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Using galvanic stimulation to explore the role of vestibulospinal inputs on lower limb motoneurons Kennedy, Paul Michael 2004

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USING GALVANIC STIMULATION TO EXPLORE THE ROLE OF VESTIBULOSPINAL INPUTS ON LOWER LIMB MOTONEURONS by PAUL MICHAEL KENNEDY B.Kin, McMaster University, 1997 M.A., University of British Columbia, 1999 A THESIS SUBMITTED IN PARITAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (School of Human Kinetics) We accept this thesis as conforming to tire required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 2004 © Paul Michael Kennedy, 2004 ii ABSTRACT Galvanic vestibular stimulation (GVS) is a technique used to activate the human vestibular system. The stimulus acts by altering the firing rate of the vestibular afferents (Goldberg et al. 1984), which leads to a change in the descending spinal inputs in the lateral vestibulospinal and reticulospinal tracts. These pathways provide a source of input onto spinal neurons that can affect the excitability of lower limb motoneurons. Galvanic stimulation, therefore, may be used to study the role of vestibulospinal input on lower limb motoneurons. The excitability of the motoneuron pool is affected by the length of the muscle fibers, so GVS may have differing effects at different muscle lengths. The strategy the nervous system adopts to activate muscles at different force producing lengths is still largely unknown. The effect of muscle length on motor unit recruitment was examined in the gastrocnemius muscle in Experiment 1. At a shortened muscle length, the onset of gastrocnemius motor unit activity occurred at significantly higher levels of plantar flexor torque. This may reflect inhibition of gastrocnemius motors units at shortened, non-optimal lengths. There are several pathways that might be involved in regulating the inhibitory inputs that act on the lower limb motoneuron pool, including the vestibulospinal tract. Experiments 2 and 3 provided evidence that a change in the vestibulospinal activity modified the amplitude of the passive ipsilateral soleus H-reflex. It was unclear, however, if this inhibition reflected a change in the activity of the la afferent pathway or motoneuron excitability. Consequently, Experiment 4 examined the effect of vestibulospinal influences on the discharge properties of single motor units in the gastrocnemius muscle. Activation of the vestibular system, using GVS, modified the onset and initial firing frequency of individual motor units at the shortened length but not at the long muscle length. This finding may reflect a change in the presynaptic inhibitory mechanisms that act on the motoneurons innervating muscle fibers that reach non-optimal force producing lengths. The results clearly demonstrate that GVS can alter the excitability of the triceps surae motoneuron pool. The descending vestibulospinal inputs operate to regulate inhibitory influences that act on the motoneuron pool. TABLE OF CONTENTS Abstract ii Table of Contents iiList of Tables vList of Figures viContribution of the Author x Abbreviations and Definitions xi List of Studies xiAcknowledgements 'i> CHAPTER 1 THESIS OVERVIEW 1.1 The vestibulospinal system 1 1.2 Exploring vestibulospinal responses 3 1.3 Regulating neuromuscular activity 4 1.4 Activating muscles at different lengths 5 1.5 Development of the experiments 7 1.6 Aims of the experiments 8 1.7 Statement of ethics 10 CHAPTER 2 OVERVIEW OF RESEARCH TECHNIQUES 2.1 General introduction 11 2.2 Galvanic vestibular stimulation 12.3 Evoking the Hoffmann reflex 3 CHAPTER 3 THE EFFECT OF MUSCLE LENGTH ON MOTOR UNIT RECRUITMENT DURING ISOMETRIC PLANTAR FLEXION IN HUMANS 3.1 Introduction 15 3.2 Methods 6 3.2.1 Experimental design 7 3.2.2 Single motor unit and EMG recordings 13.2.3 Signal processing 19 iv 3.2.4 Statistics 19 3.3 Results3.3.1 Motor unit recordings 20 3.3.2 Plantar flexor torque versus EMG thresholds 23.3.3 Plantar flexor torque 5 3.4 Discussion 23.5 Bridging summary 8 CHAPTER 4 MODULATION OF THE SOLEUS H REFLEX IN PRONE HUMAN SUBJECTS USING GALVANIC VESTIBULAR STIMULATION 4.1 Introduction 30 4.2 Methods 1 4.2.1 Experimental design4.2.2 Statistics 32 4.3 Results 4 4.3.1 GVS polarity and the H reflex 34.3.2 Interval between GVS and the H reflex 34 4.4 Discussion 38 4.5 Bridging summary 40 CHAPTER 5 INTERACTION EFFECTS OF GALVANIC VESTIBULAR STIMULATION AND HEAD POSITION ON THE SOLEUS H REFLEX IN HUMANS 5.1 Introduction 41 5.2 Methods 2 5.2.1 Experimental design5.2.2 Statistics 44 5.3 Results5.3.1 Interaction between head position and GVS 44 5.3.2 Effect of head position on the H reflex 48 5.4 Discussion 48 5.5 Bridging summary 51 CHAPTER 6 GALVANIC VESTIBULAR STIMULATION ALTERS THE ONSET OF MOTOR UNIT DISCHARGE 6.1 Introduction 52 6.2 Methods 3 6.2.1 Experimental design6.2.2 Single motor unit recordings 54 6.2.3 Galvanic vestibular stimulation6.2.4 Signal processing 56 6.2.5 Statistics6.3 Results 7 6.3.1 Influence of GVS at different muscle lengths 56.3.2 Effect of GVS on individual motor units 61 6.4 Discussion 6CHAPTER 7 GENERAL DISCUSSION AND CONCLUSIONS 7.1 Main findings 64 7.2 Activating motor units at different muscle lengths 67.3 Galvanically-evoked EMG responses 66 7.4 Future directions 69 7.5 Concluding remarks 70 7.6 Bibliography 1 LIST OF TABLES Table 3.1 The mean (± SD) plantar flexor torque and soleus EMG rms levels corresponding to the onset of medial gastrocnemius muscle (MG) single motor-unit activity at the long position for each subject. A mean of 12 (± 1.3) MG units were recorded in each condition for each subject 21 Table 3.2 The mean (± SD) plantar flexor torque and soleus EMG rms levels corresponding to the onset of medial gastrocnemius muscle (MG) single motor-unit activity at the short position for each subject. A mean of 12 (± 1.3) MG units were recorded in each condition for each subject 21 Table 5.1 The mean test reflex amplitude is expressed as baseline or zero. The relative differences in H reflex amplitude at different head positions with anodal stimulation are presented in this table. (*) Data presented from Experiment 2 47 Table 5.2 The mean test reflex amplitude is expressed as baseline or zero. The relative differences in H reflex amplitude at different head positions with cathodal stimulation are presented in this table. (*) Data presented from Experiment 2 47 Table 6.1 The mean (± SE) plantar flexor force (N) corresponding to the onset of motor unit activity in the medial gastrocnemius muscle in the long position 58 Table 6.2 The mean (± SE) plantar flexor force (N) corresponding to the onset of motor unit activity in the medial gastrocnemius muscle in the short position 58 vii LIST OF FIGURES Figure 2.1 The effect of GVS on the irregularly firing afferents of the vestibular nerve is shown for (A) no stimulation (B) cathodal stimulation on the right mastoid process and (C) anodal stimulation on the right mastoid process 12 Figure 3.1 The experimental set-up, including subject positions with the knees extended and flexed. The subject's foot was tightly secured to a footplate to minimize ankle movement. (A) concentric needle electrode inserted into medial gastrocnemius muscle (EMG recording above). (B) fine-wire intramuscular electrodes inserted into soleus (EMG recording above). (C) oscilloscope (2 Nm • s"1 ramp and force feedback) to provide the subject with visual feedback. (D) force transducer located at the distal end of the footplate 18 Figure 3.2 Concentric needle recordings of single motor-unit activity from medial gastrocnemius muscle (MG) in the extended and flexed knee position for a single subject. The vertical lines superimposed on the raw data indicate the onset of plantar flexor force production (A) and the onset of motor-unit activity (B). Dotted lines superimposed on the voluntary torque trace show the 2 Nm • s_1 ramps. Action potentials below (n = 10) were superimposed to demonstrate the continuous sampling of the same unit (gastrocnemius muscle) 22 Figure 3.3 The means ± SD (n=9) for the torque and soleus EMG rms that is required to recruit a motor unit in both the long (black bars) and short (white bars) positions. The recruitment thresholds reached a level of statistical significance between knee positions (* P < 0.05) 23 Figure 3.4 Scatter plot of the torque threshold versus the soleus EMG rms threshold for the total number of motor-unit recordings in this study (n = 229). Thresholds were plotted for motor units recorded in the long (filled circles) and short (empty circles) positions 24 viii Figure 4.1 The experimental setup is presented, showing the subject position (A) and the relationship between the galvanic stimuli (B) to the tibial nerve stimulation (C). The H reflexes that were evoked without GVS were referred to as test reflexes, while the H reflexes evoked with GVS were referred to as conditioned reflexes 33 Figure 4.2 Sample EMG responses from one subject to GVS conditioning in the right soleus muscle. The overlapping of the H reflexes demonstrates the corresponding change in amplitude of the conditioned reflex 35 Figure 4.3 In (A) the mean test reflex amplitude is expressed as baseline or zero. The relative difference in H reflex amplitude is illustrated as the change from baseline. In (B) the absolute means ± SE (n = 10) for test (white bars) and conditioned (black bars) H reflex amplitudes are presented. The changes in amplitude observed with GVS were found to be statistically significant (* P < 0.05) 36 Figure 4.4 The relative difference between the test and conditioned soleus H reflex amplitudes was plotted according to the latency between the onset of GVS and tibial nerve stimulation. The mean peak modulatory effect for both cathodal stimulation (solid circles) and anodal stimulation (empty circles) was observed at 100 ms in 4 subjects. However, the changes in H reflex amplitude did not reach statistical significance (Anode P < 0.06, Cathode P < 0.28) 37 Figure 5.1 An example of the EMG responses in the right soleus muscle from one subject is presented for GVS conditioning coupled with the head positions. The overlapping of the H reflexes, with a slight offset, demonstrates the change in amplitude of the conditioned reflex for (A) head right, anodal stimulation, (B) head left, anodal stimulation, (C) head right, cathodal stimulation, and (D) head left, cathodal stimulation 45 ix Figure 5.2 The relative means ± SE (n = 10) for the H reflexes are presented based on the response to GVS coupled with head position. The test reflex is expressed as baseline or zero. The change in H reflex amplitude is the percentage difference from the baseline. Subjects received both a (A) cathodal stimulus and an (B) anodal stimulus. The changes in amplitude observed with GVS were found to be statistically significant (* P < 0.05) 46 Figure 6.1 The experimental setup is presented, showing the location of the oscilloscope, to provide visual feedback, and the subject positions with the knees extended (A) and flexed (B), corresponding to the long and short positions respectively 55 Figure 6.2 (A) The mean (± SE) plantar flexor force (N) required to recruit a motor unit in each stimulus condition is presented for the short position (n = 7). (B) The mean initial firing frequency for those units recorded in all three conditions in the short position. Comparisons that were found to be significant by post-hoc analysis are indicated (* P < 0.05) 59 Figure 6.3 With the knee flexed, recordings of a single motor unit from the medial gastrocnemius muscle during all three stimulus conditions are presented from a single subject. The dotted lines superimposed on the raw data show the level of plantar flexor force corresponding to the onset of motor unit activity. Action potentials (n = 5) were superimposed and illustrated below for each trial to demonstrate the continuous sampling of a single-shaped unit for all three trials 60 Figure 7.1 Schematic illustrating possible vestibulospinal connections to (A) the inhibitory PAD interneuron, (B) to the la pathway, (C) to the alpha motoneuron, and (D) to the gamma motoneurons. Vestibulospinal input may regulate the excitability of the motoneuron pool by affecting the activity of the presynaptic inhibitory interneuron (black circle), alpha motoneuron (white circle - solid line) or the la afferent activity pathway (grey circle) 67 CONTRIBUTION OF THE AUTHOR This thesis contains four experiments that have been carried out by the candidate, Paul M. Kennedy, under the supervision of J. Timothy Inglis (Associate Professor, School of Human Kinetics) Romeo Chua (Associate Professor, School of Human Kinetics) and Andrew G. Cresswell (Docent, Karolinska Institute). The collection, analysis and documentation of each experiment were primarily the work of the candidate. The above statement was written by Paul M. Kennedy and agreed upon by the undersigned. J. Timothy Inglis, Ph.D. (graduate supervisor) XI ABBREVIATIONS AND DEFINITIONS CNS Central nervous system Division of the nervous system that consists of the spinal cord and the brain. Disfacilitation The action of inhibitory interneurons on the postsynaptic membrane which prevent the target neuron from firing. EMG Electromyography Technique used for recording the electrical activity in human muscles. GVS Galvanic vestibular stimulation Artificial stimulating technique used to activate the vestibular system in conscious human subjects. H reflex Hoffmann reflex Electrically-evoked muscle response considered to be the electrical equivalent of the monosynaptic stretch reflex. Inhibition Controlling the level of excitation of a nerve cell through the activity of inhibitory interneurons PAD Primary afferent depolarization Modulation of neurotransmitter release from the primary afferents by presynaptic inputs RMS Root mean square A method of processing the raw EMG signal to a single polarity. xu LIST OF STUDIES Experiment 1 Kennedy PM, and Cresswell AG. The effect of muscle length on motor-unit recruitment during isometric plantar flexion in humans. Experimental Brain Research 173:58-64, 2001. Experiment 2 Kennedy PM, and Inglis JT. Modulation of the soleus H reflex in prone human subjects using galvanic vestibular stimulation. Clinical Neurophysiology 112:2159-2163, 2001. Experiment 3 Kennedy PM, and Inglis JT. Interaction effects of galvanic vestibular stimulation and head position on the soleus H reflex in humans. Clinical Neurophysiology 113:1709-1714, 2002. Experiment 4 Kennedy PM, Cresswell AG, Chua R, and Inglis JT. Galvanic vestibular stimulation alters the onset of motor unit discharge. Muscle and Nerve 30:188-194, 2004. Xlll ACKNOWLEDGMENTS The journey is the reward (Taoist Proverb). This was a journey that was ultimately shaped by three very important individuals. First, my supervisor, Dr. J.T. Inglis provided the challenges and opportunities that allowed me to develop as a researcher, a teacher, and as an administrator. Second, Dr. A.G. Cresswell gave me the confidence I needed to not only succeed in my graduate studies, but also to pursue my aspirations at the post-doctoral level. Finally, Dr. R. Chua challenged me intellectually and offered me the opportunity to explore several research opportunities in his lab. It has been a pleasure and privilege to work with all three of these individuals. I can only hope that the end of my doctoral work marks the beginning of a long collaborative relationship with each and every one of them. I haven't made this journey alone though. I was fortunate to have been accompanied by one of the most committed, loving, and exciting individuals in my life - my partner Jen. This journey wouldn't have been as interesting, as much fun, or even possible without her support. To all those who have helped and guided me along the way, your contributions will never be forgotten. E.F.F.U. Thanks to all, and keep smiling. 1 CHAPTER 1 THESIS OVERVIEW 1.1 THE VESTIBULOSPINAL SYSTEM The vestibular system is comprised of a group of receptors located bilaterally within the inner ear. The receptors are enclosed in the labyrinth which consists of an outer bony structure and an inner membranous component. The vestibular portion of the labyrinth consists of the semicircular canals located on the bony surface and the utricle and saccule found within the membranous tissue. The space between the bony and membranous labyrinth is filled with fluid called the perilymph. Within the inner chambers of the membranous portion, there is another fluid called the endolymph. Together, the fluids provide a medium that can detect vibrations related to head movements (Iurato 1967). Hair cells are distributed along the surfaces of each of the three structures. The gravitational pull on the body causes the hairs on the receptor cells to bend in the fluid (Ross and Donovan 1984). Likewise, if the head is tilted or accelerated linearly (Fernandez and Goldberg 1976; Dickman and Correia 1989), the flow of the fluid inside the labyrinth displaces the hair cells. Movement of the hairs creates a change in the membrane potential of the receptor which will affect the firing behaviour of the vestibular nerve afferents (Flock 1965). Although the receptors are sensitive to the changes in motion and position of the head (Horak et al. 1994), the role of vestibular inputs in movement control is quite complex. Vestibular afferent information is used to control the position of the eyes as the head moves relative to the body (Leigh and Zee 1999), to determine the perception of gait trajectory (Fitzpatrick et al. 1999; Bent et al. 2000), and to activate postural reflexes that realign the body following a postural disturbance (Inglis et al. 1995). Vestibular activation can evoke a muscular response at 75 and 82 ms in the gastrocnemius (Melvill Jones and Watt 1971) and soleus muscles (Greenwood and Hopkins 1976) respectively following a fall from a height. This is because the descending vestibulospinal pathways provide a large part of the excitatory drive to the extensor muscles necessary for maintaining a stable postural position (Wilson and Peterson 1981). Indeed, loss of vestibular function can lead to a decrease in both muscle tone (Thomson et al. 1991) and a reduction in the amplitude of the ipsilateral soleus Hoffmann (H) reflex (Lacour et al. 1976). Information about the position of the head relative to the body is relayed from the vestibular receptors to the vestibular nuclear complex in the brainstem. Located along the lateral wall of the fourth ventricle, the vestibular complex is divided into four groups; the superior, inferior, medial and lateral nuclei (Barmack 2003). The lateral vestibular (Deiter's) nucleus, in 2 particular, projects to spinal segments innervating lower limb motoneurons (Wilson and Yoshida 1969). The descending inputs from the lateral vestibular nucleus may therefore be involved in regulating the activity of lower limb muscles in response to changes in head orientation with respect to the body. When a brief electrical stimulus was applied directly to the lateral vestibular nucleus, monosynaptic excitatory postsynaptic potentials were recorded in the hindlimb of the cat (Lund and Pompeiano 1968). Similarly, repetitive stimulations to the lateral nucleus have been shown to lower the threshold of individual triceps surae motoneurons, particularly the higher threshold units (Westcott et al. 1995). Damage to the lateral vestibulospinal pathway can have dramatic effects on the motor system. Vestibular patients often have difficulty realigning the body following a postural perturbation (Horak and Macpherson 1996), as the lesions disrupt the spinal reflexes that act to maintain body posture (Shupert and Horak 1996). Interestingly, higher cortical centers may actually influence the activity of neurons in the vestibular nuclei (Gildenberg and Hassler 1971; Troiani et al. 1993). Specifically, the primary motor cortex may be able to influence the level of excitability of lower limb motoneurons by modulating the activity of the descending vestibulospinal tracts. It is widely known that the cerebral cortex is involved in the processing of vestibular inputs in humans (Friberg et al. 1985; Bottini et al 1994; Vitte et al. 1996). But while the vestibular nuclei project to the primary somatosensory cortex in the parietal lobe (Grusser et al. 1990), cortical neurons from areas 2, 3a and 6 also project to the vestibular nuclei (Wilson et al. 1999). Vestibular reflexes which stabilize the head during horizontal rotation were absent when subjects were distracted by a mental task (Guitton et al. 1986). This suggests that higher centers may be involved in regulating the activity of descending vestibulospinal inputs that project towards the spinal cord. Vestibular afferent information is transmitted to the lower limb directly through the vestibulospinal tract and indirectly through the reticulospinal tract (Wilson and Yoshida 1969; Wilson and Peterson 1981). Direct projections to the motoneuron pool in the cat can exert monosynaptic excitatory effects on extensor muscles and inhibitory effects on flexor muscles of the lower limb (Grillner et al. 1970). Renshaw cells, which provide recurrent inhibitory input to motoneurons, have also been shown to be inhibited by descending vestibulospinal inputs in the cat (Pompeiano 1988). An increase in the activity of the vestibular afferents may therefore facilitate the motoneuron pool through a disfacilitation of active Renshaw cells. A more likely explanation involves the reticulospinal tract, which is believed to activate interneurons that are responsible for controlling presynaptic inhibition (Manzoni 1988). A decrease in vestibular afferent activity may increase presynaptic inhibition acting on the lower limb motoneurons, and therefore indirectly decrease motoneuron excitability. 