Journal ofVestibuJarResearch. Vol. 7. No.5. pp. 393-406.1997 Copyright II:> 1997 Elsevier Science Inc. Printed in the USA. All rights reserved

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ELSEVIER PI! S0957-4271(96)00166-8

Original COlUribution

VESTIBULAR TYPE f AND TYPE II HAIR CELLS. 1: MORPHOMETRIC IDENTIFICATION IN THE PIGEON AND GERBIL

Anthony J . Ricci,*1 Katherine J. Rennie,· Stephen L. Cochran,*:t: Golda A. Kevetter,*:t: Manning J. Correia,*t

Departments of ·Otolaryngology. tPhysiology and Biophysics. and :j:Anatomy and Neuroscience, University of Texas Medical Branch. Galveston, Texas

Reprint address: Manning J. Correia, Ph.D., Department of Otolaryngology, University of Texas Medical Branch, 7.102 Medical Research Building, Galveston, TX 77555-1063

o Abstract-Classically, type I and type II vesti­bular hair cells have been defined by their afferent innervation patterns. Little quantitative informa­tion exists on the intrinsic morphometric differ­ences between hair cell types. Data presented here define a quantitative method for distinguishing hair cell types based on the morphometric proper­ties of the hair cell's neck region. The method is based initially on fIXed histological sections, where hair cell types were identified by innervation pat­tern, type I cells having an afferent calyx. Cells were viewed using light microscopy, images were digitized, and measurements were made of the cell body width, the cuticular plate width, and the neck width. A plot of the ratio of the neck width to cuticular plate width (NPR) versus the ratio of the neck width to the body width (NBR) established four quadrants based on the best separation of type I and type II hair cells. The combination of the two variables made the accuracy of predicting either type r or type n hair ceJl~ greater than l)OC}i-.. Statistical ciuster anaiysis ~ontiime(' the quadrant separation. Similar analysiii wa!' p~r·

formed or. dissocialeci hal ;' ccii" frum st!micln:uia; canal, utricle, and la!!ena. giving results statisti­cally similar to thos(' of the fixed tissue. Additional comparisons were made hetween fixed tissue and isolated hair cells as well as across species lpigeon and gerbil) and between end organs (semicircular canal, utricle, and lagena). In each case, the same

iCorrespondence should be sent to Anthony J. Ricci, Ph.D., University of Wisconsin, Department of Neurophysiology, 273 Medical Science Center, 1300 University Ave., Madison, WI 53706; phone (608) 262-9320.

morphometric boundaries could be used to estab­Jish four quadrants, where quadrant 1 was pre­dominantly type I ce))s and quadrant 3 was almost exclusively type II hair ceJls. The quadrant sepa­rations were confirmed statistically by cluster analysis. These data demonstrate that there are intrinsic morphometric differences between type I and type I! hair ce))s and that these differences can be maintained when the hair ce))s are dissoci­ated from their respective epithelia. © 1997 Elsevier Science Inc.

o Keywords-vestibular system; hair cells; morphology; inner ear; labyrinth.

Introduction

Vestibular hair cells were first characterized as r~'pe r 0r type n haseci on their .characteri~1 ic in­ner\'atlol1 pallern \ I). Type i celis haVe' an alTer­.' 11 : t-=",'lina L the c:,!:";; thai surround, the cl1!irc. basolaleral surface of the cell. A single type j

cell can be found in a single calyx ending or gr(1up~ of type I cells can be seen in (1 single ca­lyx (2,3). lndeed, as many as 12 type I cell!> have been identified in a single avian calyx (3) . Type II hair cells have bouton-like afferent and efferent connections (1). The distribution of the two cell types varies between species, but in general, in amniotes a higher proportion of type I cells are found at the apex of the crista and along the striola of the otolith organs (2,4,5),

l<. r CElvED 30 July 1996; i\CCEI'll.:.D 16 October 1996.

393

394

while remaining regions are dominated by type II cells.

Little quantitative information is available concerning the morphology of type I and type II hair cells. Type I cells have a flask or amphora­shaped body with a broad rounded basal region, a narrow neck and a flared apical region (l). The type II hair cell is columnar or ovoidal with :.l tlat apex (5). Kevetter and colleagues (6) have described JirIerences in ned: iength. neck width. cell :)oJy iength. :ll1J apical surface wtLlth 1c-

.:~ils [rom the .rerbll. :"inJemun clOd c()ilea!.!ll~'> - -: - . using elecrrnn ·11icrnscopy. Jescribed two hair ollndle types in the vestibular epIthelium that ::u-e suggested to be correlated wilh~ype 1 ,md type II hair cells. Lapeyre and colleagues (8) have also suggested that type I hair bundles have longer stereocilia than type II hair bundles.

Correia and colleagues (9) have reported that the ratio of the neck width to the cuticular plate width (NPR) can distinguish between dissoci­ated type I and type II hair cells in vitro. Scar­fone and colleagues (10) suggested that isolated hair cells with neck regions were type I cells and those cells without necks were type IT cells. Scarfone and colleagues (10) further suggested that although hair cells existed in a variety of shapes, four basic subclasses of hair cells could be described based on the presence or absence of a neck region as well as on the neck length. The four SUbtypes consisted of a spherical cell with no neck region, an oblong cell with no neck region. and two cell types with neck re­gions that varied in length.

The ability to distinguish between dissociated type I and type II hair cells is a fundamental requirement for being able to interpret and evalu-a e e growmg 0 y 0 1 erature concernmg t e electrophysiologic, mechano-electric, pharma­cologic, morphologic, and motile properties of vestibular sensory hair cells. Also, basic mor­phometric differences between hair cell types may give insights into functional differences be­tween the two major classes of hair cells allowing for further subclassification of hair cell types.

The work presented here describes a quanti­tative method for distinguishing between type I and type IT hair cells. It uses differences in the

A. J. Ricci et al

proportions of the neck region relative to the cu­ticular plate and the cell body to define four subclasses of cells. Together, two of these con­tain more than 70% of all cells and divide these cells into groups, one of which is 96% type I hair cells and one of which is 96% type II hair cells. The method is consistent between mam­malian and avian species, between end organs. and also between histologically derived tissue ~md Jissociated hair cells. This work detines rype I and type il hair cells by :norphometric ,)f()penies intr:n:-.ic" ,0 ~ he hair c.:11. ,l~ opposed . 0 .he innervallon pallern. thus ,t1lowing for clas::.ir"ic:uion" of uissoclated hair Lells. This work ,1Is(l suggests that the dissociated hair cells ~u'e

capable of maintaining their morphometric shape found in the fixed histologic tissue.

