Principal Component Analysis and the Cumulative Gait Index: Translational Tools to Assess Gait Impairments in Rats with Olivo

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10-6-2015 “Principal Component Analysis and the Cumulative Gait Index: Translational Tools to Assess Gait Impairments in Rats with Olivocerebellar Ataxia” Chase Lambert University of South Florida, [email protected]

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“Principal Component Analysis and the Cumulative Gait Index: Translational Tools to Assess

Gait Impairments in Rats with Olivocerebellar Ataxia”

Chase S. Lambert

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Pharmacology and Physiology Morsani College of Medicine University of South Florida

Major Professor: Lynn Wecker, Ph.D. Rex Philpot, Ph.D. Seok Hun Kim, P.T., Ph.D. Paula Bickford, Ph.D. Craig Doupnik, Ph.D. Theresa Zesiewicz, M.D.

Date of Approval: September 2nd, 2015

Keywords: DigiGait analysis, olivocerebellar lesions, nicotinic acetylcholine receptor

Acknowledgements

The research required to produce this body of work would not have been possible if not for the constant support and encouragement of my family, friends, and research team members.

Thank you to my wife, Randi, for her continual support and understanding throughout the trials and tribulations of this dissertation research. Thank you for always listening and empathizing with my struggles, both internal and external, and for selflessly devoting yourself to supporting our family as a worker, mother, and wife. To my parents, thank you for your hard work to provide an environment whereby I grew up with so many opportunities for success. Thank you to my grandparents, and my brother, Cash, for their support and encouragement to complete this degree. To my daughter, Izzi: completing this dissertation while you were coming into the world was one of the hardest things I have ever done. Fortunately, the timing of your birth and early development with the completion of this degree aligned better than I could have ever planned, and now my reward for finishing this dissertation is that I get to enjoy time together with you. I hope that my hard work will pay off for you and your future siblings, and provide you and our family with opportunities and experiences that would not have been possible without these efforts.

To my lab members: Dr. Wecker, thank you for providing support, expertise, and guidance throughout my degree. The body of research that we have worked together to produce is a testament to our effectiveness as a team. Thank you for the mentoring advice and opportunities to develop extracurricular skills in leadership and at Woods Hole so that I can become the most successful version of myself. To Rex, your expertise in statistics and problem- solving, coupled with your deadpan humor, was crucial in the completion of this research. Thank you for your encouragement and for always proposing a solution to the many problems, even if

ii it wasn’t the right one. To Melanie, our “lab mother”, and my friend: you are the foundation of the lab, what kept us all afloat and sane throughout the years. Thank you for taking me under your wing early in my degree and mentoring me on how to become an effective young researcher.

Thank you for always listening, supporting, and looking out for me, and for always having my best interests at heart. Your organizational skills are exemplary, and you’re the best at getting rodents to do almost anything on a moving apparatus, too! Thank you to Bethany, Brenda, and

Kristy, for their expertise and efforts to help produce these results. Thank you to Dr. Eric

Bennett for always having the students’ best interests at heart. Finally, thank you to Dr. Karen

Liller for allowing me the opportunity to participate in the Leadership Institute, and for providing another intellectual outlet whereby I could grow as a professional while I was undertaking this dissertation research.

iii

Table of Contents

List of Tables ...... iii

List of Figures ...... iv

List of Abbreviations ...... v

Abstract...... vi

Chapter One: Introduction: Modulation of the cholinergic system for the treatment of neurodegenerative and motor disorders ...... 1 1.1. Lack of therapies for patients with ataxia ...... 2 1.2. Neuronal nicotinic acetylcholine receptors ...... 4 1.3. Neuroprotective effects of nicotine and other nicotinic agonists ...... 5 1.4. Use of nicotinic agonists for the treatment of motor disorders ...... 7 1.5. Role of the cholinergic system in gait dysfunction ...... 8 1.6. Clinical assessment of ataxia: use of a principal component analysis ...... 9 1.7. Summary ...... 10 1.8. References ...... 12

Chapter Two: Gait analysis and the cumulative gait index (CGI): translational tools to assess impairments exhibited by rats with olivocerebellar ataxia ...... 24 2.1. Abstract ...... 25 2.2. Introduction ...... 26 2.3. Materials and Methods ...... 28 2.3.1. Animals and lesions ...... 28 2.3.2. Apparatus and behavioral paradigm ...... 28 2.3.3. Data capture and analysis ...... 29 2.4. Results ...... 32 2.4.1. Ability to walk on the treadmill ...... 32 2.4.2. Effects of treadmill speed on gait parameters ...... 33 2.4.3. Effects of 3-AP on gait parameters ...... 34 2.4.4. PCA and the cumulative gait index (CGI) ...... 35 2.4.5. Effects of 3-AP on parameter relationships ...... 36 2.5. Discussion ...... 37 2.5.1. 3-AP induced gait alterations and the CGI ...... 37 2.5.2. Comparison to assessments in animal models of ataxia ...... 38 2.5.3. Comparison of gait alterations between olivocerebellar lesioned rats and humans with ataxic disorders ...... 39 2.6. Conclusions ...... 41 2.7. References ...... 42 2.8. Figures and Tables ...... 46

Chapter Three: Analysis of gait in rats with olivocerebellar lesions and ability of the nicotinic acetylcholine receptor agonist varenicline to attenuate impairments ...... 52 3.1. Abstract ...... 53 3.2. Introduction ...... 54 3.3. Materials and Methods ...... 55 3.3.1. Animals ...... 55 3.3.2. Apparatus ...... 56 3.3.3. Training and baseline determinations ...... 56 3.3.4. Lesion induction and varenicline treatment...... 57 3.3.5. Gait assessment and statistics ...... 58 3.4. Results ...... 59 3.4.1. Gait in non-lesioned animals ...... 59 3.4.2. Gait in lesioned animals ...... 60 3.4.3. Effects of varenicline on gait in lesioned animals ...... 63 3.5. Discussion ...... 64 3.6. Acknowledgements ...... 67 3.7. References ...... 68 3.8. Figures and Tables ...... 72

Chapter Four: Mecamylamine attenuates gait impairments to a similar extent as varenicline in rats with olivocerebellar lesions ...... 81 4.1. Abstract ...... 82 4.2. Introduction ...... 83 4.3. Materials and Methods ...... 84 4.3.1. Animals ...... 84 4.3.2. Apparatus ...... 84 4.3.3. Animal training and baseline assessment ...... 85 4.3.4. Lesion induction and drug treatment ...... 85 4.3.5. Assessment of gait, calculation of PCA, and statistics ...... 86 4.4. Results ...... 87 4.4.1. Effects of olivocerebellar lesions on gait ...... 87 4.4.2. Effects of saline or drug administration upon gait impairments ...... 88 4.5. Discussion ...... 89 4.6. References ...... 92 4.7. Figures and Tables ...... 95

Chapter Five: Discussion ...... 98 5.1. Translational and comprehensive approach to asses gait impairments in animals with olivocerebellar lesions ...... 99 5.2. Varenicline at a high dose is effective in improving gait impairments ...... 101 5.3. Mecamylamine can similarly attenuate gait impairments ...... 103 5.4. Implication and Limitations ...... 106 5.4.1. Methodological limitations: animals and assessment paradigm ...... 107 5.4.2. Pharmacological methodological limitations ...... 110 5.5. References ...... 112

Appendix A: Additional Figures ...... 121

Appendix B: Copyright Permissions ...... 128

About the Author ...... End Page

List of Tables

Table 2.1. Principal Component Analysis at Baseline...... 50

Table 2.2. Effects of 3-AP on Relationships Among Gait Parameters ...... 51

Table 3.1. Principal Component Analysis at Baseline...... 78

Table 3.2. Principal Component Analysis in Impaired Animals (iPCA) ...... 79

Table 3.3. Effects of Varenicline on iCGI Principal Component Deviations ...... 80

Table 4.1. Principal Component Analysis at Baseline...... 97

List of Figures

Figure 2.1. Experimental Paradigm ...... 46

Figure 2.2. Representation of Gait Parameters ...... 47

Figure 2.3. Effects of Olivocerebellar Lesions on Treadmill Performance ...... 48

Figure 2.6. Resultant PCA Factors and the Cumulative Gait Index ...... 49

Figure 3.1. Experimental Paradigm ...... 72

Figure 3.2. Gait Parameters and the CGI in Control Rats ...... 73

Figure 3.3. Gait Parameters and the CGI in Lesioned Rats ...... 74

Figure 3.4. Effects of 3-AP on CGI Principal Component Deviations ...... 75

Figure 3.5. Effects of Varenicline on the iCGI ...... 76

Figure 3.6. Effects of Saline or Varenicline on Absolute Gait Measures ...... 77

Figure 4.1. Timeline of Experimental Paradigm ...... 95

Figure 4.2. Effects of 3-AP administration Across Time on CGI Principal Components ...... 96

Appendix A: Figure 2.4. Stability of Gait Measures and Effects of Treadmill Speed ...... 121

Appendix A: Figure 2.5. Effects of 3-AP on Gait Parameters...... 123

Appendix A: Figure 4.3. Effects of Saline or Drug Administration on Absolute Gait Measures at the Final Assessment ...... 125

vii

List of Abbreviations

3-AP ...... 3-acetylpyridine

CGI ...... Cumulative Gait Index iCGI ...... impaired Cumulative Gait index

PCA ...... Principal Component Analysis iPCA ...... impaired Principal Component Analysis

CNS ...... Central Nervous System

viii

Abstract

Numerous studies suggest that modulation of the cholinergic system through the use of nicotinic agonists can improve motor function in humans or animals with motor disorders.

Specifically, although there are no approved therapeutics for patients with ataxia, the nicotinic receptor agonist varenicline has demonstrated efficacy to improve coordination and gait in several groups of patients with different subtypes of ataxia. Importantly, the mechanism underlying the varenicline’s mechanism of action to improve motor function remains to be elucidated. Thus, the purpose of these experiments was to first quantify gait impairments in rats with olivocerebellar ataxia utilizing an objective treadmill-based system to investigate temporospatial aspects of animals’ gait. These results were used to calculate an index that characterizes deviations from ‘normal’ gait, as similarly employed in clinical studies. The translational validity of this method of gait assessment was investigated by comparing gait impairments between these animals and those reported for humans with ataxia. It was next investigated whether varenicline could attenuate any gait impairments and thus improve motor functioning in these animals, as suggested by clinical findings. Finally, varenicline’s mechanism of action was investigated by attempting to block its effects by pretreating animals with the nicotinic antagonist mecamylamine. Thus, these studies demonstrate the involvement of nicotinic acetylcholine receptors in the mechanism of varenicline’s effects to improve motor functioning. Moreover, these results provide translational methods by which the efficacy of other, more selective nicotinic agonists to improve motor functioning can be tested preclinically prior to their use in humans with ataxia.

Chapter One:

Introduction

Modulation of the cholinergic system for the treatment of neurodegenerative and motor

disorders

1.1. Lack of therapies for patients with ataxia

In conjunction with other motor areas in the brain, one of the main roles of the cerebellum is the regulation of balance and motor coordination during locomotion. Thus, one of the most characteristic signs of cerebellar damage is ataxia (Morton and Bastian, 2004), derived from Latin meaning “disorderly”. Ataxia can be acquired from chemical insults including chronic ethanol administration (Hansen and Pulst, 2013), or Δ9-tetrahydrocannabinol (Smith and Dar,

2007), or following a stroke or trauma to the cerebellum. Furthermore, some subtypes of ataxia can be inherited, consisting of autosomal dominant (e.g. the spinocerebellar ataxias), autosomal recessive (e.g. Friedreich’s ataxia) (Schöls et al., 2004), X-linked (e.g. Fragile X-associated tremor ataxia syndrome), or the result of point mutations (Perlman, 2000).

Unfortunately, there is no effective pharmacological treatment for ataxia, partly due to the heterogeneity of the ataxia population. As is the case with other progressive neurodegenerative disorders, treatment options are limited to managing symptoms of the disorder, reducing side effects as a consequence of medications, and maintaining patient quality-of-life. Physical therapy exercises limited to single joints consisting of nonfatiguing slow repetitive rhythmic movements may improve motor functioning in patients with ataxia (Bastian,

1997); however, physical therapy alone will not cure the disorder and may not slow its progression, especially in patients with degenerative subtypes of ataxia that become progressively less mobile over time. Numerous studies have been undertaken in an attempt to find therapeutics for patients with ataxia of varying subtypes, including: the antiviral and antiparkinsonian drug amantadine (Peterson et al., 1988; Botez et al., 1996); serotonergic compounds including L-5-hydroxytryptophan (Trouillas et al., 1988; 1995) and buspirone

(Trouillas et al., 1997b); the carbonic anhydrase inhibitor acetazolamine (Jen et al., 1998;

Lubbers et al., 1995); and the anti-epileptic drug carbamazepine (Sechi et al., 1989).

Unfortunately, results of these studies have been mixed, with some studies demonstrating that these drugs could reduce the symptoms of ataxia and others suggesting no beneficial effect

(reviewed in Perlman, 2000). Furthermore, while these drugs may provide some benefit for treating specific symptoms of ataxia including tremor, vertigo, and nystagmus, their off-target effects can manifest numerous side effects that have hampered their use to treat patients with ataxia.

However, it has been documented that modulation of the cholinergic system may be useful for treating motor dysfunction in patients with or animal models of motor disorders, including ataxia. For example, preclinical evidence indicates that nicotine and other nicotinic agonists can reduce L-DOPA-induced dyskinesias in several animal models of Parkinson’s disease (Quik et al., 2014). Nicotine has also been shown to reduce the occurrence and severity of motor tics when given concurrently with haloperidol in patients with Tourette’s syndrome

(Silver et al., 2001). Furthermore, it has been documented that patients with cerebellar and

Friedreich’s ataxia have cholinergic deficiencies (Barbeau, 1978; Manyam et al., 1990; Hirano et al., 2008). Although several studies have demonstrated that treatment with the cholinesterase inhibitor physostigmine or the acetylcholine precursor choline may have some benefit for certain patients with ataxia, these treatments have not been effective in double-blind clinical trials

(Lawrence et al., 1980; Wessel et al., 1997), perhaps due to the heterogeneity of the ataxia population. Recently, several clinical reports and one double-blind clinical trial have documented that varenicline (Chantix™), a neuronal nicotinic acetylcholine receptor (nAChR) agonist currently prescribed for smoking cessation, improved symptoms in patients with varying subtypes of ataxia (Zesiewicz and Sullivan, 2008; Zesiewicz et al., 2009a; Zesiewicz 2009b;

Zesiewicz et al., 2012). The underlying mechanism mediating these improvements in motor function in these patients is as yet unknown; however, it is likely that it involves modulation of the cholinergic system through nicotinic receptor signaling.

1.2. Neuronal nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels found throughout the central nervous system. Neuronal nAChRs can be comprised of heteromeric combinations of α (α2-10) and β (β2-4) subtypes (e.g. α4β2, α3β4) as well as homomeric conformations (e.g. α7 ) (Millar and Gotti, 2009). Neuronal nAChRs, especially those of the α7 subtype, are highly permeable to calcium (McGehee and Role, 1995), and can desensitize following chronic exposure to nicotine and nicotinic agonists. In the rat, nAChR expression is diffuse throughout the brain, with the α4β2 subtype being the most widely and abundantly expressed (Wada et al., 1989). The expression of α7 receptors is more limited to the cerebrum, hypothalamus, hippocampus, and some brain stem nuclei (Séguéla et al., 1993). In addition to being expressed throughout the brain, other neuronal nicotinic receptor subtypes can be found in the autonomic ganglia (e.g. α3β4) (Wang et al., 2002). Conversely, non-neuronal nicotinic receptors are found within the neuromuscular junction and can be comprised of γ, ε, or

δ subtypes in combination with the α1 and β1 subtype (Millar and Gotti, 2009).

In response to binding of an agonist, neuronal nAChRs undergo a conformational change that opens the receptor pore to allow the passage of ions. Depending upon the concentration and duration of available agonists, the receptor can desensitize and become transiently inactive, or it can close, rendering the receptor functionally inactive. However, this description is overly simplistic, in that rather than always proceeding through a linear progression of these distinct conformations, nicotinic receptors can cycle through these states

(Katz and Thesleff, 1957) as a result of the concentration and duration of agonist present

(Changeux et al., 1984). Furthermore, evidence suggests that nicotinic receptors can exist in at least two distinct desensitized states in which receptors exhibit an intermediate and high-affinity for ligands (Feltz and Trautmann, 1982). Importantly, since nAChRs exist as numerous subtypes, they differ greatly in their pharmacological and physiological responses including cation permeability, rate of desensitization, and affinity for ligands.

Since many neuronal nAChRs are located presynaptically, it has been hypothesized that their primary role in the CNS is to modulate neurotransmitter release from neurons (Wonnacott,

1997, Vizi and Lendvai, 1999, Salminen et al., 2004). Indeed, when activated, presynaptic nAChRs can facilitate the release of neurotransmitters including dopamine, norepinephrine, serotonin, acetylcholine, γ-aminobutyric acid (GABA), and glutamate (Shimohama, 2009). This can occur through nAChR activation from binding of a ligand, resulting in Ca2+ influx through the receptor, by the activation of additional voltage-gated Ca2+ channels as a result of the proximal influx of Ca2+ , resulting in a depolarization, or by increasing the amount of intracellular Ca2+ through the recruitment of Ca2+ from intracellular stores (Mudo et al., 2007).

Activation of nicotinic receptors by nicotine and the corresponding Ca2+ influx has been documented to induce several downstream second messenger cascades, including protein kinase C (PKC) (Fenster et al., 1999), phospholipase C (PLC) (Pandey, 1996), Ca2+-dependent

CaM-kinase II (Damaj, 2000), and MAPK/Erk 1/2 activity (Cox et al., 1996). Also, activation of

CaM-kinase II or MAPK cascades, as well as increases in intracellular Ca2+ or cAMP levels can lead to cAMP response element-binding protein (CREB) phosphorylation and activation (Chang and Berg, 2001), which has been implicated in the transcription of a number of genes involved in learning, memory, long-term potentiation (LTP), and synaptic plasticity (Sweatt, 2001; Barco and Marie, 2011). Since nAChR activation can affect gene regulation through the activation of

Ca2+-mediated second messenger cascades, nicotinic receptors can also modulate synaptic plasticity and neuronal survival (Belluardo et al., 2000), and thus, activation of nicotinic receptors has been documented to have anti-apoptotic effects as a result of increased expression of various pro-survival factors (reviewed in Schaal and Chellappan, 2014).

