Objective.  New clinical tests of otolithic function use bone conducted vibration (BCV) and air conducted sound (ACS) stimuli and measure short latency vestibular-evoked myogenic potentials (VEMPs) over the inferior oblique muscles  (oVEMPs) or the SCM neck muscles (cVEMPS).   The interpretation of these tests relies on physiological evidence that 500Hz BCV and ACS preferentially activate otolithic afferents in comparison to semicircular canal afferents.  This study sought to identify in the guinea pig the peripheral sense organ of origin of irregular primary otolithic afferent neurons having a very sensitive response to both BCV and ACS, by labelling such neurons with juxtacellular injections of neurobiotin and histological identification of the site of origin of the labelled neurons.    

Methods.   Guinea pigs were anesthetized with single intramuscular injections of a mixture of  Ketamine (100mg/kg) and Xylazine (4mg/kg) and maintained on half that dose throughout the experiment.  The lateral cerebellum was aspirated and single primary vestibular neurons in Scarpa’s ganglion were recorded extracellularly using glass microelectrodes containing 5% neurobiotin in 2M NaCl.  Neurons were classed as regular or irregular by the variability of their resting discharge. Semicircular canal and otolith neurons were identified by their response to angular and linear accelerations.  BCV stimulation (up to 2g p-p) was delivered by a Radioear B-71 bone oscillator cemented to the skull.  ACS stimulation (up to 140dB SPL) was delivered by a TDH-49 headphone via a speculum. The usual search stimulus was 500Hz BCV and neurons which were activated by this were tested with 500Hz ACS.   Such neurons were labelled by  juxtacellular neurobiotin injections (5-15nA).  Following perfusion, the entire utricular and saccular macula were dissected from the temporal bone and processed with DAB with nickel and cobalt intensification, cleared with MSBB, to show the sense organ of origin of the labelled afferents. 

Results.  Many otolithic irregular neurons in the superior vestibular nerve were activated by 500Hz BCV at low threshold (around 0.1g) and showed a large increase in firing rate as intensity was increased.  Many of these same afferents were also activated by high intensity 500Hz ACS (threshold about 115 db SPL - about the level used in human clinical testing).  Regular otolithic neurons were not activated and semicircular canal neurons were rarely activated and then usually only at high threshold and low sensitivity.  The whole mounts of the entire macula showed a total of 22 utricular afferents which responded to both ACS and BCV originating from calyx endings around presumed Type I receptors at the region of the striola.  A total of 6 saccular afferents responding to both stimuli have been identified (Curthoys et al., 2012).  

Discussion.   This study confirms that irregular neurons originating from in or close to the striola of the utricular macula and the saccular macula are preferentially activated by 500Hz BCV and ACS in contrast to otolithic regular neurons and semicircular canal neurons. The wide presumption in clinical vestibular testing - that ACS only activates saccular afferents in the inferior vestibular nerve - is not correct. There is no “afferent specificity” by saccular afferents for ACS and utricular afferents for BCV – instead some afferents from both sense organs have low threshold, sensitive responses to both stimuli.  Differential clinical evaluation of the functional status of the utricular and saccular maculae cannot rely on stimulus type (ACS vs BCV), but the predominantly differential motor projections of the utricular and saccular maculae does allow for differential clinical evaluation of each sense organ: utricular macula from oVEMPs and saccular macula from cVEMPs. 

Conclusion. 500Hz bone conducted vibration and air-conducted sound are effective ways of activating irregular otolithic afferents and so allowing clinical testing of otolith function.  

References.
Curthoys IS, Vulovic V, Sokolic L, Pogson J, Burgess AM. Irregular primary otolith afferents from the guinea pig utricular and saccular maculae respond to both bone conducted vibration and to air conducted sound. Brain Research Bulletin, 2012; 89:16-21.  DOI:10.1016/j.brainresbull.2012.07.007