Directional response properties of saccular afferents of the toadfish, Opsanus tau

Hearing Research 111 (1997) 1^21

Directional response properties of saccular a¡erents of the toad¢sh, Opsanus tau

Richard R. Fay a

a ;b ;

*, Peggy L. Edds-Walton

b

Parmly Hearing Institute and Department of Psychology, Loyola University Chicago, 6525 North Sheridan Ave., Chicago, IL 60626, USA

b

Marine Biological Laboratory, Woods Hole, MA 02543, USA

Received 22 March 1997; accepted 8 April 1997

Abstract

The displacement sensitivity, frequency response, and directional response properties of primary saccular afferents of toadfish (Opsanus tau) were studied in response to a simulation of acoustic particle motion for which displacement magnitudes and directions were manipulated in azimuth and elevation. Stimuli were 50, 100, and 200 Hz sinusoidal, translatory oscillations of the animal at various axes in the horizontal and midsagittal planes. Thresholds in these planes defined a cell's characteristic axis (the axis having the lowest threshold) in spherical coordinates. Recordings were made from afferents in rostral, middle, and caudal bundles of the saccular nerve. The most sensitive saccular afferents responded with a phase-locked response to displacements as small as 0.1 nm. This sensitivity rivals that of the mammalian cochlea and is probably common to the sacculi and other otolith organs of most fishes. Most afferents showed lower thresholds at 100 Hz than at 50 or 200 Hz. Eighty percent of afferents have three-dimensional directional properties that would be expected if they innervated a group of hair cells having the same directional orientation on the saccular epithelium. Of the afferents that are not perfectly directional, most appear to innervate just two groups of hair cells having different orientations. The directional characteristics of afferents are qualitatively correlated with anatomically defined patterns of hair cell orientation on the saccule. In general, azimuths of best sensitivity tend to lie parallel to the plane of the otolith and sensory epithelium. Elevations of best sensitivity correspond well with hair cell orientation patterns in different regions of the saccular epithelium. Directional hearing in the horizontal plane probably depends upon the processing of interaural differences in overall response magnitude. These response differences arise from the gross orientations of the sacculi and are represented, in part, as time differences among nonspontaneous afferents that show level-dependent phase angles of synchronization. Directional hearing in the vertical plane may be derived from the processing of across-afferent profiles of activity within each saccule. Fishes were probably the first vertebrates to solve problems in sound source localization, and we suggest that their solutions formed a model for those of their terrestrial inheritors. Keywords :

Saccule; Directional sensitivity; Sound localization; Toad¢sh

1. Introduction

The inner ears of all vertebrates function in hearing and balance. The semicircular canal organs have exclusively vestibular roles, but the otolith organs may function in diverse roles including the maintenance of equilibrium (e.g., Fernandez and Goldberg, 1976; Platt, 1973), vibration detection (e.g., Young et al., 1977; Narins and Lewis, 1984), and in the detection of sound * Corresponding author. Tel.: +1 (773) 508-2714; Fax: +1 (773) 508-2719; E-mail: [email protected]

transmitted by the middle ear (e.g., McCue and Guinan, 1994; Cazals et al., 1980). Multiple functions of the otolith organs appear to be a widely shared vertebrate character (e.g., Lowenstein and Roberts, 1950; Budelli and Macadar, 1979 ; Koyama et al., 1982 ; Wit et al., 1984). The otolith organs (saccule, utricle, and lagena) include a sensory epithelium containing hair cells and support cells, and an overlying otolithic mass, or otolith, to which the hair cell cilia are coupled via an otolithic membrane. Structurally, these organs appear to be adapted as statolith organs (reviewed in Lowenstein,

0378-5955 / 97 / $17.00 ß 1997 Elsevier Science B.V. All rights reserved PII S 0 3 7 8 - 5 9 5 5 ( 9 7 ) 0 0 0 8 3 - X

HEARES 2838 11-11-97

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

2

1977) responding to the direction and magnitude of the

We have studied the primitive, accelerometer mode

Opsanus tau),

projection of the gravity vector with respect to head

of hearing in toad¢sh (

orientation. Directional response to head vibration is

apparently not specialized for sound pressure detection.

a

to

We investigated the displacement sensitivity and direc-

linear accelerations, but also requires response to fre-

tional response properties of primary saccular a¡erents

quencies above those normally associated with postural

in response to a simulation of acoustic particle motion

movements.

additional

for which displacement magnitudes and directions were

adaptations for transmitting sound energy to the endor-

manipulated and measured in three-dimensional space.

natural

consequence

of

Responses

these

to

organs'

sound

sensitivity

require

gans. Fishes are the only vertebrate group for which

We

have

chosen

to

study

the

a species that is

toad¢sh

for

several

one or more of the otolith organs are adapted to func-

reasons. There is behavioral evidence that the location

tion exclusively or predominantly in hearing. In most

of a vocalizing conspeci¢c is important for the social

¢shes, the saccule is believed to be the primary auditory

behavior of this species (Gray and Winn, 1961). Fe-

endorgan (reviewed by Popper and Fay, 1993).

males approach males that emit the `boatwhistle' call

Hearing in ¢shes has been de¢ned by sound detec-

from at least 2 m away (Fish, 1972), and males avoid

tion thresholds comparable to those of most other ver-

the territories of vocalizing males. The sound pressure

tebrates (reviewed in Fay, 1988), behavioral capacities

level of the boatwhistle has been measured to be 140 dB

for sound analysis that are qualitatively similar to those

re : 1

of other vertebrates (Fay, 1992), and an ascending audi-

its attenuation with distance has been characterized by

tory

pathway

Fine and Lenhardt (1983). Thus, we know the levels

and

Hernandez,

that

is

anatomically

(e.g.,

McCormick

WPa

at 1 m from the source (Tavolga, 1971), and

Lu

and frequency range of sounds that are known to be

and Fay, 1995) organized according to the general ver-

of biological signi¢cance. In addition, the anatomical

1996)

and

physiologically

(e.g.,

Carassius auratus)

the

organization of the saccule (Edds-Walton and Popper,

most intensively studied of the ¢shes that meet these

1995) and primary octaval nuclei (Edds-Walton, 1994 ;

criteria. Gold¢sh are highly specialized to detect sound

Highstein et al., 1992) have been described, hair cell

using a series of modi¢ed vertebrae, the Weberian os-

biophysics has been characterized (e.g., Steinacker and

sicles (Weber, 1820), that mechanically link the swim-

Perez, 1992), and the various branches of the VIIIth

bladder with the sacculi. As the swimbladder expands

nerve are accessible in vivo.

tebrate

plan.

The

gold¢sh

(

is

and contracts under the in£uence of the sound pressure

Our results indicate that most saccular a¡erents are

waveform, the ossicles transmit these motions to the

broadly tuned with a best frequency between 50 and

sacculi. Thus, gold¢sh and other species having similar

200 Hz, and that the most sensitive a¡erents respond

links are sensitive to sound pressure (reviewed by Fay,

to motions as small as 0.1 nm. By examining the re-

1988).

sponse

characteristics

of

individual

a¡erents

during

Most species of ¢sh do not have links between the

stimulation at various angles in the horizontal and mid-

swimbladder (or other gas bubble) and the ears, and

sagittal planes at biologically relevant levels and fre-

thus may or may not be sensitive to sound pressure.

quencies,

However, all ¢shes apparently share a more primitive

in the toad¢sh have response properties that provide

mode of hearing in which one or more of the otolith

information necessary for three-dimensional directional

organs respond directly, as inertial accelerometers, to

hearing.

we

have

determined

that

saccular

a¡erents

the acoustic particle motion that accelerates the ¢sh's body sound

in

a

sound

detection

¢eld.

is

This

inherently

accelerometer directional

mode

because

of the

2. Materials and methods

hair cells that transduce relative otolith movement are directional receivers (Flock, 1964) arrayed over the oto-

2.1. Preparation of the toad¢sh

lithic epithelia with their axes of best sensitivity oriented in various directions (e.g., Popper and Coombs, 1982).