1.3. Neuroprotective effects of nicotine and other nicotinic agonists

Nonselective activation of nicotinic receptors through the use of nicotine has been documented to have neuroprotective effects in diverse experimental situations. For example,

5 nicotine can protect against NMDA-mediated excitotoxicity (Kaneko et al., 1997; Semba et al.,

1996), and can protect dopaminergic neurons from degeneration when both 1-Methyl-4-phenyl-

1,2,3,6- tetrahydropyridine (MPTP) and methamphetamine are used to induce neurotoxicity and model Parkinson’s disease in animals (Janson et al., 1988; Maggio et al., 1998). Furthermore, aged mice lacking β2 nAChRs, which bind nicotine with high affinity, exhibit increased neurodegeneration and memory deficits (Zoli et al., 1999), and patients with Parkinson’s and

Alzheimer’s disease similarly exhibit declines in high-affinity nAChR expression that may partly underlie the cognitive and motor deficits exhibited in these patients (Nordberg et al., 1992; Perry et al., 1995). Cholinesterase inhibitors (e.g. donepezil and rivastigmine) currently comprise first- line therapies to alleviate cognitive impairments such as dementia in patients with Alzheimer’s and Parkinson’s disease (reviewed in: Pagano et al., 2015; Wang et al., 2015; Tan et al., 2014).

However, off-target effects of these drugs can result in unwanted side effects, and thus, the efficacy of more selective nicotinic agonists to improve cognitive and motor impairments is being investigated as another viable treatment option (Potter et al., 1999; Quik et al., 2015).

Specifically, the neuroprotective effects following nicotine administration appear to be selectively mediated through either the α4β2 or α7 subtype. For example, α4β2 activation appears to be neuroprotective in striatal and cortical neurons, as α4β2 knockout-mice do not exhibit similar neuroprotective effects following nicotine administration compared to wild-type animals

(Ryan et al., 2001; Laudenbach et al., 2002) and α7 activation is neuroprotective in hippocampal neuron cultures, an effect that can be blocked by the α7-selective antagonists methyllycaconitine or α-bungarotoxin (Dajas-Bailador et al., 2000). These findings have led to the development of more selective nicotinic agonists at α4β2 (e.g. ABT-418; ABT-089) or α7 nAChRs (e.g. GTS-21;

ABT-107) that have also been documented to be neuroprotective (Donnelly-Roberts, et al.,

1996; Sullivan et al., 1997; Nanri et al., 1997; Bordia et al., 2015). Thus, since many patients with a movement disorder also exhibit some degree of neurodegeneration (e.g. Parkinson’s disease, Friedrich’s ataxia, Huntington’s disease, and multiple system atrophy) the use of

6 selective nicotinic agonists may improve motor function or slow the progression of the disorder, with fewer side effects.

1.4. Use of nicotinic agonists for the treatment of motor disorders

In addition to having neuroprotective effects, studies have also documented that the administration of nicotinic agonists can improve motor function in patients with motor disorders.

As discussed in Section 1.1, the nicotinic agonist varenicline has shown some promise for the treatment of several subtypes of ataxia (Zesiewicz and Sullivan, 2008; Zesiewicz et al., 2009a;

Zesiewicz 2009b; Zesiewicz et al., 2012). Varenicline is a partial agonist at α4β2, α3β4, α3β2, and

α6-containing nAChRs and a full agonist at α7 receptors (Mihalak et al., 2006; Rollema et al.,

2007a; 2007b) and thus has activity at the receptor subtypes suspected to mediate the neuroprotective effects observed following treatment with nicotinic agonists (i.e. α4β2 and α7 nAChRs).

Preclinical studies have demonstrated that in animals with ataxia, chronic varenicline administration can improve performance on a rotorod and attenuate balance deficits on a beam, and similar results were found following chronic nicotine administration (Wecker et al., 2013).

Additionally, nicotinic agonists have been shown to: decrease L-DOPA-induced dyskinesias in both rodent and non-human primate models of Parkinson's disease (Quik et al., 2007, 2013;

2013b; Bordia et al 2008; Huang et al., 2011; Zhang et al., 2013), attenuate Δ9 -THC-induced ataxia in mice (Smith and Dar, 2007), attenuate motor abnormalities in a rodent model of tardive dyskinesia (Bordia et al., 2012), and reduce deficits associated with ethanol-induced ataxia in rodents (Taslim et al., 2008). Furthermore, a study from our laboratory demonstrated that chronic administration of nicotine to animals with olivocerebellar lesions could attenuate an impairment in a spatial gait parameter (Wecker et al., 2013).

It is as yet unknown which brain regions are involved in the observed improvements in motor function following the administration of nicotinic agonists. As discussed, patients with

7 some subtypes of cerebellar ataxia exhibit cerebellar degeneration, and thus modulation of nicotinic receptor signaling within the cerebellum may underlie improvements in motor function.

Indeed, direct infusion of the α4β2 selective nicotinic agonist RJR-2403 into the cerebellum of mice has been shown to attenuate ethanol-induced ataxia (Taslim et al., 2008). However, patients with Parkinson’s disease have similar, but not identical, gait abnormalities as patients with cerebellar ataxia (Ebersbach et al., 1999), suggesting the involvement of other brain regions contributing to the gait dysfunction observed in patients with movement disorders.

1.5. Role of the cholinergic system in gait dysfunction

In conjunction with the cerebellum and other motor regions, control of gait and posture has been shown to be mediated by cholinergic neurons within the pedunculopontine nucleus

(PPN) (Karachi et al., 2010). Furthermore, the loss of these cholinergic neurons has been correlated with the clinical severity of Parkinson’s disease (Rinne et al., 2008). Several clinical reports have documented that patients with lesions within the mesencephalic locomotor region, which contains the PPN, exhibit ataxia yet lack cerebellar lesions or other typical features of

Parkinsonian gait (Hathout and Bhidayasiri, 2005; Bhidayasiri et al., 2003; Masdeu et al., 1994).

Thus, evidence suggests that gait dysfunction in patients with movement disorders may not be attributed solely to cerebellar dysfunction. Since the PPN connects to numerous other brain regions involved in motor control and coordination including the cerebellum, motor cortex, and basal ganglia (Aravamuthan et al., 2007), it is plausible that the cholinergic neurons within the

PPN may mediate the motor improvements observed following the administration of nicotinic agonists. In addition, these findings highlight the importance of clinical assessments of motor function to further elucidate similarities as well as differences among the brain regions affected and symptoms manifest in patients with movement disorders.

1.6. Clinical assessment of ataxia: use of a principal component analysis

For patients with ataxia, clinical assessments of motor deficits has historically been quantified using several scales including the Scale for the Assessment of Ataxia (SARA),

International Cooperative Ataxia Rating Scale (ICARS), the Ataxia Functional Composite Scale

(AFCS), and the Berg Balance Scale (BBS) (Trouillas et al., 1997a; Schmitz-Hubsch et al.,

2006a, 2006b; Berg et al., 1989). However, several of these scales can be subjective

(Subramony et al., 2012), especially in patients with degenerative cerebellar disorders (Schmitz-

Hubsch et al., 2006b; Schoch et al., 2007). Furthermore, depending on their subtype of ataxia, these patients can vary in their symptoms and motor deficits, underscoring the inherent challenges in using these scales to quantify and compare deficits between patients, as well as in testing the effectiveness of therapies to improve these deficits.

Due to the limitations of these scales, alternative quantitative tests have been employed to more objectively and reliably measure deficits in patients with ataxia. For example, temporospatial aspects of patients’ gait can be obtained using the GAITRite Walkway System

(CIR Systems, Inc., PA, US), which can serve as a tool for monitoring disease progression and evaluating treatment effectiveness in patients (Hass et al., 2012). However, although gait analysis represents an important translatable assessment between preclinical and clinical studies, the inherent variability among patients with ataxia as well as the speed at which they ambulate can confound data interpretation among a population of patients. To address these concerns and control for these confounds, several clinical studies have used a principal component analysis (PCA) to calculate a gait index quantifying deviations from normal gait

(Wren et al., 2007; Schwartz and Rozumalski, 2008; Gouelle et al., 2013; Schutte et al., 2000).

Furthermore, although clinical studies have documented numerous gait alterations in patients with ataxia, it can be difficult to conceptualize the net result of changes in multiple gait parameters upon the overall gait cycle. This limitation is especially important due to the

9 interdependency of individual parameters upon the maintenance of the gait cycle, as alterations in several gait parameters may be due to a deficit in one key gait parameter.

The use of a PCA can address these concerns as well, since by definition a PCA reduces redundancy in the dataset by identifying correlations between dependent variables and consolidating the useful information into several principal components that categorize the dataset (reviewed in Abdi and Williams, 2010). The first principal component extracted from this analysis explains the largest amount of variance within the data, and each consecutive component extracted explains less of the total variance remaining in the dataset. This information can then be used to weight the influence of each principal component upon the dataset to produce a gait index that conceptualizes the influence of small changes in individual parameters. While the method of developing a gait index from a PCA has not been used to assess gait in the ataxia population, it has been documented that ataxic patients exhibit large increases in gait variability from normal as a result of their symptoms, which is exacerbated at speeds slower or faster than a “comfortable” walking speed (Wuehr et al., 2013; Schniepp et al.,

2012). Thus, it is plausible that a gait index measuring deviations from normal gait may be useful in comparing results between animals and humans with ataxia.

1.7. Summary

In sum, numerous studies demonstrate the efficacy of nicotinic agonists for the treatment of neurodegenerative or motor disorders in animals, underscoring their potential benefit for the treatment of similar disorders in humans. As the mechanism underlying the effects of nicotinic agonists remains to be elucidated, the purpose of these experiments is to first investigate gait impairments in animals with ataxia using the DigiGait™ treadmill system.

Ataxia was induced by the systemic administration of 3-acetylpyridine (3-AP), a competitive antagonist for Vitamin B3. Pyridines such as nicotinamide are important metabolites in the conversion of NAD+ to NADP+ for ATP synthesis within the Electron Transport Chain, and

3-AP irreversibly blocks this pathway, leading to cell death (Desclin and Escubi, 1974). While it is unknown why inferior olivary neurons in the rat are particularly susceptible to the neurotoxic effect of 3-AP, the administration of a large dose of nicotinamide 3.5 hours afterwards quenches

3-AP toxicity, resulting in a selective lesion of the inferior olive (Seoane et al., 2005).

As the goal of these studies is to provide translational results that may be of benefit to human studies, the translational potential of this method of gait assessment will be investigated by comparing gait impairments in rats with olivocerebellar lesions to those reported in humans with ataxia. These studies will also demonstrate whether the chronic administration of varenicline can attenuate any gait impairment manifest as a consequence of olivocerebellar lesions as well as whether varenicline’s effect could be blocked by the nicotinic antagonist mecamylamine, thereby further elucidating the mechanism by which varenicline can attenuate gait impairments in animals with olivocerebellar lesions.

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Chapter Two:

Gait analysis and the cumulative gait index (CGI): Translational tools to assess

impairments exhibited by rats with olivocerebellar ataxia

Note to reader:

These results have been previously published (Lambert et al., 2014) and are presented with permission of the publisher. C.S. Lambert contributed to the execution and analysis of these studies as well as their preparation for publication. R.M. Philpot and L. Wecker contributed to the design, analysis, and preparation of this study for publication. B.E. Johns, M.E. Engberg,

and S.H. Kim contributed to data analysis and manuscript editing.

C.S. Lambert, R.M. Philpot, M.E. Engberg, B.E. Johns, S. H. Kim, L. Wecker

2.1 Abstract

Deviations from ‘normal’ locomotion exhibited by humans and laboratory animals may be determined using automated systems that capture both temporal and spatial gait parameters.

Although many measures generated by these systems are unrelated and independent, some may be related and dependent, representing redundant assessments of function. To investigate this possibility, a treadmill-based system was used to capture gait parameters from normal and ataxic rats, and a multivariate analysis was conducted to determine deviations from normal.

Rats were trained on the treadmill at two speeds, and gait parameters were generated prior to and following lesions of the olivocerebellar pathway. Control (non-lesioned) animals exhibited stable hindlimb gait parameters across assessments at each speed. Lesioned animals exhibited alterations in multiple hindlimb gait parameters, characterized by significant increases in stride frequency, braking duration, stance width, step angle, and paw angle and decreases in stride, stance, swing and propulsion durations, stride length and paw area. A principal component analysis of initial hindlimb measures indicated three uncorrelated factors mediating performance, termed Rhythmicity, Thrust and Contact. Deviation in the performance of each animal from the group mean was determined for each factor and values summed to yield the cumulative gait index (CGI), a single value reflecting variation within the group. The CGI for lesioned animals increased 2.3-fold relative to unlesioned animals. This study characterizes gait alterations in laboratory rats rendered ataxic by destruction of the climbing fiber pathway innervating Purkinje cells and demonstrates that a single index can be used to describe overall gait impairments.

Keywords

Ataxia, treadmill gait analysis, olivocerebellar-lesioned rats, DigiGait™ Imaging System

Abbreviations

CGI: Cumulative Gait index; PCA: Principal Component Analysis

2.2. Introduction

Gait analysis has been used for more than 30 years to assess the effects of physical and chemical perturbations on walking and running in laboratory animals (de Medinaceli et al., 1982;

Mullenix et al., 1975). Initial assessments were conducted using the ‘painted footprint’ procedure, and typically provided measures of stride length and stance width. This led to the development of a basic apparatus that extended measures to include stride frequency and velocity (Clarke et al., 1986). Since that time, several systems have been developed that provide information at different walking speeds, critical for understanding both coordination and balance, as well as for assessing the efficacy of possible therapeutic regimens for movement disorders (Tang and Su, 2013).

Results from studies to date characterizing gait alterations in ataxic laboratory animals are difficult to reconcile because of two confounds, viz., the effect of speed on gait parameters and the interdependency of gait parameters. For example, although increased hindpaw stance width has been shown to be manifest by two unrelated transgenic ataxic mouse models (Duvick et al., 2010; Maity et al., 2012), this alteration was not present in other studies using a ‘Lurcher- like’ mouse or following cerebellar lesions (Stroobants et al., 2013; Cops et al., 2013). A careful analysis of these studies indicates that for the former, animals were tested at a treadmill speed of 35 cm/s, whereas for the latter, animals were run at 22 and 15 cm/s, respectively. The idea that walking speed is a prime consideration determining gait parameters is underscored by evidence that gait alterations in Lurcher mice, who walk considerably slower than their wild-type counterparts (<15 cm/s compared with >25 cm/s), were obviated when walking speed was taken into account (Cendelin et al., 2010).

In addition to speed, the interdependency of gait parameters must also be considered.

Although laboratory animal studies have not addressed this issue, clinical studies have indicated that many gait parameters are highly related and interdependent, and may represent similar aspects of the same general function (Goulle et al., 2013). To isolate and identify

26 combinations of gait parameters that are strongly associated with the variance in the data, but are uncorrelated to one another, a principal component analysis (PCA) may be used. Although the mathematics of this process are complex (Abdi and Williams, 2010; Jolliffe, 2002; Ringer,

2008), the purpose of a PCA is to reduce the dimensionality of a data set consisting of several interrelated variables, while retaining the variation present in the data (Jolliffe, 2002; Ringer,

2008). Thus, redundant aspects of the original data set are consolidated into a smaller number of principal components. Clinicians have used PCA to analyze gait and have demonstrated that

16 independent gait parameters could be used to describe gait patterns in humans, and based on these values, a ‘normalcy index’ could be calculated that reflected gait patterns that deviated from normal (Schutte et al., 2000), a concept that has been validated (Wren et al., 2007) and extended (Schwartz and Rozumalski, 2008) in the clinical literature. A similar strategy has not been applied to laboratory animal studies, an approach that would be highly beneficial for translational investigations.

Therefore, the goals of this study were to: characterize gait abnormalities in olivocerebellar-lesioned rats; use a PCA to identify independent aspects of gait performance; calculate a single quantifiable index of gait; and determine how this index is altered following destruction of the olivocerebellar pathway. To accomplish this, the DigiGaitTM Imaging System

(Mouse Specifics, Boston, MA) was used to determine temporal and spatial gait parameters in normal rats and rats rendered ataxic following olivocerebellar lesions (Wecker et al., 2013).

Although this system has been used to assess gait parameters in rats following spinal cord injury (Ek et al., 2010; Springer et al., 2010; Redondo-Castro et al., 2013), peripheral nerve injury or inflammation (Piesla et al., 2009; Rittenhouse et al., 2011), and damage to the brain

(Russell et al., 2011; Rane and King, 2011), this is the first study to describe a single index reflecting gait pattern impairment in laboratory rats rendered ataxic by destruction of the climbing fiber pathway innervating Purkinje cells.

2.3. Materials and Methods

2.3.1. Animals and lesions

Male Sprague-Dawley rats (225–250 g and 52–60 days old, Harlan Industries,

Indianapolis, IN) were acclimated to the vivarium for 1 week and housed two per cage in a temperature- and humidity-controlled vivarium on a 12:12 h (7 am:7 pm) light-dark cycle; experiments were conducted between 10 am and 2 pm to minimize variability in behavior. The care and use of animals were approved in accordance with guidelines set by the University of

South Florida Institutional Animal Care and Use Committee and the National Institutes of Health

Guide for the Care and Use of Laboratory Animals.

Lesions of the olivocerebellar pathway were induced by the administration of 3- acetylpyridine (3-AP; 70 mg/kg, i.p.) followed by an injection of nicotinamide (300 mg/kg, i.p.)

3.5 h later; chemicals were obtained from Sigma-Aldrich Co. (St. Louis, MO). This injection paradigm has been demonstrated to localize the resulting neurotoxicity primarily to the inferior olive, with weak effects on other brain regions involved in motor control (Seoane et al., 2005;

Llinas et al., 1975; Sotelo et al., 1975). In addition, studies in our laboratory have demonstrated that this injection paradigm leads to the loss of 85% of inferior olivary neurons in rats, and results in impaired performance on the rotorod, balance beam, and in the open field (Wecker et al., 2013).

