Functional properties of the efferent cochlear bundle of the pigeon revealed by stereotaxic stimulation

EXPERIMENTAL

NEUROLOGY

Functional

11,

1-26

(1965)

Properties

Bundle

of The

of the Pigeon Revealed Stereotaxic Stimulation

J. E. DESMEDT AND P. J. Laboratory

Efferent

of Pathophysiology Brussels,

June

by

DELWAIDE~

the Nervous Brussels, Belgium of

Received

Cochlear

System,

University

of

23, 1964

The function of the efferent cochlear bundle (ECB) has been studied in over a hundred pigeons with the spinal cord transected at C2-C3, local anesthesia and muscle paralysis. The excellent biological status of the animals was carefully assessed throughout the experiments, which frequently lasted several hours. Electrical responses evoked by click or tone pip were picked up chiefly at the exposed round-window membrane. Bipolar needles were inserted stereotaxically into the brain stem with the ECB as target (histological controls). Repetitive electrical stimulation of the ECB before delivery of the testing sound potentiates markedly the cochlear microphonic receptor potential of the hair cells and inhibits simultaneously the auditory nerve response. This dual effect results in a reduction of acoustic input to the brain. These and other observations suggest that the ECB of the bird is homologous functionally to the olivocochlear bundle of mammals. Important quantitative differences have, nevertheless, been found. The potency of the efferent inhibition is much smaller in the pigeon than it is in the cat, which raises the question of its behavioral significance in the former species. The potentiation of the cochlear microphonic component is relatively more prominent in the pigeon and it dissipates more slowly than the simultaneously recorded inhibition of the neurals. Such different kinetics might be related to the peculiar synaptic organization of the bird’s inner ear. Introduction

The efferent olivocochlear bundle (OCB) of Rasmussen (28, 29) so far has been studied physiologically in the cat where its functional characteristics as a neural system controlling transduction of sound waves 1 This States Air rological 02482. P. National

work was supported in part by the Office of Aerospace Research, United Force under Contract EOAR-64-43 and by the National Institute of NeuNational Institutes of Health under Grant NBDiseases and Blindness, J. Delwaide is Postdoctoral Research Fellow and now Aspirant of the Fonds de la Recherche Scientifique.

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into nerve impulses in the inner ear are well documented (7, 12, 16, 18). The purpose of the present study is to determine whether the efferent cochlear bundle (ECB) described by Boord (2) in the pigeon exerts similar effects in the cochlea and whether it can be considered as the functional homologue of the mammalian Rasmussen bundle. Since the bird’s cochlea is uncoiled and more simply organized (25, 26, 30-32) it was also of interest to look for quantitative and qualitative differences, if any, in the mode of action of these efferents in the pigeon as opposed to the cat. In addition, this paper describes a stereotaxic method and an avian preparation with a high spinal transection. Preliminary communications have been presented (9-l 1) . Material

and

Methods

Young adult pigeons of the common Belgian racing breed, mostly males l-3 years old (350-530 g), were used. Birds in excellent general condition with normal hearing were selected and no experiment was carried out during the molting season (September, October and March) when the bird’s vitality is impaired. They received no food during the preceding night but had access to water. Pigeons are rather brittle animals with which to work and physiological information about them is still scarce. Therefore it was necessary to make many trials in order to design the experimental technique. The pigeon was first placed in a closed Plexiglass box and anesthetized with a gas mixture of 2-4s Fluothane (Halothane, I.C.I. Ltd) in oxygen. The trachea was dissected and intubated with a PE 360 Polythene tube (Clay Adams Inc.) through which the Fluothane-oxygen mixture is introduced. The body is fixed into a Ewald (15) wooden shoe. Polythene PE 10 tubes served to cannulate a brachial vein for drug injection and a brachial artery for blood pressure recording with a mercury manometer. No heparin was used. The skull was then exposed for fixation into the stereotaxic instrument. The spinal cord was transected at C2-C3 and artificial ventilation with a Starling Ideal pump was installed at the rate of 32 per min with an adequate volume as judged from the state of cardiovascular system and brain waves. The latter were recorded through silver pins inserted into the skull posterior convexity and connected to a Grass polygraph. The electrocardiogram was picked up with needles inserted into the wings. The insertions of neck muscles on the occipital bone were removed. The spongy bone overlying the left cerebellum was gently removed with drill and rongeur up to about 3 mm in front of

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the cerebella-cerebral sulcus (Fig. 1). The right middle ear was exposed from behind by opening trabeculated air cavities (15) and a nearly tangential view was thus obtained on the membrane of the round window. Pigeons with fluid in the middle ear or other signs of a pathological condition were rejected.

FIG. 1. Stereotaxic technique in the pigeon. A. Sketch of the lateral aspect of a pigeon skull; B, dorsal view of same. The fixation points and reference planes are indicated as follows: 1, steel needle in frontal bone; 2, pointer pressing forward on the inion; 3, Plexiglas block 45” oblique with respect to the horizontal plane; 4, screws for fixation of frontal needle; 5, zygomatic bone; 6, lachrymal bone; 7, external auditory meatus; 8, exposure of the middle ear; 9, occipital crest; 10, exposure of the cerebellum for insertion of the stereotaxic needles. C. Sketch of a frontal histological section through the brain stem, with the electrode tracks. The emergence of the cranial nerves VII, and VIII, the facial genua, the abducens nucleus (VI), the trigeminal sensory nucleus (V) and the auditory nucleus laminaris (NL) are seen. The fourth ventricle is stippled, The dots indicate the approximate course of the efferents for both cochleas, according to Boord (2).

