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Longitudinal and radial differences in the subsurface cisternal system in the gerbil cochlea
llCd IIEf KI4 Hearing Research 84 (1995) 12-18
ELSEVIER
Longitudinal and radial differences in the subsurface cisternal system in the gerbil cochlea Carrie Lutz, Laura Schweitzer
*
Department of Anatomical Sciences and Neurobiology, Unicersity of LouisL,ille School of Medicine, Louis'lille, KY40292, USA
Received 12 August 1994; revised 9 December 1994; accepted 20 December 1994
Abstract
Many features of cochlear anatomy vary systematically radially and longitudinally within the organ of Corti. Thcre is limited evidence that along the longitudinal axis of the cochlea the thickness of the subsurface cisternal system in the outer hair cells (OHCs) changes. Similarly a radial gradient may exist. The thickness of the subsurface cisternal system in OHCs was measured in gerbils to determine if there are differences between the three rows of OHCs and in OHCs in different locations along the length of the organ of Corti. The results suggest that there is a longitudinal as well as a radial gradient of subsurface cisternal system thickness. These gradients are the inverse to those for efferent innervation of OHCs. It is possible that these differences may contribute to the increased susceptibility to trauma and ototoxic compounds characteristic of the innermost and basalmost OHCs. Keywords: Gradients; Outer hair cells; Subsurface cisternae; Ototoxicity; Electromotility
1. Introduction
Two primary axes can be defined in the organ of Corti. The first, and most often discussed, is the longitudinal axis (apico-basal gradient). It is along this axis that the tonotopic map within the cochlea is disposed. Second, there is a radial dimension spanning the cochlea from the modiolus (inner) to the lateral bone (outer). Weaver and Schweitzer (1994) suggested that within outer hair cells (OHCs) along the length of the organ of Corti the thickness of the subsurface cisternal system, a system of stacked membrane bound cisternae, may vary systematically. The thickness of the subsurface cisternal system has not been well investigated and has not been compared across the radial axis. This paper examines the subsurface cisternal system in outer hair cells of the gerbil to determine whether there are differences in the thickness of the system in the three rows of OHCs or at different locations along the length of the organ of Corti. The subsurface cisternae are located just inside the
* Corresponding author. Fax: (502) 852-6228. 0378-5955/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0378-5955(95)00008-9
lateral plasma membrane of OHCs and appear either as a tubular network or as flattened vesicles (Harada et al., 1986; Forge, 1989; Nagasawa et al., 1991). Up to six layers of cisternae are found in the guinea pig and the gerbil (Forge, 1989; 1991; Forge et al., 1993; Weaver and Schweitzer, 1994). There is limited evidence that the thickness of the subsurface cisternal system varies along the length of the organ of Corti so that in the longer hair cells of the apical cochlea there are more layers of cisternae (Hallworth et al., 1993; Weaver and Schweitzer, 1994). Within each cell, the subsurface cisternae when present in more than one layer, decrease in number from the subcuticular area toward its base. The greatest number of cisternal layers are found above the nucleus near the cuticular plate. Near the nucleus, there is only a single layer (Smith and Dempsey, 1957; Furness and Hackney, 1990; Weaver and Schweitzer, 1994). In the human, cat, bat and opossum the most peripheral layer of the subsurface cisternae is continuous with subsynaptic cisterns (Arnold et al., 1990; Kimura, 1975). This suggests that, in some species, the subsurface cisternae near the lateral walls of OHCs can be influenced by efferent input at the base of the cell (Kimura, 1975; Arnold et al., 1990).
c. Lutz, L. Schweitzer/Hearing Research 84 (1995) 12-18
Layers of the subsurface cisternae vary in thickness from 30 to 50 nm depending on the species (Ekstrom von Lubitz, 1981; Saito, 1983; Forge, 1989). In species that contain more than one layer, 15 to 50 nm spaces separate the cisternal layers (Smith and Dempsey, 1957; Ekstrom yon Lubitz, 1981; Saito, 1983; Forge, 1989). A dense material like that seen in rough endoplasmic reticu]um is present in the lumen of the subsurface cisternae (Smith and Dempsey, 1957; Ekstrom von Lubitz, 1981; Saito, 1983; Forge, 1991). Mitochondria are closely associated with the innermost cisternal layer, which often has ribosomes attached (Kimura, 1975; Saito, 1983). While mitochondria and the innermost cistern are in close apposition to one another they are not attached (Weaver and Schweitzer, 1994). The function of the subsurface cisternae system is unknown. An interesting observation made by Bohne (1973) was that following exposure to loud noise, the OHCs of chinchillas contained additional layers of cisternae. These 'overexposed' OHCs contained as many as thirty layers of cisternae and Bohne speculated that the additional layers might play some protective role. Since ototoxic effects occur and O H C susceptibility to noise is distributed along known longitudinal and radial gradients, a better understanding of gradients in the morphology of the subsurface cisternae system in OHCs is sought to clarify their relationship to these functional gradients.
13
2. Materials and Methods
2.1. Animals
Cochleae from six adult Mongolian gerbils (Meriones unguicalatus, Tumblebrook Farm, West Brook, MA) were used in this investigation. The care and use of animals used in this study were approved by the University of Louisville Animal Care and Use Committee. The animals were injected with an overdose of sodium pentobarbital (Nembutal - 100 mg/kg), perfused through the ascending aorta with Karnovsky's fixative (Karnovsky, 1965) supplemented with 2% tannic acid to enhance membrane visualization, and then decapitated. Immediately thereafter the cochleae were dissected free, the stapes was removed, the round window opened and a small hole was placed in the apex of each cochlea. Karnovsky's fixative supplemented with 4% tannic acid tG further enhance membrane staining was then gently perfused through the oval window using a small syringe. The cochleae were kept in fixative for 2 h and then decalcified in 10% ethylenediamine-tetraacetic acid (EDTA) with gentle agitation for 24 to 48 h at 4° C. The E D T A solution was changed every 12 h during decalcification. The progress of decalcification was assessed by gently pricking the superficial bone with an insect needle. Decalci-
Fig. 1. Outer hair cell and its subsurface cisternal system. (A) Electron micrograph (3 700 X ) of an upper middle turn [42%] OHC. Arrow points to lateral plasma membrane near the subsurface cisternae. (B) Electron micrograph of the same section (20350 x ) taken midway between the cuticular plate and cell nucleus. Arrow indicates the same location as in (A). This is the thickest portion of the subsurface cisternal system in this micrograph, the location where measurements for this study were taken.