2.3.2. Apparatus and behavioral paradigm

Prior to initial recordings, rats were trained on the DigiGaitTM treadmill for 5 consecutive days. Preliminary studies indicated that a speed of 25 cm/s represented a ‘comfortable’ walking speed for rats, in general agreement with prior reports (Gillis and Biewener, 2001; Laughlin and

Armstrong, 1982). Further, initial studies indicated that at a lower speed of 15 cm/s, rats did not perform adequately for gait analysis, whereas at a more challenging speed of 35 cm/s, representing a fast walk, performance was consistent; speeds faster than 35 cm/s may have

28 been too challenging for animals with olivocerebellar lesions to walk consistently for analysis purposes (unpublished observations). Each training day consisted of a single 2 min trial at each speed, with trials separated by 1–2 h, and with the lower speed always run first. The treadmill belt and enclosure were wiped with 70% ethanol between each trial.

For training, each animal was secured inside the apparatus and the treadmill was started immediately at 25 cm/s. For training at 35 cm/s, the treadmill belt was started at 25 cm/s, and increased to 35 cm/s at a rate of 1 cm/s. A majority of animals exhibited rhythmic gait, and quickly learned to walk comfortably on the treadmill, while some animals did not walk adequately, and exhibited avoidance behaviors, such as turning or rearing. When this occurred, these behaviors were corrected by ‘nudging’ the animals using the plastic bumper at either end of the apparatus, changing the direction of the treadmill, or slowing the treadmill speed followed by gradually increasing it as the animal learned the task.

Initial behavioral assessments were conducted 2 days following completion of training.

Rats were placed in the apparatus and a 2 min trial at both speeds was recorded. Animals who did not complete a minimum of six uninterrupted strides at either 25 or 35 cm/s were retested at that speed. At 2 days following initial recordings, animals meeting criteria were assigned randomly to either unlesioned (control) or lesioned groups (Fig. 2.1), with a balanced distribution of ages within each group. The unlesioned group was used to ascertain the stability of gait parameters over time, thereby detecting any alterations that may have occurred as a consequence of acclimation or habituation to the apparatus. Animals in the lesioned group received injections of 3-AP/nicotinamide. All animals were reassessed 7 days later for 2 min at both speeds.

2.3.3. Data capture and analysis

DigiGaitTM Imaging software (version 12.2) was used to generate temporal and spatial gait parameters. The videos generated by the system were analyzed by a rater blinded to the

29 group assignment of each animal. Sequences for analysis were chosen by the blinded rater based on: the positioning of the animal relative to the boundaries of the apparatus to ensure that the gait of the animal was unimpeded; and the ability of the rater to clearly discern all 4 paws.

The number of sequences containing ≥ 6 but ≤ 11 strides within a 2 min trial was counted, i.e., animals completing 12 consecutive strides completed two sequences. Approximately 5% of all animals (three animals) walked throughout the entire 2 min trial; the remaining animals walked sporadically throughout the trial, typically completing between 10 and 20 six-stride sequences within each trial. Strides were not counted when animals were in contact with the plastic bumpers, when animals were hopping, or when the speed of the treadmill was increased from

25 to 35 cm/s. Data were analyzed using a one-way analysis of variance (ANOVA), with

Dunnett’s test post hoc to compare final and initial assessments.

Although the DigiGaitTM system generated more than 40 indices of gait dynamics and posture, the measures analyzed included stride duration, stride frequency, stance duration, swing duration, braking duration, propulsion duration, step angle, stride length, stance width, paw angle, and paw area (Fig. 2.2). In addition, the software calculates the variability associated with each spatial parameter, which was also included for analysis; percentages and ratio calculations were not included for analysis. A representative sequence was chosen for analysis,

6–8 strides within this sequence were analyzed, and an average value generated for each parameter. Data indicated that the lesion affected gait parameters in both forelimbs and hindlimbs, albeit the magnitude of change for most parameters was greater in the latter, in agreement with our prior study (Wecker et al., 2013). In addition, for control rats, not all forelimb gait parameters were stable across measurements, likely reflecting the dual functionality of the forelimbs, which are involved not only in ambulation, but also in tactile sampling and gaining proprioceptive information (Clarke, 1995); thus, only values generated from hindlimbs were reported. Further, left and right hindlimb parameters did not differ significantly (p > 0.05 as per

30 paired t-tests) at each speed or assessment timepoint, indicating that gait was symmetrical, and thus, measures from right and left hindlimbs were pooled

Data obtained from controls or animals subsequently lesioned with 3-AP were analyzed by a 2 × 2 repeated measures ANOVA with treadmill speed (25 and 35 cm/s) and assessment

(initial and final) as variables. Subsequently, independent t-tests were used to investigate differences between treadmill speeds, and paired samples t-tests were used to determine differences between timepoints.

A PCA was performed to determine whether the individual components of gait were interrelated and whether redundant aspects of the original data set could be consolidated into principal components. This analysis provided and isolated combinations of the original measures that were uncorrelated, representing distinct components of the data set. Varimax rotation was used to derive orthogonal factor scores and the minimum Eigenvalue for extraction was set at 1. Factor loadings were analyzed using data from both treadmill speeds, and items that met a minimum loading of ≥ ±0.5 were considered relevant. The cumulative gait index (CGI) was determined as described (Schutte et al., 2000) with minor modification. Briefly, the mean

(μ) and standard deviation (σ) of each measure, at each speed, were determined using the initial values from all animals in the study. A Z-transformation (X - μ/σ) was applied to the data from each animal at both speeds and assessment timepoints. To generate the individual factor values, the associated factor loadings (α) derived from the PCA were used to weight the Z- transformed data and these values were summed and the absolute value taken (|Σαz|). The resulting values were averaged across speeds to generate a factor value at each assessment timepoint. These factor values were summed to yield the CGI, representing the total deviation from typical gait for each animal. Individual factors and the CGI were analyzed by a mixed factor

ANOVA with assessment as a repeated measure; Tukey’s test was used post hoc to ascertain group differences.

Correlational analyses (Pearson’s r) at each assessment timepoint and each treadmill speed were used to determine whether there were any relationships among gait parameters, as well as whether treadmill speed and/or 3-AP administration altered these relationships.

Statistical analyses were performed using GraphPad Prism version 6.0 (GraphPad

Software, Inc., San Diego, CA) and SPSS version 21.0 (SPSS Inc., Chicago, IL). A level of p <

0.05 was accepted as evidence of a significant effect.

2.4. Results

2.4.1. Ability to walk on the treadmill

A total of 63 animals were subjected to the 5 day training procedure and underwent initial assessment. At this time, three rats would not walk on the treadmill at either speed, one rat would not walk at 35 cm/s, and three rats failed to complete at least one sequence containing 6–11 strides at both speeds. Thus, seven of the rats were not used for further study because they failed to meet criteria upon initial assessment. The 56 remaining animals were assigned to either the unlesioned (n = 11) or lesioned (n = 45) groups with the latter containing four times as many animals as the former because a wide range of behavioral deficits occurs as a consequence of the lesion (Wecker et al., 2013).

To determine whether the ability of animals to walk on the treadmill was stable over time and whether the lesion affected walking ability, the number of sequences containing 6–11 uninterrupted strides within each trial was counted. The number of sequences upon initial assessment ranged from 1–31 at 25 cm/s and 1–44 at 35 cm/s, with averages of 11 and 18, respectively (Fig. 2.3). For unlesioned animals, performance was stable over time. For lesioned rats, 7/45 and 4/45 animals did not complete a 6–11 stride sequence at treadmill speeds of 25 and 35 cm/s, respectively. The remaining animals (38 at 25 cm/s and 41 at 35 cm/s) exhibited significantly (p < 0.05) fewer sequences at both 25 cm/s [F (3,105) = 3.173] and 35 cm/s [F

(3,108) = 14.19] with 34% and 54% decreases relative to initial values, respectively. Based on

32 studies demonstrating reduced locomotor activity of these animals in the open field (Wecker et al., 2013), it is likely that this decreased number of step sequences reflects a performance deficit and not a lack of compliance. The 11 animals that did not complete a 6–11 stride sequence at either speed (see above) did complete a 3–5 stride sequence. Analysis of gait parameters generated from these truncated sequences indicated that they did not differ significantly (p > 0.05) from those generated by animals who completed 6–11 uninterrupted strides. Thus, data were pooled for analyses of gait parameters.

2.4.2. Effects of treadmill speed on gait parameters

Unlesioned (control) animals were studied to determine the effects of treadmill speed on gait, ascertain whether gait measures were stable over time, and characterize the gait cycle in these rats. Results of a 2 × 2 repeated measures ANOVA revealed a significant (p < 0.05) main effect of speed on five temporal parameters (Fig. 2.4A). Increasing treadmill speed from 25 to

35 cm/s resulted in a 21% increase in stride frequency [F (1, 10) = 109.8], an 18% decrease in stride duration [F (1, 10) = 102.2], a 23% decrease in stance duration [F (1, 10) = 88.8], a 28% decrease (reflecting a 21% decrease for initial and a 34% decrease for final assessments) in braking duration [F (1, 10) = 12.6], and a 21% decrease in propulsion duration [F (1, 10) = 50.8]; there was no difference between swing duration at 25 cm/s and 35 cm/s (data not shown).

Although each of these parameters was altered by treadmill speed, the relative proportion of time spent in each phase of the gait cycle was constant (Fig. 2.4). Additionally, a 2 × 2 repeated measures ANOVA indicated a significant (p < 0.05) main effect of assessment timepoint (initial vs final) at 35 cm/s for stride frequency [F (1, 10) = 5.15] and braking duration [F (1, 10) = 8.00], with a 4% decrease for the former and a 22% decrease for the latter.

The spatial parameters significantly (p < 0.05) affected by speed (Fig. 2.4B) were stride length, which increased by 16% [F (1, 10) = 76.3], stance width, which decreased by 9% [F

(1,10) = 9.07), and step angle, which decreased by 22% [F (1, 10) = 8.20]. Although paw angle

33 decreased by 15%, this was not significant; paw area was unaltered. There was no main effect of speed on the calculated variability for stride length, stance width, step angle, paw angle, or paw area. In addition, there was a significant (p < 0.05) main effect of assessment timepoint on paw angle variability at both speeds [F (1, 10) = 9.81]. At 25 cm/s, paw angle variability increased 55% from 5.3 ± 0.3 to 8.2 ± 0.9 [t (10) = 3.20] and at 35 cm/s, paw angle variability increased 53% from 5.9 ± 0.5 to 9.0 ± 1.1 [t (10) = 2.63].

2.4.3. Effects of 3-AP on gait parameters

The effects of olivocerebellar lesions induced by the administration of 3-AP on gait are shown in Fig. 2.5. Results of a 2 × 2 (speed × assessment) repeated measures ANOVA indicated that the effects of 3-AP were independent of treadmill speed, i.e., there was no interaction. The following significant (p < 0.05) temporal alterations were apparent across speeds: an 9% increase in stride frequency [F (1,44) = 32.2]; an 8% decrease in stride duration

[F (1,44) = 29.4]; a 10% decrease in stance duration [F (1,44) = 38.6]; a 27% increase in braking duration [F (1,44) = 33.6]; and a 17% decrease in propulsion duration [F (1,44) = 60.5].

In addition, the lesion led to a 6% decrease in swing duration [F (1,44) = 4.11] from 138 to 131 ms and from 135 to 126 ms at 25 cm/s and 35 cm/s, respectively. Thus, the relative proportion of the gait cycle in the braking phase increased from an average of 11% to 16%, while that in the propulsion phase decreased from an average of 59% to 53%, indicating that impaired animals required greater time to achieve maximal paw contact during braking.

The spatial measures significantly (p < 0.05) altered by the lesion included: an 8% decrease in stride length [F (1,44) = 33.8]; a 25% increase in stance width [F (1,44) = 81.6]; a

32% increase in step angle [F (1,44) = 28.0]; a 233% increase in paw angle [F (1,44) = 162.6]; and a 25% decrease in paw area [F (1,44) = 176.4]. In addition, paw angle variability increased significantly [F (1, 44) = 159.3, p < 0.05] at both speeds. At 25 cm/s, paw angle variability increased by 160% from 5.8 ± 0.3 to 15.1 ± 0.9 [t (44) = 9.62] and at 35 cm/s paw angle

34 variability increased by 171% from 5.5 ± 0.3 to 14.9 ± 0.9 [t (44) = 9.97]. Paw area variability also increased significantly [F (1, 44) = 9.43, p < 0.05] by 26% at 25 cm/s from 0.35 ± 0.03 to

0.44 ± 0.03 [t (44) = 2.67] and by 18% at 35 cm/s from 0.34 ± 0.02 to 0.40 ± 0.02 [t (44) = 2.12]; stance width and stride length variability were unaffected. Thus, as a result of the lesion, animals had shorter strides, established a wider base of support, had a large and highly variable increase in lateral paw splay, and exhibited less stride-to-stride consistency in the orientation of, and contact made by, the hindpaws.

2.4.4. PCA and the cumulative gait index (CGI)

To produce a single measure of gait impairment similar to the indices used for clinical populations (Gouelle et al., 2013; Schutte et al., 2000; Schwartz and Rozumalski, 2008), the six temporal and five spatial gait parameters analyzed were subjected to a PCA to obviate redundant aspects of the original data set and ascertain whether the information could be consolidated into a smaller number of principal components. This analysis indicated that three factors accounted for 72% of the total variance in the data, explaining more variability in the data than any single gait parameter (see Table 2.1).

The first factor (e = 4.254), which was termed Rhythmicity, encompassed the temporal parameters stride duration, stride frequency, stance duration and propulsion duration, but did not include braking or swing durations, both of which were more associated with the second factor. This indicates that stride duration, stride frequency, stance duration and propulsion duration are interdependent components of gait cycle timing, whereas braking and swing duration are somewhat independent of overall stride time. Further, the valences of the loading values indicate that Rhythmicity increases as stride, stance and propulsion durations increase and stride frequency decreases.

The second factor (e = 2.406), which was termed Thrust, included braking and swing durations, as well as stride length, stance width and step angle; although paw angle was

35 positively associated with this factor, it had a greater association with the third factor. Thus,

Thrust involves regulating trajectory and momentum through adjustments in the advancement and placement of the hindlimbs. The loading valences for Thrust parameters indicate that this factor increases as braking duration, stance width, and step angle increase and swing duration and stride length decrease. The third factor (e = 1.254), termed Contact, contained paw angle and paw area, and loading valences indicated that Contact increases with increasing paw angle and decreasing paw area.

To determine whether the variability within the population was affected by the lesion, the absolute deviation from the mean for each factor for each animal was determined, and values summed to yield the CGI (Fig. 2.6). A mixed factor ANOVA indicated that the CGI for unlesioned animals did not change significantly over time, whereas the CGI for lesioned animals increased

2.7-fold (p < 0.05) relative to the initial assessment, and 2.3-fold (p < 0.05) relative to the final assessment for unlesioned animals [F (1,54) = 18.7]. Further, this difference reflected an increased absolute deviation for all three factors and differed significantly (p < 0.05) from final assessments for unlesioned rats [Rhythmicity: F (1,54) = 6.57; Thrust: F (1,54) = 19.5; Contact:

F (1,54) = 9.47]; the deviations of the three factors did not change over time for unlesioned animals.

2.4.5. Effects of 3-AP on parameter relationships

To determine whether the lesion affected relationships among gait parameters, correlations were calculated at each speed prior to and following the administration of 3-AP. The lesion did not change relationships involving swing and braking durations, stance width or step angle (data not shown). The only relationships significantly (p < 0.05) altered were those involving paw angle and paw area, i.e., components of Contact. Following the administration of

3-AP, paw angle correlated significantly (p < 0.05) with stance duration, stride duration, stride length and stride frequency at both speeds, correlations that were not present during the initial

36 assessment (Table 2.2). Further, there was a negative correlation between paw angle and propulsion duration for the initial assessment that was not affected by 3-AP. In contrast, paw area correlated significantly (p < 0.05) with stance duration, stride duration, stride length and stride frequency only at 25 cm/s; the correlations at 35 cm/s did not reach significance. Further,

3-AP altered the relationship (p < 0.05) between paw area and propulsion duration at both speeds. These results indicate that the lesion not only affects individual parameters of gait, but in several cases, alters the relationships among these parameters.

2.5. Discussion

2.5.1. 3-AP induced gait alterations and the CGI

The present study characterized alterations in temporal and spatial gait parameters at two speeds in a rodent model of olivocerebellar ataxia, determined how the relationships between gait parameters were altered in ataxic animals, and quantified the severity of impairment across multiple measures of gait using a single index. Results indicated that increasing treadmill speed resulted in significant changes in all temporal gait parameters, as well as the spatial parameters of stride length, stance width and step angle. For unlesioned animals, performance was stable across the 9 days of assessment at each treadmill speed.

Further, when treadmill speed increased, animals compensated by shortening the braking and propulsion durations of the stance phase, and by increasing stride length, alterations that contributed to all other gait parameter changes. Lesions of the inferior olive produced significant changes in multiple gait parameters that were apparent at both treadmill speeds. These alterations included increases in stride frequency, braking duration, stance width, step angle, and paw angle and decreases in stride, stance, propulsion and swing durations, as well as stride length and paw area.

A PCA, performed on gait measures from normal, healthy (unimpaired) animals to capture independent aspects of gait performance on the treadmill, identified three factors

37 termed Rhythmicity, Thrust, and Contact. Rhythmicity encompassed temporal parameters

(stride frequency, propulsion duration, and stance and stride durations), Thrust was composed of both temporal (swing and braking durations) and spatial (stride length, stance width, and step angle) parameters, and Contact reflected paw angle and area. For each of these factors, lesioned animals exhibited a 2–3 fold increased deviation from typical gait. Further, the CGI, which represents the total deviation from typical gait for all gait measures as determined by summation of the deviations for the three independent factors increased 2.7-fold following the lesion. This representation of the overall magnitude of gait impairment has been recognized by clinicians and used to assess gait impairments in humans (Schutte et al., 2000; Wren et al.,

2007; Schwartz and Rozumalski, 2008). Using a PCA to identify related measures and consolidate deviations from typical gait minimizes redundancy in the data and provides a single measure that is highly sensitive to changes in performance. Further, the CGI may have greater translational value than any single gait measure when assessing impairment and subsequent treatment effects because individual gait measures may or may not represent the same deficit across species and different species may express the same underlying deficit on different measures. This is the first study to apply this approach to laboratory animals.