3 1.2 EXPLORING VESTIBULOSPINAL RESPONSES Changes in vestibular afferent activity can be artificially achieved by using a technique called galvanic vestibular stimulation (GVS). In healthy human subjects, GVS has been used to investigate the contribution of the vestibular system during quiet stance (Fitzpatrick et al. 1994; Day et al. 1997), in locomotion (Fitzpatrick et al. 1999; Bent et al. 2000; Kennedy et al. 2003), and in the control of eye movements (Schneider et al. 2002). Unlike other methods that have been used to explore the role of the vestibulospinal inputs, galvanic stimulation provides a controlled perturbation to information arising from the vestibular system. Since the galvanic stimulus changes the discharge properties of the irregular firing afferents of the vestibular nerve (Minor and Goldberg 1991), it is believed that GVS alters the descending vestibulospinal inputs that are transmitted towards spinal motoneurons (Britton et al. 1993; Muto et al. 1995). When a low-intensity galvanic stimulus is delivered to electrodes placed over the mastoid process, a transient electromyographic (EMG) response has been recorded in the soleus muscle during quiet stance (Nashner and Wolfson, 1974; Britton et al. 1993). The galvanically-induced muscle responses have also been described in the sternocleidomastoid (Watson and Colebatch 1998), paraspinal (Ardic et al. 2000), and triceps brachii muscles (Britton et al. 1993). Interestingly, the EMG responses to GVS were larger when somatosensory information from the surface was altered either by examining patients with peripheral neuropathy (Hlavacka and Horak 2001) or by having healthy subjects stand on an unstable surface (Welgampola and Colebatch 2001). However, when subjects were either braced or seated, the galvanically-evoked EMG activity disappeared (Storper and Honrubia 1992). This has led to the suggestion that these muscle responses are task dependent (Fitzpatrick et al. 1994). At present, it is unclear why galvanically-induced EMG responses were absent when subjects were not engaged in quiet stance. Presumably, lower threshold motor units were primarily responsible for the EMG activity during quiet stance. Since smaller motor units require relatively little synaptic input to reach their respective firing thresholds (Henneman and Mendell 1981), it may be that GVS does not have a noticeable effect during low level contractions. That, however, does not mean that the change in vestibulospinal activity did not have an effect on the motoneuron pool. There is evidence that individual motor units (Westcott et al. 1995) and the amplitude of the H reflex (Lacour et al. 1976) are influenced by a change in descending vestibulospinal activity in animals. It may be possible to generate a vestibulospinal response in human subjects that are not actively engaged in the control of standing balance by examining the effect of GVS on the H reflex and single motor unit activity. If so, GVS may provide a better understanding of how vestibulospinal influences affect the excitability of the motoneuron pool. 4 1.3 REGULATING NEUROMUSCULAR ACTIVITY The discharge properties of motor units are controlled by the inhibitory and excitatory inputs transmitted to the spinal motoneurons (see Binder 2002). Descending spinal pathways, such as the vestibulospinal tract, alter motoneuron excitability through direct (Westcott et al. 1995) and indirect (Manzoni 1988) connections. Peripheral sensory inputs, such as cutaneous (Garnett and Stephens 1980; McNulty et al. 1999) muscle afferents (Macefield et al. 1991; Grande and Cafarelli 2003), Golgi tendon organs and joint afferents are also known to influence the excitability of homonymous motoneurons (Kanda et al. 1977; Crago et al. 1982). With so many varied inputs acting on the motoneuron pool, the nervous system must be able to control which inputs are transferred to the postsynaptic membrane of active motoneurons. This may be achieved by regulating the effectiveness of the incoming peripheral signals. In addition to the direct excitatory and inhibitory inputs on the motoneuron, there are additional inhibitory actions that occur in the presynaptic terminals. This type of inhibition is referred to as presynaptic inhibition and gives the nervous system the ability to control the strength of the incoming sensory afferents on the motoneuron pool. The concept of presynaptic inhibition was initially described by Frank and Fourtes (1957) when they reported a reduction in the la excitatory postsynaptic potential without changing motoneuron excitability. The inhibitory action, which is accomplished by a primary afferent depolarization (PAD) interneuron, is generated by the release of the inhibitory neurotransmitter gamma-aminobutyric acid. It is believed that activation of these inhibitory synapses on the presynaptic terminals decreases the ability of the calcium channels in the terminals to open (Rudomin and Schmidt 1999). Because calcium ions must enter the presynaptic terminals before the neurotransmitters are released, the result is a reduction in la synaptic transmission. The descending connections which may regulate presynaptic inhibition are not fully understood. Cortical stimulation, for instance, can decrease presynaptic inhibition in the lower limb, yet increase it in the upper limb (Meunier and Pierrot-Deseilligny 1998). The peripheral connections, on the other hand, appear to be a little more straightforward. Considering the la afferent excitatory input was found to be larger on lower-threshold motoneurons (Binder 2000), it was no surprise that presynaptic inhibition may selectively inhibit smaller units (Aimonetti et al. 2000). The modulation of the la excitatory pathways has been assessed with vibration (Morin et al. 1984), at the onset of voluntary contraction (Hultborn et al. 1987), during activation of the antagonist muscle (Aimonetti et al. 1999), and even during dynamic actions (Capaday and Stein 1987). Collectively, these studies suggest that presynaptic inhibition is modulated in a specific 5 manner (Meunier and Pierrot-Deseilligny 1989). That is, presynaptic inhibition is reduced to facilitate the active musculature. At the onset of a voluntary contraction, there is a decrease in presynaptic inhibition of the la terminals in the contracting muscle (Hultborn et al. 1987; lies and Roberts 1987). This observation suggests that the pathways mediating the inhibition of group la afferents may be subjected to a descending cortical influence (Nielsen and Kagamihara 1993; Quevedo et al. 1993). The cerebral cortex may be able to modulate presynaptic inhibition through several descending pathways. The reticulospinal tract, for instance, receives input from the primary motor cortex and is thought to activate presynaptic inhibition through PAD-mediating interneurons (Manzoni 1988). The reticulospinal tract, however, also transmits impulses originating from the vestibular complex to spinal segments innervating lower limb muscles. When the descending vestibular activity is removed, a reduction in the soleus H reflex was observed (Lacour et al. 1976). Therefore, a change in the vestibular afferent activity may affect the excitability of the lower limb motoneuron pool either directly via descending pathways or indirectly through a change in the level of presynaptic inhibition. The H reflex has often been used as an indirect measure of motoneuron excitability (Schieppati 1987). Briefly, the H reflex is evoked via electrical stimulation of the la fibers, which results in a contraction of the muscle (see Misiaszek 2003; Zehr 2003 for reviews). Since the muscle spindles provide the afferent limb of the reflex, the resting discharge of the la afferents will affect the size of the H reflex (Wood et al. 1996). Altering the length of the muscle will activate the muscle spindles and affect the activity within the la pathway. The discharge rates in the muscle spindle afferents tend to be lower at shorter muscle lengths and higher at longer muscle lengths (Scott et al. 1994; Cordo et al. 2002). If there is cortical regulation of presynaptic inhibition, the inhibitory inputs may be altered based on the length of the active muscle fibers. Namely, a reduction in presynaptic inhibition at longer muscle lengths may allow the excitatory la projections to facilitate active motoneurons. To investigate the role of descending vestibulospinal inputs on motoneuron excitability in this thesis, the length of the muscle was considered. It was hypothesized that GVS may have different effects on motoneuron pool excitability based on the length of muscle fibers of individual motor units. 1.4 ACTIVATING MUSCLES AT DIFFERENT LENGTHS The primary function of muscle fibers, the cells of skeletal muscles, is to generate force. These specialized cells contribute to the maintenance of the internal environment and produce movements that allow the organism to interact in the external environment. The gastrocnemius 6 muscle, for example, is comprised of cells that may extend up to 50 mm in length when the knee is fully extended and the ankle is held at 900 (Kawakami et al. 1998). The individual fibers, however, may terminate in the midfascicular region (Loeb et al. 1987; Trotter 1993). So, when a muscle changes length, non-homogeneous shortening of the muscle fibers may occur (Ettema and Huijing 1989; Van Bavel et al. 1996). Non-uniform shortening within fascicles is likely to lead to some fibers reaching non-optimal force producing length while others may generate force sufficiently. These differences in length and force producing capacity are certain to complicate how the nervous system activates the motoneuron pool. The total tension a muscle can develop depends on several factors, including the length of the muscle fibers (see Rassier et al. 1999). The fact that peak muscle tension is affected by length was well described over a century ago (Blix 1894). The decline in isometric force at muscle lengths below the optimal position was attributed to a decrease in the number of potentially bound cross-bridges (Gordon et al. 1966). Since there is also a decrease in the duration of the contraction time at shorter muscle lengths (Bigland-Ritchie et al. 1992), an increase in the frequency of activation of a muscle is needed to compensate for the reduced force-producing capability (Gandevia and McKenzie 1988). However, if a muscle reaches a critical shortened length, activating these fibers would generate minimal force while still requiring energy (Herzog 2000). So, the question is raised as to whether the CNS activates motor units whose fibers are at non-optimal length to produce force. Studies investigating the effect of changes in muscle length on the EMG activity of a muscle during a maximal voluntary contraction have provided conflicting results. While an increase in EMG activity has been reported in the shortened biceps brachii (Heckathorne et al. 1981) and biceps femoris muscles (Lunnen et al. 1981), a decrease was observed in the shortened gastrocnemius muscle (Fugl-Meyer et al. 1979; Sale et al. 1982; Pinniger et al. 2000). These differences possibly reflect the many strategies that the CNS may use to develop an efficient control of force when an active muscle resides at a shortened length. Seeing as the gastrocnemius muscle's contribution to torque output is at 60% of the maximum torque when the knee is fully flexed (Cresswell et al. 1995; Kawakami et al. 1998), the reduction in EMG may reflect a reduction in cortical drive or a strategic inhibition of certain gastrocnemius motoneurons whose fibers have a reduced capacity to contribute to force production. Presynaptic inhibition, for example, may decrease the excitability of the motoneurons whose fibers reside at shortened muscle lengths by reducing the efficacy of the excitatory volleys from la afferents (Romaiguere et al. 1993). 7 In humans, it has been shown that presynaptic inhibition projecting to lower limb motoneurons of the active muscle fibers is reduced approximately 50 ms prior to the start ofthe contraction (Nielsen and Kagamihara 1993). This suggests that the inhibition of the la afferent pathway may be regulated by descending cortical influences (Quevedo et al. 1993). One possibility is that changes in presynaptic inhibition occur because of descending vestibulospinal inputs (lies and Pisini 1992) acting on the group la afferents and/or the la interneurons. The vestibular system is known to influence motoneuron excitability directly and indirectly via the vestibulospinal and reticulospinal tracts (Wilson and Peterson 1991). If there is a presynaptic inhibitory influence on the motoneuron pool at shortened muscle lengths, we may be able to alter the discharge properties of motor units with galvanic stimulation. Specifically, the change in descending vestibulospinal activity may alter the presynaptic inhibitory inputs that are present once the muscle fibers reach a critical shortened length. First, however, we must identify how muscle length affects motor unit recruitment; then we can assess the influence of GVS on the motoneuron pool at different muscle lengths. 1.5 DEVELOPMENT OF THE EXPERIMENTS The length of the active muscle fibers will affect their force contribution to the total force output (Gordon et al. 1966). A reduction in EMG of a muscle during maximal voluntary contractions at shortened lengths may reflect a decrease in cortical drive or an increase in presynaptic inhibition (Cresswell et al. 1995) to limit the activity of the muscle fibers that reside at inefficient force-producing lengths. There are several pathways that might be involved in this process, including the vestibulospinal tracts' inhibition of la afferents (Manzoni 1988; lies and Pisini 1992). Cortical neurons are able to regulate the activity of several pathways, including the vestibulospinal tracts (Gildenberg and Hassler 1971; Troiani et al. 1993). A change in the descending vestibulospinal input may influence the level of excitability of the motoneuron pool. However, it is presently unclear as to what strategy the CNS adopts to activate a muscle fiber when it resides at an inefficient force-producing length, and whether the vestibular system may play a role in this process. To investigate the effect of muscle length on motoneuron excitability, the onset of single motor unit activity was investigated in the medial gastrocnemius muscle at different lengths. A decrease in the electrical activity of the gastrocnemius muscle has often been observed during maximal voluntary contractions with progressive shortening of the muscle fibers (Fugl-Meyer et al. 1979; Sale et al. 1982). One possible suggestion was that the EMG reduction for a specified level of plantar flexor force resulted from a change in the position of the active fibers relative to 8 the surface electrodes (Cresswell et al. 1995). Therefore, single motor unit recordings taken from the muscle provided direct analysis of the electrical activity of a muscle fiber at different force-producing lengths. A limitation of using needle electrodes is the difficulty of maintaining the same motor unit recording at different muscle lengths. The use of needle electrodes, however, presented recordings from a larger muscle volume at different force producing lengths. Galvanic stimulation is a non-painful technique that has often been used to explore vestibulospinal connections in humans (Nashner and Wolfson 1974; Britton et al. 1993; Watson and Colebatch 1997). During quiet stance, GVS produces changes in the EMG of postural muscles, such as the soleus (Nashner and Wolfson 1974; Britton et al. 1993). However, when subjects were either braced or seated, this brief EMG activity was absent (Storper and Honrubia 1992; Fitzpatrick et al. 1994). If GVS does in fact activate interneurons that are responsible for regulating presynaptic inhibitory inputs that act on the motoneuron pool (lies and Pisini 1992), it is unclear as to why galvanically-induced EMG responses were absent when subjects were not engaged in maintaining balance. It may be that GVS did not have a noticeable effect on the ongoing EMG activity when the gain of the vestibulospinal pathways was reduced. Since GVS activates descending vestibulospinal pathways (Muto et al. 1995), it may be possible to use the artificial stimulus to examine the role of the vestibular activity on the motoneuron pool by measuring the amplitude of the H reflex in a passive muscle. Certainly there is evidence that removing vestibulospinal input in the baboon can attenuate the ipsilateral soleus H reflex (Lacour et al. 1976). A change in H reflex amplitude does not necessarily imply that motoneuron excitability was altered but may represent a reduction in the discharge properties of the la afferent pathway. The H reflex was therefore only used to explore indirectly the effect of GVS on the excitability of the motoneuron pool. Motor units recorded in the presence of a conditioning stimulus were used to directly assess the effect of GVS on motoneuron excitability. By limiting the inputs that act on the la afferent/alpha motoneuron synapse, the H reflex and motor unit recordings were used to evaluate the role of descending vestibulospinal pathways that act on lower limb motoneurons. 1.6 AIMS OF THE EXPERIMENTS The proposed research sought to determine the effect of gastrocnemius muscle length on its motor unit recruitment properties and the influence of GVS on the soleus H reflex and the discharge onset of gastrocnemius motor units. Based on the rationale outlined in the previous sections, four experiments were developed. The purpose of each experiment is outlined below: 9 Experiment 1 The triceps surae muscle group, consisting of the mono-articular soleus and bi-articular gastrocnemius muscles, primarily generates plantar flexor torque at the ankle joint. Since the gastrocnemius muscle crosses the knee joint, flexion of the knee reduces the length of this muscle, therefore limiting its contribution to torque output. It is not clearly understood how the CNS activates muscles fibers that may reside at inefficient force-producing lengths. The present study was designed to determine the effect of muscle length on motor unit recruitment in the gastrocnemius muscle. Experiment 2 In Experiment 1, the onset of motor unit activity at the short muscle length occurred at higher levels of plantar flexor torque. This may reflect a change in the excitability of motoneurons innervating muscle fibers whose contribution to plantar flexor torque is reduced. The vestibular system provides a source of synaptic input to lower limb motoneurons. Galvanic stimulation has often been used to alter descending vestibulospinal impulses. It may be possible to use galvanic stimulation to examine the effect of descending vestibulospinal inputs on the motoneuron pool when the muscle is not actively engaged in the control of standing balance. Specifically, the effect of GVS on the H reflex amplitude of the passive ipsilateral soleus was examined in this study. Experiment 3 The results of Experiment 2 demonstrated that galvanic stimulation can modulate the passive soleus H reflex. This suggests that in certain situations, it may be possible to use this type of vestibular stimulation to examine the integrity of descending vestibulospinal pathways. However, the position of the head relative to the body and galvanic stimulus polarity can independently alter the ipsilateral soleus H reflex. In an attempt to understand how descending vestibulospinal inputs influence the spinal motoneurons that innervate lower limb muscles, the effect of GVS on the soleus H reflex was examined at different head positions in the prone position. Experiment 4 Experiments 2 and 3 established that, in some circumstances, GVS can modulate the amplitude of the soleus H reflex when subjects are not standing. The results suggest that the 10 vestibulospinal system regulates muscle activity by controlling presynaptic inhibitory inputs that act on the motoneuron pool. However, it was unclear if the total motoneuron pool was receiving equivalent input or if the descending vestibular inputs were directed towards certain types of motor units. Experiment 4 examined the effect of vestibulospinal influences on the discharge properties of single motor units in the gastrocnemius muscle. 