Hair bundles are critical to mechanotransduction by vestibular hair cells, but quantitative data are lacking on vestibular bundles in mice or other mammals. Here we quantify bundle heights and their variation with macular locus and hair cell type in adult mouse utricular macula. We also determined that macular organization differs from previous reports. The utricle has approximately 3,600 hair cells, half on each side of the line of polarity reversal (LPR). A band of low hair cell density corresponds to a band of calretinin-positive calyces, i.e., the striola. The relation between the LPR and the striola differs from previous reports in two ways. First, the LPR lies lateral to the striola instead of bisecting it. Second, the LPR follows the striolar trajectory anteriorly, but posteriorly it veers from the edge of the striola to reach the posterior margin of the macula. Consequently, more utricular bundles are oriented mediolaterally than previously supposed. Three hair cell classes are distinguished in calretinin-stained material: type II hair cells, type ID hair cells contacting calretinin-negative (dimorphic) afferents, and type IC hair cells contacting calretinin-positive (calyceal) afferents. They differ significantly on most bundle measures. Type II bundles have short stereocilia. Type IC bundles have kinocilia and stereocilia of similar heights, i.e., KS ratios (ratio of kinocilium to stereocilia heights) approximately 1, unlike other receptor classes. In contrast to these class-specific differences, bundles show little regional variation except that KS ratios are lowest in the striola. These low KS ratios suggest that bundle stiffness is greater in the striola than in the extrastriola.

Hair bundle structure is a major determinant of bundle mechanics and thus of a hair cell's ability to encode sound and head movement stimuli. Little quantitative information about bundle structure is available for vestibular organs. Here we characterize hair bundle heights in the utricle of a turtle, Trachemys scripta. We visualized bundles from the side using confocal images of utricular slices. We measured kinocilia and stereocilia heights and array length (distance from tall to short end of bundle), and we calculated a KS ratio (kinocilium height/height of the tallest stereocilia) and bundle slope (height fall-off from tall to short end of bundle). To ensure that our measurements reflect in vivo dimensions as closely as possible, we used fixed but undehydrated utricular slices, and we measured heights in three dimensions by tracing kinocilia and stereocilia through adjacent confocal sections. Bundle heights vary significantly with position on the utricular macula and with hair cell type. Type II hair cells are found throughout the macula. We identified four subgroups that differ in bundle structure: zone 1 (lateral extrastriola), striolar zone 2, striolar zone 3, and zone 4 (medial extrastriola). Type I hair cells are confined to striolar zone 3. They have taller stereocilia, longer arrays, lower KS ratios, and steeper slopes than do neighboring (zone 3) type II bundles. Models and experiments suggest that these location- and type-specific differences in bundle heights will yield parallel variations in bundle mechanics. Our data also raise the possibility that differences in bundle structure and mechanics will help explain location- and type-specific differences in the physiological profiles of utricular afferents, which have been reported in frogs and mammals.

Physiological studies in many vertebrates indicate that vestibular primary afferents are not a homogeneous population. Such data raise the question of what structural mechanisms underlie these physiological differences and what functional role is played by afferents of each type. We have begun to answer these questions by characterizing the architecture of 110 afferents innervating the posterior canal of Pseudemys scripta. We emphasize their spatial organization because experimental evidence suggests that afferent physiological properties exhibit significant spatial heterogeneity. The sensory surface of the posterior canal comprises paired, triangular hemicristae, which are innervated by two afferent types. Bouton afferents (66% of total afferents) are found over the entire sensory surface. They differ significantly in the shape and size of their collecting areas, number of boutons, soma size, and axon diameter; this morphological variation is systematically related to the afferent's spatial position. In addition, multivariate analyses suggest that bouton afferents may comprise two subtypes: alpha afferents have delicate processes and are found throughout the crista; beta afferents are more robust and are concentrated preferentially toward the canal center. Calyx-bearing afferents comprise two morphological subtypes: dimorphs (13% of total afferents) bear calyceal and bouton endings; calyceal afferents (21%) bear calyceal endings only. Both types occur exclusively in an elliptical region near the center of each hemicrista; their morphology varies with radial distance from the center of this elliptical region. Our data provide evidence that in Pseudemys: (1) the classical vestibular afferent types (bouton, calyx, dimorph) are structurally heterogeneous, and (2) their spatial sampling characteristics are highly structured and distinctive for each type. These spatial patterns may shed light on regional differences in physiological profiles of vestibular afferents, and they raise questions about the role of this spatial heterogeneity in signaling head movement.