Toad¢sh

15^25

cm

in

total

length

were

obtained

A representation of the axis of acoustic particle motion

from the Marine Biological Laboratory (MBL) Depart-

is thought to be computed from the responses of pri-

ment of Marine Resources and maintained at the MBL.

mary otolithic a¡erents that encode directional inputs

Fish were kept at 10^20³C with constant £ow of fresh

from hair cells (e.g., Schuijf, 1975, 1981 ; Rogers et al.,

seawater and weekly feedings with marine worms or

1988 ; Schellart and De Munck, 1987). Although prim-

squid. The toad¢sh used appeared to be sexually ma-

itive, this mode of hearing has only rarely been studied

ture : females had eggs of various sizes and stages of

quantitatively

development, and the testes of males were enlarged.

(e.g.,

Chapman

and Sand,

1974 ;

Sand

and Karlsen, 1986), primarily because it is di¤cult to

Toad¢sh were anesthetized (immersed in a bath of a

manipulate, measure, or calculate the magnitude and

1 :4000 solution of 3-aminobenzoic acid, methanesulfo-

direction

nate salt, Sigma) and immobilized (an i.m. injection of

motion.

of

the

adequate

stimulus :

acoustic

particle

0.1^0.2 ml of pancuronium bromide in a 2 mg/ml sol-

HEARES 2838 11-11-97

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

ution; Sigma) and placed in a saltwater-¢lled Plexiglas box with the cranium held in place with metal pins. The cranium was entered dorsally and the £uids surrounding the inner ear were gradually replaced with a £uoroinert liquid (FC-77, 3-M Corp.) to provide a clearer view of the saccular nerve and to promote oxygenation of the tissues. Care was taken not to injure any of the blood vessels or other tissues surrounding the ears. All recordings were made from the saccular nerve of the left ear with the electrode placed as close as possible to the epithelium. Saccular a¡erents enter the medulla in the posterior ramus of the VIIIth nerve. The anterior ramus, which innervates the utricle and the anterior and horizontal canal cristae, overlies the posterior ramus. Pieces of ¢ne surgical thread (2^3 mm long) were used to gently de£ect the anterior ramus of the VIIIth nerve to reveal the saccular nerve. In our observations, saccular a¡erents exit from their sites of innervation in various bundles, ranging in number from two (rostral and caudal bundles) to seven. To reveal the caudal bundles required the use of two pieces of surgical thread: one placed just rostral, and the other just caudal, of the saccular sensory epithelium. Upon completion of the surgery, the ¢sh was transferred to an aluminum cylinder (diameter = 23 cm, height = 8 cm, wall thickness = 0.5 cm) ¢lled with fresh seawater. The cylinder was mounted on a threedimensional shaker table (described below). A rigid holder was used to secure the head of the ¢sh in the cylinder. Water was changed when necessary to maintain temperature and appropriate dissolved oxygen levels. 2.2. Directional stimulation

The shaker table (Fay, 1984) used a system of moving-coil shakers to create sinusoidal translational motion in three-dimensional space (Fig. 1). One pair of shakers (Bruel and Kjaer #4810) was attached to the cylinder at the head and tail of the ¢sh and another pair was attached orthogonally to each side of the cylinder (Fig. 1A). Shaker pairs were used to help create linear, translatory motion in the horizontal plane without large o¡-axis (i.e., vertical) motions, or rotations. A ¢fth shaker (B and K 4809) was located below the cylinder to provide vertical stimulation (Fig. 1B). Signals to the three shaker channels were digitally synthesized (500 ms sinusoids with 20 ms rise and fall times), read out of Tucker Davis Technologies (TDT) 16-bit digital-to-analog converters (10 kHz sample rate), low-pass ¢ltered at 2 kHz, attenuated using TDT PA4 programmable attenuators, and ampli¢ed (Techron, model 5507). The ampli¢ed signals were then attenuated by 32 dB using ¢xed power resistors in order to reduce ampli¢er noise. Software was written to generate sinusoids with appro-

3

priate starting phases and amplitudes to create translational oscillatory movements of the cylinder in the horizontal and midsagittal planes of the ¢sh (Fig. 1C,D). The shaker table was supported by a pneumatic vibration isolation system. Cylinder movement was calibrated by monitoring the activity of three, orthogonally oriented accelerometers (Piezotronics model 002A10, Flexcel). A calibration program permitted all stimuli used to be speci¢ed and recorded in advance of physiological recording with the ¢sh in place. Actual stimulus directions and amplitudes were calculated from digital recordings of the accelerometer outputs. The measured stimulus directions varied less than 2³ from the nominal values for all experiments. Subsequent data analyses used the measured stimulus directions and amplitudes. Stimuli consisted of sinusoidal oscillation at 50, 100, or 200 Hz along speci¢ed translational axes. For 50 and 200 Hz, there were three stimulus directions: front-back (0³ azimuth in the horizontal plane as shown in Fig. 1C), side-to-side (90³ azimuth in the horizontal plane) and up-down (90³ elevation). These directions will be referred to as the `three orthogonal directions'. For 100 Hz stimuli, there were six azimuths in the horizontal plane (90³, 60³, 30³, 0³, 330³, 360³) and six elevations in the midsagittal plane (0³, 30³, 60³, 90³, 120³, 150³) as illustrated in Fig. 1C,D. For each axis of motion, stimulus level was varied in 5 dB steps until responses were obtained both above and below threshold (de¢ned below). 2.3. Extracellular recording

Extracellular recordings were made using 3M KCl¢lled pipettes pulled (Sutter Instrument Co., laser puller P-2000) to a tip resistance of 40^100 M6. Bundles of the saccular nerve were approached as close to the saccular epithelium as possible. In most cases, the pale outline of the entire dorsal edge of the saccular epithelium could be seen, permitting us to determine electrode location along its length. Fig. 2 shows typical branching patterns of the toad¢sh saccular nerve as it exits the epithelium. To record from a¡erents most likely innervating the rostral or caudal portions of the saccule, electrode penetrations were restricted to the rostralmost or caudalmost edges of the rostral or caudal bundle, respectively (circle and square in Fig. 2, top). Recordings from a¡erents innervating the middle portion of the saccule were made only in ¢sh that had a distinct bundle exiting from the middle region of the saccule (arrow in Fig. 2, top). Fig. 2, middle, shows the generalized pattern of hair cell orientation over the epithelial surface of the saccule (Edds-Walton and Popper, 1995). Arrowheads represent the orientation of the kinocilium on the apical surface of hair cells and indicate the most excitatory directions of stereociliar de£ection. Fig. 2,

HEARES 2838 11-11-97

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

4

Fig. 1. Schematic diagram of the three-dimensional shaker system. A : Plan view showing two pairs of shakers for x and y stimulation in the horizontal plane. Opposing shakers are 180³ out of phase, creating a push-pull action on the dish. The calibrated accelerometers shown here and in B recorded the actual motion of the dish. B : Side view of the shaker system illustrating the vertical (z-axis), and vibration isolation of the table. C : Stimulus axes in the horizontal plane. D : Stimulus axes in the midsagittal plane.

bottom, shows an innervated saccular epithelium illus-

2.4. Data analyses

trating the branching patterns typical of the di¡erent Spike times occurring during sinusoidal stimulation

saccular nerve bundles. A search stimulus was synthesized to produce nearly

were used to calculate period histograms. Period histo-

circular motion in three dimensions at 100 Hz. This

grams are frequency distributions of the stimulus phase

stimulus provided constantly changing directional stim-

angles at which spikes occur over a single sinusoidal

ulation to increase the chances of detecting a cell re-

stimulus cycle. The coe¤cient of synchronization (R)

gardless of its directional preference. Spikes were dis-

(Goldberg

criminated using a voltage criterion under software

spikes (Ns ) recorded during the 3.75 s period during

control, and spike times were recorded with 0.1 ms

which the stimulus was on at full amplitude, and the

resolution.

response phase angle (essentially, the stimulus phase

and

Brown,

1969),

the

total

number

of

All 100 Hz stimuli were presented ¢rst, followed by

angle at the peak of the period histogram) were com-

the 50 Hz and 200 Hz stimuli. If an a¡erent was lost before the full set of thresholds could be determined,

puted from period histograms. For each stimulus, the 2 statistic Z = R Ns (Batschelet, 1981) was obtained as a

partial

lowest

function of level. The threshold for each stimulus direc-

threshold in three-dimensional space, response phase

data

sets

were

used

to

estimate

the

tion was de¢ned using linear interpolation as the root

angles, and spontaneous activity. The minimum data

mean

obtained were for the three orthogonal stimuli at 100

Z = 20. The probability of obtaining a Z value of 20

Hz. If an a¡erent was held long enough to determine all

or more by chance is less than 0.001, assuming that

thresholds in the horizontal and midsagittal planes, the

all spike phase angles are equally probable (Batschelet,

axis of motion for which threshold was lowest was esti-

1981).

mated in spherical coordinates. We term this the characteristic axis, or CA.

square

(rms)

displacement

corresponding

to

Thresholds for the stimulus set were used to determine the extent to which each a¡erent was directionally

HEARES 2838 11-11-97

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

Fig. 2. The saccule of the toad¢sh,

Opsanus tau.