Materials and Methods

Avian Histology

Anesthetized pigeons (Columba Livia) were perfused by transcardiac carotid catheterization (11) with an ice cold normal Ringer's solution to clear the blood, followed by a Ringer's solu­tion supplemented with a 3% paraformaldehyde, 4% glutaraldehyde solution. Birds were decapi­tated, and the otic capsules were opened and post­fixed overnight. End organs were then removed, rinsed, and placed into 1 % osmium tetroxide for :2 h. Tissue was rinsed, dehydrated through a se­ries of increasing concentrations of methanol washes, cleared in propylene oxide, and embed­ded in Embed 812 (EM Sciences). Serial sec­tions (5 to 10 !-Lm) were placed onto lass slides, counterstame with toluidine blue, viewed un­der DIe with a 63x (0.9 NA) oil immersion lens (Zeiss Inc.) on an upright Zeiss Axiovert micro­scope. Cells were chosen for measurements only if a complete cross-section of the cell could be observed that included the nucleus and at least part of the hair bundle (to ensure that a hair cell was being measured). Examples of the histolog­ical section are given in Figure lB. The tissue was slightly overstained to enhance visibility of the neck region. Cells were chosen from across

Morphometric Identification of Vestibular Hair Cells

A

Type I Type /I ---B C

Figure 1. (A) The measurements of cuticular plate width (1), neck width (2), and cell body width (3) were made from type I and type II cells and demonstrated on examples of dissociated pigeon hair cells. The scale bar represents 10 JLm. Where a neck was not clearly defined, the neck width measurement was made directly beneath the cuticular plate. Examples of the histological sections used from the pigeon are given in (B) for the type I cell and in (C) for the type II cell. Calyx (arrow In B) identification defined the type I hair cell. Calibration in (C) also applies to B.

each end organ to give a uniform sampling over the entire width of the end organ.

Pigeon Hair Cell Isolation

White king pigeon::,. bet ween f, and 36 weeks of age. were aneqherized with pentoharbirai 150 mg/kgJ . supplelllL'nted with 1 ~ ~t ,l mi i1 e . ane then placed in " sterl'l)ta ;, i ~ i1 ": ~I G n,);cie: . -:-h~ ,:;:1~':

ear end organs were surgical !: removed an cl placed into Dulhecco ' ~ Modified Eagles Medlul11 (DMEM) augmented with 24 111M NaHC03, 15 mM HEPES. 50 mg/L ascorbate. The tissue was then treated at room temperature in the DMEM containing 0.1 mg/mL protease (type XXIV) for 10 to IS min. This treatment was to enable hair bundles to be separated from the otolithic mem­brane or from the cupula. The tissue was then transferred to a DMEM solution supplemented with 0.3% neonatal fetal calf serum. The tissue

395

was incubated in this solution for 2.S h at 3SoC in a saturated 9S% oxygen, S% carbon dioxide atmosphere. The hair cells were mechanically dissociated by vacuuming (gentle suction) the hair cells free from the epithelium with glass pi­pettes between 20 and j 00 !-Lm tip diameter. The cells were then plated onto Concanavalin A (O.S mg/mL) coated glass bottom dishes con­taining the normal extracellular Ringer' s solu­tion. The extracellular solution contained (in

HEPES, and 10 glucose. The pH was .main­tained at 7.4, and the osmolality was maintained at 31S mOsm. The isolation method was a mod­ification of previously used methods (9) and was developed to increase the yield of hair cells with morphologically intact hair bundles. Cells were chosen for imaging based on previously described criteria used for selecting electro­physiologically viable cells (9,12, l3). All ani­mal procedures were approved by the animal care and use committee of the University of Texas Medical Branch, and the studies were conducted in strict accordance with the guidelines of the American Physiological Society and the NIH.

Gerbil Tissue

Posterior semicircular canal tissue was used from the histologic work described in Kevetter and colleagues (6). Type I cells were defined by having a calyx surrounding their basolateral sur­face . Type II cells dicl not have an afferent cal~'x

and could be ciistingLi i she(~ fro m other non-hair cell type.~ hy the rres ~llc t' ( If . 1 hai r bundle . Oni: ·cll~. th:i:. · ~'0i.1ld be v.l iim!ci t:i :, \'isua Lze(~ fron.

bted fr0 111 gerbi l semi eircular canal cri sta hy method ',; described pre violls ly ( 14).

Imaging System

Hair cells were viewed with a Nikon Diaphot microscope, using either a x40 (1.2 NA) or a x60 (1.4 NA) oil immersion objective lens with either Nomarski Differential Interference Con­trast Optics or brightfield optics, and then vid-

396

eotaped at 4400 X with a super VHS VCR (NC) through an 800 line DAGE tube camera. The optical resolution (two point discrimination) was near 200 nm. Image I (Universal Imaging Corp.) software was used for digitizing the images. The plane of focus was chosen so as to maximize the surface area of the particular region being recorded. Usually, optically serial sections were made to best capture th~ cell boliy. utricular pi~He. and hair bundle. respectivel y. P,) r [he his­rologic Jata. the entire cell as well as the !lli- . .:'eus had -0 ~)e vis ib ie :n 'he '':C1f :·, e c~ i o !1 ;:cr ~ he L'eil to b~ inc!uded. The :;ystell1 's overal l magni­fication J.nl! resolution was calibrareli wilh poly­styrene beads of ~no\Vn Jiameter (nanospheres. Duke Scientific). The resolution of the digitiZ­ing system was 10 pixels per micron (when us­ing the 60x objective).

Measurem.ents

Irnagescan software (Jandel Corp.) was used for making the measurements. Similar measure­ments were made on all hair cells. As indicated in Figure 1, measurements included cuticular plate width [1], neck width [2], and cell body width [3]. The neck region was defined as the area below the cuticular plate that is thinner than either the cuticular plate or the basal region of the cell body. In instances where the cells were columnar, the neck width was measured immediately below the cuticular plate. A vari­ety of neck types were found. Some cells had neck regions where there was no initial invagi­nation, that is, neck width was equivalent to cu-

A. J. Ricl~ i et al

ticular plate width, but the cell body increased in width at the base, or cells had initial invagi­nations that never returned to widths compara­ble to the cuticular plate width. Both type I cells and type II cells were found that had neck re­gions. Measurements were made by eye from the digital images collected as described above. For the purpose of presentation, the images of the dissociated hair cells have had the back­ground digitall) .removed (Adohe Photoshop. Adobe Systems Inc.) 1'01' b~tler darity. The cells -;ho \vn in :=igure lA wre chl)~en to emphasize the lifficulty in qualiuu\ely identirying cells as type I or type II. That IS. they represent cells whose measurements place them near the bor­der of cell type classification.

