Anat Embryo1 (1991) 183:483 489 Anatomy and Embryology �9 Springer-Verlag 1991

ChAT-like immunoreaetivity of olivocochlear fibres on rat outer hair cells during the postnatal development

i

Birgit Roth, Bernhard Dannhof, and Volkmar Bruns

Zoologisches Institut der J.W. Goethe Universit/it, Siesmayerstrasse 70, W-6000 Frankfurt a.M. 11, Federal Republic of Germany

Accepted February 6, 1991

Summary. Several studies present a great deal of infor- mation about putative efferent neurotransmitters and their distribution in the adult and developing cochlea. Anatomical mapping of outer hair cell efferent fibres during ontogeny is still not available. Using quantitative electron microscopy in combination with immunocyto- chemistry, the distribution of ChAT-like immunoreac- tivity in the developing rat was investigated. Adult-like immunoreactivity in the whole cochlea is first observed in 30-day-old rats. We localized the adult-like immuno- reactivity in all efferent fibres and synapses of the outer hair cells along the entire cochlear duct. An adult-like reaction in the whole cochlea could be observed on the 25th day after birth in two out of three cases. On the 20th postnatal day, no adult-like ChAT immunoreactivi- ty was found, with the exception of one case where label- ling was seen in the basal region only. The adult-like ChAT immunoreactivity on the 30th day, 2-3 weeks after the onset of hearing, is the latest maturation of all features of the organ of Corti so far investigated. Synaptogenesis of the outer hair cell efferents reaches an adult-like appearance already on the 16th day after birth.

Key words: ChAT - Cochlea - Olivocochlear system - Development - Rat

Introduction

The efferent olivocochlear fibres belong to two separate systems: the lateral and medial according to their origins within the superior olivary complex (Warr et al. 1986). In the rat lateral olivary complex neurons project exclu- sively to the ipsilateral cochlea and terminate beneath the inner hair cells. The medial olivary complex neurons project predominantly to the contralateral cochlea and terminate at the outer hair cells (White and Warr 1983).

Offprint requests to ." V. Bruns

Several immunocytochemical investigations demonstrate acetylcholine (Ach) as the major transmitter of the later- al and medial efferent system (Rossi et al. 1986; Klinke 1986; Altschuler and Fex 1986; Eybalin and Altschuler 1990). Moreover, gamma-aminobutyric acid (GABA) is also attributed the function of a neurotransmitter (Alt- schuler and Fex 1986; Eybalin et al. 1988). Further, a large number of other substances, such as enkephaline, dynorphine, glutamate, aspartate, calcitonin gene-relat- ed peptide, as well as catecholamine, whose function is so far disputed, have been identified in the efferent fibres to the outer hair cells (for review: Altschuler and Fex 1986; Eybalin and Altschuler 1990). More recent investigations favour the presence of Ach and GABA at different regions in the cochlea. Whereas Ach was found exclusively in the basal and middle region of the cochlea (Altschuler et al. 1985; Eybalin and Pujol 1987; Eybalin and Altschuler 1990), GABA could be detected so far only in the apical region (Fex et al. 1986; Eybalin et al. 1988; Eybalin and Attschuler 1990). The thesis that there exist neurochemically different subpopulations of efferent fibres, which are connected with the regulation of the outer hair cell activities, has been derived from these results (Eybalin et al. 1988).

Information about the onset and distribution of transmitters in the efferent system during ontogeny has been very rare up to now. Sobkowicz and Emmerling (1989) investigated the enzyme activity of acetylcholine esterase (AchE) and choline acetyltransferase (CHAT) in postnatal stages of mice. They found 50% activity of ChAT and 20% activity of Ache even in the newborn organ of Corti. The enzyme activity increased strongly in the second postnatal week. The development appears mature for ChAT on the 15th day, and for AchE on the 25th day after birth. The morphological and physio- logical development of the organ of Corti is closely cor- related with the advance of immunoreactivity. With re- gard to the morphology, including synaptogenesis, the adult appearance is reached by the 20th day after birth (Wada 1923; Lenoir et al. 1980). The adult frequency range of 0A-70 kHz and the adult sensitivity are also

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reached on a r o u n d the 20th p o s t n a t a l day (Crowley and H e p p - R e y m o n d 1966; Uziel et al. 1981).

