor center crossings and distance spent in the center of the open field indicating increased stress/anxiety like behaviours. Intrathecal application of DA antagonists SCH23990 (D1- like) led to a significant reduction of locomotor activity on the open field and increased errors in

skilled locomotion. D2-like antagonists provided no significant change in the open field test, while

increasing number errors per cross the ladder rung. These data suggest that D1 like receptors play

a large role in weight bearing during locomotion while D2 like receptors may play as role in pattern generation and network coordination.

3.2 Introduction Walking is a complex motor task that requires the precise coordination of over 80 muscles in the hindlimbs of mammals. These rhythmic and stereotyped movements are generated by neuronal circuits called central pattern generators (CPG) and are located within the lumbar spinal cord. CPGs are neural networks which contain all of the necessary circuitry to produce rhythmic activity in response to tonic input (McCrea and Rybak, 2008; Rybak et al., 2015; Kiehn, 2016b). Descending projections to the spinal cord from the brain and brainstem are a key element in initiating and controlling locomotion allowing for multiple patterns to be produced so animals can adapt to diverse terrain. Monoamines like serotonin are known to contribute to the descending control of movement and disrupt the step cycle when specific receptors are blocked (Jordan et al., 2008; Cabaj et al., 2017). DA is an understudied class of catecholamine in terms of its role in modulating spinal control of locomotion. The importance of DA in modulating locomotor outputs has been demonstrated using several species such as the leech (Puhl and Mesce, 2008), lamprey (Svensson et al., 2003) and zebrafish (Fetcho et al., 2008), to name a few. Much of what we have learned about how DA controls spinal locomotor networks in mammals has been derived from in vitro preparations of the neonatal rodent spinal cord (Sharples et al., 2015). The spinal cord contains all DA receptors (Zhu et al., 2007; Sharples et al., 2014). In neonatal rats, previous work has demonstrated that L-DOPA is sufficient to evoke air-stepping (McEwen et al., 1997) while blocking of DA receptors abolishes air stepping (Sickles et al., 1992; McCrea et al., 1997). In adult mice, when descending DAergic nuclei of the brain, A11, are activated in freely behaving mice, they demonstrate increased locomotor activity. These findings indicate that DA, a part of the family of descending monoaminergic neuromodulators, acts to initiate and adapt network function (Jordan et al., 2008) and has been relatively understudied compared to other monoamine neuromodulators in the field, specifically in adult animals.

We utilized two models to study dopaminergic control of walking in the adult mouse. The first was an adult stepping decerebrate mouse preparation that allowed us to examine and perturb intact networks of the spinal cord in the absence of descending cortical control. The second preparation we utilized was intrathecal injection into freely-behaving intact animals. This allowed us to examine DA modulation of walking behaviour in freely moving animals. This preparation allows

animals to perform skilled and unskilled locomotor tasks. Our overall goal was to investigate the DAergic modulation of hindlimb locomotion by intrathecal application onto the lumbar enlargement and contrast the effects between reduced decerebrate (Nakanishi and Whelan, 2012; Meehan et al., 2017) and freely moving preparations while selectively manipulating the activity of specific DA receptor subtypes.

We show that intrathecally applied DA at the level of the spinal cord has a robust effect on weight bearing in the decerebrated adult mouse while increasing the duration of locomotor bouts (Figure 3.2A, B). In contrast, intact animals with intrathecal DA injections reduces the number of locomotor bouts with no deficit in skilled locomotion (Figure 3.6A).

3.3 Methodology and materials All experiments were approved by the University of Calgary Health Sciences Animal Care Committee (Canadian Council of Animal Care guidelines) for all procedures described here (Protocol AC15-0016, AC15-0026). Decerebration were performed on CWF Swiss Webster mice aged 8-12 weeks. Total mice used: 24 C57, 50 CFW. Intrathecal injections were performed on C57/BI6 mice at 8-12 weeks of age.

3.3.1 Experiment 1: intrathecally applied DA and agonists post decerebration in a walking procedure

3.3.1.1 Decerebration and laminectomy

The decerebration was prepared as described in chapter 2 of this thesis. This included tracheotomy, craniotomy, decerebration and laminectomy with durotomy of the lumbar segments L1-3.

3.3.1.2 EMG insertion Please see section “2.4.6.2 (B) Stepping experiments” of this thesis for a full description as well as supplementary figure 3.1 for the electrode creation.

3.3.1.3 EMG data acquisition EMG data were preamplified (Dagan X10 model: 4001 Cap comp headstage) which was then further amplified and band-pass filtered (gain=100, 100 Hz -1 kHz). The signals were then digitized (Molecular Devices: Digidata 1440; PClamp 10.4) and acquired at 3 kHz in gap free mode to acquire long durations of locomotion.

3.3.1.4 Treadmill experimental procedure. After decerebration procedures and EMG electrode insertions, mice were placed onto a motorized treadmill (Figure 3.1A). For a timeline of the experimental protocol see Figure 3.1D. This started with 5 minutes of recovery after isoflurane was stopped, followed by a recording of two bouts of sustained locomotion. A bout is defined as a period of locomotion lasting 3 or more steps, alternating between the left and right hind limbs, with proper plantar stepping. These mice were run on the treadmill during these bouts, until locomotion ceased. Animals were allowed a recuperation period of 10 minutes or until the next spontaneous initiation of locomotion occurred. After two consistent bouts of locomotion were obtained at baseline, 50 µL of 10 mM DA in aCSF was added intrathecally through a catheter. Two additional bouts were recorded after drug addition with 10 minutes of recuperation time in between each bout (Figure 3.4B).

3.3.1.5 Post hoc verification of decerebration. Post hoc analysis was performed on isolated mouse brains to verify the decerebrate cut. Post hoc analysis was also performed on spinal cords in control mice that were injected with 2% neutral red in aCSF through the intrathecal catheter at varying volumes (5, 10, 25 and 50 µL) with 2 mice in each group to establish a proper volume for injection. Mice were transcardially perfused with warm PBS (10 ml), followed by 4% PFA (10 ml). Spinal columns and brains were removed and post fixed in 4% PFA for 12 hours. The brains and spinal columns were isolated and placed into a solution of sucrose for a further 1-2 days to remove the PFA. Removed brains were then compared to atlas figures to verify the premammillary decerebration cut and the spinal cords were used to verify the location of the intrathecal catheter as well as establish the proper dose to sufficiently saturate the lumbar enlargement in the control mice.

3.3.1.6 Force transducer acquisition The force transducer (UFI, model no. 1030) was first calibrated by using a varying set of standard weights (in grams: 5, 10, 15, 20, 30, 50), and adjusted for offset. This was attached to the animal by running 4-0 suture through the gluteal muscle on the dorsal side of the sacrum. The force transducer was raised to relieve some of the animal’s load and elevate the hindlimbs. Data were digitized and time locked with EMGs for accurate time synchronization with data acquired at 3 kHz.

3.3.1.7 Exclusion criteria Bouts of walking were defined as locomotor episodes lasting longer than 3 alternating steps and consisting of proper plantar stepping. Animals exhibiting shorter than 30 seconds of walking, incorrect paw placement during stepping or body rotation during stepping locomotion were excluded and deemed non-viable (nv).