Results

Pigeon Histological Data

Hair cells were first identified by the pres­ence (type I cell; Figure lB) or absence (type II cell; Figure lC) of an afferent calyx. A total of 359 pigeon hair cells were included. Of these, 171 were type I and 178 were type II, based on the presence or absence of a nerve calyx (see Table 1 for a breakdown of end organs and cell types).

The frequency histograms for the measure­ments of neck width, body width, and cuticular plate width are given in Figure 2 (A-C). The neck width (2C) shows a separation between type I and type II hair cells. However, a great deal of overlap (filled bars) exists between dis-

Table 1. Summary of Data Obtained from Pigeon Histologic Tissue

Cell (n) Body (f.Lm) C.P. (f.Lm) Neck (f.Lm) NPR NBR

Type I (all) 171 7.3=0.1 5.3 = 0.1 2.7 = 0.1 0.55 = 0.02 0.37 = 0.01 Type II (all) 178 7.8 = 0.1 5.0 = 0.1 4.9 = 0.1 0.99 = 0.02 0.63 = 0.01 SCCI 54 6.8 = 0.2 6.5 = 0.2 2.5:!: 0.2 0040 = 0.03 0.37::: 0.02 SCC II 36 7.3 :!: 0.3 5.6 :!: 0.2 5.1 :!: 0.2 0.93:!: 0.04 0.71 :!: 0.03 UTR I 55 8.1 :!: 0.2 5.2 = 0.2 3.1 = 0.2 0.62 = 0.03 0.38 = 0.02 UTR II 86 8.5 = 0.2 5.2 :!: 0.1 5.2:!: 0.1 1.02:!: 0.02 0.83 = 0.02 LAG I 62 7.0 = 0.2 4.2 = 0.1 2.5 = 0.1 0.62 = 0.02 0.36 = 0.01 LAG II 56 7.0 = 0.2 4.3 = 0.2 4.0 = 0.2 0.99 = 0.04 0.57 = 0.03

Values are mean:!:standard error of the mean. The number of values is given in the second column (n), the cell body Width, cutic­ular plate (C.P.) width, and the neck width are given in columns 3 through 5, and the ratiometric measurements of neck width to cuticular plate width (NPR) and neck width to cell body width (NBR) are given in columns 6 and 7, respectively. The breakdown for the individual end organs, semicircular canal (SCC), utricle (UTR), and lagena (LAG) are also given.

Morphometric Identification of Vestibular Hair Cells

tributions, making the individual measurements unsatisfactory for distinguishing between type I and type II hair cells. The cuticular plate width (Figure 2B) does not show a separation between cell types. Table] summarizes the mean values obtained for each of the measurements for type I and type II hair cells between end organs. In each case, the ratio metric variables for type J cells, both combined and indi vidual end organs, are statistically significantly different (p < 0.001), using a Tukey's b ANOV A test, than their corresponding type II cell measurements. Only the individual measurement of neck width showed a statistical difference as compared to type II hair cells across each end organ. No sta­tistically significant differences were found within either the type I or type II hair cell popu-

35

30 ..!!l 25 a 20 't; 15

! '~

A 30

25

20

15

10

5

o 0

B

397

lations between end organs. The ratio of neck width to cuticular plate width (NPR) and the ra­tio of neck width to body width (NBR), given in Figure ~, panels D & E, respectively. separate type I and type II hair cells to a much greater degree than do any of the individual measure­ments. More than 60% of the cells have NPR values less than 1, suggesting the presence of a neck region. Also more than 70% of the cells Ha';e NBR values less than 1, again suggesting the presence of a neck region. The NPR values for type I and type II cells, defined b) the pres­epee or abSence of a calyx were 055 (tQ Q1) and 0.99 (:to.02), respectively. The values are mean (:tSE). For the NBR measurement, the type I and type II cells had values of 0.37 (:to.0l) and 0.63 (:to.0I). See Table 1 for a summary of

CJTypel

_Typell C _ C>.Ier1ap

50

40

30

20

10

o 2 4 6 8 10 12 14 0 2 4 6 8 10 12 o 2 4 6 8 10

Body Wdth (~) C.P. Width (~m) ~I: Width (~m)

35- D 3C\

If! ..,:: ~ ~ .... <.)20

~ 15

~ 10 :f 5

o 0.0 0.5 1.0 1.5 20

Neck to Plate Ratio (NPR)

35-'E ~ 1 ~ 20

15

10

5 o

0.0 0.5 1.0 1.5

Neck to Body Ratio (N8R)

Figure 2. Frequency histograms of cell body width, cuticular plate (C.P.) width, and neck width are given in (A) through (C) for pigeon histological data. Data from semicircular canal (SCC), utricle (UTR), and lagena (LAG) were pooled for these plots. Type I cells are indicated by the open bars, type II cells by the gray bars, and the overlap by the black bars. Frequency histograms for the NPR and NBR ratios are given in (D) a •. d (E). Both ra­tios more clearly discriminatp. betwee" ;h~ I)'pe I and type" :. -Ir cell r,op:J\alions, as indicated by the decrease in overlap between cell types.

398

measurement values for individual end organs. These results demonstrate that the NPR and NBR measurements are different between type I and type II hair cells (P < 0.001, using Stu­dent's t-test for independent variables). The probability of accurately identifying a cell as type I or type II by the NPR or NBR value could be calculated as the mm of the percentage of cells with a panicular ratio times the ?roportioI1 of type 1 (or type 10 ceils In that region. By this

:'iP~ med1l'l:. '-Ising 1 ;ulOr"f vaiue .)[ . .. -. \va~ 86(0. while [he :-iBR :11erhl)Li. with ~l '..:urnCf value of 0.58. was 79% accurate. Using In NPR

1\ J. Ricci At al

cutoff value of 0.7 created two populations of cells, one that was 97% type I and the other that was 78% type II. Using an NBR cutoff value of 0.58 again separated cells into two groups, one that was 71 % type I and the other that was 91 % type II. Although both methods show a rela­tively high accuracy, the populations of cells are different. That is, the cells in the overlap­ping region of the NPR frequency histogram (aboLlt '20 L7n) were different from the cells in the '1\"?rla!J!,in~ !"e~ion llf the NBR frequency ilisto­Jram ubout [21'" I. 8:' plotting NPR ':ersus NBR it is po~siblc' w isoiate ,he different ,)Veriapping populations described by each measurement.

open = type I, filled = type II

o see o UTR LAG

4 3 .0

•

2

0.5 1.0 1.5 2.0

Neck to Plate Ratio (NPR)

Figure 3. NBR plotted against NPR, from histologically identified pigeon hair cells, allows type I cells to be sep­arated from type II cells. Four quadrants, depicted by the solid lines, were chosen to give the best separation of cell type. Type I cells, indicated by the open symbols and identified by the presence of a calyx, were predomi­nant in quadrant 1. Type II cells, indicated by the filled circles, dominate in quadrant 3. The filled regions repre­sent the three groups derived from the hierarchical cluster analysis. These groups add statistical support to the use of the quadrants derived to separate the type I and type II hair cells.