14
C. Lutz, L. Schweitzer / Hearing Research 84 (1995) 12-18
fied cochleae were stored in 50% ethanol at 4° C for up to 30 days. For further processing, decalcified cochleae were rinsed three times in a buffered 6% sucrose solution, placed in 2% osmium tetroxide in the buffered sucrose for 2 h and then rinsed with 0.1M sodium acetate
buffer. Cochleae were divided into halves by a midmodiolar cut through a plane passing from the apex of the cochlea to a point between the oval and round windows. The half cochleae were stained with 0.5% uranyl acetate in 0.1M sodium acetate buffer, dehydrated through a graded series of methanol and em-
Fig. 2. Electron micrographs (24 300 x ) demonstrating the longitudinal and radial gradients of subsurface cisternal system thickness. Panels on the left are first (inner) row OHCs, panels on the right are in the third (outer) row. Panels on the top are from the lower middle turn [65%] and panels on the bottom are from the basal turn [90%]. (A) Subsurface cisternal system in a first row OHC of the lower middle turn [65%], 0.24/xm thick. (B) Subsurface cisternal system in a third row OHC of the same section as panel (A) (lower middle turn [65%]), 0.33 ~ m thick. (C) Subsurface cisternal system in the first row of OHCs of the basal turn [90%], 0.15/zm thick. (D) Subsurface cisternal system in the third row of OHCs of the basal turn [90%], 0.25/xm thick.
c. Lutz, L. Schweitzer/ Hearing Research 84 (1995) 12-18 bedded in L X l 1 2 resin (Ladd Research Industries, Inc., Burlington, VT). Each half was embedded with the midmodiolar face down into the flat end of a capped Beem capsule. This ensured that the cut surface of the cochlea formed the cutting face of the finished blocks. The embedding resin, L X l l 2 , was polymerized by heating for 24 h at 30 ° C, 24 h at 45°C and 24 h at 60 ° C.
A
Subsurface Cisternae Thickness Longitudinal Analysis .30 .25
o
~-
c.)
.20 \\ .15
=
2.2. Tissue sampling
15
\5
.10
u J~
Regions lying at approximately 42%, 65% and 90% (+/5 - 6 % error due to the distance between the round and oval windows) of the distance along the length of the cochlea were identified and studied in the resulting sections. These regions are referred to as the upper middle turn, the lower middle turn and the basal turn, respectively, in the remainder of this paper.
~-
.05 0
B
42~ 65% 90~ a p i c a l to basal d i s t a n c e
Subsurface Cisternae Thickness Radial A n a l y s i s .30
2.3. Microscopy .25
For light microscopy, 1 - 2 / z m sections were cut with a glass knife, mounted on slides, stained with 2% toluidine blue, and then examined to locate the appropriate regions for electron microscopic study. For electron microscopy, sections approximately 80 nm thick, adjacent to the sections studied with the light microscope, were cut with a diamond knife. The sections were supported with copper grids and stained with 4% uranyl acetate, in 50% ethanol, for 5 min, rinsed in d H 2 0 , then stained with Reynolds lead citrate for 1 rain. Sections were examined and pictures were taken using a Philips 201 electron microscope.
2.4. Morphometric Analysis In OHCs, the thickness of the subsurface cisternal system was measured from the innermost leaflet of the Subsurface Cisternae Thickness Longitudinal and Radial Analyses
g .2
..........iii i 2 ¢.J
.1
E
4~:
6d:
9~: --
Z apical to basal distance
Fig. 3. Subsurface cisternal system thickness, means and standard errors of the means. In this graph, data for individual hair cell rows (1, 2, 3) in individual locations (basalmost [90%], upper middle [42%] and lower middle [65%] turns) are shown.
g L
.20 .15
O3
c
.10
u
~-
.0b J
J
L
2 3 outer hair cell row
Fig. 4. Subsurface cisternal system (SSC) thickness, means and standard errors of the means. (A) Longitudinal analysis. The SSC is significantly thinner in the basalmost turn [90%] than in OHCs in the upper middle [42%] and lower middle [65%] turns. (B) Radial analysis. The SSC is significantly thicker in the third row of OHCs than in the first row of OHCs. The thickness of the SSC in the second row lies between that of the other two rows and is not significantly different from either. innermost cisternae to the lateral plasma membrane on electron micrographs (24300 × ) of longitudinal sections through the center of each O H C (Fig. 1). Since the thickness of the subsurface cisternal system varies along the lateral plasma membrane, high magnification micrographs were always taken at a point midway between the cuticular plate and the cell nucleus, where the system is generally thickest. Measurements were taken at the thickest point of the subsurface cisternal system within each micrograph. Micrographs were obtained from hair cells in each row, in each of the three longitudinal locations mentioned above. The measurements were obtained in one cochlea from each of the six adult gerbils.
2.5. Statistical Analysis These data were analyzed with a two-way A N O V A (radial x longitudinal locations), subsequent one way
C. Lutz, L. Schweitzer / Hearing Research 84 (1995) 12-18
16
A N O V A s and individual comparisons were analyzed with Fisher's post hoc tests (Siegel, 1956).
upper middle turn [42%], it is still almost twice as thick as that in the basal turn [90%].
