Sergei B. Yakushin

The Nodulus and Uvula: Source of Cerebellar Control of Spatial Orientation of the Angular Vestibulo-Ocular Reflex

Abstract: The nodulus and rostral‐ventral uvula of the vestibulo‐cerebellum play a critical role in orienting eye velocity of the slow component of the angular vestibulo‐ocular reflex (aVOR) to gravito‐inertial acceleration (GIA). This is done by altering the time constants of “velocity storage” in the vestibular system and by generating “cross‐coupled” eye velocities that shift the eye velocity vector from along the body yaw axis to the yaw axis in a spatial frame. In this report, we show that eye velocity generated through the aVOR by constant velocity centrifugation in the monkey orients to the GIA in space, regardless of the position of the head with respect to the axis of rotation. We also show that, after removal of the nodulus and rostral‐ventral uvula, the spatial orientation of eye velocity to the GIA is lost and that eye velocity is then purely driven by the semicircular canals in a body frame of reference. These findings are further confirmation that these regions of the vestibulo‐cerebellum control spatial orientation of the aVOR.

Control of Spatial Orientation of the Angular Vestibulo-Ocular Reflex by the Nodulus and Uvula of the Vestibulocerebellum

Abstract: Eye velocity produced by the angular vestibulo‐ocular reflex (aVOR) tends to align with the summed vector of gravity and other linear accelerations [gravito‐inertial acceleration (GIA)]. Defined as “spatial orientation of the aVOR,” we propose that it is controlled by the nodulus and uvula of the vestibulocerebellum. Here, electrical stimulation, injections of the GABAA agonist, muscimol, and single‐cell recordings were utilized to investigate this spatial orientation. Stimulation, injection, and recording sites in the nodulus were determined in vivo by MRI and verified in histological sections. MRI proved to be a sensitive, reliable way to localize electrode placements. Electrical stimulation at sites in the nodulus and sublobule d of the uvula produced nystagmus whose slow‐phase eye‐velocity vectors were either head centric or spatially invariant. When head centric, the eye velocity vector remained within ±45° of the vector obtained with the animal upright, regardless of head position with respect to gravity. When spatially oriented, the vector remained relatively constant in space in one on‐side position, with respect to the vector determined with the animal upright. A majority of induced movements from the nodulus were spatially oriented. Spatially oriented movements were generally followed by after‐nystagmus, which had the characteristics of optokinetic after‐nystagmus (OKAN), including orientation to the GIA. After muscimol injections, horizontal‐to‐vertical cross‐coupling was lost or reduced during OKAN in tilted positions. This supports the hypothesis that the nodulus mediates yaw‐to‐vertical or roll cross‐coupling. The injections also shortened the yaw‐axis time constant and produced contralateral horizontal spontaneous nystagmus, whose velocity varied as a function of head position with regard to gravity. Nodulus units were tested with static head tilt, sinusoidal oscillation around a spatial horizontal axis with the head in different orientations relative to the pitching plane, and off‐vertical axis rotation (OVAR). The direction of the response vectors of the otolith‐recipient units in the nodulus, determined from static and/or dynamic head tilts, were confirmed by OVAR. These vector directions lay close to the planes of the vertical canals in 7/10 units; many units also had convergent input from the vertical canals. It is postulated that the orientation properties of the aVOR result from a transfer of otolith input regarding head tilt along canal planes to canal‐related zones of the nodulus. In turn, Purkinje cells in these zones project to vestibular nuclei neurons to control eye velocity around axes normal to these same canal planes.