3.3.1.8 Data acquisition and analysis The duration of walking bouts was quantified using time analysis by measuring the onset and offset times of EMG signals and verified with time-matched videos. The data were analyzed from two bouts before DA addition and two bouts after DA addition.

EMG signals were rectified and smoothed using a moving average; 100x reduction factor. The signals of the 4 channels and marked the onset and offset of bursts in the previous bouts (PClamp 10.4). This allowed for the measurement of amplitude (measured in µV), burst duration (onset to offset of a burst), and cycle period (onset of a burst to the onset of the following burst). The data were then normalized to baseline. Bout duration was determined by start of a locomotor bout until the end of locomotor bout or until locomotion did not meet our exclusion criteria (section 3.3.1.7). Root mean square (RMS) was used to quantify overall EMG activity which considers frequency, amplitude and burst duration. RMS is a mathematical calculation commonly used in signal analysis

2 2 2 2 RMS=( ⅀(x1 +x2 +x3 +…+xn )/n) and affords a global view of gait changes (Sekine et al., 2013).

Force transducer√ data were acquired and measured in two ways, area under the curve as well as binned into 5 s bins across a 35 s bout of locomotion. 35 s bouts were used because of one animal

that had walked a maximum of 35 s, based on the locomotion inclusion criteria (section 3.3.1.7). An example trace is shown in figure 5A.

3.3.2 Experiment 2: intrathecal application of DA and DA antagonists in freely behaving animals

3.3.2.1 Intrathecal injection Following baseline walking acquisition (intact animals without drug intervention) of open field and ladder rung, animals were anesthetized using 1% isoflurane and 0.4 L/min oxygen (medical grade Praxair). Intrathecal injections were performed using a Hamilton syringe pre-loaded with drug (DA, SCH23390, sulpiride) + brilliant blue dye (Sigma Aldrich, cat no: B0770) with half of the animals matched with vehicle (aCSF) + brilliant blue. A 34-gauge needle was used to puncture through the dorsal side of the back of the animal between vertebra L4-L5 into the intrathecal space of the cauda equina of the spinal cord. 10 mM of drug in 7 µL of fluid was slowly injected (7 µl in 30 s) into the intrathecal space between lumbar vertebrae L3-L4. Low lumbar or sacral injections were the target site for injection and confirmed via the presence of a tail twitch following insertion of the needle. Fluid injection was closely monitored, and the injection was observable by a bubble indicator in the line to verify fluid entry into the intrathecal space. Intrathecal injections took about 2 minutes per animal and they were allowed to recover for a further 10 minutes. Following recovery time, animals were placed back into the open field to observe the effects of drug addition and vehicle.

3.3.2.2 Open field Animals were monitored in the open field for 30 minutes without any personnel in the room. Animals were not previously habituated to the open field to allow for a classification of stereotypical exploration curve and locomotor activity. The open field analysis and hardware solution (Cleversys CSI-OF; http://cleversysinc.com/CleverSysInc/csi_products/open-field/), allowed for simultaneous testing of 4 animals at a time. Videos were recorded with a HD Canon R41 video camera with 60fps and 1080p resolution. Data were analyzed post acquisition and blinded by scoring animals by number and not treatment. The open field box was classified into 3

major areas (1) box - the entirety of the open field (2) center - 25% of the total area of the box located in the exact center of the open field and (3) the remaining 75% of the area of the full area.

Following open field data collection, behaviours were classified into 6 main groups: in place activity, locomotor speed (slow, medium, fast), center crossings, and bouts of locomotion. In place activity is a measure of a pixel movement in a position in the open field while not locomoting; this includes behaviours such as grooming, sniffing, and scratching. Center crossings are defined by the number of crosses from the periphery of the open field box into the center as defined above. These classifications of behavior are measured by both distance, speed and time where applicable; locomotor speed in cm/s, locomotor bouts and center crossings measured by number, and in place activity measured by time. Data were analyzed using standard statistical tests and are discussed below.

3.3.3 Data acquisition and analysis 3.3.3.1 Ladder rung Following open field testing, after intrathecal injections, animals were run across the ladder rung again using the same recording parameters. Animals were brought up to the lab the night before and allowed to habituate to the behaviour room. The ladder rung test, which tests for skilled locomotion, was performed with a randomized rung setting across a 40 cm distance. The ladder rung corridor left a 3 cm space for the animal to cross but was narrow enough to prevent the animal turning around. A mirror was placed below the rungs at a 45 degree angle to observe and accurately visualize the paw placement onto a rung. Scoring was based on the previous SCI ladder rung scoring (Metz et. al 2013) which was modified to encompass all paw placement behaviours (see Table. 3.1). Errors were defined as a score that was at or below a threshold of 6 including wall steps but did not include backward steps. Total number of steps were counted through video analysis and were the sum of steps from both the left and right legs.

3.3.3.2 Verification of intrathecal injection In a subset of control experiments, intrathecal injections were performed with varying amounts of aCSF with 5% (wt/vol) brialliant blue dye. The dispersion over the spinal cord was verified

visually using brilliant blue dye as an indicator of fluid dispersion. Following testing, all intrathecal injections were verified immediately following sacrifice by extracting spinal cords using fluid extraction.

3.3.3.3 Fluid extraction of spinal cord Fluid extraction was used to quickly remove the spinal cord from the vertebra of the sacrificed animals. Animals were sacrificed using high doses of isoflurane (1 ml into a confined chamber). Once animals were deemed deceased, they were decapitated at the cervical level and the vertebra was isolated between L6 and the sacrum. The spinal cord was removed from the vertebral column by making an incision in the rostrocaudal axis from the previously cut vertebra. The lumbar vertebra was cut to expose the end of the cauda equina and intrathecal space (L5/6). PBS solution was loaded into a 10 ml syringe and was then placed into the intrathecal space and the fluid was used to extrude the spinal cord out of the vertebra from the caudal to the rostral segments.

3.3.4 Pharmacology 3.3.4.1 For decerebrate preparations Targeting of all DA receptors was accomplished by using DA-HCl (Sigma Aldrich, cat no: H8502) [10 mM, 50 µL, aCSF] dissolved in aCSF (in mM: 128 NaCl, 4 KCl, 1.5 CaCl2, 1 MgSO4, 0.5 Na2HPO4, 21 NaHCO3, 30 D-glucose). aCSF was used for extended duration controls to verify that the vehicle did not have an effect nor was mechanical perturbation of the spinal cord a factor.

D1-like agonist SKF81297 (Sigma aldrich, cat no: H8502) [10 mM, 50 µL, aCSF] was used to

selectively target the D1-like receptors (D1, D2), while Quinpirole (Sigma Aldrich, cat no: Q102)

[10 mM, 50 µL, aCSF] was used in separate experiments to selectively target D2-like receptors

(D2, D3, D4).