5

Rostral is to the right. Top : Innervation and recording sites along the saccule : open circle =

rostral site ; arrow = middle site ; open square = caudal site. Middle : Typical hair cell orientation pattern. The arrows point in the direction of maximum response for the hair cells of the region, i.e., the arrowhead indicates the orientation of the kinocilium with respect to the center of the stereovillae bundle. Bottom : Saccular nerve branching on the epithelium. Peroxide and a DAB reaction stained the nerve. C = caudal ; M = middle ; R = rostral.

selective, the lowest threshold, and the axis of motion in

angle (Flock, 1964 ; Hudspeth and Corey, 1977) where

spherical coordinates for which threshold was lowest

0³ is the axis that passes through the center of the hair

(the characteristic axis, or CA). Fig. 3 gives the ration-

bundle and the kinocilium (dotted line with arrowheads

ale for these determinations. Fig. 3A shows a cartoon

in Fig. 3A). In polar coordinates, the plot of this cosi-

of the apical surface of a hair cell. For hair cells, re-

nusoidal response function is a pair of circles tangent at

sponse magnitude is a cosine function of displacement

the origin, representing both the depolarizing and hy-

HEARES 2838 11-11-97

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

6

potential.

sphere in spherical coordinates is a plane. To the extent

Within a hair cell's dynamic range, the receptor poten-

that primary a¡erents are directional in the manner of

tial is proportional to the magnitude of the vectorial

hair cells, all thresholds will fall on a `threshold plane'.

stimulus

Once the threshold plane is de¢ned, the CA in spherical

perpolarizing

phases

of

component

the

that

cell's

lies

receptor

along

the

hair

cell's

coordinates is de¢ned as the azimuth (A) and elevation

most excitatory axis. In this study, directionality of primary a¡erents was

(E) of the vector that passes through the origin and is

de¢ned using displacement thresholds rather than re-

perpendicular to the plane. The best threshold in spher-

sponse magnitudes. Fig. 3B illustrates the relationship

ical coordinates is the magnitude of that vector from

between response magnitudes (circles) and thresholds

the origin to the plane. The threshold plane was de¢ned using three points :

(squares) in the horizontal plane using threshold data from

a¡erent I3.

In a linear

system, the

signal

level

thresholds

at

0³

(front-back)

and

90³

(side-side)

azi-

producing a criterion response (i.e., a threshold) is pro-

muth in the horizontal plane, and a threshold at 90³

portional to the reciprocal of response magnitude, and

elevation (vertical). To obtain these thresholds, linear

the reciprocal of the response cosine function passing

functions were ¢tted to the threshold lines in the hori-

through the origin in polar

coordinates

zontal and midsagittal planes. The x-, y-, and z-inter-

line.

a¡erent

is a straight in

cepts of these functions (illustrated by points L, F, and

the manner of hair cells, thresholds will fall on a line

U in Fig. 3E,F) de¢ned the three points used to specify

(the `threshold line'). In Fig. 3B, response magnitudes

the threshold plane. The threshold at 0³ azimuth (OF)

and thresholds are plotted at two locations : once at the

was calculated as the average of the points estimated

nominal stimulus angle, and once again at this angle

from both threshold lines, since both

threshold lines

plus

have

were

To

the

180³.

extent

This

that

an

convention

was

is

directional

adopted

because

the

this

point

in

common.

If

data

obtained

stimulus was oscillatory motion along the given axis

only for the three orthogonal stimuli, the best threshold

and

in

the

response

measured

was

an

averaged

spike

three

dimensions

was

calculated

using

these

data

one

points, but the CA could not be unambiguously de¢ned

the

with respect to its quadrant. The percent of variance

data. The lengths of the radial line segments between

accounted for by the best linear ¢t to the threshold lines

rate.

Thus,

polarity

of

there

was

oscillation

no

rationale

over

the

for

other

selecting

in

plotting

placement thresholds in nanometers. The stimulus axis

r2 ) 2 (r

at which threshold is lowest passes through the origin

either plane).

the origin and the threshold symbols represent the dis-

(

v

was

used

to

de¢ne

an

a¡erent

as

directional

0.9 in both planes) or nondirectional (

r2

6

0.9 in

and is perpendicular to the threshold lines. The length of this line between the origin and the threshold lines is the calculated lowest displacement threshold.

3. Results

Fig. 3C,D illustrate threshold data for a¡erent L6 studied

planes

Extracellular recordings were made from 257 saccu-

(D). The radial lines with arrowheads indicate the ori-

in

the

lar a¡erents. Data were obtained from 114 a¡erents of

entations

of

horizontal

the

best

(C)

and

stimulus

midsagittal

lowest

the rostral bundle of 27 ¢sh, 41 middle bundle a¡erents

thresholds in these two planes. Fig. 3E indicates the

axes

and

the

in 9 ¢sh, and 55 caudal bundle a¡erents in 9 ¢sh. The

de¢nition of the azimuth (angle A) of the best axis in

origins of 47 a¡erents from various ¢sh appeared to be

the horizontal plane, and the best threshold (OH) in

near borders between bundles and were not classi¢ed

this plane. Fig. 3F illustrates how the elevation (angle

with respect to the presumed epithelial region that they

E) of the characteristic axis (CA) and threshold (length

innervated. All vibration-responsive a¡erents responded

of CA) are determined.

The ¢gure caption gives the

with a phase-locked response giving rise to a unimodal

de¢nitions of the points, lines, and angles in Fig. 3E,F.

period histogram. Stimulus-driven spike rate increments

In three-dimensional space, a cosinusoidal response

tended to occur only for stimulus frequencies that were

function (Fig. 3A,B) is a sphere. The reciprocal of a

greater than a given a¡erent's spontaneous rate.

C

Fig. 3. Summary of the method used to estimate an a¡erent's characteristic axis (CA) and best threshold in spherical coordinates. A : Cartoon

of apical surface of a hair cell showing stereovillae and kinocilium. The circles tangent at the origin show a cosinusoidal directional response function. The arrows and dotted line indicate the most excitatory axis. B : Horizontal plane thresholds for a¡erent I3 (open squares) in polar coordinates and the best-¢tting lines. Tangent circles with ¢lled dots are reciprocals of the thresholds. The radial distance from the origin to each open square is the displacement threshold for stimulation along the axis angle indicated. The arrows and dotted line perpendicular to the threshold lines indicate the best direction for this a¡erent. C : Threshold data for a¡erent G4 in the horizontal plane. D : Thresholds for G4 in the midsagittal plane. Arrowheads indicate the most excitatory axis and the lowest threshold for the respective planes. E : Diagrammatic representation of the trigonometry used to obtain the azimuth (A) and the magnitude of the best threshold vector (OH) in the horizontal plane. Angles are in degrees. OL = threshold at 90³ azimuth ; OF = threshold at 0³ azimuth ; O = polar coordinate origin ; A = best stimulation angle in horizontal plane = atan(OF/OL) ;

3

OH = OLcos(90

A). F :

Representation of the trigonometry used to obtain the azimuth, elevation (E) and

3

best threshold (CA) in spherical coordinates. OU = threshold at 90³ elevation. E = atan(OH/OU) ; CA = OUcos(90

HEARES 2838 11-11-97

E).