3.2. Radial Differences 3. Results Our data show that there are significant differences in the thickness of the subsurface cisternal system of OHCs at different longitudinal locations and radially between those of the three rows of OHCs. Thus, both radial and longitudinal gradients of thickness of the subsurface cisternal system were found (Figs. 2, 3, 4).
3.1. Apical to basal differences There is a significant difference in the thickness of the subsurface cisternal system between OHCs in different turns of the cochlea (F = 24.14; d.f. = 2,45; P < 0.001) (Fig. 4A). The subsurface cisternal system is significantly thinner in the basal turn [90%; 0.13/xm + 0.02] than in the two more apical turns. No significant difference was found in the thicknesses in the subsurface cisternal system in OHCs of the lower middle [65%] and upper middle turns [42%] of the cochlea, which were, on average, 0.23/xm ( + 0.02) and 0.27/xm (+_ 0.02) thick, respectively. The subsurface cisternal system in the upper middle turn [42%] is approximately two times thicker than in the basal turn [90%]. Although the subsurface cisternal system in the lower middle turn [65%] is somewhat thinner than in the
IGLRADE INT LONGTIUDG N IR ALADE INT RADA ThcikneofsthseSubsurfaCc~esternae apeX~base(~ ~ ~ ~ apex
EffereInntnervatol n I~
Fig. 5. Schematic of longitudinal and radial gradients found within the cochlea. (Top) Thickness of the SSC. The SSC is thicker in the apex than in the base and is also thicker in the third row of OHCs than in the first. (Bottom) Efferent innervation. The base receives more efferent innervation than the apex and the first row of OHCs receives more efferent innervation than the third row.
A radial difference also exists between the thickness of the subsurface cisternal system in the different rows of OHCs (F = 5.946; d.f. = 2,45; P < 0.01) (Fig. 4B). This difference reached significance only for OHCs in row one (innermost; 0.18/xm +_ 0.02) versus row three (outermost; 0.25/xm + 0.02). These were not different than for OHCs in the middle row (0.20 /xm + 0.02). On average, the thickness of the system in the third row exceeded that in the first row by 36%. This difference was greatest in the most basal turn [90%] where the subsurface cisternal system in OHCs in the first row was very thin and consisted of only a single layer of cisternae which was on average only about 0.09 /am thick, as compared to the third row at the same longitudinal location, which was on average twice as thick.
4. Discussion The subsurface cisternal system in the OHCs of the gerbil have both a longitudinal (frequency-related) gradient and a radial (inner to outer) gradient of thickness (Fig. 5). The subsurface cisternal system is significantly thinner in the hair cells in the base of the cochlea as compared to the more apical regions and the system is significantly thicker in the third (outermost) row of OHCs as compared to the first (innermost) row. OHCs increase in height from the base of the cochlea to the apex and from the inner row to the outer row (Kimura, 1966) and thus the thickness of the subsurface cisternae may be related to size. There are other gradients found in the cochlea, such as in the efferent innervation, resistance to ototoxicity and response to overstimulation that have an interesting correlation to the gradients of the thickness of the subsurface cisternal system found within the cochlea (Fig. 5). As mentioned in the introduction, efferent innervation may be related to the subsynaptic cisterns which are continuous with the subsurface cisterns in OHCs in some species. In general, the efferent innervation to OHCs decreases from the base of the cochlea to the apex (Liberman et al., 1990; Dannhof and Bruns, 1993; Francis and Nadol, 1993). This is opposite to the thickness of the subsurface cisternal system which increases from the base to the apex of the cochlea. Thus, there is an increase in efferent innervation where there is a decrease in the thickness of the subsurface cisternal system, or stated differently, there is an inverse relationship between the thickness of the subsurface cisternal system and the amount of efferent innervation to the OHCs.
C. Lutz, L. Schweitzer /Hearing Research 84 (1995) 12-18
Just as there is a longitudinal gradient of efferent innervation to the OHCs, radial differences exists as well. There is a notable decrease in efferent innervation from the first (inner) row of O H C s to the third (outer) row of O H C s (Liberman et al., 1990; Nadol, 1990; Francis and Nadol, 1993). In addition, the largest terminals are found on the first row and the smallest on the third row (Xie et al., 1993), which may relate to the potentially greater influence that efferent innervation has on the first row of OHCs. This radial gradient of efferent innervation to the O H C s is opposite to the radial gradient of the subsurface cisternae. The third row of O H C s contains the thickest subsurface cisternal system and this corresponds to the row that has the least amount of efferent innervation. Therefore, an inverse relationship between efferent innervation and thickness of the subsurface cisternal system holds true both in the radial and longitudinal dimensions. Since a direct relationship exists between the amount of efferent innervation and the susceptibility to ototoxicity (Longitudinal gradients: Ylikoski et al., 1993; Forge and Richardson, 1993; Kiang et al., 1976; Radial gradients: Kaltenbach et al., 1992; Robertson, 1982; Liberman and Beil, 1979), an inverse relationship also exists between susceptibility to ototoxicity and the thickness of the subsurface cisternal system. In addition to the gradients already mentioned there is also a longitudinal gradient of electromotile properties in the O H C s (Brundin et al., 1989). O H C s along the length of the basilar m e m b r a n e display frequencyspecific sound induced electromotility. It has been suggested, therefore, that O H C s are 'tuned'. In addition, the force of O H C fast motility apparently differs between the turns of the cochlea. The O H C s in the fourth (most apical) turn of the guinea pig cochlea shorten more in response to equivalent stimulation than do cells in the third turn of the cochlea. Thus, the force generated by the apicalmost hair cells may be inferred to be greater (Gitter et al., 1993). The subsurface cisternae, perhaps in conjunction with the actin cytoskeleton, does seem to be associated with electromotility and is disrupted when electromotility ceases (Dulon et al., 1990; Holley and Kachar, 1992; Dieler et al., 1991). If the subsurface cisternae are necessary for motility, it is possible that the apicalmost hair cells, that have been suggested to generate a greater force, need a significantly thicker subsurface cisternal system than do those in the base of the cochlea. Alternatively, it is possible that the function of the subsurface cisternae is to sequester calcium along the lateral plasma membrane. Upon depolarization calcium enters the outer hair cell. Since the subsurface cisternal system is lined up so close to the plasma m e m b r a n e in the OHCs, it would seem possible that it functions to sequester calcium near the calcium sensitive potassium efflux channels. Mitochondria in the