3.3.4.2 For intact animal preparations Targeting of DA receptors, we used the DA-HCl (Sigma Aldrich, cat no: H8502) [10 mM, 7 µL, aCSF] intrathecally injected into the sacral intrathecal space. Antagonists were used to specifically target DA receptor subtypes in the intact animals. D1-like antagonist SCH23390 (Sigma Aldrich, cat no: D054) [10 mM, 7 µL, aCSF] were used to selectively target D1 and D5 receptors, while

sulpiride (Sigma Aldrich, cat no: S8010) was used to target D2-like receptors (D2, D3, D4) [10 mM, 7 µL, aCSF].

All agonists and antagonists were selected due to the strong binding affinity for their selected receptors as well as their use in the literature discussed further in section 2.3.3.1.

3.3.5 Statistics: All data were analyzed using GraphPad Prism 6 (GraphPad software Inc, www.graphpad.com).

3.3.5.1 Decerebrate All data presented here were tested and determined to be normally distributed using Shapiro-Wilk distribution tests. Statistical analysis on EMG signals was compared using parametric one-tailed paired t-tests. Statistical analysis of the RMS EMG analysis was compared using repeated measures 2-way ANOVA to compare both time effect and treatment. Breathing rates were compared using parametric paired one-tailed t-tests comparing the duty cycle of oscillatory waves (breaths). Force transducer data was compared using 2 bouts of locomotion without drug addiction compared to post drug addition values. Data were binned into 5 s intervals over a 35 s timespan of locomotion. A 2-way repeated measures ANOVA was used to compare these 2 conditions and was analyzed based on time and treatment with Bonferroni corrections used for multiple comparisons. To account for the whole locomotor bout, the area under the curve was used over the first 35 s of locomotion and was compared to control locomotor bouts using parametric single-tailed paired t- tests.

3.3.5.2 Intact intrathecal injections Following binning into 5-minute intervals, open field data were analyzed using repeated measures 2-way ANOVA for both time and treatment. Bonferroni corrections were used for multiple comparisons. When binned into 30-minute intervals, open field data were analyzed using parametric single-tailed unpaired t-tests comparing the populations of pharmaceutical vs aCSF controls. Mann-Whitney tests were used to compare ranks of average foot step score where

applicable. Errors per cross were compared using parametric single-tailed unpaired t-tests to compare pre and post drug addition in matched animals.

3.3.5.3 Significance Data were determined to be significant if the p value was less than 0.05, denoted with *, p value less than 0.01 is denoted by **. Non-significant values were abbreviated as ns in text.

3.4 Results 3.4.1 Intrathecal addition of pharmaceuticals in adult decerebrate mice Quality of the decerebration preparation is a main driver of whether an animal will sustain walking or not. Of the animals used, 25% were not viable and were either excluded or the experiment was terminated because their walking gaits did not meet the minimum requirements for sustained locomotor activity (see section 3.3.1.7).

3.4.1.1 Dopamine increases weight bearing during locomotion Following intrathecal application of DA, animals showed increased weight bearing over a 35 s time binned into 5 second bins following (Figure 3.3A: 2-way repeated measures ANOVA, p=0.0005, n=10). This is reflected as a significant increase in the area under the force curve (Figure 3.2B: one-tailed paired t-test, p=0.0066, n=10).

3.4.1.2 Dopamine increases bout duration in the adult decerebrate mouse Intrathecal application of DA at the lumbar enlargement of the adult mouse spinal cord significantly increased the length of time these mice were able to locomote (Figure 3.3A: one- tailed paired t-test, p=0.048, n=5). Stepping frequency did not increase significantly (Figure 3.3B: one-tailed paired t-test p=0.12, n=4), which was expected as animals were walking at a constant speed over the treadmill.

3.4.1.3 Dopamine did not change EMG activity in decerebrate mice Flexor amplitude did not change following intrathecal DA application (Figure 3.2C: one-tailed paired t-test p=0.2025) nor did it have an effect over time using RMS (figure 3.2D: Repeated

measures 2-way ANOVA, p=0.46, n=8). Extensor muscle amplitude did not change following DA addition (Figure 3.2E: one-tailed paired t-test, p=0.37, n=8) nor did it have a significant difference over time measured using RMS (Figure 3.2F: one-tailed paired t-test, p=0.70, n=8). Burst duration for flexor EMG activity did not have a significant difference from pre and post DA addition (Figure 3.2G: one-tailed paired t-test, p=0.27, n=6) though extensor burst duration had a trending increase following DA addition (Figure 3.2G: one-tailed paired t-test, p=0.06, n=6). Following DA addition, breathing rates of the animals increased as measured by the CO2 monitor (Figure 3.2H: one-tailed paired t-test, p=0.017, n=8).

3.4.1.4 D1-like agonist augments weight bearing in adult decerebrate mice

A D1-like agonist (SKF81297) was intrathecally added and the weight bearing was assessed. When binned into 5 s sections and measured across a 35 s bout of locomotion, it was not significant (Figure 3.4A: 2-way repeated measures ANOVA, p=0.19, n=6). However, when the area under the curve of the entire 35 s bout was examined it did show a significant increase (Figure 3.4B: paired t-test, p=0.0064, n=6,).

3.4.1.5 D2-like agonist do not have an effect on weight bearing in adult decerebrate mice

Following intrathecal administration of D2-like agonist (Quinpirole) we measured weight bearing over the first 35s of the walking bouts. There was no significant change in weight bearing

following application of D2-like agonist (Figure 3.5A: 2-way repeated measures ANOVA, p=0.4928, n=6). The area under the curve of the entire 35 seconds of locomotor activity also showed no change (Figure 3.5B: paired t-test, p=0.49, n=6)

3.4.2 Intrathecal injections in freely moving animals 3.4.2.1 DA reduces number of locomotor bouts in the open field Following intrathecal injection of DA, there was a trend for a decrease in the number of locomotor bouts (Figure 3.7A: 2-way repeated measures ANOVA, n=4, p=0.0003), along with a trend for a decrease in locomotor velocity at medium speeds of locomotion at 50-100 mm/s, with no significant change or trend in slow and fast locomotor bouts (Figure 3.7B: one-tailed unpaired t-

test, n=4, 0-50mm/s p=0.32, 50-100mm/s p=0.09, V100-200mm/s p=0.16). There was no difference in total distance travelled in the open field (Figure 3.7C: one-tailed unpaired t-test, p=0.155, n=4). There were trends for a decrease in center area distance traversed following injection (Figure 3.7D: one-tailed unpaired t-test, p=0.05, n=4,), decreased center area crossings (Figure 3.7E: one-tailed unpaired t-test, p=0.06, n=4,) and increased latency for center area crossing (Figure 3.7F: one-tailed paired t-test, p=0.09, n=4).

3.4.2.2 DA does not change ladder rung scores The reduction in overall locomotor output did not affect the stepping quality across the ladder rung test as scored by average step score (Figure 3.7G: one-tailed paired t-test, right hind p=0.30 and left hind 0.28, n=4). There was also no change in errors per cross (Figure 3.7H: one-tailed paired t-test, p=0.94, n=4) with no difference in the number of steps required to traverse the ladder rung (Figure 3.7I: one-tailed paired t-test, p=0.99, n=4).