1.7 STATEMENT OF ETHICS Experiment one was conducted in accordance with the ethical guidelines of the Karolinska Institute. Experiments two through four were approved by the clinical ethics committee of the University of British Columbia. 11 CHAPTER 2 OVERVIEW OF RESEARCH TECHNIQUES 2.1 GENERAL INTRODUCTION The purpose of this section is to provide an overview of some of the techniques used in this thesis. In particular, using GVS to activate the vestibulospinal system and the interpretations behind the Ft reflex are discussed. The specific methods, procedures and equipment used in each study are presented separately in each of the appropriate chapters. 2.2 GALVANIC VESTIBULAR STIMULATION Studies investigating the role of the vestibular system in movement control have often examined patients with vestibular deficits (see Curthoys and Halmagyi 1995). Galvanic vestibular stimulation, on the other hand, is a technique that allows researchers to study the role of vestibular inputs in organisms with an intact sensory system. Briefly, GVS involves passing a low-level (< 5 mA), constant current stimulus percutaneously to the vestibular nerve through electrodes placed over the mastoid processes. Based on animal research, the stimulus modulates the tonic discharge activity of the irregular firing afferents of the vestibular nerve (Goldberg et al. 1984). Specifically, an anodal or positive current decreases the firing rates whereas a cathodal or negative current increases the firing frequency of the irregular afferents of the vestibular system (Minor and Goldberg 1991) (Figure 2.1). Consequently, galvanic stimulation of the vestibular system can provide a controlled and consistent perturbation of vestibular information. The change in the discharge activity of the vestibular nerve brought about by GVS can induce postural changes (Day et al. 1997), ocular movements (Schneider et al. 2002), and alter the walking trajectory in humans (Fitzpatrick et al. 1999; Bent et al. 2000; Kennedy et al. 2003). Several studies have also described an EMG response to GVS in lower limb muscles (Britton et al. 1993; Watson and Colebatch 1997). In soleus, GVS evoked an EMG response approximately 60 ms following stimulus onset, followed by a second response that was opposite in polarity, beginning at about 100 ms (Nashner and Wolfson 1974). These responses have only been reported in muscles that are posturally active in a free-standing situation (Britton et al. 1993). When subjects were either braced or seated, the evoked response disappeared (Storper and Honrubia 1992). When somatosensory information from the surface was altered, either by neuropathy (Fforak and Hlavacka 2001) or by standing on an unstable surface (Welgampola and Colebatch 2001), both the GVS-evoked EMG responses were larger. This suggests that there 12 Figure 2.1 The effect of GVS on the irregularly firing afferents of the vestibular nerve is shown for (A) no stimulation (B) cathodal stimulation on the right mastoid process and (C) anodal stimulation on the right mastoid process. 13 may be a task dependent role for vestibulospinal inputs. As the relevance for vestibular information increases, so does the amplitude of the galvanic response (Fitzpatrick et al. 1999). A low-intensity galvanic stimulus delivered to electrodes placed over the mastoid process may not only activate the irregularly firing afferents of the vestibular nerve (Minor and Goldberg 1991) but could also excite local cutaneous afferents in the underlying skin. In a monopolar monaural configuration, the reference electrode may be positioned over the wrist joint. There is evidence that an electrical stimulus delivered to the superficial radial nerve can facilitate the EMG activity in the ipsilateral soleus muscle (Zehr et al. 2001). This has led to the assumption that cutaneous activation may contribute to the galvanically-evoked EMG responses in the lower limb. We can, however, eliminate the possible contribution of cutaneous afferents based on anecdotal reports from our laboratory. When the stimulating electrode was repositioned on the neck, galvanically-evoked lower limb EMG responses were absent. Sensations of movement or postural sway also disappeared if the electrode over the ear was slightly shifted off the mastoid process, even if the position of the reference electrode remained the same, suggesting that the GVS effect was dependent on the correct placement of the stimulating electrode over the vestibular nerve. Consequently, galvanically-evoked muscle responses appear to result from a change in the descending vestibulospinal inputs that are transmitted directly or indirectly to the lower limb motoneuron pool. 2.3 EVOKING THE HOFFMANN REFLEX An electrical stimulus delivered to a mixed peripheral nerve will excite the larger la afferents that innervate the muscle spindles before the smaller diameter motor fibers (Erlanger and Gasser 1968). Since the la primary afferents provide a monosynaptic excitatory influence on alpha motoneurons (Schieppati 1987), activation of the la afferents may bring about a reflexive contraction of the skeletal muscle fibers. This electrically-evoked muscle response is known as the Hoffmann (H) reflex (Hoffmann 1910, 1918) and is considered to be the electrical equivalent of the monosynaptic stretch reflex (see Zehr 2002). H reflexes are typically evoked with percutaneous electrical stimulation and recorded over the muscle using bipolar surface electrodes. As the stimulus intensity increases, there is a concomitant increase in the reflex amplitude (see Pierrot-Deseilligny and Mazevet 2000). Once the motor threshold is reached, a second motor response or M wave appears at an earlier latency. Additional increases in the stimulus intensity cause the amplitude of the M wave to increase while the amplitude of the H reflex decreases. This is because the potentials generated reflexively in the motor axon are cancelled out by collision with the action potentials directly evoked in the same motor axon (Hoffmann 1910, 14 1918). At higher stimulus levels, more cancellation occurs until the H reflex disappears and only the M wave is present. The H reflex has been characterized as the most extensively studied reflex in neurophysiology (Misiaszek 2003), largely due to the ease in which the response can be generated in both upper (Burke et al. 1989; Abbruzzese et al. 1994) and lower limb muscles (Hultborn et al. 1987; Brooke et al. 1997). The H reflex has been used to address such topics as neural changes with age (Earles et al. 2001), mechanisms regulating interlimb coordination (Zehr et al. 2003), and neural adaptations that occur with athletic training (Nielsen et al. 1993). The simplicity of the technique, however, may be misleading. As the test frequency increases, a decrease in H reflex amplitude has been observed (Fournier et al. 1984; Hultborn et al. 1996). This attenuation has been attributed to post-activation depression of the la afferent terminal. That is, depression at the la afferent/alpha motoneuron synapse may result from a reduced neurotransmitter release from the previously activated fibers. This phenomenon has also been reported when the ankle joint has been passively rotated (Crone and Nielsen 1989; Hultborn et al. 1996). Consequently, movements of the test muscle should be limited and the stimulus frequency should be reduced to minimize the effects of post-activation depression on the la afferents. If the technical issues are addressed prior to the start of the experiment, the H reflex can be a valuable technique for describing synaptic actions on spinal motoneurons. Initially, Magladery et al. (1951) suggested that the H reflex represented a monosynaptic response, since the interval between the electrical stimuli to the onset of the waveform was brief. There is evidence, however, to suggest that oligosynaptic inputs contribute to the later portions of the reflex (Burke et al. 1984). Reciprocal (Crone and Nielsen 1994), recurrent (Katz and Pierrot-Deseilligny 1999) and presynaptic inhibition (Voigt and Sinkjaer 1998) can affect the amplitude of the H reflex. Consequently, careful control over the presynaptic inputs must be taken into account when evoking and interpreting the H reflex (Zehr 2002). For instance, H reflexes should be evoked during similar levels of muscle activation. The stimulus intensity should also be adjusted to ensure that an M wave is present and that the amplitude of the motor response is constant. Finally, all measurements should be made in the same body posture to control for task dependent changes in H reflex amplitude (Misiaszek 1998; Aimonetti et al. 1999). By limiting the inputs that act on the la afferent/alpha motoneuron synapse, the H reflex can be used, in some circumstances, to asses changes in the excitability of the motoneuron pool. 15 CHAPTER 3 THE EFFECT OF MUSCLE LENGTH ON MOTOR UNIT RECRUITMENT DURING ISOMETRIC PLANTAR FLEXION IN HUMANS (Published: Experimental Brain Research 173: 58-64, 2001.) 3.1 INTRODUCTION The force that a muscle can produce is dependent not only on the neural commands generated by the central nervous system (CNS) but also on the length of the fibers and velocity of the action. For a given muscle action, the length of the muscle can vary dramatically and, based upon its length-tension relationship, may have to generate force at a length that may be less than optimal (Rack and Westbury 1969; Herzog 2000). Studies investigating the effect of muscle length on force production have focused on both the discharge properties of motor units and the level of electrical activity of the muscle. As there is a decrease in the duration of both the contraction time and half relaxation time of the twitch at shorter muscle lengths, a higher motor unit discharge would be required to produce similar levels of force in the shortened position (Bigland-Ritchie et al. 1992). This has been evidenced by Vander Linden et al. (1991) and Christova et al. (1998), who showed an increase in motor unit firing frequency at reduced muscle lengths in the tibialis anterior and biceps brachii, respectively. The changes in the surface recordings of muscle activity with progressive shortening of muscle length seem to be less clear, with both increases (Heckathorne et al. 1981; Lunnen et al. 1981) and decreases (Fugl-Meyer et al. 1979; Sale et al. 1982; Cresswell et al. 1995; Pinniger et al. 2000) in the level of EMG being reported. These differences possibly reflect the many strategies that the CNS may use to develop an efficient control of force when an active muscle is varying its length. Moreover, this is further complicated when activation of a synergistic muscle group, whose individual components are operating at differing lengths, must be controlled to provide a required joint torque. At the ankle, the functional group primarily responsible for plantar flexor torque production is the triceps surae, which consists of the bi-articular gastrocnemius and mono articular soleus muscles. Despite being classified as synergists, the relationship between these two muscles is constantly changing, depending upon the task. For example, during standing, soleus is usually active, while minimal activation of the gastrocnemius (Hodgson 1983; Duysens et al. 1991) is observed. It is with an increase in movement velocity that a corresponding increase in the activity of the gastrocnemius is observed (Hutchison et al. 1989; Duysens et al. 1991). This dissociation has been further illustrated during rapid lengthening actions, where the soleus can be rendered silent while there is a preferential increase in gastrocnemius activity (Nardone et al. 16 1989). The differential activation patterns observed in these two muscles may be explained by the composition of the respective motoneuron pools, with the soleus predominantly composed of slow twitch motoneurons and gastrocnemius having a higher number of fast twitch motoneurons. The variation in force generating capabilities between the soleus and gastrocnemius muscles may, however, be influenced more by the differences in their architectural design than their fiber type distributions (Kawakami et al. 1998; Herzog 2000). The gastrocnemius muscle crosses both the knee and ankle joints and therefore, for a given ankle angle, its contribution to torque output is considerably altered by the position of the knee (Herzog 2000), with greater torque production occurring when the muscle is lengthened via extension of the knee and or dorsiflexion of the ankle (Cresswell et al. 1995; Kawakami et al. 1998). On the other hand, the soleus muscle, with its length unchanged, would thereby be required to generate a greater or lesser percentage of the overall torque, depending on the degree of knee flexion. Despite the increased demand of the soleus muscle to plantar flexor torque with a reduction in knee angle, there may still be a minor torque contribution from the gastrocnemius, as it has been shown that even in the most flexed knee angles there is still EMG activity from gastrocnemius (Cresswell et al. 1995). In an earlier study (Cresswell et al. 1995), we found that the surface EMG amplitude from both heads of the gastrocnemius muscle during voluntary plantar flexor efforts with the knee flexed to shorten the gastrocnemius was significantly less than that produced with the knee extended. However, it was unclear whether the EMG reduction was due to electrode-muscle configuration changes or due to inhibition of gastrocnemius motor units induced by the reduction in their fiber lengths. The question therefore remains as to how the CNS activates a muscle when many of its fibers are at non-optimal force-producing lengths. The purpose of the present study was therefore to determine the effect of muscle length on motor unit recruitment for the medial gastrocnemius muscle (MG) during isometric plantar flexion. The onset of single motor unit activity with respect to plantar flexor torque and soleus EMG activity was compared with the knee extended at 180° and flexed at 90°, corresponding to long and short lengths of the MG muscle. 3.2 METHODS The following experiment included nine healthy, male participants between 25 and 42 years of age. Their mean values (± SD) for age, height, and body mass were 32.0 years (± 6.0 years), 179.7 cm (± 4.5 cm) and 78.3 kg (± 3.9 kg), respectively. All subjects regularly participated in physical activity and none of these individuals had any known neurological or motor disorders prior to testing. The experimental protocol was explained and the subjects gave 17 their informed consent to participate in the investigation. The local institutional ethics committee approved the following experimental procedures. 3.2.1 Experimental design Subjects lay in a prone position on a padded bed. Their left (n = 6) or right (n = 3) foot was tightly secured to a non-compliant footplate at a constant angle of approximately 90° (measured as the internal angle between the shank and the foot) to ensure isometric conditions. Force was measured from a load cell (unloaded frequency range 0-2.6 kHz, maximum force 2 kN; Bofors KRG-4; Nobel Electronic, Sweden), which was placed at the distal end of the footplate. The axes of the ankle and footplate were aligned as close as possible. The perpendicular distance between the load cell and axis of the footplate was used to convert force to ankle plantar flexor torque. From this initial prone position, knee angle (measured as the included angle between the thigh and shank) was manipulated by raising the trunk above the support surface (see Figure 3.1). Flexion of the hips altered the knee angle (from 180° to 90°) while the trunk and shank remained horizontal. The subjects placed their arms on the table to provide support in this elevated position. Manipulating the body in this manner shortened the length of the gastrocnemius muscle while the length of the soleus muscle remained unchanged. Levels of voluntary plantar flexor torque were produced with the aid of visual feedback. A torque ramp of 2 Nm • s_1 was displayed as a beam on an oscilloscope while a second beam, corresponding to the voluntary plantar flexor torque, were displayed simultaneously. For each trial, the subjects were instructed to align the two beams as precisely as possibly. The motor task was to gradually increase the amount of plantar flexor torque at a rate of 2 Nm • s~' until motor unit activity could be isolated at a signal-to-noise ratio of at least 2:1. At this point, the ramp was held at that force level, and the subject instructed to maintain the force output for an additional 10 to 15 s, thus maintaining motor unit firing activity. This task was performed for both the long and the short lengths of the MG muscle. 3.2.2 Single motor unit and EMG recordings The skin at all electrode sites was shaved and cleaned with 95% ethanol prior to electrode insertion. Single motor unit activity was recorded from MG muscle using a sterile concentric needle electrode (0.46 mm diameter with a 0.07 mm2 recording area; Medelec, UK) inserted percutaneously and randomly repositioned by the experimenter after each trial. Intramuscular EMG was recorded from the lateral aspect of the soleus muscle using fine-wire electrodes 18 Long Position Figure 3.1 The experimental set-up, including subject positions with the knees extended and flexed. The subject's foot was tightly secured to a footplate to minimize ankle movement. (A) concentric needle electrode inserted into medial gastrocnemius muscle (EMG recording above). (B) fine-wire intramuscular electrodes inserted into soleus (EMG recording above). (C) oscilloscope (2 Nm • s_1 ramp and force feedback) to provide the subject with visual feedback. (D) force transducer located at the distal end of the footplate. 19 (0.075 mm diameter, twisted stainless steel, Teflon coated; Leico, USA) constructed in a "double-hook" manner. A sterile hypodermic needle (0.880 mm) was used to insert the wires, and prior to this, the skin was anaesthetized by superficial injection of 0.5-1 ml prilocaine (Citanest, 5 mg • ml "'). The potential sensitive area was the uninsulated end of each wire, 2 mm in length, with an inter-electrode distance of approximately 3-4 mm. A surface reference electrode (self-adhesive Ag/AgCl electrode; Medicotest, Denmark) was placed on the lumbar spine (L3) of each subject. 3.2.3 Signal processing Soleus intramuscular EMG was amplified xlOOO and band-pass filtered between 10 and 800 Hz (NL824 and NL125; Neurolog, UK). Torque signals were low-pass filtered at 20 Hz and analogue-to-digital (A/D) converted with the soleus EMG signals (16 bit) at a sample rate of 1 kHz (Spike2 and Power 1401; Cambridge Electronics Design, UK). Motor unit activity from MG was amplified xlOOO, band-pass filtered between 0.3 and 5 kHz and similarly A/D converted at a sampling rate of 15 kHz. For each trial, soleus intramuscular EMG root mean square (rms) was calculated over the entire period using 200 ms bins. To establish the overall torque and soleus EMG rms level that corresponded to the onset threshold of MG motor unit activity, the peak of the first measurable action potential (exceeding 2:1 signal-to-noise ratio) was used to calculate the appropriate values. Single motor unit activity discrimination was performed offline using the Spike2 spike template matching software with an 80% confidence interval to ensure the analysis of single motor unit potentials. False-positive and/or negative classifications due to spurious frequency changes or action potential collisions were not observed. 