17
adult O H C s are located just within the innermost cisternal layer and may further sequester calcium. Mitochondria are known to fulfill this function in other cells (Alberts et al., 1989). H a r a d a et al. (1993) noted that an O H C could hyperpolarize even in the presence of an extracellular calcium channel blocker. This finding suggests that calcium release may well come from intracellular stores, perhaps in the mitochondria or the subsurface cisternae. Pools of calcium may enhance the ability to activate potassium channels near the base of the OHC. Intracellular isoforms of the enzyme calcium activated ATPase has been localized by Schulte (1993) along the lateral plasma membrane, the location of the cisternae. Furthermore, labelling for calcium activated ATPase in the O H C was first noted on postnatal day 14, an age close to the onset of cochlear function, when the first layers of cisternae are forming (Weaver and Schweitzer, 1994). Calcium influx is known to have an adverse effect on neurons. Excitotoxicity, a p h e n o m e n o n underlying such diverse degenerative effects in the nervous system as epilepsy and anoxic brain injury (Rothman and Olney, 1987), is mediated by calcium influx. If the subsurface cisternae serves to sequester calcium to the area adjacent to the cell membrane, protecting the intracellular space from excess calcium, it follows that ceils with a thicker subsurface cisternal system in the apex should be better protected from this type of injury. Interestingly, the cisternae are also more fenestrated in hair cells in the base of the cochlea than those in the apex (Forge and Richardson, 1993) and this increased fenestration may also increase susceptibility to injury in basal hair cells.
Acknowledgements We would like to thank Drs. James B. Longley and Fred J. Roisen for their helpful comments on the manuscript. We would also like to thank Ms. Tina Cecil for her technical assistance. These experiments were supported by a grant from the Deafness Research Foundation.
References Alberts, B., Bray, D., Lewis, J., Raft, M., Roberts, K. and Watson, J. D. (1989) Molecular Biology of The Cell. Garland Publishing, New York. Arnold, W., Anniko, M. and Pfaltz, C. R. (1990) Functional morphology of the outer hair cells of the human: new aspects. Laryngo- Rhino-otologie 69, 177-186. Bohne, B. A. (1973) Anatomical correlates of temporary shift in threshold of hearing. Acoust. Soc. Am. 53, 292.
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Brundin, L., Flock, A. and Canlon, B. (1989) Sound-induced motility of isolated cochlear outer hair cells is frequency-specific. Nature 342, 814-816. Dannhof, B. J. and Bruns, V. (1993) The innervation of the organ of Corti in the rat. Hear. Res. 66, 8-22. Dieler, R., Shehata-Dieler, W. E. and Brownell, W. E. (1991) Concomitant salicylate-induced alterations of outer hair cell subsurface cisternae and electromotility. J. Neurocytol. 20, 637-653. Dulon, D., Zajic, G. and Schacht, J. (1990) Increasing intracellular free calcium induces circumferential contractions in isolated cochlear outer hair cells. J. Neurosci. 10, 1388-1397. Ekstrom von Lubitz, D. K. J. (1981) Subsurface tubular system in the outer sensory cells of the rat cochlea. Cell Tiss. Res. 220, 787-795. Forge, A. (1989) The lateral walls of inner and outer hair cells. In: J. P. Wilson and D. T. Kemp (Eds.), Cochlear Mechanisms: Structure, Function and Models, Plenum Press, New York, pp. 29-35. Forge, A. (1991) Structural features of the lateral walls in mammalian cochlear outer hair cells. Cell. Tiss. Res. 265, 473-483. Forge, A. and Richardson, G. (1993) Freeze fracture analysis of apical membranes in cochlear cultures: differences between basal and apical-coil outer hair cells and effects of neomycin. J. Neurocytol. 22, 854-867. Forge, A., Zajic, G., Li, L., Nevill, G. and Schacht, J. (1993) Structural variability of the sub-surface cisternae in intact, isolated outer hair cells shown by fluorescent labelling of intracellular membranes and freeze-fracture. Hear. Res. 64, 175-183. Francis, H. W. and Nadol, J. B., Jr. (1993) Patterns of innervation of outer hair cells in a chimpanzee: II. Efferent endings. Hear. Res. 64, 217-221. Furness, D. N. and Hackney, C. M. (1990) Comparative ultrastructure of subsurface cisternae in inner and outer hair cells of the guinea pig cochlea. Eur. Arch. Otorhinolaryngol. 247, 12-15. Gitter, A. H., Rudert, M. and Zenner, H.-P. (1993) Forces involved in length changes of cochlear outer hair cells. Eur. J. Physiol. 424, 9-14. Hallworth, R., Evans, B. N. and Dallos, P. (1993) The location and mechanism of electromotility in guinea pig outer hair cells. J. Neurophysiol. 70, 549-558. Harada, N., Ernst, A. and Zenner, H. P. (1993) Hyposmotic activation hyperpolarizes outer hair cells of guinea pig cochlea. Brain Res. 614, 205-211. Harada, Y., Sakai, T., Tagashira, N. and Suzuki, M. (1986) Intracellular structure of the outer hair cell of the organ of Corti. Scan. Elect. Microsc. II, 531-535. Holley, M. C. and Kachar, B. (1992) The cortical cytoskeleton and cell shape changes in mammalian outer hair cells. In: Y. Cazals, K. Hornet and L. Demany (Eds.), Auditory Physiology and Perceptions. Pergamon Press, New York, 27-33. Kaltenbach, J. A., Schmidt, R. N. and Kaplan, C. R. (1992) Tone-in-