Very careful surgery under a binocular microscope was essential to minimize bleeding which is a problem with pigeons (poor hemostasis, difficulty in clamping vessels,small blood volume). Local infiltration of incision and pressure points with 1% Lidocaine (Xylocaine) effectively suppressedpain (no movement, no rise in blood pressureduring preparation) and allowed the Fluothane to be discontinued after surgery. The central temperature was maintained at 4&42 C with a heating pad. Flaxedil (Gallamine) was injected at intervals in doses of 5 mg to

4

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maintain muscle paralysis during the actual experiment. The acoustic evoked responses were picked up by platinum wires 0.1 mm in diameter insulated with varnish except at their tip. The active electrode was placed on the round-window membrane and the reference electrode on the ridge of the middle ear exposure. The potentials were displayed via P 6 Grass cathode-follower amplifiers on Tektronix 360 oscilloscopes and photographed outside the soundproof room by a Grass camera. The facilities and procedures used have been described (7). Transection of the Spinal Cord. A spinal pigeon preparation was developed to avoid the use of long-acting general anesthetics and to prevent the electrical stimulations in the brain stem from producing unwanted cardiovascular changes (Fig. 3). The latter are surprisingly prominent and troublesome in the pigeon. Thoracic spinal cord transection was performed (22), but this did not provide the best solution to our problem because removal of the posterior laminae of the dorsal vertebrae opens the air sacks contained therein and allows a rather noisy exit of respiratory air, the cervico-brachial metamers are not severed from the brainstem, and orthosympathetic discharges during stereotaxic stimulation are not completely prevented. Spinal transection at the second cervical segment was thus decided upon in spite of the increased surgical difficulties associated with the mobility of the cervical vertebrae and the presence of a big dorsal spinal vein with thin walls in intimate contact with the periosteum of the spinal canal. At this level no air sack was opened and most of the cervico-brachial metamers were below the transection. After fixation of the neck, the dorsal lamina was thinned out with a drill under binocular observation (X 16). Its innermost aspect was cut through only on the two sides and the medial part was carefully detached without tearing any small vessels. The cervical cord with roots C2 and C3 then appeared behind the big longitudinal vein with its metameric tributaries, The dura mater was incised about 1 mm from the midline. Silk threads were passed between vein and cord with watchmaker forceps and the vein was ligated, first in front and then more caudally, and cut. The cord was transected in small steps with a Keeler ophtalmic cautery. The heart rate generally slowed after cord transection, e.g., from 360 to 200 per min. (Fig. 3 E, F). The blood pressure did not drop below 70 mm Hg and it was subsequently maintained for hours at 90-120 minHg. Stereotaxic Technique. There is neither a stereotaxic atlas for the pigeon brain nor any standard principles for skull fixation, as there

EFFERENT

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:,

are for monkey, cat and rabbit. Recent experiments on the chicken or pigeon have depended on earplugs and beak holders to immobilize the skull (19, 33, 35). In studies on hearing, any damage to the eardrum or to the middle ear must be avoided and the use of conventionally designed earplugs is hazardous (7). We dispensed with earplugs and devised the following system which can be clamped, as a mechanical unit, in the transverse zero axis of a U-type stereotaxic instrument. The body of the pigeon lays only a few centimeters below the level of the head in an Ewald’s wooden shoe attached to the sidearm. The head holder incorporated three pieces for rigid fixation (Fig. 1) : a transverse horizontal steel needle transfixed the frontal bones; a sagittal pointer was screwed forward into the center of the occipital crest (inion) in the same horizontal plane; and a Plexiglas block was moved downwards along two lateral guides with a screw advance. The tranverse needle was 1.5 mm thick, 30 mm long and pointed on both ends. After a midline skin incision exposing the skull, this needle was pushed into the spongy bone at the angle limited by the zygomatic and lachrymal arches and the anterior orbital ridge. The two ends of the needle then emerging on both sides of the head were clamped between screws (4) for adjustment of the sagittal plane. The occipital pointer pressed the skull forward so that the transverse needle pushed more against the rather strong orbital ridge than on the fragile zygomatic and lachrymal bones. The horizontal plane so defined was less tilted than if earplugs were used (35). The Plexiglas block coapted to a rather flat portion of the frontal bone between the orbits on its full width and prevented rotation or displacement in the vertical axis. The 45” forward orientation of this block was chosen to facilitate exposure of the cerebral hemispheres. The method (in use since 1961) provided a firm and reproducible fixation of the pigeon’s skull. It allowed ready access to the tympanic bulla (8) and to the external auditory meatus into which a tightly fitting polythene tube, 140 mm long, conveyed the sound emitted by the earphone. Several considerations determined the best orientation of the stereotaxic electrodes. The 70” obliquity in relation to the horizontal plane (Fig. 1 A) put the electrode in a position approximately perpendicular to the axis of the brain stem and also enabled reaching the ECB and acoustical nuclei through the anterior cerebellum, thereby avoiding the vessels of the tentorium and the mesodiencephalic structures. Within this oblique transverse plane, the electrodes were further tilted ls” with respect to the vertical sagittal plane. Thus they entered the cerebellum

6

DESMEDT AND DELWAIDE

on the left side in order to reach the ECB going to the right ear (Fig. 1 B, C). Using this route there was practically no bleeding, since the trephine hole and the electrodes avoided both the sinus running along the midline over the cerebellum and the lateral venous sinus associated with the anterovertical semicircular canal. Allowance for this double obliquity was included in the estimation of the depth and lateral stereotaxic coordinates. The anteroposterior coordinate was corrected for individual variations by considering the reading for the occipital crest (9) at 2 mm from the midline and by moving the carrier 7.5 to 9 mm anteriorly. The stainless steel bipolar needles (about 50 lo at the exposed tip) used in this laboratory and other technical details can be found elsewhere (7). Results ACOUSTIC EVOKED POTENTIALS

RECORDED AT THE ROUND WINDOW

When the stimulus is an acoustic click the active electrode on the round-window membrane picks up a complex waveform (Fig. 2 F) with a cochlear microphonic potential (CM) followed by a first (Nl) and a second (Nz) neural component (21, 33). The polarity of the CM component depends on whether the air pressure at the eardrum is either increased (condensation click, Fig. 2 F) or decreased (rarefaction click, Fig. 2 E) by the first sound wave. By analogy with results on mammals, CM is interpreted as a mechano-electric transducer potential generated by the hair cells, while the neurals represent the synchronous discharge of afferent auditory nerve fibers (5, 6). As our experimental technique steadily improved in the course of time, larger responseswith lower thresholds and increasing stability were recorded, but even in the best experiments, the results differed from those obtained with similar techniques in the cat (7). We rarely saw N1 potentials of over 300 pv in the pigeon, while N1 potentials of 1 mv occur in a good cat preparation. In pigeons, CM was more prominent relative to the neurals (Figs. 2 F; 4; 6) and we think this was not a sign of deterioration. The acoustic threshold for just detectable responsewas 10 db or more higher, even in the best pigeons. The voltage of all components increasedmore or less regularly with the energy of the click (Fig. 4). The responsesevoked by the sameclick repeated every 3-6 set did not fatigue but exhibited a troublesome random variation in the pigeon, although the preparation was mechanically rigid and the recording electrodes established a clean and stable contact with the round-window membrane (21). Therefore