3.2.4 Statistics Means (± SD) were calculated for all variables. A one-way analysis of variance with repeated measures was used to analyze the torque and soleus EMG rms levels. An independent / test was used to evaluate differences between mean firing frequencies and amplitudes for MG motor units in the long and short positions. Differences between the means were considered statistically significant at a level of P < 0.05. All statistical analyses were performed using the Statistica software package (StatSoft, USA). 3.3 RESULTS After several practice trials, subjects were able to consistently match and follow the visual ramp, thereby increasing the rate of plantar flexor torque development at approximately 2 Nm • s_1, and maintain a constant level of torque once a MG motor unit began to discharge. 20 Single motor unit and EMG recordings from a single subject during such ramp contractions in the knee extended (long) and knee flexed (short) positions are presented in Figure 3.2. 3.3.1 Motor unit recordings Using the concentric recording electrode, 229 single motor units were recorded from MG. A little more than half of these recordings were achieved with the MG at the long length (121 of 229, 53%) while the remaining units (108 of 229, 47%) were recorded with the MG at the short length. For a specified level of plantar flexor torque (20 Nm), the level of soleus EMG rms observed at the short length was significantly greater than that recorded at the long length (P < 0.01). Moreover, with gastrocnemius in the shortened position, motor unit recruitment occurred at significantly higher levels of soleus EMG rms activity and torque output (P < 0.01). This behaviour was consistent across all subjects, and the difference in plantar flexor torque (torque short - torque long) that corresponded to the onset of MG single motor unit activity in the long and short lengths ranged from 17 to 38 Nm. This was similar to the difference in soleus EMG rms (rms short - rms long), which ranged from 0.09 mV to 0.30 mV. The means ± SD of the soleus EMG rms level for a torque output of 20 Nm in the knee extended and flexed positions corresponded to 0.10 ± 0.05 mV and 0.19 ± 0.09 mV (an increase of ~ 47%). The mean torque and soleus EMG rms corresponding to the onset of MG single motor unit activity are shown in Table 3.1, Table 3.2 and Figure 3.3, respectively. The mean amplitude for the MG units recruited at the short length (0.47 ± 0.43 mV) was significantly greater than the amplitude of those units recruited at the long length (0.30 ± 0.24 mV; P < 0.02). Additionally, at the onset of motor unit activity, the initial firing frequency for the first ten action potentials recorded at the short length (5.8 ± 2.2 Hz) was significantly less than the discharge rate for the same number of units recorded at the long length (7.4 ±2.6 Hz; P < 0.01). 3.3.2 Plantar flexor torque versus EMG thresholds The levels of both torque and soleus EMG rms corresponding to the onset of MG motor unit activity are plotted in Figure 3.4. In the long length, MG motor units were generally recruited at low levels of plantar flexor torque (less than 5 Nm) and soleus EMG rms levels generally less than 0.1 mV. However, there were 9 of 121 units (7.4% and for the most part from subject 7) recorded in this position that were associated with an elevated level of soleus activation and higher torque. These units were recruited at torque outputs that ranged from 10 to 35 Nm (mean 27 ± 10 Nm) and soleus EMG rms levels of 0.12 to 0.38 mV. On the other hand, there was 21 O/wef o/"MG Single Motor Unit Activity Knee Extended (Long) Subject Torque pSD Soleus EMG pSD (Nm) rms (mV) 1 1.70 1.40 0.046 0.005 2 0.79 0.63 0.028 0.010 3 0.79 0.61 0.023 0.003 4 0.72 0.54 0.018 0.003 5 1.15 1.20 0.023 0.002 6 0.94 0.83 0.023 0.002 7 8.73 13.78 0.110 0.126 8 1.41 2.28 0.018 0.027 9 0.49 1.24 0.015 0.011 Mean 2.97 0.045 SD 7.78 0.075 Table 3.1 The mean (± SD) plantar flexor torque and soleus EMG rms levels corresponding to the onset of medial gastrocnemius muscle (MG) single motor-unit activity at the long position for each subject. A mean of 12 (± 1.3) MG units were recorded in each condition for each subject. Onset of MG Single Motor Unit Activity Knee Flexed (Short) Subject Torque pSD Soleus EMG pSD (Nm) rms (mV) 1 34.81 3.76 0.344 0.074 2 34.45 3.48 0.231 0.211 3 38.16 3.29 0.316 0.084 4 37.98 5.09 0.332 0.019 5 32.04 2.28 0.190 0.016 6 31.61 3.61 0.226 0.011 7 39.07 , 9.51 0.313 0.093 8 19.00 8.50 0.112 0.072 9 32.48 9.81 0.121 0.048 Mean 32.14 0.231 SD 10.25 0.129 Table 3.2 The mean (± SD) plantar flexor torque and soleus EMG rms levels corresponding to the onset of medial gastrocnemius muscle (MG) single motor-unit activity at the short position for each subject. A mean of 12 (± 1.3) MG units were recorded in each condition for each subject. 22 Knee Extended Knee Flexed Figure 3.2 Concentric needle recordings of single motor-unit activity from medial gastrocnemius muscle (MG) in the extended and flexed knee position for a single subject. The vertical lines superimposed on the raw data indicate the onset of plantar flexor force production (A) and the onset of motor-unit activity (Fj). Dotted lines superimposed on the voluntary torque trace show the 2 Nm • s"1 ramps. Action potentials below (n = 10) were superimposed to demonstrate the continuous sampling of the same unit (gastrocnemius muscle). 23 Long Short B. 0.4 > Q 0.3 B Long Short Figure 3.3 The means ± SD (n = 9) for the torque and soleus EMG rms that is required to recruit a motor unit in both the long (black bars) and short (white bars) positions. The recruitment thresholds reached a level of statistical significance between knee positions (* P < 0.05). 24 60 50 40 E ^ 30 S" o 20 10 8 o o e coo oo°o° Oo 8 ° o » o o o o^o o 0 o oo b^o' o o o c_ o o o I c o o°o° Q 00 50 • 0oo o 0 • • e • O OO o 00 o o °o o8#° o o 0.1 0.2 0.3 0.4 Soleus EMG rms (mV) o o 0.5 0.6 Figure 3.4 Scatter plot of the torque threshold versus the soleus EMG rms threshold for the total number of motor-unit recordings in this study (n = 229). Thresholds were plotted for motor units recorded in the long (filled circles) and short (empty circles) positions. 25 a greater distribution in the recruitment levels of MG motor units in the flexed knee position, especially pertaining to the soleus EMG rms thresholds. Only a few units (3 of 108, 2.8%, mostly from subject 8) were recorded with thresholds less than 10 Nm (ranging from 2 to 8 Nm). At the short length, there were at least nine trials in which a continuous torque ramp of up to 50% maximal voluntary contraction (MVC) failed to detect motor unit activity from the MG muscle. 3.3.3 Plantar flexor torque To estimate relative torque production, data from a previous experiment on a similar population were used (Cresswell et al. 1995). In this prior study, MVC were performed in both flexed and extended knee positions. The maximal voluntary torques were 135 ± 24 Nm and 104 ± 24 Nm at the long and short lengths, respectively. As such, in the present study a mean torque recruitment of 3 ± 8 Nm in the extended knee position would be equivalent to 2 ± 6 % of the maximal voluntary effort at that same position (range from ~ 0 to 26%). Similarly for the flexed knee, a torque output of 32 ± 10 Nm equated to approximately 24 ± 9 % and 31 ± 10 % of the MVC at the long and short lengths, respectively (ranging from 3 to 46% for the short length). It is important to note, however, that these values are based on the calculated MVC for the appropriate position. 3.4 DISCUSSION The main findings of this study were the significantly increased levels of soleus EMG rms and plantar flexor torque corresponding to the onset of firing of MG motor units during ramped voluntary isometric plantar flexor efforts with the gastrocnemius muscle shortened. The observed changes are credited to increased inhibition of the MG motoneuron pool due to the diminished force-producing capabilities of the MG motor units because of their reduced length by means of knee flexion. Concentric needle electrodes were used rather than fine-wire electrodes, as used by Miles et al. (1986), Vander Linden et al. (1991) and Christova et al. (1998), for the recording of single motor unit activity in the MG muscle. The use of indwelling fine wire has an advantage in that it may result in the same motor unit being recorded even once the muscle has undergone a length change. However, a limitation is the difficulty of relocating the wires between trials, especially positioning them deeper, in order to obtain recordings from different regions within the same muscle. In the present study, the concentric needle was randomly positioned within the muscle between all trials to obtain recordings from a larger muscle volume. Using this technique, it was not possible to monitor the same motor unit at different muscle lengths; however, we were able to 26 record from at least 20 different sites in all subjects and thus were able to obtain a larger random sample of motor units. Due to the consistency of MG onset thresholds at both the long and short lengths, we are confident that the differences in onset thresholds are a direct result of changes in muscle length and are not complicated by the positioning of the needle within the muscle. The control of torque production by the triceps surae muscle group is complex, mainly because the gastrocnemius muscle is bi-articular, crossing both the knee joint and ankle, while the soleus muscle crosses the ankle only. As such, any change in knee angle will independently alter the length of gastrocnemius in relation to soleus. Furthermore, it is believed that the gastrocnemius muscle, which is like the majority of muscles that are involved in stretch-shortening actions, operates primarily on the ascending limb of the force-length relationship (Rassier et al. 1999) and that when it is maximally shortened via knee flexion, and independent of ankle angle, it is incapable of producing force (see Herzog 2000). We, and other authors, have previously demonstrated that, with a progressive shortening in the gastrocnemius muscle via knee flexion, there is a reduction in both voluntary and involuntary maximal plantar flexor torque output (Fugl-Meyer et al. 1979; Sale et al. 1982; Cresswell et al. 1995). Recently, Kawakami et al. (1998) reported that, as the knee is moved to a more flexed position, the medial gastrocnemius fascicles decrease their length by up to 24 mm and thereby increase their pennation angle by approximately 22°. Such a change may limit the amount of slack that can be taken up in the muscle, thereby resulting in the overall force transmitted to the tendon being reduced. To accommodate for the increased slack and maintain torque output in a shortening muscle, the CNS may increase the firing frequency of motor neurons (Vander Linden 1991). However, once the muscle fiber reaches a critical shortened length, the overall contribution of the muscle to torque output will be minimal, even if fully activated. In this condition, the muscle is called "actively insufficient", and it would therefore seem appropriate to reduce the drive to, or neural outflow from, spinal motoneurons whose muscle fibers reside at compromised shortened lengths (see Herzog 2000). In earlier studies (Cresswell et al. 1995; Pinniger et al. 2000) we observed a significant reduction in surface EMG from both heads of the gastrocnemius muscle during voluntary plantar flexor efforts with the knee flexed to shorten the gastrocnemius. From these studies, it was uncertain whether the EMG reduction was due to electrode-muscle configuration changes, thereby recording from different volumes and possibly different numbers of fibers within the muscle, or due to inhibition of gastrocnemius motor units induced by the reduction in their fiber lengths. To further investigate the latter of these theories, we have endeavoured to determine 27 more specifically the effect of changing gastrocnemius muscle length on the recruitment and firing behaviour of its motor units. Similar to previous findings, a greater level of voluntary drive, as evidenced by a significant increase in soleus EMG rms, was required by the plantar flexors to achieve the same level of torque in the flexed knee position. This finding was expected, as the soleus muscle must now be activated to a greater extent to compensate for the limited force contribution of the shortened gastrocnemius. It was clearly evident when trying to record single motor units from the shortened MG that the onset of their activity was significantly delayed when performing the voluntary torque ramps. This meant that the MG units at short lengths were recruited at significantly greater levels of plantar flexor drive and torque production. This finding corroborates earlier results of decreased surface EMG amplitude from the gastrocnemius and triceps surae at short as compared to long lengths when performing both submaximal and maximal voluntary isometric plantar flexions (Fugl-Meyer et al. 1979; Sale et al. 1982; Cresswell et al. 1995; Pinniger et al. 2000). Moreover, as the intramuscular recordings in this study were made from many regions within the muscle, the increased threshold of MG units is unlikely to be a result of electrode-muscle configuration changes, but due to an increased level of inhibition or disfacilitation of motor units within the MG motoneuron pool. Interestingly, other studies have demonstrated opposite changes in motor unit behaviour with changes in muscle length. For the biceps brachii, Christova et al. (1998) reported an increase in motor unit discharge rate as it underwent shortening via elbow flexion. In that study, it was unlikely that the biceps brachii was activated on the initial portion of the ascending limb of the length-tension relationship, as the upper-arm was maintained parallel to the body and not flexed, which would have additionally shortened the muscle. Similarly, Vander Linden et al. (1991) showed increased motor unit discharge rates in the shortened tibialis anterior muscle and concluded that this was due to the decreased peak tension and shorter one-half relaxation times observed in shortened muscle. However, it may well be that the tibialis anterior muscle does not become actively insufficient at their test position of 20° dorsiflexion. The underlying control mechanisms behind the increased recruitment thresholds for gastrocnemius motor units at shortened muscle lengths are not clear. It may well be that cortical drive to the MG motoneuron pool is reduced, regardless of an increased drive to the synergistic soleus. However, more likely candidates responsible for the reduced excitability of the MG motoneuron pool, or even inhibition of specific motor units whose fibers are at less than optimal lengths, are peripheral afferent receptors. Muscle spindles have the ability to reduce motoneuron 28 excitability through reduced afferent input and, as such, are potential candidates for the excitability changes we have seen. However, reduced la afferent input, via increased presynaptic inhibition of the la terminals through the possible heightened activation of group II, III and IV peripheral inputs may also result in disfacilitation of the MG motoneuron pool. Direct and indirect inhibitory effects from cutaneous and joint afferents are also potential mechanisms, as it has been shown that the former receptor can have strong influences on motoneuron excitability (Kanda et al. 1977; McNulty et al. 1999). If afferent input is responsible for modulating gastrocnemius motoneuron excitability with changes in length, then the questions still remains as to whether the total motoneuron pool is receiving equivalent inhibitory input or whether this input is directed toward specific motor units. The latter may be the case if non-uniformity of muscle fiber length exists and the CNS can adequately detect motor units or populations of motor units who are no longer capable of producing force. The result of greater MG motor unit amplitudes at shorter muscle lengths suggests that higher threshold or faster motor units were the first activated and that the slower units, which were recruited first at the longer lengths were now inhibited. The initial slower firing frequency of these units at short lengths appears to support this notion (Kudina 1999). However, it must also be pointed out that changes in muscle geometry can have an effect on motor unit amplitude, with larger amplitudes being recorded at shorter muscle lengths (Gerilovsky 1989; Garland et al. 1994) Nonetheless, this study has demonstrated that, with a reduction in muscle length, the onset of motor unit activity in the MG muscle occurs at significantly higher levels of both plantar flexor torque and soleus EMG rms activity. This alteration in recruitment may reflect a general increase in the recruitment threshold of MG motoneurons, or specific inhibition of motor units with muscle lengths that are no longer capable of producing force. At present, it is unclear whether motor units of varying types are equally affected or whether there is a somewhat selective inhibition of low-threshold motoneurons. Nonetheless, by limiting the activity of a muscle that is actively insufficient, the CNS is able to minimize metabolic costs while maximizing the force output within a functional group. 3.5 BRIDGING SUMMARY The ability to selectively control the activity of a motoneuron is important for it allows the nervous system to adjust the motor output to meet the requirements of the task. The higher recruitment thresholds observed in Experiment 1 may reflect a specific inhibition of motor units that reside at non-optimal force-producing lengths. It has been suggested that the presynaptic 29 inhibitory mechanisms that regulate the excitability of the motoneuron pool are controlled by descending cortical inputs (Quevedo et al. 1993). There are several pathways that might be involved, including the vestibulospinal tracts which regulate the activity of spinal neurons that innervate lower limb muscles (Wilson and Melvill Jones 1979). When the tonic vestibular activity is removed, there is a reduction in the amplitude of the ipsilateral soleus H reflex (Lacour et al. 1976). Therefore, a change in the vestibular afferent activity may affect the spinal mechanisms that indirectly influence the excitability of the motoneuron pool. Galvanic vestibular stimulation has often been used to artificially activate the vestibular system in human subjects (Nashner and Wolfson 1974). The low-level galvanic current is thought to activate descending spinal pathways that are responsible for controlling presynaptic inhibition of la afferents via interneurons mediating primary afferent depolarization (Manzoni 1988; lies and Pisini 1992). But while GVS has been shown to produce transient EMG responses in the lower limb during quiet stance (Britton et al. 1993; Fitzpatrick et al. 1994; Watson and Colebatch 1997), it is unclear what effect the artificial vestibular stimulus may have on a muscle that is not posturally engaged. So, the purpose of Experiment 2 was to establish if GVS could be used to activate descending vestibulospinal pathways to influence the activity of the lower limb motoneuron pool in prone lying subjects. By measuring the amplitude of the passive soleus H reflex during periods of GVS, the effect on the conditioning stimulus on the motoneuron pool was assessed. 30 CHAPTER 4 MODULATION OF THE SOLEUS H REFLEX IN PRONE HUMAN SUBJECTS USING GALVANIC VESTIBULAR STIMULATION (Published: Clinical Neurophysiology 112: 2159-2163, 2001.) 