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Physiological Studies of the Vestibulosympathetic Reflex in Humans
Elie Hammam, BMedSci (Hons I)
School of Medicine University of Western Sydney
Supervisor Prof. Vaughan Macefield
Co-Supervisor Prof. Kenny Kwok
A thesis submitted to the University of Western Sydney in candidature for the award of Doctor of Philosophy, 2014
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STATEMENT OF AUTHENTICATION
I, Elie Hammam, declare that this thesis is based entirely on my own independent work, except for sections which were performed in collaboration with colleagues as acknowledged in the study and resulted in the publication of the journal articles shown below. To the best of my knowledge this project does not contain material previously submitted in fulfillment of the guidelines and requirements for the award of Doctor of
Philosophy in the School of Medicine, University of Western Sydney, and has not been submitted for qualifications at any other academic institution.
Elie Hammam ! !
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ACKNOWLEDGEMENTS
Undertaking the highest scholarly exercise a University offers has certainly been a long, arduous, but nevertheless a fulfilling journey. Now completed, reflection has allowed me to appreciate that what I have achieved is merely a credit to my efforts. I am deeply indebted to the guidance of my mentors, encouragements from friends and support from family.
First and foremost, I wish to acknowledge my stellar supervisor Vaughan Macefield who has never shied from supporting me throughout my candidature. Vaughan, from the very beginning you believed in me and never ceased to impart your knowledge, skills and wisdom. You have ensured all throughout my candidature that I get a holistic development in preparation to a life with academic excellence. You are a highly accomplished professor but yet grounded on humility and selflessness; and that is what makes you a special individual. Words cannot sum up what you have done for me nor express my gratitude, so I will simply say - thank you.
I have had the privilege to be surrounded by a number of professors that have mentored me and saw my candidature and academic skills come to fruition. Philip Bolton you are a gentleman and a scholar. Thank you for committing to regularly making the trip down from Newcastle and more importantly for all of your scientific contribution and personal influence that has helped shape my progress as a scientist and a person. PhD supervision is not a one-man effort, but a two-man task. To my co-supervisor, Kenny Kwok, thank you for your efforts in ensuring funding and infrastructure whenever and wherever needed. Your dedication, professionalism and thoroughness set a standard of work ethos to aim for.
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“He who walks with the wise grows wise” Proverbs 13:20. Matthew Barton, it’s a blessing to have walked this journey with you. We met as colleagues and parted as life- long friends. Thank you for all your support.
Finally, this journey would have been meaningless without my family. Especially, my mum thank you for your love, unreserved support and sacrifices that shaped the man I am today. To my brothers, thank you for always believing in me, keeping me on track and seeing me grow one step at a time. Last but not least, the best outcome from these past 4 years is meeting my fiancé, Jen. Jen you were not a person to lean on because you made leaning unnecessary. You appreciate my lame jokes and sense of hunour; you are an amazing individual, thank you for your commitment and dedication in supporting me in everything that I do – I love you.
I dedicate this thesis to my family
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ABSTRACT
I have previously shown that sinusoidal galvanic vestibular stimulation (sGVS), a means of selectively modulating vestibular afferent activity, can cause partial entrainment of sympathetic outflow to muscle and skin in human subjects. However, GVS influences the firing of afferents from the entire vestibular apparatus, including the semicircular canals.
To further identify the source of vestibular input in the generation of vestibulosympathetic reflexes, I conducted a series of studies using sinusoidal linear acceleration of seated subjects (head vertical) to physiologically stimulate the vestibular system. In Study I & II,
I tested the hypotheses that selective activation of one set of otolithic organs - those located in the utricle, which are sensitive to displacement in the horizontal axis - could entrain muscle sympathetic nerve activity (MSNA) and skin sympathetic nerve activity
(SSNA). Cross-correlation analysis revealed for the 10 subjects in Study I a marked entrainment of SSNA for all types of movements: vestibular modulation was 97±3 % for movements in the X-axis and 91±5 % for displacements in the Y-axis. Furthermore, Study
II revealed partial entrainment of MSNA to the sinusoidal stimulus: vestibular modulation was 32±3 % for displacements in the X-axis and 29±3 % in the Y-axis; these were significantly smaller than those evoked in SSNA. In addition, in Study III I examined the capacity for the vestibular utricle to modulate muscle sympathetic nerve activity (MSNA) during sinusoidal linear acceleration at amplitudes below perceptual threshold. Subjects
(n=16) were exposed to a range of amplitudes presented in a quasi-random order (1.25,
2.5, 5, 10, 20 and 30 mG), at a constant frequency of 0.2 Hz. Cross-correlation analysis revealed potent sinusoidal modulation of MSNA even at accelerations subjects could not perceive (1.25-5 mG). The modulation index showed a positive linear increase with acceleration amplitude, such that the modulation was significantly higher (25.3 ± 3.7 %) at 30 mG than at 1.25 mG (15.5 ± 1.2 %). Finally, in Study IV I sought to better
! ! &! ! ! ! understand how the brain differentiates between head-only movements that do not require changes in vasomotor tone in the lower limbs from body movements that do require vasomotor changes. As a result, I tested the hypothesis that neck movements modulate
MSNA in the lower limbs of humans. Subjects (n=10) lay supine, at rest, during sinusoidal stretching of neck muscles (100 cycles, 35o peak to peak at 0.37 ± 0.02 Hz) and during a ramp-and-hold (17.5o for 54 ± 9 s) static neck muscle stretch, while their heads were held fixed in space. Cross-correlation analysis revealed cyclical modulation of
MSNA during sinusoidal neck muscle stretch (modulation index 45.4 ± 5.3%), which was significantly less than the cardiac modulation of MSNA at rest (78.7 ± 4.2%). Overall, by using slow sinusoidal physiological stimuli, evidence accumulated throughout my doctoral candidature emphasizes the role of the utricle, through the vestibulosympathetic reflex, in control of the peripheral vasculature. Moreover, these vestibulosympathetic reflexes can be evoked below perceptual threshold. In addition, through dynamic stimuli of neck proprioceptors my findings also indicate that sensory endings in the neck, as well as vestibular inputs, contribute to cardiovascular control in awake humans via their projections to the vestibular nuclei.
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!TABLE OF CONTENTS
STATEMENT OF AUTHENTICATION 2 ACKNOWLEDGEMENTS 3 ABSTRACT 5 TABLE OF CONTENTS 7 TABLE OF FIGURES 9 TABLE OF TABLES 11
Chapter 1 INTRODUCTION 12
1.1 Vestibular System 13 1.1.1. Structure and physiology
1.2. The Autonomic Nervous System 18 1.2.1. General 1.2.2. Sympathetic control of blood pressure 1.2.3. Recording sympathetic nerve activity in humans 1.2.4. Afferent and efferent pathways of the baroreflex 1.2.5. Physiological implication 1.2.5.1. Compensation 1.2.5.2. Limitations of the predominant mechanism
1.3. The vestibulosympathetic reflex (VSR) and cardiovascular control 32 1.3.1. Vestibulosympathetic reflex in animals 1.3.2. Vestibulosympathetic reflexes in humans 1.3.2.1. Caloric stimulation 1.3.2.2. Head-down neck flexion method 1.3.2.3. Off vertical-axis rotations (OVAR) 1.3.2.4. Galvanic vestibular stimulation 1.3.2.5. Linear acceleration
1.4. Aims 55
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Chapter 2 GENERAL METHODS 59
2.1. Subjects 60
2.2. Experimental Protocol 61 2.2.1. Motion Simulator 2.2.2. Neck Table 2.2.3. Microneurography 2.2.4. Recording procedures 2.2.5. Other measured parameters
2.3. Analysis 71
Chapter 3 RESULTS 74 Modulation of skin sympathetic nerve activity (SSNA) by the vestibular utricle
Chapter 4 RESULTS 84 Modulation of muscle sympathetic nerve activity by the vestibular utricle
Chapter 5 RESULTS 95 Vestibular modulation of muscle sympathetic nerve activity by the utricle during sub- perceptual sinusoidal linear acceleration in humans
Chapter 6 RESULTS 110 Modulation of muscle sympathetic nerve activity by neck proprioceptors
Chapter 7 GENERAL DISCUSSION 123
REFERENCES 141
APPENDIX 161
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TABLE OF FIGURES
Chapter 1 INTRODUCTION
Figure 1.1 Drawing of the anatomical structure of the labyrinth 14 Figure 1.2 Morphological polarization maps for saccular and utricular maculae in different species 15 Figure 1.3 Anatomical outline of the macula 16 Figure 1.4 Direct MSNA recording from an awake human subject 23 Figure 1.5 The afferent/efferent pathways of the baroreflex 25! Figure 1.6 The physiological effects of postural changes from the supine to standing position 28 Figure 1.7 Orthostasis related activation of the baroreflex (feedback mechanism) increases MSNA and leads to vasoconstriction 31! Figure 1.8 Blood pressure responses to nose-up tilt in chloralose-anesthetized and paralysed cats before and after transection of the CN VIII 33! Figure 1.9 A schematic diagram outlining the direction of GVS 42! Figure 1.10 Baroreceptor influenced MSNA is influenced by vestibular inputs 45 Figure 1.11 Correlation analysis between MSNA and cardiac and vestibular rhythms 45 Figure 1.12 Cross-correlation histograms between MSNA and GVS during sGVS 48 Figure 1.13 Bilateral recordings of muscle sympathetic nerve activity 52 Figure 1.14 Mean ± SE modulation indices for the primary (dark grey) and secondary (light grey) peaks of modulation of MSNA 53
Chapter 2 GENERAL METHODS
Figure 2.1 An illustration of the neck table displacement 64 Figure 2.2 Standard multi-unit recording of muscle sympathetic nerve activity (MSNA) 68 Figure 2.3 Continuous neural recording from a muscle fascicle of the common peroneal nerve supplying the Tibialis Anterior (TA) muscle 69
Chapter 3 RESULTS
Figure 3.1 Experimental records from one subject 79 Figure 3.2 Vestibular modulation of SSNA during platform motion 80 Figure 3.3 Modulation indices of SSNA as a function of platform motion and ECG 81
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Chapter 4 RESULTS
Figure 4.1 Multi-unit recording of muscle sympathetic nerve activity in a female subject 88 Figure 4.2 Cross-correlation histogram for one subject between MSNA and acceleration in the X-axis 89 Figure 4.3 Latency of primary and secondary bursts measured from the peak of the sinusoid 90 Figure 4.4 Modulation indices of MSNA as a function of the platform motion and cardiac activity 91
Chapter 5 RESULTS
Figure 5.1 Recording of linear potentiometer rating of perceived movement (upper trace) and acceleration (lower trace) 100 Figure 5.2 Detection reliability graph 101 Figure 5.3 Multi-unit recording of muscle sympathetic nerve activity during sinusoidal linear acceleration at 1.25 mG 102 Figure 5.4 The relationship of MSNA, blood pressure and heart rate during accelerations 104 Figure 5.5 Crosscorrelation histogram between MSNA and acceleration in the antero-posterior direction for one subject 105 Figure 5.6 Mean vestibular (A) and cardiac (B) modulation indices of MSNA as a function of acceleration amplitude 107
Chapter 6 RESULTS
Figure 6.1 Bilateral recordings of muscle sympathetic nerve activity 114 Figure 6.2 Data from one subject showing the cross-correlation histogram between MSNA and ECG 116 Figure 6.3 Mean modulation indices calculated from the cross-correlation histograms between MSNA and ECG 117 Figure 6.4 Experimental recordings during a maximal inspiratory breath-hold 119 Figure 6.5 Data from one subject showing the cross-correlation histogram between MSNA and angular displacement of the neck 120
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TABLE OF TABLES
Chapter 3 RESULTS
Table 3.1 Vestibular and cardiac modulation of SSNA during platform motion 82
Chapter 4 RESULTS
Table 4.1 Vestibular and cardiac modulation of MSNA 92
Chapter 5 RESULTS
Table 5.1 Vestibular and cardiac modulation of MSNA at different Accelerations 108
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CHAPTER 1
INTRODUCTION
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1.1 Vestibular System
1.1.1 Structure and physiology
The vestibular system, buried deep in the temporal bone on either side of the head, has important sensory functions, contributing to the perception of self-motion, head position and spatial orientation relative to gravity. In addition, it has important functions in motor control: the vestibulo-ocular reflex (VOR) provides gaze stabilization during head movements, whilst the vestibulo-spinal and vestibulo-colic reflexes provide postural stability of the body, head and neck (Highstein & Holstein 2012). Another important role of the vestibular system is in cardiovascular control, particularly during changes in posture
(Balaban & Yates 2004; James et al. 2010; Hammam et al. 2011, 2012; Holstein et al.
2011; Yates & Miller 1994; for review see Yates et al. 2014); it is contribution to the regulation of blood pressure, skin blood flow and sweat release, via vestibulosympathetic reflexes, that I will be focusing on in this thesis.
The main anatomical component of the vestibular system is an elaborate set of interconnected chambers - the labyrinth – that is continuous with the cochlea and consists of five organs: three semicircular canals and the two otolithic organs, namely the utricle and saccule (Agrawal & Minor 2010; Goldberg & Fernandez 1971). The vestibular hair cells are located in the utricle, saccule and in three jug-like swellings called ampulae at the base of the semicircular canals (Gacek 1979; Ashmore 1991) (Figure 1.1). Deflection of these hairs, which like cochlear hair cells transduce minute displacements into action potentials, results in modulation of the release of neurotransmitter from the basal and lateral surfaces of the cell. Consequently, the sensory hair cells transduce mechanical to electrochemical energy, and represent the first component of vestibular sensory information processing (Hudspeth 1983; Fettiplace et al. 1992). In the semicircular canals,
! ! "$! ! ! ! the hair cells protrude from the crista in a gelatinous mass and become enveloped in the cupula through which the endolymph cannot circulate (Goldberg & Fernandez 1971). As a result, movements of the endolymphatic fluid distort the relatively compliant cupula. So, when the head turns about the plane of the canals, the inertia of the endolymph causes movement of the cupula away from the direction of head movement and causing a displacement of hair bundles within the crista (Goldberg & Fernandez 1971; Selva et al.
2009).
Figure 1.1 Drawing of the anatomical structure of the labyrinth. The cristae ampullares of the three semicircular canals, and the maculae of the otolithic organs are highlighted in green and blue respectively (Both figure and legend modified from Highstein & Holstein 2012).
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Figure 1.2 Morphological polarization maps for saccular and utricular maculae in different species. This illustrates the different mammalian species used in vestibular research and it outlines the opposing directions in which the sensory hair cells are divided within the organ (Figure and legend modified from Lindeman 1969).
On the other hand, the macula is embedded in the utricle and saccule and consists of hair cells that lie among a bed of associated supporting cells and protrude into a gelatinous layer (Igarashi 1966). Resting on the gelatinous layer is the otholitic membrane, in which crystals of calcium carbonate (otoconia) are embedded; these crystals are key to the tilt sensitivity of the macula (Lundberg et al. 2006). Thus, when the head tilts, gravity causes the membrane to shift relative to the macula (Figure 1.3 B). The same is true with linear movements in the horizontal plane: inertia causes the otoconia and underlying gelatinous
! ! "&! ! ! ! layer to displace in the oppositive direction. For both the semicircular canals and the otolithic organs angular or linear displacements respectively cause depolarization of the hair cells and excitation of vestibular afferents.
Figure 1.3 Anatomical outline of the macula. (A) Outlines the anatomy of macula in the utricle or saccule. (B) Demonstrates the consequence of a backward head tilt (Purves 2008).
All bodies moving in three dimensions have six degrees of freedom; three of these are translational and three are rotational. The translational components may be given in terms of movements along the X, Y and Z-axis of the head. The rotational components about the axis are known as roll, pitch and yaw, respectively. The otolithic organs are primarily concerned with head translations and linear orientation in the vertical and horizontal directions by the saccule and utricle, respectively. This can be experienced, for example, when an individual travels upwards and downwards in an elevator, or in the horizontal plane in a motor vehicle. On the other hand, the receptor mechanisms of the three orthogonally oriented canals in each inner ear enable a person to respond to angular velocity or accelerations in both gravitational and inertial directions (Cathers et al. 2005;
Curthoys & Oman 1987; Selva et al. 2009), as when rotating on a device such as a merry-
! ! "'! ! ! ! go-round. However, it is important to note that the vestibular endorgans signal head movements and positions in space and not the position of the whole body. In order to complete the perception of both head and body position, additional receptor inputs are required - such as those from the neck (Wilson 1991; Bolton et al. 1998). The roles of neck afferent inputs in humans are further discussed below.
Afferent axons supplying the vestibular hair cells pass through the vestibular ganglion, where their cell bodies are located. Centrally projecting axons from these neurones travel via afferents in the vestibulocochlear nerve (cranial nerve VIII) and terminate in the brainstem, mainly in the vestibular nuclei (Minor & Goldberg 1991). This complex of four nuclei is located dorsolaterally within the medulla and caudal pons (Goldberg &
Fernandez 1984; Goldberg et al. 2012). Axons run from the vestibular nucleus to numerous areas of the CNS, such as the spinal cord, cerebellum, cerebral cortex and the nuclei controlling extrinsic eye muscles (Eatock et al. 2002; Lisberger & Pavelko 1988).
The projected axons participate in a broad range of functions subserving gaze stabilization, perception of body orientation and motion, postural and balance control and cardiovascular control (Yates et al. 2014).
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1.2 The Autonomic Nervous System
1.2.1 General
The autonomic nervous system (ANS) is the involuntary part of the nervous system that controls most visceral functions of the body. Unlike hormonal systems, one of the most striking characteristics of the autonomic nervous system is the rapidity and intensity with which it can change visceral functions. For instance, within 10 to 15 seconds the arterial pressure can be doubled; at the other extreme, the arterial pressure can be decreased low enough to cause syncope (Colman et al. 2004). The efferent outflow comprises three major subdivisions: the sympathetic nervous system (SNS), the parasympathetic nervous system (PNS) and the enteric nervous system. The enteric nervous system, located within the submucosal and myenteric plexuses of the gastrointestinal tract, is responsible for local sensorimotor control of gut function, such as peristalsis. The “fight or flight response” is the prototypical example employed to demonstrate various activities of the sympathetic nervous system under stress by producing selective energy expenditure, catabolic functions and cardiopulmonary adjustments. The overall function of the parasympathetic nerve system is restorative - to conserve energy. As for the enteric nervous system, it controls motor functions, local blood flow, mucosal transport and secretions of the gastrointestinal tract, pancreas and gallbladder (Costa et al. 2000). With respect to this thesis I will be concentrating on the sympathetic arm of the peripheral nervous system.
The sympathetic preganglionic neurones, which receive excitatory drive from the brainstem - primarily the rostral ventrolateral medulla (RVLM) (Dampney et al. 2002;
Dampney et al. 2003) - originate from the intermediolateral cell column of the thoracic and upper lumbar segments of the spinal cord from segments T1-L2 (Krassioukov &
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Weaver 1996). Myelinated axons of these preganglionic neurones exit the thoracolumbar cord via the white rami and synapse on postganglionic neurones in the spinal paravertebral ganglia (the sympathetic chain) and various prevertebral ganglia (such as the coeliac ganglion), with unmyelinated axons being sent through the peripheral nerves to innervate the target organs, including the heart, blood vessels, respiratory tract, sweat glands, sexual organs and the smooth muscle within the bladder wall and gastrointestinal tract (Alexander et al. 2009; Wallin & Charkoudian 2007; Jänig & Häbler 2003).