Despi te n u m e r o u s i m m u n o c y t o c h e m i c a l invest iga- t ions o f the pe r iphe ra l a u d i t o r y system, a n a t o m i c a l m a p - p ing o f C h A T - l i k e i m m u n o r e a c t i v i t y o f ou te r ha i r cell efferent f ibres in the deve lop ing cochlea is no t ye t avai l - able. M o s t analyses are res t r ic ted to surface p r e p a r a - t ions, which indeed indica te a ba so -ap i ca l gradient . But quan t i t a t ive d a t a a b o u t the numer ica l p a r t o f the im- m u n o s t a i n e d fibres can be de t e rmined only by us ing the e lec t ron microscope . The a im o f the presen t s tudy was to m a p spa t ia l ly and t empora l l y the d i s t r i bu t ion o f ChAT- l ike i m m u n o r e a c t i v i t y in the efferents to the ou te r ha i r cells by e lec t ron mic roscopy .

Material and methods

Sixteen rats of the Wistar strain, at the ages of 12, 16, 20, 25 and 30 days after birth, and eight adult (4-month-old) rats were investigated in this study. In total, eight experiments with animals of different ages were performed. Each experiment contained at least one animal that was 30 days old and/or one adult animal.

The rats were sacrificed with an overdose of diethyl ether. After dissection, the cochleae were fixed by immersion for 30 min in a fixative containing 4% paraformaldehyde, 0.1% glutaraldehyde and 15% saturated picric acid in 0.1 M phosphate buffer. The cochleae were stored in glutaraldehyde-free fixative for another 1.5 h. During the subsequent rinsing process with phosphate buffer, the cochleae were prepared for the immunocytochemical reaction. The bony capsules of the cochlea were first removed. The exposed cochlear spiral was then divided with a razor blade and forceps approximately into half turns, which were then transferred into reaction tubes. The length and baso-apical position of the portions of basilar membrane were measured. The tissues were then washed in phosphate buffer containing 10% sucrose. The reaction tubes containing the tissues were briefly frozen in liquid nitrogen, thawed to room temperature and washed again in phosphate buffer. All tissues were pre-incubated for 10-15 rain in phosphate buffer con- taining 10% methanol and 0.1% H202 to reduce the nonspecific staining of the background. The tissues were incubated with a monoclonal antibody to ChAT (Boehringer Mannheim) raised in rat. Incubation times and dilutions used were as follows: Anti- ChAT (dilution 1 : 5) for 48-72 h at 4 ~ C, rabbit anti-rat IgG (dilu- tion 1:40) for 1.5 h at room temperature, PAP complex from rat (dilution 1:40) for 2 h at room temperature. The antibodies were diluted in 0.1 phosphate buffer containing 0.1% normal rabbit se- rum and 0.1% NAN3. After each incubation step, the tissues were washed in phosphate buffer. The peroxidase activity was revealed by using DAB/H2Oz. The tissues were postfixed with 1% osmium tetroxide, blockstained with 1% uranyl acetate, dehydrated and embedded in epoxy resin. The first or second antibody was omitted in parallel control experiments. The embedded portions of basilar membrane were cut ultrathin in the tunnel of Corti and the three rows of outer hair cells. The uncontrasted ultrathin sections were investigated in a ZEISS EM 902 electron microscope. All the fibres and synapses of the sections were studied, so that in total, along the entire cochlear duct, approx. 4000 efferent fibres in the tunnel of Corti and approx. 250 efferent synapses on the outer hair cells were investigated in the 30-day-old rats.

The preparation technique used here is in conformity with the German law of animal protection.