Morphometric Identification of Vestibular Hair Cells 399

Table 2. Summary of Ratiometric Measurements for Both the Histologic Data and the Dissociated Hair Cells for Each Species as Well as for the Different End Organs

Animal End organ Cell group (n) NPR NBR

gerbil sec 1 (fixed) 533 0.46::': 0.01 0.30::': 0.01 gerbil sec 1 (dissociated) 88 0.40::': 0.01 0.35 = 0.01 pigeon all 1 (fixed) 140 0.47::': 0.01 0.34::': 0.01 pigeon all 1 (dissociated) 260 0.41 ::': 0.01 0.37::': 0.01 pigeon sec 1 (fixed) 48 0.34::': 0.02 0.33::': 0.02 pigeon sec 1 (dissociated) 45 0.40::': 0.01 0.35::': 0.01 pigeon UTR 1 (fixed) 38 0.48::': 0.02 0.34::': 0.01 pigeon UTR 1 (dissociated) 80 0.42 + 0.02 0.38 + 0.02 pigeon LAG , (fixed) 54 0.56::': 0.02 0.35::': 0.01 pigeon LAG 1 (dissociated) 73 0.43::': 0.02 0.39::': 0.01 gerbil sec 2 (fixed) 55 0.83 = 0.01 0.43 = 0.01 gerbil sec 2 (dissociated) 4 0.97::': 0.01 0.51 ::': 0.01 pigeon all 2 (fixed) 93 0.96::': 0.02 0.45::': 0.01 pigeon all 2 (dissociated) 41 0.95::': 0.04 0.49:: 0.01 pigeon sec 2 (fixed) 10 0.82 ::t: 0.09 0.47 = 0.03 pigeon sec 2 (dissociated) 12 1.0:: 0.08 0.48::': 0.02 pigeon UTR 2 (fixed) 47 0.96:: 0.03 0.46:: 0.07 pigeon UTR 2 (dissociated) 28 0.86:: 0.03 0.50:: 0.01 pigeon LAG 2 (fixed) 36 1.00:: 0.05 0.42:: 0.02 pigeon LAG 2 (dissociated) 18 1.02:: 0.09 0.49:: 0.02 gerbil sec 3 (fixed) 46 0.95:: 0.01 0.87::': 0.03 gerbil sec 3 (dissociated) 13 0.87::': 0.04 0.71 ::': 0.02 pigeon all 3 (fixed) 114 1.00:: 0.02 0.75:: 0.01 pigeon all 3 (dissociated) 171 1.01 :: 0.03 0.90:: 0.03 pigeon sec 3 (fixed) 30 0.96:: 0.05 0.79::': 0.03 pigeon sec 3 (dissociated) 35 1.1 ± 0.06 0.77::': 0.03 pigeon UTR 3 (fixed) 56 1.03::': 0.02 0.73 ± 0.01 pigeon UTR 3 (dissociated) 55 1.04 ± 0.03 0.79::': 0.03 pigeon LAG 3 (fixed) 28 0.97::': 0.05 0.73::': 0.03 pigeon LAG 3 (dissociated) 77 1.14 ± 0.04 0.90 ± 0.03 gerbil sec 4 (fixed) 14 0.62 ± 0.02 0.80:: 0.04 gerbil sec 4 (dissociated) 2 0.56:: 0.01 0.81 ::':0.11 pigeon all 4 (fixed) 2 0.77::': 0.00 0.72::': 0.05 pigeon all 4 (dissociated) 47 0.62:: 0.01 0.78::': 0.03 pigeon sec 4 (fixed) 2 0.71 ::': 0.00 0.72:: 0.05 pigeon see 4 (dissociated) 8 0.66:: 0.02 0.92:: 0.09 pigeon UTR 4 (fixed) 0 pigeon UTR 4 (dissociated) 19 0.62:: 0.02 0.85 ± 0.04 pigeon LAG 4 (fixed) 0000 pigeon LAG 4 (dissociated) 20 0.62:: 0.02 0.78 = 0.03

A total of 1623 cells are included. There; are no statistically significant diHerences within each quadran: between dissociated cells and fixed tissue or beiween pigeon ana gerbil. No data Indicated by - II' table

thereh~ improvlIlg the overall accurac~ 01 dl~ ­

criminatin f! between type I and type Il hair cell~ .

Figure:; plots NBR versu., NPR: the open symbols represent type I cells. identified by the presence of a calyx. and the filled symbols rep­resent type II cells, identified by the absence of a calyx. The symbol type defines the end organ from which the cells originated. Four quadrants, identified by the solid lines, were created based on the best separation between type I and type II hair cells. Quadrant 1 is defined by NPR values less than or equal to 0 .72 and NBR values less than or equal LO 0.64. It is comprised of 96.4% type I cells and 79% of all type I hair cells.

Quadrant :! l~ defIned b~ ;\IPR ntlLle~. greater than 0.72 and NBR value!- less than or equal to

(i.5 t. . Quadrctm 2 consists of 70Cic type n cell". 350/c of all type II hair cell~ and 170/( of type J hair cells. Quadrant 3 is bounded by NPR val­ues greater than 0.72 and NBR values greater than 0.58 . Type II cells comprise 95 .6% of the cells in quadrant 3. Also 61 % of type II cells are in quadrant three as compared to 2.5% of type I hair cells. Quadrant 4 is defined by NPR values less than or equal to 0.72 and NBR values greater than 0.64. QlIadrant 4 contains 0.501 .!·:111 the cells ant: ,~ cn I ~ Ii' ,~cd of 50% type 11 hail ,-d Is. This information is su mmarized in Table 2.

400 A. J. Ricci et a\

0 sec c UTR /). LAG 1.4

4 3 1.2 ~

/).

1.0 IJ

Q: 0.8 al Z

~ 0 ~o

2 . . . • J J J j j J •

0.0 il.2 0.4 O.S 0.8 1.0 1.2 1.4

NPR

Figure 4. A plot of NBR versus NPR is given for pigeon hair cells dissociated from semicircular canal (SeC, open circles), utricle (UTR, open squares), and lagena (LAG, open triangles). The solid lines indicate the quad­rants determined from the histologic data. The clusters that were statistically derived are indicated by the shaded areas. In this case a fourth cluster was added because of the increased number of cells found in this group. The original three clusters were not statistically different from those from the histologic data.