2.5.2. Comparison to assessments in animal models of ataxia

Although numerous studies have investigated gait alterations in mouse models of ataxia, the present study represents the first to characterize both temporal and spatial gait alterations in a rat following olivocerebellar lesions. Prior studies in our laboratory have shown that following the administration of 3-AP, rats exhibit shorter latency to fall from the rotorod, increased time required to cross a stationary beam, decreased velocity and distance moved in the open field, and increased stance width and decreased stride length for both hindlimbs and forelimbs during unforced locomotion (Wecker et al., 2013). The present study extends these findings to include forced locomotion, and detected multiple alterations in gait measures with hindlimbs exhibiting

38 larger alterations than forelimbs for most parameters. Because rats produce greater ground reaction force in the hindlimbs relative to the forelimbs (Clarke et al., 1997; Muir and Whishaw,

2000), with hindlimbs providing most of the early braking and supportive forces during locomotion (Clarke, 1995), gait alterations in hindlimbs are similar to changes observed in humans (Stolze et al., 2002). The finding that olivocerebellar lesions affected gait parameters during both forced and unforced or ‘natural’ movement agrees with results from studies investigating two unrelated transgenic ataxic mouse models that demonstrated gait alterations for both hindlimbs and forelimbs at speeds of either 15 cm/s or 35 cm/s, respectively (Maity et al., 2012; Cops et al., 2013). In addition, the 32% increase in step angle following 3-AP administration (Figure 5B) is consistent with ethanol-induced ataxia in rats (Hannigan and Riley,

1988). [It is critical to note that version 12.5 (and all previous versions) of the DigiGaitTM software calculates the complementary angle of the step angle as defined by Hannigan and

Riley (1988). Thus, the output from the program was subtracted from 90◦ to reflect the angle commonly referred to in the literature as step angle.]

Although studies have reported that stride length variability is increased in mice with cerebellar lesions (Stroobants et al., 2013), we did not detect any alterations in this measure in olivocerebellar lesioned rats. This difference may be attributed to methodology because the data for the former was based on a single trial following a 30 s habituation to the apparatus

(Stroobants et al., 2013), whereas data collected in the present study represented a sample of the animal’s best performance, and was dependent upon animals engaging in sustained walking for at least six successive strides.

2.5.3. Comparison of gait alterations between olivocerebellar lesioned rats and humans with ataxic disorders

Several gait alterations exhibited by rats following destruction of the climbing fiber pathway by 3-AP are very similar to those observed in patients with ataxic disorders. Studies

39 have demonstrated that, compared to healthy control subjects, ataxic patients exhibit shorter stride lengths (Palliyath et al., 1998; Morton and Bastian, 2003; Mari et al., 2012; Serrao et al.,

2013) and larger stance widths (Stolze et al., 2002; Mari et al., 2012; Serrao et al., 2013;

Hudson and Krebs, 2000; Ilg et al., 2007) during walking or turning, results similar to those in rats with olivocerebellar lesions. Further, the delayed heel-off time (i.e., approximately the beginning point of propulsion) and decreased duration from heel-off to toe-off observed in ataxic patients (Palliyath et al., 1998) are highly similar to the increased braking duration and decreased propulsion duration, respectively, exhibited by rats following the administration of 3-

AP. In addition, analysis of gait in patients with cerebellar disease using a treadmill system detected an increased foot angle splay (Stolze et al., 2002), similar to that in lesioned rats.

Increased variability of gait parameters is often described in patients with cerebellar lesions (Palliyath et al., 1998; Ilg et al., 2007; Schniepp et al., 2012; Wuehr et al., 2013).

Although stride length and stance width variability exhibited by the rats were unaffected by the lesion, paw angle variability was three times greater in lesioned than in unlesioned animals. This finding, as well as the increase in paw area variability, indicates that paw placement was less consistent following the lesion. Interestingly, stance width variability was unaltered by the lesion, similar to that reported for patients with cerebellar ataxias (Palliyath et al., 1998; Wuehr et al.,

2013), perhaps reflecting a consistent maximal increase in base support as a compensatory mechanism. In contrast, stride length variability was unaltered in lesioned animals, whereas it is increased in patients with cerebellar ataxia (Palliyath et al., 1998). The difference between our findings and those in the clinical literature may reflect differences in performance speed. Studies indicate that gait variability in ataxic patients is strongly dependent on walking speed (Schniepp et al., 2012; Wuehr et al., 2013), with minimal variability at the preferred speed of each individual (Wuehr et al., 2013). The present study was conducted during forced locomotion, which may obviate variability relative to spontaneous ambulation. Nevertheless, the overall

40 similarities in gait alterations in the present study and those from the human literature underscore the translational nature of this study and this method of gait assessment.

2.6. Conclusions

Rats rendered ataxic following olivocerebellar lesions exhibit alterations in several hindpaw gait parameters that are similar to gait alterations observed in the clinical population of ataxic patients. The use of multivariate analysis to yield a single measure of gait impairment, the

CGI, provides a sensitive and reliable indicator applicable to studies in laboratory animals. This method of assessment should be more useful than quantifying individual gait parameters when engaging in translational studies to develop treatment interventions.

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2.8. Figures and Tables

Fig. 2.1. Experimental paradigm. Rats were trained to walk on the DigiGaitTM system at 25 and 35 cm/s for 5 consecutive days. Initial behavioral assessments were conducted 2 days following training by recording baseline performance for 2 min at both speeds. At 2 days following initial recordings, animals were assigned randomly to either unlesioned (control) or lesioned groups, and the latter received injections of 3-AP/nicotinamide. All animals were reassessed 7 days later for 2 min at both speeds.

Fig. 2.2. Representation of gait parameters. Diagrammatic representation of temporal and spatial gait parameters analyzed.

Fig. 2.3. Effects of olivocerebellar lesions on treadmill performance. The number of uninterrupted sequences containing 6–11 strides within a 2 min segment at treadmill speeds of [A] 25 cm/s and [B] 35 cm/s were determined at baseline (initial assessment) and 9 days later (final assessment) for both unlesioned and 3-AP lesioned animals. Bars represent mean values + s.e.m.; the number of animals per group is shown in parentheses. The asterisks denote significant (p < 0.05) differences.

Fig. 2.6. Resultant PCA factors and the cumulative gait index. The three factors generated by the PCA (Rhythmicity, Thrust and Contact) and the CGI were analyzed across timepoints. Bars represent mean values + s.e.m. with an n = 56 for initial assessments representing the total population, n = 11 for the unlesioned group and n = 45 for the lesioned group. Data were compared using a one-way ANOVA. The asterisks denote significant (p < 0.05) differences from all other groups as determined by Tukey’s test.

Table 2.1. Principal Component Analysis at Baseline. Gait parameters from the initial assessment for all animals (n = 56) were used for a principal component analysis. Factor loadings > 0.5 are bolded, representing primary components of the factor.

Table 2.2. Effects of 3-AP on Relationships Among Gait parameters. Correlation coefficients (Pearson’s r) denoting the relationship among gait parameters at eachs peed prior to (initial) and following (final) the administration of 3-AP; significant (p < 0.05) correlations are bolded.

Chapter Three:

Analysis of gait in rats with olivocerebellar lesions and ability of the nicotinic

acetylcholine receptor agonist varenicline to attenuate impairments

Note to reader:

These results have been previously published (Lambert et al., 2015) and are presented with

permission of the publisher. C.S. Lambert contributed to the execution and analysis of these

studies as well as their preparation for publication. R.M. Philpot and L. Wecker contributed to the design, analysis, and preparation of this study for publication. B.E. Johns and M.E. Engberg,

contributed to data analysis and manuscript editing.

C.S. Lambert, R.M. Philpot, M.E. Engberg, B.E. Johns, L. Wecker

3.1. Abstract

Studies have demonstrated that administration of the neuronal nicotinic receptor agonist varenicline to rats with olivocerebellar lesions attenuates balance deficits on a rotorod and balance beam, but the effects of this drug on gait deficits have not been investigated. To accomplish this, male Sprague–Dawley rats were trained to walk on a motorized treadmill at 25 and 35 cm/s and baseline performance determined; both temporal and spatial gait parameters were analyzed. A principal component analysis (PCA) was used to identify the key components of gait, and the cumulative gait index (CGI) was calculated, representing deviations from prototypical gait patterns. Subsequently, animals either remained as non-lesioned controls or received injections of 3-acetylpyridine (3-AP)/nicotinamide to destroy the climbing fibers innervating Purkinje cells. The gait of the non-lesioned group was assessed weekly to monitor changes in the normal population, while the gait of the lesioned group was assessed 1 week following 3-AP administration, and weekly following the daily administration of saline or varenicline (0.3, 1.0, or 3.0 mg free base/kg) for 2 weeks. Non-lesioned animals exhibited a 60–

70% increased CGI over time due to increases in temporal gait measures, whereas lesioned animals exhibited a nearly 3-fold increased CGI as a consequence of increases in spatial measures. Following 2 weeks of treatment with the highest dose of varenicline (3.0 mg free base/kg), the swing duration of lesioned animals normalized, and stride duration, stride length and step angle in this population did not differ from the non-lesioned population. Thus, varenicline enabled animals to compensate for their impairments and rectify the timing of the gait cycle.

Keywords

Ataxia, varenicline, gait, principal component analysis, cumulative gait index

Abbreviations

3-AP: 3-acetylpyridine; CGI: Cumulative Gait Index; PCA: Principal Component Analysis

3.2. Introduction

Ataxia represents an array of symptoms including uncoordinated voluntary movements and deficits in balance, locomotion and gait. These characteristics typically manifest as a result of cerebellar dysfunction that can be inherited, idiopathic or result from a stroke, traumatic brain injury, lesion, or chemical insult to the brain (Perlman, 2000). Thus, the symptoms and deficits manifest can vary considerably among patients. Presently, there are no approved treatments for ataxia, although evidence suggests that modulation of the nicotinic cholinergic system within the central nervous system may provide therapeutic benefit.

Both preclinical and clinical studies have shown that nicotinic cholinergic agonists are effective in reducing motor abnormalities associated with several disorders. Nicotine has been shown to potentiate haloperidol-induced hypokinesia and catalepsy in rodent models of

Tourette’s syndrome (Elazar and Paz, 1999; Dursun et al., 1994), and decreases motor tics in patients with this disorder (Dursun et al., 1994; Silver et al., 1996; Dursun and Kutcher, 1999).

Several nicotinic agonists have also been shown to decrease L-DOPA-induced dyskinesias in both rodent and non-human primate models of Parkinson’s disease (Quik et al., 2013; Quik et al., 2007; Bordia et al., 2008; Huang et al., 2011; Zhang et al., 2014; Zhang et al., 2013), motor abnormalities in a rodent model of tardive dyskinesia (Bordia et al., 2012), and ethanol-induced ataxia in rodents (Taslim et al. 2008). In addition, balance deficits in rats following traumatic brain injury are ameliorated by the administration of the cholinesterase inhibitor physostigmine, which increases extracellular levels of acetylcholine, thereby activating nicotinic receptors

(Holschneider et al., 2011).

Clinical studies have shown that the nicotinic agonist varenicline improves gait and/or balance deficits in patients with spinocerebellar ataxia type 3 (SCA3) and type 14 (SCA14)

(Zesiewicz and Sullivan, 2008), and a recent double-blind, placebo-controlled, randomized study demonstrated that treatment with varenicline for 2 months improved axial symptoms and rapid alternating movements in adult patients with genetically confirmed SCA3 (Zesiewicz et al.,

2012). Preclinical studies from our laboratory have shown that varenicline attenuates balance deficits on the rotorod and balance beam in animals with olivocerebellar lesions (Wecker et al.,

2013), but the effects of varenicline on lesion-induced gait deficits in laboratory animals have not been investigated.

Assessments of gait abnormalities in both ataxic laboratory animals and humans is typically confounded by inherent variability within a given population, as well as walking speed during the assessment (Cendelin et al., 2010; Wuehr et al., 2013). The variability confound has been addressed in the clinical literature by the use of principal component analysis (PCA) and the subsequent determination of a gait index to express gait deviations from a normal population (Schutte et al., 2000; Wren et al., 2007; Schwartz an drozumalski, 2008; Gouelle et al., 2013). A similar approach has been used in our laboratory for preclinical studies in rats following the administration of the neurotoxin 3-acetylpyridine (3-AP), which destroys the olivocerebellar pathway (Lambert et al., 2014). These studies demonstrated that ataxic animals exhibited a nearly 3-fold increase in deviation from normal gait compared to age-matched non- lesioned animals. This finding, in concert with alterations in specific gait variables in ataxic rats that parallel those reported in clinical studies, suggested that this approach may be translatable and used to investigate the potential therapeutic benefits of nicotinic receptor agonists for the treatment of ataxia. Thus, the goals of this study were to: (1) use a PCA to isolate the key components of gait in animals walking on a motorized treadmill; (2) calculate a cumulative gait index (CGI) as a general measure of gait function relative to population norms; and (3) evaluate the dose–response effects of varenicline on 3-AP-induced gait impairments in laboratory rats following olivocerebellar lesions.

3.3. Materials and Methods

3.3.1. Animals

Adult male Sprague–Dawley rats (225–250 g and 59–66 days old; n = 63; Harlan

Industries, Indianapolis, IN) were housed two per cage (with enrichment) in a temperature and humidity-controlled vivarium on a 12:12 h light-dark cycle (6 am:6 pm) with food and water available ad libitum. Animals were acclimated for 1 week prior to study and all behavioral evaluations were conducted between 10 am and 2 pm. The care and use of animals were approved in accordance with guidelines set by the University of South Florida Institutional

Animal Care and Use Committee and the National Institutes of Health Guide for the Care and

Use of Laboratory Animals.

3.3.2. Apparatus

The DigiGait™ Imaging System and software (version 12.2; Mouse Specifics, Boston,

MA) were used to generate temporal and spatial gait parameters as described (Lambert et al.,

2014). Gait parameters, analyzed by a rater blinded to experimental condition, were generated from the hind limbs and included both temporal (swing duration, braking duration, propulsion duration, stance duration, stride duration and stride frequency) and spatial (stride length, stance width, paw angle, step angle, and paw area) measures, all of which are affected by the administration of 3-AP (Lambert et al., 2014).

3.3.3. Training and baseline determinations

Animals were trained to walk on the treadmill at speeds of 25 and 35 cm/s for 2 min at each speed for 5 consecutive days, with the 35 cm/s trial run 1.5 h after the 25 cm/s trial. The treadmill belt and enclosure were wiped with 70% ethanol between trials. At 2–3 days following completion of training, baseline performance was assessed for 2 min at each speed, and gait parameters were generated from a sequence consisting of at least 3 uninterrupted strides per paw, i.e., a total of 6 strides (Lambert et al., 2014). Animals that did not perform this sequence on a given trial were retested once at that speed. Of the 63 animals tested at baseline, 4 failed to meet this criterion and were removed from further study. For each animal, the number of

56 sequences generated within the 2 min trial at each speed was quantified, similar to a prior study

(Lambert et al., 2014).

A PCA was performed using the gait measures collected at baseline (n = 59) using both treadmill speeds. The factor loadings generated from this analysis were used to weight z- transformed gait variable data, which were then summed to produce deviation values for each identified factor. These values were summed to yield the CGI, an index representing overall deviations from prototypical gait patterns (Lambert et al., 2014). CGI values were calculated for each animal at each assessment time, and the group means determined.

3.3.4. Lesion induction and varenicline treatment

Following baseline gait assessments, animals were randomly assigned to either non- lesioned (n = 11) or lesioned (n = 48) groups (Fig. 3.1) The non-lesioned group was included to ascertain whether multiple exposures to the apparatus over time affected gait. The non-lesioned animals were handled in a manner identical to the lesioned animals, but did not receive any injections. Animals in the lesioned group received intraperitoneal (i.p.) injections of 3-AP (70 mg/kg, Sigma–Aldrich Co., St. Louis, MO) followed by nicotinamide (300 mg/kg, i.p.) 3.5 h later to selectively destroy the olivocerebellar pathway (Wecker et al., 2013; Lambert et al., 2014). At

1 week following baseline characterization, animals were reassessed on the treadmill for 2 min at 25 and 35 cm/s. Of the 48 animals who received 3-AP, 3 would not perform on the treadmill; thus, the remaining 45 animals were randomly assigned to 1 of 4 groups and received daily subcutaneous (s.c.) injections of either normal phosphate-buffered saline (PBS; 0.9%; 0.33 ml/kg) or varenicline tartrate (0.3, 1.0 or 3.0 mg free base/kg corresponding to 0.51, 1.71, and

5.13 mg tartrate salt/kg, respectively, from Tocris Bioscience, Bristol, UK or 0.43, 1.43, and 4.29 mg dihydrochloride salt/kg, respectively, kindly provided by Dr. F. Ivy Carroll, Research Triangle

Institute, Research Triangle Park, N.C.) for 2 weeks. The doses of varenicline were selected based on studies demonstrating improvement of the performance of olivocerebellar-lesioned

57 animals on the rotorod and balance beam, while the treatment duration was selected based on evidence that improvement on the rotorod plateaued following daily administration of varenicline for 2 weeks (Wecker et al., 2013). Stock solutions of varenicline were prepared weekly by dissolving the drug in PBS, and adjusting the pH to 7.4 with 1.0 N NaOH; aliquots were prepared, protected from light and stored at 4oC until use.

3.3.5. Gait assessment and statistics

As indicated, gait was reassessed for 2 min at each speed for all animals at 1, 2, and 3 weeks after baseline testing, and animals in the non-lesioned group were evaluated in parallel with lesioned animals (Fig. 3.1). Gait measures across assessment times were compared using analysis of variance (ANOVA) with speed and time as repeated measures, and a subsequent repeated measures ANOVA at each speed was used to isolate simple effects; planned comparisons to detect differences between assessments were conducted using Fisher’s least significant difference (LSD) test. Data were analyzed for violations of sphericity, and in those instances indicating significant violations, a Geisser–Greenhouse correction was utilized to adjust criteria for significance.

For lesioned animals, behavior was always assessed prior to the daily administration of varenicline to ensure that the acute locomotor effects of the drug did not influence performance on the treadmill. Because 4 of the 45 lesioned animals were unable to complete an uninterrupted 6 stride sequence at 25 cm/s, gait parameters for these animals, irrespective of treatment group, were generated from a minimum 3 stride sequence. This approach was validated by our prior study demonstrating that gait parameters generated from the truncated sequence did not differ significantly from those generated from a longer sequence (Lambert et al., 2014).