3.4.2.3 D1-like antagonists reduce overall locomotor activity and increase errors in skilled locomotor tasks

Following intrathecal injection of D1-like antagonist (SCH23390) animals showed significantly reduced distance travelled (Figure 3.8A: 2-way RM ANOVA, p=0.014, n=4), average locomotor velocity (Figure 3.8B: one-tailed paired t-test, p=0.0001, n=4) and number of locomotor bouts (Figure 3.8C: one-tailed paired t-test, p=0.0008, n=4) in the open field. We found no difference in step score when mice crossed the ladder rung (Figure 3.8D: one-tailed unpaired t-test, p=0.13, n=4). However, there was a difference in errors made traversing the ladder rung (Figure 3.8E: one- tailed unpaired t-test, p=0.047, n=4). Lastly, there was no significant change in the number of steps required to cross the ladder rung (Figure 3.8F: one-tailed unpaired t-test, p=0.96, n=4).

3.4.2.4 D2-like antagonists have no effect on locomotor activity in the open field but increase errors in skilled locomotor tasks

Following intrathecal injection of D2-like antagonist (sulpiride), we did not see any significant decrease in locomotor activity in the open field. In the open field animals were found to exhibit no difference in the distance traveled (Figure 3.9A: one-tailed paired t-test, p=0.9153, n=4) or average

velocity (Figure 3.9B: one-tailed paired t-test, p=0.1055, n=4). Animals did show a decrease in locomotor bouts in the open field test over the entire 30 minutes in open field (Figure 3.9C: one- tailed paired t-test, p=0.043, n=4). Furthermore, mice showed no change in the locomotor step score during the ladder rung test (Figure 3.9D: one-tailed paired t-test, p=0.11, n-4) although there was a significant increase in error rate (Figure 3.9E: one-tailed paired t-test, p=0.021, n=4). Mice presented no significant difference in number of steps required to traverse the ladder rung test (Figure 3.9F: one-tailed paired t-test, p=0.21, n=4).

3.5 Discussion 3.5.1 Summary of major findings I found that intrathecally DA application to the lumbar spinal cord in decerebrated mice led to augmented weight bearing during locomotion (Figure 3.3A) and increased locomotor bouts.

Addition of the D1-like agonist, SKF81297, also increased weight bearing during locomotion, but

less robustly than when DA was applied. On the other hand, D2-like agonist, quinpirole, had no significant change on EMGs or weight bearing.

Freely behaving mice intrathecally injected with DA into the lumbosacral vertebra demonstrated decreased locomotor activity along with increased stress and anxiety-like behaviours, inferred from decreased center crossing and locomotor time in the center of the open field. Intrathecal application of D1-like antagonist significantly reduced locomotor activity in the open field and

increased the number of errors as part of the ladder rung test. D2-like antagonist application had no effect on the paw placement on the ladder rung or on open field parameters but did significantly increase ladder rung crossing errors.

3.5.2 DA increases weight bearing but does not alter muscle EMG Interestingly, DA led to an increase in weight bearing in the decerebrate animal, however we did not see a significant increase in extensor amplitude or burst duration. One possibility is that EMG recordings were sampling a relatively small population of extensors of the hindlimb. For example, we did not record EMGs from quadriceps which would be expected to carry substantial loads.

The increase in weight bearing during locomotion in the decerebrate mice is best explained by

activation of excitatory D1-like receptors crucial in maintaining muscle tone during locomotor bouts and which act to stabilize locomotor networks (McCrea et al., 1997; Han et al., 2007; Schwarz and Peever, 2011; Sharples et al., 2015). These findings are further complemented by previous work performed using isolated muscle groups of the hindlimb, when isolated and

superfused with D1-like agonists, there was an increase in muscle force contraction (Reichart et

al., 2011) in both wild type and D5 knockout Mice.

One consideration is that DA may play a differential role in modulating muscular tone and burst duration in proximal and distal muscle groups. This will be established in future work by using kinematic analysis to test DAergic effects on knee and ankle joint angle.

Contrary to our initial decerebrate findings, when intact animals were administered with DA, mice reduced locomotor activity (Figure 3.7B-E) and experienced reduced locomotor bouts (Figure 3.7A) while decreasing crossings into the center area of the open field (Figure 3.7F). These animals did not change gait or experience increased errors when presented with a skilled locomotor task (Figure 3.7G-I). This is different from previous findings in our lab when the A11 nucleus was optogenetically activated, where animals increased locomotor activity in the open field (Koblinger et al., unpublished data).

We speculate that the increase in sustained locomotor bouts during locomotion in the decerebrate mice is due to a combination of lumbar network excitation and increased metabolism caused by the faster breathing (Hochman, 2015). It is surprising to see that in awake and freely behaving animals, that DA produced different effects compared with decerebrate mice. Here I present 2 potential possibilities to explain these differences:

3.5.3 Rationale for differences between intact and decerebrate animals 3.5.3.1 Cortical effects In intact animals, there is still the presence of afferent and efferent connections to the amygdala and orbitofrontal cortex (Burstein and Potrebic, 1993; Liang et al., 2011; Naitou et al., 2016). It is

possible that supraspinal control may be reducing locomotor output by affecting behavioural changes. This is reinforced by the potential anxiety and stress behaviours presented in the intact animals following DA injection (figure 3.7D-E). Though this is a possibility, our current data don’t provide evidence for this hypothesis. To expand on this idea, there are some fundamental tests which need to be carried out before we can validate such claim. The simplest and most feasible experiments would be to look at corticosterone (CORT in mice) levels in the systemic circulation, which increase following a stressor. A more robust tool, such as the elevated plus maze, would allow us to better classify these behaviours as being anxiety/stress related.

3.5.3.2 Off target activation Intrathecal injections were applied at the lumbar enlargement in decerebrate animals eventually spreading to the sacral spinal cord. While in awake and freely moving animals, the injection site was between L5-S1. This means that DA would first start acting on sacral networks before acting on the lumbar networks. This is intriguing since the sacral cord contains networks acting on a range of physiological functions, but it is unknown whether order of activation would have a differential effect. Work looking at sacral network activation by 5-HT and NMDA has shown that it is sufficient to evoke lumbar rhythmic output (Cherniak et al., 2014).

Alternatively, injections into the sacral cord may have reduced volume and concentration once it diffuses to the lumbar spinal cord. This is of specific consideration when activating DA receptors, as there is a concentration dependent effect between the DA receptor families (Clemens et al., 2012).

3.5.4 D2-like receptor contribution to locomotion

D2-like receptors seem to play less of a role in the regulation of ongoing locomotion and may serve

to stabilize the circuit (Sharples et al., 2015). Previous work looking at D2-like agonists saw an increase in frequency of fictive locomotor like rhythms without effect on stabilizing the rhythm

(Sharples et al., 2015). Data from the decerebrate preparation demonstrate that D2-like agonists did not have a clear influence to weight bearing but in intact animals, D2-like antagonists increased stepping errors across the ladder rung. This was accompanied by no changes in distance travelled

or velocity but a decrease in the number of bouts of locomotion. These results indicate that with the same amount of distance travelled, and less locomotor bouts, animals walk for longer durations. This presents an interesting finding that is opposite to what others have found in neonatal air

stepping preparations, where D2-like antagonism lead to an overall reduction in the time spent air

stepping (Sickles et al., 1992). This may indicate a differential role of D2-like receptors play from development to adulthood possibly due to changes in receptor expression during development.