Importantly, the systemic vasculature receives only sympathetic innervation and, in humans, most vascular sympathetic nerves induce vasoconstriction (Alexander et al.
2009; Wallin 2006). These sympathetic vasoconstrictor neurones regulate blood flow via their action on the arterioles (resistance vessels); vasoconstriction in skeletal muscle vascular beds causes an increase in total peripheral resistance and thus blood pressure, while vasoconstriction in cutaneous vessels reduces skin blood flow (and causes pallor) but has no significant effect on blood pressure. Microelectrode recordings from postganglionic sympathetic axons in motor fascicles of human peripheral nerves have shown that muscle sympathetic nerve activity (MSNA), which occurs as burst of activity time-locked to the cardiac rhythm, consists only of vasoconstrictor impulses (Wallin &
Charkoudian 2007). Given that the skeletal muscle vascular beds make up a significant proportion of cardiac output, neural control of the skeletal muscle circulation is essential to systemic hemodynamics (Wallin 2006). Skin sympathetic nerve activity (SSNA) is more complex, with cutaneous sympathetic nerves comprising four different fibre types: vasoconstrictor, vasodilator, sudomotor and pilomotor. Whilst SSNA activity is influenced by respiration, sleep, pain and arousal/stress it predominantly subserves thermoregulation (Wallin & Charkoudian 2007).
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1.2.2 Sympathetic control of blood pressure
Muscle sympathetic nerve activity (MSNA) is essential for normal regulation of blood pressure and tissue perfusion; MSNA has a close dynamic relationship to blood pressure by way of the arterial baroreflex. Blood pressure (BP) is a function of vascular resistance and cardiac output, two variables that are controlled by the autonomic nervous system
(Guyenet 2006). Pressure in the arterial system fluctuates with the cardiac cycle, reaching a peak during cardiac contraction (systole) and its lowest point during cardiac relaxation
(diastole). Cardiac output (CO) and total peripheral resistance (TPR) are the major factors determining arterial blood pressure (Guyenet 2006). Whilst cardiac output is the volume of blood ejected per unite time (stroke volume * heart rate), total peripheral resistance is the total vascular resistance in the systemic circulation (Mukkamala et al. 2003). This resistance is largely determined by the arteriolar diameter in the muscle vascular beds, controlled by MSNA (Fadel & Raven 2012). When the sympathetic nerve terminals innervating the arterioles release the neurotransmitter noradrenaline, it activates the adrenergic receptors to cause contraction of the vascular smooth muscle. The contraction of this smooth muscle narrows the diameter of the innervated arterioles (i.e. constricts), causing an increased resistance and, in turn, elevated blood pressure. Relaxation of vessels occurs when sympathetic vasoconstrictor drive is withdrawn, leading to a fall in arterial blood pressure. It is important to note that there is no evidence for active vasodilatation in human skeletal muscle vascular beds (Wallin 2006).
Given that the level of blood pressure is so crucial, it is necessary that it be maintained within a fairly narrow, yet adaptive range. Thus specialized receptors that monitor blood pressure are needed – these are termed baroreceptors. The negative feedback loop for the cardiovascular system is known as the baroreceptor reflex (baroreflex), where arterial
! ! #+! ! ! ! baroreceptors - spray-type nerve endings found in abundance in the aortic arch and the carotid sinus at the point of bifurcation of the carotid arteries – form the afferent limb of the reflex (Berne et al. 2004). Baroreceptors monitor blood pressure on a beat-to-beat basis and relay this information to a network of nuclei in the brainstem (Fernandes et al.
2010), responding rapidly to spontaneous fluctuations in arterial pressure (Berne et al.
2004). The neuronal relay then causes the sympathetic system to act accordingly – decreasing muscle vasoconstrictor (and cardiac sympathetic) drive when blood pressure is high, and increasing it when blood pressure falls - in order to maintain a constant blood pressure (Claydon & Krassioukov 2006; Myers et al. 2007). MSNA occurs as bursts time- locked to the cardiac cycle, and Fagius and colleagues (1985) have shown that bilateral anaesthetic block of cranial nerves IX (glossopharyngeal) and X (vagus), which convey afferents from the baroreceptors, abolishes the cardiac rhythmicity of the muscle sympathetic neurones (Fagius et al. 1985).
1.2.3 Recording sympathetic nerve activity in humans
Microneurography is a technique that allows one to record neural traffic (action potentials) in peripheral (or cranial) nerves in awake human subjects (Vallbo et al. 2004).
The first use of percutaneously inserted tungsten microelectrodes to record neural activity from peripheral nerves was performed by Hagbarth and Valbo in 1967. By inserting the tungsten microelectrodes, investigators can directly identify neural traffic in situ in myelinated and unmyelinated fibres and record both efferent and afferent impulses from fascicles supplying muscle and skin. Afferent discharges can be obtained from muscle spindles, tendon organs and muscle nociceptors travelling in muscle fascicles, or low- threshold mechanorecetors or nociceptors in cutaneous fascicles. Moreover, impulse activity from postganglionic sympathetic efferent nerves can also be recorded from
! ! #"! ! ! ! fascicles supplying muscle or skin. And while sympathetic activity can be assessed indirectly by recording blood pressure, sweat release, or blood flow, microneurography allows the direct recording of human sympathetic neural activity (Hagbarth & Vallbo
1968). As a result, it is now widely employed to measure the sympathetic outflow in conscious human subjects (Mano et al. 2006; Vallbo et al. 2004). The recording technique and identification of sympathetic fibers I have employed in this doctoral work is further detailed in Chapter 2 – General Methods.
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Figure 1.4 Direct MSNA recording from an awake human subject. A schematic representation of the microelectrode tip inserted into a muscle fascicle of the common peroneal nerve.
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1.2.4 Afferent and efferent pathways of the baroreflex
Primary afferent fibres from baroreceptors in the aortic arch travel in the vagus nerve (CN
X) in the neck whilst fibres from the carotid sinus travel in the glossopharyngeal nerve
(CN IX). The baroreceptor afferents project to the nucleus tractus solitarius (NTS) within the medulla oblongata (Sun 1995). NTS neurones conveying afferent baroreceptor signals then project to and excite, via a glutamatergic synapse, neurones within the caudal ventrolateral medulla (CVLM) (Dampney et al. 2002; Guyenet 2006), which then in turn project to and inhibit, via a GABAergic synapse, spinally projecting neurones within the rostral ventrolateral medulla (RVLM) (Dampney et al. 2003). Blockade of this inhibitory synapse in the RVLM completely abolishes the baroreflex (Guyenet 2006), demonstrating that the barosensitive sympathetic efferents appear to be regulated primarily through the
RVLM (Dampney et al. 2002). Also, studies in anaesthetized animals have shown that the tonic activity of RVLM presympathetic neurones appears to be the major determinant of tonic activity in sympathetic preganglionic vasomotor neurones. Inhibition of the RVLM leads to a decrease in sympathetic vasomotor activity (Guyenet 2006; Pilowsky &
Goodchild 2002; Dampney et al. 2003). Thus, as the negative feedback is increased during an increase in arterial blood pressure, the CVLM is excited by the increased activity in NTS and the RVLM is inhibited by the GABAergic projections from the
CVLM. This causes a withdrawal of muscle sympathetic vasoconstrictor drive and hence peripheral vasodilation, leading to a decrease in total peripheral resistance and hence arterial pressure. In addition, direct excitatory projections from NTS to the nucleus ambiguous (NA) and dorsal motor nucleus of the vagus (DMX) cause excitation of vagal motoneurones supplying the heart; release of acetylcholine at the sinus node causes a decrease in heart rate. Through both these mechanisms the baroreflex provides
! ! #%! ! ! ! continuous, beat-to-beat regulation of blood pressure. This regulatory pathway can be seen in the illustration in Figure 1.5.
Figure 1.5 The afferent/efferent pathways of the baroreflex. Sensory afferent fibres from carotid sinus and aortic arch synapse at the NTS. The NTS excites the CVLM, which in turn inhibits the RVLM, reducing muscle vasoconstrictor drive and causing peripheral vasodilation. Excitatory projections from NTS to nucleus ambiguous and the dorsal motor nucleus of the vagus cause an increase in vagal outflow to the heart, decreasing heart rate and stroke volume (Boron & Boulpaep 2009).
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1.2.5 Physiological implication
Physiologically, a means of controlling blood pressures is imperative: when the human body adopts the upright stance it undergoes orthostatic challenges and has to manage the gravitational demands imposed onto the cardiovascular system by maintaining adequate perfusion of the brain and offsetting the gravitational pooling of blood in the legs. These rapid adjustments of the vascular system are mediated by the sympathetic arm of the autonomic nervous system (Dampney et al. 2002). In contrast, when supine or prone, gravitational demands are almost evenly distributed from head to feet (Hargens &
Richardson 2009; Hinghofer-Szalkay 2011). However, a change in postural from the supine to the upright position results in an over six-fold increase in the height of the orthostatic column and gravity causes about 500-1000 ml of blood to move from veins of the upper body to the veins of the lower body. This fluid shift is primarily limited to veins of the lower abdomen, buttocks and legs which – being highly compliant vessels - expand to contain the extra volume of blood (Robertson 2008).
1.2.5.1 Compensation
The pooling of blood in the lower extremities reduces venous return and cardiac output
(Hargens & Richardson 2009; Hinghofer-Szalkay 2011). The resulting fall in blood pressure is compensated for by the baroreceptor and Bainbridge (atrial) reflexes (Sagawa
1983; Johnson et al. 1974). A decrease in negative feedback provided by baroreceptor inputs causes a decrease in cardiac parasympathetic (vagal) drive and an increase in
MSNA resulting in noradrenaline release from sympathetic nerve terminals hence, vasoconstriction (essentially acting as a “buffer”). It is the nature of this feedback mechanism that induces the characteristic cardiac rhythmicity and the inverse relationship between variations in pressure and nerve traffic that produce an increase in heart rate and
! ! #'! ! ! ! vasoconstriction in the muscle vascular bed, thereby helping to maintain an adequate blood pressure upon standing (Burke & Wallin 1974; Sundlöf & Wallin 1978; Vallbo et al. 2004; Wallin 1989). Because of the hydrostatic pressure upon standing, mean arterial and venous pressures are higher in the foot than in the head. Failure of the cardiovascular system to respond to the effects of gravity on the vascular system can produce dizziness, light-headedness and syncope (Colman et al. 2004). These symptoms can be debilitating in clinical hypotension (Mathias et al. 2001) and postural orthostatic tachycardia syndrome (POTS) (Medow & Stewart 2007). The mechanisms comprising these cardiovascular adjustments are shown schematically in Figure 1.6.
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Figure 1.6 The physiological effects of postural changes from the supine to standing position.
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1.2.5.2 Limitations of the predominant mechanism
As noted above, both the baroreceptor and Bainbridge reflexes are the predominant compensators driving peripheral blood pressure adjustments to postural challenges.
However, the atrial receptors afferents are unmyelinated (Arndt et al. 1974; Thoren 1976;
1979) and the barorecptor afferents are mostly unmyelinated or thinly myelinated
(Seagard et al. 1993; Wang et al. 1996). Thus, sensory signal transmission from baroreceptors to the brainstem is relatively slow. Indeed, Jeske and colleagues demonstrated that by stimulating the baroreceptor afferents in the aortic arch there was a long-latency (>1 sec) in activating sensory neurones prior to evoking a sympathetic response (Jeske et al. 1993), part of which is due to the slow afferent conduction to the brainstem. In addition, studies have also shown that it is not until a substantial decline in venous return to the heart that the atrial and baroreceptors are unloaded (Hargens et al.
2009; Hinghofer-Szalkay 2011). This then imposes another important limitation for the human body when overcoming venous pooling in the lower body, and indicates the existence of an additional physiological response to account for the latency of the atrial and baroreceptors.
What I will demonstrate through an assessment of the animal and human literature, and studies I have conducted during my candidature, is that the vestibular system contributes to this immediate cardiovascular adjustments imposed by the gravitational demands
(Figure 1.6). This continuous contribution of the vestibular endorgans in mediating posturally-related blood pressure adjustments, through the sympathetic nervous system is termed the vestibulosympathetic reflex and has now been studied and reviewed extensively in animals and humans (for review see Yates et al. 2014). This will be discussed in more detail below, but it is worth noting that the physiological significance
! ! #*! ! ! ! of the vestibular system with respect to cardiovascular control lies in the fact that hair cells in the otoliths organs are exquisitely sensitive to linear accelerations of the head.
While we do not know the absolute thresholds of human vestibular hair cells, I have shown that vestibulosympathetic reflexes can be generated at accelerations well below perceptual threshold. It makes sense to have a set of accelerometers in the head that can detect changes in head position, particularly to the upright position when the hydrostatic column – and hence the pressure differential between the head and the feet - is at its highest. My observations strongly support a significant role of the vestibular apparatus in the control of sympathetic outflow to the muscle vascular bed, and hence to the control of blood pressure.
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Figure 1.7 Orthostasis related activation of the baroreflex (feedback mechanism) increases MSNA and leads to vasoconstriction. This mechanism includes the influences of the otolithic organs as a result of displacements with head movements (Carter & Ray 2008).
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1.3 The vestibulosympathetic reflex (VSR) and cardiovascular control
Traditionally, the influences of the vestibular apparatus were mainly considered to be confined to regulating eye and head position as well as balance (Yates 1996). However, for some time now, the existence of anatomical pathways between the vestibular system and the autonomic nervous system have been established (Balaban & Yates 2004; Yates
1996; Yates & Miller 1994). Such anatomical evidence implicates the vestibular system in contributing to the rapid adjustments in cardiovascular homeostasis when inertial or gravitational forces are applied to the body (Gotoh et al. 2004). The evidence, from animal research, provides the anatomical substrates between the vestibular nuclei and areas within the brainstem involved in the control of blood pressure (Carter & Ray 2008;
Cui et al. 1997; Hume & Ray 1999; Kaufmann et al. 2002; Kerman et al. 2000a; Yates et al. 1993a, b; Yates & Miller 1994; Yates et al. 1991). Hemodynamic changes elicited by the vestibular stimulation are mainly accomplished through the actions of the sympathetic nervous system, and have been termed “vestibulo-sympathetic reflexes” (VSR).
1.3.1 Vestibulosympathetic reflex in animals
The important relationship between the vestibular and cardiovascular system was established from early animal studies (Conklin & Dewey 1941; Edholm 1940), in which de-afferentation of baroreceptors failed to abolish the cardiovascular responses to orthostatic challenges. Such findings suggested that receptors other than the baroreceptors might participate in initiating these postural reflexes. In 1974, the initial definitive evidence supporting the participation of the vestibular system in blood pressure regulation was published (Doba & Reis 1974). The investigators performed bilateral section of the vestibular nerves (CN VIII) in anaesthetized and paralysed cats, and showed that the
! ! $#! ! ! ! removal of vestibular inputs to the brainstem significantly impaired the animal’s ability to compensate for orthostatic challenges induced by nose-up tilt at 30°and 60° (Figure 1.8).
A finding later reported in experiments on the conscious cats (Jian et al. 1999) although the effects dissipated shortly after.
Figure 1.8 Blood pressure responses to nose-up tilt in chloralose-anesthetized and paralysed cats before and after transection of CN VIII. Data points are average blood pressure measurements in eight animals (Doba & Reis 1974).
Since then, particularly in the past two decades, a large body of animal studies employing natural and electrical stimulation has resulted in the accumulation of convincing anatomical and functional evidence for the active role of the vestibular system in controlling peripheral vasculature during postural challenges (Balaban & Yates 2004;
Yates 1996; Kerman et al. 2000a; Yates et al. 2014). Anatomical substrates outlined the patterning of the vestibulosympathetic reflex, largely depicting the differential
! ! $$! ! ! ! distribution of sympathetic activity as a result of vestibular activation, whereas functional substrates defined the neuronal circuits mediating the reflex - including pathways from higher brain regions.
Physiological stimuli have been employed to excite the vestibular afferents in order to identify which components of the vestibular apparatus are involved in generating vestibulosympathetic reflexes to cardiovascular challenges (Yates & Miller 1994;
Woodring et al. 1997). In these experiments either the animal’s body is displaced in space, or if fixed, then the head is manipulated. Though a good means of naturally stimulating the vestibular afferents, it does also recruit extra-labyrinthine inputs that influence sympathetic outflow - such as neck proprioceptors (Bolton et al. 1998). In order to overcome this limitation, investigators either denervated extra-vestibular afferents or compared the physiological responses evoked by the stimulation before and after vestibular nerve (CN VIII) resections. Jian and colleagues, for example, conducted a study where they employed whole-body movement in conscious cats (with a bilateral transaction of CN VIII). The investigators found complementary evidence to that of Doba and Reis in 1974, where the animal had a deficit in blood pressure adjustments post vestibular lesion (Jian et al. 1999; Doba & Reis 1974). Moreover, Yates and Miller recorded sympathetic activity from the cat splanchnic nerve during head rotations (20°) about a fixed body to observe blood pressure changes. To eliminate neck proprioceptor inputs and maintain the selective vestibular influence on the cardiovascular system, the cat had undergone a cervical dorsal rhizotomy. Rotations were then performed in the vertical plane (nose-up and nose-down) and roll tilts (ear-up and ear-down). The authors observed that there were increases of nerve activity during nose-up tilt and decreases during nose-down tilt. And since no change in sympathetic activity was recorded during
! ! $%! ! ! ! the roll tilts, the investigators determined that the vestibular system does indeed participate in compensating postural changes in blood pressure and that the changes in sympathetic activity are likely to be of otolithic origin (Yates & Miller 1994).
Furthermore, a related study observed similar changes to blood pressure during head rotations of the cat (50° rotations). The head-up tilt caused a marked increase in blood pressure whereas roll tilts had no clear effect. The marked blood pressure increases during head-up tilt was indeed obliterated with the transection of the vestibulocochlear nerve
(CN VIII), confirming the influence of the vestibular system on blood pressure regulation
(Woodring et al. 1997). Other studies were also conducted to examine the role of the vestibular inputs on blood flow (rather than pressure) and found that whilst the removal of the vestibular nerve caused a decrease in blood flow to the limbs of the cat (Wilson et al.
2006b, Yavorcik et al. 2009) it evoked an increase in cerebral blood flow when measured from the carotid artery (Wilson et al. 2006a). The results support the notion of differential changes in sympathetic outflow as a result of vestibular afferent activation. We know that sympathetic nerve activity is distributed differentially according to the target tissue but not location in both animals (McAllen & Dampney 1990) and humans (Rea & Wallin
1989; Vissing et al. 1994; Jänig & Häbler 2003). For example, sympathetic efferent discharges to muscle are different to those to the skin (Wallin & Charkoudian 2007).