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

3.1. Thresholds

7

illustrated in Fig. 4A,C. The interpolated displacement levels corresponding to a response magnitude of Z = 20

For each a¡erent, thresholds were estimated for each

and 80 are plotted in polar coordinates in Fig. 4B. The

stimulus axis from response versus level functions, as

thresholds for directional a¡erents tend to form a linear

HEARES 2838 11-11-97

8

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

function. Fig. 4D shows the data of Fig. 4A,C plotted as responsiveness functions in polar coordinates. It can be seen that a threshold line is equivalent to a responsiveness circle passing through the origin. For 81% of the saccular a¡erents studied in at least one plane, the directional thresholds were well ¢tted by lines (Fig. 4B). The response versus level functions for these directional a¡erents were substantially parallel (Fig. 4A,C). Thus, directionality is substantially independent of level within the a¡erent's dynamic range. Fig. 5 shows the relationships between threshold and spontaneous rate for 210 saccular a¡erents categorized with respect to the nerve bundle in which they were recorded. We de¢ned any spike activity occurring in

the absence of deliberate stimulation as spontaneous activity. Best displacement thresholds ranged from 300 to 0.1 nm (39 to 380 dB with respect to 1 Wm). There is no evidence that the di¡erent saccular nerve branches di¡ered in average sensitivity. Nonspontaneous and spontaneously active ¢bers were found in all regions of the saccule. The distribution of spontaneous spike rates is continuous from zero to nearly 200 spikes/s. A¡erents having spontaneous rates above 100 spikes/s had the lowest thresholds in all ¢sh except one, whose ¢ve caudal ¢bers were relatively insensitive, but had relatively high rates of spontaneous activity (box in Fig. 5C).

Fig. 4. A and C: Response versus level functions for a representative directional saccular a¡erent. The Z statistic is plotted as a function of signal level in dB for the six stimulation axes in the horizontal plane (stimulus azimuth is given in degrees at the top of each function). The functions in A and C were compared two ways to de¢ne directionality: horizontal comparisons de¢ne iso-response functions (thresholds) ¢tted by lines in B; vertical comparisons de¢ne iso-level functions (responsiveness) ¢tted by circles in D. The scales of the radial axes in B and D are given in units of nanometers and Z, respectively. Filled and open symbols serve to avoid confusing data points from di¡erent functions.

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R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

Fig. 5. Scatterplots of spontaneous rate (in spikes/s) versus best threshold (in dB re : 1

Wm)

9

for a¡erents from the rostral (A), middle (B), and

caudal (C) saccular nerve bundles. The box in C identi¢es data from one animal with unusually high spontaneous activity (see text).

3.2. Frequency response

near the origin indicate broad tuning (shallow slopes) while points near the upper left of the quadrant indicate

Three-point threshold tuning curves were obtained

sharper tuning (steep slopes). The single point in the

for 116 a¡erents. Ninety-¢ve a¡erents had lower thresh-

lower right quadrant indicates that one a¡erent was

olds at 100 Hz than at 50 or 200 Hz, 16 responded best

slightly more sensitive at 50 and 200 Hz than at 100

at 200 Hz, and four responded best at 50 Hz. Fig. 6

Hz. The inset shows tuning curves averaged across af-

summarizes the frequency response data. Each point

ferents for each of the rostral, middle, and caudal bun-

indicates the slopes of the tuning curve between 50

dles. The averaged slopes are

and 100 Hz (abscissa) and between 100 and 200 Hz

below 100 Hz, and are 6.5^10.3 dB/octave above 100

(ordinate). Points falling in the upper left quadrant in-

Hz. There is no evidence that a¡erents from di¡erent

dicate that the lowest threshold was at 100 Hz. Points

bundles are tuned di¡erently, on average.

3

7.8 to

3

13 dB/octave

Fig. 6. Summary of the frequency tuning characteristics of saccular a¡erents. Three-point, displacement tuning curves were formed at 50, 100, and 200 Hz. Points plot the slopes of the tuning curves between 50 and 100 Hz (abscissa) and between 100 and 200 Hz (ordinate). Inset : Average tuning curves for rostral, middle, and caudal bundle a¡erents, and the average slopes in dB/octave. Vertical lines (displaced horizontally for clarity) indicate plus and minus one standard deviation.

HEARES 2838 11-11-97

10

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

HEARES 2838 11-11-97

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

3.3. Response phase

11

angles for all directional a¡erents in response to the three

orthogonal

stimuli.

The

frequency

distributions

For a hair cell stimulated along axes in the plane of

(bars at top) indicate that response phase varies among

its apical surface, response phase should shift by 180³ at

a¡erents over a 360³ range, and there is little or no

two

evidence for two preferred response phases separated

stimulus

axes :

+

3

and

90³

with

respect

to

the

orientation of the cell's best excitatory axis. Directional

by

primary a¡erents are expected to behave similarly to

sponse phase and the threshold at the CA, spontaneous

180³.

the extent that they innervate a group of hair cells hav-

rate, azimuth, or elevation. The scatter plots indicate

ing substantially the same directional orientation (e.g.,

that response phase angles are correlated for the three

Horner et al., 1981). For any single directional stimulus,

pairs of stimulus axes shown ; there is a weak tendency

the distribution of period histogram phase angles across

for points to cluster on positive diagonals. For these

a¡erents should be bimodal with peaks separated by

a¡erents,

180³. This prediction arises from the fact that hair cells

direction tends to equal the response phase angle (+

vary widely over the sensory epithelium in their mor-

and

phologically de¢ned best excitatory axis (Edds-Walton

ing near diagonals that do not pass through the origin

and Popper, 1995). In order to evaluate this prediction,

(dotted

period histogram phase for saccular a¡erents was meas-

among a¡erents. In panel C, for example, a¡erents hav-

ured in response to the three orthogonal stimuli (0³ and

ing phase angles near 0³ for both the vertical stimulus

90³ in azimuth, and 90³ in elevation).

(abscissa) and the front-back stimulus (ordinate) could

3

No

the

correlations

response

were

phase

apparent

angle

for

between

one

re-

stimulus

180³) to the other stimulus directions. Points fall-

lines

in

Fig.

8)

re£ect

directional

di¡erences

in

be described as producing spikes at acceleration peaks

some a¡erents but not in others. Fig. 7AFig. 7B illus-

for movement upward and to the front. A¡erents hav-

trate these e¡ects for two representative a¡erents. Spike

ing phase angles near 0³ for the vertical stimulus but

rate (diamonds), the Z statistic (lines without symbols),

phase angles near + and

the coe¤cient of synchronization (Xs), and period his-

ulus

togram phase (dotted lines) are plotted as a function of

acceleration peaks for movement upward and to the

signal

Response

phase

varied

widely

with

signal

level

could

then

be

3

180³ for the front-back stim-

described

as

producing

spikes

at

a¡erent's

back. In spite of these within-a¡erent correlations, the

dynamic range, phase may advance with level by 90³

fact remains that di¡erent a¡erents vary continuously

or more (a¡erent W15), or hardly at all (a¡erent Y6).

over 360³ in response phase to a given stimulus. This

Note that a¡erent W15 is nonspontaneous and is about

variation is

45 dB less sensitive than the highly spontaneous a¡erent

stability of the phase data in Fig. 7A,B).

level

in

1-dB

increments.

Within

an

not

due to

measurement

error

(note the

Y6. Note also that a¡erent W15 synchronizes robustly at all suprathreshold levels while Y6 shows less synchronization

that

increases

throughout

the

3.4. Directional a¡erents

dynamic An a¡erent was classi¢ed as directional if the var-

range. Level-dependent

phase

shifts

are

correlated

with

r2 )

iance accounted for by linear ¢t (

to threshold lines

spontaneous rate for most a¡erents : highly spontane-

in

ous a¡erents showed little or no phase shift while low

than or equal to 0.9. Of 231 a¡erents that were eval-

spontaneous a¡erents showed the greatest shifts. These

uated for directionality in at least one plane, 187 met

e¡ects are summarized in Fig. 7C in which the slopes of

this criterion. The response of nondirectional a¡erents

the phase versus level functions are plotted with respect

will be presented in the next section. Of the 187 direc-

to

tional

spontaneous

highest the

rate

spontaneous

lowest

rates.

for

the

rates

The

34

and

mean

a¡erents

the slope

34 is

having

a¡erents 0.13

the with

deg/dB

the

horizontal

a¡erents,

and

96

midsagittal

were

studied

planes

was

completely

greater

in

both

planes. Of these, 78 were unambiguously identi¢ed as arising from either the rostral (

n = 34)

n = 22),

middle (

n = 22)

(S.E.M. = 0.16 deg/dB) for high spontaneous a¡erents,

or caudal (

and 3.7 deg/dB (S.E.M. = 0.16 deg/dB) for low sponta-

spection of electrode penetration sites on the saccular

neous a¡erents.

nerve during and following each experiment.