What Does Galvanic Vestibular Stimulation Actually Activate

Luigi Galvani spent 20 years conducting experiments to demonstrate electrical conductivity of nerves and muscles before publishing his major treatise on the subject in 1791 (Galvani, 1791). His personal friend and professional nemesis Count Alessandro Volta held a respectful but opposing view, that nerve and muscle tissues simply serve as passive conductors, and he built the first “voltaic” battery in an attempt to prove his point. Ultimately it appears that they were both partially correct, and the same bioelectric potentials sought by Galvani and debunked by Volta continue to be used in present day because they are an easy, non-invasive approach to activate the vestibular nerve(s). Yet, debate continues in contemporary medicine and science regarding the exact effect of galvanic stimulation on the nervous system. Galvanic vestibular stimulation (GVS) has been used to activate fibers of the vestibular nerve in humans and experimental animals by applying 0.1–4 mA DC currents through the skin over the mastoid processes (for reviews, see Fitzpatrick and Day, 2004; Curthoys, 2009). Steps of current are used most often, causing continuous activation of the entire vestibular nerve, particularly those fibers with irregular spontaneous firing rates (Goldberg et al., 1984; Minor and Goldberg, 1991). This stimulation excites a wide range of central vestibular neurons, including those related to both the semicircular canals and the otolith organs (Wilson et al., 1979; Peterson et al., 1980; Ezure et al., 1983; Courjon et al., 1987). However, despite this non-selective activation, it appears that only otolith-related behavioral responses are induced. Human subjects experience sensations of rocking or pitching, head and/or body tilt, and have ocular torsion – all characteristics of otolith system activation (Zink et al., 1997; Watson et al., 1998; Severac Cauquil et al., 2003; Macdougall et al., 2005; Bent et al., 2006). They do not experience sensations of rotation and do not 4display ocular nystagmus, which would occur if the semicircular canals were continuously stimulated (Mach, 1875; Cohen et al., 1965; Guedry, 1974). This apparent paradox has engendered considerable controversy: does GVS primarily or exclusively activate the otolith system, or does it activate both the otolith and semicircular canal systems equivalently? The preponderance of physiological data support the view that GVS is primarily an otolithic stimulus. A variant of GVS utilizing binaurally applied sinusoidal currents (sinusoidal GVS, sGVS) was introduced by Macefield and colleagues (Bent et al., 2006; Grewal et al., 2009; James and Macefield, 2010; James et al., 2010), and has proven to be a potent technique for inducing muscle sympathetic nerve activity (MSNA) in the legs of humans. MSNA causes peripheral vasoconstriction, which maintains adequate blood supply to the brain upon standing. This orthostatic response is clearly associated with the otolith system (Yates, 1992; Woodring et al., 1997; Kerman et al., 2003). When sGVS is applied to anesthetized rats, it can also induce sudden decreases in blood pressure and heart rate that resemble human vasovagal syncope (Cohen et al., 2011). Similar sustained drops in blood pressure have been shown in alert and anesthetized rats after linear acceleration (Zhu et al., 2007). sGVS also evokes frequency-dependent postural sway in standing subjects, further supporting the idea that the stimulus primarily activates the otolith system (Lau et al., 2003). Functional anatomical studies have also contributed to the controversy regarding the neural effect(s) of GVS. These investigations have utilized GVS to induce activation of the immediate early gene c-fos, and to visualize its protein product c-Fos, which accumulates in the nuclei of activated neurons. Steps of GVS applied unilaterally to rodents result in bilateral c-Fos expression near the ventricular wall in the medial vestibular nucleus (MVN), with muted expression in the inferior vestibular nucleus (IVN) and no c-Fos accumulation in the superior or lateral vestibular nuclei (SVN and LVN, respectively; Kaufman and Perachio, 1994; Marshburn et al., 1997; Abe et al., 2009). In this Frontiers Special Topic, Holstein and colleagues report that sGVS in rats results in c-Fos accumulation in some neurons in caudal IVN, in cells of the parasolitary nucleus, in neurons throughout MVN, and in cells located in a small medial wedge in caudal SVN. There were no activated neurons in the portions of the vestibular nuclear complex (VNC) that participate directly in the horizontal and vertical vestibulo-ocular reflexes, or the vestibulo-spinal postural reflexes. These studies reflect the same apparent contradiction evident in human physiological investigations: if GVS activates the entire vestibular nerve, why are activated neurons restricted to non-vestibulo-ocular and vestibulo-spinal regions? The most likely explanation for this discrepancy derives from a report by Courjon et al. (1987) in which a wide variety of central vestibular neurons were activated by galvanic stimulation. Units that responded to rotation promptly habituated, while those units that were non-responsive to rotation, which were presumably otolith units, continued to fire in response to GVS. We propose that this canal-specific response habituation underlies the apparent inconsistency between the global vestibular activation by GVS and the otolith-predominant neural and behavioral responses. Moreover, the c-Fos localization findings can be further interpreted in this light, since c-Fos protein is not manifest in neurons that are tonically inhibited (Chan and Sawchenko, 1994). Many vestibular neurons that participate in vestibulo-ocular and vestibulo-spinal reflexes receive formidable direct inhibition from cerebellar Purkinje cells and/or inhibitory commissural and intra-VNC fibers (for review, see Holstein, 2011). These neurons are not likely to express c-Fos protein, even though they may initially be activated by sGVS. Further still, while neurons that receive predominantly excitatory input and some cells under conditions of release from tonic inhibition show c-Fos expression in response to appropriate stimuli, other disinhibited neurons do not express c-Fos induction (for review, see Kovacs, 2008), and c-Fos is rarely observed in large motor neurons of the brainstem (Chan and Sawchenko, 1994). As a result, vestibulo-ocular, vestibulo-spinal, and vestibulo-colic motor neurons present in subregions of the VNC should not be expected to accumulate c-Fos protein. On the basis of this analysis, we conclude that while sGVS does indeed activate the entire vestibular nerve, only the otolith system expresses a persistent behavioral and neural response due to the habituation of the canal-related units and the attendant inhibition of vestibular neuronal populations. It is likely that the habituation of the semicircular canal induced activity originates in the cerebellum, but this remains to be determined.