4.1 INTRODUCTION Vestibular information has long been recognized as an important source of sensory input in balance control (Inglis and Macpherson 1995). Signals from the vestibular apparatus not only provide information about the orientation and motion of the head in space (Horak et al. 1994) but may also modulate lower limb postural responses to allow for the accurate realignment of the body following a postural perturbation (Inglis et al. 1995). These lower limb muscle responses (EMG) that arise from vestibular activation have been widely studied in humans using both natural (Melvill Jones and Watt 1971; Greenwood and Hopkins 1976) and artificial stimuli (Fitzpatrick et al. 1994; Watson and Colebatch 1997). Some of these methods include galvanic stimulation, which has often been used as a non-painful technique for activating the vestibular system (Day et al. 1997) by direct modulation of the irregularly firing afferents of the vestibular nerve (Minor and Goldberg et al. 1991). Low intensity stimulation delivered to electrodes placed over the mastoid process results in a transient EMG response predominantly recorded in the soleus muscle (Nashner and Wolfson 1974). The magnitude of this response appears to be dependent upon both the head-on-body alignment (lies and Pisini 1992) and a background level of activity in the muscle (Watson and Colebatch 1997). Moreover, increasing the duration of the galvanic stimulus can also prolong the degree of activity in the muscle (Britton et al. 1993). While galvanically induced EMG responses have been documented in human subjects it has been difficult to clarify the nature of this activity. For instance, several authors have identified a short latency component followed by a medium latency response at around 60 and 100 ms respectively (Britton et al. 1993; Watson and Colebatch 1997). A single galvanic stimulus can therefore evoke two responses in the motoneuron pool that are opposite in polarity (lies and Pisini 1992). Why there are seemingly two contrasting responses remains unclear, however the onset of the later component appears to coincide with galvanically induced postural sway in freely standing subjects (Britton et al. 1993). When subjects were either braced or seated, this brief EMG activity was absent, suggesting a task dependent role for this vestibular-evoked muscle response (Fitzpatrick et al. 1994). That is, when vestibular cues are possibly useful, as in postural stance, there is an increase in the gain of the vestibulospinal pathways facilitating this galvanic response, ln fact, if upper limb muscles are being used to maintain 31 balance, transient EMG responses evoked through galvanic stimulation can be observed in the triceps brachii muscle (Britton et al. 1993). If galvanic stimulation activates vestibulospinal pathways (Muto et al. 1995), it may be possible to elicit a response in a quiescent muscle that is not posturally engaged. The aim of this study was therefore to determine if galvanic vestibular stimulation (GVS) could evoke EMG responses in the soleus when the muscle was not being used for postural support. To achieve this, the amplitude of the soleus H reflex was measured during periods of GVS to assess the effect of the conditioning stimulus on the motoneuron pool. While evidence suggests that the position of the head can modify the galvanic response in standing subjects (lies and Pisini 1992), it is also known that rotation of the head over the shoulder activates tonic neck reflexes (TNR) which can also modulate the excitability of the soleus motoneuron pool (Hayes and Sullivan 1976). Therefore to minimize any confounding effects of head position, the soleus H reflex was measured during periods of GVS with head facing forward. 4.2 METHODS Ten healthy volunteers (5 males, 5 females) between 22 and 37 years of age participated in this study. Informed consent was obtained and the local ethics committee approved the following procedures. Since there are no significant differences in H reflex amplitudes between semi-inclined and inclined positions (Al-Jawayed et al. 1999), subjects lay in a prone position on a padded bed with both legs extended. The right foot was slightly elevated and secured to a brace to minimize ankle movement. The ankle angle was held constant at approximately 90° (measured as the internal angle between the lower leg and the foot). This created a slight flexion at the knee joint, which was measured at approximately 160° (measured as the internal angle between the thigh and the lower leg). Surface EMG was recorded from the right soleus muscle with bipolar electrodes (self-adhesive Ag/AgCl electrodes). EMG was amplified xlOOO and sampled at 10 kHz (Spike2 and CED 1401, Cambridge Electronics Design, UK). 4.2.1 Experimental design A surface-stimulating electrode was placed over the popliteal fossa while a 1 ms square wave pulse was delivered (Grass S48, GRASS Instruments, USA) over the tibial nerve at 0.2 Hz. The stimulating electrode was a steel ball placed in a frame and fastened to the leg by straps to make sure that a constant stimulus was delivered to the tibial nerve. To elicit a consistent H reflex, subjects were tested at a stimulus level that produced an M-wave equal to 15-20% of their maximum M response (between 30 and 90 V). The M wave amplitude was monitored on-line 32 using an oscilloscope (BK Precision 20 MHz Analogue Model 2522B) to ensure that the appropriate M-wave amplitude was maintained within and between trials. Monopolar monaural galvanic stimulation (4 mA, 2 s square wave pulse) was delivered to two carbon-rubber electrodes (9 cm2) placed over the subjects' right mastoid process and wrist joint, respectively. The duration of the galvanic stimulus was intentionally prolonged to reduce any possible confounding off-stimulation effects. Subjects received both cathodal (negative) and anodal (positive) galvanic stimulation. In each trial, during GVS the electrode over the mastoid was the stimulating electrode while the electrode over the wrist served as a reference. With this electrode configuration, subjects could barely perceive the 4 mA stimulus in the prone position. At no time did subjects report any painful sensations behind the ears or flashing in the eyes that may have caused a startle response during the testing periods. To measure the effect of the conditioning stimuli on the soleus H reflex, the tibial nerve was stimulated with the subject face down against the bed with their eyes closed, corresponding to a head forward position. Within each trial, the first H reflex or control was referred to as the test H reflex. The subsequent H reflex that was coupled with GVS was defined as the conditioned H reflex (Figure 4.1). In each condition, 20 consecutive H reflexes (10 test and 10 conditioned) were recorded. Based on previous reports in which transient EMG responses evoked through GVS occurred between 80 and 120 ms (Nashner and Wolfson 1974; Britton et al. 1993; Fitzpatrick et al. 1994; Hlavacka et al. 1999), the interval between the onset of GVS and the H reflex was held constant at 100 ms. While subjects showed a response to the conditioning stimuli, the interval between GVS and the H reflex was subsequently investigated in 4 test subjects to determine the optimal latency for the descending vestibular influences on the soleus motoneuron pool. This period was varied between 0 and 200 ms in 20 ms intervals. Following a control stimulus, a second H reflex was evoked during GVS. A trial therefore consisted of 10 consecutive H reflexes (5 test and 5 conditioned H-reflexes). 4.2.2 Statistics The amplitude of the H reflex was calculated as the distance between the positive and negative peak. Mean amplitudes (± SE) were calculated for both test and conditioned H reflexes. A paired t test was used to evaluate differences between test H-reflexes and conditioned H reflexes in the anode and cathode conditions. A two-way ANOVA with repeated measures was used to examine the interval between the two stimuli for anodal or cathodal galvanic stimulation. Differences between the means for all conditions were considered statistically significant at a c Test Reflex Conditioned Reflex Figure 4.1 The experimental setup is presented, showing the subject position (A) and the relationship between the galvanic stimuli (B) to the tibial nerve stimulation (C). The H reflexes that were evoked without GVS were referred to as test reflexes, while the H reflexes evoked with GVS were referred to as conditioned reflexes. 34 level of P < 0.05. All statistical analyses were performed using the Statview software package (SAS Institute Inc., USA). 4.3 RESULTS For all 10 subjects the effect of a conditioning stimulus was monitored in over 1600 H reflex recordings. Within a trial, the baseline amplitude of the soleus H reflex did not exceed 5% of the total amplitude across all subjects. In addition, there was remarkable consistency in the M wave within and between trials, as variations in absolute amplitude did not exceed 6 % of the total amplitude. Due to the consistency of the M and H responses in the test reflexes, any change in soleus H reflex amplitude must have resulted from the influence of the conditioning stimuli (see Figure 4.2). Therefore, in each trial, the mean amplitude of the conditioned reflexes was compared with the level of the mean test reflexes. This demonstrated that H reflex amplitude could be manipulated by GVS according to stimulus polarity (Figure 4.3). 4.3.1 GVS polarity and the H reflex With the stimulating electrode over the right mastoid process, anodal stimulation inhibited the right H reflex amplitude by a mean of 3.3% (SE 1.33) in comparison with the test reflex. A mean decrease in absolute amplitude from 2.65 mV (SE 0.14) to 2.59 mV (SE 0.14) was observed. Both the relative and absolute changes in H reflex amplitude were found to be significant (at P < 0.02 and P < 0.05, respectively). On the other hand, cathodal stimulation produced a mean increase of 3.2 % (SE 1.78) in amplitude. This resulted in an increase in mean amplitude from 2.57 mV (SE 0.13) to 2.63 mV (SE 0.13). Once again, the relative and absolute differences were found to be significant (at P < 0.03 and P < 0.04, respectively). 4.3.2 Interval between GVS and the H reflex Since previous studies using GVS have reported transient EMG responses in the lower limb between 80-120 ms (Nashner and Wolfson 1974; Britton et al. 1993; Fitzpatrick et al. 1994), the descending influence of GVS on the amplitude of the H reflex was investigated further in four subjects as the interval between GVS and tibial nerve stimulation was systematically altered. This was done to ensure that our initial testing interval was optimal for measuring the galvanic response. While shorter inter-stimulus intervals produced minimal changes in H reflex amplitudes, at an interval of 60 ms, cathodal stimulation had an inhibitory influence on the conditioned reflexes. At 80 ms, this same cathodal stimulus had a facilitatory effect whereas anodal stimulation also produced a slight facilitatory response. However at longer intervals of 35 Conditioned H Reflex Figure 4.2 Sample EMG responses from one subject to GVS conditioning in the right soleus muscle. The overlapping of the H reflexes, with a slight offset, demonstrates the corresponding change in amplitude ofthe conditioned reflex. 36 A 6 4 i p> _4 A -6 J Anode Cathode Anode Cathode Figure 4.3 In (A) the mean test reflex amplitude is expressed as baseline or zero. The relative difference in H-reflex amplitude is illustrated as the change from baseline. In (B) the absolute means ± SE (n = 10) for test (white bars) and conditioned (black bars) H reflex amplitudes are presented. The changes in amplitude observed with GVS were found to be statistically significant (*P < 0.05). 37 10 i 0 40 60 80 100 120 140 160 200 Time (ms) Figure 4.4 The relative difference between the test and conditioned soleus H reflex amplitudes was plotted according to the latency between the onset of GVS and tibial nerve stimulation. The mean peak modulatory effect for both cathodal stimulation (solid circles) and anodal stimulation (empty circles) was observed at 100 ms in 4 subjects. However, the changes in H reflex amplitude did not reach statistical significance (Anode P < 0.06, Cathode P < 0.28). 38 100 to 140 ms, galvanic-conditioning produced a greater cathodal response, and an anodal effect that was opposite in polarity. For both anodal and cathodal stimulation, the peak modulatory influence was observed when the onset of GVS began 100 ms prior to the H reflex stimulation (see Figure 4.4). Although the galvanic conditioning altered the amplitude of the reflex, the modulation of the soleus H reflex according to stimulus onset was not found to be significant (Anode P < 0.06, Cathode P < 0.28). This may have resulted from the variations in the conditioning responses observed between subjects. 4.4 DISCUSSION It has been shown that following a brief galvanic stimulus to the mastoid processes there is a modulation of the EMG response in the soleus muscle in quietly standing humans (Nashner and Wolfson 1974; Britton et al. 1993). Furthermore, when subjects were braced this transient EMG activity was absent (Fitzpatrick et al. 1994) suggesting that this response was task dependent. However we hypothesized in the present study that if GVS activates vestibulospinal pathways (Muto et al. 1995), it should be possible to see a modulation of the soleus H reflex in prone human subjects, even if not actively engaged in the control of standing balance. The results supported this hypothesis, as cathodal stimulation facilitated the H reflex while anodal stimulation inhibited the amplitude of the ipsilateral soleus H reflex. Galvanic stimulation during dynamic activities such as trunk tilt (Severac-Cauquil and Day 1998) or steady-state gait (Bent et al., 2000) results in larger postural adjustments than those observed during upright stance. Presumably there is a greater reliance on vestibular input during dynamic tasks, corresponding to an increased GVS response. In each of the aforementioned paradigms, when GVS activity is observed in the lower limb, subjects are often free to move either through pre-planned actions or natural postural sway. This has led some to speculate that galvanically induced EMG responses do not constitute a vestibulospinal reflex but rather reflect a compensatory action to the movement (Storper and Honrubia 1992). However, in the present study the subjects remained relaxed and prone during the testing procedures and still demonstrated a lower limb response. Therefore the modulation of the soleus H reflex must have resulted from vestibulospinal influences initiated by the galvanic stimulus. Our understanding of how GVS affects the vestibular system stems from animal research in which the firing frequency of irregular afferents of the vestibular nerve has been monitored during periods of stimulation. Recordings from the irregular afferents in the squirrel monkey demonstrate a reduction in firing frequency with anodal currents while cathodal stimulation increases the firing rates (Minor and Goldberg 1991). Lacour and colleagues (1976) have shown 39 that when this tonic vestibular activity is removed following a unilateral neurotomy, there is a reduction in the soleus H reflex in the baboon. Therefore, since the level of excitability of the ipsilateral soleus motoneuron pool is dependent upon descending vestibular influences, any change in the firing frequency of the vestibular afferents may have an effect on the amplitude of the soleus H reflex. This implies that the modulation of the soleus H reflex in the present study resulted from a change in vestibular afferent firing rate, initiated by the externally applied galvanic current. There are several possibilities as to how GVS specifically modulates the amplitude ofthe soleus H reflex. Facilitation of the motoneuron pool via a monosynaptic connection appears to be unlikely (Manzoni 1988) since the peak conditioning effect was not observed until there was a long latency between the onset of the galvanic stimulus and the peripheral nerve stimulus. The vestibulospinal tract contains large diameter fibres that have a conduction velocity of approximately 60-80 m • s"1. If GVS directly influenced the threshold of the motoneuron pool, it should have been possible to evoke an appropriate response at intervals of much less than 100 ms. Consequently, galvanically-induced modulation of the soleus H-reflex may reflect polysynaptic connections. Following a brief galvanic stimulus, there appears to be a delay in the lower limb response as the artificial vestibular input is integrated within the central nervous system (Britton et al. 1993). Once this has been achieved, vestibulospinal impulses are transmitted to the lower limb either directly through the lateral vestibulospinal tract or indirectly through the reticulospinal tract. The latter pathway is thought to control presynaptic inhibition of la afferents in the cat (Manzoni 1988). lies and Pisini (1992) have suggested that GVS might modulate inhibitory pathways responsible for presynaptic inhibition. Therefore, any change in the vestibular afferent firing rate via GVS may consequently affect the level of presynaptic inhibition on lower limb motoneurons. However, Renshaw cells, located in the ventral horn of the spinal cord, also have been shown to respond to descending vestibulospinal inputs (Pompeiano 1988). Renshaw cells have weak inhibitory synaptic connections with alpha motoneurons. Therefore, any change in Renshaw cell activity would also be capable of modulating the excitability of the motoneuron pool. While it remains unclear as to how GVS specifically modulates motoneuron excitability, the changes that we observed in the soleus H reflex resulted from the application of the conditioning stimuli. Although the modulation was significant, the small changes observed in amplitude might have resulted from the testing position of the subject. The amplitude of the soleus H reflex is lower when the subject is lying prone in comparison to when the subject is 40 completely upright (Aiello et al. 1983). Regardless, these results indicate that in some circumstances it may be possible to evoke vestibulospinal reflexes using galvanic stimulation in prone human subjects. This may provide another means through which galvanic stimulation can be used, possibly in a clinical setting, to investigate the role of the vestibular system in lower limb motor control. 4.5 BRIDGING SUMMARY Galvanic stimulation has been shown to modulate the ongoing EMG activity in the soleus muscles of standing subjects producing a short and medium latency response at 60 and 100 ms respectively (Fitzpatrick et al. 1994; Watson and Colebatch 1997). The results from Experiment 2 demonstrated, for the first time, that GVS can also modulate the amplitude of the passive soleus H reflex in prone lying subjects. This effect may represent a medium latency response (Britton et al. 1993) since the greatest change in H reflex amplitude occurred when the galvanic stimulus was delivered 100 ms prior to the tibial nerve stimulus. Although the modulation was small, the fact that GVS evoked a change in the ipsilateral soleus H reflex while subjects faced forward is in contrast to previous reports in upright subjects. Nashner and Wolfson (1974) reported that the transient EMG response in the triceps surae muscle group was absent if the head was facing forward. While the position of the head influences the GVS response in standing subjects (lies and Pisini 1992), rotation of the head independently alters the excitability of the motoneuron pool (Hayes and Sullivan 1976; Traccis et al. 1987). At present, it is not clear how GVS, may interact with head-on-body alignment in prone lying subjects. Therefore, the purpose of the subsequent study was to examine the influence of head position and galvanic stimulus polarity on the soleus H reflex. By examining how vestibular and neck afferent inputs are integrated within the nervous system, we may be able to identify how vestibulospinal inputs regulate the activity of lower limb motoneurons in a passive muscle. 41 CHAPTER 5 INTERACTION EFFECTS OF GALVANIC VESTIBULAR STIMULATION AND HEAD POSITION ON THE SOLEUS H REFLEX IN HUMANS (Published: Clinical Neurophysiology 113: 1709-1714,2002.) 5.1 INTRODUCTION Galvanic vestibular stimulation (GVS) has been widely used as a non-painful technique for activating the human vestibular system (Watson and Colebatch 1997; Day et al. 1997). Typically, a low-intensity stimulus, delivered to an electrode placed over the mastoid process, can modulate the firing frequency of the irregularly firing afferents of the vestibular nerve (Goldberg et al. 1984). An anodal or positive stimulus tends to decrease the firing frequency whereas a cathodal or negative stimulus increases the level of activity of the irregular afferents (Minor and Goldberg 1991). This change in the discharge properties of the vestibular nerve causes an upright individual to sway toward the anodal stimulus and away from the cathodal stimulus (Lund and Broberg 1983). During quiet stance, GVS produces changes in the electrical activity (EMG) of postural muscles, such as the soleus (Nashner and Wolfson 1974; lies and Pisini 1992). The galvanically induced EMG response has both a short and medium latency component that are opposite in polarity (Britton et al. 1993; Watson and Colebatch 1997). The magnitude of both responses appears to be dependent on the overall stability or steadiness of the individual (Welgampola and Colebatch, 2001). In fact, when subjects were either braced or seated, transient EMG responses in the soleus muscle were absent (Storper and Honrubia 1992; Britton et al. 1993). This has led some to speculate that the GVS-evoked muscle responses observed in the soleus muscle are task dependent (Fitzpatrick et al. 1994). Recently, it was reported that the amplitude of the soleus H reflex could be influenced by GVS in prone human subjects (Kennedy and Inglis, 2001). This was the first study to show a galvanically induced vestibulospinal response in a quiescent muscle that was not posturally engaged. Interestingly, the timing of the effect coincided with the medium latency response observed during quiet stance (Britton et al. 1993; Watson and Colebatch 1997). That is, the greatest change in H reflex amplitude occurred when the galvanic stimulus was delivered 100 ms prior to the H reflex stimulus, regardless of stimulus polarity (Kennedy and Inglis, 2001). However, unlike standing conditions, the amplitude of the soleus H reflex was modulated in the prone position with GVS by changing the stimulus polarity as subjects faced forward. 