We examined the bullfrog\u27s saccule using light and scanning electron microscopy. We found no evidence of a striola. Type A hair cells were not only distributed peripherally, but also throughout the central macula, though far less frequently than the dominant type D. We distinguished two primary hair cell types, which corresponded to the ciliary patterns: type A cilia are associated with short, conical hair cells, and type D cilia are associated with long, cylindrical hair cells. Each displays at least one subtype, which may represent developmental precursors. The otolithic membrane is crisscrossed with tunnels and topped with statoconia

Cohen, Glenn M.

Homick, Jerry

Reschke, Millard

English

Embry-Riddle Aeronautical University

The Space Congress® Proceedings 1982 (19th) Making Space Work For Mankind Apr 1st, 8:00 AM A Study in Motion Sickness: Saccular Hair Cells in the Adult Bullfrog. Glenn M. Cohen Dept. of Biological Sciences, Florida Institute of Technology Millard Reschke Neurophysiology Laboratory, Johnson Space Center, NASA Jerry Homick Neurophysiology Laboratory, Johnson Space Center, NASA Follow this and additional works at: https://commons.erau.edu/space-congress-proceedings Scholarly Commons Citation Cohen, Glenn M.; Reschke, Millard; and Homick, Jerry, "A Study in Motion Sickness: Saccular Hair Cells in the Adult Bullfrog." (1982). The Space Congress® Proceedings. 2. https://commons.erau.edu/space-congress-proceedings/proceedings-1982-19th/session-8/2 This Event is brought to you for free and open access by the Conferences at Scholarly Commons. It has been accepted for inclusion in The Space Congress® Proceedings by an authorized administrator of Scholarly Commons. For more information, please contact commons@erau.edu. A STUDY IN MOTION SICKNESS: SACCULAR HAIR CELLS IN THE ADULT BULLFROG-Dr. Glenn M. Cohen Dept. of Biological Sciences Florida Institute of Technology Melbourne, Florida 32901Dr. Millard Reschke and Dr. Jerry HomickNeurophysiology LaboratoryJohnson Space Center, NASAHouston, Texas 77058ABSTRACTWe examined the bullfrog's saccule using light and scanning electron microscopy. We found no evidence of a striola. Type A hair cells were not only distributed pe­ ripherally, but also throughout the central macula, though far less frequently than the dominant type D. We distinguished two primary hair cell types, which corresponded to the ciliary patterns: type A cilia are associated with short, conical hair cells, and type D cilia are associated with long, cylindrical hair cells. Each displays at least one subtype, which may represent developmental precursors. The otolithic membrane is crisscrossed with tunnels and topped with statoconia.INTRODUCTIONNumerous crewmen in previous American and Russian space flights have experienced motion sickness upon exposure to conditions of weightlessness. During these episodes, the performances of the severely afflicted crewmen seriously deteriorated. Although the etiology of motion sickness in space remains speculative, it is known that the vestibular apparatus, located within the inner ear, supplies the sensory input for eliciting the syndrome. According to recent interpretations, motion sickness may be caused by unusual patterns of ves­ tibular stimulation, which results in the sending of mismatched or conflicting inputs to the central nervous system. The ves­ tibular apparatus contains two divisions: the otolithic organs and the semicircular canal endorgans. The otolithic organs are believed to exert the primary influences for provoking motion sickness and the semi­ circular canals the secondary.The bullfrog is serving as the animal model for assessing functional activities of the otolithic and canal endorgans. The exper­ iments are conducted by recording from nerve branches of the saccule and hori­ zontal canal endorgan under static and dynamic conditions. The saccule, along with the smaller utricle, are two separate sense organs that comprise the otolithic organs; the horizontal canal is one of the three semicircular canals. The value of the nerve recordings, while important in them­ selves, is enhanced when correlated with the structure of the sensory organs. For example, the hair cells, the sensory re­ ceptors in the otolithic and canal end- organs, differ in sensitivity, spatial ori­ entation and temporal responsiveness. By de­ termining the hair cell populations and distributions within the organ, it becomes possible to associate patterns of nervous activity with the stimulatory mode of the hair cells.Hillman and Lewis (1971) and Lewis and Li (1973, 1975) identified types A and D ciliary patterns on hair cells in the bull­ frog's saccule, based solely upon surface morphologies. They reported that the type A, which are present in small numbers, are distributed primarily on the macular periphery, whereas type D, which are far more numerous, fill the central macula. In the present study we