The scattergram (Figure 3) demonstrates that was made between the cluster assignment and the type I hair cells are grouped much more the quadrant grouping, and the difference was closely than are the type IT hair cells. The scat- not statistically significant (P > 0.05) when tergram also compares the distributions of hair tested using the Wilcoxon matched-pairs signed cells from different end organs. The largest dif- ranks test. In only one instance is there overlap ference found between the semicircular canal between a cluster and the quadrants. It is this (SeC) hair cells and the otolith hair cells was overlapping region where the NPR and NBR that the see had fewer cells of either type in fail to distinguish between type I and type II group 2. Only 17% of type IT and 7% of type I hair cells. The fourth quadrant, not represented see hair cells are found in group 2 as com- by a cluster. did not have enough cells (only pared to 36% and 29% of utricular type II and 0.5%) of the population to warrant a~c1.u.st.e~r.lilasill-___ """' __ type I hair cells, respectively, and 16% and 46% of signment. "! lagenar type I and type II hair cells, respectively. A measure of the accuracy of the quadrants Quadrants 1 and 3 comprise 73% of all cells, with for separating all cells into the two cell types

quadrant 1 containing 96.4% type 1 c_el_ls:...an ___ d_q.:..u_a_d-_-;c'<laniffib/e~c ... afFlc;;;u~ln.at;;;e:;;dp,as;;T.;th;;:e~su:rm-;;i(rw~i t~hT;in;=-i;ea~c~h;;qf-uf;a~d:-___ ,.. __ _ _______ -Frant-3-ee~5:6%-type-U-ceH": 0 e percentage 0 1 cells found in a

A hierarchical cluster analysis using Ward's given quadrant times the percentage of the pre­method (SPSS statistical software V6.1; SPSS dominant cell type in that given quadrant. This Inc.) corroborated the separation of cells into equation then takes into account the limited sep­quadrants. In this procedure the analysis deter- arations found in quadrant 2 (68%) and quad­mines the minimum number of independent clus- rant 4 (50%) as well as the overall distribution of ters necessary to incorporate the data (15). Three cells. For all fixed-pigeon-tissue data, the accu­clusters were produced and are represented by racy is 89%, broken down by end organ for see the shaded ovals in Figure 3. The ovals were the 91 %, for utricle (UTR) 87%, and for lagena best fit that incorporated the points of cells in- (LAG) 88.4%. The differences are a reflection of cluded in the clusters. A statistical comparison the proportions of cells found in each quadrant.

Morphometric Identification of Vestibular Hair Cells

Isolated Pigeon Hair Cells

Hair cells (SeC, n = 121; UTR, 11 = 133; and LAG. 11 = 171) were dissociated from the white king pigeon. Measurements comparable to those made for the histological data presented above are given here. A plot of NBR versus NPR is given in Figure 4. No difference was observed between the individual end organs, nor were differences observed between this plot and that of the intact hair cells, given in Figure 3. A clus­ter analysis for large N (K-means cluster analy-5 ;; ., tained from the analysis in Figure 3 as starting values for the iterations. A central point was chosen for the group 4 cells, since a cluster was not produced by the smaller population of cells. A statistical comparison was made between the cluster assignment and the quadrant clustering, and the difference was not statistically signifi­cant (P > 0.05) when tested using the Wilcoxon matched-pairs signed ranks test.

Since the quadrant assignments were the same between the pigeon histological data and

401

dissociated data and neither cluster analyses dif­fered from the quadrant assignment, it can be surmised that the cluster analysis did not vary between data groups. The scattergram also shows that quadrant 3 cells show more variabil­ity in the NPR and NBR proportions than do the quadrant 1 cells. No statistical difference was found between ratiometric measures between intact and dissociated hair cells for each quad­rant or between end organ (P > 0.05 ANOV A using Tukey's b test) . Table 2 summarizes these results. The difference in the proportions of r? .. dissociated cells. This is illustrated in Figure 4. The accuracy of predicting the cell type was again calculated based on the distribution in the proportion of type I and type II cells found in each quadrant from the fixed data and the pro­portion of cells obtained from the dissociation procedure. For the total population of cells, the accuracy was 90%, and by end organ 92% for sec, 91 % for UTR, and 93% for LAG. Again the variability is determined by the proportion of cells found in each quadrant for each end organ.

.0. gerbil type I, fixed 0 gerbil type II, fixed • gerbil dissociated 1.4

1.2

1.0

c:: D.f

OJ Z 0.6

0.4

0.2

0.0

0.2 0.4 0.6 0.8

NPR

o 0 o

1.0

0 0

t:.

~ L

e.

b

2

1.2 1.4

Figure 5. A plot of NBR against NPR for gerbil posterior sec histological data as well as for dissociated hair cells. For gerbil, as with pigeon, quadrant 1 is dominated by type I hair cells, and quadrant 3 is comprised al­most exclusively of type 11 hair cells. Here, too, cluster analysis revea !o: II' major clusters corresponding to the four quadrants.

402

A 100

80 .. ; 60 ~ l. 40

20 . ~ o 1 I

Q

100 j . 80

~ 60 ai u "-IV 40 0..

20

0 1

c::J gerbil _ all pigeon ~ pigeon sec _ pigeonUTR c::J pigeon LAG

234 Quadrant

2 3 4 Quadrant

Figure 6. The bar graphs summarize the data obtained from the fixed histological data acquired from gerbil and pigeon. It points out that the majority of cells can be found in quadrants 1 and 3 and that these quad­rants are dominated by either type I or type II hair cells, respectively. The type I hair cell distribution is shown in (A) and points out that about 80% of type I hair cells are in quadrant 1. The type II hair cell distri­bution is shown in (8) and demonstrates that over 60% of type II hair cells are found in quadrant 3.