3.5.5 Sources of dopamine in the lumbar spinal cord Direct DA projections to the spinal cord come from the A11 nuclei of the hypothalamus (Barraud et al., 2010; Koblinger et al., 2014; Sharples et al., 2014), and the A10 nuclei of the VTA (German and Manaye, 1993; McRitchie et al., 1997). There is a growing body of evidence to suggest that DA may be produced locally in the spinal cord, with TH and AADC positive neurons containing necessary machinery to produce DA (Meiser et al., 2013). The spinal cord, and specifically the lumbar and sacral segments, was thought to contain TH cells in dorsal horn laminae while AADC cells were found mainly around blood vessels and the central canal but recent work has found expression in many other laminae of the spinal cord (Ren et al., 2017). Following injury, AADC and TH containing cells are activated and increase in number (Wienecke et al., 2014; Zhang, 2015). This has been postulated to be a serotonergic increase (Li et al., 2014), though it is also possible that it may lead to DA synthesis following injury (Ren et al., 2016). Further evidence for the local production of DA comes from work using SCI, where transected animals treated with L-DOPA had expressed similar concentrations of spinal DA compared to naive animals given the same treatment (Commissiong, 1985).

Previously, when DA agonists were administered systemically to DA knockout mice, there was an increase in locomotor behaviour (Kim et al., 2000) and when DAergic nuclei were ablated in the brain, administration of DA agonists were sufficient to increase circling responses (Morelli and Di Chiara, 1990). These studies looked at cortical activation but overlooked the key importance of the spinal cord in augmenting motor responses following activation.

3.5.6 Pharmacology and future experiments Clearly, there were different effects in decerebrate animals and intact animals, but one of the main considerations is the pharmacological drugs used in this study. In the decerebrate experiments,

drugs used included: DA, SKF81297 (D1-like agonist) and quinpirole (D2-like agonist). We demonstrate that D1-like receptor activation augmented weight bearing, though not as significantly as just DA. The difference in results compared to DA should be interpreted cautiously since DA may be converted to NA, or simply due to DA activating the entire complement of DA receptors I have identified 2 major experiments that will help elucidate the role of DA on locomotor networks of the lumbar spinal cord

Experiment 1: Application of D1-like and D2-like agonists simultaneously to the lumbar spinal

cord in decerebrate mice. With activation of both D1-like and D2-like receptors together can we replicate results seen from just adding DA alone? Theoretically, a combinatorial approach should target all of the same receptors, while reducing the metabolites of DA, however in practice this is difficult to do. Alternatively, experiment 2: Application of DA along with a DβH inhibitor. A DβH inhibitor will prevent DA being converted to NA while allowing all DA receptors to be activated.

3.5.7 Next steps We aim to increase sample sizes of our agonist data set. Furthermore, there are other experiments that could use the decerebrate preparation. I have shown that optogenetic activation of VGAT cells in the spinal cord are sufficient to abolish H-Reflex in the adult decerebrate mouse (Figure 2.12). With this approach, we aim to target A11 DAergic cell populations projecting from the brain to further dissect the role of descending control of locomotion.

Some of our findings using intrathecal injections in intact animals show clear trends, but we need to increase the number of animals to increase power. Presently, there are some interesting findings that we cannot fully explore using these tests presented here. Namely, the presence of the anxiety or stress-like behaviours that are exhibited by animals intrathecally injected with DA demonstrates an interesting direction for looking at spinally mediated effects on behaviour. As discussed above (section 3.5.3), we will need to verify that the behaviours we have observed are in fact fear or

stress-like by using more robust testing methods (elevated plus maze) and looking at blood cortisol levels.

3.6 Conclusions We have found that DA contributes to weight-bearing support in the decerebrate preparation. Intrathecally applied DA is sufficient to increase weight bearing of the hindlimbs correlated with flexor amplitude increases but no change in gastrocnemius amplitude or burst duration.

Furthermore, when D1-like receptors are activated, there is a change in weight-bearing support,

but not as significant as when all receptors were activated by DA. D2-like receptors seem to have no effect on locomotor activity, weight bearing and pattern generation.

Our second set of experiments of intrathecal injections into the lumbar spinal cord in awake and freely behaving animals yielded some interesting results that open the field for further experiments. Most notable is when DA is applied it reduced locomotor activity in the awake animals while decreasing the center crossings and time spent in center of the open field while presenting with n increase motor deficits when crossing the ladder rung test. These findings, though contrary to our decerebrate results, are interesting since the major difference between preparations is the presence of the cerebrum. As previous experiments have demonstrated, there are direct afferent and efferent projections from the amygdala and orbitofrontal cortex which may play a role in development of both fear and anxiety responses. Another possibility is that these differences may be caused by activation of the autonomic nervous system acting on cortical structures.

Overall, the findings presented above are a unique opportunity for future treatment of many diseases and ailments, but most importantly, it provides insight into the role DA plays within the lumbar spinal cord.

3.7 Figures

Figure 3.1: Data collection methods, parameters and timeline of decerebrate preparation (A) Schematic representation of the head fixed decerebrate preparation and locomotor wheel. (B) Electromyogram recording locations in the tibialis anterior and gastrocnemius of the mouse hindlimb including force transducer placement. (C) Schematic Lego diagram of the clutch system; when engaged it leads to treadmill driven locomotion and can be quickly disengaged for spontaneous locomotion. (D) Schematic representation of the timeline of preparation and recording of locomotion.

Figure 3.2: Dopamine increases weight support during bouts of locomotion (A) Weight support binned into 5 second intervals. -5 indicates 5 seconds before locomotion (baseline), 0 indicates initial 5 seconds of locomotion, etc. Comparison between 2 bouts of locomotion from animals treated with aCSF (Black) with the same animals when DA (Green) was administered intrathecally. Data are presented as mean ± SEM. (B) Animal-by-animal graph of area under the curve of a set of 10 animals given intrathecal DA (Right) compared to locomotion before drug administration (Left).

Figure 3.3: Dopamine increases bout duration but does not increase EMG activity (A) Data showing that intrathecally applied DA augments locomotor bout duration when animals run over a treadmill. (B) Frequency of stepping does not significantly change when DA is intrathecally administered. (C) Flexor amplitude is not significantly different compared across baseline (Black) to DA addition (Green). (D) RMS measurement of the flexor EMG signal shows no significant change. (E) Extensor amplitude is not significantly different when baseline (Black) to DA addition (Green) are compared. (F) RMS measurement of the EMG signal from the extensor muscles shows no significant change. (G) Burst duration does not change following addition of DA (Green) but there is a trend for an increase in extensor duration. (H) Dopamine addition produces an increase in breathing rates after intrathecal administration. Data are presented as mean ± SEM.

Figure 3.4: D1-like agonist augments postural support during locomotion (A) Weight support binned into 5 second intervals over a period of 35 seconds. Comparison

between 2 bouts of locomotion before drug addition (aCSF, Black) and 2 bouts post D1-agonist addition (SCH23390, Red). Data are presented as mean ± SEM. (B) Area under the curve of 6 animals from 35 second bout durations when D1-like agonist was intrathecally administered (Right) compared to baseline locomotion (aCSF, Left).