duced stereocilia lesions as a function of exposure level and duration in the hamster cochlea. Hear. Res. 60, 205-215. Karnovsky, M. J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137A. Kiang, N. Y. S., Liberman, M. C. and Leving, M. D. (1976) Auditory-nerve activity in cats exposed to ototoxic drugs and high-intensity sounds. Ann. Otol. 85, 752-768. Kimura, R. S. (1966) Hairs of the cochlear sensory cells and their attachment to the rectorial membrane. Acta Otolaryngol. 61, 55-72. Kimura, R. S. (1975) The ultrastructure of the organ of Corti. Intl. Rev. Cytology 42, 173-222. Liberman, M. C. and Bell, D. G. (1979) Hair cell condition and auditory nerve response in normal and noise-damaged cochleas. Acta Otolaryngol. 88, 161-176. Liberman, M. C., Dodds, L. W. and Pierce, S. (1990) Afferent and efferent innervation of the cat cochlea: quantitative analysis with light and electron microscopy. J. Comp. Neurol. 301,443-460. Nadol, J. B., Jr. (1990) Synaptic morphology of inner and outer hair cells in the human organ of Corti. J. Elect. Microsc. Tech. 15, 187-196. Nagasawa, A., Harrison, R. V., Mount, R. J. and Harada, Y. (1991) Three dimensional intracellular structure of the cochlea using the A-O-D-O method. Scan. Microsc. 5, 747-754. Robertson, K. (1982) Effects of acoustic trauma on stereocilia structure and spiral ganglion cell tuning properties in the guinea pig cochlea. Hear. Res. 7, 55-74. Rothman, S. M. and Olney, J. W. (1987) Excitotoxicity and the N M D A receptor. Trends Neurosci. 10, 299-302. Saito, K. (1983) Fine structure of the sensory epithelium of guinea-pig organ of Corti: subsurface cisternae and lamellar bodies in the outer hair cells. Cell Tiss. Res. 229, 467-481. Schulte, B. A. (1993) Immunohistochemical localization of intracellular Ca-ATPase in outer hair cells, neurons and fibrocytes in the adult and developing inner ear. Hear. Res. 65, 262-273. Siegel, S. (1956) Non-parametric Statistics for the Behavioral Sciences. McGraw-Hill Inc., New York City, NY. Smith, C. A. and Dempsey, E. W. (1957) Electron microscopy of the organ of Corti. Am. J. Anat. 100, 337-367. Weaver, S. P. and Schweitzer, L. (1994) Development of gerbil outer hair cells after the onset of cochlear function: An ultrastructural study. Hear. Res. 72, 44-52. Xie, D. H., Henson, M. M., Bishop, A. L. and Henson, O. W. J. (1993) Efferent terminals in the cochlea of the mustached bat: Quantitative data. Hear. Res. 66, 81-90. Ylikoski, J., Pirvola, U., Moshnyakov, M., Palgi, J., Arumae, U. and Saarma, M. (1993) Expression patterns of neurotrophin and their receptor mRNAs in the rat inner ear. Hear. Res. 65, 69-78.
ELSEVIER
Longitudinal and radial differences in the subsurface cisternal system in the gerbil cochlea Carrie Lutz, Laura Schweitzer
*
Department of Anatomical Sciences and Neurobiology, Unicersity of LouisL,ille School of Medicine, Louis'lille, KY40292, USA
Received 12 August 1994; revised 9 December 1994; accepted 20 December 1994
Abstract
Many features of cochlear anatomy vary systematically radially and longitudinally within the organ of Corti. Thcre is limited evidence that along the longitudinal axis of the cochlea the thickness of the subsurface cisternal system in the outer hair cells (OHCs) changes. Similarly a radial gradient may exist. The thickness of the subsurface cisternal system in OHCs was measured in gerbils to determine if there are differences between the three rows of OHCs and in OHCs in different locations along the length of the organ of Corti. The results suggest that there is a longitudinal as well as a radial gradient of subsurface cisternal system thickness. These gradients are the inverse to those for efferent innervation of OHCs. It is possible that these differences may contribute to the increased susceptibility to trauma and ototoxic compounds characteristic of the innermost and basalmost OHCs. Keywords: Gradients; Outer hair cells; Subsurface cisternae; Ototoxicity; Electromotility
1. Introduction
Two primary axes can be defined in the organ of Corti. The first, and most often discussed, is the longitudinal axis (apico-basal gradient). It is along this axis that the tonotopic map within the cochlea is disposed. Second, there is a radial dimension spanning the cochlea from the modiolus (inner) to the lateral bone (outer). Weaver and Schweitzer (1994) suggested that within outer hair cells (OHCs) along the length of the organ of Corti the thickness of the subsurface cisternal system, a system of stacked membrane bound cisternae, may vary systematically. The thickness of the subsurface cisternal system has not been well investigated and has not been compared across the radial axis. This paper examines the subsurface cisternal system in outer hair cells of the gerbil to determine whether there are differences in the thickness of the system in the three rows of OHCs or at different locations along the length of the organ of Corti. The subsurface cisternae are located just inside the
* Corresponding author. Fax: (502) 852-6228. 0378-5955/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0378-5955(95)00008-9
lateral plasma membrane of OHCs and appear either as a tubular network or as flattened vesicles (Harada et al., 1986; Forge, 1989; Nagasawa et al., 1991). Up to six layers of cisternae are found in the guinea pig and the gerbil (Forge, 1989; 1991; Forge et al., 1993; Weaver and Schweitzer, 1994). There is limited evidence that the thickness of the subsurface cisternal system varies along the length of the organ of Corti so that in the longer hair cells of the apical cochlea there are more layers of cisternae (Hallworth et al., 1993; Weaver and Schweitzer, 1994). Within each cell, the subsurface cisternae when present in more than one layer, decrease in number from the subcuticular area toward its base. The greatest number of cisternal layers are found above the nucleus near the cuticular plate. Near the nucleus, there is only a single layer (Smith and Dempsey, 1957; Furness and Hackney, 1990; Weaver and Schweitzer, 1994). In the human, cat, bat and opossum the most peripheral layer of the subsurface cisternae is continuous with subsynaptic cisterns (Arnold et al., 1990; Kimura, 1975). This suggests that, in some species, the subsurface cisternae near the lateral walls of OHCs can be influenced by efferent input at the base of the cell (Kimura, 1975; Arnold et al., 1990).