Results

In the case o f posi t ive C h A T - i m m u n o r e a c t i v i t y , all tun- nel r ad ia l f ibres and all efferent synapses on the ou te r

ha i r cells were s ta ined wi thou t any except ion (Fig. 1). Label l ing o f afferent f ibres or o the r non -ne rvous s truc- tures was no t found. N o i m m u n o s t a i n e d f ibre or synapse was obse rved in one o f the con t ro l exper iments per- fo rmed. In to ta l approx . 4000 efferent fibres in the tun- nel o f Cort i , and approx . 250 efferent synapses on the ou te r ha i r cells were eva lua ted in the 30-day-o ld rats , for example . A n age-specif ic dependence o f i m m u n o r e - act ivi ty cou ld be de t e rmined (Fig. 4).

12 and 16 days after birth

I m m u n o r e a c t i v i t y was no t f o u n d in any o f the nine speci- mens. Two an imals each were eva lua ted per stage.

20 days after birth

A t this s tage no i m m u n o r e a c t i v i t y was found in 13 ou t o f 14 samples (Fig. 2). The cochleae o f four an imals were eva lua ted over on average more than 50% o f the length o f bas i l a r m e m b r a n e . Clear ly posi t ive i m m u n o s t a i n i n g was obse rved in one cochlea at the pos i t i on 0 % to 15% length o f bas i la r m e m b r a n e . N o i m m u n o r e a c t i v i t y was found in the apical reg ion (65% to 100%) o f the same cochlea.

25 days after birth

A clear posi t ive i m m u n o r e a c t i o n was f o u n d at all exam- ined bas i la r m e m b r a n e pos i t ions in two ou t o f three an imals f rom different exper iments . In one an ima l im- m u n o s t a i n i n g was no t asce r ta ined in any bas i l a r mem- b rane region.

30 days after birth and adult

ChAT- l ike i m m u n o r e a c t i v i t y was found at these stages a long the ent ire cochlear duct . Ou t o f 13 an imals exam- ined (5 at 30 days af ter b i r th ; 8 adul ts) all tunnel r ad ia l f ibres and synapses showed posi t ive, C h A T - l i k e immu- nos ta in ing (Figs. 1 and 3).

Discussion

Method

The C h A T - a n t i b o d y used here is cons ide red to be a spe- cific m a r k e r for chol inerg ic nerve fibres, and has been successfully app l i ed by Eckens te in and T h o e n e n (1982), Eyba l in and Pujo l (1985, 1987), Bia lowas and F ro t sche r (1987).

Despi te the s t anda rd i zed and rou t ine ly p e r f o r m e d ex- per iments , va r i a t ions regard ing the in tensi ty o f the im- m u n o s t a i n i n g occur red be tween the exper iments . I f no i m m u n o s t a i n i n g cou ld be ascer ta ined in the adu l t or the 30-day-o ld an imal , the exper imen t was no t t aken into

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Fig. 1 a-c. ChAT immunoreactivity of efferent fibres and synapses of the outer hair cells in 30-day-old rats. Note that all fibres are labelled, a Tangential section of one row of outer hair cells showing immunostained synapses (arrou,s). • 4400. b Tangential section through the tunnel of Corti showing ChAT immunoreaction in all tunnel radial fibres. BM, basilar membrane; PC, pillar cells. x 4400. c Schematic drawing of the organ of Corti. Bar demon- strates the position of section shown in b

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Fig. 2a-d. 20 days after birth. At this stage no adult-like ChAT immunoreactivity is seen in efferent fibres and synapses, a, e Base of an outer hair cell contacted by unstained afferent (asterisks)

and efferent synapses, x 15000. b, d Unreactive tunnel radial fibres of the outer hair cells, x 24000. Plane of sectioning as shown in Fig. 1 c

account for the evaluation. Moreover, the positive reac- tion in 30-day-old rats offered a comparison with the labelling of the younger animals of the same experiment.

