Segregation of efferent projections to different turns of the guinea pig cochlea

69

Hearing Research, 25 (1987) 69-76 Elsevier

HRR

00839

Segregation of efferent projections to different turns of the guinea pig cochlea * Donald Department

Robertson,

Caitrin-Jane

Anderson

and Kenneth

S. Cole

ofPhysiology, University of Western Australiu, Nedlands, Western Australia, 6009 Australia (Received

23 April 1986; accepted

8 July 1986)

Localized intracochlear injections of the fluorescent retrograde label diamidino yellow were used to investigate the organization of efferent projections from the brainstem to different turns of the cochlea, in the guinea pig. It was found that the location of small neurones within the lateral superior olive ipsilateral to the injection varied in a systematic manner when injections proceeded from base to apex of the cochlea. In addition, a cruder form of cochleotopic organization was present in that most of the large medial system efferent neurones were labelled only after injection into the 3 most basal turns of the cochlea. The decline of medial system efferent innervation proceeding from base to apex was most striking for the contralateral efferent neuronea. The details of base to apex innervation density were different for the different nuclei of origin of the medial system, implying the existence of complex subsystems within the medial neurone population. cochlea,

efferent,

differential

projection

Introduction The efferent projections to the mammalian cochlea can be conveniently divided into a small neurone or lateral system and a large neurone medial system (Guinan et al., 1983; Warr, 1978). In the guinea pig, the small neurone lateral system arises almost exclusively from within the lateral superior olivaty (LSO) nucleus and more than 99% of these cells project to the ipsilateral cochlea (Robertson, 1985; Strutz and Beilenberg, 1984). Within the cochlea, this lateral system is believed to terminate largely on the dendrites of the primary afferent neurones beneath the inner hair cells (Brown, 1985; Guinan et al., 1983). The larger neurone medial system .has been shown in the guinea pig to arise from cell bodies in various nuclei; the ventral and medial nuclei of the trapezoid body (VNTB and MNTB), the dorsomedial periolivary complex (DMPO) and the ventral nucleus of the lateral lemniscus (VLL). Approxi* Supported by grants from the National Health and Medical Research Council, the Australian Research Grants Scheme and the University of Western Australia. 0378-5955/87/$03.50

0 1987 Elsevier

Science Publishers

mately two thirds of the extra-LSO system neurones project to the contralateral cochlea, where they probably terminate mainly on the outer hair cells (Robertson, 1985). Recent studies in two species provide evidence that at least some of these efferent neurones project differentially to the different turns of the cochlea. In the cat, autoradiographic studies have demonstrated that the medial system innervates largely the more basal, high frequency turns of the cochlea, whilst the small neurone lateral system, which in this species appears to arise from the margins of, rather than within the LSO, innervates all turns (Guinan et al., 1984). The work of Stopp (1983), employing intracochlear injection of the retrograde tracer true blue, indicated that in the guinea pig, the cell bodies of the small neurone LSO system tended to be found within this nucleus in locations which suggested a correspondence between their site of termination within the cochlea and the LSO afferent iso-frequency contours. Injections into the basal turn labelled neurones largely in the dorsomedial lobe of this nucleus whilst more api-

B.V. (Biomedical

Division)