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the means of several responses were plotted in the diagrams, and they were consistent over several hours. When the stimulus was a tone pip with IOmsec rise time, a CM response was recorded, the voltage of which varies with the frequency (33, 38). No detailed analysis of the audiogram was made, but CM responses occurred up to 16 kc, i.e., for frequencies higher than generally stated in the literature (21, 38). The best frequencies of brain stem units of pigeon ranged up to 4 kc (34) for which frequency the behavioral threshold rose abruptly (20) although the pigeon still reacted to tones 10-12 kc (20, 26, 37). EFFECT

OF BRAINSTEM

STIMULATION

ON

AUDITORY

EVOKED

RESPONSES

We first defined the stereotaxic coordinates of the brain stem regions which on stimulation induced changes in the round-window responses to click. Bipolar needle electrodes were inserted through the cerebellum into the brain stem and for each position the effect of delivering thirty electric pulses at 300 per set just before the testing click was noticed. The positive points were concentrated in a critical region along the cephalocaudal and dorsoventral coordinates. Figure 2 B illustrates one of the strongest effects on the neurals, which were substantially reduced with respect to control (Fig. 2 A). Frame C shows that the N1 spike, evoked by an unconditioned click attenuated - 6 db, presented about the same voltage as that in Fig. 2 B, so that the inhibitory effect can be estimated as a - 6 equivalent db change. When CM was prominent in the round-window response (Fig. 2 F) the efferent stimulation not only inhibited N1 and Nz but it potentiated the CM potential (Fig. 2 G) . Among the ninety-one pigeons tested under satisfactory conditions, seventy-two allowed demonstration of modifications in the auditory evoked potentials. When present, these changes were always in the same direction, viz., decrease in N1 and Nz and increase in CM. Such dual efferent effects are also seen during activation of the OCB in cat (7, I 2, 16, 17) in which the inhibition of the neural is, however, much stronger (7). We have been much concerned with the marked difference in potency of efferent inhibition in the two species (8), but our present control experiments led us to consider that it is genuine. Histological Controls. Strong gating effects can only be demonstrated in the cat when the stimulating electrodes are in close contact with the OCB and the relative smallness of the phenomenon in the pigeon might be ascribed to inadequate stereotaxy. The electrodes were moved in small steps to attempt maximal activation of the efferent system. Displacement

8

DESMEDT

AND

DELWAIDE

of ,the electrodes by 1 mm in any direction from an optimal location led generally to a clear reduction in the efferent effect. When stimulating the cat’s OCB on the midline a displacement of electrodes by only 0.25 mm reducesthe efferent gating (7), but the dependenceon electrode location is less critical when the cat’s OCB is stimulated more laterally (11) where the fibers fan out to enter the vestibular root (29). On this

-20

db

-20

-23

-15

FIG. 2. Changes in round-window responses induced by ECB activation. As in the other figures, the energy of the clicks is given in db relative to our arbitrary level. The white dot in some of the frames indicates that the ECB has been stimulated. Negativity of the active electrode records upwards. A-D. Pigeon P47, 42Og, transection of thoracic (Dl) spinal cord and Flaxedil paralysis; cloaca1 temperature 40 C. In B, 40 pulses at 400 per set were delivered to ECB 10msec before the testing click, and the neural potentials N, and N, were reduced in voltage. D. 100 pv step function and time scale in msec. E-I. Pigeon P49, 510 g, transection of cervical (C3) spinal cord and Flaxedil paralysis; cloaca1 temperature 39.5 C. E. Rarefaction click to show phase reversal of the CM component (arrow). F-I. Condensation clicks of stated intensities. G. 30 pulses at 300 per set were delivered to ECB 10 msec before the testing click (see text).

basis we believe that, in the pigeon, the efferent fibers are rather loosely arranged even on the midline. In any case the main fact is that the efferents were strongly stimulated in most experiments, In serial paraffm section, lo-20 p thick, the electrode tracks were reconstructed. They terminated in the dorsal brain

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stem under the fourth ventricle at the level of the facial genua (Figs. 1 C; 10 A, B). The rostra1 part of the abducens nucleus appeared in many of these sections. Identification of structures was facilitated by reference to anatomical studies of the bird’s nervous system (1, 3, 14, 27, 36). The electrodes penetrated only 1 mm or less into the brain stem and caused little damage. Under high magnification the fine prussian blue precipitate related to the iron deposited from the electrode tip at the end of the experiment (Fig. 10 C) appeared either in the dorsal raphe or slightly more laterally above the abducens nucleus or between the latter and the facial genu. According to Boord (2) the efferent fibers run precisely at this level.” The Electrical Stimulation. The efferent effects illustrated can be regarded as maximal since they were not augmented by any change in the stimulation program. As a rule, twenty to forty pulses at 300-400 per set represented an optimal combination. A substantial decrease or increase in number of shocks or the frequency, or both, produced lesser effects. The optimal duration of the square pulses was 30-50 usec and longer pulses did not appear more effective at the same voltage. Pulses of 5-8 v frequently produced detectable changes in the cochlear potentials evoked by click. The effects increased with voltage of the conditioning pulses and reached a maximum at lo-40 v. No further changes occurred for larger voltages, which suggests that fibers of a single functional type are recruited as the pulses get stronger until the entire ECB is thrown into action. Central Temperature. The cloaca1 temperature of a conscious pigeon sitting quietly in its cage is 41-42.5 C. In our experiments the thermoregulatory mechanisms were upset by the high spinal section, the curarization and the artificial ventilation at a fixed rate, but the central temperature was maintained by a heating pad. Since the normal rectal temperature of the cat is 39 C, thus 3 C lower than the pigeon, we wondered whether this factor might account for the discrepancy of results. However, when the pigeon’s central temperature was allowed to cool to 38 C by switching off the heating, the effects of maximal ECB activation were not increased. Indeed they tended to be depressed at temperatures lower than 37 C. Muscle and Masking Effects. In the cat, contraction of middle ear 2 Dr. Grant L. Rasmussen kindly confirmed the above statements.