c. Lutz, L. Schweitzer/Hearing Research 84 (1995) 12-18
Layers of the subsurface cisternae vary in thickness from 30 to 50 nm depending on the species (Ekstrom von Lubitz, 1981; Saito, 1983; Forge, 1989). In species that contain more than one layer, 15 to 50 nm spaces separate the cisternal layers (Smith and Dempsey, 1957; Ekstrom yon Lubitz, 1981; Saito, 1983; Forge, 1989). A dense material like that seen in rough endoplasmic reticu]um is present in the lumen of the subsurface cisternae (Smith and Dempsey, 1957; Ekstrom von Lubitz, 1981; Saito, 1983; Forge, 1991). Mitochondria are closely associated with the innermost cisternal layer, which often has ribosomes attached (Kimura, 1975; Saito, 1983). While mitochondria and the innermost cistern are in close apposition to one another they are not attached (Weaver and Schweitzer, 1994). The function of the subsurface cisternae system is unknown. An interesting observation made by Bohne (1973) was that following exposure to loud noise, the OHCs of chinchillas contained additional layers of cisternae. These 'overexposed' OHCs contained as many as thirty layers of cisternae and Bohne speculated that the additional layers might play some protective role. Since ototoxic effects occur and O H C susceptibility to noise is distributed along known longitudinal and radial gradients, a better understanding of gradients in the morphology of the subsurface cisternae system in OHCs is sought to clarify their relationship to these functional gradients.
13
2. Materials and Methods
2.1. Animals
Cochleae from six adult Mongolian gerbils (Meriones unguicalatus, Tumblebrook Farm, West Brook, MA) were used in this investigation. The care and use of animals used in this study were approved by the University of Louisville Animal Care and Use Committee. The animals were injected with an overdose of sodium pentobarbital (Nembutal - 100 mg/kg), perfused through the ascending aorta with Karnovsky's fixative (Karnovsky, 1965) supplemented with 2% tannic acid to enhance membrane visualization, and then decapitated. Immediately thereafter the cochleae were dissected free, the stapes was removed, the round window opened and a small hole was placed in the apex of each cochlea. Karnovsky's fixative supplemented with 4% tannic acid tG further enhance membrane staining was then gently perfused through the oval window using a small syringe. The cochleae were kept in fixative for 2 h and then decalcified in 10% ethylenediamine-tetraacetic acid (EDTA) with gentle agitation for 24 to 48 h at 4° C. The E D T A solution was changed every 12 h during decalcification. The progress of decalcification was assessed by gently pricking the superficial bone with an insect needle. Decalci-
Fig. 1. Outer hair cell and its subsurface cisternal system. (A) Electron micrograph (3 700 X ) of an upper middle turn [42%] OHC. Arrow points to lateral plasma membrane near the subsurface cisternae. (B) Electron micrograph of the same section (20350 x ) taken midway between the cuticular plate and cell nucleus. Arrow indicates the same location as in (A). This is the thickest portion of the subsurface cisternal system in this micrograph, the location where measurements for this study were taken.
14
C. Lutz, L. Schweitzer / Hearing Research 84 (1995) 12-18
fied cochleae were stored in 50% ethanol at 4° C for up to 30 days. For further processing, decalcified cochleae were rinsed three times in a buffered 6% sucrose solution, placed in 2% osmium tetroxide in the buffered sucrose for 2 h and then rinsed with 0.1M sodium acetate
buffer. Cochleae were divided into halves by a midmodiolar cut through a plane passing from the apex of the cochlea to a point between the oval and round windows. The half cochleae were stained with 0.5% uranyl acetate in 0.1M sodium acetate buffer, dehydrated through a graded series of methanol and em-
Fig. 2. Electron micrographs (24 300 x ) demonstrating the longitudinal and radial gradients of subsurface cisternal system thickness. Panels on the left are first (inner) row OHCs, panels on the right are in the third (outer) row. Panels on the top are from the lower middle turn [65%] and panels on the bottom are from the basal turn [90%]. (A) Subsurface cisternal system in a first row OHC of the lower middle turn [65%], 0.24/xm thick. (B) Subsurface cisternal system in a third row OHC of the same section as panel (A) (lower middle turn [65%]), 0.33 ~ m thick. (C) Subsurface cisternal system in the first row of OHCs of the basal turn [90%], 0.15/zm thick. (D) Subsurface cisternal system in the third row of OHCs of the basal turn [90%], 0.25/xm thick.
c. Lutz, L. Schweitzer/ Hearing Research 84 (1995) 12-18 bedded in L X l 1 2 resin (Ladd Research Industries, Inc., Burlington, VT). Each half was embedded with the midmodiolar face down into the flat end of a capped Beem capsule. This ensured that the cut surface of the cochlea formed the cutting face of the finished blocks. The embedding resin, L X l l 2 , was polymerized by heating for 24 h at 30 ° C, 24 h at 45°C and 24 h at 60 ° C.
