Galvanic Stimulation of the Vestibular Periphery in Guinea Pigs During

23 Irregular vestibular afferents exhibit significant phase leads with respect to angular velocity of 24 the head in space. This characteristic and their connectivity with vestibulospinal neurons 25 suggest a functionally important role for these afferents in producing the vestibulo-collic 26 reflex (VCR). A goal of these experiments was to test this hypothesis using weak galvanic 27 stimulation of the vestibular periphery (GVS) to selectively activate or suppress irregular 28 afferents during passive whole body rotation of guinea pigs that could freely move their 29 heads. Both inhibitory and excitatory GVS had significant effects on compensatory head 30 movements (CHV) during sinusoidal and transient whole body rotations. Unexpectedly, GVS 31 also strongly affected the vestibulo-ocular reflex (VOR) during passive whole body rotation. 32 The effect of GVS on the VOR was comparable in light and darkness and whether or not the 33 head was restrained or unrestrained. Significantly, there was no effect of GVS on 34 compensatory eye and head movements during volitional head motion, a confirmation of our 35 previous study that demonstrated the extra vestibular nature of anticipatory eye movements 36 that compensate for voluntary head movements. 37 38 Introduction: 39 Application of galvanic currents to the vestibular periphery has been shown to affect a 40 subpopulation of afferent fibers innervating vestibular hair cells in all of the vestibular sense 41 organs (Kim and Curthoys 2004; Goldberg 2000). Specifically, irregularly firing axons show a 42 high degree of sensitivity to weak galvanic currents, whereas regular afferents are relatively 43 unaffected (Goldberg et al. 1984; Baird et al. 1988). An afferent’s sensitivity to electrical 44 stimulation is one of several properties used to distinguish between the two types (for review 45 see Goldberg 2000; Eatock and Songer 2011). Afferents vary in their anatomy, response 46 dynamics, firing regularity and response gains (Baird et al. 1988). Irregular afferents are 47 characterized by calyceal endings (but may be dimorphic), are associated with more centrally 48 located hair cells, and have spontaneous irregular firing rates. With respect to their dynamic 49 responses, irregular units fall into two categories: those with relatively low gains at higher 50 frequencies and others, which are part of a continuum of regular, intermediate and irregular 51 units with linearly increasing gains as a function of discharge regularity. The low gain units 52 have calyx endings whilst the high gain units tend to be dimorphs (Baird et al. 1988; Goldberg 53 2000). 54 Previous experiments have shown that GVS does not affect the vestibulo-ocular reflex (VOR) 55 during sinusoidal motion or brief velocity steps in primates (Minor and Goldberg 1991; 56 Angelaki and Perachio 1993; Chen-Huang et al. 1997). Angelaki and Perachio (1993) did find 57 that anodal GVS reduced eye velocity during prolonged velocity steps lasting several seconds 58 and during off-axis angular rotation (Angelaki et al. 1992). Based on these results, they 59 concluded that irregular otolith afferents might be essential for the generation of steady state 60 nystagmus during off-vertical axis rotations (OVAR) and for velocity storage and the VOR at 61 sinusoidal frequencies less than 0.1 Hz. The limited effects of irregular afferent “functional” 62 ablation are surprising, as experimenters have also shown that there is no preferential 63 innervation of second-order vestibular neurons by the different afferent types (Highstein et al. 64 1987; Boyle et al. 1992; Chen-Huang et al. 1997). One possible explanation for this apparent 65 paradox is the influence of extra-vestibular factors such as target distance or attention (Chen66 Huang et al. 1997; Chen-Huang and McCrea 1998) on the responses of neurons within the 67 vestibular nucleus. Alternatively, irregular afferents might predominantly influence vestibular 68 control of head stability (Angelaki and Perachio 1993; Bilotto et al. 1982) rather than the VOR, 69 although this idea has never been directly tested in head-unrestrained animals. 70 In order to directly test the latter hypothesis, we performed galvanic stimulation in head71 unrestrained guinea pigs. The current study presents evidence for effects of GVS on vestibular 72 compensatory head movements during passive whole body rotations. Furthermore, here we 73 show that in the guinea pig, GVS has a significant effect on the VOR during both sinusoidal and 74 transient head motion over a broad range of movement frequencies and velocities. In contrast, 75 there is no effect of GVS on the anticipatory compensatory eye movements that occur during 76 self-generated (active) head movements of the same animals (Shanidze et al. 2010b). 77 The approach of the current study differs in two critical ways from previous work. First, our 78 experiments were done in the guinea pig, unlike the seminal primate studies of Minor and 79 Goldberg (1991). Second, both eye and head responses to horizontal vestibular stimulation 80 were measured in animals whose heads were unrestrained, a more natural preparation. 