Gerbil Semicircular Canal Hair Cells

z\ P9*&0&4::888 IdslOlUgtcdftJ IdSliilileG gerbil hair cells and 107 dissociated gerbil hair cells was analyzed in a manner similar to that used for the pigeon data. Again, groups I and 3 represent the majority of hair cells and can sep­arate type I and type II hair cells. The mean ac­curacy value for the NPR method, using a cut­off value of 0.7, was 88.8%, while the NBR method. with a cutoff value of 0.48, was 92.6% accurate, supporting the conclusion that the ra­tio measurements can distinguish between cell types. The mean NPR and NBR values from each quadrant for gerbil cells (either fixed or dissociated) were not statistically different from either the histologic data or the dissociated pi-

A. J. Ricci et al

geon hair cells data (see : uble 2). A plot of NBR versus NPR is given in Figure 5. Just as with the pigeon data, the distributions separate type I and type II cells into two major popula­tions that are statistically distinct from each other. Statistical clusters, represented again by the shaded regions, were not statistically differ­ent from those established by the pigeon data. A summary of the percentages of cells found in each quadram is given in Figure 6 for direct comparison with the pigeon data. Also. Table 2 ,ummarize:; rhe :,;crbil dala for both fi ':ed :.md J issocialcu hair cells. Two Jifferences exist be­t\Vecn :he ~igc()11 Jam Lll1U ,he gerbil data. Tile fixed pigeon data resulted in three clusters while the gerbil fixed data reveals four. The three clusters are nor different from those of the fixed pigeon data, whereas the fourth cluster is equivalent to the fourth cluster described for the pigeon dissociated cells and in fact represents only about 5% of the population. The second difference is that for the fixed gerbil data, group 2 and cluster two are comprised largely of type I hair cells, while the pigeon data has a more mixed distribution that is dominated by type II hair cells. Again this group represents only about 5% of the total population of cells. A measure of the accuracy of the quadrants for separating the cells into the type I and type II categories could be calculated for the gerbil data, as it was for the pigeon. Both the histolog­ical and the dissociated cells gave an accuracy of 96%.

A summary of the NPR and NBR values for each quadrant and for each end organ is given in '8b12 2. I ne Pigeon and gerou see IS some-what biased toward cells from the first quad­rant; however, this may be a reflection of the proportion of this cell type found in the intact end organ. No statistical difference was seen between the NPR values or the NBR values be­tween end organs or between species (Table 2). Since the NPR and the NBR values are consis­tent between histologic data and dissociated hair cell data, and these measurements are capa­ble of discriminating between type I and type II hair cells in the histologic sections, it can be concluded that these measurements can differ­entiate between hair cell types when the hair cells are dissociated.

Morphometric Identification of Vestibular Hair Cells

Figure 6 summarizes the distribution of type I and type IT hair cells between quadrants for pi­geon and gerbil. The pigeon data are further separated by end organs. In all cases, group 1 represents the major type I hair cell population. and group 3 represents the major type Il popula­tion. The major difference between gerbil and pigeon is in the proportion of type Il cells found in quadrant 2. Virtually no (2%) gerbil type IT hair cells are found in this quadrant as com­pared to 35% for the pigeon. This difference is partly due to the otolith organs of the pigeon contributing a large percentage of these type II cells (the gerbil data is only from posterior SCC). It also may be a reflection of a small sampling size for the gerbil data.

Table 2 summarizes the ratiometric values for each quadrant for each end organ for each species. No statistical differences (Tukey's b test ANOV A P > 0.5) were observed for mea­surements within a quadrant. Each quadrant had at least one statistically distinct variable, as would be predicted if the quadrants represented four distinct groups. Quadrant 1 was statistically sig­nificantly different (ANOV A, Tukey's b test P < 0.001) from quadrant 3 in both the NPR and the NBR measurements.

Over 1600 cells were included in this study. The cells come from two different species, from three different end organs, and were assessed from either fixed histological tissue or from freely dissociated hair cells. In all cases, two dominant morphometric classifications could be made. the sum of which constituted more than 70% of the hair cell populations. One of these groups was cOI11po~ed of 96(ir typ'';: I hair eelb and tilt other of 969( rype II hair cell;,. demon~trating the strength 01 the ratiometric method of hair cell identification.

Discussion

The data presented here first used histological data from .the pigeon to define morphometric criteria, intrinsic to the hair cells, to distinguish between hair cell types. The morphometric ra­tios of NPR and NBR can be used to separate vestibular hair cells into four quadrants, t: two

403

major populations comprising about 70% of all cells, quadrants 1 and 3. These quadrants also separated type I (quadrant 1) and type IT cells (quadrant 3) with an accuracy of greater than 90%. Cluster analysis was used to statistically corroborate the quadrant assignment. Dissoci­ated pigeon hair cells were shown to separate similarly, based on hierarchical cluster analysis. Fixed histological and dissociated gerbil hair cells from the semicircular canals were shown to follow the same trend in NBR and NPR as the pigeon data, suggesting that this method may be useful across species.

Separation of cells into four quadrants based or type I or type Il classification was statisti­cally corroborated by cluster analysis, which grouped cells into four similar patterns as the quadrant separation. A small overlap exists be­tween a statistical cluster and quadrants 2 and 3. This overlap serendipitously points to the area of the NPR, NBR plot that fails to distinguish between the cell types. This area represents less than 5% of the total cell population. Cells in this area have neck regions that can be found in both type I and type IT hair cells. An electron micro­graph example of this type of type I cell is given in Figure 43 of Wersall and Bagger-Sjoback (16). Another example of a type I hair cell that falls into the 5% of ambiguity was given in Fig­ure 4A of Schessel and colleagues (17). This example, from the lizard, shows a hair cell with an NBR and NPR that would place the cell be­tween quadrant~:2 and 3. If this example is typi­cal for the lizard, then the method described here would not be adequate fOi distinguishing between hair cell tyne~ in tht lizard.

A mino:' di~(..'renan~·\ i.' rha: the fi>..ec! D'!2eOn . . data were clustered Into three group!.. Wh . Ie all other data. pigeon dissociated a~ well as gerbil fixed anci dissociated. were clustered into four groups. Importantly, the three clusters from the fixed pigeon data correspond to three of the four clusters from the other data sources. This dis­crepancy, which includes at most 5% of the cells studied, may be explained in a variety of ways. There may be a species difference. There may be a bias in the dissociation process. There may be a subpopulation of cells that have changed shape with the dissociation process or the fixa­l" ' 11 pl'Ocess.