looked

at some

of our

histological

material

and

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DESMEDT

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muscles attenuates both the CM and the neural potentials evoked by sound (18, 39). These muscles have been cut in studies on the OCB (7, 13, 17). In the bird a muscle is inserted on the extracolumella and on the edge of the eardrum; it passes out of the middle ear through a foramen and attaches itself to the basioccipital bone (4, 24, 25). This muscle is probably homologous to the stapedius of mammals (4, 23). It may be mainly concerned with stretching the eardrum (4, 24), but its function has not been studied with modern techniques. In view of the hazards involved, the muscle was not cut in our experiments but must have been inactive in view of the curarization. Masking by unwanted sounds could be excluded since the pigeon was in a soundproofed room and since the electrical stimulations and the other procedures produced no movement in the animal (spinal transection, Flaxedil) nor any unwanted sound at the cochlea studied. In any case, spurious muscular or masking effects would have tended to depress further the neural responses and would thus not lead one to underestimate the potency of efferent inhibition. Anesthetics, Flaxedil. Spinal pigeons prepared under Fluothane gave better results than the other preparations tried. With pentobarbital, the anesthetic phase was preceded by troublesome agitation and dosage was critical. Urethane (1 g/kg, ip) was more satisfactory for the pigeon (2 1, 34) and allowed demonstration of ECB effects (Fig. 5). The latter however seemed less powerful on the whole than in unanesthetized spinal pigeons. Furthermore, with the neuraxis intact, the pigeon displayed marked cardiovascular changes upon brain stem stimulation in spite of the general anesthesia. The heart rate accelerated (Fig. 3 A) and the blood pressure rose, which precipitated bleeding at the surgical exposures. A fatal cardiovascular shock frequently followed the cardiac tachy-arrythmia (Fig. 3 B) and terminated the experiment. After high spinal transection a similar sitmulation in the brain stem either did not affect the cardiovascular system (Fig. 3 C) or it elicited a brief vagal bradycardia of no consequence (Fig. 3 D). Fluothane proved to be a convenient and smooth anesthetic for the initial surgery and it did not affect the efferent inhibition. Other volatile anesthetics such as ether or Trilene produced vomiting, excess salivation, clogging of tracheal cannula and agitation. We stimulated the ECB before and after Flaxedil infusion in four pigeons but detected no significant changes in the amount of CM potentiation. Under these particular conditions the noise produced by

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muscle contractions masked the neurals which could not be assessed. Thus Flaxedil as a drug did not appear to antagonize the ECB effect and its use to prevent any muscle movement was justified. General Status of the Preparation. In spite of 3 hours of surgical preparation, the biological status of the pigeons exhibited little deteriora-

FIG. 3. Polygraph records of the electrocardiogram (upper trace) and of the spontaneous electrical activity of the cerebral hemispheres (lower trace) under various conditions. For records A through D the arrows indicate delivery of a train of electric pulses to the region of the ECB in the brain stem. A and B. Pigeon P.59 anesthetized with urethane, spinal cord intact; A, before, and B, after muscle paralysis with Flaxedil. C. Pigeon P80 with transection of cervical cord; the stimulation elicited a telencephalic arousal but no cardiac changes. D. Pigeon P88 with transection of cervical cord; the stimulations elicited bradycardia through the vagus nerve. E, F and G. Pigeon P82 prepared under Fluothane anesthesia; E, before and F, 5 min. after transection of the cervical cord; G, same 5 hours later at the end of the experiment. Vertical calibration, 100 pv; horizontal calibration, 1 sec.

tion. To estimate blood volume, 1 ml of red blood cells marked with 51Cr and suspended in Krebs-ATP-glucose solution was injected intravenously. After 15 min mixing was complete and the isotopic dilution was estimated in a blood sample. The blood volume was 8.5-10 ml per 100 g body weight before surgery and it decreased by 13% or less after S-7 hours of experimentation. Hematocrit values were 40-42 and also changed little. The artificial ventilation when nicely adjusted did not upset the acid-base equilibrium. Blood pH was maintained at 7.35-7.40, bicarbonate standard at 25-26 mEq/l and pCO2 increased only by about

12

DESMEDT AND DELWAIDE

10%. At no time did the systolic blood pressure drop below 70mmHg even during transection of the spinal cord. The electrocardiogram and the spontaneous electrical activity of the cerebral hemispheres maintained normal patterns for hours (Fig. 3 F, G). The low threshold of acoustic evoked potentials and their large voltage also bear witness to the good condition of the animals: TITRATION

OF EFFERENT

EFFECTS

By comparing the ECB-conditioned responseswith the intensity function of the responsesto sound alone, the efferent effects can be expressed as equivalent decibel changesin apparent sound energy, but this titration method is meaningful only if its results are independent of the energy chosen for the testing sound (7). Neurals. The responsesto click illustrated in Fig. 4 suggest that in the pigeon the neurals, but not the CM component, can be titrated in this manner. The first row of records shows the responseselicited by clicks over a range of 30 db. The second row showsthe effect of maximal ECB activation on the same. In the third row the testing click (without ECB activation) was attenuated by -4 or -5 db in such a way that the voltage of N1 matches nicely with that of the corresponding N1 in the second row. In this experiment the intensity function of N1 was steep for clicks between -10 and -25 db relative to our arbitrary level; it flattened out between -25 and -35 db and resumeda definite slope for still smaller clicks (Fig. 4 M). The changesinduced by ECB in N1 voltage corresponded remarkably to this peculiar pattern. When the testing click was chosenin the steepportion of the N1 function, the ECB induced a clear reduction in N1 voltage (Fig. 4 B, H, K). By contrast when the testing click corresponded to a flat portion of the N1 function, the ECB did not change the voltage of N1 (Fig. 4 E) although it had certainly been fully activated, as shown by the concomitant doubling of the voltage of the CM component (arrow). Thus the absolute or percentage changes in voltage of neurals induced by ECB varied markedly with the steepnessof the intensity function at the level chosen for the testing click, whereas the centrifugal effects appeared consistent throughout when they were titrated in equivalent decibels. The inhibitory effect could, indeed, be described as equivalent to a shift of the intensity function to the left by about -5 db. In many good experiments the maximal inhibition of neurals amounted to no more than a -2 or -3 equivalent decibel change (Fig. 2 G, H). While the largest effect observed was

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equivalent to -6 equivalent db (Fig. 2 B, C), in several pigeons the neurals were apparently unaffected by ECB, although the CM was potentiated (Fig. 6). This curious result, never obtained in cat, will be considered further below. In four experiments the potentials evoked by the click in the auditory -40

-40

db

-45

M 50( I-

*Emi!!! -30

-30

db

-35

40( IP" 3oc I-

-20

-20

db

-25

9.. . : 7,’ . .. +. . . . .

