Summary. Using anatomical criteria, the olivo-coch- lear fibers ending in the organ of Corti (efferent fibers) have recently been separated into two sys-.
Experimental BrgJnResearch
Exp Brain Res (1987) 65:261-270
9 Springer-Verlag 1987
Choline acetyltransferase (CHAT) immunoelectron microscopy distinguishes at least three types of efferent synapses in the organ of Corti M. Eybalin and R. Pujol INSERM- U. 254, and Universityof Montpellier II Laboratoire de Neurobiologiede l'Audition, CHR H6pital St. Charles, F-34059 Montpellier C6dex, France
Summary. Using anatomical criteria, the olivo-cochlear fibers ending in the organ of Corti (efferent fibers) have recently been separated into two systems: a lateral system innervating principally the inner hair cell (IHC) area and a medial system innervating mainly the outer hair cells (OHCs). Electrophysiological and biochemical experiments suggest that acetylcholine may be a neurotransmitter of these efferent fibers. However, efferent synapses that use acetylcholine as neurotransmitter have not yet been identified at the electron microscopic level. Using a pre-embedding immunoelectron microscopic technique with a monoclonal antibody against choline acetyltransferase (CHAT), we localized ChAT-immunostained fibers below both the IHCs and OHCs. In the inner spiral bundle, one type of ChAT-immunostained fibers was vesiculated and formed axo-dendritic synapses with the afferent auditory dendrites contacting the inner hair cells. A second type of ChAT-immunostained fibers seen in the inner spiral bundle was unvesiculated. Unstained vesiculated varicosities synapsing with the auditory dendrites were also seen in the inner spiral bundle. At the OHC level, ChAT immunostaining was found in nearly all the terminals synapsing with the OHCs. The finding of two types of ChAT-immunostained efferent synapses in the organ of Corti, i.e. axodendritic synapses in the inner spiral bundle and axosomatic synapses with the OHCs, supports the hypothesis that both the lateral and the medial olivocochlear systems use acetylcholine as a neurotransmitter. The finding of numerous unstained synapses in the inner spiral bundle, and some below OHCs, together with previous data about putative cochlear neurotransmitters, suggests the possibility of additional non-cholinergic olivo-cochlear systems. It Offprint requests to: M. Eybalin, INSERM- U. 254, CHR H6pital St. Charles, F-34059 Montpellier C6dex, France
might soon appear useful to reclassify efferents according to the nature of the different neurotransmitters/co-transmitters found in the various efferent synapses of the organ of Corti.
Key words: Acetylcholine - Efferent cochlear systems - Choline acetyltransferase - Immunoelectron microscopy - Rat
Introduction It has been demonstrated that the olivo-cochlear (efferent) fibers belong to two separate systems, respectively called lateral and medial according to their origins within the superior olivary complex (see: Warr et al. 1986). The lateral olivo-cochlear system projects principally into the ipsilateral cochlea whereas the medial system projects bilaterally, with a preference for the contralateral side (see: Warr et al. 1986). Previous studies indicate that the lateral system innervates mainly the inner hair cell (IHC) area (see: Warr et al. 1986) where it probably forms most of the efferent "en passant" axo-dendritic synapses with afferent dendrites contacting the IHCs of the inner spiral bundle (see: Iurato 1974; Pujol and Lenoir 1986). The medial efferent system mainly innervates the outer hair cells (OHCs; see: Warr et al. 1986), probably via the axosomatic synapses made by large efferent terminals with the OHCs (see: Iurato 1974; Pujol and Lenoir 1986). Well before the abovementioned neuroanatomical distinction was made, experimental data favored a neurotransmitter role for acetylcholine at the level of efferent endings in the organ of Corti. For instance, electrophysiological experiments demonstrated the effects of acetylcholine and cholinergic ligands on various cochlear potentials (Amaro et al. 1966; Fex 1968, 1973; Brown et al. 1969; Guth and