Adult-like ChAT immunoreactivity

Deviating f rom the results presented here, to the effect that there is immunoreact ivi ty in all efferent fibres to the outer hair cells, ChAT-like staining has been de- scribed only locally and partially in the literature. Immu- noreactivity decreasing baso-apically was ascertained in the guinea-pig by Altschuler et al. (1985), who examined ChAT-like immunoreactivi ty by light microscopy. The number of efferent fibres, their diameter as well as the

size of the fibre bundle in the tunnel of Corti, decreases towards the apex (Dannhof 1990). For this reason the detection of immunostained fibres in the light micro- scope comes up against the limits of the resolving capaci- ty and thus labelling could remain undetected. Electron microscopic analyses by other workers showed only ap- prox. 80% of all efferents to the outer hair cells were immunostained (Eybalin and Pujol 1985, 1987; Eybalin and Altschuler 1990). Eybalin and Pujol use the same ChAT-ant ibody on the same strain of rats as we do, but the method used by them deviates decisively from that presented here. In contrast to ours, their fixative contains no picric acid and no glutaraldehyde, and the tissues are treated with Triton-X-100. So we think that the presence of unstained fibres in their preparat ions is the result of the difference in method.

Fig. 3a-f. 30 days after birth. In 30-day-old rats all outer hair cell efferents show adult-like ChAT immunostaining, a Three immuno- labelled efferent synapses contacting an outer hair cell. x 15000. b, e Base of outer hair cells contacted by an immunoreactive effer- ent synapse and an unreactive afferent (asterisks) synapse.

• 15000. d Tunnel radial fibres showing ChAT immunoreactivity in all fibres, x 24000. e Tunnel radial fibres of a control experiment showing no immunostaining, x 24000. f Unreactive efferent syn- apses of a control experiment. • 15000. Plane of sectioning as shown in Fig. 1 c

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20 DAB i . . . . . . . . . . . . . . . . . . . .

01 .... ..... : : : 25 DAB

2 7- . . . . . . . . . . . . . . . . . . . . . . " . . . . . . . . . . . . . .

1 O I I | I I I

30 DAB

ADULT 8 -

6

4 -

2 -

0 0

I I I I I 20 40 60 80 100

BM- LENGTH [%] Fig. 4. Baso-apical distribution of the investigated portions of basi- lar membrane in the developing rat. For each investigated animal the position and length of the basilar membrane portions are shown. Adult-like immunoreaction: solid lines; non-reaction: dot- ted lines

ChA T-like immunoreactivity during ontogeny

Despite numerous immunocytochemical investigations, little is described in the literature concerning the onset and the distribution of transmitter substances during on- togeny. Sobkowicz and Emmerling (1989) examined the enzyme activity of ChAT and Ache at postnatal stages in mice. There was 20% activity of AchE and 50% activ- ity of ChAT in comparison to the adult value even in the newborn organ of Corti. The activity of ChAT rose to the adult level from birth up to the 15th postnatal day, and remained unchanged afterwards. The activity of AchE increased up to the 25th day after birth. In contrast to the present study, Sobkowicz and Emmerling used in their investigation homogenates of individual turns, which contain both the region of the outer and of the inner hair cells. In the lateral olivocochlear system, which projects to the inner hair cells, Ach was identified as neurotransmitter (Godfrey and Ross 1985; Rossi et al. 1986; Klinke 1986; Altschuler and Fex 1986; Bledsoe et al. 1988; Eybalin and Altschuler 1990). The maturity of the inner hair cells precedes that of the outer hair cells (Pujol 1986), so that the high ChAT enzyme activity in the younger stages could be due to the inner hair cells. It is further known that growth cones of cholinergic nerve fibres release Ach even before the contact to the

target cell is established (Vaca 1988). This indicates that the expression of cholinergic enzymes precedes the mor- phological maturity of the synapses. The early presence of Ach may be connected with a regulative function. In any case a non-synaptic function for cholinergic en- zymes may be present (Vaca 1988). Thus the results of Sobkowicz and Emmerling do not contradict the present results.

Nevertheless it is striking that the adult expression of ChAT occurs late in comparison with the structural differentiation of the organ of Corti. Even the synaptic contacts of both hair cell populations appear to be ma- ture for some time (Lenoir et al. 1980; Pujol and Lenoir 1986). However, the presence of CHAT, possibly in smaller concentrations, cannot be ruled out in 12- to 20-day-old rats. The late onset of the synthesizing en- zyme ChAT implies also late onset of the neurotransmit- ter substance of acetylcholine, which would have func- tional consequences for the outer hair ceils.