70

cal injections labelled cells in more lateral and ventral regions. The present study in the guinea pig was undertaken to elucidate the differential nature of the efferent projections to the different cochlea regions for both the small and large neurone systems. The tracer substance used was the retrograde fluorescent label diamidino yellow (DY) (Keizer et al., 1983). This dye forms a fine colloidal suspension in aqueous media and preliminary experiments indicated that it does not diffuse over large distances from its injection site. It therefore appeared ideally suited for localized injections into the individual cochlea turns. Methods 15 young pigmented guinea pigs (180-260 g) were anaesthetized by intraperitoneal injection of Pentothal (thiopentone sodium), supplemented by an intramuscular injection of Innovar when needed. For injections into the hook, or most basal region of the cochlea, the tympanic bulla was exposed via a dorsal approach and DY was injected through the round window. For all other injections, the tympanic membrane was removed to gain access to the cochlea. A 100 pm hole, through which the DY was injected was drilled in the Scala tympani of one turn and an outlet hole of the same size was drilled in the Scala tympani of the next more apical turn (Fig. 1). When an injection was made into the 4th turn, an outlet was made by making a small hole in the cochlea apex. DY (obtained from Dr. Illing GmbH & Co.) was injected as a 2% suspension in distilled water, via glass pipettes of tip diameter 50 pm, using either a small hydrostatic pressure head, or an oil-filled Hamilton syringe. The volume injected was 1 ~1 or less. After injection, the perfusion holes were covered by small skin grafts from the external meatus, all wounds were sutured with surgical silk and the animals were allowed to recover from the anaesthetic. After 5 days, they were deeply anaesthetized by intraperitoneal injection of Nembutal, and fixed by intracardiac perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were embedded in gelatinalbumin and soaked overnight in 30% sucrose in

inlet

-apex

turn1

2

3

4

Fig. 1. Schematic representation of the technique of localized intracochlear injection of aqueous suspensions of diaminido yellow. In the example shown an injection is being made from the scala tympani of the 2nd turn to the Scala tympani of the 3rd turn.

0.1 M phosphate buffer, before cutting 40 pm serial sections on a Leitz Kryomat. Sections were viewed by epi-illumination with an Olympus Vanox fluorescence microscope using the V excitation and barrier filter combination. The bright yellow nuclei of labelled cells could be easily seen. The location of all labelled cells was recorded, and photographs of the distribution of cells within the LSO were taken using Kodak technical 2415 film. Results Inspection of the injected cochleas after fixation, showed that the injected dye had formed a solid cast within the Scala tympani between the inlet and outlet perfusion holes (Fig. 2). In 12 of the 15 animals used, the dye was located within the Scala tympani of the injected turn. In the other animals, which were not included in subsequent analysis, the perfusion had either damaged the Scala media or had spread into the Scala vestibuli of the adjacent turn, presumably because of mechanical damage to the bony septum separating each turn. Fluorescence microscopy on dissected portions of the organ of Corti from the successful cochleas, revealed that the dye had spread and iabelled cell nuclei for up to l/2 turn basal and apical of the solid dye cast. Fig. 3A-C, shows the appearance of labelled

71

Fig. 2. Example lying throughout dye cast.

of dissected cochlea showing solid cast of dye the 3rd turn. Thick arrows indicate limits of

cells in the various brainstem nuclei. The dye preferentially labelled the cell nucleus, though some faint granular staining of the cell cytoplasm was sometimes evident. The dye appeared to be quite resistant to fading during prolonged viewing.

LSO neurones In agreement with previous studies on the guinea pig, large numbers of small labelled cells were found within the LSO ipsilateral to the injected cochlea in all animals. However, there was a striking systematic variation in the location of labelled cell nuclei within the body of the LSO according to the injection site (Figs. 3D-F, 4). Injection through the round window into the most high frequency basal portion of the cochlea resulted in labelling which was mainly limited to the most dorsomedial lobe of the LSO on the side ipsilateral to the injection. As the site of injection moved systematically more apically, the location of labelled cells progressed ventrally and then laterally around the LSO until for the 4th turn to apex injection, labelled cells

were found largely in the dorsolateral extreme of the nucleus. In most instances, despite this clear turn-by-turn organization within the LSO, there were one or two labelled cells found in regions of the nucleus quite separate from the vast majority of cells. The largest numbers of cells within the LSO were found in the case of 1st to 2nd turn injections with the 4th to apex injections showing the least labelled cells. Table 1 shows the mean numbers of labelled cells found for all injections. The total mean number was 1649. Fig. 5A shows that there was a systematic increase in the preponderance of LSO neurones expressed as a percentage of all labelled cells, when progressing from base to apex. The number of labelled neurones found in the contralateral LSO was extremely small, in agreement with previous studies. Despite the small numbers however, there was a clear difference between basal and apical injections. For hook region injections, no labelled cells were found. In the most apical injections, however, these rare neurones comprised up to 2% of the total. Such rare contralateral LSO cells were always found in the same region of the LSO as the majority of ipsilateral labelled cells.