262 A m a r o 1969; Bobbin and Konishi 1971, 1974; Galley et al. 1971, 1973; Konishi 1972; Fex and A d a m s 1978; Robertson and Johnstone 1978; Comis and Leng 1979). Tetanizations of the efferent fibers also induced a release of acetylcholine in perilymph (Fex 1973; Norris and Guth 1974). W h e n the V I I I t h nerve was sectioned, a hypersensitivity to acetylcholine occurred in the denervated cochleas (Daigneault and Brown 1969). However, a type of efferent synapse (i.e. either axo-dendritic or axo-somatic), whose neurotransmitter is mimicked by agonists of acetylcholine or blocked by its antagonists, has never been definitely identified in electrophysiological experiments. Similarly, biochemical experiments have only been able to associate acetylcholine with the efferent system. Thus, a reliable anatomical mapping of cholinergic fibers and synapses in the organ of Corti has been needed. Acetylcholinesterase cytochemistry has already been applied by several authors to the organ of Corti (see: Iurato 1974). All the efferent fibers were stained for acetylcholinesterase, but this enzyme is not considered to be a specific m a r k e r of cholinergic neurons. Choline acetyltransferase (CHAT), the synthesizing enzyme of acetylcholine, is thought to represent a m o r e specific m a r k e r of cholinergic neurons. Furthermore, its activity has already been demonstrated in the organ of Corti (Jasser and Guth 1973; Fex and Wenthold 1976; Godfrey et al. 1976; Godfrey and Ross 1985). Recently, a light microscopic immunohistochemical mapping of C h A T has been carried out in the organ of Corti (Altschuler et al. 1985b). Although the immunohistochemical localization of C h A T confirmed the findings of acetylcholinesterase histochemistry, no conclusive data could be obtained, at this light microscopic level, about a putative cholinergic nature of the efferent axodendritic synapses in the inner spiral bundle. M o r e precise data regarding C h A T immunolocalization in the organ of Corti were thus necessary, particularly concerning the inner spiral bundle. We have examined C h A T immunocytochemistry in the organ of Corti at the electron microscopic level in order to gain new information as to the cholinergic nature of both kinds of efferent synapse and hence of the two olivo-cochlear systems. Preliminary reports of these findings were presented as an abstract (Eybalin and Pujol 1984), and a short note (Eybalin and Pujol 1985). Material and methods
The immunocytochemical experiments were performed on the cochleas of 10 Wistar rats (200-250 g). In preliminary experiments (Eybalin and Pujol 1985), fixation by immersion (30 min, room temperature) with 4% paraformaldehyde in 0.1 M phosphate
Fig. 1. Semi-thin transverse section of a rat organ of Corti (basal turn). Choline acetyltransferase (CHAT) immunostaining is seen in the inner spiral bundle (ISB) (arrowhead), in fibers crossing the tunnel (thin arrow), and at the base of the outer hair cells (OHCs; arrows), The base of the OHC in row 3 is slightly out of focus and thus does not reveal ChAT immunostaining. Nomarski optics. Bar: 50 ~tm
buffer, pH 7.2, as fixative was found to give a good preservation of both the cochlear morphology and the ChAT antigenicity. Consequently, we used this fixation protocol throughout these experiments. We did not add glutaraldehyde to the fixative since, in contrast to what we observed with anti-enkephalin antisera (Eybalin et al. 1983, 1984, 1985b), glutaraldehyde did not significantly improve the quality of the morphology and, conversely, strongly reduced ChAT antigenicity, even at low concentrations (e.g. 0.05%, Eybalin and Pujol 1985). Similarly, fixation by intraaortic perfusion was avoided since the morphology was not as clear as with fixation by immersion (Eybalin and Pujol 1985). After fixation, the cochleas were rinsed in several baths of phosphate buffered saline (PBS). The bony otic capsule and the stria vascularis were removed. The immunocytochemical procedure has been previously described (Eybalin and Pujol 1985). Briefly, the cochleas were: (1) pre-ineubated