Baclofen, motion sickness susceptibility and the neural basis for velocity storage

Reduction of the dominant time constant (T(VOR)) of the angular vestibulo-ocular reflex (aVOR) by habituation is associated with a decrease in motion sickness susceptibility. Baclofen, a GABA(b) agonist, reduces the time constant of the velocity storage integrator in the aVOR in a dose-dependent manner. The high frequency aVOR gain is unaltered by baclofen. Here we demonstrate that the reduction in T(VOR) produced by oral administration of 20 mg of baclofen causes a significant reduction in motion sickness susceptibility, tested with roll while rotating (RWR). These data show that motion sickness susceptibility can be pharmacologically manipulated with a GABA(b) agonist and support our conclusion that motion sickness is generated through velocity storage. We also show how baclofen acts on velocity storage at the neural level. A vestibular-plus-saccade (VPS) neuron was recorded in the rostral medial vestibular nucleus (rMVN) of a cynomolgus monkey, an area where we postulate that velocity storage is generated. The cell had a time constant during steps of velocity that was close to that of the T(VOR). After parenteral administration of baclofen, there was a similar decrease in the time constants of the VPS neuron and the T(VOR). This is the first demonstration of the concurrence of unit and aVOR time constants before and after baclofen. The data support the hypothesis that the velocity storage integrator is generated through activity of vestibular-only (VO) and VPS neurons in rMVN and suggest that GABA(b) synapses on VO and VPS neurons are likely to be involved in the baclofen-induced reduction in motion sickness susceptibility.