A second PCA (termed iPCA) was performed using data generated 1 week post 3-AP to identify the principal components of impaired gait in the lesioned population. This analysis was

58 used to calculate the iCGI, an index reflecting gait deviations within the impaired population, and used to ascertain whether varenicline affected prototypical impaired gait. The iCGI values were calculated at 1 week following the administration of 3-AP, and following the administration of saline or varenicline. To control for differences in the degree of impairment in each animal, values from saline- or varenicline-injected animals were expressed relative to their 1 week values. Data were compared using a 2 × 4 repeated-measures ANOVA (assessment × treatment condition) with subsequent planned comparisons using Fisher’s LSD test to determine if varenicline administration altered the gait of lesioned animals. A p-value <0.05 was accepted as criterion for statistical significance for all analyses.

3.4. Results

3.4.1. Gait in non-lesioned animals

Non-lesioned animals completed 11 ± 2.1 and 18 ± 2.9 sequences consisting of 6–11 strides during the 25 and 35 cm/s baseline assessments, respectively, and the number of sequences did not change across weeks. Analysis of gait indicated that several temporal and spatial measures differed significantly (p < 0.05) from baseline at a treadmill speed of 25 cm/s

(Fig. 3.2). Stride, stance, propulsion and swing durations increased 12–17% and stride length increased 12–14% over time, with a plateau apparent at 2 weeks [for stride duration, F(3,28) =

9.84; for stance duration, F(3,28) = 7.70; for propulsion duration, F(3,28) = 9.11; for swing duration, F(3,28) = 7.69; for stride length, F(3,28) = 9.33]. Stance width and step angle decreased over time, with significant (p < 0.05) effects only at 3 weeks [for stance width, F(3,28)

= 3.68; for step angle, F(3,28) = 4.12]; no other gait parameters exhibited time-dependent alterations. At a treadmill speed of 35 cm/s, all gait parameters exhibited changes similar to, but less than those observed at 25 cm/s (data not shown).

A PCA was conducted using baseline gait measures from all animals at both treadmill speeds to reduce the dimensions of the dataset. This analysis identified four principal

59 components that accounted for 82% of the variance in the data (Table 3.1). The first factor or

Rhythm [eigenvalue (e) = 4.9] included the temporal parameters stride duration, stride frequency, stance duration, and propulsion duration as primary measures (absolute loading value >0.5), while the second factor or Thrust (e = 2.1) included braking duration, stride length, stance width, and step angle as primary measures. Although the absolute loading value for paw angle was >0.5 for the Thrust component, the magnitude of this measure was greater for the third factor or Contact (e = 1.14), which also included paw area, identical to prior observations

(Lambert et al., 2014). However, unlike prior observations, the PCA identified a fourth component, termed Gain (e = 1.1), that contained the single measure of swing duration; although step angle had a loading value >0.5 for Gain, the magnitude of this measure was greater for Thrust. Additional analyses performed using the 1, 2 and 3 week gait measures from non-lesioned animals indicated that the factors identified at baseline were unaffected by repeated treadmill exposure. Therefore, these factors were used to calculate a CGI for non- lesioned animals at each assessment time, reflecting alterations at both treadmill speeds. In agreement with a prior study (Lambert et al., 2014), the CGI did not differ significantly between baseline and assessment week 1 (Fig. 3.2). However, at 2 and 3 weeks, the CGI increased significantly [F(3,28) = 4.91, p < 0.05] by 74% and 61%, respectively, indicating an increased deviation from baseline within the non-lesioned population. Further, analysis of each principal component indicated that this divergence within the population could be attributed to a greater than 2-fold increase in Rhythm [F(3,28) = 4.77, p < 0.05]. Although Thrust and Contact increased by 35–57%, and contributed to the overall increase in the CGI, these increases were not significant; Gain did not change over time.

3.4.2. Gait in lesioned animals

The effects of olivocerebellar lesions on gait parameters were assessed in rats at weekly intervals at 1 week after the administration of 3-AP and following injections of saline for 2

60 weeks. One lesioned animal failed to run on the treadmill at 2 and 3 weeks, and thus, was removed from study. The number of 6–11 stride sequences decreased at 1 week following 3-AP administration and remained stable thereafter. At 25 cm/s, the number of sequences at baseline, 1, 2, and 3 weeks after 3-AP administration was 12 ± 1.1, 7.0 ± 0.94, 6.3 ± 0.8, and

7.0 ± 1.1, respectively. At 35 cm/s, the number of sequences at baseline, 1, 2, and 3 weeks after 3-AP administration was 18 ± 1.4, 8.8 ± 1.1, 11 ± 1.5, and 11 ± 2.1, respectively.

Gait parameters that differed significantly (p < 0.05) over time at a treadmill speed of 25 cm/s are shown in Fig. 3.3. At 1 week after the administration of 3-AP, stride, stance and propulsion durations decreased significantly (p < 0.05) by 10%, 14%, and 19%, respectively [for stride duration, F(3,27) = 5.39; for stance duration, F(3,27) = 8.41; for propulsion duration,

F(3,27) = 7.40]. These temporal parameters exhibited recovery over time with stride duration at

3 weeks not significantly different from baseline; stance and propulsion durations at this time were still significantly less (p < 0.05) than baseline. Several spatial parameters were also significantly (p < 0.05) affected at 1 week following the administration of 3-AP with a 10% shorter stride length, a 42% larger stance width, a 34% smaller paw area, and a 3.6-fold increase in paw angle [for stride length, F(3,27) = 5.03; for stance width, F(3,27) = 9.15; for paw area, F(3,27) = 20.2; for paw angle, F(3,27) = 33.3]. By 3 weeks, stride length recovered and values were not significantly different from baseline. Although paw area exhibited some recovery, values at 2 and 3 weeks were significantly (p < 0.05) different from those at both baseline and 1 week following the administration of 3-AP. In contrast, neither stance width nor paw angle exhibited recovery, with values at 2 and 3 weeks that remained significantly (p <

0.05) different from baseline. At a treadmill speed of 35 cm/s, all gait parameters exhibited changes similar to those at 25 cm/s, albeit the magnitude was less, similar to observations from non-lesioned animals (data not shown).

Lesion-induced alterations in gait parameters at both treadmill speeds are reflected in the CGI, shown in the lower right corner of Fig. 3.3. The lesion resulted in a significant (p <

0.05) 2.9-fold increase in the CGI at 1 week [F(3,27) = 21.1], and this increase persisted for the following 2 weeks. Analysis of each principal component (Fig. 3.4) indicated that this increase was attributed to significant (p < 0.05) increases in both Thrust [F(3,27) = 16.3] and Contact

[F(3,27) = 23.7]. Also depicted in the stacked histogram is a comparison of principal components in lesioned versus non-lesioned groups, further illustrating that the increased CGI following the lesion differed from that in non-lesioned rats, which was attributed to increased

Rhythm.

This finding, in concert with results indicating that gait changes in lesioned animals persisted over time, led to a second analysis to determine the principal components of gait in impaired animals, termed the iPCA. This analysis identified factors that accounted for 86% of the total variance in the data (Table 3.2) and indicated that for lesioned animals, iRhythm (e =

4.9) included all the temporal parameters identified in non-lesioned animals with the addition of swing duration; although paw area had a loading value >0.5 for iRhythm, the magnitude of this measure was greater for iContact. iThrust (e = 1.9) included those measures identified in non- lesioned animals with the exception of stance width; the loading value for step angle was larger for iContact. iContact (e = 1.56) was composed of paw angle and area, which were identified in the non-lesioned population as part of this factor, but also included both stance width and step angle. Although iGain (e = 1.04) reflected stride length, which was not identified as part of this factor in the non-lesioned population, the loading value for this measure was greater for iThrust.

Thus, the principal components of gait in the lesioned population reflected slightly different relationships and loading factors for gait measures than in the non-lesioned population. The iPCA was used to calculate the iCGI to evaluate the effects of varenicline on gait in lesioned animals. Two animals had iCGI values that were greater than 2 standard deviations from the mean and thus were excluded, resulting in a mean iCGI for all lesioned animals at 1 week following 3-AP administration of 6.91 ± 0.40 (n = 43).

3.4.3. Effects of varenicline on gait in lesioned animals

The effects of varenicline on the iCGI, expressed relative to values determined 1 week after the administration of 3-AP, are shown in Fig. 3.5 and illustrate that the daily administration of 3.0 mg/kg varenicline increased the iCGI, an effect that was significant [F(3,37) = 2.98, p <

0.05] relative to all other groups following 2 weeks of drug administration (3 week assessment).

Further, at 2 and 3 week assessments, the proportion of animals with increased iCGIs, denoting a greater deviation from the impaired state, was greater for all varenicline-injected groups than for the saline group. For the saline group, 50% of the animals (5/10) exhibited a (+) and 50% exhibited a (−) iCGI change at 2 and 3 weeks following the lesion, whereas in all varenicline- injected groups, greater than 50% (60–90%) of the animals exhibited a (+) change indicating a tendency to change from impaired performance.

Analysis of the factors comprising the iCGI indicated that the increased deviation from impaired gait following varenicline (3.0 mg free base/kg) treatment (Table 3.3) was attributed to a significant (p < 0.05) 3-fold increase in iRhythm [F(3,37) = 3.61] and an almost 2-fold increase in iThrust relative to saline-injected group values. Although the overall ANOVA for the latter was not significant [F(3,37) = 1.62], a planned comparison of saline versus varenicline (3.0 mg free base/kg) indicated a significant (p < 0.05) difference between groups. As a consequence of these findings, individual gait parameters from non-lesioned and lesioned animals following the administration of saline or varenicline (3.0 mg free base/kg) for 2 weeks were compared at 3 weeks. Results are shown in Fig. 3.6. The administration of varenicline normalized swing duration, i.e., swing duration for varenicline-injected animals was significantly (p < 0.05) different than for saline-injected animals, and not significantly different from the non-lesioned group

[F(2,29) = 6.50]. In addition, saline-injected animals exhibited significant (p < 0.05) alterations in stride duration, stride length, and step angle relative to non-lesioned animals, whereas these gait parameters did not differ between varenicline-injected and non-lesioned animals [for stride duration, F(2,29) = 5.79; for stride length, F(2,29) = 5.44; for step angle, F(2,29) = 5.45].

Thus, varenicline ‘tended to normalize’ stride duration, stride length, and step angle.

3.5. Discussion

The goal of this study was to determine the dose–response effects of varenicline on 3-

AP-induced gait impairments in laboratory rats following olivocerebellar lesions using a PCA and consequent calculation of a single index to characterize gait deviations. Results demonstrate that the gait of ataxic animals differs from that of non-lesioned animals, and that the administration of 3.0 mg free base varenicline for 2 weeks leads to gait adaptations of several parameters. Importantly, these adaptations attenuate 3-AP-induced gait impairments at a walking speed of 25 cm/s, suggesting that these animals compensate for their impairment by altering several components of gait.

The present study extends prior findings (Lambert et al., 2014) and demonstrates that weekly experience on a treadmill for up to 3 weeks, albeit limited to 4 min per time, leads animals to acclimate or habituate to the apparatus, resulting in a significant increased deviation of gait relative to baseline determinations. Analysis of the factors that comprised the CGI revealed that the maximal 74% increased deviation over time could be attributed primarily to alterations in Rhythm, which reflected changes in the temporal measures of stride, stance, and propulsion durations. Thus, increased treadmill experience leads to alterations in gait cadence in normal animals over time.

In contrast to the role of temporal measures (Rhythm) underlying the increased CGI in non-lesioned animals over time, analysis of the factors contributing to the increased CGI as a consequence of the lesion indicated a role for Thrust and Contact, reflecting primarily stride length, stance width, step angle, and paw area and angle. Thus, the primary deficit in lesioned animals may be attributed to spatial alterations, and are likely responsible for the inability of these animals to maintain the temporal parameters involved in normal rhythm. Due to the major differences in gait between lesioned and non-lesioned animals, a second PCA was performed

64 on lesioned animals to identify components of the principal factors in the impaired population, and thus serve as a reference for evaluating the effects of varenicline.

Results from the iPCA determined from gait measures in lesioned animals were similar, but not identical to those from the PCA determined from baseline gait measures from non- lesioned animals. The major difference between these analyses was a change in the association of swing duration from Gain for the PCA to iRhythm for the iPCA. In addition, stance width and step angle, which were associated only with Thrust for the PCA, were associated with both iThrust and iContact for the iPCA, with the loadings for iContact greater than those for iThrust. Thus, following 3-AP administration, the timing of the gait cycle (iRhythm) is limited by the duration of the swing phase, and the contribution of stance width and step angle to Thrust is reduced. Importantly, the large increase in association of stance width with iContact likely indicates a postural change to increase the base of support of the animal. Because step angle is directly influenced by stance width, the increased association of step angle with iContact is most likely due to this change in stance width. Thus, the iPCA identified a new basis that best represents the dynamics of gait in the lesioned population relative to the non-lesioned population, and as such, represents a valid reference for evaluating the effects of varenicline.

When the iCGI was calculated from the iPCA and the dose–response effects of varenicline determined, results indicated that the highest dose of drug (3.0 mg free base/kg) administered for 2 weeks led to a nearly 2-fold increase in the iCGI, indicating that gait following the administration of this dose of varenicline was unlike that of the impaired population. Further, this increase was attributed to increases in iRhythm and iThrust. Analysis of individual gait parameters indicated that the increase in iRhythm reflected a drug-induced increase in swing duration and consequent increased stride duration to values that did not differ from the non- lesioned population. Similarly, the increase in iThrust reflected an increase in stride length and consequent decrease in step angle to values that were not significantly different from those in the non-lesioned population. Thus, varenicline enabled animals to compensate for their

65 impairment by normalizing swing duration and stride length, thereby rectifying the timing of the gait cycle.

Although several studies have used a treadmill system to investigate alterations in both temporal and spatial gait parameters in animal models of disease (Berryman et al., 2009; Piesla eta l., 2009; Song et al., 2012; Hansen and Pulst, 2013; Jiang et al., 2015), it can be difficult to conceptualize how alterations in the parameters generated affect overall gait, especially because of differences in magnitude and direction. Using a PCA to identify the key components of gait enables calculation of an index representing gait deviations from a defined population.

Although this method has been used clinically to characterize gait deficits in several clinical conditions (Schutte et al., 2000; Wren et al., 2007; Schwartz and Rozumalski, 2008; Gouelle et al., 2013), it has not been adopted for use in preclinical studies. This approach has tremendous power in that it provides a single value from a large and often redundant dataset that is sensitive to small changes in individual gait measures, and should be utilized because of its translational potential to provide a succinct and reliable indicator of gait deviation.

Results from this preclinical study, as well as clinical evidence (Zesiewicz et al., 2012), clearly indicate that varenicline is efficacious for alleviating both balance and gait deficits in ataxia. It remains to be determined whether these actions: (1) are mediated by the varenicline- induced activation or desensitization of neuronal nicotinic receptors, and which specific receptor subtypes are involved; and (2) occur as a consequence of a direct effect on the cerebellum or other anatomic regions. Indeed, varenicline has been shown to have full and/or partial agonist activity at α4β2, α3β2, α3β4, α6-containing, and homomeric α7 nicotinic receptors (Mihalak et al.,

2006; Rollema et al., 2007a; Rollema et al., 2007b; Chatterjee et al,. 2011), all of which are expressed in the cerebellum (Turner and Kellar, 2005; Turner et al., 2011; Caruncho et al.,

1997). Alternatively, it is possible that the direct effects of varenicline are mediated by other neuroanatomical regions. This idea is supported by studies indicating that the loss of balance resulting from cortical impact injury leads to compensatory changes in brain regions remote to

66 the site of injury, and that administration of the cholinesterase inhibitor physostigmine, which alleviated the impairment, increased the activity of these remote pathways (Holschneider et al.,

2013).

In sum, results indicate that varenicline improves gait in laboratory animals with olivocerebellar lesions, a finding similar to that reported for patients with SCA3 (Zesiewicz et al.,

2012); the cellular mechanisms involved remain to be elucidated. Results also underscore the benefit of using a PCA to consolidate specific gait measures into a limited number of factors to characterize gait. This analysis can then be used to provide a meaningful index that is sensitive to subtle changes in gait function. This approach represents a powerful tool that has been underutilized in both preclinical and clinical studies.

3.6. Acknowledgments

Research reported in this publication was supported by the National Institute of Neurological

Disorders and Stroke of the National Institutes of Health under award number R01NS072114.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank Dr. Brenda Marrero-Rosado and Mrs. Kristy Menning for their critical evaluation of this manuscript, and Dr. F. Ivy Carroll for providing some of the varenicline used in this study.

3.7. References

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3.8. Figures and Tables

Fig. 3.1. Experimental paradigm. Rats were trained to walk on the DigiGait™ system for 5 consecutive days and gait assessments were conducted at baseline and weekly thereafter for up to 3 weeks. Non-lesioned (control) animals were assessed in parallel with lesioned animals, the latter receiving injections of 3-AP following baseline assessment. At 1 week after the lesion and reassessment, rats received daily injections of either saline or varenicline (0.3, 1.0, or 3 mg free base/kg); gait was reassessed following 1 or 2 weeks. A PCA was conducted using gait data generated from all animals at baseline, and results used to calculate a CGI for non- lesioned and lesioned/saline-injected rats. Similarly, an analysis was also performed with gait data from all lesioned animals (iPCA), and results used to calculate the iCGI for lesioned/saline- injected and lesioned/varenicline-injected rats. The CGI or iCGI were calculated using z- transformations of each gait measure and the loadings from the PCA or iPCA, respectively.

Fig. 3.2. Gait parameters and the CGI in control rats. Temporal and spatial gait parameters were determined in rats for 2 min at treadmill speeds of 25 and 35 cm/s at weekly intervals; data shown is from 25 cm/s. Each point is the mean ± S.E.M. of determinations from 11 animals. Data were analyzed by a one-way ANOVA and Fisher’s LSD test. The asterisks denote significant (p < 0.05) differences from baseline (Base).

Fig. 3.3. Gait parameters and the CGI in lesioned rats. Temporal and spatial gait parameters were determined in rats for 2 min at treadmill speeds of 25 and 35 cm/s at baseline (Base) and weekly intervals following the administration of 3-AP; data shown is from 25 cm/s. Each point is the mean ± S.E.M. of determinations from 10 animals. Data were analyzed by a one-way ANOVA and Fisher’s LSD test. The single asterisks denote significant (p < 0.05) differences from baseline; the double asterisks denote significant (p < 0.05) differences from 1 week determinations.