However, there is evidence demonstrating that the vestibular system can distribute sympathetic outflow differentially according to tissue type and location. Tracing of sympathetic fibers via the prevertebral ganglia demonstrated that axons in the upper thoracic cord modulate sympathetic outflow to the upper body and similarly in the lower thoracic cord regulating sympathetic outflow to the lower limbs (Strack et al. 1988; Lee et al. 2007). In addition, RVLM neurones have been shown to individually project to these differential areas of the spinal cord (Gowen et al. 2012). This then begs to question how
! ! $&! ! ! ! does this anatomical distribution behave physiologically. Electrical stimulation of the vestibular afferents in the cat decreased sympathetic activity to the muscle in the hindlimb and elicited increases to the forelimb and facial areas (Kerman et al. 2000b). This evidence, based on postganglionic recordings, proves differential patterning of vestibular mediated sympathetic outflow. Preganglionic recordings of RVLM neurones projecting to different segments of the spinal cord, regulating upper and lower limbs, exhibited comparable excitation to vestibular stimulation (Sugiyama et al. 2011). This suggests that whilst peripherally there is a differential patterning, it requires the integration of vestibular inputs in spinal neurones in addition to the RVLM (Sugiyama et al. 2011). This phenomenon has not been examined in the human and is still a subject for investigation.
Adding to our knowledge on vestibulosympathetic reflexes in the quadruped, other investigators used the rat in their experiments during natural stimulation under gravitational stress. Whilst a vestibular-compromised animal failed to adjust blood pressure during free fall, the vestibular-intact rodent evoked the normal physiological pressor response (Abe et al. 2007, 2008b). Moreover, vestibular compromised rodents exposed to greater gravitational stressor (centrifuged at 3G) also demonstrated an inability to appropriately adjust blood pressure, compared to the vestibular intact rat (Abe et al.
2008a; Gotoh et al. 2004; Matsuda et al. 2004). In summary, the aforementioned studies demonstrated, using different animal models (rodents and cats) and means of natural stimulation, that the vestibulosympathetic reflex indeed is potent in haemodynamic alterations during postural challenges. In addition, the vestibulosympathetic response complements the baroreceptor reflex in maintaining blood pressure and elicits differential patterning in the quadruped and any disturbances in the labyrinths cause disturbances to the cardiovascular system. It is important to note though, that whilst there was a strong
! ! $'! ! ! ! correlation between removal of vestibular inputs and impairments in hemodynamic control, these were shown to be short-term deficiencies (Wilson et al. 2006a, b; Yavorcik et al. 2009). Long-term examination of the animals demonstrated the attenuation of impaired vascular control, suggesting a non-labyrinthine compensatory mechanism overtaking the role of vestibulosympathetic reflexes.
As previously mentioned, there are several extracranial sources of information activated during natural stimulation that could play a role in signalling cardiovascular changes in posture. Although the specific receptors and pathways have not been identified,
Mittelstaedt and colleagues have demonstrated that shifts in blood volume may provide clues that the body’s position has changed relative to gravity. They refer to this perception as ‘somatic graviception’ (Mittelstaedt 1992; Vaitl et al. 1997, 2002). It is also well known that mechanoreceptors in the neck play an important role in somatomotor reflexes that maintain posture and interact with vestibulosomatic reflexes (Lindsay et al. 1976;
Wilson 1991). When performing head-up tilt, for example, the neck is being stretched and hence proprioceptors in the neck muscles excited. It was Bolton and colleagues in 1998 who indeed demonstrated that after an upper cervical dorsal rhizotomy to eliminate neck afferents the sympathetic response to head-up tilt was evident, but not before the deafferentation. This finding affirms the input of the vestibular system into cardiovascular control but also proves the dynamic relationship the labyrinths has with neck inputs
(representing whole body location) in the vestibular nuclei (Kasper et al. 1988; Neuhuber
& Zenker 1989) to accurately adjust peripheral vasculature (Bolton et al. 1998). As discussed, natural stimulations provided a good insight into physiological responses but do include the limitations of extra-vestibular afferent stimulation such as neck afferents or graviceptors (Bolton et al. 1998; Mittelstaedt 1992; Vaitl et al. 1997, 2002). As a result,
! ! $(! ! ! ! other studies were conducted employing electrical stimulations of the vestibular afferents to avoid inputs from outside this system, and thereby examine more closely the contributions of vestibular afferents exclusively. These methods yielded results in which both inhibitory and excitatory changes, in sympathetic nerve activity, were documented
(Cobbold et al. 1968; Ishikawa & Miyazawa 1980; Ishikawa et al. 1979; Kerman & Yates
1998; Yates et al. 1993a; Yates et al. 1995). For example, the study led by Kerman and colleagues (2000), using electrical stimulation of the vestibular afferents in the cat, confirmed that muscle vasoconstrictor fibres in the face, forelimb and hindlimb could be modified by the stimulus, though the nature of modulation was different depending on location in the body, strongly suggesting different patterning of efferent vestibular mediated control (Kerman et al. 2000b). As discussed earlier, this finding of differential patterning of sympathetic activity is strongly supported by findings during natural stimulations of conscious cats (Wilson et al. 2006b; Yavorcik et al. 2009).
1.3.2 Vestibulosympathetic reflexes in humans
Whilst the studies in animals clearly provided overwhelming evidence for the anatomical and physiological pathways describing the influence of the vestibular apparatus on the cardiovascular system, its role in humans has been more difficult to explore and establish.
Much like the animal work, using different methodologies, the research conducted on human participants employed physiological and electrical stimulation, with the inherent strengths and weaknesses associated with these approaches.
1.3.2.1 Caloric stimulation
Caloric stimulation is a technique that delivers cold or warm water to the tympanic membrane via the ear canal, producing nystagmus (involuntary eye movements), and
! ! $)! ! ! ! hence indicating that vestibulo-occular reflexes have been activated. In short, this method produces a thermal gradient within the semicircular canals (the horizontal canals in particular) that leads to increased endolymphatic flow, ergo, vestibular stimulation.
Employing this natural stimulation, Costa and colleagues (1995) recorded muscle sympathetic nerve activity to unilateral caloric stimulation using warm water irrigation.
The investigators found no evidence of increased sympathetic outflow to the leg (Costa et al. 1995). On the other hand, Cui and colleagues employed bilateral caloric stimulation, using both hot and cold-water irrigation, and concluded that caloric stimulation decreases
SSNA (Cui et al. 1999) and momentarily increases MSNA and that this response is proportional to the degree of nystagmus (Cui et al. 1997). The discrepancy in the findings of these two groups is unclear, but it is possible that the differences are due to the dissimilar means of caloric stimulation the investigators used (Carter & Ray 2008). It is also worth pointing out Ray and colleagues (Ray et al. 1998; Wilson et al. 2004) found no modulation of either for sympathetic nerve activity to either muscle or skin during active horizontal rotations of the head, another method that stimulates the horizontal canals.
Finally, it is important to note that the aforementioned studies limited the stimulations to the horizontal canals, as the vertical canals cannot be selectively stimulated in human subjects. Of course, whilst this means that there is a possibility that the vertical canals play a role in cardiovascular control, studies in the animals strongly argue against this
(Yates & Miller 1994).
1.3.2.2 Head-down neck flexion method
Another means of stimulating the vestibular apparatus physiologically in humans is head- down neck flexion (HDNF). The method entails laying the subject prone with the head and body aligned, and by tilting the head downward the maneuver creates an altered
! ! $*! ! ! ! gravitational input to the otolithic organs. As a response to this stimulus, Essendoh,
Normand and their respective colleagues demonstrated vascular circulatory changes outlining decreases in arterial pressure and blood flow to the limbs (Essandoh et al. 1988;
Normand et al. 1997). However, it was Shortt and Ray (1997) who directly recorded intraneural sympathetic activity and demonstrated that the method leads to an increase in
MSNA (burst frequency) and heart rate; an increase that was sustainable throughout the
10 min of neck flexion (Shortt & Ray 1997). The same response was not evident during recording of sympathetic activity innervating the skin (SSNA) (Ray et al. 1997), further outlining the independency of the two systems (MSNA and SSNA). Furthermore, studies outlined that the response in MSNA elicited is dependent on the magnitude of the stimulus (Hume & Ray 1999) and is the same to the upper limbs as it is to the lower limbs
(Monahan & Ray 2002), contrary to what is reported in animal studies (Kerman et al.
2000b; Wilson et al. 2006b; Yavorcik et al. 2009). However, in addition to stimulating both the utricle and saccule, head-down neck flexion activates several non-vestibular inputs capable of increasing sympathetic outflow – in particular, afferents from muscle
(and other) receptors in the neck (Bolton & Ray 2000).
1.3.2.3 Off vertical-axis rotations (OVAR)
Another approach to investigate the roles of the otolithic organs in regulating MSNA is off vertical-axis rotations (OVAR), which involve continuous rotation of the body at a constant velocity about its longitudinal axis, with the axis of rotation tilted by 15 degrees from Earth vertical. After a short period of time (12 seconds), the signals from the semicircular canals are lost as they adapt to constant velocity rotations (Barmack 2003), allowing the otolithic organs to be activated; by keeping the neck aligned with the body axis this eliminates any neck afferent input. OVAR in the head-up position revealed an
! ! %+! ! ! ! increase in muscle sympathetic nerve activity to the lower limbs, whilst in the head-down position a decrease in MSNA occurred (Kaufmann et al. 2002), results opposite to that seen in HDNF (Shortt and Ray 1997). Discrepancies shown in these studies may be due to the methodology of stimuli used to activate the vestibular apparatus, such as posture relative to gravity, use of dynamic versus static stimuli and neck displacements in one study but not in the other. This generates a need for an experimental design to selectively activate vestibular inputs without acting on other non-vestibular inputs.
1.3.2.4 Galvanic vestibular stimulation
Galvanic vestibular stimulation (GVS) is an electrical form of vestibular stimulation that was initially used to investigate the role of the vestibular system in ocular reflex, locomotion and posture (Cathers et al. 2005; Fitzpatrick et al. 2006). It was later adopted in vestibulosympathetic reflex research as it offers a selective form of stimulation to the vestibular apparatus (Goldberg et al. 1984; Minor and Goldberg 1991; Fitzpatrick & Day
2004). It works through the application of a weak current across the mastoid processes that result in an overall activation of the vestibule (Carter & Ray 2008). There has been no direct recording from human vestibular afferents during GVS, but we can speculate on their behaviour by data obtained from a wide range of species. Goldberg and colleagues
(1984) made direct recordings from vestibular afferents in primates during application of
GVS. They showed that, in the squirrel monkey, when cathodal GVS was applied in the perilymphatic space and anodal GVS applied at a more proximal point, both caused excitatory responses in vestibular afferents (Goldberg et al. 1984). Thus, GVS can be considered to stimulate the hair cell axon terminals of the vestibular afferents and alter their firing (Goldberg et al. 1984). In general, cathodal currents depolarise and thus increase the firing rate of vestibular afferents, whereas anodal currents hyperpolarise and
! ! %"! ! ! ! thereby decrease their firing rate (Wardman & Fitzpatrick 2002). A schematic diagram demonstrating the direction of the electrical current can be seen in Figure 1.9. A limitation of GVS is that it cannot discriminate between the vestibular endorgans (semicircular canals or otolith organs). However, animal research has shown that the response to GVS is predominantly otolithic (Cohen et al. 2012, 2013; Holstein et al. 2012) and the evidence strongly argues against a contribution from the semicircular canals in the modulation of sympathetic activity (Costa et al. 1995; Ray et al. 1998; Wilson et al. 2004). Thus, one can conclude that any changes in sympathetic outflow during GVS can be attributed to activation of the otolithic organs (Carter & Ray 2008; Cohen et al. 2012, 2013; Holstein et al. 2012). Other advantages of GVS is that it is selective to the vestibular system and it does not modulate neck afferents (as in head-down neck flexion), or cause fluid shifts in the body (as in off-vertical axis rotations), or any other inputs that may affect sympathetic outflow - such as heart rate, blood pressure or respiration (Carter & Ray 2008; Cathers et al. 2005).
Figure 1.9 A schematic diagram outlining the direction of GVS. Anodal current (+) is placed on the right and the cathodal current (-) on the left. (Both figure and legend was modified from Cathers et al. 2005).
! ! %#! ! ! !
It was Bolton and colleagues (2004) who first applied GVS in the form of brief (1 s) pulses to examine the vestibular contributions to cardiovascular control, in particular its effect on sympathetic outflow to muscle vascular beds in the lower limbs. The investigators found that the application of a 2 mA current across the mastoid processes in a binaural, bipolar fashion adequately modified the firing of vestibular afferents because subjects reported strong perceptual illusions of sway towards the anode. However, despite being delivered at different times following the R-wave, GVS failed to cause a net change in MSNA, but did cause short-latency bursts of skin sympathetic nerve activity (SSNA).
Therefore, it was concluded that the short duration of electrical vestibular stimuli did not interact with the baroreceptors, nor did they cause modulation of muscle sympathetic nerve activity, but did excite cutaneous vasoconstrictor and sudomotor neurones (Bolton et al. 2004).
Alternatively, Voustianiouk and colleagues (2006) employed dynamic stimuli in the form of brief trains (30 ms) of 10 pulses of GVS, and found a clear modulation of MSNA.
Whilst many animal studies, using trains of electrical stimuli to the vestibular system demonstrated the elicited cardiovascular change (Ishikawa & Miyazawa 1980; Kerman et al. 2000c; Uchino et al. 1970), Voustianiouk and colleagues provided evidence for the presence of short-latency vestibulosympathetic reflexes in humans. The authors concluded that these reflexes might contribute to the control of arterial blood pressure, especially during rapid postural changes (Voustianiouk et al. 2006). To further investigate, Bent et al (2006) used continuous (as opposed to intermittent) dynamic galvanic vestibular stimulation to assess the capacity of vestibular inputs to modulate sympathetic outflow. Vestibular afferents were stimulated using continuous sinusoidal
(0.5–0.8 Hz, 60–100 cycles, ±2 mA) bipolar, binaural GVS (Bent et al. 2006). Participants
! ! %$! ! ! ! experienced strong perceptual illusions of “rocking in a boat” or “swinging from side to side in a hammock”, at a frequency matching that of the stimulation. Interestingly, this study showed that overall MSNA increased by 156% and that sinusoidal GVS was able to cyclically modulate MSNA (Figure 1.10). Furthermore, there was evidence of generation of de novo sympathetic bursts to the dynamic GVS, producing two bursts of MSNA per cardiac interval (Bent et al. 2006), with one burst being temporally coupled to the vestibular rhythm and the other being time-locked to the cardiac rhythm (Figure 1.11).
This conclusively demonstrated that the human vestibular apparatus exerts a significant influence on MSNA that may operate independently of the baroreceptor system in the control blood pressure (Bent et al. 2006).
! ! %%! ! ! !
Figure 1.10 Baroreceptor influenced MSNA is influenced by vestibular inputs. Experimental records from one subject. A Rest activity used as a control. B-D represent consecutive sequences recorded during the course of sinusoidal GVS (0.5Hz, 60-100 cycles, 2mA). The rectangles displays the relationship of the sympathetic burst to the cardiac and vestibular input (Bent et al. 2006).
! ! %&! ! ! !
Figure 1.11 Correlation analysis between MSNA and cardiac and vestibular rhythms. (A) Cross-correlation histograms of the relationship between MSNA and ECG and autocorrelogram of ECG. (B&C) Cross correlation of ECG and GVS and respiration and GVS. (D&E) Cross-correlation histogram between MSNA and the GVS (Bent et al. 2006).
! ! %'! ! ! !
To further explore the effect of sinusoidal GVS, in separate studies, the Macefield laboratories exposed participants to a wider range of frequencies (0.2 – 2.0 Hz, 200 cycles, ±2 mA) during recordings of both muscle and skin sympathetic nerve activity
(Grewal et al. 2009; James et al. 2010). Similarly, in these studies all subjects reported the evoked robust vestibular illusion of “rocking in a boat” or “swinging from side to side”.
Cross-correlation analysis revealed partial phase locking to both muscle and skin with the vestibular modulation of MSNA found to be greatest at 0.2 Hz and lowest at 0.8 Hz – the latter being the frequency closest to the cardiac rhythm. Unlike the vestibular modulation of MSNA, that of SSNA was high at all frequencies of stimulation. This prompted further investigation to better understand the nadir modulation of MSNA. In an extension of the latter study, sinusoidal GVS (sGVS) was delivered at the resting heart rate of a given subject, and at frequencies (0.1, 0.2, 0.3 and 0.6 Hz) above or below this central frequency. Results confirmed that vestibular modulation of MSNA was significantly reduced when it coincides with the cardiac rhythm, confirming the competitive nature of vestibular and baroreceptor inputs. This further highlights the dominance of the arterial baroreceptors in modulating MSNA (James & Macefield 2010).
Furthermore, as the highest modulation of sympathetic outflow in the study by Grewal et al (2009) was reported to be 0.2 Hz, in my pre-doctoral research I further explored the vestibular modulation during sGVS delivered at frequencies lower than those used previously (0.08 – 0.18 Hz). These are frequencies specifically associated with very slow postural displacements (i.e. such as those experienced during tall building sway, evoking motion sickness) (Hammam et al. 2011, 2012). Analysis of the neural discharge during the periods of stimuli revealed for the first time that low-frequency sinusoidal GVS induces two peaks of modulation of MSNA for each cycle of stimulation (Figure 1.12)
! ! %(! ! ! !
(Hammam et al. 2011). This observation also held true when recording of sympathetic activity to the skin (Hammam et al. 2012).
Figure 1.12 Cross-correlation histograms between MSNA and GVS during sGVS. Vestibular modulation of MSNA during sGVS at 0.08 Hz (A) 0.13 Hz (B) and 0.18 Hz (C). Two bursts response, in MSNA, for each cycle of sGVS – one related to the positive phase of the sinusoid and the other to the negative phase. Two peaks of modulation are apparent at 0.08 and 0.13 Hz, and partially at 0.18 Hz (Hammam et al. 2011).
! ! %)! ! ! !
In order to explain the two-peak response it is noteworthy that all experiments in
Hammam et al. (2011; 2012) recorded sympathetic nerve activity from the left common peroneal nerve. In addition, the anode was always placed over the right mastoid. As will be shown below, these are important to bear in mind when interpreting the results. As the cross-correlation analysis revealed two bursts of MSNA per cycle of sinusoidal GVS, we proposed that the primary peak (Figure 1.12), defined as the larger burst, was related to the positive phase of the sinusoid (i.e. 0 to 2 mA), which occurs over the right mastoid process. It is known that hyperpolarisation occurs at the anode (Fitzpatrick & Day 2004), so we concluded that hyperpolarisation of the vestibular nerve occurred on the right side during the positive phase of stimulation. However, given that the stimuli were applied in a binaural bipolar fashion (i.e. across both mastoid processes), hyperpolarisation of the right vestibular nerve will be occurring concurrently with depolarization of the left vestibular nerve – i.e. when the left side is experiencing the negative phase of the sinusoid (0 to -2 mA). As the current slowly alternates, shifting back towards the right side it then causes hyperpolarisation of the left, but depolarisation of the right vestibular nerve. It is this secondary depolarization that is believed to be responsible for the secondary burst. The secondary peak – defined as the smaller burst - corresponds to the negative phase of the sinusoid over the right vestibular nerve. This phenomenon - a primary peak associated with the positive phase of the sinusoidal stimulus, followed shortly afterwards by a second peak associated with the negative phase - matched the stimulus frequency but was never observed at the higher frequencies of stimulation used previously (Bent et al. 2006;
Grewal et al. 2009; James et al. 2010), presumably because at such frequencies there is insufficient time for a second peak to be expressed. Nevertheless, cross-correlation data of
MSNA and sGVS show a high prevalence of secondary peaks of modulation at all frequencies (0.08 – 0.18 Hz), though they were absent in some subjects at the higher
! ! %*! ! ! ! frequencies. Indeed, on average the incidence of secondary bursts was higher at lower frequencies of sGVS (0.08 Hz) than at the higher frequencies (0.18 Hz), where the shorter interval between the sinusoidal peaks curtailed expression of this secondary phase of modulation.