Lenoir and Puel (1987) could show a frequency-de- pendent development of oto-acoustic emissions in rats, which lasts up to the 28th day. Oto-acoustic emissions are produced by the cochlea, and can usually be recorded in the ear canal. They occur spontaneously but can also be evoked by acoustic stimuli. The evoked oto-acoustic emissions contain more energy, produced by the cochlea, than the originally applied stimulus. It is generally as- sumed that the oto-acoustic emissions are due to an ac- tive biomechanical process in the cochlea, which is local- ized in the outer hair cells. Active motor properties of outer hair cells have been described recently (Brownell 1986; Zenner 1988). Isolated outer hair cells react to an adequate stimulus, e.g. the iontophoretic application of acetylcholine (Brownell et al. 1985; Slepecky et al. 1988), with contraction and elongation along their length axis (motility). This motility induces an amplifica- tion of the travelling wave, and may be controlled by efferent nerve fibres (Zenner and Gitter 1987). The late onset of ChAT leads to the conclusion that the efferent control of the outer hair cell motility is not completely mature before the 30th day after birth. This is in agree- ment with the development of oto-acoustic emissions as described above (Lenoir and Puel 1987).

Puel and Uziel (1987) found further long-lasting physiological processes in the development of the sensi- tivity, latency and frequency selectivity of cochlear ac- tion potentials. They subdivided the development of the action potentials into three phases, the last phase (after the 18th postnatal day) being characterized by slow ma- turity of the adult thresholds, latencies and frequency selectivity. The authors assume that a number of mutual- ly independent developmental processes play a part in the formation of the cochlear action potentials. Thus the development of sensitivity must be closely correlated with the morphological maturity of the organ of Corti. The delayed maturity of the frequency selectivity is close- ly connected, according to Puel and Uziel, with the "functional development of active processes within the cochlea" and thus, we conclude, also with the onset of the transmitter substance.

Despite the assumption that acetylcholine is the ma- jor efferent transmitter in the cochlea (Rossi et al. 1986;

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Kl inke 1986; Al t schu le r a n d Fex 1986; Fex and Al t - schuler 1986; Eyba l in and Al t schu le r 1990), it should still be cons ide red tha t the func t ion o f fur ther , i m m u n o - cy tochemica l ly ident i f ied subs tances in the efferents is largely unclar i f ied. Here the role o f G A B A is pa r t i cu la r - ly d i sputed . A p a r t f rom its func t ion ing as n e u r o t r a n s m i t - ter, the role o f n e u r o m o d u l a t o r or o f a t r o p h i c fac tor is also discussed for G A B A (Fex and Al t schu le r 1984; Fex et al. 1986; Bledsoe et al. 1988; Merchan -Pe rez et al. 1989; W h i t l o n and Sobkowicz 1989). D u r i n g the on to - geny o f the o rgan o f Cor t i , G A B A i m m u n o r e a c t i v i t y in the reg ion o f the ou te r ha i r cells cou ld be obse rved in ra ts for the first t ime on the 15th d a y af ter b i r th (Merchan-Pe rez et al. 1989) and in the mouse in the sec- ond p o s t n a t a l week ( W h i t l o n and Sobkowicz 1989). The adu l t prof i le is a l r eady reached on the 17th to 18th day o f d e v e l o p m e n t (Merchan-Pe rez e t a l . 1989). Since G A B A label l ing in the efferent fibres to the inner ha i r cells is p resen t before the onse t o f the a u d i t o r y funct ion , Merchan-Pe rez et al. deduce the role o f a t r o p h i c fac tor o f G A B A , at least in ear ly on togeny . W h i t l o n and Sob- kowicz (1989) also t ake a t roph ic inf luence u p o n the d i f fe rent ia t ing nervous sys tem into cons idera t ion . A quan t i t a t ive e lec t ron mic roscop ic analys is c o m p a r a b l e to the p resen t s tudy is very much needed to c lar i fy the func t ion o f G A B A .

Acknowledgements. The authors would like to thank Prof. M. Frotscher (Frankfurt/Freiburg) for his advice on immunocyto- chemistry and Prof. H. Zimmermann (Frankfurt) for helpful dis- cussion. This investigation was supported by the Deutsche For- schungsgemeinschaft SFB 45/B21.

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