A
Subsurface Cisternae Thickness Longitudinal Analysis .30 .25
o
~-
c.)
.20 \\ .15
=
2.2. Tissue sampling
15
\5
.10
u J~
Regions lying at approximately 42%, 65% and 90% (+/5 - 6 % error due to the distance between the round and oval windows) of the distance along the length of the cochlea were identified and studied in the resulting sections. These regions are referred to as the upper middle turn, the lower middle turn and the basal turn, respectively, in the remainder of this paper.
~-
.05 0
B
42~ 65% 90~ a p i c a l to basal d i s t a n c e
Subsurface Cisternae Thickness Radial A n a l y s i s .30
2.3. Microscopy .25
For light microscopy, 1 - 2 / z m sections were cut with a glass knife, mounted on slides, stained with 2% toluidine blue, and then examined to locate the appropriate regions for electron microscopic study. For electron microscopy, sections approximately 80 nm thick, adjacent to the sections studied with the light microscope, were cut with a diamond knife. The sections were supported with copper grids and stained with 4% uranyl acetate, in 50% ethanol, for 5 min, rinsed in d H 2 0 , then stained with Reynolds lead citrate for 1 rain. Sections were examined and pictures were taken using a Philips 201 electron microscope.
2.4. Morphometric Analysis In OHCs, the thickness of the subsurface cisternal system was measured from the innermost leaflet of the Subsurface Cisternae Thickness Longitudinal and Radial Analyses
g .2
..........iii i 2 ¢.J
.1
E
4~:
6d:
9~: --
Z apical to basal distance
Fig. 3. Subsurface cisternal system thickness, means and standard errors of the means. In this graph, data for individual hair cell rows (1, 2, 3) in individual locations (basalmost [90%], upper middle [42%] and lower middle [65%] turns) are shown.
g L
.20 .15
O3
c
.10
u
~-
.0b J
J
L
2 3 outer hair cell row
Fig. 4. Subsurface cisternal system (SSC) thickness, means and standard errors of the means. (A) Longitudinal analysis. The SSC is significantly thinner in the basalmost turn [90%] than in OHCs in the upper middle [42%] and lower middle [65%] turns. (B) Radial analysis. The SSC is significantly thicker in the third row of OHCs than in the first row of OHCs. The thickness of the SSC in the second row lies between that of the other two rows and is not significantly different from either. innermost cisternae to the lateral plasma membrane on electron micrographs (24300 × ) of longitudinal sections through the center of each O H C (Fig. 1). Since the thickness of the subsurface cisternal system varies along the lateral plasma membrane, high magnification micrographs were always taken at a point midway between the cuticular plate and the cell nucleus, where the system is generally thickest. Measurements were taken at the thickest point of the subsurface cisternal system within each micrograph. Micrographs were obtained from hair cells in each row, in each of the three longitudinal locations mentioned above. The measurements were obtained in one cochlea from each of the six adult gerbils.
2.5. Statistical Analysis These data were analyzed with a two-way A N O V A (radial x longitudinal locations), subsequent one way
C. Lutz, L. Schweitzer / Hearing Research 84 (1995) 12-18
16
A N O V A s and individual comparisons were analyzed with Fisher's post hoc tests (Siegel, 1956).
upper middle turn [42%], it is still almost twice as thick as that in the basal turn [90%].
3.2. Radial Differences 3. Results Our data show that there are significant differences in the thickness of the subsurface cisternal system of OHCs at different longitudinal locations and radially between those of the three rows of OHCs. Thus, both radial and longitudinal gradients of thickness of the subsurface cisternal system were found (Figs. 2, 3, 4).
3.1. Apical to basal differences There is a significant difference in the thickness of the subsurface cisternal system between OHCs in different turns of the cochlea (F = 24.14; d.f. = 2,45; P < 0.001) (Fig. 4A). The subsurface cisternal system is significantly thinner in the basal turn [90%; 0.13/xm + 0.02] than in the two more apical turns. No significant difference was found in the thicknesses in the subsurface cisternal system in OHCs of the lower middle [65%] and upper middle turns [42%] of the cochlea, which were, on average, 0.23/xm ( + 0.02) and 0.27/xm (+_ 0.02) thick, respectively. The subsurface cisternal system in the upper middle turn [42%] is approximately two times thicker than in the basal turn [90%]. Although the subsurface cisternal system in the lower middle turn [65%] is somewhat thinner than in the
IGLRADE INT LONGTIUDG N IR ALADE INT RADA ThcikneofsthseSubsurfaCc~esternae apeX~base(~ ~ ~ ~ apex
EffereInntnervatol n I~
Fig. 5. Schematic of longitudinal and radial gradients found within the cochlea. (Top) Thickness of the SSC. The SSC is thicker in the apex than in the base and is also thicker in the third row of OHCs than in the first. (Bottom) Efferent innervation. The base receives more efferent innervation than the apex and the first row of OHCs receives more efferent innervation than the third row.
A radial difference also exists between the thickness of the subsurface cisternal system in the different rows of OHCs (F = 5.946; d.f. = 2,45; P < 0.01) (Fig. 4B). This difference reached significance only for OHCs in row one (innermost; 0.18/xm +_ 0.02) versus row three (outermost; 0.25/xm + 0.02). These were not different than for OHCs in the middle row (0.20 /xm + 0.02). On average, the thickness of the system in the third row exceeded that in the first row by 36%. This difference was greatest in the most basal turn [90%] where the subsurface cisternal system in OHCs in the first row was very thin and consisted of only a single layer of cisternae which was on average only about 0.09 /am thick, as compared to the third row at the same longitudinal location, which was on average twice as thick.