These series of studies showed that cyclically changing vestibular inputs could exert a potent excitation of muscle vasoconstrictor drive (Bent et al. 2006; Grewal et al. 2009;
James et al. 2010; Hammam et al. 2011). Presumably, this excitation acts through the rostral ventrolateral medulla (RVLM) - the primary output nucleus for muscle vasoconstrictor neurones (Dampney et al. 2003a, b). This nucleus has been shown to receive excitatory inputs from the vestibular apparatus, primarily from the otolith organs
(Yates et al. 1993a; Yates et al. 1991). Accordingly, we speculated that the frequency- dependant modulation of MSNA during sGVS reflects the operation of vestibular inputs from both sides of the head projecting onto the RVLM. This was later confirmed during experiments involving bilateral recordings of MSNA, where cross-correlation analysis did indeed reveal a reversal of modulation in the primary and secondary peaks recorded from the left and right sides: a primary peak on the left was associated with a secondary peak on the right, and a secondary peak on the left was associated with a primary peak on the right (Figure 1.13 and 1.14). This is probably of greater interest physiologically, given that it supports the idea that sympathetic control of blood pressure and blood flow is lateralised, at least with respect to the vestibulo-sympathetic reflexes studied. It is generally accepted that sympathetic outflow is symmetrical: resting burst rates and amplitude distributions of muscle sympathetic nerve activity have been shown to be similar on the two sides (Sundlof & Wallin 1977; Sverrisdottir et al. 1998); the same has been shown for skin sympathetic nerve activity (Bini et al. 1981). However, whilst the
! ! &+! ! ! ! evidence by El Sayed (2012) supports the notion of lateralization, only one other study has specifically addressed lateralisation of sympathetic control. Diedrich et al. (2009) showed that loading of carotid sinus baroreceptors by sinusoidal neck suction caused a differential expression of MSNA on the left and right sides, abolishing the normally right- sided dominance of MSNA. Nevertheless, those are interesting findings that merit further physiological and anatomical investigations.
As previously discussed, it is believed that vestibulosympathetic reflexes are mediated by the otolith organs rather than the semicircular canals (Costa et al. 1995; Ray et al. 1998) and, while GVS affects the firing of afferents originating in all parts of the vestibular apparatus (Goldberg et al. 1984; Minor & Goldberg 1991), recent evidence supports the idea that it is only the otolithic organs that participate in vestibulosympathetic reflexes induced by sinusoidal GVS (Cohen et al. 2012; Holstein et al. 2012). However, what we do not know is whether it is the utricular or saccular components of the otolithic organs that are mediating the vestibulosympathetic reflexes. This requires a different means of vestibular afferent stimulation with the ability to eliminate the semicircular canals and differentiate between the otolithic organs.
! ! &"! ! ! !
Figure 1.13 Bilateral recordings of muscle sympathetic nerve activity, together with ECG, blood pressure and respiration, during sinusoidal galvanic vestibular stimulation (GVS) at 0.08 Hz in one subject. Overall, sympathetic outflow was similar between the two sides, but close inspection revealed subtle differences. In the expanded section, the sympathetic bursts have been shifted back 1.25 s in time to account for peripheral conduction delays, allowing those bursts aligned with the cardiac cycle (‘c’) or vestibular stimulus (‘v’) to be identified (El Sayed et al. 2012).
! ! &#! ! ! !
Figure 1.14 Mean ± SE modulation indices for the primary (dark grey) and secondary (light grey) peaks of modulation of MSNA. Data obtained from 10 subjects (El Sayed et al. 2012).
! ! &$! ! ! !
1.3.2.5 Linear acceleration
Linear acceleration is a natural means of activating the vestibular apparatus providing an insight into the physiological role of vestibulosympathetic reflexes in humans. Previous studies have employed linear accelerations to demonstrate the contribution of the otolithic organs to cardiovascular control. Yates and colleagues (1999) showed increases in blood pressure and heart rate during linear accelerations (200 mG); findings that were absent in patients with idiopathic profound bilateral reduction in vestibular function. Similar results were found by Jauregui-Renaud et al (2006): control subjects showed a sustained increase in heart rate, and a transient increase in respiration, when exposed to brisk linear accelerations (260 mG), while patients with chronic bilateral vestibular dysfunction did not. These studies, amongst others (Jauregui-Renaud et al. 2003, 2005; Radtke et al. 2000,
2003; Yates et al. 1999) strongly support the contribution of the otolithic organs to cardiovascular control. Furthermore, Cui et al (2001) recorded MSNA in subjects that were exposed to five cycles of high acceleration, of varying amplitudes (100, 150, 200 mG), in the antero-posterior direction on a magnetically levitated sled and found that higher magnitudes of accelerations decreased total MSNA, in both antero-posterior and horizontal directions (Cui et al. 1998; 2001). However, all of these studies mentioned employed linear displacements at high accelerations (100-260mG) activating extra-cranial receptors – such as those activated by fluid shifts.
! ! &%! ! ! !
1.4 Aims
Based on the body of evidence presented, in animals and humans, the general purpose of my experiments is to better identify the role of the vestibular system and of the neck afferents in influencing sympathetic outflow. I performed a series of experiments during my candidature examining the role of vestibular utricle (otolithic endorgan) and neck afferents in mediating sympathetic outflow.
! Study I: Utricular influence on SSNA
As discussed, previous studies have employed linear accelerations at high amplitudes
(100-260mG), which may have compromised the selectivity of vestibular stimulation and recruited extra-cranial afferents. In this study, employing slow sinusoidal linear accelerations (~4mG) as a selective means of stimulation, I tested the hypothesis that one set of otolithic afferents - those located in the utricle, which are sensitive to displacement in the horizontal axis - could entrain skin sympathetic nerve activity.
Study II: Utricular influence on MSNA
The aim of this study is to assess whether physiological activation of the utricle has an effect on sympathetic outflow to muscle of the lower limbs. I tested the hypothesis that selective activation of utricular afferents does not evoke vestibulosympathetic reflexes to the muscle vascular bed of the lower limbs, given that it is likely that the vestibular contribution to blood pressure regulation is more important in the upright position and hence is governed by afferents within the saccule, which are sensitive to acceleration in the vertical (gravitational) plane.
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Study III: Subperceptual accelerations and its effect on MSNA
In Study III, we delivered sinusoidal linear accelerations to seated subjects at amplitudes extending from 1.25 to 30 mG to assess the effects of amplitude on the magnitude of the modulation of MSNA. In addition, using the same stimuli, we assessed the capacity of subjects to detect motion, and to know the direction of that motion, with a view to identifying whether there is any relationship between perceptions of motion and the expression of vestibulosympathetic reflexes. Specifically, we set out to test the hypothesis that vestibular modulation of MSNA can be observed even during sinusoidal movements that cannot be perceived.
Study IV: Neck proprioceptors and their contribution to MSNA modulation
The aim of Study IV is to further investigate extra-vestibular receptors by examining the effects of a dynamic mechanical stimulus of the neck - sinusoidal lateral stretch of the neck muscles - on sympathetic outflow to the muscle vascular bed in the lower limbs of humans. In particular, I tested the hypothesis that neck movements modulate MSNA in the absence of changes in vestibular afferent input.
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Note:
Please find attached in the Appendices section the following papers (published or in review) corresponding to research that I have carried out prior to or during my candidature. More specifically, Appendices 1-4 correspond to Chapters 3-6, respectively.
Appendix 1: • This paper corresponding to Chapter 3 has been accepted for publication in the journal Experimental Brain Research on the 01st of May 2012 entitled “Low-frequency physiological activation of the vestibular utricle causes biphasic modulation of skin sympathetic nerve activity in humans”. It was authored by Grewal T, Dawood T, Hammam E, Kwok K, Macefield VG. Exp Brain Res 220:101-108.
Appendix 2: • This paper corresponding to Chapter 4 has been accepted for publication in the journal Experimental Brain Research on the 28th of June 2013 entitled “Modulation of muscle sympathetic nerve activity by low-frequency physiological activation of the vestibular utricle in awake humans”. It was authored by Hammam E, Kwok K, Macefield VG. Exp Brain Res 230:137-142.
Appendix 3: • This paper corresponding to Chapter 5 has been accepted for publication in the journal Experimental Brain Research on the 25th of January 2014 entitled “Vestibular modulation of muscle sympathetic nerve activity by the utricle during sub- perceptual sinusoidal linear acceleration in humans”. It was authored by Hammam E, Chui LVH, Wong KS, Kwok K, Macefield VG. Exp Brain Res 232: 1379-1388.
Appendix 4: • This paper corresponding to Chapter 6 has been accepted for publication in the journal Experimental Brain Research on the 12th of March 2014 entitled “Neck proprioceptors contribute to the modulation of muscle sympathetic nerve activity to the lower limbs of humans”. It was authored by Bolton PS, Hammam E, Macefield VG. Exp Brain Res 232(7): 2263-2271.
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Appendix 5: • The following attachment is paper that discusses the influence of the saccular hair cells on modulation of MSNA. It is entitled: “Modulation of muscle sympathetic nerve activity by the vestibular otoliths during sinusoidal linear acceleration in supine humans”. It is authored by Hammam E, Bolton PS, Macefield VG and is currently submitted (29/05/2014) and under review in the Journal: Frontiers in Neuroscience, section Autonomic Neuroscience.
Appendix 6:
• Attachment 6 is work that was conducted during my early candidature and has been discussed in my introduction. The study examines the bilateral modulation of MSNA and SSNA by vestibular hair cells. It is entitled: “Evidence from bilateral recordings of sympathetic nerve activity for lateralization of vestibular contributions to cardiovascular control”. It was authored by El Sayed K, Dawood T, Hammam E, Macefield VG and accepted for publication on July 3, 2012 in the journal Experimental Brain Research – 221:427-436.
Appendix 7:
• Attachment 7 is vestibular research that I completed during my Bachelor of Medical Science (Honours). The study is entitled: “Low-frequency sinusoidal galvanic stimulation of the left and right vestibular nerves reveals two peak of modulation in muscle sympathetic nerve activity”. It was authored by Hammam E, James C, Dawood T, Macefield VG and accepted for publication on July 11, 2011 in the journal Experimental Brain Research – 213:507-514.
Appendix 8:
• Attachment 8 is also another study conducted for my Honours. The study published is entitled: “Low-frequency galvanic vestibular stimulation evokes two peaks of modulation in skin sympathetic nerve activity”. It was authored by Hammam E, Dawood T, Macefield VG and accepted for publication on March 28, 2012 in the journal Experimental Brain Research – 219:441-446.
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CHAPTER 2
GENERAL
METHODS
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2.1. Subjects
All studies were approved by the Human Research Ethics Committees of the University of Western Sydney and, for the experiments in Study III the Hong Kong University of
Science and Technology and satisfied the Declaration of Helsinki. All participants provided informed written consent to the experiments and were healthy human volunteers aged 18-55 years. Prior to the experiments, participants were asked to avoid any caffeinated beverages and to abstain from smoking, so as not to affect sympathetic nerve activity. It is known that caffeine causes an increase in MSNA (Corti et al. 2002), while smoking increases sympathetic outflow to both muscle and skin (Hering et al. 2010;
Narkiewicz et al. 1998).
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2.2 Experimental Protocol
2.2.1 Motion simulator
In Study I & Study II, experiments were performed at the University of Western Sydney on 11 subjects (6 male, 5 female; 18 to 44 years) and 12 subjects (8 male, 4 female; 19 to
31 years) respectively. Subjects were seated in a comfortable chair, with the legs supported horizontally and the head resting vertically against a padded support. To maintain utricular hair cell activation, the participants’ head was then stabilized with a
Velcro strap to avoid rotations and other movements. A blindfold, earplugs and earmuffs were used to avoid any visual or audible clues. The chair was surmounted on a motorised platform driven by two linear motors that had a maximal excursion of ±20 cm in the X
(antero-posterior) and Y (medio-lateral) directions. Accelerations were measured using two high-sensitivity accelerometers, with a threshold of <10 µG (QA650, Honeywell,
USA), fixed to the platform – one orientated in the X-axis, the other in the Y. Sinusoidal movements of the platform were delivered separately in the X or Y directions at 0.08 Hz,
4mG for 100 cycles. The extent of the displacements was ±15 cm. The subjects were blindfolded during the stimulation and were asked to provide their perceptions at the conclusion of each stimulation sequence.
Study III was conducted at the Hong Kong University of Science and Technology.
During the experiment, 13 subjects (7 male, 6 female; 18 to 33 years) were seated in a padded armchair with their feet on the floor and the head supported by a padded and enclosed frame that kept the head and neck in a vertical position. The head was then stabilized with a Velcro strap to avoid rotations and other movements, this enables us to selectively activate the utricular hair cells in the vestibular apparatus. The chair was
! ! '"! ! ! ! placed against the rear wall of a 4 x 3 m room. The room was built on a motion simulator platform that contained two sets of linear motors capable of accelerating the room in the horizontal plane with maximum amplitude of 30 mG (0.3 ms-2). The room contained no windows, so there were no visual cues of movement. In this study, the motion was limited to the antero-posterior direction (X-axis). Subjects were provided with earplugs and were exposed to white noise. Six amplitudes of acceleration - 1.25, 2.5, 5, 10, 20 and 30 mG - and a static condition (no motion) were presented in a quasi-random fashion, at a constant frequency of 0.2 Hz, with a 2-minute interval between each. For the motion detection experiments sinusoidal movements of the room were delivered as sets of 25 cycles. The maximal extent of the displacements was ±800 mm. Subjects were not informed when the motion would start, and were instructed to move a linear potentiometer, oriented in the axis of motion, with forward movement of the slider indicating perception of motion in the forward direction and movement backward perception of motion in the posterior direction. Measurements of the potentiometer signal were made in each hemi-cycle of the movement, resulting in 50 points of measurement. Motion detection was measured according to the following criterion: if the movement of the potentiometer occurred within a given hemi-cycle of acceleration this was counted as a perceived movement. If movement of the potentiometer occurred irrespective of direction it was counted as a perception of motion; if movement of the potentiometer occurred in the same direction as the direction of actual movement it was counted as a percept of the knowledge of the direction of movement, as well as a perception of movement. The threshold for correct detection was set at 67% for both measures.
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2.2.2 Neck Table
In Study IV, where the relationship of neck proprioceptors and MSNA were investigated twelve normotensive healthy volunteers (8 male, 4 female; 18-25 years) participated in the study conducted at the University of Western Sydney. Participants lay supine on a purpose built table that fixed the head in space but allowed passive neck muscle stretch by displacing the body about the fixed head with the axis of rotation centered at the level of the 4th and 5th cervical vertebra. The table was fitted with a calibrated potentiometer
(PE30, Vishay Sfernice, Malvern, PA, USA) that recorded table position (neck angle) and motion relative to the fixed head. Two minutes of rest was recorded with the head and body aligned in the mid-sagittal plane and then during sinusoidal displacement of the body (100 cycles; range 0.27 to 0.43 Hz) at 35o peak-to-peak (right-left), followed by a rest period with the body aligned, in the midline, with the head. In addition, recordings were the made during a ramp (8.9 ± 1.3o/s (mean ± SD)) and hold (17.5o for 54 ± 9s
(mean ± SD)) displacement of the body, about the fixed head, to the right and then to the left with 1 minute rest period between the two lateral displacements. All recordings were made with the subjects eyes closed.
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Figure 2.1 An illustration of the neck table displacement. Participants lay supine on a table that fixed the head in space but allowed passive neck muscle stretching by displacing the body under the fixed head (35° peak-to-peak at 0.33 – 0.57 Hz).
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2.2.3 Microneurography
Microneurography was used to record oligounitary muscle sympathetic nerve activity in our awake subjects. No attempt was made to record single unit activity. The protocol of employing this technique began with brief pulses (0.2 ms) of cathodal (depolarising) stimuli (<5 mA) delivered at 1 Hz to the skin at the fibular head via a 1 mm search probe connected to a computer–controlled, constant-current isolated stimulator (Stimulus
Isolator, ADInstruments, Sydney, Australia); a Ag-AgCl surface electrode on the skin on the opposite side of the knee served as the anode. This allowed us to identify sites on the skin immediately overlying the nerve: by definition, the nerve is closer to the skin if the current required is lower to activate the nerve (and evoke muscle twitches in the supplied muscles and paraesthesiae in the cutaneous distribution of the nerve). After identifying the best sites and marking the skin, the area was sterilised with an alcohol swab and a tungsten microelectrode (FHC, Bowdoinham, ME, USA) with a 200 !m diameter and a 2-
5 !m uninsulated tip inserted into the skin. An uninsulated reference microelectrode was inserted subcutaneously ~2 cm from the recording microelectrode. The microelectrodes were connected to the input terminals of an isolated amplifier headstage (NeuroAmp EX,
ADInstruments, Sydney, Australia). Intraneural stimulation (0.2 ms, 1 Hz, 1 mA) through the recording microelectrode, relative to the reference electrode, was performed whilst advancing the microelectrode towards the nerve (Figure 2.3). The subject was asked to report any sensations, especially radiating paraesthesiae (“pins and needles”) in the cutaneous distribution of the common peroneal nerve (dorsum of the foot or the lateral aspect of the leg). These sensations were used to guide the microelectrode tip into a cutaneous fascicle of the nerve, whereas muscle twitches indicated that the microelectrode tip was approaching a muscle fascicle. Stimulus currents were reduced progressively as the cutaneous sensations or muscle twitches increased in intensity, until responses could
! ! '&! ! ! ! be evoked at currents <0.05 mA. The stimulating leads were then removed and the preamplifier and amplifier switched on; the surface electrode on the opposite side of the knee served as the ground electrode. Small advances of the microelectrode caused the tip to penetrate the wall of the fascicle, heralded by “insertion discharges” – bursts of action potentials induced by mechanical irritation of myelinated axons. A period of 4 weeks minimum was allowed to elapse prior to recording from the same nerve in the same.
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2.2.4 Recording procedures
Neural activity was recorded (Figure 2.2), together with the other physiological parameters described below, on a computer-based data acquisition system (PowerLab
16SP, ADInstruments, Sydney, Australia). The neurogram was sampled at 10 kHz and displayed in real-time on a computer monitor and routed to external speakers for audio feedback. Cutaneous fascicles were defined by the generation of paraesthesiae during intraneural stimulation and afferent responses to stroking the skin in the fascicular innervation territory. Muscle fascicles were defined by the generation of discrete muscle twitches of the innervated muscles (Delius et al. 1972).
Muscle sympathetic nerve activity (MSNA) was identified according to the following criteria (Sundlof & Wallin 1977):
• Regular spontaneous bursts synchronised to the cardiac cycle;!
• Sustained increases in activity during either an inspiratory-capacity or
progressive increases in activity during a prolonged end-expiratory apnoea !
• No responses to arousal stimuli!
