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We review recent progress in using numerical models to relate utricular hair bundle and otoconial membrane (OM) structure to the functional requirements imposed by natural behavior in turtles. The HEAD MOVEMENTS section reviews the evolution of experimental attempts to understand vestibular system function with emphasis on turtles, including data showing that accelerations occurring during natural head movements achieve higher magnitudes and frequencies than previously assumed. The STRUCTURE section reviews quantitative anatomical data documenting topographical variation in the structures underlying macromechanical and micromechanical responses of the turtle utricle to head movement: hair bundles, OM, and bundle-OM coupling. The MACROMECHANICS section reviews macromechanical models that incorporate realistic anatomical and mechanical parameters and reveal that the system is significantly underdamped, contrary to previous assumptions. The MICROMECHANICS: HAIR BUNDLE MOTION AND MET CURRENTS section reviews work based on micromechanical models, which demonstrates that topographical variation in the structure of hair bundles and OM, and their mode of coupling, result in regional specializations for signaling of low frequency (or static) head position and high frequency head accelerations. We conclude that computational models based on empirical data are especially promising for investigating mechanotransduction in this challenging sensorimotor system. [ABSTRACT FROM AUTHOR]

Finite element models of otoconial membrane (OM) were developed to investigate the effects of three geometric variables on static and modal response of the OM: (1) curvature of the macular surface, (2) spatial variation in thicknesses of three OM layers, and (3) shape of the macular perimeter. A geometrically accurate model of a turtle utricle was constructed from confocal images. Modifying values for each variable formed variants of this model: modeling the macula surface as flat, OM layer thicknesses as spatially invariant, and the macular perimeter as a rectangle. Static tests were performed on each modified OM model, and the results were compared to determine the effects of each geometric variable on static mechanical gain (deflection per unit acceleration). Results indicate that all three geometric variables affect the magnitude and directional properties of OM static mechanical gain. In addition, through modal analysis, we determined the natural frequencies and displacement modes of each model, which illustrate the effects of the three geometric variables on OM dynamics. This study indicates the importance of considering three-dimensional OM geometry when attempting to understand responses of the OM and, therefore, the modulation of hair cell signals to accelerations during head movements. [ABSTRACT FROM AUTHOR]

Hair bundle structure is a major determi- nant 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 stereo- cilia) 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 extra- striola), striolar zone 2, striolar zone 3, and zone 4 (medial extra- striola). 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. [ABSTRACT FROM AUTHOR]

Finite element models of otoconial membrane (OM) were developed to investigate the effects of three geometric variables on static and modal response of the OM: (1) curvature of the macular surface, (2) spatial variation in thicknesses of three OM layers, and (3) shape of the macular perimeter. A geometrically accurate model of a turtle utricle was constructed from confocal images. Modifying values for each variable formed variants of this model: modeling the macula surface as flat, OM layer thicknesses as spatially invariant, and the macular perimeter as a rectangle. Static tests were performed on each modified OM model, and the results were compared to determine the effects of each geometric variable on static mechanical gain (deflection per unit acceleration). Results indicate that all three geometric variables affect the magnitude and directional properties of OM static mechanical gain. In addition, through modal analysis, we determined the natural frequencies and displacement modes of each model, which illustrate the effects of the three geometric variables on OM dynamics. This study indicates the importance of considering three-dimensional OM geometry when attempting to understand responses of the OM and, therefore, the modulation of hair cell signals to accelerations during head movements.

We have developed an extrinsic Fabry-Perot interferometer (EFPI) to measure displacements of microscopic, living organelles in the inner ear. The EFPI is an optical phase-shifted instrument that can be used to measure nanometer displacements. The instrument transmits a coherent light signal to the end of a single glass optical fiber where the measurement is made. As the coherent light reaches the end of the fiber, part of this incident signal is reflected off the internal face of the fiber end (reference reflection) and part is transmitted through the end of the fiber. This transmitted light travels a short distance and is reflected off the surface whose displacement is to be measured (the target). This sensing reflection then reenters the fiber where it interferes with the reference reflection. The resulting interference signal then travels up the same optical fiber to a detector, where it is converted into a voltage that can be read from an oscilloscope. When the target moves, the phase relation between reference and sensing reflections changes, and the detector receives a modulated signal proportional to the target movement. Reflections of as little as 1% at both the sensor tip and target surfaces produce good results with this system. We use the EFPI in conjunction with fine glass whiskers to measure the stiffness (force per unit deflection) of stereociliary bundles on hair cells of the inner ear. The forces generated are in the tenths of picoNewton range and the displacements are tens of nanometers. Here we describe the EFPI and its development as a method for measuring displacements of microscopic organelles in a fluid medium. We also report experiments to validate the accuracy of the EFPI output and preliminary measurements of ciliary bundle stiffness in the posterior semicircular canal.