in 30% normal rabbit serum in PBS (30 min), (2) incubated with the anti-ChAT monoclonal antibody (2-3 gg/ml; Boehringer Mannheim, lot n~ 1493101) for 60--67 h at 4~ C and (3) incubated in rabbit IgGs antirat IgGs conjugated to HRP (1:50, 1 h, Nordic Labs, Tilburg). In stages (2) and (3), PBS containing 0.2% bovine serum albumin was used to dilute the antibodies. In stage (2), Triton X-100 was present at concentrations ranging from 0.04% to 0.08%. The peroxidatic activity was revealed using DAB/H202. The cochleas were then osmicated and classically processed for electron microscopy. In the control experiments, the anti-ChAT monoclonal antibody was replaced in stage (2) by rat IgGs. Results
Light microscopy Along the entire organ of Corti, scattered C h A T immunostaining was observed in the inner spiral bundle, and there were patches of immunostaining below the O H C s (Fig. 1). W h e n the upper tunnel crossing fibers were present in the section, they were also immunostained. All the above immunostaining
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Fig. 2. Low magnification of the base of an inner hair cell (IHC) and the inner spiral bundle. Both vesiculated (arrows) and unvesiculated (arrowheads) ChAT-immunostained fibers can be seen together with vesiculated (thin arrows) and unvesiculated unstained fibers (most of them probably afferent (a)), Bar: 5 ~tm
was absent in control cochleas, where the anti-ChAT monoclonal antibody was replaced by rat IgGs. As observed in our previous immunocytochemical experiments (Eybalin et al. 1983, 1984, 1985a, b), the tectorial membrane and sometimes the basilar membrane displayed unspecific staining.
Electron microscopy At the electron microscopic level, specific ChAT immunostaining was found in the areas of the organ of Corti that were immunopositive at the light microscopic level, i.e. in the inner spiral bundle, the
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Fig. 3. Unvesiculated ChAT-immunostained fibers in the inner spiral bundle. Fibers immunostained for ChAT (arrowheads) display a clear ~immunoreactivity on the plasma membrane, the neurotubules, and the outer membrane of the mitochondria. No vesicle like structure can be found in their cytoplasm. One of the fibers is in close contact (arrow) with a vesiculated unstained varicosity. Bar: 0.5 ~m Fig. 4. Synapses between a vesiculated ChAT-immunostained varicosity (arrows) and two auditory dendrites (a) in the inner spiral bundle. Unvesiculated immunostained fibers (arrowheads) are in contact with the immunostained varicosity. Bar: 0.5 ~tm Fig. 5. High magnification of a synapse between a vesiculated ChAT-immunostained varicosity and an afferent auditory dendrite (a) within the inner spiral bundle. The presynaptic spicules can be clearly seen (arrow). Bar: 0.5 ~m Fig. 6. Synapses (arrows) between vesiculated unstained varicosities and auditory dendrites (a) in the inner spiral bundle. Unvesiculated ChAT-immunostained fibers can also be seen (arrowheads). Bar: 1 gm t u n n e l of C o r t i a n d at t h e O H C level. I n t h e s e a r e a s , t h e r e l a t i v e n u m b e r of C h A T i m m u n o s t a i n e d a n d C h A T u n s t a i n e d fibers w e r e r o u g h l y t h e s a m e in all t h e cochleas, w h a t e v e r t h e c o n c e n t r a t i o n s of T r i t o n X-100 u s e d (0.04% to 0 . 0 8 % ) . A t this u l t r a s t r u c t u r a l
level also, no i m m u n o s t a i n i n g was f o u n d in c o n t r o l cochleas. In t h e i n n e r spiral b u n d l e (Fig. 2), two p o p u l a tions of fibers w e r e i m m u n o s t a i n e d with t h e antiChAT monoclonal antibody. Numerous immuno-
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Fig. 7a, b. A ChAT-immunostained fiber in the OHC area. a A ChAT-immunostained fiber courses between Deiters' cells (D) and synapses with the base of an OHC in the 2rid row. Small afferents (a) contact the OHC. b A serial section of the area framed in a clearly shows a branching of the immunostained fiber: the small branch is unvesiculated and also appears in a (arrowhead). Bar: 1 Ixm Fig. 8. Branching of a ChAT-immunostained fiber coursing between Deiters' cells (D). Bar: 1 ~m