Skin sympathetic nerve activity (SSNA) was identified according to the following criteria
(Delius et al. 1972):
• Irregular spontaneous bursts unsynchronised to the cardiac cycle;!
• Reflex activation in response to loud/surprising sensory stimuli (such as a
sudden loud yell or clap)!
• No increases in activity during a voluntary inspiratory apnoea!
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Figure 2.2 Standard multi-unit recording of muscle sympathetic nerve activity (MSNA). This is achieved via an insulated tungsten microelectrode inserted into a muscle fascicle of the common peroneal nerve. Note that bursts of MSNA are composed of negative- going spikes that occur with a clear cardiac rhythmicity.
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Reference electrode
Pre- amplifier headstage
Recording tungsten Vaccum microelectrode Cushion
Figure 2.3 Continuous neural recording from a muscle fascicle of the common peroneal nerve supplying the tibialis anterior (TA) muscle. The Pre-amplifier headstage is used to amplify the raw nerve recording 100 X. The recording tungsten microelectrode is shown, as is the subdermal reference electrode. The green lead is attached to a surface Ag/AgCl ground electrode (not shown). The vaccuum cushion on which the upper leg rests provides mechanical stability to the thigh and allows the exposure of the fibular head.
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2.2.5 Other measured parameters
Electrocardiographic activity (ECG) was recorded with Ag-AgCl surface electrodes on the chest and sampled at 2 kHz. Respiration was recorded using a piezoelectric transducer wrapped around the chest, sampled at 100 Hz. This generates a linear voltage proportional to the changes in thoracic circumference associated with breathing (Pneumotrace, UFI,
Morro Bay, CA, USA). Non-invasive blood pressure was recorded continuously via digital arterial plethysmography (Finometer; Finapres Medical System, Amesterdam, The
Netherlands), which incorporates correction for the height of the hydrostatic column (i.e. difference in position of the finger sensor relative to the position of the heart). In addition, this system calculates haemodynamic parameters such as stroke volume, total peripheral resistance and cardiac output. The Finometer repeatedly calibrates the reconstructed blood pressure wave at set intervals against brachial arterial measurements using an upper arm cuff. Arterial pressure was sampled at 400 Hz. Skin blood flow was measured from the index finger using an infrared photoelectric pulse plethysmograph (ADIsntruments,
Sydney, NSW, Australia). The sensor records pulsatile changes in cutaneous blood volume, as the arterial pulse expands and contracts the microvasculature, bringing about changes in cutaneous blood flow. This signal was sampled at 400 Hz. Sweat release was indicated by changes in skin potential (BioAmplifier, ADInstruments, Sydney, Australia), measured with Ag-AgCl surface electrodes on the volar and dorsal aspects of a hand and sampled at 400 Hz.
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2.3 Analysis
Sympathetic nerve activity (muscle) was displayed as an RMS-processed (root mean squared, moving average time-constant 200 ms) signal. However, the primary analyses were performed using the raw, negative-going, sympathetic spikes to avoid any contamination from spikes produced by positive-going myelinated axons (such as spontaneously active muscle spindles) (Bent et al. 2006; Grewal et al. 2009). Negative going spikes in the neurogram, R-waves of the ECG and the positive peaks of the sinusoidal stimulus were detected using window discriminator software (Spike Histogram for Macintosh v2.2, ADInstruments, Sydney, Australia). This same software was used to construct cross-correlation and autocorrelation histograms. For each event the program plots the time of the current event (time 0) and the times of events before (negative times) and after (positive times) the current event. In this way, the periodicity of the signal can be illustrated in the form of an autocorrelation histogram, and compared with the periodicities revealed by the cross-correlation histograms. Cross-correlation analyses are used to examine the temporal relation between a particular physiological variable, such as the spikes comprising the MSNA signal, and another – in our case the R-waves of the
ECG or the positive peaks of the sinusoidal signal recorded from the motion simulator accelerometers or neck table potentiometer. Accordingly, this may reveal the influence of one physiological variable upon the pattern of activity of another. Cross-correlation histograms (cross-correlograms) are constructed by selecting in turn, each impulse in the analysis channel (MSNA) that occurs at a time defined with respect to events in the reference channel (R-waves or positive peaks of the sinusoids) and allocating to appropriate bins in the histogram all the impulses occurring within a selected analysis time (50 ms bins). Discriminator levels of the neural activity were adjusted (Spike
Histogram, ADInstruments, Sydney, Australia) so that negative-going (C-fibre) spikes
! ! ("! ! ! ! exhibited a robust cardiac modulation, as revealed by cross-correlation between the neural activity and the ECG. These same discriminator settings were used for construction of cross-correlograms between the MSNA and the positive peaks of the sinusoidal stimulis
(motion simulator or neck table).
The histogram data were exported as numerical text to a statistical and graphical analysis program (Prism 5 for Windows v 5.03, GraphPad Software, USA), to fit the data to a mathematical function - a smoothed polynomial. Lower-order polynomials were used to fit curves to the cardiac cross-correlation histograms while higher-order polynomials were required to fit curves to the slower vestibular cross-correlation histograms. The purpose for smoothing cross-correlation histograms between sympathetic nerve activity and platform/neck-table motion is to eliminate any cardiac related peaks. This enables us to further examine the nerve activity more accurately with respect to the sinusoidal motion.
The modulation of sympathetic nerve activity was quantified by measuring the difference in the number of spikes on the smoothed curve at the peak of the modulation and at the trough. This figure was then expressed as a percentage by employing the following formula:
Modulation Index % = [(Peak - Trough)] x100 (MI) Peak
Comparisons were then made between the mean modulation values of each type of stimuli across all individuals. A one-way analysis of variance (ANOVA) was used to determine if there was a significant difference (P < 0.05).
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Statistical Analyses:
Study I
A one-way analysis of variance (ANOVA) was used to determine if there was a significant difference (P < 0.05) between any of the platform motions; this was coupled with a Mann-Whitney test (Prism 5.0 for Macintosh, GraphPad Software, USA).
Study II
In addition to the above, a paired t-test was to determine any statistically significant differences between the primary and secondary peaks and if there were any variances in their latencies.
Study III
Ordinary One-way analysis of variance (ANOVA) was used in conjunction with a paired t-test to determine whether there was a significant difference (p < 0.05).
Study IV
In addition to the above statistical tests, the RMS-processed MSNA signal was used to count individual bursts to calculate burst frequency (bursts/min), burst incidence (bursts per 100 heart beats) and cumulative burst amplitude during the 60 s immediately preceding (rest) and during neck muscle stretch, thereby allowing comparison with previous studies that have used a burst analysis protocol (Shortt & Ray 1997; Ray &
Hume 1998; Hume & Ray 1999). Statistical analyses were performed using Prism v6.01
(GraphPad software, USA). Data are presented as mean ± SEM, and a significant statistical difference (paired t-test) was considered to exist if p < 0.05.
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CHAPTER 3
Modulation of skin sympathetic nerve activity
(SSNA) by the vestibular utricle
This chapter has been edited from the original paper that was published in the journal
Experimental Brain Research on the 1st of May 2012, titled “Low-frequency physiological activation of the vestibular utricle causes biphasic modulation of skin sympathetic nerve activity in humans”. It was authored by Grewal T, Dawood T,
Hammam E, Kwok K, Macefield VG. Exp Brain Res 220:101-108
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SUMMARY
We have previously shown that sinusoidal galvanic vestibular stimulation (sGVS), a means of selectively modulating vestibular afferent activity, can cause partial entrainment of sympathetic outflow to muscle and skin in human subjects. However, GVS influences the firing of afferents from the entire vestibular apparatus, including the semicircular canals. In Study I, I tested the hypothesis that selective stimulation of one set of otolithic organs - those located in the utricle, which are sensitive to displacement in the horizontal axis - could entrain sympathetic nerve activity. Skin sympathetic nerve activity (SSNA) was recorded via tungsten microelectrodes inserted into cutaneous fascicles of the common peroneal nerve in 10 awake subjects, seated (head vertical, eyes closed) on a motorised platform. Slow sinusoidal accelerations-decelerations (~4 mG) were applied in the X (antero-posterior) or Y (medio-lateral) direction at 0.08 Hz; composite movements in both directions were also applied. Subjects either reported feeling a vague sense of movement (with no sense of direction), or no movement at all. Nevertheless, cross- correlation analysis revealed a marked entrainment of SSNA for all types of movements: vestibular modulation was 97±3 % for movements in the X-axis and 91±5 % for displacements in the Y-axis. For each sinusoidal cycle there were two major peaks of modulation – one associated with acceleration as the platform moved forward or to the side, and one associated with acceleration in the opposite direction. These observations, I believe, are reflecting inertial displacement of the stereocilia within the utricle during acceleration, which causes a robust vestibulosympathetic reflex.
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INTRODUCTION
As discussed previously (Introduction: Chapter 1), it is believed that vestibulosympathetic reflex is mediated by the otolith organs rather than the semicircular canals (Costa et al. 1995; Ray et al. 1998; Yates et al. 1993a). We have previously shown that sinusoidal galvanic vestibular stimulation (sGVS), applied bilaterally to the mastoid processes at 0.5-0.8 Hz, induces a potent modulation of muscle sympathetic nerve activity
(MSNA), with the occasional generation of de novo bursts time-locked to the vestibular input (Bent et al. 2006). Subsequent studies from our laboratory showed that continuous sinusoidal GVS at 0.2-2.0 Hz causes partial phase-locking of sympathetic nerve activity directed to both muscle (Grewal et al. 2009) and skin (James et al. 2010). The vestibular modulation of MSNA appears to be independent of the cardiac modulation (Bent et al.
2006) but competes with inputs from the arterial baroreceptors (Grewal et al. 2009; James et al. 2010).
Most recently, I demonstrated that sinusoidal GVS applied at 0.08-0.18 Hz generates two bursts of modulation of MSNA, which I interpreted as reflecting bilateral projections from the vestibular nuclei to the medullary nuclei responsible for the generation of MSNA
(Hammam et al. 2011). Moreover, application of these low frequencies also induced two peaks of modulation of skin sympathetic nerve activity (SSNA; Hammam et al. 2012).
While GVS affects the firing of afferents originating in all parts of the vestibular apparatus (Goldberg et al. 1984; Minor & Goldberg 1991), recent evidence supports the idea that it is only the otolith organs that participate in vestibulosympathetic reflexes induced by sinusoidal GVS (Cohen et al. 2012; Holstein et al. 2012). However, what we do not know is whether it is the utricular or saccular components of the otolith organs that can evoke vestibulosympathetic reflexes. So, the purpose of this study is to test the
! ! ('! ! ! ! hypothesis that, in awake human subjects, selective stimulation of afferents in the utricle can induce a potent modulation of skin sympathetic nerve activity (SSNA) as we know that this shows a very robust modulation during sinusoidal GVS (James et al. 2010;
Hammam et al. 2012). Subjects were seated on a motorised platform with the head supported vertically: in this position, horizontal displacements of the body will stimulate receptors exclusively in the utricle. In addition, we used the same low frequency – 0.08
Hz – with which I had recently documented two peaks of modulation of MSNA and
SSNA during sinusoidal GVS (Hammam et al. 2011, 2012) in order to determine whether two peaks of modulation could also be evoked during physiological stimulation of vestibular afferents.
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RESULTS
In our previous studies using sinusoidal galvanic vestibular stimulation all subjects reported robust vestibular illusions of “rocking in a boat” or “swinging in a hammock” that matched the stimulation frequency. However, in the current study, which utilised physiological activation of the utricle, the majority of subjects reported no sensation of movement of the platform at frequencies of 0.08 Hz; those that could perceive the slight movements reported difficulty in determining the direction of movement. The majority of subjects felt relaxed during the platform motion. Somewhat surprisingly, nausea was rare, although one subject had to terminate the experiment after only a few minutes of platform motion. For the remainder of the subjects, 8 reported no nausea while 2 reported some slight nausea (average discomfort level = 1.03± 0.02/10); none felt they were going to vomit.
Experimental records from one subject, at rest and during XY platform motion, are shown in Figure 3.1. In the period of platform movement illustrated there were no overt changes in blood pressure, heart rate, respiration, sweat release or skin blood flow. The negative going sympathetic spikes have been discriminated and represented as standard pulses
(spikes); these were used to generate cross-correlation histograms between SSNA and the
X and Y acceleration signals, and between SSNA and ECG. Figure 3.2 shows the cross- correlation histogram between SSNA and the Y acceleration signal during sinusoidal platform movement in the medio-lateral axis. It can be seen that there is a clear modulation of SSNA in this subject, with two clear peaks of modulation for each sinusoidal cycle: one large burst associated with the acceleration phase in one direction and another associated with the acceleration phase in the opposite direction; smaller peaks related to the peak and trough (deceleration in each direction) are also present.
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Figure 3.1 Experimental records from one subject. Spontaneous skin sympathetic nerve activity, shown as the raw signal (nerve) and RMS-processed signal (RMS nerve) recorded from a cutaneous fascicle of the common peroneal nerve. Resting activity is shown in the left panel; the right panel shows activity during application of composite movements in the X-Y plane. Negative-going sympathetic spikes are shown discriminated in the top trace (spikes); these were used to generate the crosscorrelation histograms between the vestibular (accelerometer) or cardiac (ECG) signals.
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Figure 3.2 Vestibular modulation of SSNA during platform motion. Smoothed cross- correlation histogram between SSNA and acceleration in the Y axis. The Y sinusoidal curve is superimposed over the histogram. Time zero corresponds to the peak acceleration of the platform motion: earlier peaks in the stimulus train are shown to the left, later peaks to the right. The sinusoidal curve above represents the acceleration-deceleration profile. Note that each cycle of acceleration causes two primary peaks of modulation, with two smaller peaks also apparent.
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Figure 3.3 Modulation indices of SSNA as a function of platform motion and ECG. Modulation indices (primary peak) were significantly higher for the vestibular stimulation (black columns) than for the ECG during vestibular stimulation (white columns). Mean ± SE data from 10 subjects for platform movements in the X and Y directions, and for 5 subjects for the XY (composite) stimuli.
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Mean data for vestibular and cardiac modulation are shown graphically for all 10 subjects in Figure 3.3. There were no significant differences in the magnitude of the vestibular modulation in any direction, nor were there any differences in the magnitude of cardiac modulation. However, for all directions of platform motion, vestibular modulation of
SSNA was significantly larger than the cardiac modulation; this is not surprising, given that cardiac modulation of SSNA is known to be weak. Mean data are presented numerically in Table 3.1.
Table 3.1 Vestibular and cardiac modulation of SSNA during platform motion
SSNA X axis Y axis X axis (XY) Y axis (XY)
Vestibular 96.7 ± 2.5 91.0 ± 4.7 87.6 ± 4.1 86.6 ± 4.1 modulation % Cardiac 55.4 ± 7.2% 47 ± 1.9% 37.2 ± 2.3% 39.6 ± 6.4% modulation % **** **** **** **** n 10 10 5 5
Modulation index = [(peak-trough)/peak] " 100, calculated from the cross-correlation histograms between SSNA and the vestibular or cardiac inputs. Mean ± SEM data from 10 subjects for X and Y motion, and from 5 subjects for the composite XY motion. No significant difference between the modulation indices. In each condition vestibular modulation was greater than cardiac modulation, but there were no differences in the size of either the vestibular modulation or cardiac modulation as a function of the direction or type of motion. n = number of subjects. **** = p<0.0001.
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DISCUSSION
Using a motorised platform, I have shown for the first time that physiological activation of the vestibular utricle at low frequencies causes a robust modulation of skin sympathetic nerve activity in awake human subjects. Two primary peaks of modulation occurred during each cycle of sinusoidal acceleration: one associated with acceleration of the body forwards or to the left - and presumably inertial movement of the vestibular hairs backwards (or to the right) - and a second peak that presumably corresponds to movement of the hairs forwards (or to the left) during acceleration backwards (or to the right).
Evidently, these movements are sufficient to evoke robust modulation of sympathetic outflow to the skin.
However, as I had previously discussed (Hammam et al. 2012), it is not known why vestibular modulation of sympathetic outflow to the skin should be so pronounced.
Nevertheless, given the need for coupling between the control of blood flow in skin and muscle, it may well be that vestibular modulation of SSNA is part of the same mechanism by which the vestibular system contributes to the control of blood flow in muscle and hence arterial pressure. Still, we do know that slow rhythmic movements of the body can lead to motion sickness, and that two of the peripheral markers of incipient motion sickness are cutaneous vasoconstriction and sweat release. Indeed, during low-frequency sinusoidal GVS I found that the vestibular modulation of SSNA was significantly higher for those subjects who experienced nausea than for those who did not (Hammam et al.
2012). Nausea was surprisingly rare in the current study, so I cannot perform a similar analysis with the present material, but one can predict that vestibular modulation of SSNA during platform motion would be higher in a group of participants who are susceptible to motion sickness than in a group who have no history of motion sickness.
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CHAPTER 4
Modulation of muscle sympathetic nerve activity
by the vestibular utricle
This chapter has been edited from the original paper that was published in the journal
Experimental Brain Research on the 28th of June 2013, titled “Modulation of muscle sympathetic nerve activity by low-frequency physiological activation of the vestibular utricle in awake humans”. It was authored by Hammam E, Kwok K, Macefield VG. Exp
Brain Res 230:137-142
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SUMMARY
I have shown that selective stimulation of one set of otolith organs - those located in the utricle, sensitive to displacement in the horizontal axis – causes a marked entrainment of skin sympathetic nerve activity (SSNA) (Study I; Grewal et al. 2012). Here, in Study II, I assess whether muscle sympathetic nerve activity (MSNA) is similarly modulated. MSNA was recorded via tungsten microelectrodes inserted into cutaneous fascicles of the common peroneal nerve in 12 awake subjects, seated (head vertical, eyes closed) on a motorised platform. Slow sinusoidal accelerations-decelerations (±4 mG) were applied in the X (antero-posterior) or Y (medio-lateral) direction at 0.08 Hz. Cross-correlation analysis revealed partial entrainment of MSNA: vestibular modulation was 32±3 % for displacements in the X-axis and 29±3 % in the Y-axis; these were significantly smaller than those evoked in SSNA (97±3 % and 91±5 %, respectively). For each sinusoidal cycle there were two peaks of modulation – one associated with acceleration as the platform moved forward or to the side, and one associated with acceleration in the opposite direction. I believe the two peaks reflect inertial displacement of the stereocilia within the utricle during sinusoidal acceleration, which evokes vestibulosympathetic reflexes that are expressed as vestibular modulation of MSNA as well as of SSNA. The smaller vestibular modulation of MSNA can be explained by the dominant modulation of MSNA by the arterial baroreceptors.
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INTRODUCTION
In Study I I used a motorized platform on which the subject sat with the head vertical, and applied sinusoidal linear acceleration at 0.08 Hz to selectively activate otolith organs afferents sensitive to horizontal displacement – i.e. those in the utricle (Grewal et al.
2012). I showed that physiological stimulation of the utricule caused a robust modulation of skin sympathetic nerve activity (SSNA) when delivered in both the antero-posterior and medio-lateral directions. The aim of the current study (Study II) is to assess whether physiological activation of the utricle has a similarly marked effect on sympathetic outflow to muscle. We tested the hypothesis that selective activation of utricular afferents does not evoke vestibulosympathetic reflexes to the muscle vascular bed of the lower limbs, given that it is likely that the vestibular contribution to blood pressure regulation is more important in the upright position and hence is governed by afferents within the saccule, which are sensitive to acceleration in the vertical (gravitational) plane.
