Christopher K. Lam. A Thesis presented to The University of Guelph

Transcription

1 Does Synaptic Depression Of The Cerebellar Vermis Modulate The Reflexive Muscular Or Full- Body Postural Responses To An Electrical Vestibular Perturbation In Healthy Young Adults? by Christopher K. Lam A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Doctor of Philosophy in Human Health and Nutritional Sciences Guelph, Ontario, Canada Chris Lam, 2016

2 ABSTRACT DOES SYNAPTIC DEPRESSION OF THE CEREBELLAR VERMIS MODULATE THE REFLEXIVE MUSCULAR OR FULL- BODY POSTURAL RESPONSES TO AN ELECTRICAL VESTIBULAR PERTURBATION IN HEALTHY YOUNG ADULTS? Christopher Ka- yin Lam University of Guelph, 2016 Advisor: Professor L. R. Bent This thesis is an investigation of the relationship between the cerebellum and the vestibular system in postural control. When an electrical current is passed behind the ears, it results in a biphasic reflexive response in the muscles used for balance as well as a postural response, which occurs in the direction perpendicular to head orientation. Although our understanding of the postural and EMG responses to a vestibular perturbation is already quite detailed and continues to expand, there is still much to learn. One area of particular interest that remains irresolute is the origin of the vestibular reflexes and the involvement of other supraspinal structures in the modulation of these responses. The aim of this thesis is to further investigate the role of the cerebellar vermis in the modulation of the reflexive muscle responses and the direction of the postural responses to a vestibular perturbation. The current thesis is comprised of three studies, all of which had a similar experimental design. Each study consisted of two conditions: a TEST group, who received continuous theta burst stimulation (ctbs) to cause temporary synaptic depression of the cerebellar vermis; and a SHAM group, who received paired pulse transcranial magnetic stimulation (pptms), which had no effect on cerebellar function. Baseline vestibular responses were evaluated with an unaltered cerebellum before the intervention of either ctbs or pptms, then vestibular responses were reevaluated immediately after. The first experiment utilized a square wave, electrical vestibular stimulus to evaluate the electromyographic (EMG) reflex responses in the soleus muscles with a depressed cerebellum. It was found that the medium latency (ML) response of the EMG reflex significantly increased with cerebellar depression (ctbs), whereas there was no significant change with pptms. This demonstrated that the cerebellar vermis is involved in the modulation of the EMG reflexive response to a vestibular perturbation. In the second experiment, a stochastic vestibular stimulus was used to evaluate the effects of extra- vestibular sensory input (vision and light tactile input from the finger) on the ML vestibular reflex, while the cerebellar vermis was temporarily depressed. It was found that the dampening of vestibular response amplitudes, caused by the availability of vision and touch, was significantly lessened with cerebellar depression (ctbs), where there were no changes with pptms. These results demonstrate the involvement of cerebellar vermis and its role in sensory integration, an essential part of the response to a vestibular perturbation. In the final experiment, the objective was to determine the role of the cerebellum in developing appropriately directed postural responses to a vestibular perturbation under different orientations of head turn. We found that with the head facing over the shoulder, the postural response was no longer directed perpendicular to head orientation when the vermis was depressed with ctbs. The sway direction became more mediolateral, as the cerebellum was unable to integrate neck proprioceptive input with the vestibular signal to generate a postural response directed orthogonal to head orientation. Collectively, the current work contributes novel findings that the cerebellum is significantly involved in the modulation of both the muscle reflex and postural response to a vestibular perturbation.

3 Acknowledgements iii I would like to thank my committee, Dr. Leah Bent, Dr. Craig Tokuno, and Dr. Richard Staines for their continued guidance and support throughout the course of my Ph.D. and their invaluable contribution to the completion of this thesis. I would also like to thank the members of my comprehensive exam committee as well as my defense committee, including Dr. Steven Prentice of the University of Waterloo, Dr. Steve Brown, Dr. Lori Vallis and Dr. John Zettel of the University of Guelph and Dr. Andrea Green of the University of Montreal. Without their direction and expertise, I would not have gained the knowledge or experience of which I have now. I would like to acknowledge the other graduate students of the department of Human Health and Nutritional Sciences, from the labs of Drs. Brown, Vallis, Zettel and Srbely. They have always been around for moral support, to talk about science, and most importantly, they let me pass electrical currents through their mastoids to evoke reflexive vestibular responses. A special recognition to the members of Dr. Bent s lab: Nick Strzalkowski, Catherine Lowrey, Chris Hayes, Adam Toth, Gagan Gill, Meghan Yip, Robyn Mildren, Erika Howe and all of the other fourth year students. They have been there for me, to help with data collection, to discuss (argue about) science, and to support me for all of my presentations. I would like to thank my family, my sister, Jenny, and her husband, Mohan, who I look up to and who were always there to listen and give advice; my dog, Toby, who was the fluffiest, happiest, best looking best friend; my father, Joe, who has been the greatest support I could have ever asked for; providing me with a house, food, money, laundry service and personal food delivery to my lab; and finally my mother, who passed away on March 5 of 2016, who was my greatest inspiration, who would always make sure that everything that I did was to the best of my ability, and made me practice piano for the majority of my childhood (which I now appreciate, even though I made it very difficult for her). I miss her very much and would not have been able to complete my degree without her. In addition to my family, I am very grateful for the support of my partner, Jessica Bannon, who put up with my entire emotional spectrum, everywhere from happy to sad, and everything in between. She was always there to listen and give advice, and would even take my side when I was clearly at fault. I am very lucky to have her and am excited to move with her to Calgary, onto the next step of our lives. Finally, I would like to thank my advisor, Dr. Leah Bent. Without her guidance, the freedom she provided me, and the opportunities she gave me, I would not be the same person that I am today. I would not have had the same experiences in science, nor would I gain the same social or communication skills. I have learned so much from her and am certain I will benefit from all of these experiences to further my career in the scientific field.