examined the saccule with regard to: (1) the distribution of hair cells within the macula; (2) the correspondence between ciliary patterns and hair cell types; and (3) the rela­ tionships between both the otolithic mem­ brane and the underlying hair cells and overlying statoconia. Portions of this data have been previously presented (Cohen, et al., 1978).8-57MATERIALS AND METHODSAdult bullfrogs (Rana catesbeiana) were an­ esthetized with tricaine methanesulfonate (MS-222). The labyrinth was approached dorsally. The skulls of young adults, which are largely cartilaginous, were relatively easy to penetrate but those of older adults, which are bony, presented more surgical difficulties. Upon removal, the inner ears were immediately immersed in cold (4 C) 2% glutraldehyde that was buffered with 0.1M phosphate or cacodylate contain­ ing 1-2 mM Ca and postfixed in buffered 1% osmium for one hour. Because stronger so­ lutions caused considerable cellular shrink­ age, their use was discontinued.The otolithic membrane was removed most sat­ isfactorily by first mechanically cleaning the surface with forceps and then flushing the saccular surface with a fine stream of fluid forced from a syringe. Mechanical cleaning alone was not successful. At first commercial mouthwashes were employed to clean the macular surfaces because they contain surfactants that might have dis­ lodged the otolithic membrane. However, their use was discontinued when we found that soaking not only damaged the surface structures but also did not facilitate the removal of the otolithic membrane. The macula was identified as a slightly denser area within the central saccule. After post- fixation, the macula was clearly visible as a dark spot; nerve fibers coursed beneath. Specimens were dehydrated in a graded series of ethyl alcohols (20, 50, 75, 90, 95, 1001) and then critically-point dried in C02, coated with a gold-palladium vapor using an Edwards E12E vacuum coater and ex­ amined in an ETEC Auto-scan scanning elec­ tron microscope at several accelerating voltages. Original magnifications of micro­ graphs ranged from 500 to 10,000. For light microscopy, specimens were embedded in epoxy (Araldite 502) and sectioned on an ultra- microtome (Sorvall MT-1) equipped with glass knifes. The sections were stained with either toluidine blue (Dawes, 1979) or para- phenylenediamine (Estable-Puig, et al., 1956), then examined with an Olymus microscope under bright field and phase microscopy.Fixation. Glutaraldehyde, when used as the sole fixative, did not satisfactorily pre­ serve the integrity of delicate surface structures. As a result, microvilli col­ lapsed and disappeared unless the tissue was first stiffened in osmium tetroxide, after which the microvilli formed a dense carpet.Ciliary Flexibility. When the macular sur­ face was swept with a fine brush made from human hair in order to remove otolithic debris, the stereocilia snapped off at their necks, but the kinocilia generally remained intact. The differences in flexibility between the two ciliary forms are attribut­ able to their proteinaceous cores. The stereocilia contain longitudinal arrays of actin filaments, whereas the kinocilia are filled with paraxially arranged micro- tubules.Hair Cells. The macula (sensory epithelium) lies in a shallow basin that is encir: .ed by a slightly elevated rim. There waj no evidence of a striola or any topographic contours that are characteristic of its mam­ malian counterpart. The hair cells are flanked by supporting cells. The two cell types are sealed together at their apical ends by tight junctions. Lewis and Li (1975) identified two distinctive kino- and stereociliary patterns in the bullfrog's saccule, based upon surface morphologies, findings which we have largely confirmed (Cohen, et^ a.1., 1978). However, we noted that type A cilia were not only distri­ buted in the macular perimeter but also mingled throughout the central macula, albeit far less frequently than the dom­ inant type D. The type A displays re­ latively short stereocilia and a kinocilium that is about three-fold longer (Fig. 1). The subtype exhibit slightly longer stereo- cilia; the subtype exhibits a kinocilium that is taller than the stereocilia. Kin­ ocilia from both types A and D have large bulbous terminals at their apical ends (Fig. 1). However, the bulbous terminals of type A hair cells are usually torn off during the removal of the otolithic membrane. Their bulbous terminals are unusually susceptible because they tower above the smaller stereocilia and therefore lack the protection afforded the type D, in which the shorter kinocilium is tucked in at the side of the domed ciliary bundle.Upon examining semithin (ly) sections, we distinguished two primary hair cell types; the short, conical type, and the long, cy­ lindrical type. Each displays subtypes which in the adult bullfrog may represent developmental precursors. In order to combine