s t a i n e d fibers w e r e u n v e s i c u l a t e d , w i t h d i a m e t e r s r a n g i n g f r o m 0.3 g m to 1.6 gin. T h e y c o n t a i n e d immunostained neurofilaments and neurotubules a n d u n s t a i n e d m i t o c h o n d r i a (Figs. 2 - 4 , 6). T h e s e u n v e s i c u l a t e d i m m u n o s t a i n e d fibers w e r e l o c a t e d m a i n l y in t h e l o w e r p a r t of t h e i n n e r s p i r a l b u n d l e . T h e i r o r i e n t a t i o n , r a d i a l o r spiral, c o u l d n o t b e f i r m l y
established, but several of them were found coursing r a d i a l l y t o w a r d t h e i n n e r pillar. E v e n w h e n this t y p e of immunostained fiber contacted other types of fiber, v e s i c u l a t e d o r n o t , in t h e i n n e r spiral b u n d l e (Fig. 3, 4), n o s y n a p t i c - l i k e d i f f e r e n t i a t i o n was n o t e d . I n no case, d i d i m m u n o s t a i n e d u n v e s i c u l a t e d fibers c o n t a c t an I H C .
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Fig. 9. Synapsebetween a ChAT-immunostainedterminal and an OHC. The arrows point at presynapticdensities. Afferent dendrites (a). Bar: 1 ~m Fig. 10. Vesiculated terminals synapsingwith an OHC. One of the terminals is ChAT-immunostainedwhereas the other is unstained. Afferent dendrite: a. Bar: 1 txm
The vesiculated varicosities represented the second type of anti-ChAT immunostained fibers (Fig. 2). These immunostained varicosities were less numerous than the unvesiculated immunostained fibers described above. Their diameters ranged from 0.4 t~m to 1.6 ~m, and contained numerous immunostained clear vesicles and unstained mitochondria (Figs. 4, 5). Granular vesicles could not be clearly discerned within these immunostained varicosities. The immunostained vesiculated varicosities were located mainly in the upper part of the inner spiral bundle; they apparently spiraled within the bundle. Contacts were found between these immunostained vesiculated varicosities and (1) afferent auditory dendrites (Figs. 4, 5); (2) unstained vesiculated varicosities; (3) immunostained unvesiculated fibers (Figs. 2, 4) and (4) the IHCs. But, synapfic differentiations were seen only in the case of contacts b e t w e e n immunostained vesiculated varicosities and auditory dendrites (Figs. 4, 5). Unstained vesiculated varicosities were also observed within the inner spiral bundle (Figs. 2, 6). In the absence of quantitative data, we can only indicate that these unstained vesiculated varicosities were at least as numerous as the ChAT immunostained vesiculated varicosities. The unstained vesiculated varicosities contacted all other types of fiber and varicosity as well as the
IHCs, but as in the case of immunostained vesiculated varicosities, synaptic differentiations were seen only in the case of axo-dendritic contacts with the afferent auditory dendrites (Fig. 6). Both types of immunostained fibers (but principally unvesiculated) were found crossing the tunnel of Corti in its upper level. Unstained fibers, again mainly unvesiculated, were also seen crossing the tunnel at this level. No immunostained fibers were found crossing the tunnel on its floor. Both types of ChAT immunostained fibers spiraled with unstained fibers between the Deiters' cells (Fig. 7). At this level, the immunostained fibers sent branches to the OHCs (Figs. 7, 8). In these cases, we observed that the unvesiculated immunostained fibers became filled with clear vesicles. Except for the afferent dendrites, all the fibers contacting the OHCs were vesiculated. Nearly all these vesiculated endings were immunostained (Figs. 7, 9, 10). They were filled with numerous clear vesicles, more or less intensely immunostained. As in the case of the vesiculated immunostained varicosities in the inner spiral bundle, no granular vesicles were clearly distinguishable. The immunostained terminals also contained numerous unstained mitochondria. Facing the OHCs, these terminals had densities bordering the membrane (Fig. 9). Opposite