P97

Nl

2oc )-

IOC IL

G!lilMl'~ -10

J

db

-10

K

-14

N 150

* *

*

CM *

100 l :*.*. .* . . l **wQ... P” l

+

+

‘d.,*

50 I----

hi----

-30

db

-50

FIG. 4. ECB effects in relation to the intensity function of responses to click. Pigeon P97, 485 g, transection of cervical (CZ) spinal cord, Flaxedil paralysis, cloaca1 temperature 40.5 C. A to L. Responses evoked by rarefaction clicks of stated intensities; for the second row of frames, 40 pulses at 400 per set were delivered to ECB 10 msec before the testing click. M, N. Same experiment, intensity functions for N, (M) and for CM (N). Abscissa, click intensity in decibels relative to arbitrary level. Ordinate, voltage of component in uv. Each dot corresponds to the mean of three to seven responses. The stars indicate the voltage of responses conditioned by ECB activation. The letters in diagram M help correlation with the records.

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nuclei of the brain stem were recorded through a second pair of stereotaxic electrodes (Figs. 8, 10 D). The brief, l-2 msec, initial component of short latency, which represented activity in auditory afferents or in the second-order neurons, allowed precise measurements to be made. It increased regularly in voltage as the click got louder. It was reduced but not suppressed by activation of the ECB whose effect on the inner ear thus resulted in an actual reduction in imput into the central acoustic pathway. The changes induced by the ECB in the responses could be expressed consistently as equivalent decibels over a range. They never amounted to more than a -7 equivalent decibel change. This evidence was thus entirely consistent with the conclusion based on round-window records. Cochlear Microphonic. Although the intensity function of the CM response to click was frequently monotonic (Fig. 4 N) the potentiation of CM by ECB could vary markedly with click intensity. In the diagram N, the stars do not arrange themselves in a row parallel to the intensity function and a sort of ceiling effect appears on the left side. The CM change in Fig. 4 B would appear equivalent to +20 equivalent decibels (compare with Fig. 4 G) but for other click intensities in the same experiment changes as large as $30 or even +35 equivalent decibels would have to be accepted. In other pigeons the potentiation would appear much less (Fig. 2). In fact this titration method was neither useful nor consistent as far as the CM evoked by click was concerned. The round-windowpositive phase of CM was frequently potentiated more than the roundwindow-negative phase (Fig. 5) no matter which came first (Fig. 6) but in several animals both phases were increased. The potentiation. by ECB of the CM evoked by tone pips agrees more closely with the results in cat (7). When pips of various frequencies were considered (Fig. 5 A-C) the increase in CM was larger when the corresponding intensity function was steeper and the voltage of CM could nearly be doubled at a well-chosen pip frequency (Fig. 5 E, F). The effect could be expressed consistently as an equivalent decibel increase over a range of pip intensities, viz., as a shift to the right of the intensity function (Fig. 5 D). The matching of potentiated CM to the function may be somewhat tricky, as the ECB increased the early oscillations more than the later ones (Fig. 5 F) and a choice had to be made as to which portion was going to be matched. In any case the maximum effect on CM evoked by pip did not exceed +7 equivalent decibels in the pigeon (S), a figure which is definitely larger than for the cat (7).

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I

kc

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4

kc

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IO

kc

FIG. 5. ECB effects in relation to the intensity function of responses to tone pips. A-C. Pigeon P8, 450 g, urethane anesthesia and Flaxedil paralysis; potentiation of CM for tones of 1, 4 and 10 kc. D-G. Pigeon P40, 380 g, transection of thoracic (Dl) spinal cord and Flaxedil paralysis; tone pips of 2.5 kc with 10 msec rise time. D. Intensity function with response voltage in microvolts as ordinate and pip intensity in decibels relative to arbitrary level as abscissa. Each dot corresponds to the mean of three to five responses; the stars to CM conditioned by maximal ECB activation. E. Control response in same experiment. F. ECB stimulation 10 msec before testing pip. G. ECB stimulation 200 msec before testing pip.

FIG. 6. A typical Pigeon P80, 425 g, cloaca1 temperature In B and D, the Vertical calibration,

experiment in which the ECB affects the CM component only. transection of cervical (C2) spinal cord, Flaxedil paralysis, 41.5 C. A, B. Rarefaction clicks. C, D. Condensation clicks. ECB was maximally stimulated just before the testing click. 100 pv.

16

DE~E~EDT AND DELWAIDE PARAMETERS

OF STIMULATION

As for the cat’s OCB, the single shock to the pigeon’s ECB produced no detectable change in the round-window response to click and the ECB thus appears as an iterative system. The optimum frequency of stimulation was also similar, 300-400 per set (7), and the minimum number of shocks eliciting an efferent effect was three to five at such a frequency. In a few cases a small CM potentiation appeared for fewer shocks than needed to produce a small depression of N1, and this was not unexpected since N, inhibition was slight in a few pigeons (Fig. 6). With more shocks both effects got larger up to about forty. In pigeon P76 increasing the number of pulsesfrom 40 to 1.50at 400 per set potentiated more and more CM (from +4OoJo to +60%) while the N1 inhibition simultaneously decreasedfrom its maximum (from -30% to -14%). The different kinetics of ECB effects on CM and Nr, respectively, in the pigeon, stood out more clearly when the testing clicks were elicited at various intervals after maximal faradizations. Both CM and N1 displayed the largest changesat the shortest train-click intervals (Fig. 7 B) . The changes dissipated progressively as the train-click intervals got longer (Fig. 7 C, D) but the N1 inhibition disappeared for intervals of 40-100 msec while the CM potentiation was still quite marked for intervals of 200-300 msec (Fig. 7 F). When tone pips instead of clicks were delivered to the ear, a clear potentiation of CM also appeared for trainpip intervals of 200 msec (Fig. 5 G). In the diagrams of Fig. 7, the voltages of the CM (H) and the N1 (I) responsesto click are plotted as a function of the intervals between the end of the ECB maximal stimulation and the testing click. The mean voltages of ninety-four control responses to an identical but unconditioned click were 1.55 +- 8 uv (SD) for CM and 178 + 14 pv for N1. The dissipation of CM potentiation ,after a maximal activation of ECB appeared about four times as slow as that of neural reduction. We wonder whether, for the intervals of 100-200 msec, the N1 voltage was not paradoxically increased above its control value in Fig. 7 I. This point was looked into more closely in pigeon P97. The histograms of Fig. 7, each based on about thirty tests, confirm the clear reduction of N1 for short, 20 msec, train-click interval (Fig. 7 L). For the interval of 200 msec (Fig. 7 K), the histogram is shifted to the right but presents a double peak, one corresponding to that of the control histogram (Fig. 7 J) and another peak corresponding to larger responses. This may suggest that the probability of responsewas increased in only a fraction of the population of neural units considered. In so far as