the findings from light and scanning electron microscopy, the macular surface gently tugged with forceps to separate cells along their seams and expose a lateral face. When viewed with SEM, the conical and cylindrical cell types, cor­ responded to types A and D ciliary patterns, respectively (Fig. 2). Nonetheless, three factors complicate this assignment. First,8-58hair cells located at macular periphery are smaller than their counterparts in the central macula. Second, the hair cell bodies at opposite sides of the periphery tilt sharply and centripetally, but acquire a vertical alignment a short distance away. However, the ciliary orientation seem un­ affected by the tilt of the hair cell body. Third, kino- and stereociliary heights change from the periphery to the central macula.Otolithic Membrane. The otolithic membrane is composed of two major components, the gelatinous layer and the statoconia. The gelatinous layer covers the macula in the living frog. However, it is very difficult to preserve the original dimensions and spatial relationships of the gelatinous layer because of its severe shrinkage. As a re­ sult, the gelatinous layer of preserved specimens often loses its precise align­ ment with the underlying ciliary bundles. The shrinkage is more severe peripherally than centrally. In addition, as the layer lifts up, it tears and stretches the thin veiled meshwork that connected the lumenal surfaces of the supporting cells to the gelatinous layer. The gelatinous layer is crisscrossed with anastomosing tunnels. The undersurface formed domes over the ciliary bundles and often enlarged in bulbous cham­ bers. When viewed from beneath or from above in partially cleaned specimens, the gela­ tinous layer vaguely resembled a honeycomb but clearly lacked its geometric symmetry.Statoconia. Saccular statoconia vary in size and shape depending upon their dis­ tribution within the otolithic membrane. Because most of the otolithic membrane was removed in order to expose the under­ lying hair cells, the normal statoconial arrangement was lost: the largest stato­ conia lie on the bottom and the smallest on top. Instead, only statoconia of about the same sizes remained (Fig. 3). However, lodged within the gelatinous layer, smaller "internal" statoconia rested immediately above the ciliary dome and filled the upper portions of the tunnel (Fig. 4). These "internal" statoconia did not directly touch the ciliary bundles, which were shielded by thin veils of fibrous material and by wedges of the dense gelatinous layer.DISCUSSIONLewis and Li (1973, 1975) identified two ciliary types, the A and D, in saccular hair cells of the bullfrog, based upon surface morphologies. We have confirmed and extended their findings. First, wefound the type A hair cells were not only distributed in the macular periphery but also in the central macula, though they were less common than the dominant type D. Second, the type A kinocilia bore bulbous terminals, which were usually lost during preparative procedures. Third, we were able to match ciliary types with hair cell bodies in most instances. For example, the type A cilia were associated with the short, conical hair cells. The morphology of the conical hair cells changed between the periphery and central macula. At the periphery, these hair cells were centri­ petally tilted and possessed the longest kinocilia and shortest sterocila (Fig. 1), However, central conical hair cells were larger and vertically aligned. Their kinocilia were shorter and steriocilia were taller, resulting in a resemblance to type D. On the other hand, the type D cilia were associated with tall, cylindrical hair cells. The latter also varied in size and shape. Thus, the peripheral cylin­ drical hair cells, in addition to their centripetal tilt, were short and bulbous. They gradually lengthened and assumed a more cylindrical shape and vertical align­ ment as they approached the central macula. However, we are unable to ascribe the lineage of several intermediates which cluster around the cylindrical hair cells, and bear type D cilia. Although they seem to be transitional forms, they also might be unrelated. Fourth, we do not believe that type A are developmental pre­ cursors that transform into the dominant type D during macular growth (Lewis and Li, 1973), though the evidience is admittedly suggestive. The fact that conical hair cells occurred throughout the central macula of the adult bullfrog without having undergone major morphological modification indicates that they are neither developmental precursors to type D nor are they develop- mentally arrested. The ambiguity results from the typing of hair cells based upon surface morphologies alone. Because A and D ciliary morpholgies differ at the per- phery but resemble each other in the central macula, they must be matched with their respective hair cell bodies to ensure ac­ curate identification.The gelatinous layer of