42 While there is evidence to suggest that the position of the head can influence the GVS response in standing subjects (Nashner and Wolfson 1974; lies and Pisini 1992), it is also known that rotation of the head can independently modulate the excitability of the motoneuron pool (Hayes and Sullivan 1976; Traccis et al. 1987). Interestingly, vestibular and neck afferents activate the same neurons within the vestibular nuclei (Wilson 1992). Consequently, these sensory afferents produce opposite responses, which cancel one another out at the level of the motoneuron pool (Kasper et al. 1988). However, it is not clear how GVS, an artificial vestibular input, may interact with a natural stimulus, such as head-on-body alignment when subjects are lying in a prone position. Therefore, the purpose of the present study is to examine the interaction between head position and galvanic stimulus polarity. We wished to establish under what, if any, conditions could natural head movements and artificial vestibular stimuli produce an optimal response at the level of the soleus motoneuron pool. The amplitude of the soleus H reflex was measured in prone human subjects during periods of galvanic stimulation. In each condition, the head was either rotated towards the left or right shoulder. This allowed us to assess the impact of GVS and head-on-body alignment on the amplitude of the soleus H reflex. The results suggest that although GVS can influence the excitability of the soleus motoneuron pool in prone human subjects, the overall effect is dependent upon the position of the head. 5.2 METHODS This experiment involved ten healthy volunteers between the ages of 24 to 40 years (mean age = 31.2). Written, informed consent was obtained and the clinical research ethics board at the University of British Columbia approved the following procedures. 5.2.1 Experimental design Subjects lay prone on a padded bed with both legs extended. The right foot was elevated and secured to a brace to limit ankle movement. The ankle angle was held constant at approximately 900, measured as the internal angle between the lower limb and the foot. This created a slight flexion at the knee joint, which was measured at approximately 1600 between the internal angle of the thigh and lower leg. Similar to the procedures described in a previous experiment (Kennedy and Inglis, 2001), a surface-stimulating electrode was placed behind the knee to deliver a 1 ms square wave pulse (Grass S48, GRASS Instruments, USA) over the tibial nerve at 0.2 Hz. The stimulating electrode was a steel ball strapped to the leg to ensure that a constant stimulus was delivered to the nerve. 43 Subjects were tested at a stimulus level (between 25-100V) that produced an M-wave equal to 10-15% of their maximum M response. To ensure that the appropriate M-wave amplitude was maintained within and between trials, the M-wave was monitored on-line using an oscilloscope (BK Precision 20 MHz Analogue Model 2522B). Surface EMG was recorded from the right soleus muscle with bipolar electrodes (self-adhesive Ag/AgCl electrodes). Soleus EMG was amplified xlOOO and sampled at 10 kHz (Spike2 and CED 1401, Cambridge Electronics Design, UK). Galvanic stimulation (4 mA, 2 s square wave pulse) was applied using a monopolar monaural configuration with the stimulating electrode (9 cm2) on the right mastoid process and the reference electrode (9 cm2) on the right wrist joint. This allowed us to assess the influence of a galvanic stimulus applied to the right side of the body on the right soleus H reflex. In addition, this electrode configuration was used so that the subjects would barely perceive the 4 mA stimulus. Subjects did not report any painful sensations behind the ears or flashing in the eyes that may have evoked a startle response during the testing periods. In fact, most participants did not feel the GVS stimulus at all, although they did report feeling a perception of movement. The duration of the galvanic stimulus was intentionally prolonged to reduce any possible confounding off-stimulus effects. Subjects received both a cathodal (negative) stimulus and an anodal (positive) galvanic stimulus. To measure any possible interaction between head-on-body alignment and galvanic stimulation, GVS was randomly coupled (anode or cathode) with head position (left or right). The head was turned approximately 900 to the left or right by placing the ear on a marker on the bed. Within each trial, the first H reflex or control was referred to as the test H reflex. The next H reflex was evoked during GVS and defined as the conditioned H reflex. In each trial, twenty consecutive H reflexes (10 test and 10 conditioned) were recorded. In accordance with our previous study (Kennedy and Inglis, 2001), the interval between the onset of GVS and the H reflex was held constant at 100 ms. In an attempt to quantify the overall effect of the conditioning stimuli, the right soleus H reflex was evoked at different head positions without galvanic vestibular stimulation. Subjects placed their forehead on a marker on the bed, corresponding to the head forward, or baseline position. The head was turned approximately 90° to the left or right by placing the ear on a marker on the bed. This time, 10 consecutive H reflexes were recorded for each trial, with the head either facing to the left, forward, or to the right. 44 5.2.2 Statistics The amplitude of the H reflex was measured as the difference between the positive and the negative peak. Mean amplitudes (± SE) were calculated for both test and conditioned H reflexes in all trials. The mean values of each trial for each subject were then compared to evaluate the effect of the conditioning stimuli on the amplitude of the H reflex. A one-way analysis of variance (ANOVA) with repeated measures was used to examine the effect of head position on H reflex amplitude. To assess differences related to head position and GVS, a two-way ANOVA with repeated measures was performed. A Tukey's post hoc analysis was used in the case of significant effects. Differences between the means for all conditions were considered statistically significant at a level of P < 0.05. All statistical analyses were performed using the Statview software package (SAS Institute Inc., USA). 5.3 RESULTS Changes in the amplitude of the soleus H reflex in response to the conditioning stimuli are illustrated in Figure 5.1 for a representative subject. Superimposing the test and conditioned reflexes shows that the overall effect of GVS on the H reflex in a prone individual is modulated by head-on-body alignment. This effect was monitored in 1200 H reflexes from all ten subjects. Between trials, the absolute amplitudes of both the M-waves and test H reflexes did not exceed five percent of the total amplitude. Therefore, we are confident that any change in the amplitude of the conditioned H reflexes must have resulted from the influence of the conditioning stimuli. Accordingly, the mean amplitudes of the test reflexes were compared with the mean amplitudes of the conditioned H reflexes for each subject. 5.3.1 Interaction between Head Position and GVS A comparison of the mean H reflex amplitudes showed that there was a significant interaction between GVS polarity and the position of the head (F19 = 11.15, P < 0.009). A Tukey's post hoc analysis was then used to determine the statistical significant conditions. With the stimulating electrode over the right mastoid process and the head turned towards the right shoulder, anodal stimulation inhibited the right soleus H reflex from 2.61 mV (SE ±0.13) to 2.52 mV (SE ± 0.13). This corresponded to a statistically significant mean decrease of 4.2% (SE ± 1.59) (see Figure 5.2 for the relative changes). Data from a previous experiment on a similar population has shown that with the head facing forward, an anodal stimulus inhibits the H reflex by a mean of 3.3 % (SE ± 1.33) (Kennedy and Inglis, 2001). When subjects turned their head to the left, anodal stimulation only inhibited the H reflex by a statistically insignificant mean of 45 Figure 5.1 An example of the EMG responses in the right soleus muscle from one subject is presented for GVS conditioning coupled with the head positions. The overlapping of the H reflexes, with a slight offset, demonstrates the change in amplitude of the conditioned reflex for (A) head right, anodal stimulation, (B) head left, anodal stimulation, (C) head right, cathodal stimulation, and (D) head left, cathodal stimulation. 46 A. Cathode 11 6 -5 -4 3 2 1 0 -1 .0 Left Right Figure 5.2 The relative means ± SE (n = 10) for the H reflexes are presented based on the response to GVS coupled with head position. The test reflex is expressed as baseline or zero. The change in Ft reflex amplitude is the percentage difference from the baseline. Subjects received both a (A) cathodal stimulus and an (B) anodal stimulus. The changes in amplitude observed with GVS were found to be statistically significant (* P < 0.05). 47 Relative Difference between test and conditioned H reflexes (%) Effect of Anodal Stimulation Subject Left Forward (*) Right 1 1.0 0.5 -4.5 2 3.9 -4.1 -12.7 3 -3.8 -14.3 -12.3 4 0.2 -4.9 0.9 5 -0.3 -0.3 -1.4 6 0.1 -1.3 0.8 7 -0.1 -2.9 -5.1 8 -3.2 -2.5 -3.0 9 -0.2 -0.6 1.0 10 -2.5 -2.5 -5.8 Mean -0.5 -3.3 -4.2 SE 0.71 1.33 1.59 Table 4.1 The mean test reflex amplitude is expressed as baseline or zero. The relative differences in H reflex amplitude at different head positions with anodal stimulation are presented in this table. (*) Data presented from Experiment 3. Relative Difference between test and conditioned H reflexes (%) Effect of Cathodal Stimulation Subject Left Forward (*) Right 1 3.8 4.9 1.3 2 -5.4 -6.1 -1.9 3 14.8 14.0 -3.8 4 0.4 0.5 -2.4 5 1.6 2.0 -1.1 6 4.0 1.0 -3.8 7 4.5 3.4 -4.0 8 2.2 0.1 -0.9 9 1.4 1.4 1.1 10 12.0 10.5 7.5 Mean 3.9 3.2 -0.8 SE 1.82 1.78 1.10 Table 4.2 The mean test reflex amplitude is expressed as baseline or zero. The relative differences in H reflex amplitude at different head positions with cathodal stimulation are presented in this table. (*) Data presented from Experiment 3. 48 0.5% (SE ± 0.71) (Table 5.1). A mean decrease in absolute amplitude from 2.64 (SE ± 0.13) to 2.62 (SE ± 0.12) was observed. With the head turned to the right, a cathodal stimulus delivered to the right mastoid process produced a minor change in the right soleus H reflex from 2.52 mV (SE ± 0.13) to 2.51 mV (SE ± 0.13). This resulted in a statistically insignificant mean decrease of 0.8% (SE ± 1.1). It has been previously shown, when subjects faced forward, a cathodal stimulus facilitates the amplitude of the H reflex by a mean of 3.2% (SE ± 1.78) (Kennedy and Inglis, 2001). When subjects turned their heads to the left, a cathodal stimulus facilitated the H reflex by a statistically significant mean of 4.0% (SE ± 1.8) (Table 5.2). This resulted in an increase in mean amplitude from 2.54 mV (SE ± 0.13) to 2.62 mV (SE ± 0.13). 5.3.2 Effect of head position on the H reflex Surprisingly, the greatest facilitation of the right soleus H reflex was found when a cathodal stimulus was presented with the head turned to the left while the largest inhibitory effect was found when anodal stimulation was paired with the head turned to the right. This is in contrast to previous reports in which it has been shown that turning the head to the left inhibits the amplitude of the right soleus H reflex while turning the head to the right facilitated the H reflex in prone lying subjects (Hayes and Sullivan 1976). Since the amplitude of the soleus H reflex can be manipulated by both head-on-body alignment (Traccis et al. 1987) and GVS polarity (Kennedy and Inglis 2001), we wanted to measure the specific contribution of head position on the conditioned reflexes. Therefore, the amplitude of the H reflex was measured according to head position in all 10 subjects without galvanic stimulation. When the subjects were faced down against the bed, corresponding to a head forward position, the mean amplitude of the H reflex was 2.59 mV (SE ± 0.28). Interestingly, there was a decrease in the absolute amplitude to 2.53 mV (SE ± 0.30) when subjects turned their head to the right. This resulted in a mean decrease of 1.9% (SE ± 2.8). Conversely, when subjects turned their head to the left, there was an increase in the absolute amplitude to 2.67 mV (SE ± 0.28). This corresponded to a mean increase of 3.7% (SE ± 3.2). Despite the changes in H reflex amplitude based on head position, due to the variations in the conditioning responses between subjects, these changes were not found to be statistically significant (F2,is = 1.605, P < 0.23). 5.4 DISCUSSION This study shows that while it is possible to elicit a vestibulospinal response with GVS in prone lying individuals (Kennedy and Inglis, 2001), this response is highly dependent upon the position of the head relative to the trunk. For instance, anodal stimulation decreased the 49 amplitude of the H reflex when the head was positioned to the right. Conversely, cathodal stimulation significantly increased the amplitude of the conditioned reflex when the head was turned to the face the left shoulder. However, when cathodal stimulation was administered while the head was rotated to face the right shoulder, or when anodal stimulation was delivered while the head was facing left, only minimal changes in H reflex amplitude were observed. Galvanic stimulation has been widely used as an artificial method for activating the vestibular system in humans (Fitzpatrick et al. 1994; Watson and Colebatch 1997). A low intensity stimulus delivered to electrodes placed over the mastoid process may not only activate the irregularly firing afferents of the vestibular nerve (Minor and Goldberg 1991) but may also excite local cutaneous afferents in the underlying skin. Since no control trials were performed to assess the impact of stimulating an area of skin on the amplitude of the soleus H reflex, we cannot exclude the possible contribution of cutaneous activation on the observed lower limb response. However, the electrodes were placed over the mastoid process and wrist joint to minimize the cutaneous sensations associated with the galvanic stimuli. Secondly, the level of stimulation was strong enough to evoke the perception of movement associated with galvanic stimulation (Day et al. 1997) without a strong skin response. In fact, after several trials, none of the subjects could perceive any skin sensations with the galvanic stimuli, but all subjects did report feeling a perception of movement. Therefore, we are confident that the GVS stimulus was targeting the vestibular nerve and that the changes in the H reflex amplitude were largely due to the vestibular stimulus. While it remains unclear as to how GVS and head movements specifically modulate the excitability of the soleus motoneuron pool, it is likely that there is a common underlying mechanism responsible for the changes seen in H reflex amplitude. Neurons in the vestibular nucleus receive inputs from both neck and peripheral vestibular afferents (Wilson 1991). The vestibular system provides a tonic descending influence that may modulate presynaptic mechanisms (Manzoni 1988; Pompeiano 1988) that are capable of affecting the threshold of lower limb motoneurons. Therefore, any change in the discharge properties of the vestibular afferents would alter the descending spinal inputs and modify the amplitude of the soleus H reflex. In fact, when vestibular inputs are removed altogether, there is a large reduction in the amplitude of the H reflex (Lacour et al. 1976). Galvanic stimulation artificially influences the discharge rate of the irregular afferents in the vestibular nerve (Goldberg et al. 1984). Information about the position of the head has also been shown to influence the discharge properties of vestibular afferents (Gdowski and McCrea, 2001). When a cathodal stimulus is presented, there is an increase in the firing frequency of the 50 vestibular afferents (Minor and Goldberg 1991), which facilitates the amplitude of the soleus H reflex in prone lying individuals (Kennedy and Inglis, 2001). In this study, turning the head to the left had a similar effect, as the amplitude of the H reflex was greater in this position in comparison to when subjects faced forward. It may well be that rotation of the head towards the left shoulder also created an increase in the firing behaviour of the irregular afferents. By pairing cathodal currents with head left, the resulting combined response is a larger conditioned H reflex since both stimuli increase the firing frequency of the vestibular afferents. However, when two contrasting signals are put together, such as anodal stimulation and head left, the two signals cancel out one another, and only minimal changes in the conditioned reflexes were observed. Head position has long been known to play a role in modulating the excitability of the soleus motoneuron pool. Turing the head to the left has been shown to inhibit the right soleus H reflex while turning the head to the right facilitates the response in subjects who were seated upright (Traccis et al. 1987) and lying prone (Hayes and Sullivan 1976). In contrast to previous reports, we have demonstrated opposite changes in H reflex amplitude with different head positions. In the present study, turning the head to the left facilitated the response while turning the head to the right inhibited the right soleus H reflex. While the changes in H reflex amplitude with head position were small and non-significant, similar changes in amplitude were seen in all ten participants. The consistency of the M-wave within and between trials cannot account for the changes observed at different head positions. Since movement of the head relative to the trunk not only activates tonic neck reflexes but vestibulospinal reflexes as well (Kasper et al. 1988), it may be that the modulation in H reflex amplitude recorded at different head positions resulted from a change in the discharge properties of the vestibular afferents. Regardless, the present study has demonstrated that externally applied galvanic currents to the right mastoid process can, in some circumstances, modulate the amplitude of the right soleus H reflex in prone human subjects. Furthermore, it appears that the response is largely dependent upon the position of the head relative to the trunk. Accordingly, the largest inhibition of the soleus H reflex was observed when anodal stimulation was paired with the head turned to the right. Interestingly, this same stimulus facilitated the soleus EMG response during quiet stance (lies and Pisini 1992; Fitzpatrick et al. 1994). This change in the vestibulospinal response between prone and upright conditions may reflect changes in the neural pathways based on the task at hand, thus changing the characteristics of the vestibulospinal response. 