Figure 3.5: D2-like agonist injection does not change on postural support (A) Weight support binned into 5 second intervals. Comparison between 2 bouts of locomotion

from animals treated with aCSF (Black) to the same animals when D2-like agonist (Quinpirole, Brown) was administered intrathecally. Data are presented as mean ± SEM. (B) Animal-by -

animal analysis of area under the curve of a set of 5 animals given intrathecal D2-like antagonist (Quinpirole, Right) compared to animals treated with aCSF (Left).

Figure 3.6: Intrathecal injection protocol (A) Representative illustration of the location and method of intrathecal injection into the lumbar space. (B) Example diagram of the injection protocol and times used for each animal cohort on intrathecal injection in intact animals. (C) Example of the open field test with 4 boxes oriented in a clockwise orientation. Each box contains a smaller blue box that is 25% of the total box size and referred to as the center with the remaining 75% of area around the edges of the box referred to as the periphery. (D) Schematic used with permission from Metz et al. 2007 to represent our current ladder rung test. Below the figure is an example of the randomized pattern of alternating rungs and empty spaces used.

Figure 3.7: Intrathecal DA addition decreases bouts of locomotion in the open field and reduces center activity and crosses. (A) Comparison of the number of bouts from two time points: pre injection and post injection. DA shows a significant decrease in number of bouts following intrathecal administration. (B) velocity during the locomotor bouts binned into 3 velocity ranges: 0-50 mm/s (slow), 50-100mm/s (medium) and 100+ mm/s (fast) compared between aCSF control injected groups (Black) and DA injected groups (Green). (C) total distance traversed (mm) during a 30-minute time period. Comparisons of animals pre and post drug addition with aCSF controls (Black) compared to DA injected animals (Green). (D) total distance traversed over a 30-minute time period before and after intrathecal injection. Post injection comparisons of control animals given aCSF (Black) and treatment animals given DA (Green). (E) Number of center crosses performed by the animals pre and post injections. Pre-injection: comparisons of animals not treated with any drug. Post injection: comparisons between animals treated with aCSF (Black) and DA (Green). (F) Time taken from start of testing until the first center crossing with time displayed in seconds. Comparisons between treated animals with aCSF (Black and DA (Green). Post injection animals treated with DA were found significantly different than animals treated with aCSF. (G) average step score measured from paw placement when crossing the ladder rung test. Animals scored following injection of aCSF (Left) or DA (Right). Both hindlimbs are represented as RH (Right hind) and LH (Left hind). Inset: simplified step score scale represented by colours, green (Score: 8-6) being normal locomotion and red (score: 0-2) being severe deficit. (H) average number of errors following 3 consecutive crosses of animals post injection. Comparisons of aCSF (right) and DA (Green). (I) average number of steps following 3 consecutive crossings following injection of either aCSF (Right) and DA (Left). All error bars represented as mean ± standard deviation.

Figure 3.8: Intrathecal D1-like addition abolishes locomotor activity but does not affect step score (A) Total distance (mm) covered in the open field box over 30 minutes binned into 5 minute

intervals 0-30. 30 minutes post injection of aCSF (Black) and D1-like antagonist (Purple). (B) Average velocity (mm/s) in the open field box over 30 minutes binned into 5 minute intervals. 30 minutes post injection of aCSF (Black) and D1-like antagonist (Purple). (C) Total number of locomotor bouts in the open field box over 30 minutes binned into 5 minute intervals. Comparisons

between aCSF (Black) and D1-like antagonist (Purple). (D) average step score measured from paw

placement when crossing the ladder rung test using modified paw classification score (Table 3.1).

Animals scored following injection of aCSF (Left) or after injection of D1-like antagonist (Right). Both hindlimbs are represented as RH (Right hind) and LH (Left hind). Inset: simplified step score scale represented by colours, green (Score: 8-6) being normal locomotion and red (score: 0-2) being a severe deficit. Comparisons between animals injected with aCSF (Right) and D1-like antagonist (Left). (F) average number of steps to cross the ladder rung test following 3 consecutive

crossings. Comparisons made post injection of either aCSF (Left) and D1-like antagonist (Right). All error bars represented as mean ± standard deviation.

Figure 3.9: Intrathecal addition of D2-like antagonist does not change motor activity in the open field but increases errors per cross over the ladder rung test. (A) Total distance (mm) covered in the open field box over 30 minutes binned into 5 minute intervals. 30 minutes post injection 0-30 minutes, with comparisons between acsf (Black) and D2- antagonist (Sulpiride, Brown) (B) Average velocity (mm/s) in the open field box over 30 minutes binned into 5 minute intervals. 30 minutes post injection 0-30 minutes, with comparisons between acsf (Black) and D2-antagonist (Sulpiride, Brown). (C) Total number of locomotor bouts in the

open field box over 30 minutes binned into 5 minute intervals. 30 minutes post injection 0-30 minutes, with comparisons between acsf (Black) and D2-antagonist (Sulpiride, Brown). (D) average step score measured from paw placement when crossing the ladder rung test using modified paw classification score (Table 3.1). Animals scored follwing injection of aCSF (Left) and after injection of D2-like antagonist (Right, Sulpiride). Both hindlimbs are represented as RH (Right hind) and LH (Left hind). Inset: simplified step score scale represented by colours, green (Score: 8-6) being normal locomotion and red (score:0-2) being severe deficit. (E) Average errors per cross after 3 consecutive crossings of the ladder rung. Comparisons between animals injected with aCSF (Right) and D2-like antagonist (Left). (F) average number of steps to cross the ladder rung test following 3 consecutive crossings. Comparisons made post injection of either aCSF

(Left) and D2-like antagonist (Right). All error bars represented as mean ± standard deviation.

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Table 3.1: Ladder rung step scoring

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CHAPTER 4

4.1 General discussion While there are many implications of DAs role in mood, hunger and motor control the importance that DA plays at the level of the spinal cord developed systems in adult mice has not always been appreciated. Previous studies have looked at the receptor contribution in isolated neonatal spinal cords, but here I present a bridge in the current gap of knowledge to explore the DA contribution to spinal networks in adult mice.

4.1.1 Development of the decerebrate preparation The first main piece of this thesis was the development of decerebrate preparation. This was a major hurdle and lead to a significant accomplishment in my master's thesis; a publication of a working protocol and potential uses in collaboration with 3 labs across other international Universities (Denmark, France and USA). The development of this preparation took many years across many strains of mice. With the successful implementation of this technique within our lab, we are now able to follow development of spinal networks from neonatal to adulthood. This affords a unique opportunity in the field that aims to bridge the many gaps in the literature, spinal control of locomotion being one of them. This also presents an opportunity to utilize transgenic models previously unobtainable in other model animals such as rats and cats. With viral vectors and gene therapies becoming increasingly popular it is foreseeable that the decerebrate preparation in mice will be utilized to target specific cell populations in the spinal cord to better understand the roles of these spinal neurons with the reduction of descending connectivity.