Multiple pathways for the transmission of visual information from retina to brain have been described in reptiles, but little is known about their functional organization. These parallel channels begin at the retina, and we have therefore begun to study the functional organization of retinal ganglion cells in the turtle, Pseudemys scripta elegans. This paper describes the numbers and distribution of cells in the ganglion cell layer. To develop criteria for the identification of ganglion cells, we labelled them retrogradely by applying horseradish peroxidase (HRP) to the optic nerve. Ganglion cells were found to vary substantially in size and cytology. In low density areas of the retina, ganglion cells typically have cytoplasm with well developed Nissl substance, a distinct, pale nucleus, and a large nucleolus. In high density areas of retina, ganglion cells are small, densely staining, and gliaform. The average minimum proportion of ganglion cells in the ganglion cell layer is 75--80% of total profiles. No more than five or six percent of profiles in the ganglion cell layer are neurons which do not send an axon into the optic nerve (displaced amacrine cells or intraretinal association cells). The ganglion cell layer of P. s. elegans can be divided into a number of regions on the basis of cell density. Isodensity maps constructed from Nissl-stained, wholemounted retinas indicate that there is an elongated region of high ganglion cell density, the visual streak, which extends from nasal to temporal retina and is oriented such that its long axis follows the horizontal axis of the eye. The streak is aligned with the externally visible iris line. Seen in cross-section, the ganglion cell layer in the streak is three to four cells thick; in nonstreak retina, ganglion cells form only a monolayer of somas. Ganglion cell density drops off more rapidly above the streak than below it. The temporal arm of the streak is both shorter and broader than the nasal arm. There is a peak in ganglion cell density at the midpoint of the streak, in the approximate center of the retina. Here, ganglion cell densities exceed 20,000 cells mm-2. The total number of ganglion cells in the retina is 350,000--390,000.

Recent evidence suggests that ganglion cell size and its regional variation may be an important feature of vertebrate retinas. Accordingly, we have examined Nissl-stained, whole-mounted Pseudemys scripta retinas to determine the soma size spectrum of ganglion cells at different retinal loci. Cell size histograms reveal that at any given point on the retina, a majority of ganglion cells are small (6-10 microns), and in peripheral samples there is some evidence for a second, larger size class (12-15 microns). Comparison of samples along the dorsoventral and nasotemporal axes suggests that there are two major trends in soma size variation. Along the dorsoventral axis, ganglion cell diameter increases sharply from the visual streak (6-7 microns, cf. Peterson and Ulinski, '79) to the dorsal and ventral periphery (9-10 microns). These changes reflect a tendency toward increased size for the entire distribution as well as a relative decrease in the frequency of small ganglion cells. This soma size variation is significantly correlated with changes in ganglion cell density. Along the nasotemporal axis, temporal ganglion cells are significantly larger than those at more nasal retinal loci. This difference reflects an overall increase in the size of ganglion cells in temporal retina and a small but significant increase in the percentage of neurons larger than 15 microns.

We have described a population of neurons in the retinal of a shark, Heterodontus francisci, which is precisely aligned within the inner plexiform layer (IPL) and which differs from neurons in the ganglion cell layer (GCL) in soma size and topographical distribution. GCL neurons are relatively small and form a horizontally oriented visual streak; IPL neurons are significantly larger and form a circular specialization in the far temporal retina. Thus, it appears that there are two distinct retinal specializations in Heterodontus: one subserving frontal vision and one which provides a panoramic view of the lateral visual field.