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In this paper we characterize the architecture and segmental innervation, histochemical composition, muscle spindle populations, and motor pool organization of rat spinal accessory (SA) muscles: sternomastoid (SM), cleidomastoid (CM), cleidotrapezius (CT), and acromiotrapezius (AT). We also consider whether individual rat neck muscles are supplied by more than one population of motor neurons as they are in turtles and cats and whether in SA muscles motor neuron size scales with target muscle fiber type. SM, CM, and CT are ventral, parallel strap muscles. Each can be divided into grossly visible white and red compartments. AT is a dorsolateral sheetlike muscle that shows no gross compartmentalization. All four muscles are dominated by fast-twitch glycolytic (FG) and fast oxidative glycolytic (FOG) fibers, and FG fibers are significantly more numerous than the FOG type in three out of four muscles. Thus SA muscles in rats appear to be specialized for rapid, phasic head movements. Topographical analyses revealed that there is a striking compartmentalization of fiber types in the ventral muscles that corresponds to the red and white segments seen grossly. Spindles are found only in regions containing slow-twitch oxidative (SO) fibers. Cross-muscle comparisons indicate that there are significant differences between SA muscles in their fiber type composition. The motor pools of SA muscles form a single column from lower medulla to C5. Rostral cells lie dorsomedially in the ventral horn and, at the C1/C2 junction, the column shifts ventrolaterally. Within this column, each motor pool occupies a characteristic rostrocaudal position in the order SM:CM:CT:AT. Thus SM and (in part) CM motor neurons lie more medially than cells supplying the trapezius complex, suggesting that they may be under different patterns of synaptic drive. We saw no evidence that rat SA muscles are supplied by more than one population of motor neurons. Direct comparisons between the soma sizes of motor neurons that supply muscles or parts of muscles with significantly different histochemical compositions indicate that these size differences are in the direction predicted from their histochemical profiles, thus suggesting that in these muscles motor neuron soma size may scale with muscle fiber type.

A common observation in studies of neuronal structure is that axons differ in the size of their synaptic boutons. The significance of this size variation is unclear, in part because we do not know how the size of synaptic boutons is related to their internal organization. The present study has addressed this issue by using three-dimensional reconstruction of serial thin sections to examine the ultrastructure of synaptic boutons that vary in size. Our observations are based on complete or near-complete reconstructions of 53 synaptic boutons contacting large neurons in the ventromedial gray matter of the upper cervical spinal cord (probable neck motor neurons). We characterized bouton size in terms of volume and total area of membrane apposed to the motor neuron surface (apposition area). Boutons vary in apposition area by a factor of 40, and there is a significant positive correlation between our two measures of bouton size. In addition, bouton size is systematically related to four ultrastructural variables: 1) total active zone area, 2) number of active zones, 3) individual active zone area, and 4) number of synaptic vesicles. The correlations between these variables and both of our measures of bouton size are positive and significant. These data suggest that bouton size may be an index of ultrastructural features that are thought to influence transmitter storage and release.

. A major outstanding goal of vestibular neuroscience is to understand the distinctive functional roles of type I and type II hair cells. One important question is whether these two hair cell types differ in bundle structure. To address this, we have developed methods to characterize stereocilia numbers on identified type I and type II hair cells in the utricle of a turtle, Trachemys scripta. Our data indicate that type I hair cells, which occur only in the striola, average 95.9 Ϯ16.73 (SD) stereocilia per bundle. In contrast, striolar type II hair cells have 59.9 Ϯ 8.98 stereocilia, and type II hair cells in the adjacent extrastriola average 44.8 Ϯ 10.82 stereocilia. Thus type I hair cells have the highest stereocilia counts in the utricle. These results provide the first direct evidence that type I hair cells have significantly more stereocilia than type II hair cells, and they suggest that the two hair cell types may differ in bundle mechanics and peak mechanoelectric transduction currents.

The ability of hair bundles to signal head movements and sounds depends significantly on their structure, but a quantitative picture of bundle structure has proved elusive. The problem is acute for vestibular organs because their hair bundles exhibit complex morphologies that vary with endorgan, hair cell type, and epithelial locus. Here we use autocorrelation analysis to quantify stereociliary arrays (the number, spacing, and distribution of stereocilia) on hair cells of the turtle utricle. Our first goal was to characterize zonal variation across the macula, from medial extrastriola, through striola, to lateral extrastriola. This is important because it may help explain zonal variation in response dynamics of utricular hair cells and afferents. We also use known differences in type I and II bundles to estimate array characteristics of these two hair cell types. Our second goal was to quantify variation in array orientation at single macular loci and use this to estimate directional tuning in utricular afferents. Our major findings are that, of the features measured, array width is the most distinctive feature of striolar bundles, and within the striola there are significant, negatively correlated gradients in stereocilia number and spacing that parallel gradients in bundle heights. Together with previous results on stereocilia number and bundle heights, our results support the hypothesis that striolar hair cells are specialized to signal high-frequency/acceleration head movements. Finally, there is substantial variation in bundle orientation at single macular loci that may help explain why utricular afferents respond to stimuli orthogonal to their preferred directions.