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RESULTS
As in Study I, during sinusoidal displacements of 0.08 Hz and peak accelerations of 4 mG subjects could not reliably detect that they were moving with their eyes closed. If they could perceive motion, they were not aware of its direction, either in the antero-posterior
(X) or medio-lateral (Y) directions. No subject reported discomfort, and none reported nausea during the sinusoidal displacements. Stable recordings of muscle sympathetic nerve activity (MSNA) were obtained in 12 subjects, in 11 of whom we obtained data in the X-axis and 7 in the Y-axis. Experimental records from one subject, at rest and during platform motion in the medio-lateral (Y) axis, are shown in Figure 4.1. The negative going spikes have been discriminated and represented as standard pulses (spikes); event markers have also been produced for the R-waves of the ECG and the positive peaks of the accelerometer (Y), for constructing the cross-correlation and autocorrelation histograms shown for another subject in Figure 4.2.
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Figure 4.1 Multi-unit recording of muscle sympathetic nerve activity in a female subject. In addition, ECG, blood pressure and respiration were recorded during sinusoidal activation of the motion simulator in the medio-lateral axis. The gap separates the recording at baseline activity and during acceleration in the Y axis. Negative-going sympathetic spikes are shown discriminated in the top trace (spikes); these were used to generate the cross-correlation histograms between the vestibular (accelerometer) or cardiac (ECG) signals.
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Figure 4.2 Cross-correlation histogram for one subject between MSNA and acceleration in the X-axis. The histogram has been fitted with a smoothed polynomial. The superimposed sinusoid represents the acceleration of the platform in the antero- posterior direction. There are two peaks of sympathetic outflow for each cycle of the sinusoid, corresponding to the anterior and the posterior acceleration respectively. The left vertical axis refers to the number of spikes per bin for the smoothed curve, the right axis to the actual number of spikes per bin.
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Figure 4.2 shows the cross-correlation histogram between MSNA and the X acceleration signal during sinusoidal platform movement in the antero-posterior axis. It is evident that two peaks of modulation are produced for each cycle of the sinusoidal stimulus: the large peak corresponds to the positive peak (anterior displacement) of the sinusoid and the smaller peak is coupled with the trough of the sinusoid (posterior displacement). The secondary peak consistently occurred after the primary peak, as shown in Figure 4.3, but there were no differences in latency between primary peaks in the X and Y-axes or between secondary peaks in the two axes.
Figure 4.3 Latency of primary and secondary bursts measured from the peak of the sinusoid. It clearly shows that the primary peaks occur earlier than the secondary. There was no statistical difference when comparing the primary or secondary peaks in the two axes.
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Mean data for the cardiac and vestibular modulation of MSNA are shown graphically in
Figure 4.4. There were no significant differences in the magnitude of the vestibular modulation in any direction, nor were there any differences in the magnitude of cardiac modulation. However, as expected, the magnitude of the cardiac modulation of MSNA was significantly larger than the vestibular modulation. Mean data are presented numerically in Table 4.1.
Figure 4.4 Modulation indices of MSNA as a function of the platform motion and cardiac activity. Modulation indices were significantly higher for cardiac modulation (white column) than for the vestibular stimulation (black columns). Mean ± SE data from 11 subjects in the X direction and 7 for the Y stimuli.
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Table 4.1 Vestibular and cardiac modulation of MSNA
MSNA X-axis Y-axis
Vestibular modulation % 32.4 ± 2.5 28.9 ± 2.9
Cardiac modulation % 86.4 ± 3.6 86.1 ± 2.5 n 11 7
Modulation index = [(peak-trough)/peak] X 100, calculated from the smoothed cross- correlation histograms between MSNA and the vestibular or cardiac inputs. Mean ± SEM data from 11 subjects for X and 7 for Y motion. There was no significant difference between the respective modulation indices. In each condition, the cardiac modulation was greater than vestibular modulation. n represents the number of subjects.
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DISCUSSION
This study has shown that, contrary to my prediction, selective stimulation of the vestibular utricle causes a robust modulation of MSNA, although it was not as large as that of SSNA (Study I). The vestibular utricle is a highly sensitive array of mechanoreceptors that transduce linear acceleration in the horizontal plane. Using a stimulus that specifically targets this organ eliminates engagement of the semicircular canals, as well as of the saccule. The results showed a clear modulation of MSNA by acceleration in both the X and Y-axes. There was no statistically significant difference in the modulation indices in either direction. Moreover, there were no differences in latency of either the primary or secondary peaks in each direction, indicating that whilst the utricle evokes a strong sympathetic modulation there is no preferred directional sensitivity with respect to its capacity to modulate MSNA.
The magnitudes of the modulation indices of the physiologically-evoked vestibulosympathetic reflexes were comparable to those evoked by sinusoidal GVS
(Hammam et al. 2011), delivered at the same frequency (0.08 Hz). This finding further affirms the claim of Cohen et al. (2012) and Holstein et al. (2012) - that the otolith organs and not the semicircular canals produce vestibulosympathetic reflexes evoked by sinusoidal GVS. However, the vestibular modulation of MSNA (32±3 % and 29±3 % for the X and Y directions) was much weaker than that of SSNA; it was approximately a third of the magnitude of SSNA modulation (97±3 % and 91±5 %, respectively; Study I
Grewal et al. 2012). I believe this is due to the fact that the dominant modulator of muscle vasoconstrictor drive is the arterial baroreceptors. Nevertheless, as shown previously, the relatively slow projections from the unmyelinated baroreceptor afferents impose a limitation on gravitational blood pooling. The need for instantaneous compensatory
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CHAPTER 5
Vestibular modulation of muscle sympathetic nerve activity by the utricle during sub-perceptual
sinusoidal linear acceleration in humans
This chapter has been edited from the original paper that was published in the journal
Experimental Brain Research on the 25th of January 2014, titled “Vestibular modulation of muscle sympathetic nerve activity by the utricle during sub-perceptual sinusoidal linear acceleration in humans”. It was authored by Hammam E, Chui LVH, Wong KS, Kwok K,
Macefield VG. Exp Brain Res 232: 1379-1388.
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SUMMARY
I assessed the capacity for the vestibular utricle to modulate muscle sympathetic nerve activity (MSNA) during sinusoidal linear acceleration at amplitudes extending from imperceptible to clearly perceptible. Subjects (n=16) were seated in a sealed room, eliminating visual cues, mounted on a linear motor that could deliver peak sinusoidal accelerations of 30 mG in the antero-posterior direction. Subjects sat on a padded chair with their neck and head supported vertically, thereby minimizing somatosensory cues, facing the direction of motion in the anterior direction. Each block of sinusoidal motion was applied at a time unknown to subjects and in a random order of amplitudes (1.25, 2.5,
5, 10, 20 and 30 mG), at a constant frequency of 0.2 Hz. MSNA was recorded via tungsten microelectrodes inserted into muscle fascicles of the common peroneal nerve.
Subjects used a linear potentiometer aligned to the axis of motion to indicate any perceived movement, which was compared with the accelerometer signal of actual room movement. On average, 67% correct detection of movement did not occur until 6.5 mG, with correct knowledge of the direction of movement at ~10 mG. Cross-correlation analysis revealed potent sinusoidal modulation of MSNA even at accelerations subjects could not perceive (1.25-5 mG). The modulation index showed a positive linear increase with acceleration amplitude, such that the modulation was significantly higher (25.3 ± 3.7
%) at 30 mG than at 1.25 mG (15.5 ± 1.2 %). I conclude that selective activation of the vestibular utricle causes a pronounced modulation of MSNA, even at levels well below perceptual threshold, and provides further evidence in support of the importance of vestibulosympathetic reflexes in human cardiovascular control.
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INTRODUCTION
Many studies have explored the capacity to detect movements of the body subjected to passive accelerations, and the approaches used have differed, but most have used displacements of seated subjects (head vertical) in the horizontal plane, thereby targeting the contributions from the utricle. The types of displacements employed have differed across studies (Gundry 1978), but it has been argued that the perception of whole-body linear movement is sensitive to a combination of the acceleration and rate of change of the motion, having similar dynamics to the "irregular" sensory receptors of the otolith organs
(Benson et al. 1986). For example, Gianna et al. (1996) examined detection thresholds to three different types of motion - involving either a step, linear or parabolic acceleration profile – and showed that detection thresholds were lowest for the step profiles.
Moreover, thresholds for these profiles were not significantly different in patients with bilateral loss of vestibular function, indicating that somatosensory afferents could contribute to the perception of motion with brisk acceleration profiles, which feature a large jerk component. Similarly, Soyka et al. (2011) showed that detection thresholds were lowest with trapezoidal stimuli and highest with triangular ramps, while thresholds for detecting sinusoidal stimuli were intermediate. Most studies using sinusoidal stimuli have used frequencies of 1 Hz or less, frequencies at which the vestibular receptors are believed to be the most important afferents responsible for the detection of motion in the absence of vision (Guignard & King 1972).
In addition to its sensory roles, and its contributions to the control of posture and locomotion, the vestibular apparatus has been shown to play significant roles in the control of blood pressure. Indeed, there is a wealth of anatomical data showing connections between the vestibular nuclei and areas of the brainstem involved in control
! ! *(! ! ! ! of the sympathetic vasoconstrictor drive (Yates et al. 1991, 1993a; Yates & Miller 1994;
Kerman et al. 2000b), and lesions of vestibular afferents have been shown to reduce the compensatory increases in blood pressure during nose-up tilt in the cat (Doba & Reis
1974; Jian et al. 1999). In Study I & II, I targeted the otolith organs exclusively by stimulating the utricle physiologically: by delivering low-amplitude, low-frequency sinusoidal movements of the seated body (head vertical), I showed modulation of MSNA and SSNA of comparable magnitude to that produced by sinusoidal GVS (Study I:
Grewal et al. 2012; Study II: Hammam et al. 2013). Interestingly, this vestibular modulation of sympathetic outflow occurred even though most subjects did not know the direction in which they were moving, suggesting that vestibulosympathetic reflexes can be generated at sub-perceptual levels. In the current study, Study III, I delivered sinusoidal linear accelerations to seated subjects at amplitudes extending from 1.25 to 30 mG to assess the effects of amplitude on the magnitude of the modulation of MSNA. In addition, using the same stimuli, I assessed the capacity of subjects to detect motion, and to know the direction of that motion, with a view to identifying whether there is any relationship between perceptions of motion and the expression of vestibulosympathetic reflexes. Specifically, I set out to test the hypothesis that vestibular modulation of MSNA can be observed even during sinusoidal movements that cannot be perceived.
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RESULTS
Motion detection
When examining the detection of motion, not surprisingly, it was difficult with low amplitude accelerations. At lower amplitudes, subjects found it more difficult to discriminate the direction in which they were being moved. Raw records from one subject are shown in Figure 5.1. It can be seen that the subject reliably detected the motion, and was quite confident of the direction of movement, at amplitude of 20 mG, but performed poorly at 5 mG. Mean data from 16 subjects are shown in Figure 5.2 (A). There was a sigmoidal relationship between acceleration amplitude and both detection of movement and knowledge of the direction of motion, the two curves being significantly different from each other (p<0.01; ANOVA). Figure 5.2 (B) shows the data from 2.5 to 30 mG plotted semi-logarithmically; a sigmoidal curve has been fitted to both sets of data. On average, 67% correct detection of movement did not occur until 6.5 mG. Conversely, correct knowledge of the direction of movement did not occur until 10.2 mG.
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Figure 5.1 Recording of linear potentiometer rating of perceived movement (upper trace) and acceleration (lower trace). This is during sinusoidal linear accelerations of 2.5 mG (A), 5 mG (B) and 20 mG (C). The subject could not reliably detect either the motion or direction of motion until the 20 mG stimulus was applied.
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Figure 5.2 Detection reliability graph. (A) Percentage correct (mean ± SE) detection of motion, and correct detection of the direction of motion, for 16 subjects exposed to sinusoidal linear acceleration at a constant rate of 0.2 Hz but at accelerations ranging from 1.25 to 30 mG. (B) semi-logarithmic plot of data from 2.5-30 mG, with fitted sigmoidal curves shown superimposed. It can be seen that, on average, subjects could not reliably detect (67% correct) that they were moving until the acceleration reached 6.5 mG; moreover, they did not know whether they were moving forwards or backwards until the acceleration was much higher (~10.2 mG).
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Figure 5.3 Multi-unit recording of muscle sympathetic nerve activity during sinusoidal linear acceleration at 1.25 mG. Below the nerve recording is a root-mean square processed version of the signal. Negative-going sympathetic spikes are shown discriminated in the top trace (spikes); these were used to generate the cross-correlation histograms between the vestibular (accelerometer) or cardiac (ECG) signals.
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MSNA
Despite the variability in the amplitude of acceleration, no subjects reported discomfort or nausea as a result of the experiment. Successful sympathetic recordings were obtained in
13 subjects, in seven of whom all six amplitudes were studied; in the remaining five subjects only five amplitudes were completed due to technical difficulties. Experimental records from one subject are shown in Figure 5.3. The negative going sympathetic spikes have been discriminated and represented as standard pulses (spikes) for constructing cross-correlation histograms between the positive peaks of the acceleration signal
(vestibular modulation) or the R-waves of the ECG (cardiac modulation). There was no overt modulation of respiration, heart rate or blood pressure, despite the frequency of acceleration (0.2 Hz) being close to that of respiration (~0.25 Hz). Moreover, at each of the acceleration there were no significant changes in MSNA burst count, mean blood pressure or heart rate, compared to the static condition (Figure 5.4). Vestibular cross- correlation histograms are shown for one subject in Figure 5.5. It is evident that at both the lowest amplitude of 1.25 mG and at the highest amplitude of 30 mG there is a peak of modulation produced for each cycle of the sinusoidal stimulus.
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Figure 5.4 The relationship of MSNA, blood pressure and heart rate during accelerations. Mean changes in normalized MSNA burst count (A) mean blood pressure (B) and heart rate (C) during the static condition (0 mG) and at each level of acceleration. There were no significant differences in each parameter as a function of acceleration (ANOVA).
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Figure 5.5 Crosscorrelation histogram between MSNA and acceleration in the antero- posterior direction for one subject. Accelerations of 1.25 mG are represented in (A) and 30 mG in (B). The histograms have been fitted with a smoothed polynomial. The superimposed sinusoid schematically represents the acceleration profile of the platform: motion in the forward direction is indicated by the positive phase of the sinusoid, which includes the period of acceleration before the peak and deceleration after the peak. It can be seen that at both the lowest (A) and highest (B) accelerations there is a single peak of sympathetic modulation corresponding to each cycle of the sinusoid.
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Mean data for the vestibular and cardiac modulation of MSNA are presented graphically in Figure 5.6 and numerically in Table 5.1. ANOVA revealed a significant effect of acceleration amplitude on the vestibular modulation of MSNA (F=2.342 [5,64]; p=0.05), with a significant positive linear slope across amplitudes (slope=0.79; p=0.006). The modulation index was significantly higher (25.3 ± 3.7 %) at 30 mG than at 1.25 mG (15.5
± 1.2 %; p=0.02, paired t-test, n=10). Unlike the vestibular modulation of MSNA there was no effect of acceleration amplitude on the cardiac modulation of MSNA (F=0.138
[5,67]; p=0.98). As expected, at all amplitudes vestibular modulation of MSNA was significantly lower than the cardiac modulation (F=173.7 [11,131]; p<0.0001).
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Figure 5.6 Mean vestibular (A) and cardiac (B) modulation indices of MSNA as a function of acceleration amplitude. 0 mG = static condition (vestibular modulation = 0 in the absence of a sinusoidal vestibular input). Mean ± SE data from 13 subjects, but only partial data were obtained at certain amplitudes (see Table 5.1).
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Table 5.1 Vestibular and cardiac modulation of MSNA at different accelerations
MSNA 1.25mG 2.5 mG 5 mG 10mG 20mG 30mG
Vestibular modulation (%) 15.5±1.25 18.9±1.86 19.7±1.74 17.2±1.36 22.1±2.42 25.3±3.75
Cardiac modulation (%) 86.25±1.89 85.58±3 83.66±3.4 85.71±2.9 84.58±2.5 84.12±3.5
n 10 11 12 13 12 12
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Modulation index = [(peak-trough)/peak] " 100, calculated from the smoothed cross- correlation histograms between MSNA and the vestibular or cardiac inputs. Mean ± SEM; n = number of subjects. There was no significant difference between the respective modulation indices (ANOVA).
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DISCUSSION
In Study I & II I showed that low frequency (0.08 Hz), low-amplitude (4 mG) sinusoidal linear acceleration of the seated body in the horizontal plane causes a robust entrainment of sympathetic nerve activity to both skin (Study I) and muscle (Study II). Interestingly, as most subjects volunteered that they could not feel the motion, one purpose of the current study was to quantify the capacity to detect motion during sinusoidal linear acceleration at different amplitudes; the second purpose was to examine the effects of acceleration amplitude on the modulation of MSNA. This introduces a novelty to the study as previous research has differed in terms of whether they measured the detection of motion (e.g. Naseri & Grant 2012) or detection of the direction of movement (Benson et al. 1986; Gianna et al. 1996; Soyka et al. 2011; Kingma 2005). Subjects were required to follow the perceived movements using a linear potentiometer, allowing me to measure whether subjects were able to detect the motion per se, as well as whether they had any knowledge of the direction of motion they were experiencing. From the results obtained, I have shown that the threshold required to be able to detect the motion is 6.5 mG and the level to accurately determine the direction of motion is 10.2 mG. It is worth reiterating that this study was conducted at the frequency of 0.2 Hz and I would expect detection thresholds for movement direction to be lower at higher frequencies of stimulation (1 Hz vs 0.2 Hz). Also, I have shown that vestibular modulation of sympathetic outflow to the muscle vascular bed occurs during physiological activation of the utricle at levels well below perceptual threshold for detecting motion (6.5 mG), the magnitude of the modulation showing a positive linear relationship with acceleration amplitude. Finally, the potent and sensitive modulation of MSNA occurring at very low amplitude of acceleration (1.25 mG) emphasises the physiological need for a reflex to compliment the predominant systems (Baroreceptor/Bainbridge) in controlling peripheral blood pressure.
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CHAPTER 6
Modulation of muscle sympathetic nerve activity
by neck proprioceptors
This chapter has been edited from the original paper that was published in the journal
Experimental Brain Research on the 12th of March 2014, titled “Neck proprioceptors contribute to the modulation of muscle sympathetic nerve activity to the lower limbs of humans”. It was authored by Bolton PS, Hammam E, Macefield VG. Exp Brain Res
232(7): 2263-2271
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SUMMARY
Several different strategies have now been used to demonstrate that the vestibular system can modulate muscle sympathetic nerve activity (MSNA) in humans, and thereby contribute to the regulation of blood pressure during changes in posture. However, it remains to be determined how the brain differentiates between head-only movements that do not require changes in vasomotor tone in the lower limbs from body movements that do require vasomotor changes. I tested the hypothesis that neck movements modulate
MSNA in the lower limbs of humans. MSNA was recorded in 10 supine young adult subjects, at rest, during sinusoidal stretching of neck muscles (100 cycles, 35o peak to peak at 0.37 ± 0.02 Hz) and during a ramp-and-hold (17.5o for 54 ± 9 s) static neck muscle stretch, while their heads were held fixed in space. Cross-correlation analysis revealed cyclical modulation of MSNA during sinusoidal neck muscle stretch
(modulation index 45.4 ± 5.3%), which was significantly less than the cardiac modulation of MSNA at rest (78.7 ± 4.2%). Interestingly, cardiac modulation decreased significantly during sinusoidal neck displacement (63.0 ± 9.3%). By contrast, there was no significant difference in MSNA activity during static ramp-and-hold displacements of the neck to the right or left compared to that with the head and neck aligned. These data suggest that dynamic, but not static, neck movements can modulate MSNA, presumably via projections of muscle spindle afferents to the vestibular nuclei, and may thus contribute to the regulation of blood pressure during orthostatic challenges.