404

Little quantitative data exist concerning the morphologic differences between type I and type IT hair cells. The ratio of neck width to cu­ticular plate width has been used previously to distinguish between the two cell types (9). As can be seen from Figure 3, an NPR cutoff value of 0.7 would select 85% of the type I hair cells and would be contaminated by 5% of the type II hair cell population. The type II hair cell popu­lation would misidentify the remaining 15% of [ \'De I hair cell .... "" ·t!thl1lluh ,~ffil..:;e"r ip -.:e!ec t -

ing type _ hal i" .:eib. ~he !,)PR il1ea~urel11enl

!acks ~ pec :fi L'i[ \ when ,elecring lype fl hall'

,;e!ls. Also. :-JPR alone would ~"(cilide J ,Jopula­[ion \)f type I ce!ls. It is difficult [0 assess [he ' qualitative methods used in separating type I and type II hair cells. It is possible that some of the electrophysiologic differences reported in the literature are due to either misidentifying a type II cell as a type I cell or, in the case of NPR identification, being limited to a particular class of type I hair cells (14,18-21). The plot of NBR versus NPR suggests that the neck properties exist in a continuum so that only the cells on the extremes would be clearly distinguishable qual­itatively. The concept of hair cells existing in a morphologic continuum has been suggested for the cells of the avian papilla (22). The plots of NBR versus NPR also suggest a morphometric continuum in which type I and type II hair cells are subsets. It will be interesting to determine if the morphometric continuum can be correlated to an electrophysiologi.c, functional continuum. The plots further demonstrate that the type I cells of quadrant 1 are more homogeneous than the type II cells of quadrant 3, based on the vari­ability observed in the scattergrams.

The functional significance of the morpho­metric variations between. type I and type II hair cells as well as between the subtypes of each population is unknown. Electrophysiologic dif­ferences have been described between type I and type II hair cells (18,20.21). The majority of the work that has studied the electrophysio­logic properties of the type I hair cell has used morphometric parameters, in some instances NPR, while in other cases the parameters are more subjective (that is, flask-shaped), to differ­entiate between type I and type IT cells. Where NPR was used for distinguishing cell types,

A. J. RiCCI el di

cells from the overlapping region of the fre­quency histogram were avoided (14,18). In these cases, it is possible that a population of type I cells were excluded from investigation, for ex­ample, group 2. The case where more qualita­tive cell type differentiation was used is more difficult to assess (19_21,23,24); however, given the overlap in morphometric properties it is pos­sible either that a population of type I hair cells was excluded or that a population of type II ':e lk '.V:1S !11i~ide:ltified 'lS type! cells. Either of lhe explanations could explain ,>ome of the elec­trophysiolngic Jisc:-epancies reported in [he iit­erature. The data presented here allow for addi­tional quantitative description and differentlation of type I and type II hair cells.

Kevetter and colleagues (6) suggested that dissociated hair cells could be morphologically distorted due to the loss of support from the ca­lyx or the surrounding epithelium, mechanical distortion due to trituration or maceration, os­motic differences, or the progressive degeneration of the isolated cells. Scarfone and colleagues (25) suggested that the morphology of the iso­lated hair cells was a function of the anesthesia used on the animal or the enzyme level used in the dissociation process. Data presented here suggest that the dissociated hair cells chosen for this study maintained their morphometric integ­rity at least in terms of the three measurements made here. There were no differences between the histologic values and the dissociated values for any of the variables measured. With the iso­lation procedures used here. cells that appeared to be electrically viable at the onset of the ex­periment maintained their shape as well as their membrane characteristics for several hours. Most of the cells included in this study were video­taped within the first 90 minutes of observation. This is not to say that the above distortions do not occur, but rather that it is possible to iden­tify cells where changes have occurred and to avoid including them in the analysis.

The presence of a neck region in isolated hair cells suggests that this thinning is intrinsic to the cell cytoskeletal structure and not im­parted by the surrounding calyx. More than 60% of the hair cells had neck regions as de­fined by either the NPR or the NBR. In general, type I hair cells had a more prominent neck in

Morphometric Identification of Vestibular Hair Cells

that both the NPR and NBR values were low. Selecting cells based on the presence or absence of a neck region is at best an oversimplification of the morphometric description of hair cells. The functional significance of the neck region is not clear, however, a motile function has been ascribed to this area (24,26-31). The neck re­gion of type I hair cells is capable of elongating and contracting in response to voltage changes. Since the measurements from the histologic data agree with those from the dissociated cells, the motile ability does not appear to influence the dissociated cell's morphology. However, this motile ability may be correlated to the neck shape and the prominence of a neck region in over 80% of the type I hair cells.

Scarfone and colleagues (l0) describe four subclasses of hair cells: two are type I cells. those with neck regions, and two are type II hair cells, those without neck regions. The work pre­sented here demonstrates that these qualitative descriptions and classifications are accurate only when describing extreme examples of hair cell types. It is clear from the data presented here that the majority of type I cells (88%) have neck regions that show a more defined initial taper and a more pronounced flaring at the basal end of the neck region than do type II hair cells; however, more than 70% of all cells showed some type of invagination below the cuticular plate and some type of flaring at the basal end of the cell. In addition, the spherical cell de­scribed by Scarfone and colleagues (I m was not found in the semicircular canaL neithe-r the dis­sociated celb nor the histologic section" of ei­ther tile gerbil or pIgeon. Ti1i~ cell type \\'a~.

found in the otoiith W~"!l~ se~'- c(lm/al~' . :.~~­

per), from which tissue \';a!-, obtained in tht· stud­ies of SCUlfone and colleagues (10). Howe\,er. as a general class of type II cells, the spherical

405

cell type is limited, since it involves only a small percentage of cells.

In conclusion, the data presented here de­scribe a method for discriminating between type I and type II hair cells that is consistent between end organs, between avian (White King pigeon) and mammalian (Mongolian gerbils) species, and between fixed hair cells and dissociated hair cells. It also demonstrates that dissociated hair cells can maintain theix: jntljvsic moq:>hologir integrity and that these morphometric properties can be used to identify cell type. Technically, the method is simple to employ, relying on three measure­ments that could be made at low power to dis­criminate between hair cell types. Since species differences may exist and dissociated cell mor­phology may vary depending on the procedures. it may be necessary initially to corroborate the basic NPR versus NBR plot. Given this, the mea­surements can be routinely made before electro­physiologic recording from the cell and may serve to eliminate some of the discrepancies presently existing concerning the electrophysio­logic properties of vestibular type I and type II hair cells. These results may serve to bridge the existing gap between studies on intact vestibu­lar end organs and those done on dissociated el­ements of the vestibular epithelium.

AckllOlvledgmems - Our thanks to Susanne 10hnston for her assistance in manuscripl preparation and for her ~eneral organi7.allonal skill,. and l[ Michelle Pacheco for her assistance with fi~urL' productilln. Thi~ \\ Clrk \\ a ~ · ,uppone(1 \:1~ a Nl H/NIDCD Rc) I A ward i DO)) :'.'7:)) \(. r\~. 1. :::-0!Tci .... .tdR i'. a l'\ ~.S.:.. J.!esearch i-·~;idV .. j:.JI~ I. ... dn l\lr; ;\.:.; ........ ah . .:n rcIlO\\ :-.upponeu 0)'

grant DCOO 16s!-02. GAK I~ supported by an RCDA (DCOO():'i2). SLC i,. supponed \:1~ i'iASA gran! J\AG 2-780. [,1.1C is a NlH Claude Pepper Investigalor.