Fig. 3.4. Effects of 3-AP on CGI principal component deviations. Z-transformations of each gait measure and the loadings from the PCA were used to calculate the deviations attributed to each factor. Values for each factor from non-lesioned (NL) and lesioned (Les) animals were calculated following weekly assessments and compared using a 2 × 4 repeated-measures ANOVA (treatment × assessment) and Fisher’s LSD test. Data represent mean ± S.E.M. of determinations from 11 (NL) or 10 (Les) animals per group. The asterisks denote significant (p < 0.05) differences from corresponding group baseline values.

Fig. 3.5. Effects of varenicline on the iCGI. Temporal and spatial gait parameters were determined in rats for 2 min at treadmill speeds of 25 and 35 cm/s following the administration of 3-AP, and a PCA was conducted using gait data generated from all lesioned animals (iPCA), with results used to calculate the iCGI. Following the 1 week gait assessment, animals received daily injections of saline or varenicline (0.3, 1.0 or 3.0 mg free base/kg), and gait reassessed weekly for the following 2 weeks. The mean iCGI of all lesioned animals at 1 week following the administration of 3-AP was 6.91 ± 0.40 (n=43) and did not differ significantly by group assignment. The iCGI values at 2 and 3 weeks after 3-AP were: 7.2 ± 0.47 and 7.7 ± 1.3, respectively, for the saline group; 8.5 ± 1.4 and 8.0 ± 0.8, respectively, for the 0.3 mg/kg varenicline group; 8.2 ± 0.77 and 7.9 ± 1.1, respectively, for the 1.0 mg/kg varenicline group; and 8.8 ± 1.2 and 10.5 ± 1.2, respectively, for the 3.0 mg/kg varenicline group. Data are expressed relative to pretreatment (1 week assessment) values to control of individual differences in impairments. The bars represent group mean values ± S.E.M., and the ratio in each bar is the number of animals exhibiting a (+) change relative to initial lesioned values relative to the total number of animals (n) in each group. Data were analyzed by a 2 × 4 ANOVA (assessment × treatment) and Fisher’s LSD test. The asterisk denotes significant (p < 0.05) differences from saline group values.

Fig. 3.6. Effects of saline or varenicline on absolute gait measures. Temporal and spatial gait parameters were determined in lesioned rats following the daily administration of saline (Sal) or varenicline (Var, 3.0 mg free base/kg) for 2 weeks, and in corresponding non-lesioned (NL) rats. Bars represent the mean ± S.E.M. of determinations from 10 (Sal or Var) or 11 (NL) animals/group. Data were analyzed by ANOVA and Fisher’s LSD test. The single asterisk denotes significant (p < 0.05) differences from NL group values and the double asterisk denotes significant (p < 0.05) differences from Sal group values.

Table 3.1. Principal Component Analysis at Baseline. Note: A principal component analysis was conducted using gait measures from baseline assessments of all animals (n = 59). For each measure, the largest absolute loading value across the 4 factors is bolded, representing primary components of each factor.

Table 3.2. Principal Component Analysis in Impaired Animals (iPCA). Note: A principal component analysis was conducted using gait parameters generated from animals (n = 45) at 1 week after the administration of 3-AP, termed the iPCA. For each variable, the largest absolute loading value across the 4 factors is bolded, representing primary components of each factor.

Table 3.3. Effects of Varenicline on iCGI Principal Component Deviations. Note: Animals received 3-AP followed 1 week alter by daily injections of saline or varenicline for 2 weeks. The z-transformations of each gait measure and the loadings from the iPCA were used to calculate the deviations attributed to each factor. Values for each factor were calculated and compared using ANVOA, and Fisher’s LSD test was used for planned comparisons of saline versus varenicline. Data represent mean ± S.E.M. of determinations from 10 (saline and varenicline, 1.0 and 3.0 mg/kg) or 11(varenicline, 0.3 mg/kg) animals per group. The asterisks denote significant (p < 0.05) differences from corresponding saline group values.

Chapter Four:

Mecamylamine can attenuate gait impairments in rats with olivocerebellar lesions to a

similar extent as varenicline

Note to reader:

C.S. Lambert contributed to the execution and analysis of these studies as well as their preparation for publication. R.M. Philpot and L. Wecker contributed to the design, analysis, and

preparation of this study for publication.

C.S. Lambert, R.M. Philpot, L. Wecker

4.1. Abstract

Balance and gait dysfunction are common deficits manifest in patients with ataxic disorders, and treatment options are currently limited for these patients. Recent studies from our laboratory have demonstrated improvements in balance and gait in animals with olivocerebellar lesions following chronic administration of nicotinic receptor agonist varenicline. As the mechanism underlying this phenomenon is unknown, the objective of this study was to determine if varenicline’s effect could be blocked using the nicotinic antagonist mecamylamine.

Male Sprague-Dawley rats were trained to walk on a motorized treadmill, and temporospatial gait parameters were obtained and subjected to a principal component analysis to identify primary gait components and produce a cumulative gait index (CGI) that characterized typical gait. Animals were then injected with 3-acetylpyridine followed by nicotinamide to lesion the olivocerebellar pathway and produce ataxia. Gait performance was investigated following induction of the lesion and one and two weeks following chronic administration of saline, varenicline, mecamylamine and varenicline, or mecamylamine alone. As a result of the lesion, animals exhibited a 2.4-fold increase in gait deviation. Following two weeks of chronic treatment, varenicline-injected animals exhibited attenuations of gait impairments. Furthermore, animals receiving mecamylamine injections exhibited attenuations of gait impairments in a similar manner as varenicline-injected animals, whereas when mecamylamine and varenicline were used in combination, only the low dose of mecamylamine blocked the effects of varenicline. Thus, evidence suggests that the mechanism underlying varenicline’s ability to attenuate gait impairments is mediated through nicotinic receptor inactivation.

Keywords

Ataxia, gait, principal component analysis, varenicline, mecamylamine

Abbreviations

CGI: Cumulative Gait Index; PCA: Principal Component Analysis

4.2. Introduction

Evidence from preclinical studies demonstrates that modulation of cholinergic signaling can improve motor functioning in animals with motor dysfunction. For example, the administration of the cholinesterase inhibitor physostigmine can ameliorate balance deficits in rats as a result of traumatic brain injury (Holschneider et al., 2011) and nicotine can attenuate ethanol-induced ataxia on the rotorod in mice (Al-Rejaie and Dar, 2006; Dar et al., 1993).

Studies from our laboratory have demonstrated that chronic administration of the nicotinic agonist varenicline to rats with ataxia attenuates balance deficits on a beam, and both nicotine and varenicline can improve performance on a rotorod (Wecker et al., 2013). Additionally, gait impairments as a result of olivocerebellar lesions in rats can be attenuated by chronic administration of nicotine when animals’ gait is assessed by a static gait analysis (Wecker et al.,

2013) as well as by chronic administration of varenicline when rats walk on a motorized treadmill (Lambert et al., 2015). Other studies have demonstrated the ability of chronic administration of nicotine, varenicline, and other nicotinic agonists to reduce L-DOPA-induced dyskinesias in several preclinical models of Parkinson’s disease (Bordia et al., 2010; Zhang et al., 2013; Quik et al., 2013; Zhang et al., 2014a; Zhang et al., 2014b).

Importantly, the underlying mechanism by which nicotinic agonists may improve balance, gait, or motor dysfunction is presently unknown. Results from a prior study demonstrate the effects of chronic nicotine administration to attenuate an impairment in stance width could be blocked by the nonspecific nicotinic antagonist mecamylamine, implicating nicotinic receptor activation as the mechanism (Wecker et al., 2013). However, it has also been shown that chronic administration of nicotinic antagonists such as mecamylamine can similarly improve motor deficits. For example, chronic administration of mecamylamine can reduce the severity of motor tics in patients with Tourette’s syndrome (Silver et al., 2000) and can reduce L-

DOPA-induced dyskinesias to a similar extent as nicotine in rats with 6-hydroxydopaminergic lesions (Bordia et al., 2010). Notably, the results of the latter study suggest that both nicotine

83 and mecamylamine attenuate L-DOPA-induced dyskinesias through a similar mechanism

(Bordia et al., 2010).

The mechanism by which both nicotinic agonists and antagonists can improve motor dysfunction is unclear. Furthermore, varenicline’s mechanism of action to attenuate gait impairments in rats with ataxia is also unknown, and thus, the goal of this study was to further elucidate the mechanism by varenicline can improve motor function by using the nicotinic antagonist mecamylamine. It was investigated whether mecamylamine could block varenicline’s effects, thereby suggesting that this phenomenon is medicated through nicotinic receptor activation.

4.3. Materials and Methods

4.3.1. Animals

Adult male Sprague-Dawley rats (225-250g and 52-59 days old; n =85; Harlan

Industries, Indianapolis, IN) were housed two per cage (with enrichment) in a temperature and humidity-controlled vivarium on a 12:12 hour light-dark cycle (6 AM: 6 PM). Food and water were available ad libitum and animals were acclimated to the vivarium for 1 week prior to the onset of the study. All behavioral assessments were conducted between 10 AM-2 PM. The care and use of animals were approved in accordance with guidelines set by the University of South

Florida Institutional Animal Care and Use Committee and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

4.3.2. Apparatus

The DigiGait™ Imaging System and software (version 12.5; Mouse Specifics, Boston,

MA) were used to assess the gait of animals throughout the experiment. Gait parameter values were generated from animal’s hindlimbs and included the temporal and spatial gait measures

84 affected by 3-AP (Lambert et al., 2014) that were investigated in a prior study (Lambert et al.,

2015).

4.3.3. Animal Training and Baseline Assessment

Animals were trained to walk on the treadmill at 25 and 35 cm/s for two minutes at each speed for five consecutive days, and each animal walked at the faster speed 1.5 hours following the trial at the slower speed. The treadmill belt and enclosure were cleaned with 70% ethanol between each trial. Three days following completion of training, animals’ baseline performance was assessed for 2 minutes at both speeds, and hindpaw gait parameters were generated from a representative successful trial, which consisted of at least 3 uninterrupted strides per paw for a total of 6 strides (Lambert et al., 2014; 2015). A representative sequence generated from each animal within a 2-minute trial that met this criterion was used to generate temporospatial gait measures, similar to prior studies (Lambert et al., 2014; 2015). Animals that did not successfully generate a sequence that met criterion at either speed were reassessed once at that speed. Of the 85 animals tested, 8 failed to meet this criterion and were removed from further study.

4.3.4. Lesion induction and drug treatment

One day following baseline assessment, animals were given intraperitoneal (i.p.) injections of 3-AP (70 mg/kg, Sigma-Aldrich Co., St. Louis, MO) and nicotinamide (300 mg/kg, i.p.) 3.5 hours later to selectively lesion the olivocerebellar pathway (Wecker et al., 2013).

Animals’ performance was reassessed on the treadmill one week later to quantify gait impairments manifest as a result of the lesion. Of the 77 animals that received 3-AP, 9 did not walk on the treadmill one week following 3-AP administration and were removed from further study.

The remaining 68 animals were randomly assigned to one of six treatment groups and received two daily subcutaneous (s.c.) injections for a total of two weeks of either: phosphate-

85 buffered saline (PBS; 0.9%; 0.33 ml/kg) (n=15); PBS followed by 3 mg free base/kg varenicline

(n=17); 0.8 mg free base/kg mecamylamine followed by either saline (n=9) or 3 mg free base/kg varenicline (n=10); or 3.2 mg free base/kg mecamylamine followed by either saline (n=8) or 3 mg free base/kg varenicline (n=9). Injections for each animal were always separated by 30 minutes, regardless of treatment assignment. For all animals, behavior was always assessed prior to daily injections to ensure that the acute locomotor effects of the drug did not influence treadmill performance. Gait was reassessed for all eligible animals 2 and 3 weeks following baseline assessment, or 6 and 13 days following induction of saline or drug treatment (Fig. 4.1).

The dose of 3 mg free base/kg of varenicline tartrate (corresponding to 5.13 mg tartrate salt/kg from Tocris Bioscience, Bristol, UK) was selected based on prior findings that only this dose led to significant attenuations of 3-AP-induced gait impairments (Lambert et al., 2015).

Mecamylamine used was mecamylamine hydrochloride (corresponding to 0.97 or 3.9 mg hydrochloride salt/kg, respectively, from Sigma-Aldrich Co., St. Louis, MO). The low dose of 0.8 mg free base/kg mecamylamine and the time-course were selected based on a prior study from our laboratory demonstrating that a 30-minute pretreatment with 0.82 mg free base/kg mecamylamine blocked nicotine’s effects to attenuate gait impairments (Wecker et al., 2013).

Additionally, the higher dose of 3.2 mg free base/kg mecamylamine was used to investigate dose-related effects at a dose that has been shown to block varenicline’s effects to enhance electrical brain-stimulation reward (Spiller et al., 2009). Stock solutions of the drugs were prepared weekly by dissolving the drug in PBS and adjusting the pH to 7.4 with 1.0N NaOH; aliquots were prepared, protected from light, and stored at 4oC until use.

4.3.5. Assessment of gait, calculation of PCA, and statistics

Although animals were assessed at two treadmill speeds (25 & 35 cm/s), only gait parameters obtained from the slower treadmill speed were used for analysis. This was due to prior findings that there were no significant attenuations of gait impairments when animals

86 walked at the faster speed of 35 cm/s (Lambert et al., 2015). A PCA was performed using the gait parameters collected from animals at baseline (n = 77) using data from 25 cm/s only. The factor loadings obtained from this analysis were used to weight z-transformed data for each gait parameter, and these values were summed to yield the CGI, a cumulative gait index that represented deviations from normal gait (Lambert et al., 2014; 2015). CGI values were determined for each animal at each assessment as described (Lambert et al., 2015), and the group means were calculated for each timepoint. CGI values across assessment timepoints were analyzed by a repeated-measures ANOVA and Fisher’s least significant difference (LSD) test.

At the gait assessments 1 week following the administration of 3-AP and afterwards, gait parameters were generated from a representative walking sequence consisting of a minimum of

3 total uninterrupted strides as several animals were unable to complete a total of 6 strides due to their impairment. Four and six animals did not walk sufficiently for gait analysis on the treadmill at the 2 and 3 week assessment timepoints, respectively, and were excluded from the remainder of the study. Gait measures across assessment timepoints were compared using analysis of variance (ANOVA) with time as a repeated measure. Planned comparisons were used to detect differences between assessment timepoints using Fisher’s LSD. Data were analyzed for violations of sphericity, and a Geisser-Greenhouse correction was used to adjust criteria for significance when significant violations occurred.

Absolute gait measures for each gait parameter at the 3 week timepoint were compared using a 2 x 3 MANOVA [(+/- varenicline) x (no mecamylamine, low dose, or high dose mecamylamine)] to determine whether any dose or combination of the drugs attenuated 3-AP- induced gait impairments. Subsequent planned comparisons between each treatment group and saline-injected animals were conducted using Fisher’s LSD post-hoc. A p-value of < 0.05 was accepted as criterion for statistical significance for all analyses.

4.4. Results

4.4.1. Effects of olivocerebellar lesions on gait

A PCA was conducted using gait data from all animals at baseline at a treadmill speed of

25 cm/s. Results of this analysis were similar to prior findings (Lambert et al., 2015) and revealed three principal components that explained 80% of the variance within the data (Table

4.1) and was comprised of the factors Rhythmicity (e = 5.61), Thrust (e = 1.72), and Contact (e

= 1.47). The lesion resulted in a significant (p < 0.05) 2.4-fold increase in the CGI at the week 1 assessment timepoint that persisted across the remainder of assessments [F (3, 33) = 9.07]

(Fig. 4.2). This increase could be attributed to significant increases in both Rhythmicity [F (3, 33)

= 5.68] and Thrust [F (3, 33) = 8.25], but not Contact.

Gait impairments as a result of 3-AP administration were of similar magnitude to those described (Lambert et al., 2014; 2015), and included significant (p < 0.05) differences between the baseline and 1 week assessment for swing, propulsion, stance, and stride durations, as well as for stride length, paw angle, stance width, step angle, and paw area.

4.4.2. Effects of saline or drug administration upon gait impairments

At the final assessment timepoint and in agreement with prior findings (Lambert et al.,

2014), saline-injected animals exhibited recovery of some temporal and spatial gait impairments, as the absolute gait measures were not significantly different from baseline (data not shown). However, further analyses revealed that saline-injected animals did not recover to the same extent as animals in other treatment groups. Results of the overall MANOVA revealed that there were several significant interactions between the treatment conditions including: stance duration [F (2, 52) = 3.58], stride duration [F (2, 52) = 3.26], stride length [F (2, 52) =

3.27], and step angle [F (2, 52) = 3.26]. The results of subsequent planned comparisons are shown in Fig. 4.3 and illustrate significant (p < 0.05) differences between saline-injected and varenicline-injected groups for stance and stride durations, as well as for stride length. Values

88 for swing duration from varenicline-injected animals approached, but did not meet the criterion for statistical significance (p = 0.08).

Similarly, either dose of mecamylamine alone led to animals exhibiting a significantly (p

< 0.05) greater recovery from impairments for stance and stride durations and stride length when compared to saline-injected animals. Regarding the combinations of drugs, only animals in the 3.2 mg free base/kg mecamylamine and varenicline group exhibited significant (p < 0.05) attenuations of 3-AP-induced gait impairments for stance and stride durations and stride length.

Conversely, animals in the 0.8 mg free base/kg mecamylamine and varenicline group did not exhibit statistically significant attenuations (p > 0.05) when compared to saline-injected animals and thus did not exhibit a similar recovery. Thus, when used in combination, the low dose of mecamylamine blocked the effects of varenicline to attenuate gait impairments, while a relatively high dose of varenicline, mecamylamine alone, and a higher dose of mecamylamine combined with varenicline led to significant attenuations of several 3-AP-induced gait impairments when compared to saline-injected animals.

4.5. Discussion

The goal of this study was to use the nicotinic antagonist mecamylamine to determine if the effect of varenicline to attenuate 3-AP-induced gait impairments was mediated by activation of nicotinic receptors. These results demonstrate that mecamylamine alone was equally as effective as varenicline in attenuating impairments in one spatial and two temporal gait parameters and resulted in normalization of the timing of the gait cycle. Notably, chronic mecamylamine at a low dose antagonized the effects of varenicline to attenuate gait impairments, whereas mecamylamine at a higher dose did not antagonize the effect of varenicline. Indeed, animals injected with the latter combination showed equivalent attenuations of gait impairments as animals that received injections of mecamylamine or varenicline alone.