Non-LSO neurones The innervation of the different cochlear turns by the medial system neurones showed complex trends which differed from nucleus to nucleus. Overall however, it was clear that the hook region and basal turn injections gave the highest proportion of labelled neurones within these extra-LSO nuclei. Surprisingly however, only the nuclei contralateral to the injected cochlea showed a systematic reduction in the proportion of these neurones from base to apex (Fig. 5B-D). In consequence, whereas the contralateral projection was clearly greater than the ipsilateral for the basal turn, within the more apical turns, the ipsilateral and contralateral projections were more or less equal. Not shown in Fig. 5, owing to the relatively small number of cells involved, are the details of labelled neurones in the MNTB and the lateral nucleus of the trapezoid body (LNTB). Labelled neurones were found in the MNTB only for the

Fig. 3. A-C Examples of labelled neurones in (A) LSO, (B) DMPO and (C) VLL. Note the bright staining of the cell nuclei and the obvious size difference between LSO and other cells. D-F examples of the location of labelled newones in ipsilateral LSO after injections into (D) hook, or most basal region, (E) 1st to 2nd turn. and (F) 4th to apical turn. The medial aspect of the LSO lies to the right in all cases.

73

2-3

3+4

&-+APEX

Fig. 4. Twical distribution of labelled neurones in ipsilateral locations: Medial edge of the LSO is to the right in all cases.

LSO shown

hook, first and second turn injections. Conversely, LNTB neurones were only labelled after injection into third, fourth and apical turns. In contrast to the pattern within the LSO, there was no striking tendency for the spatial location of labelled cells within a given medial system nucleus to vary systematically according to the injection site. Fig 6 shows examples of the location of cells within 8 superimposed serial sections of the contralateral DMPO and MNTB. Labelled cells are seen scattered throughout the dorsoventral extent of the DMPO for all 3 injection sites. If there is any trend, it lies in the mediolateral plane, with cells labelled from hook injecTABLE

I

MEAN

NUMBERS

OF LABELLED

IPSILATERAL

for 5 superimposed

sections

for each of the 4 injection

tions tending to be found in the more medial regions of the DMPO, although the small number of labelled cells for the more apical injections makes quantitative investigation of this question difficult. The only clear difference seen in Fig. 6, which has been mentioned above, lies in the total absence of MNTB cells in the case of more apical injections. Attempts to find a systematic tonotopic organization along the rostro-caudal plane were equally fruitless. Fig. 7 shows that in the contralateral DMPO there is no clear grouping of cells in this nucleus except for the 3rd to 4th turn injection, in which no labelled cells are found in the most

LSO CELLS

Injection

Hook + 1

1+2

2+3

3-4

4 + Apex

Mean number

290 (N=2)

578 (N=2)

439 (N=2)

237 (N=4)

105 (N=2)

Total = 1,649

CONTRALATERAL

DMPO-MNTB

0

caudal HOOK

I-2

2-3

3-14

L,APEX

y._, DMPO

length

10’0

rostra1

Fig. 5. Turn-by-turn distribution of efferent neurones found in the various brainstem nuclei. Bars represent the number of cells expressed as a percentage of the total number of labelled cells in each animal. Results are means from 2 or more animals. Error lines show range. Stippled bars. ipsilateral nuclei: open bars, contralateral nuclei.

Fig. 7. Distribution of labelled neurones along the rostro-caudal extent of the contralateral DMPO for 4 different injection locations.

rostra1 one third of the DMPO. Here however, the number of labelled cells is so small as to place doubts on the significance of this result. These findings were qualitatively similar for all the medial system nuclei.