Given this level dependence, a¡erents were compared

saccular bundles based on visual in-

Fig. 9 shows thresholds in polar coordinates for four

with respect to period histogram phase angles interpo-

representative

lated at a criterion response magnitude of Z = 20. Fig. 8

for determining an a¡erent's CA in spherical coordi-

shows scatter plots and frequency distributions of phase

nates. As an example, the thresholds and threshold lines

saccular

a¡erents,

illustrating

the

logic

6

Fig. 7. E¡ects of level on response phase. A and B : Response magnitude (Z statistic), phase (period histogram phase), spike rate, and coe¤cient of synchronization (R) versus stimulus level for two a¡erents representing those with very low or zero spontaneous rates (A), and those with high spontaneous rates (B). C : Scatterplot of phase-shift slopes versus spontaneous rate for the 34 a¡erents with the lowest spontaneous rates and the 34 a¡erents having the highest spontaneous rates. Negative slopes indicate that phase lagged as signal level was raised. Symbols plotting slope for low spontaneous a¡erents are displaced horizontally for clarity.

HEARES 2838 11-11-97

12

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

for a¡erent K16 can be interpreted as follows. The horizontal plane data indicate a CA that is oriented at about 20³ from the side-to-side axis, but cannot determine whether the CA projects up to the left and back, or up to the right and front. The midsagittal plane data indicates that the CA projects either up to the back or down to the front. Combining these data indicates that the CA most likely projects up to the left and back. The other threshold data in Fig. 9 illustrate a¡erents that project up into each of the three other quadrants. Directionality of a¡erents in spherical coordinates is illustrated in Fig. 10. This plot shows a view looking down onto the northern hemisphere of a globe with the toad¢sh at its center. Concentric circles indicate elevations of 0, 15, 30, 45, 60, and 75³. The north pole (90³ elevation) is at the center of the innermost circle. Symbols indicate the locations on the northern hemisphere's surface through which an a¡erent's CA passes. It should be noted that for each point in the northern hemisphere, there is a corresponding point in the southern hemisphere through which a given CA passes. The present data do not permit us to determine whether the CAs project upward or downward. With few exceptions, CAs for rostral a¡erents (Fig. 10A) tend to penetrate the northern hemisphere close to the equator (i.e., with low elevations), and tend to cluster around an axis oriented at about 345³ azimuth. This axis is approximately equal to the orientation of the sensory epithelium of the left saccule (Fay et al., 1996). Note that points projected to the animal's left and right in Fig. 10A actually represent similar CAs that di¡er primarily with respect to elevation (i.e., the points penetrating the northern hemisphere to the animal's right de¢ne CAs that tilt just below the equator in the forward and leftward directions). The majority of CAs for middle bundle a¡erents (Fig. 10B) tend to cluster near this same azimuthal axis but di¡er from the rostral a¡erents in having higher elevations (median = 56³), and in projecting upward to the animal's rear and to the right. The majority of caudal a¡erents (Fig. 10C) also tend to have CA azimuths clustered near the axis of the sensory epithelium, and also tend to project upward to the animal's rear and to the right. However, unlike both rostral and middle bundle a¡erents, the caudal a¡erents have a relatively wide distribution of elevations that ranges from nearly horizontal (points near the equator) to nearly vertical (points near the north pole). In general, saccular a¡erents from the left ear have CAs that vary widely in elevation, with a tendency to cluster at azimuths near that of the saccular epithelium. Of all directional a¡erents, the modal azimuthal axis is 345³ with 76% of a¡erents falling within þ 11.25³ of this mode. It is expected that the distributions of CAs for saccular a¡erents of the right ear would be bilater-

ally symmetrical with those of the left ear. Thus, the neurally coded outputs of the sacculi appear to be capable of representing the azimuth and elevation of acoustic particle motion axes via interaural and intraaural patterns of neural activity (see Section 4). 3.5. Nondirectional a¡erents

Of the 231 a¡erents evaluated for directionality, about 20% (n = 44) were classi¢ed as nondirectional. These exhibited threshold patterns in one or both planes that could not be well ¢tted with straight lines, and thus were not consistent with the highly directional response patterns presented above (Figs. 9 and 10). In order to begin to understand deviations from simple directionality, a model was constructed that predicted the threshold patterns expected under the assumption that a single a¡erent may innervate two groups of hair cells having di¡erent orientations. Fig. 11 illustrates some of these predictions. Fig. 11A shows a polar plot of the responsiveness (circular ¢gure) and thresholds (symbols) expected from a single input that is directional in the manner of a single hair cell. Response magnitude varies as the cosine of the stimulus angle, and thresholds are proportional to the reciprocal of the cosine function. The most excitatory axis is oriented about 50³ to the left in the horizontal plane, and the threshold lines are perpendicular to this axis. This model accounts well for the threshold data from the directional a¡erents presented above (Figs. 9 and 10). Fig. 11B depicts two cosine input functions with a directional di¡erence of about 30³ and with di¡erent sensitivities. Thresholds were obtained for the addition of these two cosines with the assumption that a response would occur when the stimulus magnitude exceeded whichever threshold was lowest along a given axis of stimulation. The threshold pattern in this case forms a parallelogram. The two nearly vertical sides of the threshold parallelogram arise from the less sensitive input with the most excitatory axis near + and 390³ azimuth (dotted line circles). The two oblique sides are the threshold line segments derived from the more sensitive input (solid line circles). Fig. 11C illustrates that the threshold parallelogram may be a square if the two inputs have the same sensitivity and are oriented 90³ with respect to one another. Of the 44 a¡erents studied in two planes and classi¢ed as nondirectional, the polar threshold patterns of 40 of them (91%) could be reasonably well ¢tted (by eye) with an appropriate parallelogram. Fig. 12 shows three representative nondirectional a¡erents of this type. A¡erent N1 (from the rostral bundle) shows a parallelogram shape in both the horizontal (top) and midsagittal (bottom) planes. A¡erents L12 (middle bundle) and H14 (caudal bundle) exhibit threshold lines in

HEARES 2838 11-11-97

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

13

Fig. 8. Scatterplots (symbols) and frequency distributions (bars) of response phase (phase of the period histogram peak with respect to peak acceleration) interpolated at threshold (Z = 20) for directional a¡erents. A : Response phase to the side-side stimulus (90³ azimuth) versus response phase to the front-back stimulus (0³ azimuth). B : Response phase to the up-down stimulus (90³ elevation) versus response phase to the sideside stimulus (90³ azimuth). C : Response phase to the front-back stimulus (0³ azimuth) versus response phase to the up-down stimulus (90³ elevation). Frequency distributions of response phase angle are plotted for the stimuli indicated at the abscissae in A, B, and C. Dotted diagonal lines are displaced from the solid diagonal line by 180³.

one plane and a parallelogram in the other plane. A

so) with the plane of the null for one of the inputs. For

likely explanation for the observation of a combination

example, a¡erent H14 appears to have one input ori-

of inputs in one of the planes but not the other is that

ented essentially vertically. In this case, this input's null

one plane of observation may be congruent (or nearly

plane will be on the horizontal and therefore unobserv-

Fig. 9. Threshold points and best-¢tting lines in the horizontal (top row) and midsagittal (bottom row) planes for four a¡erents (A^D) selected from the data shown in Fig. 10. These illustrate the orientations of threshold lines and the axis of best sensitivity (arrows) that determine the appropriate northern hemisphere quadrant in which the CAs are to be plotted. The lengths of the horizontal and vertical axes, as drawn, correspond to the scale in nanometers given for each a¡erent.