the bullfrog's otolithic membrane is thicker and denser than the laboratory mammal's (Lim, 1973, 1979). It also differs by the presence of a network of tunnels crisscrossing through it and extending downward to the hollow domes of Its undersurface, thereby providing the opportunity for fluid movement through the gelatinous layer. We do not functional significance of this structural relationship. On its undersurface,8-59gelatinous layer overlies the hair and sup­ porting cells. The ciliary bundles extend upward into the hollowed domes. Although the correspondence is distorted in fixed specimens because of shrinkage, the cili­ ary bundles fit loosely rather than snugly into the large hollowed domes. The ciliary bundles do not seem to touch the ceilings of the domes. A thin meshwork or veil fills the space between the ciliary bundles and their domes. Dohlman (1971) described the loose anchoring of the cilia by fibrous strands. Although the sizes and shapes of the domes are specific for ciliary types, they do not directly correspond to the configuration of the ciliary bundles.The statoconia are essential for proper functioning of the otolithic organs (Lim, 1973). The bullfrog's saccular stato­ conia vary in size and shape depending upon their location. The smaller statoconia lie on top and the larger ones below them. (Fig. 3). We were intrigued by the pre­ sence of statoconia within the gelati­ nous layer. These smaller, "internal" statoconia filled the top of the domes (Fig. 4) but did not appear to touch the underlying ciliary bundles, which were pro­ tected by the veil and by wedges of the denser gelatinous layer. The spatial se­ paration of the statoconia from the ciliary bundles was obvious in sectioned specimens but was ambiguous when viewed under SEM because of the introduction of preparative artefacts.In short, the analysis of saccular struc­ ture will be combined with neurophysio- logical analysis to understand its con­ tributions to balance and motion sickness.ACKNOWLEDGEMENTSWe want to thank Ms. Patricia Ryan for her technical assistance,One of us (G.M.C.) conducted this researchas a recipient of a NASA Summer Faculty Research Fellowship at. the Johnson Space Center, Houston, Texas.REFERENCESCohen, G. M. , M. Reschke, and P. Ryan. 1978. Saccular hair cells in the adult bullfrog Rana catesbeiana. Midwinter Res. Meeting Assoc. Res. Otolarynology Abstracts. Pg. 4.Dawes, C. J. 1979. Biological Techniques for Transmission and Scanning Electron Microscopy. Ladd ResearchIndustries, Inc., Burlington, VT. Pg. 207-208.Dohlman, G. F. 1971. The attachment of the cupulae, otolith, and tectorial membranes to the sensory cell area. Acta Otolaryngol. 71,89-105.Estable-Puig, J. F., W. C. Bauer, and J. M. Blumberg. 1965. Para- phenylenediamine staining of osmium fixed, plastic embedded tissue for light and phase microscopy. J. Neuropathol. Exp. Neurol. 24, 531-535.Hillman, D. E. and E. R. Lewis. 1971.Morphological basis for a mechanical linkage in otolithic receptor trans- duction in the frog. Science 174 , 416-419.Lewis, E. R. and C. W. Li. 1973, Evidence concerning the morphogenesis of saccular receptors in the bullfrog (Rana catesbeiana) , J. Morphol. 139, 351-363.Lewis, E. R. and C. W. Li. 1975. Hair cell types and distributions in the otolithic and audirotyr organs of the bullfrog. Brain Res. 83, 35-50.Lim, D. J. 1973, Formation and fateof the otoconia. Ann. otol. 83, 23-35.Lim, D. J. 1979. Fine morphology of the otoconial membrane and its relation­ ship to the sensory epithelium. SEM/79, ITT Research Institute, Chicago, IL. Pgs. 929-938.8-60Fig. 1. Different ciliary patterns at ma- cular periphery. Type A (A) at macular margin list its bulbous kinociliary ter­ minal. Type D (D), the most common, lie in­ side the margin. Intermediate (I) types may be precursors to other types or repre­ sent a separate lineage. X3,780.Fig. 3. Statoconia on bottom of mound. The statoconia are pointed at both ends as a result of the sharply angled 'sides converg­ ing. The statoconia vary little in size at this location. Statoconia are smoothly sur­ faced in fresh preparations but these show etching from fixation. X2,840.Fig. 2. '.uaceral view of hair cells in cen­ tral macular. The long, cylindrical hair cell bodies (C) correspond to the type D ciliary pattern. The short, bulbous hair cells (I) match the intermediate ciliary pattern of Fig. 1. A human red blood cell (R) provides a size perspective. X3,Q25.Fig. 4. "Internal" statoconia. These sta­ toconia fill the tunnel that lies directly above the ciliary dome. The internal sta­ toconia are smaller than those comprising the statoconial mount. They are also etched from fixation. X6,62Q.8-61

A Study in Motion Sickness: Saccular Hair Cells in the Adult Bullfrog.

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A Study in Motion Sickness: Saccular Hair Cells in the Adult Bullfrog.

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