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the densities, the OHC membrane was thicker and had a subsurface cystern. These membrane characteristics of immunostained terminals and OHCs have been extensively described with classical electron microscopic techniques and have been demonstrated to be constant features of axo-somatic synapses at the OHC level (see: Iurato 1974; Pujol and Lenoir 1986). Scarce unstained vesiculated terminals were also observed synapsing with the OHCs (Fig. 10). They had the same morphological features as the ChAT immunostained terminals, i.e. numerous clear vesicles, mitochondria and presynaptic densities. Discussion
In this study, we ultrastructurally immunolocalized ChAT in vesiculated and unvesiculated fibers of the rat organ of Corti. In addition, we demonstrated that ChAT immunostained vesiculated fibers form two kinds of synapse. The first is axo-dendritic and occurs in the inner spiral bundle, between immunostained varicosities and auditory dendrites. The second is axo-somatic, between immunostained terminals and OHCs. Since no staining was observed in control cochleas, we consider that the abovementioned ChAT immunostaining represents specific recognition sites of the anti-ChAT monoclonal antibody. The localization of the ChAT immunostaining observed in the rat organ of Corti (see also our preliminary results: Eybalin and Pujol 1984, 1985) agrees with the light microscopic immunolocalization of ChAT by others in the guinea pig organ of Corti, using a polyclonal anti-ChAT antiserum and antiChAT monoclonal antibodies (Altschuler et al. 1985b). Similar to our findings, Altschuler et al. (1985b) reported the localization of ChAT immunostaining in fibers of the inner spiral bundle and at the base of the OHCs. The tunnel spiral bundle, which does not exist in the rat, also displayed ChAT immunostaining in the guinea pig organ of Corti. Although light microscopy is sufficient for identifying ChAT-immunostained fibers either in the inner spiral bundle or below the OHCs, electron microscopy is needed for more precise data, particularly regarding the synaptic connections. For instance, it is noteworthy that the inner spiral bundle area is the pathway for all fibers, unmyelinated at this point, going to or coming from the IHCs or the OHCs, and not all these fibers are cholinergic. In this respect, we revealed four populations of fibers in the inner spiral bundle which would not have been identified using only light microscopy: (1) vesiculated ChAT immunostained varicosities; (2) unvesiculated ChAT immunostained fibers; (3) unstained vesiculated varicosities and (4) unstained unvesiculated
fibers. The two types of varicosity clearly belong to the efferent innervations of the organ of Corti since they contain vesicles, a characteristic feature of cochlear efferent fibers (see: lurato 1974; Pujol and Lenoir 1986). The ChAT-immunostained unvesiculated fibers can also be considered to be efferent since there is no evidence for acetylcholine being a neurotransmitter of the primary auditory neurons (see: Wenthold 1980). The fourth type of inner spiral bundle fiber, the unstained unvesiculated, probably corresponds to the afferent auditory dendrites. However, one cannot exclude the possibility that some of these unstained unvesiculated fibers are efferent in nature. With electron microscopy, we also observed that vesiculated varicosities synapsing with the auditory dendrites contacting the IHCs were either immunostained or not. To interpret this result one may first consider that the concentrations of Triton X-100 we have used was too low to ensure a specific CHATimmunostaining of all the efferent varicosities of the inner spiral bundle. In this way, previous immunocytochemical data available on the cochlea have shown both at the electron microscopy (Eybalin et al. 1984) and at the light microscopy level (Altschuler et al. 1984b) that increasing the concentration of Triton X100 increased the number of Met-enkephalin immunostained profiles in the inner spiral bundle. However, even with the highest concentration of