In agreement with these findings, prior results from our laboratory using a static gait analysis demonstrated that a low dose of mecamylamine (at a comparable dose of 0.82 mg free base/kg) did not attenuate an impairment in the spatial gait parameter stance width (Wecker et al., 2013). Furthermore, a low dose of mecamylamine blocked nicotine’s effects to attenuate an impairment in stance width, implicating nicotinic receptor activation as the mechanism (Wecker et al., 2013). Results from this study, including the finding that a low dose of mecamylamine similarly blocked the effects of varenicline to attenuate an impairment in stride length, extend prior findings by suggesting that nicotinic receptor inactivation is the mechanism underlying varenicline’s effects to attenuate gait impairments.

Mecamylamine has been shown to dose-dependently block varenicline’s effects to enhance brain-stimulated reward mediated through receptor activation (Spiller et al., 2009).

Thus, if receptor activation was the underlying mechanism for varenicline’s effects to attenuate gait impairments, it would be expected that this effect would be dose-dependently antagonized by mecamylamine. Furthermore, the effects of mecamylamine and varenicline in combination were not synergistic, suggesting that their effects to attenuate gait impairments when administered individually are mediated through a similar mechanism. In agreement with these results, both mecamylamine and nicotine have been documented to ameliorate L-DOPA- induced dyskinesias in 6-hydroxydopamine-lesioned rats, but combining the drugs did not result in synergistic effects (Bordia et al., 2010).

Studies have documented that chronic administration of both nicotine and mecamylamine increases nicotinic receptor expression within the plasma membrane (i.e. upregulation) (Marks et al., 1992; Perry et al., 1999; Collins et al., 1994; Pauly et al., 1996).

Thus, it is plausible that the similar mechanism shared by nicotine and mecamylamine underlies nicotinic receptor upregulation following chronic administration of these drugs. Although receptor upregulation was not investigated in this study, this interpretation is supported by findings that chronic administration of varenicline similarly upregulates nicotinic receptors in the

90 brains of rodents (Hussmann et al., 2012; Marks et al., 2015). Increased expression of nicotinic receptors following chronic administration of nicotinic agonists has been shown to increase nicotinic receptor sensitivity as well as increase receptor desensitization in response to the binding of agonists (Henderson and Lester, 2015). Thus, upregulation of nicotinic receptors that are more easily desensitized may further explain the interpretation of Bordia et al., (2010) that the effects of nicotinic receptor agonists and antagonists to reduce L-DOPA-induced dyskinesias is mediated through nicotinic receptor desensitization.

In sum, results from this study and others suggest that to achieve improvements in motor functioning in animals with motor disorders, nicotinic drugs must be administered for a prolonged duration, further supporting the hypothesis of nAChR inactivation as the underlying mechanism. However, further studies confirming nAChR inactivation under these experimental conditions are needed to confirm this interpretation. Furthermore, the specific brain regions and nicotinic receptor subtypes involved in this adaptation of gait following treatment with mecamylamine or varenicline remain to be elucidated, especially to confirm whether nicotinic receptor inactivation results in upregulation of nicotinic receptors that may ultimately underlie the behavioral recovery observed when these drugs are administered chronically.

4.6. References

[1] Al-Rejaie S, Dar MS. Possible role of mouse cerebellar nitric oxide in the behavioral interaction between chronic intracerebellar nicotine and acute ethanol administration: observation of cross-tolerance. Neuroscience. 2006;138:575-85.

[2] Bordia T, Campos C, McIntosh JM, Quik M. Nicotinic receptor-mediated reduction in L-

DOPA-induced dyskinesias may occur via desensitization. J Pharmacol Exp Ther.

2010;333:929-38.

[3] Collins AC, Luo Y, Selvaag S, Marks MJ. Sensitivity to nicotine and brain nicotinic receptors are altered by chronic nicotine and mecamylamine infusion. J Pharmacol Exp Ther.

1994;271:125-33.

[4] Dar MS, Li C, Bowman ER. Central behavioral interactions between ethanol, (-)-nicotine, and

(-)-cotinine in mice. Brain Res Bull. 1993;32:23-8.

[5] Henderson BJ, Lester HA. Inside-out neuropharmacology of nicotinic drugs.

Neuropharmacology. 2015;96:178-93.

[6] Holschneider DP, Guo Y, Roch M, Norman KM, Scremin OU. Acetylcholinesterase inhibition and locomotor function after motor-sensory cortex impact injury. J Neurotrauma. 2011;28:1909-

19.

[7] Hussmann GP, Turner JR, Lomazzo E, Venkatesh R, Cousins V, Xiao Y, et al. Chronic sazetidine-A at behaviorally active doses does not increase nicotinic cholinergic receptors in rodent brain. J Pharmacol Exp Ther. 2012;343:441-50.

[8] Lambert CS, Philpot RM, Engberg ME, Johns BE, Kim SH, Wecker L. Gait analysis and the cumulative gait index (CGI): Translational tools to assess impairments exhibited by rats with olivocerebellar ataxia. Behav Brain Res. 2014;274:334-43.

[9] Lambert CS, Philpot RM, Engberg ME, Johns BE, Wecker L. Analysis of gait in rats with olivocerebellar lesions and ability of the nicotinic acetylcholine receptor agonist varenicline to attenuate impairments. Behav Brain Res. 2015;291:342-50.

[10] Marks MJ, O'Neill HC, Wynalda-Camozzi KM, Ortiz NC, Simmons EE, Short CA, et al.

Chronic treatment with varenicline changes expression of four nAChR binding sites in mice.

Neuropharmacology. 2015;99:142-55.

[11] Marks MJ, Pauly JR, Gross SD, Deneris ES, Hermans-Borgmeyer I, Heinemann SF, et al.

Nicotine binding and nicotinic receptor subunit RNA after chronic nicotine treatment. J Neurosci.

1992;12:2765-84.

[12] Pauly JR, Marks MJ, Robinson SF, van de Kamp JL, Collins AC. Chronic nicotine and mecamylamine treatment increase brain nicotinic receptor binding without changing α4 or β2 mRNA levels. J Pharmacol Exp Ther. 1996;278:361-9.

[13] Perry DC, Davila-Garcia MI, Stockmeier CA, Kellar KJ. Increased nicotinic receptors in brains from smokers: membrane binding and autoradiography studies. J Pharmacol Exp Ther.

1999;289:1545-52.

[14] Quik M, Mallela A, Ly J, Zhang D. Nicotine reduces established levodopa-induced dyskinesias in a monkey model of Parkinson's disease. Mov Disord. 2013;28:1398-406.

[15] Silver AA, Shytle RD, Sanberg PR. Mecamylamine in Tourette's syndrome: a two-year retrospective case study. J Child Adolesc Psychopharmacol. 2000;10:59-68.

[16] Spiller K, Xi ZX, Li X, Ashby CR, Jr., Callahan PM, Tehim A, et al. Varenicline attenuates nicotine-enhanced brain-stimulation reward by activation of α4β2 nicotinic receptors in rats.

Neuropharmacology. 2009;57:60-6.

[17] Wecker L, Engberg ME, Philpot RM, Lambert CS, Kang CW, Antilla JC, et al. Neuronal nicotinic receptor agonists improve gait and balance in olivocerebellar ataxia.

Neuropharmacology. 2013;73:75-86.

[18] Zhang D, Bordia T, McGregor M, McIntosh JM, Decker MW, Quik M. ABT-089 and ABT-

894 reduce levodopa-induced dyskinesias in a monkey model of Parkinson's disease. Mov

Disord. 2014a;29:508-17.

[19] Zhang D, Mallela A, Sohn D, Carroll FI, Bencherif M, Letchworth S, et al. Nicotinic receptor agonists reduce L-DOPA-induced dyskinesias in a monkey model of Parkinson's disease. J

Pharmacol Exp Ther. 2013;347:225-34.

[20] Zhang D, McGregor M, Decker MW, Quik M. The α7 nicotinic receptor agonist ABT-107 decreases L-Dopa-induced dyskinesias in parkinsonian monkeys. J Pharmacol Exp Ther.

2014b;351:25-32.

4.7. Figures and Tables

Fig. 4.1. Timeline of experimental paradigm. Rats were trained to walk on the DigiGait™ system for five consecutive days and assessed weekly thereafter for a total of four assessments. A PCA was performed using gait data from all animals at baseline (n=77) at 25 cm/s only, and the loading factors from this analysis were used to calculate CGI values across assessments. One week following the administration of 3-AP/nicotinamide, animals’ performance was reassessed to quantify gait impairments. One day later, animals were randomly assigned to one of six experimental conditions, receiving daily subcutaneous injections for a total of 13 days of either: saline + saline (n=15), saline + 3 mg varenicline free base/kg/day (n=17), 0.8 mg mecamylamine free base/kg/day + saline (n=9), 0.8 mg mecamylamine free base/kg/day + 3 mg varenicline free base/kg/day (n=10), 3.2 mg mecamylamine free base/kg/day + saline (n=8), or 3.2 mg mecamylamine free base/kg/day + 3 mg varenicline free base/kg/day (n=9).

Fig. 4.2. Effects of 3-AP administration across time on CGI principal components. Z-scores were calculated for each animal’s gait parameter values and multiplied by the loading factors from the PCA to calculate the CGI. Overall CGI values and values for each principal factor were compared using a repeated-measures ANOVA and Fisher’s LSD post-hoc. Data represent mean ± S.E.M. of values from eligible saline-injected lesioned animals (n =12) and the asterisks denote significant differences from baseline determinations for each respective component.

Table 4.1. Principal component analysis at baseline. A principal component analysis was performed on gait parameter values obtained at baseline from all animals (n=77) at 25 cm/s only. For each parameter, the largest absolute loading value across the Factors is bolded, representing the primary components comprising each factor.

Gait Measure Rhythmicity Thrust Contact Stride duration 0.972 0.225 -0.018 Stride frequency -0.967 -0.192 0.045 Stance duration 0.880 0.401 -0.119 Propulsion duration 0.937 -0.022 0.092 Braking duration -0.354 0.666 -0.353 Swing duration 0.821 -0.219 0.198 Stride length 0.973 0.223 -0.015 Stance width -0.298 0.499 0.635 Step angle -0.371 0.510 0.542 Paw angle -0.276 0.628 -0.228 Paw area 0.183 -0.145 0.728 % Total variance 51.0 15.6 13.3

Chapter Five:

Discussion

5.1. Translational and comprehensive approach to assess gait impairments in animals with olivocerebellar lesions

In a prior study, our laboratory utilized a static gait analysis and demonstrated that rats with olivocerebellar lesions exhibit deficits in stride length and stance width (Wecker et al.,

2013). While this method of gait assessment provides important insights into gait adaptations exhibited by animals with ataxia, it is limited, both in the number of gait parameters that can be investigated, as well as its inability to measure temporal aspects of gait.

Due to these limitations, we have undertaken a comprehensive approach to characterize gait deficits in animals with olivocerebellar lesions through the use of a motorized treadmill system (i.e. the DigiGait™). While this treadmill system has been used to assess gait deficits in other rodent models of motor disorders including spinal cord injury (Springer et al., 2010; Ek et al., 2010) and Parkinson’s disease (Glajch et al., 2012; Paumier et al., 2013), or in transient models of ataxia in mice (Hansen and Pulst, 2013), these studies represented the first use of this system in rats with persistent olivocerebellar ataxia as a result of 3-AP administration. This system confers numerous advantages over a static gait analysis, in that all animals can be forced to walk at a desired speed, obviating the confounding influence of speed on gait parameters (Cendelin et al., 2010). Moreover, this system can extensively capture more gait parameters than the static gait analysis method, allowing the collection of both temporal and spatial aspects of animals’ gait at baseline as well as following impairment to characterize gait deficits manifest as a result of ataxia.

The results of this study illustrated that animals with olivocerebellar lesions exhibit alterations in their gait as a result of their impairment that persisted for several weeks.

Importantly, results were obtained from two walking speeds, one which is a comfortable walking speed for rats (25 cm/s) (Gillis and Biewener, 2001; Laughlin and Armstrong, 1982), and a faster speed (35 cm/s) that is more challenging. This approach was especially important, given

99 that patients with ataxia exhibit more rhythmic gait with minimal variability at ‘preferred’ walking speeds, but at speeds outside of this range, gait variability and gait performance worsen

(Schniepp et al., 2012; Wuehr et al., 2013). Thus, the use of two treadmill speeds allowed for a more comprehensive assessment of gait and expanded the comprehensive nature of this method of gait analysis. Furthermore, the use of a PCA to compact data from eleven temporospatial gait parameters revealed three primary factors that comprised gait and explained the majority of the variance in the dataset, including Rhythmicity, Thrust, and Contact

(Lambert et al., 2014). These three factors were combined to yield a cumulative gait index (CGI) which illustrated that animals with olivocerebellar lesions exhibited a nearly 3-fold increase in gait deviation from ‘normal’.

Importantly, the results of gait analysis of animals with olivocerebellar ataxia match findings from clinical studies of patients with ataxia, in that both species exhibit shorter stride lengths (Palliyath et al., 1998; Mari et al., 2012; Morton and Bastian, 2003), larger stance widths

(or base of support) (Stolze et al., 2002; Mari et al., 2012; Ilg et al., 2007; Hudson and Krebs,

2000; Serrao et al., 2013), increased foot/paw angle splay (Stolze et al., 2002), and increased variability in spatial measures (Palliyath et al., 1998; Stolze et al., 2002). In animals with olivocerebellar lesions, the magnitude of impairment was similar at both treadmill speeds.

Thus, these findings demonstrate the translational validity of this method of gait assessment, as well as its potential usefulness to test the effectiveness of therapeutics to improve gait deficits preclinically before they are tested in patients with ataxia. This conclusion is especially relevant given that gait analysis can be performed in both clinical and preclinical subjects and represents a translatable and objective method of assessing motor function and coordination. Thus, results of this study provided a foundation for future experiments to test the effectiveness of nicotinic drugs to improve gait dysfunction in animals with ataxia.

100

5.2. Varenicline at a higher dose is effective in improving gait impairments

With a comprehensive understanding of the gait adaptations manifest by rats with olivocerebellar ataxia and methodological tools to assess deviations from normal gait in these animals, it was next investigated whether the nicotinic agonist varenicline could attenuate gait impairments. The rationale for this approach originated from clinical studies documenting improvements in the gait and coordination of patients with varying subtypes of ataxia, including: fragile X tremor/ataxia syndrome (Zesiewicz et al., 2009a), spinocerebellar ataxia types 3 & 14

(Zesiewicz and Sullivan, 2008), atypical Friedrich’s ataxia (Zesiewicz et al., 2009b), and spinocerebellar ataxia type 3 (Zesiewicz et al, 2012). As our prior study validated that the motorized treadmill system could detect gait adaptations as a result of ataxia in rats that matched those in humans with ataxia, it was investigated whether similar beneficial effects of varenicline could be obtained in animals with olivocerebellar lesions. Additionally, non-lesioned animals were employed to investigate the effects of repeated treadmill exposure on gait over the course of four weeks.

Results of this study demonstrated that normal, non-lesioned animals exhibit gait adaptations from normal as a consequence of repeated treadmill exposure, underscoring the commonsense, yet important finding that gait is dynamic and adaptable. These animals exhibited up to a 74% increase in gait deviation from normal merely two weeks following baseline assessment (Lambert et al., 2015). These gait adaptations included a longer total stride duration, longer stride lengths, and smaller stance widths, illustrating that animals became more efficient at walking on the treadmill following repeated exposure.

For lesioned animals, results demonstrated that the daily administration of a high dose of varenicline for two weeks could attenuate both temporal and spatial gait impairments that rectified the timing of the gait cycle. Importantly, varenicline did not improve all gait impairments,

101 as deficits in several spatial parameters (i.e. paw angle, paw area, stance width) were still apparent following two weeks of varenicline administration. Rather, animals injected with the highest dose of varenicline adapted temporal aspects of their gait that rectified the timing of the gait cycle. Importantly, these attenuations of gait impairments were only present when animals were given the highest dose of varenicline tested (3 mg free base/kg), suggesting that a high dose is necessary to elicit improvements in motor function.

Importantly, results from these studies demonstrate that the effects of varenicline to attenuate gait impairments were speed-dependent, in that significant attenuations of gait impairments were only detected at the slower treadmill speed and not at the faster one. This could be due to increased gait variability at the faster speed, an interpretation that is supported by clinical findings that ataxic patients’ gait variability is speed-dependent, in that their variability is minimal at their preferred walking speed and increased at slower or faster speeds (Wuehr et al., 2013); (Schniepp et al., 2012). Thus, perhaps the increased variability at speeds faster than rats’ ‘comfortable’ walking speed (i.e. 25 cm/s) may have made it more difficult for animals to adapt measures of their gait in response to impairments.

Also notable is that lesioned saline-injected animals exhibited recovery from the impairment in several gait parameters that was equivalent to values obtained at baseline. These results suggest that repeated treadmill exposure following impairment can improve deficits in gait, a finding that has been documented in other models of motor disorders including spinal cord injury (Wang et al., 2015; Cristante et al., 2013), Parkinson’s disease (Dutra et al., 2012;

Lau et al., 2011), and stroke (Ke et al., 2011; Matsuda et al., 2011). However, as discussed, when compared to age-matched non-lesioned animals, lesioned saline-injected animals did not recover from the impairment to the same degree as animals injected with the highest dose of varenicline.

102 In this study, a principal component analysis was also employed to characterize the components of impaired gait (iPCA), illustrating that the relationships among gait parameters are affected following the lesion as well as the gait parameters themselves. Results of this iPCA were used to generate a cumulative gait index that characterized impaired gait (iCGI). On this measure, lesioned animals injected with the highest dose of varenicline uniquely altered their gait from the ‘normal’ impaired state as a result of attenuations of their gait impairments that was not present in lesioned saline-injected animals or the two lower doses of varenicline.