HEARES 2838 11-11-97

14

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

Fig. 10. Directionality of toad¢sh saccular a¡erents for which threshold lines were obtained in both the horizontal and midsagittal planes. View is looking down onto the northern hemisphere of a globe with the toad¢sh at its center. Concentric circles indicate elevations of 0³ (= `equator'), 15³, 30³, 45³, 60³, 75³, and 90³ (= north pole). Symbols: representation in spherical coordinates of the location on the northern hemisphere at which an a¡erent's CA penetrates the globe's surface. The four a¡erents shown in Fig. 9 are identi¢ed with arrows.

able by stimulating anywhere on the horizontal plane. For a¡erent L12, one of the inputs appears to be oriented near the horizontal plane at approximately + and 390³ azimuth (nearly vertical sides of the parallelogram). This input's null plane thus falls near the midsagittal plane and would not be observable using stimulus axes in that plane. In summary, about 80% of saccular a¡erents have directional response properties consistent with the hypothesis that their e¡ective inputs arise from hair cells with very similar directional orientations. Of the remaining 20% of a¡erents, 91% have directional response properties suggesting e¡ective inputs from just two hair cell groups having di¡erent orientations and sensitivities. 4. Discussion

The present experiments indicate that primary a¡erents of the toad¢sh saccule encode acoustic particle motions with su¤cient sensitivity, frequency response, and directionality to account for behavioral detection thresholds (Fish and O¡utt, 1972) and for the detection

and localization of vocalizing conspeci¢cs (e.g., Gray and Winn, 1961; Fish, 1972). 4.1. Sensitivity

Saccular a¡erents are widely distributed with respect to displacement threshold at the characteristic axis (CA); thresholds range between 300 and 0.1 nm, rms. This wide threshold variation among a¡erents corresponds to that for saccular a¡erents of the gold¢sh (Fay and Ream, 1986; Fay, 1984) and may be characteristic of ¢shes in general. It appears that the limited dynamic range of individual a¡erents (e.g., Fig. 7) is compensated for by this wide threshold distribution so that sounds of widely di¡erent level may be encoded within the dynamic range, or near threshold, of at least a subset of a¡erents. Among mammals, cochlear a¡erents tend to fall into two groups with respect to threshold and spontaneous activity: low spontaneous a¡erents tend to have higher thresholds than moderate or high spontaneous a¡erents (reviewed by Ruggero, 1992). Our results on the toad¢sh do not show a bimodal distribution of thresholds, but do resemble the results for mammals to the extent that low spontaneous

HEARES 2838 11-11-97

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

15

guinea pig is about 0.2 nm and cilia displacement is 0.025 nm. In studies on the vibration sensitivity of the bullfrog sacculus, Koyama et al. (1982) reported phase-

36 G at 50

locked responses to accelerations as low as 10

Hz. The displacement corresponding to this acceleration is about 0.1 nm. Thus, the phase-locked response of saccular a¡erents of both toad¢sh and bullfrog appear

to

have

sensitivities

comparable

to

that

of

the

mammalian cochlea. The behavioral audiogram for the toad¢sh (Fish and O¡utt, 1972) shows moderate sound pressure sensitivity to frequencies below 150 Hz (lowest thresholds at about 100

dB

re :

1

WPa),

and

declining

sensitivity

toward

higher frequencies. This audiogram is characteristic of ¢sh

species

without

special

adaptations

linking

the

swimbladder and ear (reviewed in Fay, 1988). In the acoustic than

far¢eld

Z

1/2

of

a

wavelengths

sound from

source the

(usually

source

greater

(Rogers

and

Cox, 1988)), acoustic particle motion at the threshold for toad¢sh hearing (at 100 Hz) is approximately 0.1 nm. It should be noted, however, that these sound pressure thresholds may be misleading since, as we suggest, the toad¢sh ear is better viewed as a detector of acoustic particle motion than of sound pressure. Although the toad¢sh has a swimbladder that is used for sound production (Fine, 1983), there is no specialized mechanical link to the ears, and no evidence that it provides an additional, pressure-dependent motional stimulus to the otolith organs. Thus, the present results indicate that the normal mode of hearing for the toad¢sh probably involves the direct detection of acoustic particle motion by the saccule. The direct detection of particle motion may also explain the `mismatch' reported by Fine (1981) between the tuning of saccular a¡erents (characteristic frequencies below 100 Hz) and the fundamental frequency of the boatwhistle call (130 to 250 Hz). Fine de¢ned tuning with respect to the sound pressure levels in a small labFig. 11. Threshold points and best-¢tting lines or parallelograms for three hypothetical a¡erents illustrating the patterns expected given

oratory tank. For sound sources in small tanks, particle displacements tend to be greater than in the far¢eld for

inputs from one hair cell (A) ; two hair cells having di¡erent orien-

equal sound pressure levels (Rogers and Cox, 1988) and

tations

predicted

may be unpredictable due to possible standing waves

thresholds for B and C arise from two directional inputs indicated

and proximity to the source and to air-water interfaces.

by

(B

solid

and

and

C)

dashed

and

di¡erent

circular

sensitivities

¢gures.

For

(B).

these

The

¢gures,

sensitivity

was de¢ned by the circular (cosine) ¢gures, and thresholds were estimated as proportional to the reciprocal of sensitivity.

The present data on sensitivity to particle motion indicate an excellent match between the frequency response of a¡erents (lower thresholds at 100 Hz than at 50 or 200 Hz) and the spectrum of the boatwhistle call (a

a¡erents tend to have higher thresholds. As discussed

fundamental frequency of 130 Hz for toad¢sh of the

below, low and high spontaneous saccular a¡erents also

Woods Hole, MA region (Watkins, 1967)).

di¡er with respect to the e¡ects of stimulus level on the

Displacement sensitivities of a¡erents of the saccule, lagena, and utricle have been determined for the gold-

phase angle of synchronization. toad¢sh

¢sh (Fay, 1984). The saccules of the gold¢sh and toad-

saccular a¡erents (thresholds as low as 0.1 nm) is re-

¢sh are organized very di¡erently (Platt, 1977 ; Edds-

markable.

dB

Walton and Popper, 1995) and the toad¢sh saccular

sound pressure level (near the human threshold of hear-

otolith has a far greater mass and very di¡erent shape

ing at 1 kHz), basilar membrane displacement in the

than that of the gold¢sh (Popper and Coombs, 1982).

The

extreme

Allen

displacement

(1996)

has

sensitivity

estimated

of

that

at

0

HEARES 2838 11-11-97

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

16

Fig. 12. Thresholds for three nondirectional saccular a¡erents in polar coordinates (A^C). The horizontal plane data for a¡erent L12 resembles the model predictions in Fig. 11B. The midsagittal plane data for a¡erent H14 resembles the model predictions in Fig. 11C. The lengths of the horizontal and vertical axes, as drawn, correspond to the scale in nanometers given for each a¡erent.

Nevertheless, the lowest thresholds for both toad¢sh

Several other behavioral experiments on the detec-

and gold¢sh otolith a¡erents were found to be similar

tion of particle motion have been reported for ¢shes

(0.1 nm). These comparisons indicate that otolith or-

(Carassius

auratus :

Fay and Patricoski, 1980 ;

gans can have similar sensitivities regardless of the sizes

morhua :

and shapes of their otoliths.

man and Hawkins, 1974 ;

Lu et al. (1996) measured behavioral thresholds for 100 Hz particle motion in the oscar (Astronotus

tus).

ocella-

Gadus

Sand and Karlsen, 1986 ; O¡utt, 1974 ; Chap-

manda limanda :

Pleuronectes platessa

and

Li-

Chapman and Sand, 1974). Determina-

tions of particle displacement at threshold were inferred

Displacement thresholds in the horizontal plane

from pressure and other measures in some of these ex-

and on the vertical axis were essentially similar at

periments, and the lowest thresholds were calculated to

1.2^1.6 nm, rms. These thresholds are about 22^24 dB

be 0.04 nm in the frequency range between 80 and 300

above the thresholds for the most sensitive a¡erents of

Hz. This value is 8 dB below the lowest physiological

both gold¢sh and toad¢sh. It is not yet clear how to

thresholds obtained for saccular a¡erents of the gold-

interpret these di¡erences since both behavioral (Lu et

¢sh (Fay, 1984) and toad¢sh (present study), and is 30

al.,

results)

dB below the behavioral thresholds determined for the

thresholds were de¢ned somewhat arbitrarily. For ex-

oscar by Lu et al. (1996). In general, the best available

ample, the lowest neurophysiological thresholds were

physiological and behavioral data place the lowest dis-

obtained from spontaneously active neurons using the

placement thresholds of otolith organs in the range be-

coe¤cient of synchronization in the de¢nition of the

tween about 0.1 and 1 nm.

1996)

and

neurophysiological

(present

response. Synchronization in the neural response can be measured at signal levels 10^15 dB below those

4.2. Frequency response

that cause spike rate increments in spontaneously active a¡erents (e.g., see Fig. 7B). Fay and Coombs (1983)

Frequency selectivity was not a primary focus of the

have shown for the gold¢sh that the thresholds for

present experiments since thresholds were determined

tones masked by noise correspond to the signal-to-noise

only at 50, 100, and 200 Hz. On average, thresholds

ratios at which spike rate increases above the noise-

were lowest at 100 Hz with a few a¡erents having lower

driven rate. Thus, behavioral thresholds for the toad¢sh

thresholds at 50 or 200 Hz. This frequency response is

may be 10^15 dB above those measured for saccular

consistent with the behavioral audiogram for the toad-

a¡erents.