We have attempted to develop an objective, semiquantitative classification of fiber types in turtle neck and limb muscle using microphotometry and multivariate statistical techniques. We first stained serial sections for myosin adenosine triphosphatase (ATPase) (with acid and alkaline preincubation and without preincubation), NADH-diaphorase, and two glycolysis-associated markers, alpha-glycerophosphate dehydrogenase (alpha-GPDH) and glycogen phosphorylase A (GPA). This allowed us to characterize individual muscle fibers in terms of their contraction speed and metabolic properties. Next we used microphotometry to measure the optical density of the reaction product in each fiber, and we subjected the resulting optical density matrix to cluster and discriminant function analyses in order to assign fibers to groups (fiber types) and to determine which stains contribute most to the distinction between groups. As a control, we processed a well characterized mammalian muscle (rat sternomastoid) simultaneously. Our results suggest that both neck and limb muscle in Pseudemys can best be described as falling into three groups: 1) slow oxidative (SO) fibers; 2) fast oxidative glycolytic (FOG) fibers, with relatively high oxidative and glycolytic capacities; and 3) fast glycolytic (Fg) fibers, with low oxidative, low/intermediate alpha-GPDH, and high GPA activities. These three fiber types differ from like-named types in rat muscle both in the pH lability of their myosins and in their metabolic profiles.

As part of a project aimed at elucidating mechanisms of vestibulocollic control in the red-eared turtle Pseudemys scripta, we have calculated the planar relations of its semicircular canals using principal-components analysis. This information is prerequisite to understanding the pattern of canal activation that is set up by head movement of any spatial form. In addition, we have developed a method for monitoring canal orientation in an awake, behaving animal, and we have used this technique to assess canal position in resting turtles. Our results indicate that ipsilateral canals in Pseudemys are not mutually orthogonal, nor are complementary canals precisely coplanar, although they approach this idealized condition more closely than do the canals of several other vertebrates for which quantitative data exist. One significant departure from the perfectly orthogonal configuration is that both verical canals are rotated slightly toward the frontal plane; thus, Pseudemys should be somewhat more sensitive to head roll than to head rotation in other planes. Radiographic analyses of awake, resting turtles indicate that the anterior interparietal suture is held aligned with the earth horizontal and midsagittal plane. The horizontal canal is pitched up (open anterior) 3-4 degrees relative to the earth horizontal.

Regional variation in the composition of the ganglion cell layer is a characteristic feature of vertebrate retinas. This suggests that corresponding patterns of regional variation might be expected in CNS structures which receive a direct, topographic projection from the retina. To investigate this possibility, I studied the retinotectal projection in an iguanid lizard (Dipsosaurus dorsalis), which shows a marked degree of regional variation in its retinal ganglion cell layer. Results of these experiments indicate that in D. dorsalis the optic tectum is differentiated horizontally into five regions which can be distinguished on the basis of cytoarchitecture and the patterning of retinal afferents, as visualized with silver degeneration and autoradiographic tracing techniques. These five areas are named according to their location: 1) Ventral Rim, 2) Lateral Wall, 3)Dorsolateral Area, 4) Dorsal Wall and 5) Medial Rim. Following enucleation, the Medial and Ventral Rim are characterized by fascicles of coarse degenerated fibers in layers 13, 12, and 9, by a relative absence of fine particulate debris in reduced silver material, and by the absence of silver grains in autoradiographs following intraocular injections of tritiated proline. They differ in the density of large calibre fibers and in the temporal course of degeneration. In the Lateral Wall the cellular layers are best differentiated, and nine retino-recipient layers can be distinguished below the stratum opticum. Degeneration is particularly heavy over the plexiform layers 9, 11, and 13. There is a superficial to deep gradient of degeneration density and of silver grains within layers 13 and 11. In the lower half of layer 11, argyrophilic debris and silver grains are aligned in vertical columns. The Dorsolateral Area is a circular region which covers the dorsolateral convexity of the tectum just caudal to mid tectal levels. It is characterized by a relatively low density of degenerated fibers in layers 13, 12, and especially in layer 9. Finally, the Dorsal Wall is similar to the Lateral Wall in the laminar distribution of degeneration and silver grains, except that there is an additional band of fine debris over layers 12 and 13 which is not visible in the Lateral Wall. Rostrally, the Dorsal Wall is greatly expanded (the Dorsal Expansion) and in this region a well developed plate of neurons lies immediately subjacent to the stratum opticum. This is the only tectal area which shows substantial degeneration at short survival times. These data suggest that in D. dorsalis there is horizontal variation in the morphology and patterning of retinal ganglion cell axons to the superficial tectal layers. Since Dipsosaurus retina exhibits a substantial degree of regional variation in the ganglion cell layer, and since the retina projects topographically onto the tectum, it is suggested that regional variation in the optic tectum may reflect regional variation in the ganglion cell layer of the retina.