4 Table of Contents iv ABSTRACT... ii Acknowledgements... iii Table of Contents... iv CHAPTER 1 General Introduction and Literature Review Summary of dissertation significance Dissertation overview Summary of dissertation objectives and testable hypotheses Overall Research objective Experiment I: The medium latency muscle response to a vestibular perturbation is increased after depression of the cerebellar vermis Experiment II: The ability of vision and touch to reduce the medium latency shear response to a stochastic vestibular perturbation is attenuated with depression of the cerebellar vermis Experiment III: The direction of the postural response to a vestibular perturbation is mediated by the cerebellar vermis Statement of Ethics LITERATURE REVIEW Why study vestibular contributions to balance? Vestibular Anatomy and methods to assess its contributions to balance Vestibular Anatomy Vestibular Apparatus Eighth Cranial Nerve Vestibular Nuclei Galvanic Vestibular Stimulation Postural responses to GVS Electromyographic responses to GVS Short Latency response Medium Latency response Differences between galvanic vestibular stimulation and stochastic vestibular stimulation How sensory input affects the postural and electromyographic responses to galvanic vestibular stimulation Cerebellar anatomy, Transcranial magnetic stimulation and its effects on cerebellar function Cerebellar Anatomy Functional Zones of the Cerebellar Cortex Neuronal Circuitry Transcranial Magnetic Stimulation TMS effects on the cerebellum Rationale behind why the cerebellum may influence the response to a vestibular perturbation Anatomical connections between the vestibular system and cerebellum Functional connections between the vestibular system and the cerebellum Summary CHAPTER 2 Experiment I: The medium latency muscle response to a vestibular perturbation is increased after depression of the cerebellar vermis INTRODUCTION Subjects Experimental Overview Experimental Setup Galvanic Vestibular Stimulation... 35

5 2.2.5 Transcranial Magnetic Stimulation Threshold Test protocol Sham protocol Data analysis Electromyography Shear Forces Statistical analysis RESULTS Background EMG Short Latency (SL) Medium Latency (ML) Shear Forces DISCUSSION Modulation of the ML but not the SL response Force Plate Shear Responses Did we depress the Vermis? CONCLUSION Bridge Summary CHAPTER 3 Experiment II: The ability of vision and touch to reduce the medium latency shear response to a stochastic vestibular perturbation is attenuated with depression of the cerebellar vermis INTRODUCTION METHODS Participants Participant Setup Stochastic vestibular stimulation Sensory Conditions Transcranial Magnetic Stimulation Threshold Testing Continuous Theta Burst Stimulation Paired Pulse Stimulation Protocol Data Analysis Statistics RESULTS Cumulant Density Amplitude Differences in amplitude for sensory conditions Habituation Effects of cerebellar depression on sensory condition amplitudes Coherence Estimates DISCUSSION Differences between Sensory Conditions Did we depress the cerebellum? Cerebellar depression affects sensory integration Habituation CONCLUSION Bridge Summary CHAPTER 4 Experiment III: The direction of the postural response to a vestibular perturbation is mediated by the cerebellar vermis INTRODUCTION METHODS v

6 4.2.1 Participants Experimental Setup Stochastic Vestibular Stimulation Setup Optotrak and Force Plate Setup Transcranial Magnetic Stimulation Continuous Theta Burst Stimulation (TEST Stimulation) Paired Pulse TMS (SHAM Stimulation) Data analysis Head orientation Cumulant density Statistics RESULTS Head- Referenced Sway Direction DISCUSSION What sensory input is being integrated? Areas of Sensory Integration The cerebellum Other supraspinal areas involved in sensory integration Were there other postural influences on the sway direction? Limitations CONCLUSION Chapter 5: General Discussion Summarizing paragraph Rationale for switching from GVS to SVS after experiment I Habituation of vestibular reflex amplitudes in experiment II but not in experiment I Head Orientation Why do we need to modulate reflexes? Limitations Significant Contributions and Future Directions Techniques Other regions REFERENCES vi