The decerebrate preparation is not without caveats though. The decerebrate preparation in adult mice is a unique tool that allows the dissection of spinal networks with reduced input from the brain allowing the direct manipulation of spinally driven networks. One of the caveats to this preparation is that with reduced descending control, stereotypical locomotion is limited to single plane walking either forwards or backwards. This leads to abolishment of visual control to perform some complex movements such as adaptations to changing terrain that use afferent signaling to adapt. We attempted to overcome these caveats of the decerebrate preparation by utilizing an intact

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preparation with intrathecal drug applications. It was our goal for these experiments to observe DA receptor activation to compliment the decerebrate data. The intact animal is capable of demonstrating freely behaving locomotor patterns with cortical control. It was our intention to use a preparation in the adult mouse to further disseminate the role DA plays on the lumbar locomotor networks. This preparation though has its limitations as well. Mainly, the experiments were unable to sample the exact same data parameters as the decerebrate animals (ie. EMG, weight bearing). As well, animal behaviour is variable in contrast to decerebrate animals.

In this thesis, I examined the contribution of DA receptors in the lumbar spinal cord of adult mice both a reduced decerebrate preparation and in freely behaving animals. Here I have dissected the roles that the different DA receptor subtypes play in the control of locomotor patterns and in weight bearing. Using the decerebrate preparation with the animal head fixed over a rotating treadmill, we are able to exogenously apply DA through either a catheter or through a three vertebrae laminectomy directly to the lumbar spinal cord. By applying DA to the lumbar cord, we have shown that there are increases in the duration of bouts of locomotor activity. Furthermore, animals had significantly higher weight bearing coinciding with an increase in extensor EMG tone. When

D1-like agonists were applied to the lumbar spinal cord, there is an increase in weight bearing, though it is not as significant as adding DA. This alludes to the importance of both receptor subtypes in locomotion. Alternatively, agonists inherently have higher selectivity when compared

to DA, and the lack of metabolites which may be activating NA systems. When D2-like agonists were added to the lumbar spinal cord, there was no significant increase in weight bearing. One caveat of these experiments is that quinpirole has a higher selectivity of D2 and D3 over D4. This may have been a contributing factor to these results, though it has also been shown that D2-agonism has modest effects during fictive locomotion in isolated neonatal spinal cords (Sharples et al., 2015). Currently, we still need to explore other more fine nuances in the EMG recordings during stepping locomotor to fully understand the D2-like contribution.

We also found that animals had reduced center crossing in the open field and decreased center area locomotion, suggestive of a rather surprising increase in stress/anxiety-like behaviours. Interestingly though, DA appeared to have no effect on the stepping pattern, average foot score, or

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time to cross for skilled locomotion across the ladder rung. On the other hand, by using D1-like antagonists, there was a major decrease of locomotor activity both in open field and in the ladder rung. Though when steps were observed there was no significant changes to locomotor stepping score or error rate across the ladder rung. D2-like receptors seem to play a lesser role in locomotion as D2-like antagonists did not have an effect on the locomotor activity in the open field or on skilled locomotion across the ladder rung test. Interestingly though, 3 out of 4 animals given D2-like antagonists, demonstrated a caudal orientated hyperextension of the hindlimb in 1 or more of their ladder rung crossings, suggesting a possible effect on sensory transmission (data not presented).

Since D2 activation has been shown to depress afferent transmission, an interesting possibility is that increased afferent transmission during complex tasks produces these effects (Prochazka et al., 1988; Llewellyn et al., 1990).

4.1.2 Opposing results may be attributed to different recording parameters The results of these multiple tests on application of DA onto the lumbar spinal cord seem to be conflicting, specifically when DA is applied exogenously onto the spinal cord. From the decerebrate preparation, DA appears to have a robust effect on weight bearing and duration of locomotion, whereas in intact animals it appears to have an opposite effect. This may be due to the fact that we are measuring 2 different parameters by measuring motivational locomotor activity in an open field whereas on a treadmill we are measuring time to exhaustion or cessation of locomotor activity. The same goes for weight bearing. We directly measured the weight supported during locomotion when using the decerebrate preparation while the ladder rung measures skilled locomotor tasks, looking at paw placement and errors in the step cycle.

4.1.3 State may be responsible for the differential effects in modulating networks Another possibility is that there are inherent differences in the excitability states of spinal networks in decerebrate mice compared to intact freely behaving animals. Our lab has recently demonstrated that neuromodulators have drastically different effects on network output depending on the underlying state of the network. In this case, dopamine had relatively modest effects on network output during high excitability state of fictive locomotion compared to a lower excitability state that they described as being represented by spontaneous network activity (Sharples and Whelan,

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2017). This idea may account for the differential effect we saw when comparing the modest effect of intact animals to the robust effect in decerebrate animals. As the decerebrate preparation is a reduced model of walking involving the removal of the cerebrum, it may afford a different state of the downstream network. The act of decerebration is a physical intervention onto the brain of animals and may change the excitability effects on downstream neurons and in this case, change excitability states of the locomotor CPG. This is supported in other works in humans following cervical SCI showing motoneuronal excitation increases distally following cervical injury (Thomas et al., 2017).

4.1.4 Cranial control may cause differential effects in intact vs decerebrate animals Alternatively, there are a couple explanations that are less likely, focused around DAergic controls of other systems including the autonomic nervous system. Our data suggest that animals experience increased stress and anxiety, while reducing locomotor activity. We speculate that activation of the sympathetic nervous system may be producing an increase in cortisol which would then act on the HPA axis to increase anxiety (Varghese and Brown, 2001; Adinoff, 2004; Stephens and Wand, 2012). The thoracolumbar spinal cord contains networks directly associated with sympathetic nervous system having thoracolumbar outflow therefore may be inducing global activation through indirect systemic transmission. This is a strong possibility and this may not been seen in decerebrate animals lacking the cerebrum. Stress/anxiety responses can be activated in 2 main ways: direct activation and indirect activation. Another more hypothetical explanation to this finding is the direct activation of behavioural circuits. With the presence of ascending projections from the spinal cord (L1-6 and sacral) to the amygdala and orbital cortex (Burstein and Potrebic, 1993; Liang et al., 2011), the activation of spinal networks that project to the amygdala may be activating a stress response leading to reducing locomotor activity and center field crossings.

4.1.5 Differences in injection location There is another explanation for the difference in results focused on the location of DA injection and CPG activation. In the decerebrate animals, the injection is over the L3-5 segments of the spinal cord. Whereas in the intact animal the drug bolus was injected in the sacral cord to reduce the potential for spinal cord injury. This difference in pharmaceutical application may explain this

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discrepancy of results. In the intact animal, drug addition makes contact first to the sacral cord would lead to activation of this center preceding the activation of the lumbar cord. This would lead to activation of the parasympathetic pathways of the animal which include networks necessary for bowl and sexual function (Naitou et al., 2016), as well as networks that control tail function (Etlin et al., 2013; Cherniak et al., 2014). While motor networks of the sacral spinal cord can generate rhythmic activities and project rostrally to the lumbar spinal cord, thus interacting with lumbar circuits (Cherniak et al., 2017). However; we do not have full understanding of how dopamine may modulate these sacral networks, let alone the DA receptor concentration in the sacral cord.