Triton X-100 (0.2%) we used, numerous unstained varicosities were still present suggesting that Metenkephalin unstained profiles really existed in the inner spiral bundle (Eybalin et al. 1984). In the present ChAT experiments if we have used Triton X100 at lower concentrations (0.04% to 0.08%) than in enkephalin experiments (0.1% to 0.2%), we must consider that incubation time was considerably longer (67 h versus 15 to 30 rain). With such a long time of incubation even a low concentration of Triton X-100 might be enough to allow the access of the anti-ChAT monoclonal antibody to all the fibers, actually we did not find any difference between the lowest (0.04%) and the highest (0.08%) concentration used. Thus, we are inclined to think that most of the ChAT unstained varicosities are not cholinergic. Another finding in this study was the presence in the inner spiral bundle of numerous unvesiculated fibers with ChAT-immunostained neurofilaments and neurotubules. This ultrastructural localization of the immunostaining may reflect axonal transport of CHAT. Of course these unvesiculated CHATimmunostained fibers do not form a separate group. They could belong to the lateral system and they later become vesiculated before synapsing with afferent auditory dendrites in the inner spiral bundle. They
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also could be medial efferent fibers passing through the inner spiral bundle to reach the OHCs, since it has previously been demonstrated that efferent fibers to the OHCs cross the inner spiral bundle, or travel to some extent in it, before reaching the OHCs (Wright and Preston 1973; Perkins and Morest 1975). At the OHC level, nearly all the vesiculated terminals synapsing with the OHCs were CHATimmunostained. To account for the few unstained terminals at this level, we again hypothesize that these unstained terminals are not cholinergic. A reclassification of the olivo-cochlear efferents into a lateral system and a medial system has recently been proposed (see: Warr et al. 1986). Neurons of the lateral system, mostly project ipsilaterally from the lateral superior olive to the inner spiral bundle (see: Warr et al. 1986), where axo-dendritic efferent synapses are localized (see: Iurato 1974; Pujol and Lenoir 1986). Neurons of the medial system project bilaterally, mainly contralaterally, from medial nuclei of the superior olivary complex on the OHCs (see: Warr et al. 1986), forming axo-somatic synapses (see: Iurato 1974; Pujol and Lenoir 1986). However, it has also been reported (see: Warr et al. 1986) that the distribution of the two efferent systems in the organ of Corti is more complex than previously thought, since a minor part of the lateral system seems to end in the OHC area whereas a minor part of the medial system may end in the inner spiral bundle. Available data on the putative neurotransmitters of both olivo-cochlear systems add more to this complexity. The presence of a ChAT immunostaining in numerous axo-dendritic synapses of the inner spiral bundle and nearly all the axo-somatic synapses at the OHC level suggests that part of the lateral and most of the medial olivo-cochlear systems are cholinergic. This hypothesis is strongly supported by findings of ChAT-immunostained (Altschuler et al. 1984a) and acetylcholinesterase-positive perikarya (see: Warr et al. 1986) in both the lateral superior olive and the medial nuclei of the superior olivary complex. However, this does not mean that the two efferent systems in the organ of Corti are solely cholinergic in nature. Immunocytochemical experiments have demonstrated that Met-enkephalin immunostaining is present in numerous, but not all, perikarya immunostained with ChAT in the lateral superior olive, and absent in ChAT-immunostained perikarya in the medial nuclei of the superior olivary complex (Altschuler et al. 1984a). Thus, many synapses of the lateral olivo-cochlear system may be either enkephalinergic or cholinergic or mixed enkephalinergic/cholinergic, whereas most of the synapses of the medial system are probably cholinergic but not enkephalinergic.