7 List of Figures vii Figure 2.1: Rectified, smoothed and spike triggered averaged trace of muscle activity averaged over all subjects (n=11). Cathode right and Anode right responses were overlaid to facilitate response detection. The shaded area represents the pre- stimulus background activity used to normalize response amplitudes and the thick downward arrow represents the stimulus onset. The first response occurring at approximately ms is the short latency response, labeled SL, and the latter response, labeled ML is the medium latency response starting at the termination of the SL response (approximately ms). Double arrow lines represent amplitude measurement. Vertical dotted lines represent onsets and offsets of the responses, determine by visual inspection, when traces diverged from each other or the baseline.38 Figure 2.2: A. Cumulative average of all contralateral EMG responses (n = 11) before and after TMS. The solid traces represent the test condition and the dotted traces are the SHAM condition. The grey traces represent the SL and ML responses before TMS and the black traces are the SL and ML responses after TMS. The downward arrow is the onset of the electrical stimulus. B. Normalized short latency reflex amplitudes (% of background activity) of pre (black) and post (checkered) TMS for both TEST and SHAM conditions. C. Normalized medium latency reflex amplitudes (% of background activity) of pre (black) and post (checkered) TMS for both TEST and SHAM conditions. Error bars represent standard error and significance is represented with a * when p < Figure 2.3: A. Cumulative average of all shear responses (n = 11) before and after TMS. The solid traces represent the test condition and the dotted traces are the SHAM condition. The grey traces represent the shear response before TMS and the black traces represent the shear after TMS. The downward arrow is the onset of the electrical stimulus. B. & C. SL and ML Fx shear force (N) of pre (black) and post (checkered) TMS for both TEST and SHAM conditions. Error bars represent standard error Figure 3.1: A. The top row represents the cumulative average of pre and post shear force cumulant density responses for SHAM stimulation (n = 10) and the second row represents the responses for TEST stimulation (n = 10). The four traces represent the four sensory conditions CTRL condition (no eyes no touch; black), EO (eyes open, no touch; light blue), T (no eyes, touch; dark blue) and EOT (eyes open, touch; orange). The vertical arrow represents the onset of vestibular stimulation, where shear force output and SVS input have 0 lag. The downward and upward inflections represent the short latency and medium latency reflex response, respectively. B. Medium latency reflex amplitudes from pre to post for SHAM then TEST. All values within the SHAM group are normalized to the SHAM pre CTRL condition; for example, SHAM post EO response amplitude is presented as a percentage of the SHAM pre CTRL condition amplitude. Normalization is similar for the TEST group: all amplitudes are normalized to the TEST pre CTRL condition. C. Medium latency reflex amplitudes from pre to post for SHAM then TEST. All pre TMS values are normalized to the pre CTRL amplitude, while all post TMS values are normalized to the post CTRL amplitude. This is also done for the TEST group 64

8 viii Figure 3.2: A. Pooled coherence plots (n = 10) of all sensory conditions (CTRL, EO, T, EOT) for shear force relative to SVS for pre TMS. Horizontal dashed lines indicate the 95% confidence limit. Significant coherence levels were observed for all conditions at frequencies below 15 Hz. B. Difference of Coherence traces of all conditions relative to CTRL (for example: CTRL- EO: CTRL coherence subtract EO coherence). No significant DOC levels were found for any condition relative to CTRL. 66 Figure 3.3: Pooled coherence plots between shear force and SVS for SHAM (n = 10) and TEST (n = 10) stimulations of all conditions (CTRL, EO, T, EOT). Black traces represent pre TMS coherence, grey traces represent post TMS and dotted lines represent the difference of coherence (DoC) between pre and post. The straight horizontal dashed lines indicate the 95% confidence limit and significant coherence levels were observed for all conditions of frequencies below 15 Hz, whereas the DoC never exceeded that line.. 67 Figure 4.1: A. Stochastic vestibular stimulation (SVS) was applied via the mastoids. Cumulant density traces were established between Fx shear output from the force plate and the SVS input. The Fx shear axis was rotated over 180 o and cumulant density was calculated at each angle. The peak medium latency amplitude was measured at each angle and the angle associated with the highest correlation determined the direction of the postural response. B. The head orientation angle was established by kinematic data of a rigid body, secured to the right side of the forehead. The dependent variable of head- referenced sway direction was calculated by subtracting the sway direction from the head orientation angle...84 Figure 4.2: Raw head orientation and raw sway direction angles are presented on the figures, while head- referenced sway direction values (which appear on the bar graphs in figure 4.3) are displayed next to each corresponding figure. The larger grey arrow represents the raw head orientation and the two smaller arrows protruding from the head, orthogonally, represent the sway direction. The white arrow represents the sway direction before TMS application and the striped arrow represents the sway direction after TMS. The two figures on the left (A) display the head forward condition and the two on the right (B) present the head left condition. The top two figures and bottom two figures are SHAM and TEST Treatments, respectively 87 Figure 4.3: Head- referenced sway direction (degrees) for TEST and SHAM Treatments for head facing forward (A: top) and head facing left (B: bottom). The light grey bar represents the sway angle before TMS application and the dark grey bar represents the sway angle after TMS. Significance is denoted by an asterix and is set to p <