¢sh (Fish and O¡utt, 1972) showing a loss of sensitivity

HEARES 2838 11-11-97

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

17

above 100 Hz. However, quantitative comparisons are

Directional responses of toad¢sh saccular a¡erents

di¤cult to make because the behavioral thresholds were

are correlated with the pattern of hair cell orientation

de¢ned with respect to sound pressure while the present

over the epithelial surface (Edds-Walton and Popper,

thresholds

dis-

1995). Rostral and caudal hair cell orientations sweep

placement. The present results are generally in accord

from front-back to nearly vertical toward the middle

with the behavioral displacement thresholds and band-

region

were

de¢ned

respect

to

Pleuronectes platessa)

particle

Gadus morhua)

(Fig.

were reported by Horner et

present results, lowest thresholds were found between 160

(Fig.

2B).

Similarly,

rostral

a¡erents of the middle bundle have higher elevations

al. (1981) using vertical stimulation. Consistent with the

and

epithelium

Li-

and dab (

Particle velocity tuning curves for saccular a¡erents

80

the

(Chapman and Sand, 1974), two spe-

cies lacking a swimbladder.

of the cod (

of

and caudal a¡erents tend to have low elevations while

widths for plaice (

manda limanda)

with

Hz

with

sharper

roll-o¡s

toward

10).

In

general,

the

azimuths

a¡erents tend to cluster near

3

of

most

saccular

45³. This is qualitatively

consistent with the oblique angle of the sensory epithelium in the horizontal plane (Fay et al., 1996). Within each of the three regions designated along the

higher

saccule, there are ¢bers whose CA would not be pre-

given.

dicted for that region. In Fig. 10, for example, note the

These authors noted that, as we have found for the

single a¡erent from the rostral saccule that has an ele-

toad¢sh, rostral and caudal branches of the saccular

vation of nearly 75³ and the few a¡erents with very low

nerve did not di¡er with respect to tuning or best fre-

elevations from the middle region. It is possible that

quency. In addition, our tuning results are in accord

these a¡erents may have innervated the borders of the

with studies of isolated saccular hair cells of the toad-

regions to which they were assigned. That is, the rostral

¢sh. Steinacker and Perez (1992) reported resonances in

a¡erents with the highest elevations may have inner-

some cells between 107 and 175 Hz with a mean of 142

vated nearly vertically oriented hair cells adjacent to

Hz. Since cells with these resonances were isolated from

the middle region where all hair cells are vertically ori-

all

epithelium

ented. Similarly, the a¡erents with low elevations from

(Steinacker and Romero, 1992), there was no evidence

the middle bundle may have innervated hair cells in the

for tonotopy in the saccule of the toad¢sh.

transitional area between the rostral and middle regions

frequencies.

areas

Absolute

over

the

stimulus

length

of

levels

the

were

sensory

not

Fay and Olsho (1979) and Fay and Patricoski (1980)

(see Fig. 2). The few a¡erents with low elevations and

studied particle displacement tuning curves for saccular

azimuths near 90³ (i.e., whose CA is nearly perpendic-

and lagenar a¡erents of the gold¢sh (

Carassius auratus).

ular to the long axis of the ¢sh) were also unexpected

Saccular a¡erents tended to have best frequencies at

based on the general orientation of the saccular epithe-

400 Hz or above, but tuning curves of lagenar a¡erents

lium in the horizontal plane. We suggest that a combi-

resemble

best fre-

nation of the curvature of the epithelium and irregular-

quencies between 70 and 200 Hz (see also data pre-

ities or wrinkles in the otolith/epithelium surface may

sented in Fay, 1981). In general, the lagena of the gold-

result in some hair cells having CAs that are most sen-

¢sh

orientation

sitive to side-side motion. In general, the directionalities

pattern to the saccule of the toad¢sh, while the gold¢sh

of saccular a¡erents correspond qualitatively with the

saccule has a small, elaborately shaped otolith and hair

orientation of the sensory epithelium and with regional

cells

dorsal-ventral

patterns of hair cell orientation. However, it appears

axis. In general, response properties of toad¢sh saccular

that the gross morphology of the saccule cannot quan-

a¡erents

titatively predict the azimuths of all a¡erents.

is

those

of

the

comparable

oriented

in

along

resemble

toad¢sh

size

saccule with

and

hair

cell

a single, primarily

those

of

the

gold¢sh

lagena

more

than those of the gold¢sh saccule. This seems to be true with respect to directionality as well (see below).

Directional data have been obtained from saccular a¡erents of the cod in the horizontal plane (Hawkins and Horner, 1981) and from a¡erents of the saccule,

4.3. Directionality

lagena,

and

midsagittal,

utricle and

of

the

frontal

gold¢sh

planes

in

(Fay,

the

horizontal,

1984).

In

both

Eighty-one percent of the saccular a¡erents studied

species, all a¡erents investigated were highly directional

were `highly directional' in the sense that the threshold

in the manner expected of hair cells. As we have ob-

function of stimulus angle tends to be a straight line in

served in toad¢sh, most saccular a¡erents of the cod

polar coordinates in both the horizontal and midsagit-

had best responses on an axis near that of the saccule's

tal planes. These are the threshold and responsiveness

gross orientation in the horizontal plane. In gold¢sh,

functions

saccular hair cells are oriented along a single, primarily

expected from

a

single

hair

cell

stimulated 1977).

vertical axis (Platt, 1977) and the CAs of saccular a¡er-

Thus, most of the a¡erents studied behave as if they

ents cluster at about 15³ azimuth and 55³ elevation,

received input from one hair cell or from a group of

consistent with the saccular hair cell orientation pat-

hair cells having the same or very similar directional

terns. The lagenar epithelium of the gold¢sh is similar

orientation on the sensory epithelium.

in size and gross orientation to the saccular epithelium

similarly

(Flock,

1964 ;

Hudspeth

and

Corey,

HEARES 2838 11-11-97

18

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

of the toad¢sh. In addition, its hair cells have morphologically de¢ned orientations that range widely in elevation (Platt, 1977). The CAs of lagenar a¡erents range widely in elevation with a tendency to cluster in azimuth at about 30³. Thus, the toad¢sh saccule and the gold¢sh lagena are similar in size, epithelial orientation and hair cell orientation pattern, and their a¡erents have similar thresholds, frequency responses, and directionalities. We hypothesize that these organs have similar roles in directional hearing. 4.4. Nondirectional a¡erents

About 20% of toad¢sh saccular a¡erents had very low correlation coe¤cients for directionality (i.e., the thresholds were not well ¢tted by straight lines in polar coordinates). The thresholds of about 91% of these nondirectional a¡erents could be accounted for by assuming that they received directional input from two hair cells (or two groups of hair cells) having di¡erent directional orientations on the epithelium. This is the ¢rst observation that otolithic a¡erents may receive input from hair cells of di¡erent orientations. Nondirectional a¡erents are essentially omnidirectional to the extent that they show no directional `null plane' (i.e., they respond reasonably well in all planes of stimulation). These a¡erents may function simply as sound detectors without directional selectivity. Given our observations that individual saccular a¡erents may make synaptic contact with over 100 hair cells in some cases (Edds-Walton et al., 1996), it is remarkable that most a¡erents function as if the innervated hair cells formed only one or two groups with respect to directional orientation. 4.5. Synchronization phase angle