The technique of injection of a colloidal suspension of retrograde tracer undoubtedly leads to a less than ideal localization within the cochlear spiral. Despite the fact that a solid cast of dye was found between inlet and outlet holes (Fig. 2), there

HOOK

Discussion

Z-+3

o Cl0

0 6’0 /oo 0 0 00

8

0

0

/‘O o o

DMPO

/

0 0 00 ‘0

$00 0

0

0 ‘-

0

MNTB ,/

_.__

--

-~~~_

/

Fig. 6. Drawings of contralateral DMPO and MNTB in 3 different animals with different cochlear inJectIon sites, showing the location of labelled neurones in 8 superimposed sections. Note presence of MNTB cells only for hook injection. Medial edge of DMPO is to left in all cases.

can be little doubt that the dye spreads for some distance apically and basally of this cast. For this reason, little reliance can be placed on the absolute numbers of labelled cells found for each injection, since there is probably a degree of overlap from one site to the next. Our estimate of the total number of ipsilateral LSO neurones in the entire cochlea derived from the sum of all single turn injections was 1649. This is well in excess of the highest estimate from HRP experiments in this species of about 400 (Robertson, 1985). However, recent unpublished experiments in our laboratory place the number of labelled cells in ipsilateral LSO, determined by injection of DY throughout the whole cochlea, at closer to 1000. Thus, while the degree of overlap is clearly considerable, it may not be as extensive as some comparisons would imply. Despite these limitations however, it has proved possible to clearly show that there is a differential projection to the different cochlear turns using this technique. The results obtained for the LSO are in sufficient agreement with those obtained using physical barriers between the different cochlear turns (Stopp, 1983) to draw reasonable inferences about the relative distribution of projections from other brainstem nuclei. Several important points emerge from the present data. There is what appears to be a tonotopitally related projection to the different cochlear turns from different parts of the LSO, as found by previous authors in cat and guinea pig (Guinan et al., 1984; Stopp, 1983). This implies that the cochlear projection area of the lateral system neurones is at least sufficiently restricted spatially to reveal such tonotopicity. An upper limit of approximately one cochlear turn might therefore be placed on the length of cochlea over which most lateral efferents form synapses beneath the inner hair cells. The fact that scattered labelled neurones can be found in regions of the LSO apparently unrelated to the injection location also suggests that some of these neurones have a more widespread terminal arborization. The very existence of a tonotopically related projection for the lateral system opens up the possibility that these neurones may receive input from limited frequency regions of the ascending or descending auditory pathway, as has been clearly

demonstrated for the medial system efferent neurones (Liberman and Brown, 1985; Robertson, 1984; Robertson and Gummer, 1985). The data also show that the small population of efferents in the contralateral LSO projects primarily to the apical turns (2% of the 4th turn projection), and comprises less than 1% of the total only when the entire length of the cochlea is considered. This finding is in qualitative agreement with results obtained in the cat, though it must be borne in mind that in this latter species the small neurones are reportedly found only on the margins of the LSO. As far as the medial system is concerned, interesting details emerge from this study. Firstly, the overall medial projection, as a percentage of the projection to each turn, declines dramatically from base to apex, being more than 50% of the total in the hook region, and less than 30% in the apex. Secondly, the ipsilateral projection behaves differently from the contralateral one, showing very little decline in relative numbers from base to apex (Fig. 5B-D). Indeed, in the apical turn, the ipsilateral projection may be equal to or greater in importance than the contralateral, depending on the nucleus of origin. The ipsilateral projections may therefore not be simply a less numerous mirror image of the contralateral projection, but may have to be considered as a separate system subserving possibly different functions. This concept of distinct subsystems within the medial efferent population is reinforced by detailed consideration of the turn-by-turn pattern for each of the medial system nuclei. The MNTB only sends projections to the first and second turns. The LNTB only sends projections to the third and fourth turns. The VNTB and VLL contralateral projections only begin to decline in relative importance in the third turn and beyond, whereas the DMPO declines beyond the basal turn and is totally absent from the apex. It may be that a parallel exists here with the findings by others that different transmitter candidates appear to be associated with sub-components of the medial efferent systems in the basal and apical cochlear turns (Altschuler et al., 1985; Eybalin and Pujol, 1986). These results also sound a note of caution for single neurone studies attempting to relate the