9 CHAPTER 1 General Introduction and Literature Review Summary of dissertation significance Passing an electrical current behind the ears has become a common method to evaluate vestibular contributions to balance. The current causes a change in firing of the peripheral vestibular afferents, which typically result in a predictable full- body postural response and electromyographic (EMG) reflexes in the muscles used for balance (Nashner and Wolfson 1974; Lund and Broberg 1983; Britton et al. 1993; Fitzpatrick et al. 1994; Fitzpatrick and Day 2004). Over the years, researchers have dedicated their careers to examining and characterizing these responses, in hopes to better understand the way in which the body uses vestibular input to effectively maintain balance. This information, in turn, can be applied in a clinical setting as a diagnostic tool or in the design of rehabilitation programs. To date, many aspects of the postural and EMG responses have been characterized in a variety of different ways. Direct neural recordings in primates and decerebrate cats have shown us the physiological effects of electrical stimulation at the level of the vestibular afferents (Goldberg et al. 1982; Cullen et al. 2009). In humans this work has been expanded to investigate changes in stimulus duration and intensity. Research has highlighted that these alterations can modulate onset, magnitude, and direction of the EMG responses (Britton et al. 1993; Fitzpatrick et al. 1994; Rosengren and Colebatch 2002). Additionally, by manipulating feedback from extra- vestibular sensory sources, it is possible to further characterize both EMG and postural responses, and determine how the body integrates vestibular input with other sources (Nashner and Wolfson 1974; Lund and Broberg 1983; Britton et al. 1993; Fitzpatrick et al. 1994; Welgampola and Colebatch 2001; Muise et al. 2012).

10 Although our understanding of the vestibular reflexes (postural and EMG responses to a 2 vestibular perturbation) continues to expand, there is still much to learn. One area of particular interest that remains irresolute is the origin of the vestibular reflexes and the involvement of other brain regions in the modulation of these responses. As the vestibular responses are evidently affected by sensory input (Britton et al. 1993; Fitzpatrick et al. 1994; Welgampola and Colebatch 2001; Muise et al. 2012), some researchers have postulated that the cerebellum may be involved in the modulation of the vestibular reflexes (Cathers et al. 2005); The cerebellum is a supraspinal structure well known for its role in sensory integration and balance control (Itō 1984; Morton and Bastian 2004). There is plenty of evidence as to why the cerebellum may modulate the vestibular responses. Firstly, there are direct anatomical connections, such that vestibular input is sent to the cerebellum via primary and secondary vestibular afferents (Fitzpatrick and Day 2004). Additionally, animal studies have shown that the cerebellum is essential in integrating vestibular input with other sensory input to interpret body orientation, movement (Itō 1984; Manzoni et al. 1999; Morton and Bastian 2004; Cullen et al. 2009) and the control of eye movements (Zink et al. 1998; Schneider et al. 2000). There is an entire field of study examining the vestibular influence on the control of eye movements (termed the vestibular ocular reflex), however this thesis will only focus on the vestibular reflexes seen in lower limb EMG and full- body postural responses. Single- unit neural recordings from the cerebellum have revealed the convergence of neck proprioceptive and vestibular signals, central to the awareness of body orientation and movement (Manzoni et al. 1997; Manzoni et al. 1999; Brooks and Cullen 2009). Finally, a recent study by Kammermeier et al. (2013) showed the direction of the postural response to a vestibular perturbation was significantly different in cerebellar patients than a healthy population. However, what has yet to be shown is whether the cerebellum is involved in

11 modulating the reflexive EMG responses in the muscles of the lower leg or direction of the 3 postural response to a vestibular perturbation in healthy young adults. The primary objective of this thesis was to determine if the cerebellar vermis is involved in the modulation of the response to a vestibular perturbation in healthy human subjects. We explored this connection through a series of experiments focusing on the reflexive muscle and postural responses to vestibular stimuli. 1.2 Dissertation overview Over the next few sections, I outline the significance of this thesis and provide an overview of the objectives and hypotheses of the thesis. Following the general introduction is a literature review of relevant topics. The literature review is comprised of the following four sections: i) why study vestibular contributions to balance?; ii) vestibular anatomy and methods to assess vestibular contributions to balance; iii) cerebellar anatomy and its contributions to balance; and iv) rationale behind why the cerebellum may influence the response to a vestibular perturbation. Chapters 2 through 4 report the three experiments conducted during the PhD thesis, Chapter 2: Experiment I, Chapter 3: Experiment II, Chapter 4: Experiment III. Chapter 5 integrates the findings from the separate chapters and provides a general discussion of the thesis conclusions. 1.3 Summary of dissertation objectives and testable hypotheses Overall Research objective The objective of the thesis was to determine if the cerebellar vermis is involved in the modulation of the responses to a vestibular perturbation in healthy human subjects. Using a form

12 4 of repetitive transcranial magnetic stimulation, we were able to temporarily depress the function of the cerebellar vermis and examine electromyographic and shear force reflexive responses to both square wave and stochastic stimuli. Three studies were completed over the course of this thesis and four hypotheses were developed based on the objectives. They are outlined below: Experiment I: The medium latency muscle response to a vestibular perturbation is increased after depression of the cerebellar vermis Objective: Experiment I was performed to establish whether the reflexive responses to square wave vestibular stimuli are modulated with a depressed cerebellar vermis. Subjects received square wave vestibular stimuli both prior to and after a depressed cerebellar vermis. The following hypothesis was proposed: Hypothesis 1: During acute, transient cerebellar depression, there will be a significant increase in medium latency (ML) response amplitude but no change in SL response amplitude. We believe the short latency (SL) involves more direct pathways to the muscle but the ML response travels through and is modulated by the cerebellum prior to reaching the muscle Experiment II: The ability of vision and touch to reduce the medium latency shear response to a stochastic vestibular perturbation is attenuated with depression of the cerebellar vermis.