Fos expression in neurons of the rat vestibulo-autonomic pathway activated by sinusoidal galvanic vestibular stimulation

The vestibular system sends projections to brainstem autonomic nuclei that modulate heart rate and blood pressure in response to changes in head and body position with regard to gravity. Consistent with this, binaural sinusoidally modulated galvanic vestibular stimulation (sGVS) in humans causes vasoconstriction in the legs, while low frequency (0.02–0.04 Hz) sGVS causes a rapid drop in heart rate and blood pressure in anesthetized rats. We have hypothesized that these responses occur through activation of vestibulo-sympathetic pathways. In the present study, c-Fos protein expression was examined in neurons of the vestibular nuclei and rostral ventrolateral medullary region (RVLM) that were activated by low frequency sGVS. We found c-Fos-labeled neurons in the spinal, medial, and superior vestibular nuclei (SpVN, MVN, and SVN, respectively) and the parasolitary nucleus. The highest density of c-Fos-positive vestibular nuclear neurons was observed in MVN, where immunolabeled cells were present throughout the rostro-caudal extent of the nucleus. c-Fos expression was concentrated in the parvocellular region and largely absent from magnocellular MVN. c-Fos-labeled cells were scattered throughout caudal SpVN, and the immunostained neurons in SVN were restricted to a discrete wedge-shaped area immediately lateral to the IVth ventricle. Immunofluorescence localization of c-Fos and glutamate revealed that approximately one third of the c-Fos-labeled vestibular neurons showed intense glutamate-like immunofluorescence, far in excess of the stain reflecting the metabolic pool of cytoplasmic glutamate. In the RVLM, which receives a direct projection from the vestibular nuclei and sends efferents to preganglionic sympathetic neurons in the spinal cord, we observed an approximately threefold increase in c-Fos labeling in the sGVS-activated rats. We conclude that localization of c-Fos protein following sGVS is a reliable marker for sGVS-activated neurons of the vestibulo-sympathetic pathway.

Head Stabilization by Vestibulocollic Reflexes During Quadrupedal Locomotion in Monkey

Little is known about the three-dimensional characteristics of vestibulocollic reflexes during natural locomotion. Here we determined how well head stability is maintained by the angular and linear vestibulocollic reflexes (aVCR, lVCR) during quadrupedal locomotion in rhesus and cynomolgus monkeys. Animals walked on a treadmill at velocities of 0.4-1.25 m/s. Head rotations were represented by Euler angles (Fick convention). The head oscillated in yaw and roll at stride frequencies (approximately 1-2 Hz) and pitched at step frequencies (approximately 2-4 Hz). Head angular accelerations (100-2,500 degrees/s2) were sufficient to have excited the aVOR to stabilize gaze. Pitch and roll head movements were <7 degrees , peak to peak, and the amplitude was unrelated to stride frequency. Yaw movements were larger due to spontaneous voluntary head shifts and were smaller at higher walking velocities. Head translations were small (< or =4 cm). Cynomolgus monkeys positioned their heads more forward in pitch than the rhesus monkeys. None of the animals maintained a forward head fixation point, indicating that the lVCR contributed little to compensatory head movements in these experiments. Significantly, aVCR gains in roll and pitch were close to unity and phases were approximately 180 degrees over the full frequency range of natural walking, which is in contrast to previous findings using anesthesia or passive trunk rotation with body restraint. We conclude that the behavioral state associated with active body motion is necessary to maintain head stability in pitch and roll over the full range of stride/step frequencies encountered during walking.

Vestibular experiments in space.

Experiments were performed while monkeys flew in space in the ‘‘Cosmos/ Bion’’ Missions to determine the effect of microgravity on the oculomotor and vestibular systems. Eye-head coordination during gaze shifts to lateral targets (gaze fixation reaction, GFR) and multiunit activity in the medial vestibular nuclei (MVN) and cerebellar flocculus were studied in rhesus monkeys in the Bion 6 (Cosmos 1514) through Bion 11 projects. In the first few days of space flight, gaze displacement onto lateral targets became hypermetric, and the amplitude of head movements decreased. This was compensated for by 112

Adaptation of Orientation Vectors of Otolith-Related Central Vestibular Neurons to Gravity