51 5.5 BRIDGING SUMMARY The results from the previous two studies demonstrated that GVS can be used to activate descending vestibulospinal pathways that influence the excitability of the motoneuron pool in prone lying subjects. However, the ability to evoke a vestibulospinal response with GVS was dependent upon the position of the head. The change in the excitability of the motoneuron pool may reflect a general modulation in the threshold of the pool or a specific inhibition of individual motor units. Certainly, there is evidence that higher threshold or larger motor units receive greater synaptic currents from descending vestibulospinal inputs (Westcott et al. 1995). The purpose of the next study was therefore to identify whether GVS can affect the onset of single motor units in the lower limb muscles. Subjects performed slow isometric plantar flexor actions until single motor unit activity was detected. During randomly selected trials, a 1-mA bipolar, binaural galvanic stimulus was triggered just prior to the start of plantar flexor activity. However, based on the results of Experiment 1, when a plantar flexor muscle action is initiated at a shorter muscle length, the onset of gastrocnemius motor unit activity occurs at significantly higher levels of plantar flexor torque. This may reflect a change in the gain of the presynaptic inhibitory mechanisms that act on the motoneuron pool once a muscle reaches a shortened, non-optimal force-producing length. The vestibulospinal system may regulate muscle activity by controlling the presynaptic inhibitory inputs that act on the motoneuron pool. So, the aim of this study was extended to establish the effect of GVS on single motor units in the gastrocnemius muscles at different muscle lengths. 52 CHAPTER 6 GALVANIC VESTIBULAR STIMULATION ALTERS THE ONSET OF MOTOR UNIT DISCHARGE (Muscle and Nerve 30:188-194, 2004.) 6.1 INTRODUCTION The level of excitability of a motoneuron is not constant and is influenced by a variety of synaptic inputs (Binder et al. 1996; Henneman and Mendell 1981). Both peripheral sensory input (Garnett and Stephens 1980; Macefield et al. 1991) and descending corticospinal input (Lemon et al. 1986) alter the excitability and discharge properties of motor units (MU). The reticulospinal tract, for example, receives input from the cerebral cortex and is thought to activate segmental interneurons that are responsible for controlling presynaptic inhibitory mechanisms (Manzoni 1988). The reticulospinal tract also receives inputs from the vestibular system, which is an important source of sensory information in regulating lower limb motoneuron activity (Westcott et al. 1995). When tonic vestibular activity is removed, the excitability of the soleus motoneuron pool is reduced (Lacour et al. 1976). Therefore, a change in vestibular afferent activity may affect the excitability of the lower limb motoneuron pool either directly via descending pathways or indirectly through a change in presynaptic inhibitory pathways. Changes in vestibular afferent input can be achieved in humans by delivering a low-intensity stimulus to electrodes placed over the mastoid process. This non-painful galvanic vestibular stimulation (GVS) (Nashner and Wolfson 1974; Day et al. 1997) can evoke a transient electromyographic (EMG) response in the soleus during quiet stance (Britton et al. 1993; Watson and Colebatch 1997) and affect the amplitude of the soleus H-reflex in subjects lying prone (Kennedy and Inglis 2001). It is unclear whether the galvanically induced EMG responses observed in the soleus results from a general change in excitability of the motoneuron pool or a specific modulation of individual MUs. When plantar flexion is initiated at a shorter muscle length, the onset of gastrocnemius MU activity occurs at significantly higher levels of plantar flexor torque (Kennedy and Cresswell 2001) and soleus drive. Consequently, the aim of the present study was to establish whether GVS can affect the discharge onset of single MUs in the medial gastrocnemius muscle, and if so, whether GVS has differing effects at different muscle lengths. 53 6.2 METHODS Ten healthy subjects (5 men, 5 women) aged 22-31 years (mean 26.2 years) participated in this study. None of these individuals had any known neurological or motor disorders prior to testing. The experimental protocol was explained and the subjects gave their written, informed consent to participate in this investigation. The clinical research ethics board at the University of British Columbia approved the following experimental procedures and the study was performed according to the declaration of Helsinki. 6.2.1 Experimental design Subjects lay in a prone position on a padded bed with their head resting on their chin and facing forward. Both legs were extended and the right foot was placed against a forceplate (4060 Series, Bertec Corporation, Columbus, OH) that was mounted vertically. The foot was positioned on the forceplate with an ankle angle of approximately 90° and secured to minimize any ankle movement. An oscilloscope was placed in front of the subjects to display the voluntary plantar flexor force. A second beam on the oscilloscope was used to display a force ramp that increased at a rate of 1 N • s"'. Subjects were instructed to align the 2 beams as accurately as possible. The motor task was to increase gradually the amount of plantar flexor force at the required rate of 1 N • s"1 until MU activity could be visually identified at a signal-to-noise ratio of at least 2:1. At that time the ramp was discontinued and the subject was required to maintain force at, or slightly above, the level at which MU activity was isolated. This force level and the unit's discharge were maintained for approximately 10 seconds, after which the subject was allowed to relax. Timing of the motor task was controlled by giving a verbal command to indicate the start of the trial. A second command was presented approximately 3 seconds later to signal the start of the force ramp. At the completion of each trial the recording electrode was randomly repositioned in the medial gastrocnemius muscle before the start of the next trial. During specific trials, a galvanic stimulus was triggered following the first verbal command. The galvanic stimulus was intentionally activated 3 seconds before the start of the force ramp and remained on for the duration of the trial. This was done to reduce any possible confounding on- or off-effects of the galvanic stimulus on the subject's ability to perform the motor task (Watson et al. 2003). Trials were randomly assigned so that subjects received either no stimulus or an anodal (positive) or cathodal (negative) stimulus on the right mastoid process. Anodal stimulation decreases and cathodal stimulation increases the discharge properties of the vestibular nerve (Goldberg et al. 1984). Consequently, changing the stimulus polarity alters the impulses that are transmitted from the vestibular nerve to spinal segments innervating lower limb muscles. We 54 speculated that a reduction in vestibular activity, by anodal stimulation, would delay the onset of MU activity in the gastrocnemius muscle. Each subject received several practice trials to gain familiarity with the task. They were also exposed to galvanic stimulation prior to the testing period so that they felt comfortable in following the ramp in the presence of the on-going stimulus. The first experimental trial began as soon as the subject could consistently follow the ramp and maintain a constant level of force once a MU had been activated. Trials were performed in the initial prone position as well as in a second kneeling position. In this position the internal knee angle was reduced from 180° to 90°, thereby shortening the gastrocnemius muscle. Accordingly, the kneeling position will be referred to as the short position, and the prone position as the long position, in reference to the length of the gastrocnemius muscle (Figure 6.1). At the end of each testing session, subjects were placed in the short position one final time in an attempt to examine the effect of the conditioning stimuli on an individual MU. This entailed recording the same action potential for 3 successive contractions. One limitation of needle electrodes is the difficulty in recording a single MU for a prolonged period of time. As such, the subjects were encouraged to remain as still as possible during the trials to minimize electrode movement within the muscle. As the onset threshold of a single MU progressively decreases following repetitive, isometric actions (Denier et al 1985; Suzuki et al. 1990), a 1 minute rest interval was given between trials. Stimuli were randomly presented to each subject, and after 3 consecutive trials the electrode was randomly repositioned in the medial gastrocnemius muscle. 6.2.2 Motor Unit Recordings The skin at all recording electrode sites was cleaned using 70% isopropyl alcohol. Single MU activity was recorded from the medial gastrocnemius muscle using a sterile needle electrode (0.2 mm diameter, 35 mm length, standard profile tip; Fred Haer Inc., Bowdoinham, ME). A sterile reference needle electrode was placed 5 cm below the popliteal fossa between the proximal heads of the gastrocnemius muscle at an approximate depth of 15 mm. A surface ground electrode (self-adhesive Ag/AgCl electrode) was placed on the lateral malleolus of the right leg. 6.2.3 Galvanic Vestibular Stimulation Galvanic stimulation (1-mA square-wave pulse) was applied using a bipolar, binaural configuration. Two carbon-rubber electrodes (9-cm2) were placed behind the subject's mastoid 55 A. Long Position (Knee Extended) Short Position (Knee Flexed) Figure 6.1 The experimental setup is presented, showing the location of the oscilloscope, to provide visual feedback, and the subject positions with the knees extended (A) and flexed (B), corresponding to the long and short positions respectively. 56 processes. An output signal was sent from the computer through a 1401-micro interface (Cambridge Electronics Design, Cambridge, UK) which delivered a pulse through a constant-current analog stimulus isolation unit (AM Systems 200; Carlsborg, WA) that provided an output current of 1 mA for each volt input. Although we cannot be certain about the effectiveness of the 1-mA GVS pulse, the level of stimulation was strong enough to evoke the perception of movement Day et al. 1997, without evoking a strong skin response. Therefore, we are confident that the GVS stimulus was targeting the vestibular nerve and that the changes in the onset of MU activity were largely due to the vestibular stimulus. 6.2.4 Signal Processing Single MU recordings were amplified 25,000 times and band-pass filtered between 0.3 and 10 kHz (custom-built Yale amplifier). This signal was analog-to-digital converted (16 bit) at a sample rate of 15 kHz (Spike2 and 1401-micro interface, Cambridge Electronics Design, Cambridge, UK). A speaker system (Grass AM8, Grass Instruments, Astro-Med Inc., West Warwick, RI) was used for audio presentation of the EMG signal. The force signal was amplified 1000 times and converted along with the vestibular stimulus signal at a sample rate of 1 kHz. For each trial, the plantar flexor force level was used to determine the threshold of activation for each MU. To ensure that each trial was based on the activity of a single-shaped action potential, single MU activity discrimination was performed off-line using commercially available template matching software. Trials in which it was difficult to identify the shape of the MU relative to the background EMG activity were omitted from analysis. 6.2.5 Statistics Each subject participated in a minimum of 12 trials that combined at least 4 of each stimulation condition (no-stimulus, anode or cathode) at both muscle lengths. The mean force level corresponding to the onset of MU activity was calculated for every subject at both knee positions. Then, a grand mean (± SE) was calculated for each condition using the mean values from every subject. A repeated-measures analysis of variance (ANOVA) was used to examine the effect of vestibular stimulation on the onset of activation at 2 different muscle lengths. A repeated-measures ANOVA was also used to evaluate differences between mean firing frequencies between all 3 stimulus conditions. A Tukey's HSD post hoc analysis was used in the case of significant effects. Differences between the means for all conditions were considered to be statistically significant at P < 0.05. 57 6.3 RESULTS A total of 333 single MUs were recorded from the medial gastrocnemius muscle. From the entire sample, 205 units (62%) were recorded in the long position, and the remaining 128 MUs were recorded in the short position (38%). The mean plantar flexor force required to initiate single MU firing was compared for each galvanic stimulation condition and muscle length. Accordingly, for the long muscle length, 66 units (32%) were recorded without stimulation, 70 units (34%>) with anodal stimulation, and 69 units (34%o) with cathodal stimulation. In the short position, 42 units (33%) were recorded without stimulation, 44 units (34%) with anodal stimulation, and 42 units (33%) with cathodal stimulation. The mean plantar flexor force corresponding to the onset of single MU activity for each stimulus condition and muscle length is shown in Tables 6.1 and 6.2. 6.3.1 Influence of GVS at different muscle lengths With the gastrocnemius muscle in the long position, MU activity was recorded from all 10 subjects. During the no-stimulus condition, the mean force threshold corresponding to the onset of single MU activity was 8.70 N (SE ± 1.22). When a cathodal stimulus was presented prior to muscle activation, the mean onset threshold was 10.43 N (± 1.91). With an anodal stimulus, MU activity was generated at mean force of 10.59 N (± 1.66). Although the influence of galvanic stimulation on motor unit recruitment was not consistent with expectations, the absolute differences in the recruitment thresholds at the long muscle length were not statistically significant (F2,l8 = 1-129, P = 0.345). Even though they followed the ramp to maximal force levels, in 3 of 10 subjects, we failed to detect recruitment of MUs from the medial gastrocnemius muscle at the short muscle length, despite recordings from 4 different sites within the muscle. While these 3 subjects had no difficulty in activating MUs at low force thresholds in the long length, only low-level intramuscular EMG was recorded in the medial gastrocnemius when the muscle was at the shortened length. Since no MU activity was recorded in 3 of the subjects in the short position, the effect of the conditioning stimuli was examined in the remaining 7 participants (Figure 6.2A). Compared to the no-stimulus condition, cathodal stimulus resulted in a mean decrease in the recruitment threshold of approximately 14%. In contrast, compared to the no-stimulus condition, anodal stimulation resulted in a mean recruitment threshold increase of approximately 17%. Unlike the long condition, the differences in the forces required to generate MU activity between the stimulation conditions were statistically significant when the knee was flexed 58 Onset of Single Motor Unit Activity (N) Knee Extended (Long Position) Subject Number of No Stimulus Cathodal Anodal Recordings Stimulus Stimulus 1 14 8.6 10.1 10.6 2 22 2.9 3.2 3.9 3 25 12.0 16.5 12.7 4 26 7.0 6.1 7.6 5 24 3.3 4.0 5.1 6 21 15.3 12.5 22.8 7 30 7.9 7.6 13.3 8 14 9.5 13.6 10.6 9 15 8.1 7.9 9.3 10 14 12.3 22.8 10.1 Mean 20.5 8.7 10.4 10.6 SE 1.9 1.2 1.9 1.7 Table 6.1 The mean (± SE) plantar flexor force (N) corresponding to the onset of motor unit activity in the medial gastrocnemius muscle in the long position. Onset of Single Motor Unit Activity (N) Knee Flexed (Short Position) Subject Number of No Stimulus Cathodal Anodal Recordings Stimulus Stimulus 1 14 88.3 53.3 101.0 2 12 12.4 12.6 19.0 3 14 63.3 57.9 67.0 4 0 5 40 18.9 18.7 21.9 6 0 7 0 8 12 55.1 54.8 57.0 9 24 37.5 20.0 45.0 10 12 90.4 89.0 97.0 Mean 12.8 52.2 43.7 58.2 SE 3.9 11.8 10.5 12.4 Table 6.2 The mean (± SE) plantar flexor force (N) corresponding to the onset of motor unit activity in the medial gastrocnemius muscle in the short position. 59 No Stimulus Cathode Anode No Stimulus Cathode Anode Figure 6.2 (A) The mean (± SE) plantar flexor force (N) required to recruit a motor unit in each stimulus condition is presented for the short position (n = 7). (B) The mean initial firing frequency for those units recorded in all three conditions in the short position. Comparisons that were found to be significant by post-hoc analysis are indicated (* P < 0.05). No Stimulus Cathode Anode Threshold Figure 6.3 With the knee flexed, recordings of a single motor unit from the medial gastrocnemius muscle during all three stimulus conditions are presented from a single subject. The dotted lines superimposed on the raw data show the level of plantar flexor force corresponding to the onset of motor unit activity. Action potentials (n = 5) were superimposed and illustrated below for each trial to demonstrate the continuous sampling of a single-shaped unit for all three trials. 61 (F2,i2= 4.813, P = 0.03). Post-hoc analysis indicated that significant differences occurred between the cathode and anode stimulus conditions. 6.3.2 Effect of GVS on individual motor units In 5 of the 7 subjects, the same MU was recorded in all of the 3 stimulation conditions at the short muscle length. Template matching software was used to ensure that the same motor unit was present in each trial. A total of 20 MUs were successfully recorded in this manner. The mean plantar flexor force required to activate a MU during the no-stimulus condition was 77.2 N (SE ± 18.2). With a cathodal stimulus, MU activity was generated at a mean force of 63.8 N (± 16.0), thereby resulting in a mean threshold decrease of 18 %. When an anodal stimulus was used, the mean onset threshold of MU activity was 94.9 N (± 19.0 N), which corresponded to a mean increase of approximately 9%. For these 20 MUs, the differences in recruitment thresholds with different stimulation conditions were found to be statistically significant (F2,n - 10.208, P = 0.001). The significant differences were found between the cathodal and anodal conditions. A representative single MU recording during ramp contractions for all 3 conditions is presented in Figure 6.3. The initial firing frequencies of the same MUs recorded in all 3 stimulus conditions at the short muscle length were also significantly altered by the galvanic stimulus (F2j8 = 3.107, P = 0.05). The mean firing frequency of the first 10 action potentials recorded during the no-stimulus condition was 7.5 Hz (SE ± 0.4). When cathodal and anodal stimulations were given, the discharge rates were 7.9 Hz (± 0.5) and 6.9 Hz (± 0.3), respectively. Once again, the differences between the cathode and anode stimulus conditions were significant. 