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INTRODUCTION
While the baroreflex is able to modulate muscle vasomotor tone, we know from a significant body of research (for review Chapter 1: Introduction) that signals from the vestibular system reduce the impact of postural change on cardiovascular function.
However, there are some circumstances when the head moves and hence the vestibular system is activated but there is no change in body position – such as lifting the head while lying supine. In these circumstances there is no need to increase vasomotor tone to the lower limbs. So the question arises, how does the brain know when it is appropriate to modulate vasomotor tone to the lower limbs of the habitually upright human?
It is also well known that mechanoreceptors in the neck play an important role in somatomotor reflexes that maintain posture and interact with vestibulosomatic reflexes
(Lindsay et al. 1976; Wilson 1992). However, a previous study suggests that neck afferents do not modulate sympathetic outflow in humans (Ray & Hume 1998).
Interestingly, this study examined the involvement of static neck posture but animal studies have shown that electrical stimulation of the low threshold, presumably muscle spindle, afferents from neck muscles can modulate sympathetic outflow (Bolton et al.
1998). Furthermore, it is known from animal studies that information from muscle spindles in the neck project to the vestibular nuclei and that their natural stimulation influences vestibular neurone interactions (Kasper et al. 1988; Neuhuber & Zenker 1989).
Therefore, the aim of the current study, Study IV, was to investigate the effects of a dynamic mechanical stimulus of the neck - sinusoidal lateral stretch of the neck muscles - on sympathetic outflow to the muscle vascular bed in the lower limbs of humans. In particular, I tested the hypothesis that neck movements modulate MSNA in the absence of changes in vestibular afferent input.
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RESULTS
Subjects lay supine with eyes closed and the head firmly clamped over the temporal bones, which prevented any movement of the head. At times unknown to the subjects the body was sinusoidally displaced in the horizontal plane about the neck, with a maximal angular excursion of ±17.5o from the midline (35o peak-to-peak) and frequencies ranging from 0.27 to 0.45 Hz (mean ± SEM 0.37 ± 0.02 Hz); 100 cycles of stimuli were delivered to each subject. Subjects reported they were aware of when their body was moving but five were unable to perceive the direction in which they were moving, while three could perceive the direction of movement in at least one direction and one could only perceive the direction of movement if she “really concentrated”. All but one subject reported they did not perceive head movement at any point during the recording sessions; this subject felt as if the head and body were “moving together as one.”
Experimental records from one subject are shown in Fig. 1. In this subject recordings of muscle sympathetic nerve activity (MSNA) were made from the left and right common peroneal nerves concurrently. It can be seen that, while sympathetic outflow was similar between the two sides, there were subtle differences in burst morphology, as indicated by the asterisks in the bursts enclosed by the rectangles; we had observed such differences previously during bilateral recordings of MSNA during galvanic vestibular stimulation
(El Sayed et al. 2012).
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Figure 6.1 Bilateral recordings of muscle sympathetic nerve activity. Other parameters recorded include ECG, blood pressure, respiration and neck angle during sinusoidal angular displacement about the neck in one subject. Overall, sympathetic outflow was similar between the two sides, but the rectangles highlight subtle differences between the two sides, with differences in burst peaks or expression being indicated by the asterisks.
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Cross-correlation analysis of the data from this same subject, shown in Figure 6.2B, indicated that MSNA was cyclically modulated with the sinusoidal changes in neck angle, though it is clear that the magnitude of this modulation was lower than the coupling to the cardiac cycle (Figure 6.2A). Across subjects, the modulation index during sinusoidal body displacement about the neck varied significantly (p=0.002) between subjects (n=10), ranging from 24.7 to 76.4% (45.4 ± 5.3 %). Analysis of the RMS-processed nerve signal revealed no significant differences between values at baseline and during sinusoidal neck stimulation for burst frequency (27.6 ± 4.9 vs 27.3 ± 4.6 bursts/min; p=0.8480, paired t- test), burst incidence (43.8 ± 8.1 vs 42.4 ± 7.5 bursts/100 heart beats; p=0.5162) or cumulative burst amplitude (12.1 ± 2.1 vs 11.0 ± 1.6 mV/min; p=0.2313).
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Figure 6.2 Data from one subject showing the cross-correlation histogram between MSNA and ECG. (A) and angular displacement of the neck about the fixed head (B). Autocorrelation histograms of ECG and angular displacement are shown in C and D. Note that MSNA displayed a very pronounced cardiac rhythmicity (A) but it was also modulated by neck displacement (B). The vertical lines at time 0 indicate the peak of the R-wave (C) and the positive peak of the sinusoidal angular displacement of the neck (D). Same subject as illustrated in Figure 6.1; data from only one side are shown.
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As shown in Figure 6.3, across subjects the neck-related modulation of MSNA was significantly smaller (p=0.0002) than the cardiac modulation at rest (78.7 ± 4.2s %).
Interestingly, cardiac modulation was significantly lower (p=0.0273) during the sinusoidal body displacement than at rest (63.1 ± 9.3 %). The neck-related modulation was also significantly lower than the respiratory modulation of MSNA at rest (67.7 ± 7.6 %; p=0.0231), but there was no significant effect of sinusoidal body displacement on the magnitude of the respiratory modulation (59.0 ± 7.3 %; p=0.6957).
Figure 6.3 Mean modulation indices calculated from the cross-correlation histograms between MSNA and ECG. This is represented at rest (cardiac), MSNA and neck angle during sinusoidal neck displacement (neck) and between MSNA and ECG during sinusoidal neck displacement (cardiac + neck). Neck modulation of MSNA was significantly lower than cardiac modulation at rest, and cardiac modulation was significantly lower during neck stimulation. *=P<0.05; ***=P<0.001.
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While the frequency at which the body was displaced was close to the respiratory frequency (0.23 to 0.39 Hz, 0.33 ± 0.02 Hz), a matched pairs analysis indicated that the frequencies were significantly different (p=0.0078). The magnitude of the modulation of
MSNA to respiration at rest (67.7 ± 7.6 %) was not significantly different to that during sinusoidal body displacement (59.0 ± 7.3 %; p=0.8215), suggesting that there was no interaction between body displacement and respiration. Nevertheless, to demonstrate that the modulation of MSNA was independent of respiration, in one subject we performed sinusoidal body displacement during a maximal inspiratory breath-hold (inspiratory- capacity apnoea), as shown in Figure 6.4. It is clear that modulation of MSNA occurred in the absence of cyclic respiration (Figure 6.5).
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Figure 6.4 Experimental recordings during a maximal inspiratory breath-hold. Muscle sympathetic nerve activity, together with ECG, blood pressure, respiration and neck angle are recorded during sinusoidal angular displacement about the neck in one subject while he performed a maximal inspiratory breath-hold. This manouevre allowed examination of the effects of sinuoidal neck displacement in the absence of respiration.
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Figure 6.5 Data from one subject showing the cross-correlation histogram between MSNA and angular displacement of the neck. This can be seen about the fixed head at rest (A) and during a single maximal inspiratory breath-hold (B). Autocorrelation histograms of angular displacement are shown in C and D. The vertical lines at time 0 indicate the positive peak of the sinusoidal angular displacement of the neck (same subject as shown in Figure 6.4). Despite the lower number of cycles studied during the breath-hold, MSNA still showed cyclic modulation to the sinusoidal neck displacement.
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In addition to the sinusoidal displacements of the body to stretch the neck muscles, in nine subjects we applied static ramp-and-hold stretch by displacing the body from the midline to the right or to the left. The mean ramp rate was 8.9 ± 1.3o/s, which was held at 17.5o for
54 ± 9 s. Analysis of the RMS-processed nerve signal revealed no significant differences between values at baseline and during static neck displacement for burst frequency (30.1
± 5.1 vs 29.4 ± 4.0 bursts/min; p=0.8149, paired t-test), burst incidence (47.0 ± 8.5 vs
47.2 ± 7.6 bursts/100 heart beats; p=0.9692) or cumulative burst amplitude (13.0 ± 2.3 vs
11.2 ± 2.1 mV/min; p=0.1030).
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DISCUSSION
Two new findings arise from this study. Firstly, that neck proprioceptors can modulate
MSNA in the lower limbs of humans and, secondly, that the cardiac modulation of
MSNA is reduced in the presence of neck modulation of MSNA. Whilst previous studies had examined the influence of neck afferent inputs on MSNA (Ray & Hume 1998, 1999), a significant difference in the present investigation is that I analysed MSNA of subjects during dynamic stretching of the neck as oppose to static flexion or extension of the head and neck. In addition it has been well observed that dynamic stimulus is effective on
MSNA (Bent et al. 2006; Voustianiouk et al. 2005) but static (single pulse) stimuli does not (Bolton et al. 2004). Moreover, cardiac modulation of MSNA was reduced in the presence of dynamic neck stretch. The cross-correlation analysis in our study showed cyclical modulation of the MSNA in all subjects during neck muscle stretching induced by sinusoidal body displacement about the fixed head. This most likely involved the pathways that mediate vestibulosympathetic reflexes (Yates 1996). The magnitude of the neck modulation of MSNA in this study was significantly less than the cardiac and respiratory modulation at rest. However, all of our subjects were supine during our study - a posture that reduces MSNA to the lower limbs (Burke et al. 1977). Interestingly, Sauder et al (2008) showed greater sensitivity of the vestibulosympathetic reflex in the upright posture. Therefore, it is likely that neck modulation of MSNA will also be different in different postures since neck inputs are likely to be acting via the vestibular nuclei. In summary, our data show that neck afferents can modulate MSNA, presumably through the known projections of neck mechanoreceptor afferents to the vestibular nuclei.
Moreover, neck muscle afferents may also compete with the baroreceptors to modulate muscle sympathetic outflow.
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CHAPTER 7
GENERAL
DISCUSSION
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The aim of this work was to use physiological stimuli to identify the source of vestibular input in the generation of vestibulosympathetic reflexes in human subjects and to examine whether neck proprioceptors also play a role. Specifically, I was interested in extending the work I had done in my Bachelor of Medical Science (Honours) year, in which I used low-frequency sinusoidal Galvanic Vestibular Stimulation (sGVS) to quantify the vestibular modulation of muscle sympathetic nerve activity (MSNA) and skin sympathetic nerve activity (SSNA) to the lower limbs (see Appendices VII & VIII). While sGVS has revealed potent modulation of sympathetic nerve activity to both muscle and skin, it is limited in that the electrical stimulation of the vestibular nerves affects afferents originating in the entire vestibular apparatus – the semicircular canals as well as the otolithic organs. In the set of experiments reported in Studies I & II, I used the same low frequencies (0.08 Hz) I had used in my studies with sGVS, but rather than electrical stimulation, for my doctoral work I used physiological stimuli. It is worth remembering the exquisite sensitivity of the vestibular endorgans with an emphasis on the two otolith organs: the saccule and the utricle are roughly organised perpendicular to one another, ensuring sensory inputs to linear acceleration in both planes. Whilst utricular hair cells are mainly responsive to acceleration in the horizontal plane, the saccular hair cells are predominantly concerned with vertical displacements. By virtue of directionally-sensitive hair cell displacement, head movement and position are detected with respect to gravity
(Lundberg et al. 2006). Importantly, by using low-amplitude, low frequency, sinusoidal linear acceleration of the body I could target the utricular component of the otolith organs by delivering stimuli to the upright (head vertical) body: the utricle is sensitive to linear acceleration in the horizontal plane. Until now, the origin of the vestibular modulation of sympathetic outflow has been uncertain, though it is generally accepted that the otolithic organs provide the most important source of sensory input. It is important to note that my
! ! "#%! ! ! ! studies are not the first to have used sinusoidal linear acceleration: previous studies have employed linear accelerations to demonstrate the contribution of the otolithic organs to cardiovascular control. For instance, Yates and colleagues (1999) employed a chair- surmounted upright on a sled to deliver linear acceleration (200 mG) followed by deceleration. With the head fixed in different positions, thereby targeting different vestibular hair cells, they examined the cardiovascular response of 3 patients with idiopathic profound bilateral reduction in vestibular function and 10 healthy “normal” participants. The normal subjects exhibited increases in systolic blood pressure and decreases in R-R interval (elevated heart rate) for few seconds whereas the vestibular compromised patients had a reduction in cardiovascular response. This highlights the importance of a normal functioning vestibular system in cardiovascular control. Similarly,
Jauregui-Renaud et al (2006) employed head fixed, linear acceleration on a sled in normal participants and patients with chronic bilateral (idiopathic) vestibular dysfunction. The stimuli were delivered predictably and unpredictably, coinciding with end of inspirations and expiration. They too found in their results that control subjects showed a sustained increase in heart rate (within the 1st or 2nd heart beat), and a transient increase in respiration, when exposed to brisk linear accelerations (260 mG), while patients with chronic bilateral vestibular dysfunction did not at all. These studies, amongst others
(Jauregui-Renaud et al. 2003, 2005, Radtke et al. 2000, 2003; Yates et al. 1999) strongly support the contribution of the otolithic organs to cardiovascular adjustments to changes in body position. Furthermore, Cui et al (2001) recorded MSNA during five cycles of sinusoidal linear accelerations of varying amplitude (100, 150, 200 mG) in the horizontal plane and found that higher magnitudes of acceleration decreased total MSNA, in both the antero-posterior (X) and medio-lateral (Y) axes (Cui et al. 1999, 2001). It is important to reiterate that the studies mentioned employed linear displacements at high accelerations
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(100-260mG) activating extra-cranial receptors – such as those activated by fluid shifts - and that sympathetic nerve responses to vestibular stimulation can vary tremendously on the basis of the properties of the stimulus that is delivered. The novel aspect of my research is that much lower accelerations (4 mG) were used, with most stimuli being far below perceptual threshold. This emphasizes the physiological role of the vestibular apparatus, specifically the utricular hair cells, in the control of sympathetic outflow to the muscle vascular bed. In addition to looking at direct vestibular inputs, below and above perceptual threshold, I also looked at potential modulation of muscle sympathetic nerve activity via neck muscle afferent input.
In the first study I carried out (Study I- Chapter 3), I have shown, for the first time, that physiological activation of the utricular component of the vestibular apparatus, which is sensitive to movements of the head in the horizontal plane, causes a robust modulation of skin sympathetic nerve activity (SSNA) (97±3 % and 91±5 %, for the X and Y axes). This modulation was comparable in magnitude to that demonstrated using sinusoidal galvanic vestibular stimulation (sGVS) at frequencies ranging from 0.2-2.0 Hz (James et al. 2010).
However, GVS changes the firing of afferents originating in the entire vestibule – the utricle, saccule and semicircular canals – so we do not know whether afferents from each of these structures can modulate sympathetic outflow equally, although recent evidence indicates that only the otolith organs participate in the vestibulosympathetic reflexes induced by sinusoidal GVS (Cohen et al. 2012; Holstein et al. 2012). Interestingly, in the rat, sGVS causes vasovagal responses, characterized by reductions in heart rate and blood pressure (Cohen et al. 2011).
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Furthermore, in Study II (Chapter 4), with subjects seated vertically, I demonstrated, contrary to my prediction, that selective stimulation of the vestibular utricle causes robust modulation of MSNA (32±3 % and 29±3 % for the X and Y axes), although it’s magnitude was approximately one-third that I had shown for SSNA in Study I. I believe this is due to the fact that the predominant modulator of muscle vasoconstrictor drive is the arterial baroreceptors – cardiac modulation of MSNA is much greater than vestibular modulation. Nevertheless, my results showed a clear modulation of MSNA by acceleration in both the X and Y axes and there was no statistically significant difference in the modulation indices in either axis.
In addition, in a study that I carried out that has been submitted for publication and is currently under revision (Appendix V) I extended the work in Study I & II to target a different pool of otolithic hair cells. By laying the subjects supine, with the neck aligned with the spine and the nose vertical, the saccular hair cells are activated (although not exclusively) during slow physiological displacements produced by sinusoidal linear acceleration to the supine body in the rostro-caudal (X) direction (longitudinal axis of the body). The results outlined a marked modulation of MSNA. However, the magnitude of this modulation (29% in the X-axis) was no different to that produced by selective stimulation of the utricle (32% in the X axis, 29% in the Y axis; Study II), nor was it significantly different from delivery, to the supine body, in the medio-lateral (Y) axis
(32%), in which both saccular and utricular hair cells are activated). This argues against a dominant role of the saccule in modulating MSNA in humans, a hypothesis I predicted in
Study II; rather, my results show that both saccular and utricular organs contribute.
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Because the acceleration was applied at 0.08 Hz, the same frequency I had used when delivering sinusoidal GVS (Hammam et al. 2011, 2012), two peaks of modulation were also found, but the mechanism by which the two peaks are generated is different. With
GVS, the sinusoidal current is passed across the mastoid processes, causing depolarization of one vestibular nerve and at the same time hyperpolarization of the contralateral nerve; it is this cyclical fluctuation in current that we believe accounts for the primary and secondary peaks of the modulation of SSNA and MSNA (Hammam et al. 2011, 2012), an interpretation supported by bilateral recordings of SSNA or MSNA I had performed during sinusoidal GVS (El Sayed et al. 2012). However, the origin of the two peaks, during physiological activation of the otolith organs, is, I believe, peripheral. As the subject accelerates in the forward direction, inertia causes the utricular otoconial membrane to shear and the hair bundles to deflect in the posterior direction, returning to the neutral position at the point of zero acceleration and then in the anterior direction as the platform is displaced posteriorly. The same process also occurs during acceleration in the Y-axis, deflecting hairs medially and laterally as a response to the direction of motion.
I believe that anatomical (Holstein et al. 2011) and physiological (Yates et al. 1991,
1993a) evidence for projections from the vestibular nuclei to the rostroventrolateral medulla (RVLM) - the primary output nucleus for MSNA - can explain the sinusoidal modulation of MSNA during sinusoidal linear acceleration: the two phases of motion in which the hairs are displaced evokes the two peaks of MSNA.
In contrast, in Study III (Chapter 5), MSNA expressed only one peak, which I attribute to the higher frequency of stimulation (0.2 Hz) than used in Studies I and II (0.08 Hz).