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statistical methods designed for normal distribution are applicable, the mean voltage of N1 in Fig. 7 K is significantly larger than the control in Fig. 7 J at p = 0.01. The dissipation was also studied by recording central responses which are uncontaminated by a CM component, and this allowed assessment of actual input to the brain (Fig. 8). The data presented the same pattern and the same time scale as for N1 recorded at the round window. The ECB-conditioned response was back to control

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FIG. 7. Dissipation of the centrifugal effects after stimulation of ECB. All the responses were recorded from the round window. A-I. Pigeon P49 (see Fig. 2) ; A and G, control responses to click alone; B-F, testing click preceded by ECB stimulation with 30 pulses at 300 per set at the intervals indicated above each frame; H and I, diagrams with voltage in microvolts of CM (H) and N, (I) as ordinates and with train-click intervals in seconds as abscissa. The horizontal lines mark the mean voltages f SD for ninety-four control responses to the unconditioned click. J to L. Pigeon P97 (see Fig. 4). Histograms of the voltage of about thirty N, potentials evoked either by the testing click alone (J) or by the ECB-conditioned click with train-click intervals of 200 msec (K) or of 20 msec (L).

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voltage for intervals of 100 msec (Fig. 8 E) and a few potentials were above the normal range for intervals of 200 msec (Fig. 8 F’, H). When the central temperature was allowed to drop from 42 to about 36 C, both the CM potentiation and the N1 inhibition dissipated more IOmsec

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FIG. 8. Dissipation of ECB-induced changes in central responses. Pigeon PlOO, 480 g, transection of cervical (C2) spinal cord, Flaxedil paralysis, cloaca1 temperature 41.5 C. Potentials recorded in the region of the nucleus laminaris (Fig. 1OD) and evoked by an homolateral click. A and G. Control responses to the click alone; B-F, maximal ECB stimulation preceding click at the intervals indicated above each frame. H. Diagram with voltage in millivolts of response as ordinate and with train-click interval in seconds as abscissa.

slowly but the difference between the time scale of either effect was still evident (Fig. 9). In this experiment the maximum neural inhibition was no more than 2.5 equivalent decibels and it proved more convenient to plot the percentage voltage change instead in the ordinate. The increase in CM evoked by click was also expressed in percentage, since this could not be estimated consistently in equivalent decibels. The tendency of the neurals to increase slightly above the control occurred for longer trainclick intervals. The drop in central temperature did not significantly

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change the size of the efferent effect for the shortest train-click intervals. The experimental data were too scarce for estimation of a temperature coefficient of dissipation of ECB effects. At 36 C the latency of the CM potential estimated from the time of delivery of the click was unchanged hut the latency of the N1 spike increased by about 10% and that of the 7

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FIG. 9. Influence of hypothermia on the dissipation of ECB effects. Pigeon P76, 480 g, transection of cervical (C2) spinal cord and Flaxedil paralysis. Abscissa, interval in msec between ECB stimulation and the testing click. Ordinates, percentage changes of the CM (in A) and the N, (in B) components of the round-window response. The zero corresponds to the control response to click alone. Dots, cloaca1 temperature 42 C. Circles, cloaca1 temperature 36 C.

20

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FIG. 10. Histological controls. Frontal paraffin sections, IO-20 p thick, through the brain stem; Kliiver-Barrera stain. Scales of 100 p in A, B and D ; scale of 10 p in C. A. Pigeon P24. B. Pigeon P49. The arrows point to the tip of the stimulating cathode, close to the raphe in the dorsal brain stem. Many fibers are seen to decussate at that level and some of them belong to the ECB. C. Prussian blue precipitate (arrows) at the tip of the stimulating electrode in pigeon P49. D. Pigeon PloO showing recording electrode in the region of nucleus laminaris. Abbreviations: NJ, noyaux jumeaux of Cajal; NL, nucleaus laminaris; R, raphe; Ve, vermis anterior of the cerebellum; V, descending root of the trigeminal sensory nucleus; VI, abducens nerve; VII, facial nerve.

Nz spike by 20%. All the above changes proved reversible upon rewarming the animal. Discussion

The bird’s ear discloses substantial differences with respect to the mammalian ear (25, 26, 30, 31, 34) and it is important to find out whether

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the efferent innervation of the cochlea (29, 2) presents similar or different functional properties in the two groups. We chose to work with pigeons because they are readily available and their ECB has been described by Boord (2). Pigeons do not withstand physiological experimentation as reliably as cats or monkeys and much of our work was concerned with the design of technical procedures (anesthesia, stereotaxy, transection of cervical spinal cord, . . .) in order to optimize the avian preparation in relation to our problem (Figs. 1, 10). With regard to the nature of the influence exerted by the efferent innervation on the cochlea in pigeons, our data indicate substantial similarities with the OCB of carnivores. Electrical stimulation of the ECB in pigeon elicits a dual effect (8) : It potentiates the CM potential thought to be generated at the apex of the hair cells; and it simultaneously reduces the voltage of the auditory nerve response to sound (Figs. 2, 4). The latter effect can be considered as inhibitory since it results in an actual reduction of the acoustic input to the ascending pathway as shown by direct recording of the potentials evoked by click in the first auditory relay of the brain stem (Fig. 8). In a large number of animals this dual effect has been consistently recorded over periods of hours, under experimental conditions which excluded any spurious interference (no systemic cardiovascular changes, no muscle contraction, no auditory masking, . . .). Both the potentiation of CM and the inhibition of the neurals can be eliminated by administration of strychnine or brucine, but they are not affected by other convulsants (9) which means that the neuropharmacological properties of the efferents to the cochlea are similar in pigeon and cat (12, 13). Finally the parameters of electrical stimulation of the ECB agree surprisingly well with those of the cat’s OCB. It is thus clear that the pigeon’s ECB does influence the transduction of sound waves into nerve impulses in the inner ear with the net result of reducing the acoustic input to the brain. On the basis of the above evidence we propose to consider that the ECB of the bird is homologous functionally to the mammalian olivocochlear system. Clear and specific differences have nevertheless been found in the operational characteristics of the pigeon’s ECB and they have been closely analyzed in view of their potential significance for both the fine organization of the ear and the acoustic behavior of the bird. In the first place the potency of efferent gating appears very limited in the pigeon, no matter how it is estimated. The neural responses evoked by click are never completely suppressed and the percentage reduction is