6.4 DISCUSSION The main findings of our study were that galvanic stimulation of the vestibular system modified the onset of activation and the initial firing frequency of MUs at shortened muscle lengths of the medial gastrocnemius. This may reflect a change in the gain of the presynaptic inhibitory mechanisms that act on the motoneuron pool once a muscle reaches a non-optimal force-producing length. As the recording needle was randomly repositioned in the medial gastrocnemius muscle between trials, a large sample of MUs at both long and short muscle-lengths was obtained. Due to the low variability of onset threshold within each condition, we are confident that any differences in onset thresholds are a direct result of the change in muscle length and the effect of the galvanic vestibular stimuli. A limitation of using needle electrodes is the difficulty of 62 maintaining the recording of a single MU for a prolonged period of time. However, to reinforce our finding we attempted to record the same MU during all 3 stimulation conditions at the short muscle length only, where population differences were already significant. The result of this additional test on 5 subjects and 20 MUs supported the earlier population finding that a modulation of recruitment threshold in medial gastrocnemius MUs occurs via GVS, particularly at the shortened, non-optimal force-producing length. Although it has been argued that a muscle must be posturally engaged for an EMG response to be present following GVS (Britton et al. 1993), this study provides evidence that it is possible to evoke a change in the excitability of the gastrocnemius motoneuron pool of a muscle that is voluntarily activated. A decrease in EMG activity has been reported in the gastrocnemius muscle at short compared to long muscle lengths (Fugl-Meyer et al 1979; Sale et al. 1982; Cresswell et al. 1995) despite maximal voluntary effort. The effect of muscle length on MU excitability and ultimately force output is complicated and probably depends upon several factors, including the contractile properties of the muscle fibers and the effect of excitatory and inhibitory influences from peripheral and spinal sources in addition to regulation by descending inputs. Of the latter, the vestibular system is known to provide a source of input to segmental pathways that can affect the activity of lower limb motoneurons. Direct projections can exert monosynaptic excitatory effects on extensor muscles of the lower limb (Wilson and Yoshida 1969; Grillner et al. 1970) and can also inhibit recurrent inhibitory input on the motoneuron pool by inhibition of Renshaw cells (Ross and Thewissen 1987). As such, an increase in the activity of vestibular afferents may facilitate the gastrocnemius motoneuron pool through either a direct vestibulospinal influence or an inhibition of Renshaw cells. This is partially consistent with our observations. Cathodal stimulation over the right mastoid process, resulting in an increase in vestibular afferent activity (Goldberg et al. 1984), lowered the onset threshold of MU recruitment in the right medial gastrocnemius muscle below the level of the no-stimulus condition for all subjects in the short position. Typically, galvanic currents applied to the mastoid process in quiet stance produce transient EMG responses in the lower limb at approximately 60 and 100 ms (Britton et al. 1993, Watson and Colebatch 1997). Cathodal stimulation delivered to subjects while lying prone increases the ipsilateral soleus H-reflex 100 ms after the onset of the conditioning stimuli (Kennedy and Inglis 2001). As the vestibulospinal tract contains large-diameter axons that have a conduction velocity of approximately 60-80 m • s"', galvanically induced modulation of the MU recruitment threshold most likely reflects polysynaptic connections between the vestibular system and the gastrocnemius motoneuron pool. The lateral vestibulospinal and reticulospinal tracts are 63 the main pathways for vestibular signals to the spinal segments controlling limb muscles (Pompeiano and Brodal 1957). Activation of these pathways via cathodal stimulation could provide an inhibitory influence on presynaptic inhibitory mechanisms that act on the lower limb motoneurons (Manzoni 1988; Pompeiano 1988), thereby indirectly increasing motoneuron excitability. Conversely, anodal stimulation should decrease the activity of the descending vestibulospinal inputs (Goldberg et al. 1984), which may result in an elevated MU recruitment threshold through increased presynaptic and recurrent inhibition of the motoneuron pool. It appears from our results that, at least in a short gastrocnemius muscle, where inhibition of the motoneuron pool appears to be taking place (Cresswell et al. 1995; Kennedy and Cresswell 2001), GVS can additionally modulate the onset threshold of voluntarily activated MUs by a descending input. The reason that the effect is only observed at shorter muscle lengths remains unclear. Once a muscle fiber reaches a critical shortened length, its overall contribution to force output will be minimal. So, there may be an increase in the presynaptic inhibitory mechanisms acting on the motoneuron pool to limit the activity of a muscle at non-optimal force-producing lengths. Consequently, changes in activity of the vestibular afferents induced by galvanic stimulation may alter gain of the presynaptic inhibitory mechanisms acting on the motoneuron pool once the muscle fibers reach a compromised force-producing length. Alternatively, there is evidence that higher-threshold (larger) MUs receive greater synaptic currents from descending vestibulospinal inputs (Westcott et al. 1995). These larger MUs may be activated preferentially at shorter muscle lengths (Kennedy and Cresswell 2001), which may explain why GVS appeared to have a greater effect when the knee was flexed. At present, it is unclear whether MUs of varying types are equally affected by GVS or whether there is selective modulation of specific motoneurons. Presumably, lower-threshold MUs were recruited first in the long position (Henneman and Mendell 1981). Since smaller MUs require relatively little synaptic input to reach their firing thresholds, it may be that GVS did not have a noticeable effect on the onset of MU activity in the long position. Nevertheless, the changes in the initial firing frequency and onset of medial gastrocnemius MU activity suggest that vestibulospinal mechanisms are able to influence the force output of individual motoneurons. Thus, galvanic stimulation of the vestibular system may offer a new method for exploring the role of vestibulospinal inputs on the motoneuron pool in human subjects. 64 CHAPTER 7 CONCLUSIONS AND GENERAL DISCUSSION 7.1 MAIN FINDINGS This thesis presents evidence that GVS can evoke an EMG response in a muscle that is not actively involved in maintaining balance. Galvanic stimulation modulated the amplitude of the H reflex in a passive soleus muscle and the recruitment properties of motor units in the active medial gastrocnemius muscle. There were several findings to suggest that the change in descending vestibulospinal activity indirectly modulated motoneuron excitability. First, the peak conditioning effect occurred when the galvanic stimulus was delivered 100 ms prior to the H reflex stimulus. Since GVS can evoke a short-latency EMG response during quiet stance at around 60 ms (Britton et al. 1993; Fitzpatrick et al. 1994; Watson and Colebatch 1997), it is unlikely that the galvanically-evoked changes in H reflex amplitude represented a direct vestibulospinal influence on that motoneuron pool. Second, the fact that the onset thresholds and initial firing frequencies of gastrocnemius motor units were significantly altered by GVS suggests that there was a change in the excitability of the motoneuron pool. Interestingly, motor unit activity was only affected by GVS when the muscle was at a shortened length. At the shortened position, gastrocnemius motor units were recruited at higher levels of plantar flexor torque and soleus drive. This may reflect an inhibition of motor units having shortened, non-optimal fascicle lengths. A change in the descending vestibulospinal input may alter the presynaptic inhibitory mechanisms that act on individual motor units whose contribution to force output may be limited. 7.2 ACTIVATING MOTOR UNITS AT DIFFERENT MUSCLE LENGTHS Studies investigating the effect of muscle length on the discharge properties of motor units have shown that there is a reduction of the contraction and/or half relaxation time of a muscle twitch at shorter muscle lengths (Bigland-Ritchie et al. 1992). This means that higher motor unit frequencies are needed to produce the same absolute amount of force. For example, the stimulus frequencies required to generate 50% of the maximum tetanic force from the abductor digiti minimi muscle were almost 30% higher at shorter muscle lengths in comparison to the control length (Gandevia and McKenzie 1988). Once a muscle fiber reaches a critical shortened length, its contribution to force output will be limited. At this point, an increase in descending drive to the motoneuron pool will have no further effect on force output. Therefore, the nervous system may reduce the excitability of spinal motoneurons whose muscle fibers reside 65 at non-optimal force-producing lengths (Herzog 2000) in an attempt to minimize metabolic expenditure. The results from Experiments 1 and 4 support this hypothesis as the onset of single motor units from the gastrocnemius were detected at higher plantar flexors forces at a shortened muscle length. This finding was not altogether surprising since a decrease in the surface EMG of the gastrocnemius muscle has often been observed with progressive shortening of the muscle fibers (Fugl-Meyer et al. 1979; Sale et al. 1982). It was suggested that the EMG reduction at a specified level of plantar flexor force may have been attributable to a decrease in the number of fibers within the electrode recording range (Cresswell et al. 1995). However, our findings suggest there may be a reduction in the excitability of the gastrocnemius motoneuron pool arising from a change in the synaptic inputs that regulate the excitability of the motoneuron pool. There are several possible mechanisms that might be responsible for modulating motoneuron excitability at different force producing lengths. For instance, the cortical drive directed to the motoneuron pool or portions of the motoneuron pool may be reduced when gastrocnemius muscle fibers reach a critical shortened length. Muscle spindles provide afferent information about the changes in length of the muscle fibers. The primary motor cortex may use la afferent feedback from muscle spindles to redirect synaptic input away from specific motor units whose fibers are at less than optimal lengths. Alternatively, the CNS may regulate motoneuron excitability by using feedback from the lb afferents innervating the Golgi tendon organs. The lb pathways can inhibit the homonymous motoneuron pool (Crago et al. 1982), although it is unclear how this mechanism may apply in this situation. Muscle spindles not only provide sensory information about the length of the muscle fibers, but influence the excitability of the motoneuron pool as well (Matthews 1966; Macefield et al. 1991). At the start of a muscle action, muscle spindles excite homonymous motoneurons (Schieppati 1987) and inhibit antagonistic motoneurons (Crone and Nielsen 1994). The inhibitory action is referred to as reciprocal inhibition. Although EMG activity was not recorded from the ankle dorsiflexors in either Experiment 1 or 4, Pinniger et al. (2003) observed no significant electrical activity in the tibialis anterior using a similar protocol. Therefore, it is unlikely that reciprocal inhibition was responsible for the changes in motor unit discharge properties at the short muscle length. There may, however, have been a reduction in excitatory la afferent firing in the gastrocnemius muscle at the shortened length. The muscle spindle's discharge properties reflect the length of the muscle (Cordo et al. 2002). If there is reduced la firing at the shortened lengths for the same descending cortical input, there will be a decrease in 66 the excitatory inputs transmitted to the gastrocnemius motoneurons. So, reduced la afferent input may have contributed to a reduction in motoneuron excitability. If reduced la afferent firing is responsible for lowering gastrocnemius motoneuron excitability at shorter lengths, then a question arises as to why GVS modulated motor unit recruitment properties at the short muscle length only. Galvanic stimulation is believed to activate interneurons responsible for presynaptic inhibition (Manzoni 1988; lies and Pisini 1992). Inhibitory interneurons that contact the la afferent terminal can reduce the amount of neurotransmitter that is released from the bouton. Presynaptic inhibition reduces motoneuron excitability by decreasing the la excitatory postsynaptic potentials that are conveyed to the alpha motoneuron (Frank and Fourtes 1957). When the internal knee angle was reduced, it may be that there was an increase in the presynaptic inhibitory inputs to disfacilitate gastrocnemius motoneurons whose contribution to force output is limited. Interestingly, the la afferent excitatory current directed to low threshold motoneurons tends to be larger than the inputs acting on higher threshold motoneurons (Binder 2002). The presynaptic inhibitory inputs may be more effective in disfacilitating the low threshold motoneurons. The result is that higher threshold motor units may be preferentially recruited at shorter lengths. 7.3 GALVANICALLY-EVOKED EMG RESPONSES During quiet stance, galvanic stimulation can activate the vestibular system and evoke changes in the ongoing EMG of the soleus muscle at around 60 ms followed by a second component, that is opposite in polarity, at around 120 ms (Britton et al. 1993; Watson and Colebatch 1997). The short latency component has been attributed to a direct facilitation of the motoneuron pool (Britton et al. 1993). The medium latency response, on the other hand, may reflect an indirect change in the presynaptic inhibitory mechanisms that act on spinal motoneurons (lies and Pisini 1992). When subjects were lying prone, GVS had the greatest effect on the amplitude of the ipsilateral soleus H reflex when the onset of the conditioning stimuli began 100 ms prior to the tibial nerve stimulus (Kennedy and Inglis 2001). The timing of the effect is similar to the medium latency response observed during quiet stance (Britton et al., 1993; Watson and Colebatch, 1997). Since the conduction velocity of the vestibulospinal tract is approximately 60-80 m • s"', it is unlikely that the changes in H reflex amplitude resulted from a direct facilitation of the motoneuron pool. Consequently, the galvanically-induced responses presented in this thesis presumably reflect polysynaptic influences. There are several possibilities as to how GVS specifically modulates the amplitude of the soleus H reflex and gastrocnemius motor unit activity (see Figure 7.1). Galvanic stimulation 67 Vestibulospinal Inputs ( A B CD Figure 7.1 Schematic illustrating possible vestibulospinal connections to (A) the inhibitory PAD interneuron, (B) to the la pathway, (C) to the alpha motoneuron, and (D) to the gamma motoneurons. Vestibulospinal input may regulate the excitability of the motoneuron pool by affecting the activity of the presynaptic inhibitory interneuron (black circle), alpha motoneuron (white circle - solid line) or the la afferent activity pathway (grey circle). 68 artificially alters the discharge rate of the irregular afferents in the vestibular nerve (Goldberg et al. 1984). A cathodal stimulus, for instance, increases the level of activity of the irregular afferents (Minor and Goldberg 1991). The corresponding change in activity is then transmitted to the lower limb either directly through the vestibulospinal tract or indirectly through the reticulospinal tract. The vestibulospinal pathway activates Renshaw cells; cells which provide recurrent inhibitory input to motoneurons (Pompeiano 1988). An increase in the activity ofthe vestibular afferents may facilitate the motoneuron pool through inhibition of Renshaw cells. Then again, the reticulospinal tract is also believed to activate interneurons that are responsible for controlling presynaptic inhibition (Wilson and Peterson 1981; Manzoni 1988). As a result, cathodal stimulation may disfacilitate the presynaptic inhibitory mechanisms that act on the lower limb motoneurons, and indirectly increase motoneuron excitability. Presynaptic inhibition is mediated by interneurons that influence the depolarization (PAD) of the la terminals. These PAD interneurons inhibit the release of neurotransmitter from the la axon by reducing the amplitude of the propagated signals or by blocking incoming action potentials (see Rudomin 1999). Since the la afferents make excitatory connections to alpha motoneurons (Schieppati 1987) a decrease in presynaptic inhibition will allow the la afferents to contribute to the facilitation of their corresponding motoneurons during a voluntary action. If vestibulospinal pathways project to PAD interneurons, GVS may indirectly modulate the excitability of the motoneuron pool by influencing the activity inhibitory influences. However, it is possible that the changes in the H reflex and motor unit recruitment properties did not involve presynaptic inhibition at all. It may be that the GVS had a direct influence on the la afferent discharge properties, possibly by influencing gamma motoneuron activity. There is evidence that an increase in muscle spindle discharge can lower recruitment thresholds and firing rates of voluntarily activated motor units (Grande and Cafarelli 2003). Cathodal stimulation, for instance, may increase la afferent activity by activating the gamma motoneurons. If the gamma motoneurons are stimulated, there would be an increase in la afferent activity which may lead to a larger H reflex and lower recruitment thresholds at shortened muscle lengths. At present it is unclear whether GVS can affect the activity of the gamma motoneurons or PAD-mediating interneurons. Finally, it must be considered that the galvanically-evoked changes in H reflex amplitude and motor unit discharge properties represent two different synaptic influences acting on the motoneuron pool. Attenuation of the H reflex may represent an indirect effect on the motoneuron pool brought about by an increase in the activity of either Renshaw cells (Pompeiano 1988) or inhibitory PAD interneurons (Manzoni 1988; lies and Pisini 1992). Alternatively, changes in the 69 onset of gastrocnemius motor units brought about by GVS may represent a direct vestibulospinal influence on the motoneuron pool. There is evidence that the ipsilateral vestibulospinal input is preferentially directed to higher-threshold motoneurons (Westcott et al. 1995). The increased motor unit amplitudes and slower initial firing frequencies in the shortened position suggest that these larger motor units were activated preferentially at shorter muscle lengths. As a result, GVS may have appeared to have a greater effect when the knee was flexed because of a direct change in the discharge behaviour of these larger motoneurons. 7.4 FUTURE DIRECTIONS There are several approaches that could be used to identify the mechanisms responsible for the GVS-evoked changes in the H reflex amplitude and motor unit onset. Electrical stimulation of cutaneous afferents has been shown to reduce the influence of presynaptic inhibition in a voluntarily activated muscle (lies and Roberts 1987; lies 1996). If there was an increase in the level of presynaptic inhibition of the gastrocnemius motoneuron pool at a shortened muscle length, a brief electrical stimulus applied to the sural nerve may lower the recruitment threshold of gastrocnemius motor units when the knee was flexed. Furthermore, electrical stimulation delivered with cathodal galvanic stimulation may have a greater effect than electrical stimulation alone. Alternatively, if la afferent input was involved, it would be important to provide direct evidence that GVS could affect the discharge properties of muscle spindle afferents. Specifically, GVS may alter la afferent activity through gamma motoneurons. The technique of microneurography could record the discharge properties of muscle spindles directly from peripheral nerves in conscious human subjects (see Gandevia and Hales 1997). Needle electrodes inserted through the popliteal fossa and into the tibial nerve could record from large diameter afferents innervating muscle spindles in the gastrocnemius and soleus muscles during periods of galvanic stimulation. In comparison to a control condition without stimulation (a) anodal galvanic stimulation may lower the firing rates of muscle spindles and (b) cathodal stimulation may increase the muscle spindle afferent activity. Even if GVS could modulate the background discharge of primary muscle afferents, it would still need to be determined if the change in muscle spindle firing properties was due to a direct effect on the la pathway or mediated by presynaptic inhibitory interneurons. Studies of presynaptic inhibition in man have often used a conditioning volley such as vibration to activate first order inhibitory interneurons. When a short duration (~ 10 ms) repetitive stimulus is applied to a muscle tendon, a long lasting depression in the level of excitability of the motoneuron pool, 70 attributed to presynaptic inhibition, has been described (Hultborn et al. 1987; Nielsen and Petersen 1994). It may be possible to compare the effects of GVS and vibration on the discharge properties of individual muscle spindles to assess the presence of presynaptic inhibitory inputs on the sensory receptors. 7.5 CONCLUDING REMARKS Our understanding of how GVS affects the vestibular system, and corresponding pathways, is largely based on animal research (Goldberg et al. 1984; Minor and Goldberg 1991). While it is unclear how a percutaneous stimulus affects the vestibular system in humans, the responses to GVS during quiet stance have been well described (Nashner and Wolfson 1974; Britton et al. 1993; Fitzpatrick et al 1994; Watson and Colebatch 1997). Now, it appears that externally applied galvanic currents can, in some circumstances, modulate the excitability of the motoneuron pool in a muscle that is not actively engaged in maintaining balance. The responses have been identified in the triceps surae muscle group and are consistent with animal studies examining the influence of vestibulospinal inputs on skeletal motoneurons. That is, by decreasing the firing frequency of the vestibular afferents through anodal stimulation (Minor and Goldberg 1991), there is a decrease in the excitability of the motoneuron pool. Up to now, most studies using GVS have focused on how the electrical stimulus affects postural control. 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