81 Previous experiments have described the effects of GVS on vestibular nerve activity in the 82 guinea pig. Kim and Curthoys (2004) replicated, in anesthetized guinea pigs, the preferential 83 effect of GVS on irregular afferents found in chinchilla (e.g. Baird et al. 1988) and monkey (e.g. 84 Goldberg et al. 1984). To control for any possible influence of head restraint on the 85 effectiveness of GVS, we also report data from both head restrained and head unrestrained 86 guinea pigs. 87 Materials and Methods: 88 Experimental and surgical procedures were performed in accordance with the National 89 Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by 90 the University of Michigan’s University Committee on Use and Care of Animals (UCUCA). 91 Surgical Procedures and Testing. For eye coil, head post and electrode implantation, the 92 animals were anesthetized using an intramuscular cocktail of Ketamine (0.40 ml/kg) and 93 Xylazine (0.50 ml/kg) and were administered saline solution (20 cc) and atropine (0.125 94 ml/kg) subcutaneously for each surgical procedure. A heating pad was used to maintain the 95 animals’ body temperature. Vital signs were monitored until the animal became mobile and 96 could stand upright. Three guinea pigs were prepared for chronic eye and head movement 97 recording by implanting a 2D search coil in the right eye and a head post on the skull using 98 dental acrylic (C&B Metabond, Parkell Inc., Edgewood, NY). A second search coil was affixed to 99 the head post to record head movements. The guinea pigs’ bodies were restrained but their 100 heads were free to move during vestibular stimulation. Passive whole-body angular rotation 101 about an earth vertical axis centered between the animals’ ears was used to evoke reflex 102 responses. Animals were rotated sinusoidally at frequencies ranging from 0.2 to 10 Hz all 103 tested at velocities ranging from 20 to 90 deg/s (20, 30, 40, 60, 80, 90 deg/s; accelerations of 104 300 to 3000 deg/s/s). To mimic voluntary head movements, abrupt transient “velocity steps” 105 between 20 and 90 deg/s with peak accelerations up to 2000 deg/s/s were used (for details 106 see Shanidze et al. 2010a). 107 Galvanic Stimulation. Stimulating electrodes were implanted bilaterally in the middle ear. The 108 electrodes and leads were assembled prior to the surgery and gas sterilized. Each electrode 109 consisted of two Teflon-coated, 32-gauge, platinum-iridium wires (A-M Systems, Sequim, WA) 110 soldered to a set of stainless steel connectors. Each electrode had a ball, 0.05 mm in diameter, 111 on the implanted end. A retro-auricular incision was made and the dorsal bulla exposed. The 112 bulla was drilled to provide access to the middle ear. Using a surgical microscope, the ossicles, 113 cochlea and round window were observed through the opening as landmarks for placement of 114 the stimulating electrodes. One electrode was placed near the round window, wedged in place 115 and fixed with Vetbond (3M, St. Paul, MN). The second electrode, serving as a ground, was 116 implanted in the bulla distal to the first electrode and fixed in place with Vetbond. Metabond 117 was used to seal the bulla opening. The leads from both electrodes were led subcutaneously to 118 the skull and attached to a previously constructed acrylic pad. Currents ranging between 20 119 and 80 μA and comparable to those used previously in guinea pig (Kim and Curthoys, 2004) 120 were applied either cathodally or anodally (to achieve either excitation or inhibition of the 121 vestibular periphery, respectively) and were timed to occur in relation to the vestibular 122 stimulus. Currents were applied bilaterally and in temporal synchrony using separate 123 constant current isolated pulse stimulators (Model 2100, A-M Systems) for each ear. For each 124 experiment, currents were balanced between the two ears. Current was applied to each ear to 125 determine thresholds at which vestibular nystagmus was elicited. Currents were then applied 126 bilaterally for 1-2 seconds and adjusted so that no eye movements were invoked in the 127 absence of rotational stimuli with either cathodal or anodal bilateral stimulation. 128 During the experiment, fully awake animals were restrained and placed on a servo-controlled 129 turntable (Neurokinetics, Inc, Pittsburgh, PA, see Shanidze et al. 2010a). Video camera 130 recordings using infrared illumination were used to ensure the animals remained alert and to 131 confirm that head position remained relatively upright and aligned with the body axis during 132 stimulation. Eye and head movements were recorded using the electromagnetic search coil 133 technique (e.g. Robinson 1963 in human; Fuchs and Robinson 1966, Judge et al. 1980 in 134 monkey; Shanidze et al. 2010a in guinea pig). A Primelec search coil system (D. Florin, Ostring, 135 Sw; model CS681) generated three orthogonal electromagnetic fields around the guinea pig. 136 The Primalec field coils were stationary relative to the world and the animals were rotated 137 within the generated fields. In this configuration, measured eye and head movement signals 138 were eye-in-space and head-in-space relative to the earth fixed coordinate frame established 139 by the field coils. Eye position, head position and body velocity data were each sampled at 14