22

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at most -60% and frequently less. A more meaningful estimation as equivalent decibel change of the sound stimulus has been shown to apply to the pigeon’s neural responses (Fig. 4). In most experiments the ECB inhibition amounts to -2 or -3 equivalent decibels and the largest effect seen does not exceed -7 equivalent decibels (8). In the cat tested with the same methods, a complete suppression of acoustic input is commonly observed when the testing sound is within 20 db from threshold and inhibitions amounting to -25 equivalent decibels are not exceptional (7). The above figures relate to experimental situations involving maximal faradization of the bundle of efferents and they probably set a mean upper limit to the amount of gating that can occur in a behaving animal. The function of the efferents in behavior is still obscure but it can at least be speculated about what the carnivore could do with such a strong centrifugal inhibitory system whereas it is difficult to see the advantage for the pigeon of being able to change its acoustic input by only a few decibels. The pigeon performance is dominated by vision and it would be most interesting to know whether such specialization might involve a reduction in the potency of the ECB with respect to that of other birds, If so the behavioral significance of the ECB would be considerably stressed. This question cannot be answered now and requires a similar study to be performed on birds which rely predominantly on auditory cues. Another important difference is the relative prominence of the potentiation of the CM receptor potential in the pigeon. For the cat’s OCB the increase of the CM potential evoked by click or tone pip frequently amounts to no more than +l or +2 equivalent decibels, even when a -25 equivalent decibel inhibition of neurals is simultaneously produced (7). The reverse situation occurs with the pigeon for which the CM potentiation by ECB stimulation is more conspicuous than neural inhibition. When a tone pip is used as the testing sound the CM may increase by as much as +7 equivalent decibels (Fig. 5). For obscure reasons the CM potentials evoked by clicks, though markedly increased, have not allowed consistent estimation in equivalent decibels to be made. However, in a few paradoxical experiments, the CM component was potentiated markedly without there being a noticeable reduction of the neurals (Fig. 6). As a practical implication, liminal efferent effects are detected more easily by watching the changes induced in the CM component in the pigeon, and by watching the changes in the neural component in the cat. These observations are further emphasized by the unexpected difference

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in kinetics of ECB effects on CM and neurals, respectively (10). The maximum changes are observed in both components when the testing sound is made to occur just after the ECB faradization. As the interval between the ECB stimulation and the testing sound increases, the changes dissipate-at a different rate and the potentiation of CM is more enduring than the inhibition of the neural components (Fig. 7, 8). These results contrast sharply with those in cat where the OCB effects on CM and neural components dissipate at the same rate (7). The foregoing observations may be considered in relation to the hypothesis of Desmedt and Monaco (12, 13) suggesting that the efferent fibers to the cochlea release a K- or Cl-permeability-increasing transmitter at their terminals, thus presenting an example of postsynaptic inhibition. The action of such a transmitter on the membrane of the base of the hair cells would account for the potentiation of the CM receptor potential generated by mechanoelectrical transduction at the top of the hair cell. The suppression of the neural response to sound would result along this line from the action of the same transmitter on the membrane of the peripheral auditory dendrite. These mechanisms will be considered further at a later time. In any case a similar type of neurochemical transmitter can be postulated for the pigeon’s ECB since its effects on the ear are likewise antagonized specifically by a suitable dose of strychnine or brucine (9). The question of how the pigeon’s efferents release their transmitter in the inner ear must be carefully considered in view of the different kinetics of the changes induced in CM and neurals, respectively (Fig. 7). We do not favor the uneconomical postulate that the pigeon might possess two sets of efferent fibers with slightly different properties, one affecting the hair cells only and the other acting on the dendrites. We think that a single and homogeneous set of efferents can do the job and that the prominence and slower dissipation of CM potentiation results from peculiarities in synaptic organization. Thus the efferents might establish particularly extensive and elaborated synaptic contacts onto the hair cells which would thereby receive a larger share of transmitter. The prolonged CM potentiation would follow if diffusional hindrances slowed down the removal of transmitter from the synaptic cleft between efferent endings and hair cell. An alternative explanation is that enzymic inactivation of the released transmitter, if any, would operate more slowly at the hair cell membrane than at the auditory dendrites. It is generally held that the CM receptor potential represents one link in the mechanism leading to the triggering of an action potential in the

24

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afferent auditory fiber (5). If so, potentiation of CM without concomitant “stabilization” or inhibition of the afferent dendrite should result in a recruitment of additional units in the auditory nerve. The data of Figs. 7 and 8 suggest that such a situation may, indeed, obtain about 200 msec after a burst of activity in the ECB since the neural response evoked by sound tends to be increased after the inhibitory phase. This unexpected transient increase can at least tentatively be related to the differential rate of removal of the efferent transmitter released on hair cell and dendrite, respectively. Such a phenomenon has not been recorded at any stage of the action of the olivocochlear fibers on the cat’s inner ear (7). The above speculations certainly do not exhaust the problems raised and their purpose is mainly to stimulate further investigations on ‘the electron microscopy and biophysics of the bird’s inner ear.3 References

4. 5. 6.

8.

9. 10.