The phase angles at which toad¢sh saccular a¡erents synchronize to the acceleration waveform illustrate two e¡ects that require comment. First, response phase angles are level-dependent for zero or low spontaneous a¡erents, but are independent of level for highly spontaneous a¡erents. The level dependence can be understood as a nonlinear threshold e¡ect. For low spontaneous a¡erents, it is hypothesized that spikes tend to be initiated at the time (or phase) within the stimulus cycle when the excitatory waveform reaches the a¡erent's threshold. For stimuli near threshold, this time would be the peak of the excitatory waveform. At levels above this, the excitatory sinusoid would reach threshold earlier in its cycle. Thus, as we have observed, phase would advance with stimulus level up to about 90³. The lack of level-dependent phase behavior among highly spontaneous a¡erents may be explained as follows. The threshold for these a¡erents is below a spontaneous synaptic drive (hence, high spontaneous activity), and

the addition of a sinusoidal stimulus simply modulates the probability of spikes in a linear manner. Within the linear range of levels, the peak of the excitatory drive would always result in the greatest spike probability (i.e., a peak in the period histogram) regardless of stimulus level. The second phase e¡ect is that the distributions of iso-response phase angles for the three orthogonal stimuli are broad and continuous over a 360³ range. This is inconsistent with the simple prediction that phase angle distributions should be bimodal with peaks separated by 180³. Similar observations on the response phases have been reported for cod saccular a¡erents (Horner et al., 1981) and for gold¢sh otolithic a¡erents (Fay, 1981, 1984; Fay and Olsho, 1979). We hypothesize that this result arises from a¡erents di¡ering with respect to the component of relative otolith movement that is excitatory to the hair cells innervated (Fay, 1997). Hair cells with cilia tightly coupled to the otolith are expected to respond in proportion to otolith displacement while those not in direct contact with the otolith will respond in proportion to otolith velocity. Since displacement and velocity are 90³ out of phase, a phase angle distribution with four peaks spaced 90³ apart would be expected. With su¤cient variance about each mean, the distribution of phase angles would appear uniform. This variation could arise from cases in which hair cells are coupled to the otolith in a manner intermediate between displacement and velocity coupling. A second possibility is that for a given stimulus frequency, the di¡erent resonant (¢ltering) properties of saccular hair cells (Steinacker and Perez, 1992; Furukawa and Sugihara, 1989) may introduce phase shifts that vary among hair cells with resonant frequency. In any case, these continuous phase-angle distributions indicate levels of complexity in the a¡erent response that are not adequately incorporated in current theories of sound localization by ¢shes (e.g., Rogers et al., 1988). 4.6. Directional hearing

Based on the present results, we hypothesize that the axis of acoustic particle motion in the horizontal plane (azimuth) can be computed based on the relative levels of response from a¡erents from the two sacculi, and that elevation is computed by the relative levels of response of a¡erents from the rostral/caudal versus the middle regions of the saccule. Interaural di¡erences in overall response arise from the tendency of most a¡erents to have a CA that is parallel to the gross orientation of the saccular otoliths in the horizontal plane: the left otolith points about 40³ to the left, and the right otolith points 40³ to the right. With the two sacculi oriented at about 80³ with respect to one another, the di¡erence of their overall response levels will vary with the axis of particle motion in a way that could be used

HEARES 2838 11-11-97

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

19

to compute azimuth. The signed di¡erence between the

other words, the axis of motion may be resolved, but

overall responses of the two sacculi would produce a

which

unique value for every axis between + and

(1975) has shown that this ambiguity can be resolved

3

90³ azi-

end

of

the

axis

points

to

the

source ?

Schuijf

ex-

in the near¢eld through the encoding of the phase rela-

pected to occur because each azimuthal axis of particle

tionships between particle motion and sound pressure.

motion in the range between

The present data do not address the question of sound

muth.

However,

produce 180³

3

the

back-front

same

signed

confusions

would

be

þ 90³ (e.g., +30³) would

di¡erence

as

an

axis

at

30³, or +150³ azimuth. This problem is hypothe-

pressure encoding in toad¢sh, but we point out that both

the

have

level-

and

observed

cell-dependent

sized as well for terrestrial animals (Woodworth, 1938)

we

pose

potential

and appears to be solved by direction-dependent ¢lter-

phase-processing hypothesis.

phase

di¡erences

di¤culties

for

this

ing of the pinnae, head movements, and other factors such as visual cues and likelihood estimates based on knowledge

of

the

local

object

scene

(Hartmann

4.7. Summary and conclusions

and

Rakerd, 1989 ; Wightman and Kistler, 1993). It is pres-

Saccular a¡erents of the toad¢sh are exquisitely sen-

ently unknown whether these or related cues operate as

sitive to linear oscillation of the ¢sh's body, some a¡er-

well for ¢shes.

ents showing phase-locked responses to rms displace-

Our

¢nding

of

a

robust

dependence

of

response

ments

less

than

0.1

nm

at

100

Hz.

This

sensitivity

phase on level (in a 25 dB range of levels and a 90³

rivals that of the mammalian cochlea and is probably

range of phase angles) for low spontaneous a¡erents

common to the otolith organs of most ¢shes. The mech-

suggests that di¡erences in response magnitude between

anisms

the two sacculi are also represented as di¡erences in the

appeared ¢rst among the ¢shes and has been retained.

response phase or time. For an 100 Hz sinusoid, the

Detection

level-dependent interaural response time di¡erence can

tions are probably mediated by the saccule functioning

be as large as 2.5 ms (i.e., a quarter cycle). In the ver-

as

tical planes, elevation is represented by the pro¢le of

particle motion. The `accelerometer mode' of hearing is

activity across a¡erents within a saccule, since a¡erents

the

from the middle region have characteristic axes with

brates.

higher elevations than those of the rostral and caudal

an

responsible

and

for

processing

accelerometer

most

hair

primitive

cell

of

sensitivity

the

responding

mode

yet

probably

toad¢sh's

directly

vocaliza-

to

identi¢ed

acoustic

for

verte-

Saccular a¡erents are frequency selective with highest e¡ective frequencies ranging between about 100 and 200

regions. These hypotheses suggest that mechanisms for direc-

Hz, or higher. Di¡erences among cells in frequency tun-

tional hearing by ¢shes in both azimuth and elevation

ing may be due to broad resonances shown for isolated

may be essentially similar to those identi¢ed for terres-

saccular hair cells and to the modes and sti¡ness of

trial vertebrates. In humans, for example, the represen-

their ciliary attachments to the otoliths. This latter ef-

tation of sound source azimuth derives from interaural

fect has been suggested as the most primitive basis for

di¡erences in level and time, while elevation may be

peripheral

represented in terms of spectral shape (Wightman et

brates (Fay, 1997). The overall frequency range is ad-

al., 1991). Spectral shape is a stimulus feature repre-

equate

sented

`boatwhistle'

monaurally

in

mammals

as

an

across-a¡erent

to

encode

analysis

the

but

is

frequency

probably

of

not

higher harmonics of this call. Thus, we conclude that

rather organized directly with respect to elevation. Fi-

the fundamentally important aspect of this courtship

nally, we note that the directional response of the sac-

vocalization is its pulse repetition rate, re£ected in the

cule to acoustic particle motion gives ¢shes the equiv-

fundamental

alent

that

phase lock within their responsive bandwidth, the fun-

have been demonstrated for the ears of some amphib-

damental frequency is encoded within all a¡erents in

ians and birds (reviewed by Fay and Feng, 1987), and

the time domain.

of

monaural

`pressure

gradient'

organized,

receivers

in the lateral line system (Denton and Gray, 1983). The accelerometer could

be

particle

mode

viewed

motions

as are

of

otolith

stimulation

pressure-gradient normally

by

Since

all

saccular

of

the

a¡erents

The great majority of saccular a¡erents have direc-

¢shes

tional properties that would be expected if they inner-

since

vated a single hair cell or a group of hair cells having

pressure

the same directional orientation. This is remarkable giv-

in

detection

produced

frequency.

phases

the

wide

enough

tonotopically

and

verte-

but

not

amplitudes

for

toad¢sh

is

the

indicated

fundamental

vocalization,

encode

yet

(tonotopic) pro¢le of activity. We have found that the saccule

to

frequency

en that a single a¡erent may innervate over 100 hair

gradients (Rogers and Cox, 1988). One issue of directional hearing by ¢shes remains :

cells. We hypothesize that an a¡erent must be selective

theoretical work indicates that a system of particle mo-

with regard to which hair cells are ¢rst innervated dur-

tion receivers is subject to an essential ambiguity about

ing development, and how new hair cell connections are

the direction of sound propagation (e.g., Schuijf, 1975 ;

made during growth and hair cell proliferation through-

Schellart and De Munck, 1987 ; Rogers et al., 1988). In

out the lifespan. Of the a¡erents that are not perfectly

HEARES 2838 11-11-97

R.R. Fay, P.L. Edds-Walton / Hearing Research 111 (1997) 1^21

20

directional, most appear to innervate just two hair cells

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