Behavioral experiments indicate that central pathways that process otolith-ocular and perceptual information have adaptive capabilities. Because polarization vectors of otolith afferents are directly related to the electro-mechanical properties of the hair cell bundle, it is unlikely that they change their direction of excitation. This indicates that the adaptation must take place in central pathways. Here we demonstrate for the first time that otolith polarization vectors of canal-otolith convergent neurons in the vestibular nuclei have adaptive capability. A total of 10 vestibular-only and vestibular-plus-saccade neurons were recorded extracellularly in two monkeys before and after they were in side-down positions for 2 h. The spatial characteristics of the otolith input were determined from the response vector orientation (RVO), which is the projection of the otolith polarization vector, onto the head horizontal plane. The RVOs had no specific orientation before animals were in side-down positions but moved toward the gravitational axis after the animals were tilted for extended periods. Vector reorientations varied from 0 to 109 degrees and were linearly related to the original deviation of the RVOs from gravity in the position of adaptation. Such reorientation of central polarization vectors could provide the basis for changes in perception and eye movements related to prolonged head tilts relative to gravity or in microgravity.

Adaptive Changes in the Angular VOR: Duration of Gain Changes and Lack of Effect of Nodulo‐Uvulectomy

Alterations in the gain of the vertical angular vestibulo‐ocular reflex (VOR) are dependent on the head position in which the gain changes were produced. We determined how long gravity‐dependent gain changes last in monkeys after four hours of adaptation, and whether the adaptation is mediated through the nodulus and uvula of the vestibulocerebellum. Vertical VOR gains were adaptively modified by rotation about an interaural axis, in phase or out of phase with the visual surround. Vertical VOR gains were modified with the animals in one of three orientations: upright, left‐side down, or right‐side down. Monkeys were tested in darkness for up to four days after adaptation using sinusoidal rotation about an interaural axis that was incrementally tilted in 10° steps from vertical to side down positions. Animals were unrestrained in their cages in normal light conditions between tests. Gravity‐dependent gain changes lasted for a day or less after adaptation while upright, but persisted for two days or more after on‐side adaptation. These data show that gravity‐dependent gain changes can last for prolonged periods after only four hours of adaptation in monkeys, as in humans. They also demonstrate that natural head movements made while upright do not provide an adequate stimulus for rapid recovery of vertical VOR gains that were induced on side. In two animals, the nodulus and uvula were surgically ablated. Vertical gravity‐dependent gain changes were not significantly different before and after surgery, indicating that the nodulus and uvula do not have a critical role in producing them.

What does galvanic vestibular stimulation actually activate: response.

2010; Hammam et al., 2011, 2012; Grewal et al., 2012). The sense of roll is consist-ent with a host of other studies using GVS (Fitzpatrick et al., 1994; Inglis et al., 1995; Day et al., 1997; Zink et al., 1997; Day and Cole, 2002; Scinicariello et al., 2002/2003; Wardman et al., 2003a,b; see Fitzpatrick and Day, 2004 for review). Modeled on this research, we used 2–3 mA currents in lightly anesthetized rats and found strong activation of the sympathetic nervous sys-tem and vasovagal responses, but not tonic deviations of the eyes (Cohen et al., 2011). We have also stimulated the vestibular nerve with trains of pulses to activate MSNA in humans (Voustianiouk et al., 2006), some-times utilizing currents of 5 mA. MSNA was facilitated, but ocular deviations were never induced.The semicircular canals can exert a powerful influence on eye movements through the vestibulo-ocular reflex. Head turns induce eye movements with veloci-ties of up to 400/s (Atkin and Bender, °1968), and with response characteristics up to 20 Hz ( Grossman et al., 1988; Tabak and Collewijn, 1994; Armand and Minor, 2001). Furthermore, a change in body temperature of only 7C during caloric °stimulation with either air or water read-ily induces nystagmus with slow phase velocities of 30–40/s in monkeys (° Arai et al., 2002) and 10–20°/s in humans (M. Dai, personal communication). Thus, at best, the semicircular canal activation induced using strong GVS by Curthoys and MacDougall, which produced average slow phase eye velocities of 5° /s, was weak and functionally inconsequential.The question of function is particularly relevant in this regard. The semicircular canals stabilize gaze in space during head