BECCARI, N. 1943. “Neurologia comparata.” Sansoni Edizioni Scientifiche, Firenze. Booan, R. L. 1961. The efferent cochlear bundle in the caiman and pigeon. Exptl. Neurol. 3: 225-239. BOORD, R. L., and G. L. RASMUSSEN. 1963. Projection of the co&ear and lagenar nerves on the cochlear nuclei of the pigeon. J. COW@. Neural. 120: 463-475. BREUER, J. 1908. Ueber das GehSrorgan der Vogel. Sitzber. Kaiserl. Akad. W&s. Wien, Kl. Math. Naturw. 116: 249-292. DAVIS, H. 1961. Some principles of sensory receptor action. Physiol. Rev. 41: 391-416. DAVIS, H., I. TASAKI, and R. GOL~TEIN. 1952. The peripheral origin of activity with reference to the ear. Cold Spring Harbor Symp. Quant. Biol. 17: 143-154. DESM~DT, J. E. 1962. Auditory-evoked potentials from cochlea to cortex as influenced by activation of the efferent olivo-cochlear bundle. J. Acoust. Sot. Am. 94: 1478-1496. DESMEDT, J. E., and P, J. DELWAIDE. 1963. Activation of efferent cochlear bundle in the pigeon. J. Acoust. Sot. Am. 36: 809. DESMEDT, J. E., and P. J. DELWAIDE. 1963. Neural inhibition in a bird: effect of strychnine and picrotoxin. Nature ZOO: 583-585. DESMEDT, J. E., and P. J. DELWAIDE. 1964. Particularites fonctionnelles de l’inhibition efferente cochleaire chez le pigeon. Arch. Intern. Pkysiol. Biochem. 72: 341-346.

a A recent electron microscope study of the organ of Corti in the pigeon indicates that efferent terminals establish extensive synaptic contacts with the hair cells (R. Cordier, 1964, Sur la double innervation des cellules sensorielles dans I’organe de corti du pigeon. Corn@. Rend. Acad. Sci. Paris 266: 6238-6240).

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DESMEDT, J. E., and V. LAGRUTTA. 1963. Function of the uncrossed efferent olivo-cochlear fibres in the cat. Nature 200: 472-474. DESMEDT, J. E., and P. MONACO. 1961. Mode of action of the efferent olivocochlear bundle on the inner ear. Nature 192: 1263-1265. DESMEDT, J. E., and P. MONACO. 1962. The pharmacology of a centrifugal inhibitory pathway in the cat’s acoustic system. Proc. 1st. Zntnat. Pharmacol. Meeting 8: 183-188. ERULKAR, S. D. 1955. Tactile and auditory areas in the brain of the pigeon. J. Camp. Neurol. 103: 421-457. EWALD, J. R. 1892. “Physiologische Untersuchungen iiber das Endorgan des Nervus Octavus.” J. F. German, Wiesbaden. FEX, J. 1962. Auditory activity in centrifugal and centripetal cochlear fibres in cat. Acta Physiol. &and. Supp. 55 189: l-68. GALA-OS, R. 1956. Suppression of auditory nerve activity by stimulation of efferent fibers to cochlea. J. Neurophysiol. 19: 424-437. GALAMBOS, R., and A. RUPERT. 1959. Action of the middle ear muscles in normal cats. J. Acoust. Sot. Am. 31: 349-355. GOCAN, P. 1964. Dispositif de contention stereotaxique rigide du pigeon. Arch. Ital. Biol. 102: 36-39. HEISE, G. A. 1953. Auditory thresholds in the pigeon. J. Psychol. 66: 1-18. Electrical responses to acoustical HEISE, G. A., and W. A. ROSENBLITH. 1952. stimuli recorded at the round window of the pigeon. J. Camp. Physiol. Psychol. 45: 401-412. KAYSER, C. 1929. Regulation thermique apres section medullaire dorsale chez le pigeon. Compt. Rend. Sot. Biol. 100: 286-288. KILLIAN, G. 1890. Zur vergleichenden Anatomie und vergleichenden Entwickelungsgeschichte der Ohrmuskeln. Anat. Anz. 5: 226-229. POHLMAN, A. G. 1921. The position and functional interpretation of the elastic ligament in the middle-ear region of gallus. J. Morphol. 36: 229-262. PORTMANN, A. 1950. Systeme nerveux, Organes des sens, pp. 185-220. In “Trait& de zoologie.” P. P. Gras& [ed.]. Masson, Paris. PUMPHREY, R. J. 1961. Sensory organs: hearing, pp. 69-86. In “Biology and comparative physiology of birds,” vol. 2. A. J. Marshall led.]. Academic Press, New York. RAMON Y CAJAL, ‘5 1907. Les ganglions terminaux du nerf acoustique des oiseaux. Trab. Inst. Cajal. Invest. Biol. Madrid 5: 195-225. RASMUSSEN, G. L. 1946. The olivary peduncle and other fiber projections of the superior olivary complex. J. Comp. Neural. 84: 141-219. RASMUSSEN, G. L. 1960. Efferent fibres of the cochlear nerve and cochlear nucleus, pp. 105-115. In “Neural Mechanisms of the Auditory and Vestibular Systems.” G. L. Rasmussen and W. F. Windle led.]. Thomas, Springfield, Illinois. RETZIUS, G. 1884. “Das Gehororgan der Wirbelthiere: Morphologisch-Histologische Studien. II. Das Gehororgan der Reptillien, der Vogel und der Saugethiere.” Samson and Wallin, Stockholm. SATOH, N. 1917. “Der Histologische Bau der Vogelschnecke und Ihre Schadigung durch akustische Reize und durch Detonation.” Benno Schwabe, Basel.

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J. 19.54. Schallsinnesorgane, ihre Funktion und biologische Bedeutung bei Vdgeln. Acta Congr. Intern. Ornithol., Ilth, Basel: pp. 374-379. 1963. Methode de derivation des SCHWARTZKOPFF, J., and J. C. BREMOND. potentiels cochleaires chez l’oiseau. J. Physiol. Paris 66: 495-518. STOPP, P. E., and I. C. WHITFIELD. 1961. Unit responses from brain-stem nuclei in the pigeon. J. Physiol. London 158: 165-177. VAN TIENHOVEN, A., and L. P. JUHASZ. 1962. The chicken telencephalon, diencephalon and mesencephalon in stereotazic coordinates. J. Camp. Newel. 118: 185-198. WALLENBERG, A. 1904. Neue Untersuchengen iiber den Hirnstamm der Taube. SCHWARTZKOPFF,

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M. P. 1933. Ueber das Unterscheidungsvermagen der Viigel fiir die hohen Tone. Zeitschr. Vergleich. Physiol. 19: 424-438. 38. WEVER, E. G., and C. W. BRAY. 1936. Hearing in the pigeon as studied by the electrical responses of the inner ear. J. Camp. Psychol. 22: 353-363. 1956. The control of sound transmission by 39. WEVER, E. G., and J. A. VERNON. the middle ear muscles. Ann. Otol. Rhinol. Laryngol. 65: 7-14. 37.

WASSILJEW,