13 Objective : Experiment II involved the application of a stochastic vestibular stimulus and 5 examined the short and medium latency reflexes in the cumulant density plots between shear force output and the stochastic stimulus. The objective was to establish whether acute depression of the cerebellar vermis would blunt the ability of sensory integration (vision and touch) to reduce the response amplitude. We developed the following hypothesis regarding the influence of cerebellar input on the sensory attenuation of vestibular reflexes: Hypothesis 1: After acute cerebellar depression, the attenuating effects of sensory input on the response amplitude would be lessened, resulting in relatively larger amplitude responses. We hypothesized that this effect would exist in all conditions with additional sensory input (eyes open condition, touch condition and eyes open and touch condition), however it would be the largest in the condition with the most sensory input (eyes open and touch), as it is this condition that is attenuated the most with sensory integration Experiment III: The direction of the postural response to a vestibular perturbation is mediated by the cerebellar vermis Objective: The objective of this final experiment was to establish whether the direction of the postural response evoked by a stochastic vestibular stimulus is altered with synaptic depression of the cerebellar vermis. Ordinarily, the direction of the postural response is perpendicular to head orientation, and it is believed that the head direction is assessed through sensory integration from spindles in the neck. With evidence of the convergence of neck proprioceptive and vestibular signals within the cerebellar vermis (Manzoni et al. 1999; Kleine 2004), we established the following hypothesis:

14 6 Hypothesis 1: Following cerebellar depression the sway direction to a vestibular perturbation will no longer be aligned with the direction of head turn. We believe this is because the depressed cerebellar vermis will have a reduced capacity to properly integrate neck proprioceptive input with the vestibular signal in order to generate an appropriate postural response. 1.4 Statement of Ethics All of the experiments documented in this thesis were conducted in accordance with the ethical guidelines of the University of Guelph, University of Waterloo and Brock University and conformed to the standards set by the Declaration of Helsinki. Ethical approval was obtained from the research ethics boards at each institution prior to data collection. 1.5 LITERATURE REVIEW Why study vestibular contributions to balance? Vestibular input is the hidden sense that tells us about how our head is moving in space. This sense occurs subconsciously until the system experiences a problem, which can result in dizziness and debilitating balance disorders (Fitzpatrick and Day 2004). Data from the National Health and Nutrition Examination Survey indicated that 35% of Americans over the age of 40 suffer from balance disorders of vestibular origin (Agrawal et al. 2014) and these

15 disorders have been associated with healthcare costs exceeding $4 billion dollars (Saber Tehrani et al. 2013). 7 The connection between the inner ear and balance has been studied as early as the 1700 s when Volta applied an electrical current behind his ears and experienced a sense of imbalance before falling over (Hoepli, 1923; Fitzpatrick and Day 2004). Since then the effects of vestibular stimulation on postural control have been extensively analyzed. Decades of research have scrutinized the reflexive muscular responses and whole- body postural sway in response to vestibular stimulation, however, even with the current breadth of knowledge there is still a number of questions that have yet to be answered. One noticeable gap is the lack of knowledge regarding the involvement of different brain regions in the development or modulation of the vestibular balance responses. Understanding the role of various cortical contributions will not only provide a more complete insight into vestibular function but it may also lead to more effective rehabilitation and technological advancements in vestibular research. There are a number of supraspinal structures that receive vestibular input, including different areas of the somatosensory and parietal cortices, the thalamus and the cerebellum (Itō 1984; Lobel et al. 1999; Lopez and Blanke 2011). The cerebellum is of particular interest as it is involved in balance control, motor coordination and sensory integration, all characteristics that align with the functions of the vestibular system. The overall objective of this thesis is to examine if, or to what extent the cerebellar vermis modulates the reflexive muscular and postural responses to a vestibular perturbation. The following sections of the literature review will provide a background into the functions of both

16 8 the vestibular system and cerebellum, and will discuss the effects of stimulation on each location; concluding with the known anatomical and functional connections and influences of cerebellum on the vestibular output Vestibular Anatomy and methods to assess its contributions to balance Vestibular Anatomy Vestibular Apparatus The vestibular system, commonly referred to as the inner ear, is sensitive to accelerations that act on the head. These accelerations include the constant acceleration due to gravity as well as those generated through angular or translational movement. The vestibular apparati are imbedded in the skull bilaterally and encompass two sub- organs: the otolith organs, which include the utricle and saccule; and three semicircular canals termed the horizontal, superior and posterior canals. The otolith organs are plate- like structures which report on linear accelerations in 3 planes as they are arranged, one horizontally (utricle), and the other vertically (saccule). The semicircular canals are also oriented so they are sensitive to accelerations in 3 dimensions, however due to their morphological shape they are primarily sensitive to angular accelerations. The horizontal canal lies flat and is most sensitive to rotations in the yaw axis, while the other two are arranged at approximately 45 degrees from the intra- aural axis and pick up angular accelerations in the pitch and roll axes.