4.1.6 Redundant systems of the CNS Another, more theoretical, explanation is that there are numerous redundant pathways the the nervous system and specifically the spinal cord (Loy et al., 2002). This is a “failsafe” measure used within these networks to prevent against abnormal or unusual activity used to increase control of the motor output. In the freely behaving animal, these afferent and efferent projections work in tandem to provide this safety measures to prevent against disruption to the step cycle and gait. In the reduced decerebrate preparation, this descending control is reduced and therefore may not have the same efficacy in controlling “out of range” locomotor activity.

Overall, these data provide insight into the role that each receptor subtype plays in locomotion and points to significant differences in effects in intact and reduced preparations. In awake animals,

DA seems to reduce speeds of locomotion and decrease the overall activity. D1-like antagonists significantly reduced locomotor activity and increased stepping errors across the ladder rung,

indicating that D1-like receptors are necessary for normal locomotion. D2-like antagonists did not seem to have a robust effect. By using the reduced preparation, we see that DA has a robust effect

on weight bearing and muscle tone during stepping. Using D1-like agonists, we can conclude that

D1-like receptors play a role in contributing to muscle tone and therefore weight bearing. On the

other hand, D2-like receptors play a role in gait stabilization and network coordination.

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4.2 Therapeutics for Pathologies There are many motor diseases that plague the world currently, and with most lacking current treatments, it affords a clear reason to understand the spinal contribution to stepping locomotion. Most notably, spinal cord injury provides a potential usage for this new information. As previously stated, SCI is caused by damage to the spinal cord and is followed by motor and sensory deficits and in most cases, presents with neuropathic pain. This presents a large hurdle to overcome, to activate motor circuits without affecting pain afferents. Furthermore, following SCI injury, presence of TH+ neurons increase in the spinal cord (Hou et al., 2016) and DA is necessary for neuronal regrowth following injury and development. This increase in DAergic cells following injury give a clear insight into the importance of DA following injury and specifically the role it plays in locomotor circuits.

Currently, the best treatment for SCI is rehabilitation which activates spinal networks, helping with neuronal regrowth and restructuring within the spinal cord (van den Brand et al., 2012). With DA being inherently antinociceptive (Jensen and Yaksh, 1984; Barasi and Duggal, 1985; Morgan and Franklin, 1991; Altier and Stewart, 1998; Meyer et al., 2009), it may also act as an analgesic (Hong et al., 2014), though this is currently debated (Naithani et al., 2017). Loss of DAergic drive is postulated to be the reason that Parkinson's patients present with increases in pain (Wood, 2008) and following injury (SCI) there is an upregulation of DAergic nuclei within the spinal cord. With these current findings, there presents a potential therapeutic for intrathecal activation of motor circuits while reducing nociceptive circuits may therefore help overcome these barriers. To the best of our knowledge, this idea has not been explored, this may serve as an invaluable tool for allowing patients to increase rehabilitation while boosting beneficial plasticity within the spinal cord. Another possibility is the activation of downstream neuronal circuitry. As shown, decerebrate animals can exhibit stepping locomotion with reduced descending control. Other groups around the world have allowed a rat to regain control using electrical stimulation at the spinal cord (Lavrov et al., 2008) and indeed administration of monoamines has been shown to be effective in separate studies. This comes with many hurdles to translation to the human condition as electrical stimulation and the targeted population of spinal neurons are not specific and may have off target effects (ie. pain)(Minassian et al., 2016; Rejc et al., 2017). With neurotransmitter release locally

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in the spinal cord using input signals from the brain and brainstem, it may be a more feasible and less invasive treatment possibility for SCI. Though there does not seem to be one simple cure for motor diseases and specifically SCI, it presents a unique opportunity for combinatorial therapies formulated from previous literature and our current findings.

4.3 Contribution to technology development Here I have presented our work looking at the DAergic contribution to spinally derived locomotor control. Through this study, we have outlined the interactions and contributions of the major DA receptor subtypes. I have created a micro LED implantable technology for optogenetically activating spinal circuits in transgenic animals (Figure 2.12A, B). Previously, it was difficult to use optogenetics in the spinal cord due to the size of the lazer and inability to secure it in place. This current method allows the stable implantation and provides selective activation of specific cell types projecting from the brain in transgenic adult mice. This will allow scientists to further probe into the circuitry of the spinal cord by directly targeting projecting cell types without the use of pharmaceuticals, that may have off target effects. Here we demonstrate the “proof of concept” activation of inhibitory interneurons by using a transgenic mouse model VGAT-ChR2 (H134R)- eYFP-BAC. In this mouse model, all inhibitory neurons in the spinal cord express Chr2 and when activated, inhibit networks within the spinal cord. This was tested by using the monosynaptic reflex (H-Reflex) as a measure of spinal cord excitability. We compared the H-reflex to the M-wave ratio following electrical stimulation of the sciatic nerve (Figure 2.12C). I found that the micro-LED implantation was indeed able to activate these inhibitory interneurons of the spinal cord, therefore reducing the H-reflex to M-wave ratio (Figure 2.12D). Though this is a proof of concept experiment, it is an exciting approach to studying spinal circuits that I believe will be invaluable in future research within the spinal cord field.

Furthermore, I am currently in progress in the development of an in cage wireless monitoring system. One issue that many researchers face is the unquantifiable and unknown amount of locomotor activity when not in testing conditions. Animals such as rats and mice, spend most of their time in the home cage when not in testing conditions. This presents a unique issue as most

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researchers do not know how much activity occurs outside of the testing paradigms. We have created an in cage wireless locomotor monitoring system that uses wheel counting to quantify this previously unknown variable. This technology consists of a 3d printed wheel base with proprietary electronics mounted in the base. This monitors ambient temperature within the cage, amount of distance travelled on the wheel and most importantly allows for many animals to be housed in the same cage at one time. Many studies involving SCI and other neuropathologies single house animals to reduce fighting, decrease suture removal and ease of monitoring for abnormal behaviours. With our system, we aim to reduce the stress, induced by single housing animals while actively monitoring locomotor activity outside of the common testing paradigms. This will provide researchers with a useful piece of data that will extrapolate past the common measurement paradigms of traditional testing. It is my hope that this technology will be a commonly used item across all labs studying mice.

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https://s100.copyright.com/AppDispatchServlet 1/1 9/4/2018 Frontiers | Dopamine: a parallel pathway for the modulation of spinal locomotor networks | Frontiers in Neural Circuits

Keywords: dopamine, monoamines, central pattern generator, locomotion, spinal cord

Citation: Sharples SA, Koblinger K, Humphreys JM and Whelan PJ (2014) Dopamine: a parallel pathway for the modulation of spinal locomotor networks. Front. Neural Circuits 8:55. doi: 10.3389/fncir.2014.00055

Received: 18 March 2014; Accepted: 11 May 2014;

Published online: 16 June 2014.

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My name is Kyle Mayr and I am currently completing my Masters degree in Neuroscience. I am looking to use a figure from the article cited as:

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My name is Kyle Mayr and I am currently comple ng my Masters degree in Neuroscience at the University of Calgary. My supervisor is Dr. Patrick Whelan, the author of the ar cle:

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