Thus, this study confirmed that varenicline (at a high dose) could attenuate gait impairments in animals with olivocerebellar lesions, matching the clinical findings of Zesiewicz et al., (2012). In the double-blind clinical trial of Zesiewicz et al., (2012), beneficial effects were observed when varenicline was given at a dose of 1 mg/b.d., and required up-titration from 0.5 mg/day over several weeks. This was recommended due to unwanted side effects of varenicline, including nausea, vomiting, and other gastrointestinal disruptions (Leung et al.,

2011), likely due to varenicline’s activity at other nAChR subtypes such as the α3β4 subtype present in autonomic ganglia or the 5-HT3 receptor (Lummis et al., 2011). It is unclear how the dose of 3 mg free base/kg to rats used in these studies (s.c., once daily) translates to the therapeutic dose of 1 mg (p.o., twice daily) in humans (Zesiewicz et al., 2012), especially since varenicline has a much shorter half-life in rats of 4 hours compared to 20-24 hours in humans

(Obach et al., 2006). However, varenicline exhibits similarities between the two species in that it exhibits high bioavailability once absorbed, has good brain penetration, and demonstrates equivalent binding affinities at the α4β2 nAChR (Faessel et al., 2010; Rollema et al., 2007).

5.3. Mecamylamine can similarly attenuate gait impairments

As results demonstrated that a high dose of varenicline improved motor deficits in animals with olivocerebellar lesions, it was next investigated whether mecamylamine could

103 block the effects of varenicline to attenuate gait impairments, thus providing insight into the mechanism of action of varenicline. Results demonstrated that chronic mecamylamine administration was equally effective as varenicline to attenuate gait impairments, and a similar, but not synergistic, effect was observed when high doses of the two drugs were used in combination, suggesting that mecamylamine and varenicline attenuate gait impairments through a similar mechanism.

Since varenicline activates nicotinic receptors while mecamylamine blocks them, it seems paradoxical that both of these drugs resulted in attenuations of gait impairments.

However, these drugs have a similar mechanism that may explain this finding. For example, chronic exposure to both nicotine and varenicline results in increased cell surface expression of nicotinic receptors, termed upregulation (Perry et al., 1999; Marks et al., 1992; Marks et al.,

2015, Hussmann et al., 2012). Similarly, chronic exposure to nAChR antagonists such as mecamylamine and the α4β2 competitive nAChR antagonist dihydro-β-erythroidine (DHβE) can upregulate nAChRs (Collins et al. 1994; Pauly et al. 1996; Abdulla et al., 1996; Zambrano et al.,

2015). Thus, it is likely that the mechanism of nAChR upregulation following prolonged exposure to nicotinic drugs is more complex than simple nicotinic receptor activation (i.e. an

“outside-in” mechanism) (Kishi and Steinbach, 2006; Henderson and Lester, 2015).

Rather, evidence suggests that the mechanism of nicotinic receptor upregulation is mediated primarily through intracellular mechanisms (i.e. an “inside-out” mechanism). For example, chronic exposure to nicotine increases the number and rate of assembly of immature nicotinic receptors within the endoplasmic reticulum (ER) (Henderson et al., 2014; Sallette et al.,

2005) as well as the insertion of mature receptors within the plasma membrane (Henderson et al., 2014). Immature intracellular α4β2 nAChRs pooled within ER can exist as two stochiometries that exhibit high-sensitivity (α4)2(β2)3 and low-sensitivity (α4)3(β2)2 for nicotine. Nicotine can readily penetrate the plasma membrane and act as a “maturation enhancer” (Sallette et al.,

104

2005), promoting increased expression of this high sensitivity conformation (Nelson et al., 2003) within the ER at the expense of the low-sensitivity conformation. This interpretation is strengthened by the finding that nAChR upregulation does not require de novo protein synthesis

(Wang et al., 1998); rather, the existing pool of nicotinic subunits residing within the ER can be recruited and trafficked to the plasma membrane for insertion.

Therefore, following chronic exposure to nicotinic drugs over time, increased cell surface expression of this high-sensitivity conformation can result in increased nicotinic receptor sensitivity as well as increased receptor desensitization in response to the binding of agonists

(Henderson and Lester, 2015). Indeed, nicotinic receptors have at least a 20-fold higher affinity for ligands when they are desensitized than when they are in the resting state (Quik and Lester,

2002). Thus, while an “outside-in” mechanism of nicotinic receptor activation or desensitization following chronic exposure to nicotinic agonists alone is not sufficient to explain the phenomenon of nicotinic receptor upregulation, this “inside-out” approach provides a mechanism by which other findings suggesting that the effects of nicotinic drugs are mediated through desensitization can be interpreted (Henderson and Lester, 2015). For example, chronic administration of mecamylamine can attenuate L-DOPA-induced dyskinesias in a similar manner as nicotine, likely mediated through receptor desensitization (Bordia et al., 2008; 2010).

Since by definition, desensitization occurs in response to prolonged exposure to an agonist, but not an antagonist (Ochoa et al., 1989), the interpretation of desensitization underlying the improvements in L-DOPA-dyskinesias can be explained through this “inside-out” mechanism.

Therefore, it is plausible that the effects of both varenicline and mecamylamine to attenuate gait impairments as documented in this study are ultimately mediated through increased expression of nicotinic receptors.

105 5.4. Implications and Limitations

These experiments provide strong evidence that the nicotinic agonist varenicline and the nicotinic antagonist mecamylamine can improve gait impairments manifest as a result of olivocerebellar lesions in rats, and suggest nicotinic receptor inactivation as the underlying mechanism. Furthermore, these results lay the foundation for future experiments to be conducted using selective nicotinic agonists and antagonists to determine the identity of the nicotinic receptors facilitating this behavioral recovery. It is also worthwhile to investigate the specific brain regions involved in the motor improvement, be it within the cerebellum or its connections to other brain regions involved in the control of coordinated movement and gait.

Additionally, these results demonstrate a method by which a cumulative gait index can be derived from the use of a principal component analysis to conceptualize the overall net effect of alterations in specific gait parameters upon gait as a whole and to investigate deviations from both normal and impaired states.

A strength of these studies employing 3-AP administration to induce motor deficits in rats is that these animals can vary markedly in the degree of their impairment (Wecker et al., 2013;

Lambert et al., 2014), and thus, this population of animals more closely resembles the heterogeneous human ataxia population. Results demonstrated that regardless of the degree of their impairment, nearly all animals (90%) given the highest dose of varenicline exhibited significant deviations from “normal” impaired gait on the iCGI that led to attenuations of gait impairments (Lambert et al., 2014). Thus, this finding further demonstrates the importance of employing a population of animals with varying degrees of impairment for studies that may yield relevant and translational findings.

Furthermore, these studies demonstrate the usefulness and translational importance of employing a PCA to conceptualize the net effects of alterations in gait parameters. For example, while olivocerebellar lesions resulted in small, yet significant 10-20% changes in most gait

106 parameters, the use of a PCA to derive a CGI revealed that animals with ataxia exhibit a nearly

3-fold increase in gait deviation as a consequence of their impairment (Lambert et al., 2014).

The calculation of a PCA represents an objective method to weight the contribution of each gait parameter upon the gait cycle while maintaining the variability within the experimental population and reducing redundancy within the dataset.

The first use of a PCA to produce a gait index that gained widespread clinical acceptance was generated by Schutte et al., (2000) and yielded a ‘normalcy index’ that was used to investigate gait alterations in patients with cerebral palsy. This method of using a PCA to conceptualize gait alterations has been shown to be effective in comparing a heterogeneous group of patients to one another, to test the progression of motor deterioration over time, and to investigate the efficacy of clinical interventions upon gait impairments (Cimolin and Galli, 2014).

Additionally, a ‘gait deviation index’ has been employed to investigate deviations from ‘normal’ gait (Schwartz and Rozumalski, 2008), and has been used to quantify impairments in patients with Parkinson’s disease (Galli et al., 2012), muscular dystrophy (Thomas et al, 2010), and rheumatoid arthritis (Esbjörnsson et al., 2014). The results presented herein represent the first use of a PCA to calculate a cumulative gait index (CGI) to quantify deviations from normal gait in animals with ataxia. Thus, although comparisons between this specific index in animals and humans with ataxia are lacking, numerous reports documenting that ataxic patients exhibit increased gait variability compared to control subjects (Palliyath et al., 1998; Ebersbach et al.,

1999; Stolze et al., 2002) suggest that similar findings would be obtained if this index was employed in studies investigating the gait of patients with ataxia.

5.4.1. Methodological limitations: animals and assessment paradigm

Because male Sprague-Dawley rats were used in these experiments, it is unknown if similar effects can be obtained from female animals. Additionally, since these experiments required rats to undergo forced locomotion on a treadmill, sufficient data for gait analysis could

107 not be obtained from 20-30% of animals due to animal noncompliance. Thus, while it is unknown whether similar beneficial effects of varenicline can be obtained from other strains of rats or mice, it may be of benefit to employ other rodent models of ataxia for future experiments.

Perhaps mice or other strains of rats are more compliant when subjected to forced locomotion, and thus, this may reduce the number of animals necessary for successful completion of the study. However, careful selection of the strain to be tested is warranted, since a study investigating treadmill performance among several strains of mice found considerable variability in animals’ compliance and gait parameter data depending upon the age, strain, and genetic background of animals used (Wooley et al., 2009). Thus, prior to conducting studies assessing gait on a treadmill, it is crucial to determine whether a large portion of the animal population to be used will be compliant during forced locomotion so that adequate gait measures can be obtained. For comparison purposes, it is also important to employ age-matched normal or wild- type animals to investigate how gait parameters change over time as a consequence of repeated treadmill exposure.

As discussed, animals were assessed at two treadmill speeds: 25 and 35 cm/s.

Therefore, the effects of olivocerebellar lesions and treatment with nicotinic agonists and antagonists at speeds outside of this range cannot be determined from these results. A slower treadmill speed of 15 cm/s was employed during preliminary studies, but as this speed was not sufficiently challenging, most animals exhibited noncompliant behaviors such as turning or rearing that made data collection and subsequent analysis difficult. Thus, another recommendation for future studies is to employ treadmill speeds that are sufficiently challenging for animals so as to maximize compliance, but not fast enough that animals are overwhelmed by the rapidity of the treadmill belt and exhibit learned helplessness. Again, this caveat is especially important to consider when employing animals with progressive degenerative motor deficits, as their performance may worsen over time.

108 Furthermore, these experiments only provide information for gait parameters obtained from the hindlimbs of rats and not the forelimbs. This was for several reasons, including: animals reliably exhibited deficits in hindlimb parameters, whereas data from the forelimbs was less consistent, and whereas the hindlimbs in rats are used primarily for locomotion, the forelimbs have a dual functionality in locomotion as well as in foraging and environment exploration (Clarke, 1995). Thus, the use of a different species of animals (such as mice) where all four paws serve similar functions for locomotion may yield important insights into forelimb alterations in animals with gait, including whether the forelimbs compensate for hindlimb gait impairments or are similarly affected. Additionally, improvements in the temporal and spatial resolution of the DigiGait™ analysis software may streamline the process of gait analysis with fewer errors that need to be corrected by the experimenter post-hoc.

Furthermore, the results of these studies are limited to the available gait parameter output provided by the proprietary DigiGait™ software, and mainly focus on the kinematics of animals’ movements (e.g. velocity, time). Other relevant gait information including the kinetics of animals’ movement (e.g. muscle function (EMG) or force displacement) cannot be quantified by this system. This is important given that patients with ataxia exhibit increased amplitude and duration of EMG bursts as well as greater force variability throughout the gait cycle in comparison to healthy controls (Martino et al., 2014). Thus, although results from these studies represent a comprehensive analysis of animals’ gait, the addition of other important aspects of gait such as the mechanisms that produce movement and the timing and actions of muscles involved may produce a more complete understanding when assessing gait abnormalities

(Cimolin and Galli, 2014).

109 5.4.2. Pharmacological methodological limitations

While a beneficial effect of varenicline was documented following chronic administration for two weeks, a longer time course of administration was not investigated. This was due to prior findings that both varenicline and nicotine exert a maximal effect to improve rotorod performance in animals with olivocerebellar lesions following two weeks of administration that is not improved by an additional two weeks (Wecker et al., 2013). Thus, it is possible that a longer time course of varenicline administration could result in greater improvements in motor function, such as improvements in spatial gait parameters that were not observed following two weeks of varenicline administration. Additionally, because the half-life of varenicline in rats is four hours

(Rollema et al., 2007), it is possible that a greater improvement in motor function or attenuations of more gait impairments could be observed if varenicline was administered multiple times per day or chronically through osmotic minipumps.

Although the highest dose of varenicline used in these studies (3 mg free base/kg) is among the highest doses given to rats reported in the literature, other studies have also used this dose to investigate the behavioral effects of varenicline (Spiller et al., 2009; O’Connor et al.,

2010). One study administered varenicline at a much higher dose of 10 mg free base/kg to rats and reported that this resulted in plasma levels of varenicline that are >50-fold higher than levels recommended for humans (Rollema et al., 2009). However, higher doses of varenicline can be given to rodent species, since they cannot vomit, a common unwanted side effect following the administration of high doses of varenicline in humans (Rollema et al., 2009; Leung et al., 2011). Due to these side effects, varenicline is only well-tolerated in humans at doses up to 2 mg/day (Faessel et al., 2006).

While varenicline is FDA-approved for smoking cessation, its effects at 5-HT3 and autonomic α3β4 nAChRs may explain the emergence of reports of unwanted side effects including nausea, gastrointestinal disturbances, and suicidal ideations (Lummis et al., 2011).

110 These off-target effects may discourage its general use for the treatment of the symptoms of ataxia, especially since these effects are more likely to occur when varenicline is administered at a high dose (as recommended by the findings presented here). Fortunately, the field of nicotinic receptor research is continually discovering and testing novel nicotinic compounds that are more selective, including sazetidine-A (a potent α4β2 partial agonist) (Xiao et al., 2006) and

ABT-089 (a α4β2 and α7 receptor partial agonist) (Rueter et al., 2004). Once the neuronal nicotinic receptor subtypes facilitating the effect of varenicline are elucidated, these more selective nicotinic compounds may provide equivalent or improved benefits in motor function with fewer side effects, and thus may be more beneficial for the heterogeneous group of patients with ataxia.

111

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Appendix A: Additional Figures

Fig. 2.4. Stability of gait measures and effects of treadmill speed. Both [A] temporal and [B] spatial gait parameters were determined from a representative stride sequence exhibited by control (unlesioned) animals at treadmill speeds of both 25 and 35 cm/s at 2 days following training and 9 days later. Bars represent mean values + s.e.m. of determinations from 11 animals. Data were analyzed using a 2 × 2 (speed × assessment timepoint) repeated measures ANOVA with t-tests post hoc to assess differences. The asterisks indicate significant (p < 0.05) differences between speeds for: stride frequency, initial [t (20) = 5.97] and final [t (20) = 5.97]; stride duration, initial [t (20) = 6.02] and final [t (20) = 5.84]; stance duration, initial [t (20) = 8.28] and final [t (20) = 6.99]; braking duration, initial [t (20) = 2.23] and final [t (20) = 3.06]; propulsion duration, initial [t (20) = 5.34] and final [t (20) = 5.20]; stride length, initial [t (20) = 5.58] and final [t (20) = 4.29]; and step angle, final [t (20) = 2.56]. The double asterisks indicate significant (p < 0.05) differences between assessments for stride frequency [t (10) = 2.25] and braking duration [t (10) = 2.52].

121 122 Fig. 2.5. Effects of 3-AP on gait parameters. Both [A] temporal and [B] spatial gait parameters were determined from a representative stride sequence exhibited by animals 2 days prior to (initial) and 7 days following (final) the induction of an olivocerebellar lesion by the administration of 3-AP; parameters were determined at treadmill speeds of both 25 and 35 cm/s. Bars represent mean values + s.e.m. of determinations from 45 animals. Data were analyzed using a 2 × 2 (speed × assessment timepoint) repeated measures ANOVA with t-tests post hoc to assess differences. The asterisks indicate significant (p < 0.05) differences between initial (pre-lesion) and final (post-lesion) assessments for: stride frequency, 25 cm/s [t (44) = 3.77] and 35 cm/s [t (44) = 5.73]; stride duration, 25 cm/s [t (44) = 3.53] and 35 cm/s [t (44) = 6.82]; stance duration, 25 cm/s [t (44) = 4.37] and 35 cm/s [t (44) = 7.68]; braking duration, 25 cm/s [t (44) = 3.86] and 35 cm/s [t (44) = 5.32]; propulsion duration, 25 cm/s [t (44) = 5.37] and 35 cm/s [t (44) = 11.0]; stride length, 25 cm/s [t (44) = 3.47] and 35 cm/s [t (44) = 6.80]; stance width, 25 cm/s [t (44) = 8.87] and 35 cm/s [t (44) = 7.08]; step angle, 25 cm/s [t (44) = 4.18] and 35 cm/s [t (44) = 3.71]; paw angle, 25 cm/s [t (44) = 11.7] and 35 cm/s [t (44) = 11.3]; and paw area, 25 cm/s [t (44) = 9.83] and 35 cm/s [t (44) = 10.7].

123 124 Fig. 4.3. Effects of saline or drug administration on absolute gait measures at the 3 week assessment. [A] Temporal and [B] spatial gait parameters at the final assessment timepoint were compared using a 2x3 MANOVA with Fisher’s LSD post-hoc. Data are presented as means ± S.E.M. from animals injected with “Sal”: saline + saline (n=12), Var: saline + 3 mg varenicline free base/kg/day (n=15), 0.8MEC: 0.8 mg mecamylamine free base/kg/day + saline (n=8), 3.2MEC: 3.2 mg mecamylamine free base/kg/day + saline (n=7), 0.8MEC+VAR: 0.8 mg mecamylamine free base/kg/day + 3 mg varenicline free base/kg/day (n=7), or 3.2MEC+VAR: 3.2 mg mecamylamine free base/kg/day + 3 mg varenicline free base/kg/day (n=9).

125 126 127 Appendix B: Copyright Permissions

128 129 130 131

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139 140 141 About the Author

Chase S. Lambert received his Ph.D. in Behavioral Pharmacology in 2015. While at USF, he served on the Association of Medical Science Graduate Students (AMSGS) as Recruitment

Chair and Financial Officer for a total of three years. Chase also completed a two-year

Fellowship as a student in USF’s Doctoral Student Leadership Institute. Chase is married to

Randi McCallian and is the father of Isette Luna Lambert, 4 months old at the time of the dissertation defense. Chase has one brother, Cash, and is the son of Tammy and Steve

Lambert.