17 The vestibular system is able to code for accelerations thanks to hair cells, which are 9 imbedded in each organ. Atop each hair cell and suspended in a gelatinous substance are dozens of cilia, which bend when inertial forces act on the head. Depending on the direction of the acceleration, calcium channels on the cilia will either open or close, resulting in either a depolarization or hyperpolarization of the primary vestibular afferent. Therefore, orientation of the hair cells is critical to the organ s sensitivity to accelerations. In the otolith organs, hair cells are oriented such that they are sensitive to accelerations in every angle of the plane. In contrast, each of the hair cells within the semicircular canals face the same direction so they will all respond identically to a particular angular acceleration. Thus, having each of these two vestibular organs bilaterally in the head is critical to the acute detection of accelerations (Fitzpatrick and Day 2004) Eighth Cranial Nerve The nerve that transmits vestibular input is the eighth cranial nerve, also known as the vestibulocochlear nerve. It contains cochlear neurons, which are associated with hearing, as well as primary vestibular afferents, which convey vestibular input from the hair cells to the vestibular nuclei. At rest, when the head is upright and motionless, the vestibulocochlear nerve has a reported average tonic firing rate of approximately 100 Hz (Fitzpatrick and Day 2004). Individual primary vestibular afferents have been classified as having either a regular firing pattern, or an irregular firing pattern based on their discharge regularity and research has shown that only the firing of irregularly firing afferents are modulated with electrical stimulation (Goldberg et al. 1984).

18 Vestibular Nuclei 10 The vestibular nuclei are found bilaterally within the brainstem, and anterior to the cerebellum. They receive input from the primary vestibular afferents as well as some input from the cerebellum (Itō 1984; Morton and Bastian 2004). The nuclei are partitioned into four distinct parts, the superior, inferior, medial, and lateral nuclei. The superior and inferior nuclei send projections to other areas of the cortex, while the medial and lateral nuclei are associated more with motor output. The medial vestibulospinal tract originates in the medial vestibular nucleus, and can control head, neck and eye movements whereas the lateral vestibulospinal tract originates in the lateral vestibular nucleus and is used to control extensor and axial muscles below the neck. The vestibular nuclei also send vestibular input to the cortex via the thalamus. There are three main areas that receive vestibular input and process cognitive aspects of the vestibular signal: the PIVC (parieto- insular vestibular cortex), the area 2V and the 3aV within the somatosensory cortex (Lobel et al. 1999) Galvanic Vestibular Stimulation Galvanic vestibular stimulation (GVS) is a technique used to examine the vestibular system s contribution to balance. It works by passing a binaural, bipolar, electrical current behind the ears, which alters the firing of primary vestibular afferents. It has been postulated that the body interprets the change in firing as a movement of the head and body and thus compensates for the sense of imbalance with a postural response. There are two distinct responses associated with GVS, the postural response and the electromyographic (EMG) response. During quiet stance, the postural response is a sway in the intra- aural axis (Nashner and Wolfson 1974; Lund and Broberg 1983; Britton et al. 1993; Fitzpatrick et al. 1994).

19 Associated with the sway is a quantifiable muscle response (electromyographic response), 11 evoked in the posturally active muscles (Britton et al. 1993). Vestibular stimulation can be delivered in a few different ways: electrical stimulation behind the ears (Nashner and Wolfson 1974; Lund and Broberg 1983; Britton et al. 1993; Fitzpatrick et al. 1994), caloric stimulation (passing cold water in the ears; Jałocha- Kaczka et al. 2014; Taki et al. 2014; Ferrè et al. 2015)) or via head movements (ie. tilts, translations or rotations; Cullen et al. 2009; Angelaki et al. 2010). For the purpose of this thesis, passing an electrical current through the mastoids was the only form of vestibular stimulation used so it will be the main focus of this literature review. There are three ways bipolar, binaural electrical stimulation can be delivered to the subject: square wave direct current stimulation, sinusoidal stimulation and stochastic stimulation. i) Square wave direct current stimulation: The first and most common form of vestibular stimulation is the square wave stimulus, which is referred to as GVS. The stimulus is a direct current and results in a sustained postural response and a quantifiable electromyographic reflex response when averaged over a number of trials (Britton et al. 1993). This form of stimulation has been used to provide insight into much of the vestibular research to date including knowledge on reflexive vestibular functions associated with balance (Britton et al. 1993; Fitzpatrick et al. 1994), sensory integration and sensory reweighting (Britton et al. 1993; Fitzpatrick et al. 1994; Welgampola and Colebatch 2001; Fitzpatrick and Day 2004; Muise et al. 2012). The square wave stimulus was used to perturb the vestibular system in the first experiment of this thesis; to examine the influence of the cerebellar vermis on the electromyographic reflex response.

