Reciprocal Synapses Between Outer Hair Cells and their Afferent Terminals: Evidence for a Local Neural Network in the Mammalian Cochlea
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- Thiers, F.A., Nadol, J.B. & Liberman, M.C. JARO (2008) 9: 477. doi:10.1007/s10162-008-0135-x
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Cochlear outer hair cells (OHCs) serve both as sensory receptors and biological motors. Their sensory function is poorly understood because their afferent innervation, the type-II spiral ganglion cell, has small unmyelinated axons and constitutes only 5% of the cochlear nerve. Reciprocal synapses between OHCs and their type-II terminals, consisting of paired afferent and efferent specialization, have been described in the primate cochlea. Here, we use serial and semi-serial-section transmission electron microscopy to quantify the nature and number of synaptic interactions in the OHC area of adult cats. Reciprocal synapses were found in all OHC rows and all cochlear frequency regions. They were more common among third-row OHCs and in the apical half of the cochlea, where 86% of synapses were reciprocal. The relative frequency of reciprocal synapses was unchanged following surgical transection of the olivocochlear bundle in one cat, confirming that reciprocal synapses were not formed by efferent fibers. In the normal ear, axo-dendritic synapses between olivocochlear terminals and type-II terminals and/or dendrites were as common as synapses between olivocochlear terminals and OHCs, especially in the first row, where, on average, almost 30 such synapses were seen in the region under a single OHC. The results suggest that a complex local neuronal circuitry in the OHC area, formed by the dendrites of type-II neurons and modulated by the olivocochlear system, may be a fundamental property of the mammalian cochlea, rather than a curiosity of the primate ear. This network may mediate local feedback control of, and bidirectional communication among, OHCs throughout the cochlear spiral.
Keywordsinner ear auditory nerve spiral ganglion
Mammalian outer hair cells (OHCs) serve a dual function: as sensory receptors, transducing sound-evoked vibrations into electrical signals, and as biological motors, reverse-transducing these electrical signals into mechanical motion to amplify cochlear vibrations (Dallos and Fakler 2002). OHCs are innervated by terminals of the olivocochlear (OC) bundle (Kimura and Wersall 1962), a feedback system, which can modulate the gain of the cochlear amplifier (Guinan 1996), and by the peripheral terminals of type-II spiral ganglion cells (Kiang et al. 1982), which send central axons to the cochlear nucleus (Brown et al. 1988).
Despite insights into the motor function of OHCs, little is known about their sensory role because type-II spiral ganglion cells comprise only 5–10% of the auditory nerve (Spoendlin 1972), and their central and peripheral axons are small and unmyelinated (Kiang et al. 1982). Thus, single-fiber recordings from type-II neurons are rare (Brown 1994; Robertson et al. 1999), and little is known about transmission at OHC/type-II synapses, except that it probably does not involve the same AMPA-type glutamate receptors at type-I synapses on inner hair cells (Matsubara et al. 1996; Pujol and Puel 1999).
Synapses between OHCs and their afferent terminals appear less robust than those at the IHC (Dunn and Morest 1975), where a prominent pre-synaptic ribbon with its halo of synaptic vesicles is almost always present (Liberman 1980b; Khimich et al. 2005; Stamataki et al. 2006). In contrast, ribbons are usually absent in adult OHCs (Dunn and Morest 1975; Liberman et al. 1990), and the OHC/type-II synapse is defined only by a small cluster of pre-synaptic vesicles and a small plaque of modest pre- and post-synaptic membrane thickening (Liberman et al. 1990).
Ultrastructural studies have shown reciprocal synapses between type-II terminals and their OHC contacts (Nadol 1981; Francis and Nadol 1993; Thiers et al. 2002b), in which a classic “afferent” synapse, with its vesicle cluster in the OHC, is paired with an “efferent” synapse from the same terminal, characterized by a vesicle cluster in the type-II terminal positioned opposite a subsynaptic cistern in the OHC. This subsynaptic cistern is similar morphologically to that opposite classical efferent synapses from the vesicle-filled terminals of the olivocochlear bundle, which also project to the bases of OHCs (Kimura and Wersall 1962). These reciprocal synapses could mediate bidirectional signaling, and, given the extensive branching of type-II projections in the OHC area (Simmons and Liberman 1988a), such bidirectional signaling would constitute a local neural network through which OHCs communicate with each other over essentially the entire cochlear spiral, without engaging the central nervous system or the olivocochlear efferent pathway. However, reciprocal synapses in the OHC area have only been described in human (Nadol 1981; Thiers et al. 2002b) and chimpanzee (Francis and Nadol 1993) and in a neonatally de-efferented cat (Pujol and Carlier 1982), where their presence was viewed as an anomaly resulting from the loss of olivocochlear innervation.