REFERENCES

I. Wersall 1. Studies on the structure and innervation of the sensory epithelium of the crista ampullares in the guinea pig. Acta Otolaryngol 1956; 126S: I.

2. Jorgensen J, Anderson T. On the structure of the avian maculae. Acta Zoologica 1973;54: 121-30.

3. Correia MJ, Lang DG, Eden A. A light and electron microscope study of the Ilc.tl:,i i lcesses within tl. · pigeon anterior sf":licircular canal neuroepithelium. In:

Correia M, Perachio A. editors. Contemporary sensory neurobiology. Alan Liss Inc; 1985. p247-62.

4. Lindeman H, Reith A, Winther F. The distribution of type I and type II cells in the cristae ampullares of the guinea pig. Acta Otolaryngol 1981 ;92:315-21.

5. Lewis ER. Leverenz EL, Bialek W. The vertebrate inl1er ,"'r Boca Raton, Florida: CRe Press; 1985.

6. ~'l .dlCI IJA, Correia MJ, Martinez PRo Morphometric

studies of type I and type II hair cells in the gerbil's posterior semicircular canal crista. J Vestib Res 1994;4:429-36.

7. Lindeman HH, Ades H, West RW. Scanning electron microscopy of the vestibular end organs. In: Proceed­ings 5th symposium on the vestibular organs in space exploration. Washington: NASA; 1973. pI45-56.

8. Lapeyre P, Guilhaume A. Cazals Y. Differences in hair bundles associated with type I Jnd type II vestibular hair ~ells of the guinea pig sat:t:ule. Acta Otolaryngol 1991: I I :!:635-i2.

9. Cllrreia MJ. Christensen BN. "'Ioore LE. LUlg DG. Studies ,)f solitary ,emKircular ~anal hair ~elb 111 ihe adult pigeon. I: Fn':ljllcn<::, and tilll!! domain .Inai'. \1' ·)f Jt.:(1 \ C .Inti pas~I" l.: 1ll.:1l1tiralh.: :}n)pcri!c~;. I 'It.!url Q)I1., ,­

iol 1989:62:924- ~~. 10. S~arr"om: S. UlfenJahi vI. Lul',lranJ P. Finck .\. :_I:,!11l

and de~tron ITIIl.:rnscopy 'If isolated vestibular hair ·~dls

from the ~uinea 1ig. Cdl Tissue ~es 1991 a:266:5 I-~. II. Eden A., Correl.! YI1. Improved fixation llf the pigeon

brain by transcardiac carotid catheterization. Physiol Behav 1981;27:947-9.

12. Housley GD, Norris CH. Guth PS. Electrophysiologic propenies and morphology of hair cells isolated from the semicircular canal of the frog. Hear Res 1989;38:259-76.

13. Ricci AJ, Erostegui C, Bobbin RP, Norris CH. Compar­ative electrophysiologic propenies of guinea pig (Cavia cobaya) outer hair cells and frog (Rana pipiens) semi­circular canal hair cells. Comp Biochem Physiol 1994a; 107 A: 13-21.

14. Rennie lO, Correia MJ. Potassium currents in mamma­lian and avian isolated type I semicircular canal hair cells. J NeurophysioI1994;71:317-29.

15. Ward J. Hierarchical grouping to optimize an objective function. J Am Stat Assoc 1963;558:236-44.

16. Wersall J, Bagger-Sjoback D. Morphology of the vesti­bular organ. In: Kornhuber HH, editor. Handbook of sensory physiology. vol 6, pan I. New York: Springer­Verlag; 1974.

17. Schessel DA. Ginzberg R. Highstein SH. Morphophys­iology of synaptic transmission between type I hair cells and vestibular primary afferents: an intracellular study employing horseradish peroxidase in the lizard. Calores versicolor. Brain Res 1991 :544: 1-16.

A. J. Ricci et al

18. Correia MJ, Lang DG. An electrophysiologic compari­son of solitary type I and type II vestibular hair cells. Neurosci Lett 1990;116: 106-111.

19. Griguer C, Sans A, Valmier J, Lehouelleur J. Inward potassium rectifier current in type 1 vestibular hair cells isolated from the guinea-pig. Neurosci Lett 1993; 149:51-5.

20. Rennie KJ, Ashmore IF. Ionic currents in isolated vesti­bular hair cells from the guinea-pig crista ampullaris. Hear Res 1991 :51 : 279-9:!.

21. Eatock RA. Hutzler MJ. Ionic currents in vestibular hair cells. 1n: Cuht!n B. Tnmko D. Guedry F. l.!ditors. Sensing and controlling motion . NY Acad Sci 1992:656:38-74.

22. 3ri" 1. Fi,hl.!r FP. \'Ianky GA. The -,:ulIl:lllar plate of he Iwir ';~il in relall,)n n morphological .,;radients "r

the chicken basi liar papi lIa. Hear Res 1994:75:2-4-56. 23. '/.liat J. Devall .J. DIIIIlIl D. 5alb -\. Vl.!slIilular hair

Cl.!lIs isolated rnJlil the guinea pig labynnth. Hear Res ! 9Xl):-Hl:~55-olJ.

24. Sans A. Grigut!r C, Lehouelleur 1. The ve!>lIbular type [ hair cells: a self regulating system? Acta Otolaryngol SuppI1994:513:11-4.

25. Scarfone E, Ulfendahl M, Figueroa L, Flock A. Anes­thetics may change the shape of isolated type I hair cells. Hear Res 199Ib;54:247-50.

26. Didier A, Decory L. Cazals Y. Evidence for potassium­induced motility in type I vestibular hair cells in the guinea-pig. Hear Res 1990;46: 171-6.

27. Lapeyre P, Cazals Y. Guinea-pig vestibular type I hair cells can show reversible shonening. J Vestibular Res 1991;1:241-50.

28. Zenner H, Zimmermann U. Motile responses of vestib­ular hair cells following caloric, electrical or chemical stimuli. Acta OtolaryngoI1991;111:291-7.

29. Ogata y, Sekitana T. Motility of vestibular hair cells in the chick. OtorhinolaryngoI1993;55:135-9.

30. Griguer C, Lehouelleur J, Valat J, Sahuquet A, Sans A. Voltage-dependent reversible movements of the apex in isolated guinea pig vestibular hair cells. Hear Res 1993;67:110-16.

31. Rennie lO, Ricci AJ, Correia MJ. Potential driven movements of type I vestibular hair cells [Abstract]. Biophys J 1994:66(2):AI69.