4. Discussion The subsurface cisternal system in the OHCs of the gerbil have both a longitudinal (frequency-related) gradient and a radial (inner to outer) gradient of thickness (Fig. 5). The subsurface cisternal system is significantly thinner in the hair cells in the base of the cochlea as compared to the more apical regions and the system is significantly thicker in the third (outermost) row of OHCs as compared to the first (innermost) row. OHCs increase in height from the base of the cochlea to the apex and from the inner row to the outer row (Kimura, 1966) and thus the thickness of the subsurface cisternae may be related to size. There are other gradients found in the cochlea, such as in the efferent innervation, resistance to ototoxicity and response to overstimulation that have an interesting correlation to the gradients of the thickness of the subsurface cisternal system found within the cochlea (Fig. 5). As mentioned in the introduction, efferent innervation may be related to the subsynaptic cisterns which are continuous with the subsurface cisterns in OHCs in some species. In general, the efferent innervation to OHCs decreases from the base of the cochlea to the apex (Liberman et al., 1990; Dannhof and Bruns, 1993; Francis and Nadol, 1993). This is opposite to the thickness of the subsurface cisternal system which increases from the base to the apex of the cochlea. Thus, there is an increase in efferent innervation where there is a decrease in the thickness of the subsurface cisternal system, or stated differently, there is an inverse relationship between the thickness of the subsurface cisternal system and the amount of efferent innervation to the OHCs.
C. Lutz, L. Schweitzer /Hearing Research 84 (1995) 12-18
Just as there is a longitudinal gradient of efferent innervation to the OHCs, radial differences exists as well. There is a notable decrease in efferent innervation from the first (inner) row of O H C s to the third (outer) row of O H C s (Liberman et al., 1990; Nadol, 1990; Francis and Nadol, 1993). In addition, the largest terminals are found on the first row and the smallest on the third row (Xie et al., 1993), which may relate to the potentially greater influence that efferent innervation has on the first row of OHCs. This radial gradient of efferent innervation to the O H C s is opposite to the radial gradient of the subsurface cisternae. The third row of O H C s contains the thickest subsurface cisternal system and this corresponds to the row that has the least amount of efferent innervation. Therefore, an inverse relationship between efferent innervation and thickness of the subsurface cisternal system holds true both in the radial and longitudinal dimensions. Since a direct relationship exists between the amount of efferent innervation and the susceptibility to ototoxicity (Longitudinal gradients: Ylikoski et al., 1993; Forge and Richardson, 1993; Kiang et al., 1976; Radial gradients: Kaltenbach et al., 1992; Robertson, 1982; Liberman and Beil, 1979), an inverse relationship also exists between susceptibility to ototoxicity and the thickness of the subsurface cisternal system. In addition to the gradients already mentioned there is also a longitudinal gradient of electromotile properties in the O H C s (Brundin et al., 1989). O H C s along the length of the basilar m e m b r a n e display frequencyspecific sound induced electromotility. It has been suggested, therefore, that O H C s are 'tuned'. In addition, the force of O H C fast motility apparently differs between the turns of the cochlea. The O H C s in the fourth (most apical) turn of the guinea pig cochlea shorten more in response to equivalent stimulation than do cells in the third turn of the cochlea. Thus, the force generated by the apicalmost hair cells may be inferred to be greater (Gitter et al., 1993). The subsurface cisternae, perhaps in conjunction with the actin cytoskeleton, does seem to be associated with electromotility and is disrupted when electromotility ceases (Dulon et al., 1990; Holley and Kachar, 1992; Dieler et al., 1991). If the subsurface cisternae are necessary for motility, it is possible that the apicalmost hair cells, that have been suggested to generate a greater force, need a significantly thicker subsurface cisternal system than do those in the base of the cochlea. Alternatively, it is possible that the function of the subsurface cisternae is to sequester calcium along the lateral plasma membrane. Upon depolarization calcium enters the outer hair cell. Since the subsurface cisternal system is lined up so close to the plasma m e m b r a n e in the OHCs, it would seem possible that it functions to sequester calcium near the calcium sensitive potassium efflux channels. Mitochondria in the
17
adult O H C s are located just within the innermost cisternal layer and may further sequester calcium. Mitochondria are known to fulfill this function in other cells (Alberts et al., 1989). H a r a d a et al. (1993) noted that an O H C could hyperpolarize even in the presence of an extracellular calcium channel blocker. This finding suggests that calcium release may well come from intracellular stores, perhaps in the mitochondria or the subsurface cisternae. Pools of calcium may enhance the ability to activate potassium channels near the base of the OHC. Intracellular isoforms of the enzyme calcium activated ATPase has been localized by Schulte (1993) along the lateral plasma membrane, the location of the cisternae. Furthermore, labelling for calcium activated ATPase in the O H C was first noted on postnatal day 14, an age close to the onset of cochlear function, when the first layers of cisternae are forming (Weaver and Schweitzer, 1994). Calcium influx is known to have an adverse effect on neurons. Excitotoxicity, a p h e n o m e n o n underlying such diverse degenerative effects in the nervous system as epilepsy and anoxic brain injury (Rothman and Olney, 1987), is mediated by calcium influx. If the subsurface cisternae serves to sequester calcium to the area adjacent to the cell membrane, protecting the intracellular space from excess calcium, it follows that ceils with a thicker subsurface cisternal system in the apex should be better protected from this type of injury. Interestingly, the cisternae are also more fenestrated in hair cells in the base of the cochlea than those in the apex (Forge and Richardson, 1993) and this increased fenestration may also increase susceptibility to injury in basal hair cells.
Acknowledgements We would like to thank Drs. James B. Longley and Fred J. Roisen for their helpful comments on the manuscript. We would also like to thank Ms. Tina Cecil for her technical assistance. These experiments were supported by a grant from the Deafness Research Foundation.
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