In the present study, we used serial-section electron microscopy to show that such reciprocal synapses are as prevalent among OHC/type-II contacts in the normal cat cochlea as in the primate. Thus, bidirectional signaling throughout the OHC area may be a fundamental feature of the mammalian inner ear, rather than a curious specialization of the primate. Adding to the complexity of the neural circuitry in the OHC area, we also document an extensive direct innervation of the type-II system by olivocochlear efferent terminals.
Three cochleas from three cats were used for the ultrastructural analysis. Two cats were from control animals, which, as part of previous physiological studies (Liberman 1978; Liberman and Beil 1979), underwent single-fiber recordings from the auditory nerve and had normal responses at all frequency regions [data from MCL94L (Fig. 3 in Liberman 1978) and MCL109L (Fig. 3 in Liberman 1978; Liberman and Beil 1979)]. Another cat, # 93042L, underwent unilateral surgical de-efferentation during the first post-natal week, as described elsewhere (Liberman et al. 2000). All animal procedures were approved by the IACUC of the Massachusetts Eye and Ear Infirmary. Tissues procured prior to 1980 (when institutional animal care committees were not yet in existence) was done so according to procedures that have subsequently been approved by the local IACUC.
All animals were perfused intravascularly with 2.5% glutaraldehyde and 1.5% paraformaldehyde in 0.065M phosphate buffer at pH7.3, followed by intralabyrinthine perfusion of the same fixative and post-fixation at 4°C for several days. After post-fixation, cochleas were perfused with 1% osmium tetroxide for 1h and then rinsed, dehydrated, and embedded in epoxy resins. After polymerization, cochleas were drilled down, and dissected partial turns of the organ of Corti were re-embedded, thinned, and mounted on slides for light-microscopic evaluation. Cochlear lengths were measured by computerized planimetry and a frequency map computed for each ear (Liberman 1982).
The completeness of de-efferentation was assessed by measuring the summed diameters of efferent fascicles crossing to the OHC region through the tunnel of Corti using high-N.A. objectives and Nomarski optics. Efferent fibers are distinguished from afferents by their position in the tunnel. These data were obtained in osmium-stained cochlear whole mounts at 12 evenly spaced locations along from the cochlear spiral: Diameter measurements of all tunnel-crossing efferents in one microscopic field from each location were compared among a group of control and surgically de-efferented ears (Liberman et al. 2000). Previous work showed that this metric is well correlated with the volume of olivocochlear terminals remaining on OHCs (Liberman and Gao 1995).
For immunohistochemistry, cochleas from CBA/CaJ mice (6–8 weeks) were extracted and fixed in chilled 4% paraformaldehyde for 10 min prior to dissection into half turns without decalcification. Cochlear pieces were blocked for 2h at 4°C in 5% normal horse serum with 1% Triton X-100 and then incubated overnight at 4°C in the primary antibodies (rabbit anti-GluR2/3 from Chemicon at 1:500 and mouse anti-CtBP2 from BD Transduction Labs at 1:200) diluted in 1% normal horse serum with 1% Triton. Secondary antibody incubations followed: the first, overnight at 4°C, included biotinylated donkey anti-mouse (1:200, Jackson ImmunoResearch) plus fluorescent-conjugated chicken anti-rabbit (AlexaFluor 488, Invitrogen, 1:1,000): the second, for 1 h at room temperature, included streptavidin-conjugated AlexaFluor 568 (Invitrogen, 1:1,000) plus a goat anti-chicken conjugated to AlexaFluor 488 (Invitrogen, 1:1,000).