The finding of ChAT immunostained efferent synapses anatomically supports the whole body of results suggesting that acetylcholine is an efferent neurotransmitter in the organ of Corti (Amaro and Guth 1966; Fex 1968, 1973; Brown et al. 1969; Daigneault and Brown 1969; Guth and Amaro 1969; Bobbin and Konishi 1971, 1974; Galley et al. 1971, 1973; Iurato 1974; Konishi 1972; Jasser and Guth 1973; Norris and Guth 1974; Fex and Wenthold 1976; Godfrey et al. 1976; Fex and Adams 1978; Robertson and Johnstone 1978; Comis and Leng 1979; Altschuler et al. 1984a, 1985b; Brownell et al. 1985; Godfrey and Ross 1985). The existence of two different types of ChAT-immunostained synapse (axo-dendritic and axo-somatic) may possibly account for the effectiveness of intra-cochlear perfusions of either nicotinic or muscarinic antagonists in blocking the effects of electrical stimulation of the efferent fibers crossing the midline at the floor of the IVth ventricle (Galley et al. 1971, 1973; Konishi 1972; Fex 1973; Bobbin and Konishi 1974; Fex and Adams 1978). This electrical stimulation has already been shown to be mimicked by a combination of acetylcholine and eserine instilled in the cochlea (Bobbin and Konishi 1971, 1974; Robertson and Johnstone 1978; Comis and Leng 1979). For reasons still obscure, the same electrophysiological effects are obtained with the two classes of cholinergic ligands. To explain this unusual finding, the existence of a mixed muscarinic-nicotinic cholinergic receptor at the cochlear efferent synapses has been postulated (Fex and Adams 1978). However, a binding of [3HI quinuclidinyl benzilate, a muscarinic antagonist, has been reported in sensory areas of the cochlea (James et al. 1983). This binding was not displaced by dtubocurarine, a nicotinic antagonist, suggesting that receptors that are exclusively muscarinic exist in the organ of Corti. It has recently been found, in cultures of dissociated cochlear hair cells, that the addition of acetylcholine to the culture media elicits a decrease in the length of OHCs, similar to the effect of a depolarizing current applied intracellularly (Brownell et al. 1985). This in vitro finding may well account for what occurs in vivo when the cholinergic axo-somatic synapses with OHCs are activated. Using either light or electron microscopic techniques, other putative neurotransmitters have been localized in efferent synapses and fibers, such as enkephalins (Eybalin et al. 1983, 1984, 1985a, b; Altschuler et al. 1984b), dynorphins (Altschuler et al. 1985a) and GABA (Richrath et al. 1974; Schwartz and Ryan 1983; Fex et al. 1986; Parnaud et al. 1986; Thompson et al. 1986) together with its synthesizing enzyme, glutamate decarboxylase (Fex and Altschuler 1984). As may already be the case for
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acetylcholine and enkephalins in the lateral efferent system (Altschuler et al. 1984a), future studies may or may not indicate a co-localization of acetylcholine with G A B A and/or dynorphins. Thus, the specific physiological effect of each type of efferent synapse may differ, depending on the neurotransmitter and possible co-transmitters it contains. These physiological differences may have been overlooked in previous electrophysiological experiments. The kind of stimulation currently used (faradization of the efferent fibers at the floor of the IVth ventricle) is far from selective. Moreover, in the species studied (cat and, to a lesser extent, guinea pig), the efferent fibers that cross the midline at the floor of the IVth ventricle belong to both the medial and the lateral systems (see: Warr et al. 1986). In conclusion, although acetylcholine can be considered to be the major efferent neurotransmitter of the organ of Corti, it appears that portions of both the lateral and the medial system are not cholinergic. In the future, it may be necessary to reclassify the efferent olivo-cochlear systems using neurochemical criteria. Acknowledgements. The authors wish to thank Liliane AbouMadi, Sabine Ladrech and Pierre Sibleyras for their technical assistance, Sam Weiss and Anne Bara for editorial assistance.
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