Indeed, at frequencies between 0.2-2.0 Hz, sinusoidal GVS produces only one peak of modulation, while at frequencies <0.2 Hz two peaks are generated: as explained
! ! "#)! ! ! ! previously (Hammam et al. 2011), I believe that the second peak does not occur at the higher frequencies because the next cycle of the sinusoidal stimulus curtails its expression. Moreover, we know that individual neurones within the vestibular nuclei possess directional sensitivity (Fernandez & Goldberg 1976), so it is likely that a given set of afferents responding to movement in one direction is not the same set that responds to movement in the opposite direction. Nevertheless, it would appear that - as a population - utricular afferents display no preferential directional sensitivity, at least with respect to the generation of vestibulosympathetic reflexes. This is also evident in the results from the composite sequences reported in Study I, in which sinusoidal displacements were produced in both the X and Y axes: the magnitude of the modulation of skin sympathetic nerve activity was relatively similar regardless of whether the modulation index was computed with respect to acceleration in the X axis or acceleration in the Y axis. Again, this would suggest that, as a population, utricular afferents – regardless of the optimal directional sensitivity of individual mechanosensors – can generate robust vestibulosympathetic reflexes, as evidenced by the sinusoidal entrainment of SSNA and
MSNA to sinusoidal acceleration applied in the horizontal plane in one or two axes.
Another noteworthy finding from these studies is that the magnitude of modulation, for both SSNA and MSNA, by the utricle is similar to that observed during electrical stimulation of the vestibular nerves with sinusoidal GVS at 0.08 Hz (Hammam et al.
2011, 2012). This affirms the observations by Cohen et al (2012) and Holstein et al
(2012), that the vestibulosympathetic reflex evoked by galvanic stimulation originates in the otolithic organs, not the semicircular canals. Furthermore, whilst the mean modulation indices produced by sinusoidal linear acceleration in the X and Y axes showed similar distributions across subjects, individual subjects could exhibit larger changes in
! ! "#*! ! ! ! sympathetic modulation in one axis. We do not know why this is the case, but it has been hypothesised by Yates and colleagues (1999) that “shaping” and “tuning” of hair cell responses based on experiences may impact on individual effects; this hypothesis remains to be tested.
Importantly, during low-frequency (0.08 Hz) low-amplitude (4 mG) sinusoidal linear acceleration of the body in the horizontal plane (Study I & II) most subjects volunteered that they could not feel the motion, and certainly could not tell the direction in which they were moving – forwards, backwards or side to side. So in Study III, I sought to quantify the capacity to detect motion during sinusoidal linear acceleration at different amplitudes.
Another aim was to examine the effects of acceleration amplitude (0-30 mG; 0.2 Hz) on the modulation of MSNA. Previous studies have differed in terms of whether they measured the detection of motion (Naseri & Grant 2012) or detection of the direction of movement (Benson et al. 1986; Gianna et al. 1996; Soyka et al. 2011; Kingma 2005).
Here I used the one experimental paradigm to assess both the threshold for detection of motion, and the threshold for knowing of the direction of motion. Earlier studies had been designed to examine one or other parameter, never the two together, so this study (Study
III) provides novel data on how well subjects can perceive sinusoidal accelerations in the horizontal plane without any visual cues. Sinusoidal linear stimulation is advantageous as it allows one to provide a reproducible, constant peak-velocity stimulus - defined in frequency and amplitude - to the subject and does not require a long track over which the acceleration is applied. Another advantage is that the sinusoidal stimulus is bidirectional, allowing one to assess the detection of direction of movement as well as the detection of motion. In the study by Naseri and Grant (2012), the lowest amplitudes used were 0.5 ms-
2 (~50 mG), which is higher than our maximum amplitude (30 mG). However, their
! ! "$+! ! ! ! experimental design was a forced-choice paradigm to measure the just noticeable difference (absolute threshold) between two sets of stimuli: the mean difference limen for a reference amplitude of 0.5 ms-2 was 0.050 ms-2 (~5.0 mG) for a 0.4 Hz stimulus and
0.062 ms-2 (~6.2 mG) for a 0.6 Hz stimulus. Nevertheless, these thresholds were comparable to the threshold found in the present study (6.5 mG).
Using sinusoidal accelerations of up to 0.4 ms-2 (~40 mG) at 1 Hz, Kingma (2005) examined thresholds for detection of the direction of motion and found that the mean detection threshold in the antero-posterior direction was 0.085 ms-2 (~8.5 mG). This is lower than the level found in the current study (10.2 mG), again emphasizing differences in the ways in which studies are conducted; we would expect detection thresholds for movement direction to be lower at higher frequencies of stimulation (1 Hz vs 0.2 Hz in the current study). It is also worth pointing out that different investigators have used different criteria for determining thresholds: we chose a level of 67% correct as the threshold, as did Gianni et al (1996), whereas Benson et al (1986) and Soyka et al (2011) used 62.5% and Nasseri and Grant (2012) adopted 70%; this creates difficulty in comparing thresholds across studies.
As for the effect of acceleration amplitude on the vestibulosympathetic reflex, the magnitude of modulation tended to increase with increasing amplitude of acceleration, though this failed to reach statistical significance when all amplitudes were considered together. Nevertheless, there was a significant positive slope of the magnitude of modulation as a function of acceleration amplitude, and the modulation was significantly higher at 30 mG than at 1.25 mG when only these two amplitudes were compared. From the work conducted on the squirrel monkey by Fernandez and Goldberg (1976) one would
! ! "$"! ! ! ! certainly expect a greater vestibular input with the greater acceleration amplitudes and hence a greater modulation of sympathetic outflow: these investigators showed that an increase in force introduced onto the hair cells increases the afferent discharge rates. It is also noteworthy that in their study the range of accelerations extended to 5 G, whereas our maximum acceleration was 2 orders of magnitude lower. This tells us that the vestibular afferents certainly do respond to the highest acceleration (30 mG) employed in the current study but, more importantly, they also respond at the lowest amplitude (1.25 mG). Indeed, the observation of robust modulation of MSNA during this very low amplitude sinusoidal acceleration – well below the mean perceptual threshold for detection of motion (6.5 mG)
- indicates that the modulation of MSNA was not due to any conscious awareness or arousal-related component, and purely reflects the expression of a vestibulosympathetic reflex. However, while these reflexes are robust, it is worth pointing out that they are certainly smaller than the baroreceptor-mediated reflexes: as noted above, cardiac modulation was much higher than the vestibular modulation, and was not affected by the amplitude of acceleration. Indeed, Figure 5.6 demonstrates the modulation of MSNA at the lowest of amplitudes (1.25 mG) and significantly greater at the highest (30 mG).
Moreover, whilst there was an increase in modulation there was no significant change in burst count, blood pressure or heart rate (Figure 5.4). Nevertheless, despite the low stimulus, the fundamental neural substrates were demonstrated in humans, and this highlights the exquisitely rapid detection of fluctuations by the vestibular system but only be of importance at larger postural changes when immediate blood pooling is compensated, and until the relatively slower unmyelinated baroreceptor fibres unload
(Wang et al. 1996; Hargens et al. 2009; Hinghofer-Szalkay 2011).
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In Study IV (Chapter 6), where I examined the influence of neck afferents on MSNA, two new findings arose from this work. Firstly, I showed that neck proprioceptors can modulate MSNA in the lower limbs of awake humans and, secondly, that the cardiac modulation of MSNA is reduced in the presence of neck modulation of MSNA. Whilst a previous study by Ray and Hume did examine the influence of neck afferent input on
MSNA, they found, in contrast to our study, that neck afferents did not modulate lower limb MSNA (Ray & Hume 1998). In another study by the same group, the influence of neck afferent activity on lower limb MSNA was excluded as a possible modulator of
MSNA because head and neck extension in their supine subjects failed to modulate lower limb MSNA (Hume & Ray 1999). However, a significant difference between these studies and ours is that the earlier studies analysed the MSNA of subjects during static flexion or extension of the head and neck while subjects were in the lateral decubitus or supine position, respectively. In contrast, we recorded MSNA during dynamic stretching of the neck. That the stimulus may need to be dynamic in order to have an effect on
MSNA has been observed with respect to vestibular modulation of MSNA in humans. I, as part of the Macefield group, and others, have demonstrated that dynamic (sinusoid or pulse train) galvanic vestibular stimulation modulates MSNA in the human (Bent et al.
2006; Voustianiouk et al. 2005) but that static (single pulse) stimuli fails to (Bolton et al.
2004). This makes teleological sense since changes in posture (body position) are more likely to require modulation of vascular tone than during static states. Importantly, in this study (Study IV) I also recorded MSNA activity during static stretch of the neck in our subjects and, like Ray & Hume (1998), found that the MSNA burst activity did not change. Together, these findings suggest that, at least when horizontal (i.e. supine or lateral recumbent position) dynamic neck stretches can significantly modulate MSNA,
! ! "$$! ! ! ! although, as for the vestibular modulation, the magnitude of this modulation is lower than the cardiac (and respiratory) modulation of MSNA.
It should be pointed out that sinusoidal displacement of the body about the neck might change transmural pressures at the carotid sinus and hence change the input from the arterial baroreceptors. Not withstanding the fact that the phasic cardiac modulation of
MSNA was much greater than that produced by sinusoidal neck movements in Study IV, or by electrical or physiological activation of the otolithic organs in our previous studies,
Shortt and Ray (1997) discounted changes in carotid baroreceptor input in their experiments by arguing that there were no changes in blood pressure and, because there were no changes in thoracic volume, discounted any contribution from the low-pressure baroreceptors. In addition, Hume and Ray (1999) showed that, unlike neck flexion in the prone position, neck extension in the supine position or yaw rotations of the neck caused no increases in MSNA, arguing against activation of non-specific receptors in the head associated with gravitationally-induced vascular congestion. However, it has still not been explained why MSNA did not decrease during head-down neck extension (Hume & Ray
1999), which is what one would expect from a system that could linearly translate changes in head position to changes in MSNA. Nevertheless, to exclude any contributions from the carotid arterial baroreceptors would require performing these experiments in individuals who have undergone bilateral anesthetic block of the glossopharyngeal (and vagus) nerves, which is not a trivial procedure (Fagius et al. 1985). It would be interesting to assess the magnitude of any neck-induced or vestibular-induced modulation of sympathetic outflow in the complete absence of baroreceptor-mediated entrainment of
MSNA; this may well be addressed in future studies.
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Interestingly, I also found that the cardiac modulation of MSNA was reduced in the presence of dynamic neck stretch. Animal studies suggest the vestibular nuclei and regions of the brainstem are involved in integrating information from both somatic and visceral sources and higher centres in order to regulate blood pressure in different body positions and contexts (Yates & Stocker 1998; Yates & Miller 2009; DeStefino et al.
2011). The cross-correlation analysis in our study showed cyclical modulation of MSNA in all subjects during neck muscle stretching induced by sinusoidal body displacement about the fixed head. This most likely involved the pathways that mediate vestibulosympathetic reflexes (Yates 1996). Experimental evidence from animal studies have shown that neck muscle spindle afferents, which are exquisitely sensitive length sensors (Richmond & Abrahams 1979), project to the medial and descending vestibular nuclei and can modulate vestibular neuronal activity (Kasper et al. 1988; Neuhuber &
Zenker 1989). Electrical stimulation of nerves innervating muscles of the neck at currents just sufficient to stimulate muscle spindle (and Golgi tendon organ) afferents has been shown to change activity in respiratory and sympathetic nerves in the cat (Bolton et al.
1998). Moreover, it has been shown that the presence of intact neck afferents inhibits the influence of the vestibular system on sympathetic outflow during natural stimulation of the labyrinths (Bolton et al. 1998).
Across all of the experimental studies I conducted during the course of my PhD (I-IV), as well as the additional studies included in the Appendices, cardiac modulation of MSNA was much higher than the vestibular or neck afferent modulation; this is simply due to the strong coupling of MSNA to the cardiac rhythm via the baroreflex - unlike SSNA, which has only weak cardiac rhythmicity (Delius et al. 1972; Bini et al. 1981; Macefield &
Wallin 1999; James & Macefield 2010; Fatouleh & Macefield 2013). Nevertheless, as
! ! "$&! ! ! ! noted previously, the vestibular modulation of MSNA does appear to be independent of the cardiac modulation, but does compete with inputs from the arterial baroreceptors when the frequencies of vestibular stimulation are closer to that of the cardiac rhythm
(James & Macefield 2010). In studies I & II the frequency of the sinusoidal stimulus
(0.08 Hz) was far removed from the cardiac frequency (~1 Hz), as it was also during
Study III (0.2 Hz). So evidently, the utricular inputs can exert a potent modulation of muscle vasoconstrictor drive, which presumably acts through projections from the vestibular hair cells to the vestibular nuclei onto the rostral ventrolateral medulla
(Holstein et al. 2011), which has been shown to receive excitatory inputs from the vestibular apparatus, primarily from the otolithic organs (Yates et al. 1991, 1993a). Again, the magnitude of the neck modulation of MSNA in Study IV was significantly less than the cardiac and respiratory modulation at rest. However, all of our subjects were supine during our study - a posture that reduces MSNA to the lower limbs (Burke et al. 1977).
Interestingly, Dyckman et al (2007) have shown that, in the presence of increased MSNA induced by baroreceptor unloading with lower-limb negative pressure, vestibular-induced drive to MSNA remains but that during a decrease in MSNA produced by an increase in mean arterial pressure (baroreceptor loading using lower-limb positive pressure) the vestibular drive is abolished. The same group also reported greater sensitivity of the vestibulosympathetic reflex in the upright posture (Sauder et al. 2008). Therefore, it is likely that neck modulation of MSNA will also be different in altered postures since neck inputs are likely to be acting via the vestibular nuclei. However, it remains to be determined if the magnitude of neck modulation of MSNA in postures that are likely to induce an orthostatic challenge is sufficient to increase vasomotor tone in the lower limbs and thereby reduce the likelihood of orthostatic hypotension.
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Methodological considerations
In Studies I-III I believe that I specifically activated the utricular component of the vestibular apparatus. Of course, I cannot rule out the possibility that other mechanoreceptors may contribute, including non-vestibular afferents sensitive to acceleration (graviceptors), particularly at the higher amplitudes of accelerations employed in Study III, such as 20 and 30 mG. But the most parsimonious explanation is that the otolithic organs specifically sensitive to acceleration in the horizontal plane - i.e. those in the utricle - are responsible for the modulation of both MSNA and SSNA. This interpretation is supported, in Study III, by the fact that the magnitude of the modulation of MSNA tended to increase with the increase of the orthogonal shearing of the utricular hair cells, a finding well documented in the study by Fernandez and Goldberg (1976).
As for Study I in particular, the vestibular modulation of SSNA seen cannot be explained as an arousal-related phenomenon: the sinusoidal stimulation was of long duration (100 cycles @ 0.08 Hz = 27 min) and subjects would have habituated to the novel stimulus very quickly (within seconds). Likewise, the modulation of SSNA is not the indirect result of respiratory coupling (Delius et al. 1972), as the entrainment of either the respiratory or cardiac cycles to sinusoidal GVS failed to be demonstrated (Bent et al. 2006). Unlike
MSNA, which is entrained to the cardiac rhythm via the baroreflex, SSNA has only weak pulse-related rhythmicity (Delius et al. 1972; Bini et al. 1981; Macefield & Wallin 1999;
James et al. 2010). Moreover, at a comfortable (essentially thermoneutral) room temperature the SSNA recorded from our subjects would have been dominated by the activity of cutaneous vasoconstrictor neurones, so cardiac rhythmicity would not be expected to be high. However, consistent with previous studies, some cardiac rhythmicity was observed: construction of cross-correlation histograms of sympathetic spikes can
! ! "$(! ! ! ! reveal cardiac modulation even when this is not apparent in multi-unit recordings
(Macefield & Wallin 1999).
In addition, one of the challenges in determining the physiological influences of neck afferent input in humans is that head movements, which activate the labyrinthine organs, usually accompany neck movements. This is a particular challenge in determining the influence of neck afferent information on muscle sympathetic nerve activity since the activation of the vestibular system can induce a potent facilitatory influence on MSNA in humans (Bent et al. 2006). In Study IV, I have overcome this by moving the body about a fixed head. Since the subjects did not perceive any head movement, the only vestibular influence on MSNA in the study is that arising from the static influence of gravity on the otolithic organs. One can also exclude a contribution from extra-vestibular (somatic)
‘graviception’ (Mittelstaedt 1992; Yates et al. 2000) since the stimulus was not sufficient to induce a head-up sensation that accompanies centrifugal blood shifts occurring when subjects are positioned horizontally (Mittelstaedt 1992; Vaitl et al. 1997, 2002).
Furthermore, it has been shown in paraplegic persons that somatic ‘graviception’ does not involve pressure or sheer activation of mechanoreceptors in the body (Mittelstaedt 1992).
The stimulus-induced stretch of the lateral neck muscles involved displacements (8.5 ±
1.2o/s) well above the 1.4o/s threshold for activation of neck proprioceptors during passive rotation of the body under a fixed head (Taylor & McCloskey 1988; Mergner et al. 1991).
The activation of neck mechanoreceptors was confirmed by the fact that the subjects reported the perception of the body movement occurring at or about the neck. This dynamic neck stretch is unlikely to have mechanically distorted the baroreceptors because there was no change in the MSNA during static neck displacement, nor was there any
! ! "$)! ! ! ! change in mean arterial blood pressure or heart rate during either dynamic (sinusoidal) or static stretch of the neck. Similarly, other studies have reported that there was no change in MSNA, mean arterial blood pressure or heart rate in subjects in whom the neck was stretched (Ray & Hume 1998; Ray et al. 1998; Monahan & Ray 2002).
In addition, although MSNA was clearly modulated by the neck stretch in Study IV, I cannot rule out the possibility of a respiratory contribution to the modulation of MSNA occurring during neck stretch since there may, on occasion, have been temporal overlap - even though the frequency of the neck stretch stimulus was significantly different to the respiratory frequency in the subjects. There is indirect evidence that suggests that neck afferents are not involved in increases in breathing frequency in humans (Monahan et al.
2002). The bilateral recording of MSNA I carried out provided evidence of lateralisation of sympathetic outflow, with differences in burst morphology occurring on either side of the body as the body was displaced side to side; I had reported such differences previously during sinusoidal galvanic vestibular stimulation (El Sayed et al. 2010), and one would expect that, if the modulation during neck stimulation was secondary to respiration, then there would not be any side-to-side differences in the modulation of
MSNA. Finally, MSNA was modulated during sinusoidal neck stretch during a sustained breath-hold, thereby ruling out any contribution from respiration.
Conclusions
It is unequivocally clear in the literature, from both animal and human studies, the need for an additional system to immediately compensate for rapid postural adjustments of blood pressure. Vestibulosympathetic reflexes are enhanced when blood pressure is low and diminished when blood pressure increases. This evidence supports the notion that the
! ! "$*! ! ! ! two responses are complementary, and act together to maintain cardiovascular homeostasis during postural challenges. While the baroreflex is able to significantly modulate vasomotor tone and thereby blood pressure in response to changes in posture, signals from the vestibular system, in contrast, are able to indicate changes in posture prior to an orthostatic hypotensive event and thereby reduce the impact of postural change on cardiovascular function and brain perfusion. By using slow sinusoidal physiological stimuli, evidence accumulated throughout my doctoral candidature emphasizes the role of the utricle, through the vestibulosympathetic reflex, in peripheral vascular control above and below perceptual threshold. In addition, through dynamic stimuli of neck proprioceptors my findings also indicate that sensory endings in the neck, as well as vestibular inputs, contribute to cardiovascular control in awake humans via their projections to the vestibular nuclei.
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APPENDICES
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