Classifying and identifying the sources of synaptic terminals in the OHC area
Subsequent light-microscopic studies of peripheral branching patterns, using whole-cell filling via neuronal tracers such as HRP, showed that the large OC terminals arise from radially directed terminal branchlets, which cross the middle of the tunnel of Corti and immediately give rise to a small cluster of five to ten endings on neighboring OHCs in all three rows (Liberman and Brown 1986; Brown 1987a). In contrast, the peripheral dendrite of a type-II spiral ganglion cell crosses at the floor of the tunnel and spirals towards the base of the cochlea in the outer spiral bundles while slowly rising towards the bases of the OHCs and ultimately giving rise to small terminals contacting ten to 100 OHCs, typically contacting cells from a single OHC row (Brown 1987b; Simmons and Liberman 1988a). As illustrated in Figure 2B and C, these spiraling type-II dendrites are plentiful in the region under the OHCs and are particularly rich in neurotubules.
Based on this exhaustive morphometric analysis, we subsequently identified terminals as originating from either type-II or OC neurons on the basis of their size, vesicle content, and connection to radial vs. spiral dendrites.
Prevalence of reciprocal synapses in cat
Previous ultrastructural work in human and non-human primates described reciprocal synapses between type-II terminals and OHCs (Nadol 1981; Thiers et al. 2002b). These synapses included, at the zone of apposition, both (1) an afferent specialization, characterized by modest pre- and post-synaptic membrane thickenings and a small cluster of pre-synaptic vesicles in the OHC, with or without a synaptic ribbon; and (2) an efferent specialization characterized by a flattened subsynaptic cistern within the OHC, similar to that seen opposite olivocochlear terminals (Fig. 2A), a modest degree of pre- and post-synaptic membrane thickening and a small cluster of pre-synaptic vesicles within the type-II terminal.
Reciprocal synapses were found among type-II contacts on all three rows of OHCs. As shown in Figure 6B, which displays the afferent/efferent ratio for each terminal, arrayed in 18 columns to represent the 18 hair cells reconstructed, the efferent components of the reciprocal synapses were slightly more common on third-row OHCs. It may be significant, in this regard, that third-row OHCs in this cochlear region in cat also receive far fewer classic efferent synapses from OC terminals (Liberman et al. 1990 and data from this study not shown).
To further verify the type-II origin of these reciprocal terminals, we examined semi-serial sections (every fifth) through five second-row OHCs from the 1.0-kHz region of the de-efferented cat described above. Indeed, as shown in Figure 7A, the fraction of terminals with reciprocal synapses was similar in the chronically de-efferented ear to that seen in the corresponding regions of two different normal ears.
Reciprocal circuits formed by type-II fibers
The schematic in Figure 8C summarizes this local neuronal circuitry under these 18 OHCs. Salient points are (1) all type-II fibers make reciprocal synapses with multiple OHCs, (2) the ratios of afferent/efferent specialization are randomly distributed across type-II fibers, and (3) if reciprocal synapses enable bidirectional communication between OHCs and type-IIs, the local neural network under the OHCs provides a robust vehicle for intercommunication among OHCs.
OC synapses onto type-II fibers and terminals
Previous ultrastructural studies have described synapses from large, vesicle-filled terminals (presumably of OC origin) onto small (presumed type-II) terminals under OHCs and/or onto the neurotubule-rich presumed dendrites of type-II fibers in the outer spiral bundles (Thiers et al. 2002a) or in the tunnel of Corti (Liberman 1980a). However, previous studies have provided little insight into the prevalence of this type of synaptic interaction.
Ultrastructural studies of OHC synaptic morphology
Morphological signs of bidirectional signaling between hair cells and their sensory neurons have been reported in other systems and in a range of species. Reciprocal synapses have been found on IHCs in mouse, both in adult ears (Sobkowicz et al. 2003) and in cultured neurons reinnervating cochlear explants (Sobkowicz et al. 1993). They have also been reported between afferent neurons and hair cells of the vestibular macula in rat (Ross 1997), crista ampullaris in chinchilla (Lysakowski and Goldberg 1997), crista ampullaris in bullfrog (Dunn 1980), and the paratympanic organ in chicken (Giannessi 1989).
Reciprocal synapses in the OHC area were first reported in a serial-section ultrastructural study of an adult human cochlea (Nadol 1981) and then confirmed in a chimpanzee (Francis and Nadol 1993) and a human infant (Thiers et al. 2002b). The pattern of reciprocal synapses observed in primates was similar to that observed here in cat. All studies note an increased prevalence in apical vs. basal halves of the cochlea and among third-row vs. first-row OHCs, i.e., in gradients complementary to that for innervation of OHCs by terminals of olivocochlear efferents (Liberman et al. 1990), the other source of terminals in the OHC area.