20 ii) Sinusoidal vestibular stimulation: True to its name, this method of stimulation is in the 12 form of a sinusoid, an alternating current at a single frequency. It results in a postural response that follows the shape of the stimulus, and is said to evoke the sensation of rocking on a boat. This form of GVS was not used for any experiment in this thesis. iii) Stochastic stimulation: Stochastic vestibular stimulation (SVS) is a white noise, alternating current stimulus. This paradigm is fairly new, being introduced in the 1990 s. It evokes a very different response than the square wave stimulus, which provides a different insight into the vestibular output. It adds information about frequency content and how the sub- organs may respond differently to various frequencies. It also provides a more reliable response that is not affected by posture or natural sway (Pavlik et al. 1999). This form of vestibular stimulation was used in experiments 2 and 3. The differences in the responses between SVS and GVS will be discussed in detail after the electromyographic responses to GVS section Postural responses to GVS The application of GVS generates a full- body, inverted pendulum sway response during quiet stance. The sway is directed in the intra- aural axis, meaning it is directed perpendicular to head orientation (ie. with head forward, the sway is mediolateral; and when the head is facing over the shoulder, the sway is anteroposterior). Sway is directed towards the side of the head where the current is hyperpolarizing the vestibular afferents (anodal stimulus). The sway is a top- down response, beginning with a tilt of the head then cascading to lower segments with smaller magnitudes (Fitzpatrick et al. 1994). It can be quantified with center of pressure (COP) or shear force measurements from a force plate, as well as whole body kinematic measurements (center of mass measurements; COM) (Fitzpatrick et al. 1994). The postural response begins

21 approximately half a second after stimulus onset with subjects rapidly swaying towards the 13 anode electrode. Shortly after stimulus termination, the body responds to the change in firing in a equivalent manner to the onset of the stimulus, only this time swaying in the opposite direction (Séverac Cauquil et al. 2000; Watson et al. 2003) Electromyographic responses to GVS Vestibular stimulation alters the firing of vestibular afferents, which results in a reflexive modulation of alpha motor neuron pool excitability in posturally active muscles. This is referred to as the EMG reflex response to a vestibular perturbation. After processing and averaging over several trials, a distinct biphasic response becomes evident, consisting of two separate phases. The first phase of the response is termed the short latency (SL) response, occurring at approximately ms after stimulus onset. The second, beginning upon the termination of the SL and opposite in polarity, is called the medium latency (ML) response, which occurs at approximately ms (Britton et al. 1993; Welgampola and Colebatch 2001). These responses represent a modulation in muscle activity, increasing or decreasing relative to the tonic background muscle activation. During quiet, unsupported stance this response is only apparent in the posturally active lower leg muscles such as the soleus and gastrocnemius. However, when other muscles contribute to the maintenance of balance (ie. the biceps and triceps gripping a stable support while standing on a wobble board, or the trunk muscles while seated), the responses in the legs are attenuated and are then expressed in these other muscles (arms or trunk).

22 14 There has been plenty of research to characterize the EMG responses to GVS, however the origins and roles of the SL and ML are still not fully understood. It has been postulated that the SL and ML responses could be transmitted via different spinal tracts (vestibulospinal vs reticulospinal: Britton et al. 1993; Fitzpatrick et al. 1994; Marsden et al. 2002; Cathers et al. 2005) and that they may also originate in different sub- organs (otolith organs vs. semicircular canals: Cathers et al. 2005). It has also been proposed that the responses may be modulated by input from other brain regions such as the cerebellum (Cathers et al. 2005), however to date there has been a lack of conclusive data to support these hypotheses Short Latency response Not much is known about the SL reflex response. Its role is unclear as it does not correspond to the whole- body sway response and is opposite in polarity to the ML (ie. if the ML is an increase in muscle activity, the SL is a decrease). The SL response begins between ms after stimulus onset and requires a slightly stronger stimulus to evoke clear, consistent responses (above 2 ma: Fitzpatrick et al. 1994; Ali et al. 2003). For quite a few years, it was thought that the SL response could not be modulated by anything other than the stimulus intensity (Britton et al. 1993; Fitzpatrick et al. 1994). Many conditions that would significantly modulate the ML response, such as the addition of visual input and increasing the duration of the stimulus, had seemingly no effect on the SL response (Baldissera 1990; Britton et al. 1993; Fitzpatrick et al. 1994). However these assumptions were contested in the early 2000 s when researchers demonstrated that the SL response could be modulated with a ramp- like stimulus (Rosengren and Colebatch 2002) and that the addition of vision and touch did in fact significantly modulate the magnitude of the SL response (Welgampola and Colebatch 2001).