16

distribution of efferent neurone response types to their possible nuclei of origin. Clearly, since the pattern of nucleus-by-nucleus innervation differs in each cochlear turn, unless account is taken of the region of the cochlea which the efferent innervate, it would be hazardous to assign a particular population of response type to any particular medial system nucleus. Our failure to find convincing evidence for tonotopic order in the spatial distribution within the individual medial system nuclei is surprising. It contrasts with the autoradiographic work of Guinan and co-workers in the cat brainstem, and is at variance with what might be expected from the sharp tuning of these neurones to acoustic stimuli and the demonstrated tonotopicity of their terminal fields within the organ of Corti (Liberman and Brown, 1985; Robertson, 1984; Robertson and Gummer, 1985). In the data shown from the DMPO in Fig. 6 there is some tendency for the more basal projecting cells to cluster dorsomedially, which is qualitatively in agreement with the description in cat based on autoradiography. It may be that the small number of cells, and the undoubted spread of diamidino yellow apical and basalward of the injection site do not allow an existing tonotopic arrangement to be clearly revealed. Acknowledgements The authors thank B.M. Johnstone for helpful discussion.

and R. Pujol

References Altschuler. R.A., Hoffman, D.W., Reeks, K.A. and Fex, J. (1985) Localization of dynorphin B-like immunoreactivities in the guinea pig organ of Corti. Hear. Res. 17, 249-258.

Brown, M.C. (1985) Peripheral proJections of labelled efferent nerve fibres in the guinea pig cochlea: an anatomical study. In: Abstr. VIIIth Midwinter AR0 Meet., pp. 9-10. Eybalin, M. and Pujol, R. (1986) Choline acetyltransferase (ChAT) immuno-electron microscopy distinguishes at least three types of efferent synapse in the organ of Corti. Exp. Brain Res. (in press). Guinan, J.J., Jr., Warr, W.B. and Norris. B.A. (1983) Differential projections from the lateral v. medial zones of the superior olivary complex. J. Comp. Neurol. 221, 358-370. Guinan, J.J.. Jr., Warr, W.B. and Norris, B.A. (1984) Topographic organization of the olivocochlear projections from the lateral and medial zones of the superior olivary complex. J. Comp. Neurol. 226, 21-27. Keizer, K.. Kuypers, H.G.J.M., Huisman, A.M. and Dann, 0. (1983) Diamidino yellow dichloride: a new fluorescent retrograde neuronal tracer which migrates only very slowly out of the cell. Exp. Brain Res. 51, 179-191. Liberman, M.C. and Brown, M.C. (1985) Intracellular labelling of olivocochlear efferents near the anastomosis of Oort in cats. In: Abstr. VIIIth Midwinter AR0 Meet., pp. 13-14. Robertson, D. (1984) Horseradish peroxidase injection of physiologically characterized afferent and efferent neurones in the guinea pig spiral ganglion. Hear Res. 15. 113-121. Robertson, D. (1985) Brainstem location of efferent neurones projecting to the guinea pig cochlea. Hear. Res. 20, 79-84. Robertson, D. and Gummer, M. (1985) Physiological and morphological characterization of efferent neurones in the guinea pig cochlea. Hear. Res. 20, 63-77. Stopp, P.E. (1983) The distribution of the olivocochlear bundle and its possible role in frequency/intensity coding. In: Hearing-Physiological Bases and Psychophysics, pp. 176179. Editors: R. Klinke and R. Hartmann. Springer. Berlin. Strutz, J. and Beilenberg, K. (1984) Efferent acoustic neurones within the lateral superior olivary nucleus of the guinea pig. Brain Res. 299, 174-177. Warr, W.B. (1978) The olivocochlear bundle: its origins and terminations in the cat. In: Evoked Electrical Activity in the Auditory Nervous System, pp. 43-62. Editors: R.F. Naunton and C. Fernandez. Academic Press, New York.