Prior studies in primates relied on circumstantial evidence that the small, vesicle-poor terminals making reciprocal synapses with OHCs arise from peripheral dendrites of type-II neurons. Here, we provide more direct evidence by demonstrating a similar prevalence of reciprocal synapses in an ear after surgical transection of the OC bundle. Prior studies in guinea pig (Jones and Eslami 1983) and cat (Ginzberg and Morest 1983, 1984; Simmons and Liberman 1988b) noted efferent specializations (e.g., a subsynaptic cistern) at terminals which otherwise appeared to be afferent, i.e., small and vesicle poor. However, in the absence of serial-section analysis, they were viewed as curiosities or anomalies. Reciprocal synapses between type-II terminals and OHCs were reported in both chinchilla (Iurato et al. 1978) and cat (Pujol and Carlier 1982) after cutting the OC bundle, just as seen here. However, the finding in both cases was interpreted as pathological, although thorough comparisons with control ears were not presented. Other de-efferentation studies have reported survival of efferent specializations in the OHC area (Smith and Rasmussen 1963; Spoendlin and Gacek 1963; Nakai and Igarashi 1974; Morrison et al. 1975); however, without systematic analysis of terminal morphology and independent assessment of the degree of de-efferentation, it is difficult to separate effects of incomplete OC removal from the presence of type-II reciprocal synapses.
A major peripheral target of the medial OC pathway are OHCs, where cholinergic signaling through α9/α10 nicotinic receptors (Vetter et al. 2007) and the resultant decrease in OHC contribution to cochlear amplification have been well studied (Guinan 1996). Previous ultrastructural studies have described synapses from vesicle-rich, presumably OC, terminals onto type-II neurons. In primates, such synapses have been seen in the spiral ganglion onto type-II somata (Kimura et al. 1987; Thiers et al. 2000). In cat, such synapses have been noted in the tunnel of Corti (Liberman 1980a), and in cat, chinchilla, and primate, they have been described in the outer spiral bundles (Iurato et al. 1978; Ginzberg and Morest 1984; Nadol and Burgess 1990).
Few previous studies have described the prevalence of such contacts, and their existence is often overlooked when considering peripheral effects of activating the OC system (but see Maison et al. 2007a). In the apical half of the cat cochlea, an average of ∼30 such synapses were seen under each first-row OHC, on either the type-II terminals themselves or their terminal branchlets (Fig. 9C). Given that OC innervation density grows towards the base (Liberman et al. 1990), the prevalence of such OC/type-II contacts may also increase dramatically in higher frequency regions.
Cochlear neurons do not express α9 or α10 but do express a number of other nicotinic ACh receptors including α7 and β2 (Bao et al. 2005), which could mediate cholinergic signaling at these synapses; GABAergic transmission also must be considered, given evidence for GABA in OC terminals in the OHC area (Maison et al. 2006) and for GABAB receptors in type-II terminals (Maison et al. 2007b). Regardless of the transmitters and receptors involved, the prevalence of the OC/type-II synapses and OHC/type-II reciprocal synapses provides the hardware for complex local neural processing in the OHC area of the mammalian ear.
Type-II responses and synaptic transmission at their reciprocal synapses
The peripheral process of each bipolar type-II neuron contacts up to 100 OHCs (Simmons and Liberman 1988a), typically within a single OHC row. The type-II central axon projects to the cochlear nucleus, where it bifurcates into ascending and descending branches, as do the type-Is (Brown et al. 1988). The spiraling fibers and terminal branchlets of type-IIs in the organ of Corti are immunopositive for the Na+ channels (Nav1.6), required to generate and propagate action potentials (Hossain et al. 2005). However, the peripheral and central axons of type-II neurons remain unmyelinated from cochlea to cochlear nucleus (Brown et al. 1988) and constitute only 5–10% of the auditory nerve (Spoendlin 1972); thus, almost nothing is known about their response properties in vivo (Robertson et al. 1999).