23 15 The proposed origin of the SL response was within the otolith organs (Cathers et al. 2005). In a study by Cathers et al. (2005), subjects who received GVS with their head pitched down continued to produce SL responses with complete attenuation of the ML response. The head pitch was specifically oriented to eliminate the contributions of the semicircular canals (as the resultant vector of all canals would point directly vertical, thus eliminating the postural component associated with the ML response) Medium Latency response The ML response, which begins between ms after stimulus onset corresponds to the whole- body postural sway response evoked by GVS (Fitzpatrick et al. 1994). It is suggested that the ML originates in the semicircular canals, as it is eliminated when the head is pitched (see explanation above; Cathers et al. 2005). The ML response can be evoked at slightly lower stimulus intensities (as low as 0.5 ma: Fitzpatrick et al. 1994) and can also be modulated by the duration of the stimulus, increasing along with the elongation of the stimulus (Britton et al. 1993). Unlike the SL response, the ML is undeniably modulated by the addition or removal of sensory input (Britton et al. 1993; Fitzpatrick et al. 1994; Welgampola and Colebatch 2001; Muise et al. 2012). When more sensory input is available during GVS, for example, vision and or tactile input from the hand, the ML response amplitude is significantly reduced (Britton et al. 1993; Fitzpatrick et al. 1994; Welgampola and Colebatch 2001). In contrast, when sensory feedback is removed, such as hypothermia- induced anesthesia of the foot sole, the amplitude of the ML response is significantly increased (Muise et al. 2012).

24 Differences between galvanic vestibular stimulation and stochastic vestibular 16 stimulation Both GVS and SVS are used in this thesis and there are certain differences that are inherent in the responses evoked by each type of stimulation. First, the postural response is vastly different between GVS and SVS. GVS will normally produce a prolonged inverted pendulum sway response (described in the Postural responses to GVS section above) whereas SVS does not generate a noticeable full- body postural response. The stochastic stimulus is constantly changing polarities, rapidly pushing the subject bilaterally, however the movements are so quick and trifling that it has been shown that a subject receiving subthreshold SVS can actually be more stable (reduced RMS of COP) than they are in quiet stance (Goel et al. 2015). Although it is not visible, nor can it be detected by COP or COM measurements, there is still a postural response that can be quantified with SVS. SVS can evoke small movements, which can be detected in the shear force output of a force platform, and identified with a cumulant density function between SVS and shear output. Similar to the postural responses evoked by GVS, these responses are generated in the intra- aural direction and can be modulated with the addition or removal of sensory input (Mian and Day 2014). Another difference between GVS and SVS is the way in which data must be processed and analyzed. To analyze the responses from GVS, rectified and smoothed data must be spike trigger averaged over many trials (ie. minimum 40; Britton et al. 1993). With SVS, however, rather than averaging, the muscle and shear force output collected during stimulation is essentially cross- correlated with the stochastic stimulus input using a cumulant density function. The responses of SVS and GVS, although acquired in a very different way, have very similar characteristics. They both have a biphasic short and medium latency response with similar onset latencies (however

25 the SVS onset is approximately ms sooner than GVS; Welgampola and Colebatch 2001; 17 Dakin et al. 2007), they can provide an indication of the direction of the postural response (Mian and Day 2014), and they can both be modulated by sensory input (Welgampola and Colebatch 2001). Finally, with SVS, you can get another measure that cannot be obtained with GVS, the measure of coherence. Coherence is the measure of the linear relationship between two processes across various frequencies (Dakin et al. 2007). It provides insight into the correlated frequencies between the SVS signal and the electromyographic or shear output, something cannot be obtained when using GVS. Also, the signal is unbiased from magnitude of background activity in muscles, which allows for inter- and intra- subject comparisons as well as between healthy individuals and patient populations (Dakin et al. 2007) How sensory input affects the postural and electromyographic responses to galvanic vestibular stimulation As already alluded to in the sections above, the magnitude of the SL and ML reflex responses can be modulated by sensory input from vision and somatosensory sources; increasing or decreasing in size with the gain placed on vestibular input (Welgampola and Colebatch 2001). Sensory input is also critical for the postural response, changing not only the magnitude but also altering the direction of the response (Nashner and Wolfson 1974; Lund and Broberg 1983; Britton et al. 1993).

26 The direction of the postural response is modulated depending on how the head is 18 oriented. When the head is facing forward, the postural response is directed mediolaterally, whereas if the head is turned to face over the shoulder, the postural response becomes anteroposterior, remaining orthogonal to head orientation. This occurs regardless of the orientation of the rest of the body (Lund and Broberg 1983), implying that proprioceptive input from the entire body is taken into account in the postural response. Although it is not the only source of body proprioception, somatosensory input from the neck (ie. muscle spindles, joint receptors, skin receptors) plays a critical role in the postural response, as the neck proprioceptive signals are important to transform vestibular signals into a body- centered reference frame. Experiments have examined the integration of neck and vestibular input through single- unit nerve recordings in cerebellar nuclei and have taught us a lot about multimodal sensory integration required for the development of the postural responses (Manzoni et al. 1997; Manzoni et al. 1999; Kleine 2004). These experiments will be discussed later in the final section of the literature review, Functional connections between the vestibular system and the cerebellum. Perhaps even more critical than the afferent feedback from neck proprioceptors, is the perception of body orientation. A study by Gurfinkel et al. (1989) discovered that if the head was held in a position, looking over the shoulder, for an extended period of time (up to 10 minutes), it would evoke the illusion that the head would return to a forward- facing position. Surprisingly, they found that the postural response changed according to the perception of head orientation, even though the head had not moved throughout the trial. However, there was an immediate recovery of the postural response to the appropriate intra- aural direction as soon as the subjects opened their eyes to realize that their head had not moved since the start of the trial. This refuted