Similarly, the transmitters and receptors involved at the OHC/type-II synapse are not well understood, even for the afferent component. Immunohistochemistry shows voltage-gated calcium channels (Cav1.3) at the basal pole of adult OHCs, and voltage-dependent calcium currents have been measured in isolated OHCs at least as late as P30 (Knirsch et al. 2007). Immature OHCs (at P3) show capacitance changes elicited by voltage steps, consistent with vesicle release (Beurg et al. 2008). Thus, conventional synaptic transmission may take place at the OHC/type-II contact. There is ample pharmacological evidence that IHC/type-I synapses are glutamatergic and that type-I terminals express AMPA-type receptors (Ruel et al. 2007). In vitro, neonatal type-IIs show a glutamate response that is enhanced by cyclothiazide (Jagger and Housley 2003), consistent with AMPA-type transmission; however, this response appears to be from somatic receptors and may represent a transient developmental stage. In the adult, immunoelectron microscopic studies fail to show glutamate receptors at the type-II synapse (Matsubara et al. 1996), and the prominent excitotoxic response elicited at the IHC/type-I synapse after cochlear perfusion of glutamate agonists is absent in the OHC area (Pujol and Puel 1999). Immunohistochemistry in adult mouse cochleas confirms that AMPA-type glutamate receptors colocalize with synaptic ribbons in the IHC area but are absent in the OHC area (Fig. 10).
Transmitter(s) and receptor(s) at the efferent component of the reciprocal synapse are completely unknown. The subsynaptic cistern at the type-II reciprocal synapse appears identical to that opposite OC terminals on OHCs. This membranous sac, along with ryanodine receptors, has been implicated in calcium-induced calcium release underlying slow-onset, long-lasting suppressive effects of activating the OC bundle (Sridhar et al. 1997; Lioudyno et al. 2004). Thus, similar mechanisms may be at play here; indeed, some of the OC-evoked slow effects could involve interactions mediated via the OC/type-II synapse and the OHC/type-II reciprocal synapse.
Reciprocal synapses or reciprocal circuits have been well studied in the olfactory bulb (Chen et al. 2000) and retina (Vigh and von Gersdorff 2005), respectively. In both these systems, there is evidence for glutamatergic transmission via NMDA receptors on the afferent side coupled with GABAergic transmission via GABAA and/or GABAC receptors. NMDA receptors are present in the cochlea (Niedsielski and Wenthold 1995): At the IHC/type-I synapse, they have been implicated in the excitotoxic response (Puel et al. 1994). However, it is not clear whether type-II neurons also express NMDA receptors. There is strong evidence for GABAergic transmission in the OHC area, particularly the involvement of GABAA receptors (Maison et al. 2006); and targeted deletion of either α5 or β2 GABAA subunits is associated with cochlear threshold shifts consistent with OHC dysfunction. Thus, it is possible that the type-II reciprocal synapses also couple glutamatergic excitation and GABAergic inhibition.
Significance of local neural circuits in the OHC area
If viewed as a sensory neuron, the functional role of the type-IIs lies only in the contributions of their central projections to brainstem circuitry. If viewed as a local neural circuit, with reciprocal synapses that mediate local feedback control and/or communication among neighboring OHCs, the functional significance of the type-II system takes on an added dimension, i.e., modulation of normal OHC function. The contribution of OHCs to cochlear threshold sensitivity requires their somatic electromotility, which is driven in vivo by receptor potentials produced by the OHC’s stereociliary bundle (Dallos et al. 2006). Proper function of this cochlear amplifier presumably requires the coordinated action of neighboring groups of OHCs near the peak of the traveling wave, as well as precise control of stereociliary operating points to optimize the potentials driving somatic motors. It has been proposed that the OC system provides such feedback (LePage 1989); however, maintenance of normal threshold sensitivity and sharpness of neural tuning after complete de-efferentation of the adult ear effectively rules out that hypothesis (Liberman 1990). The local neural circuitry of the type-II system could also perform such feedback, i.e., controlling and coordinating the resting position of neighboring OHCs, without involving the central nervous system or its descending projection to OHCs.
Consistent with a role in fine-tuning of cochlear micromechanics, type-II neurons in the apical half of the cochlea branch profusely among Hensen’s cells (Fechner et al. 1999), where their terminals form synapses that appear efferent (Burgess et al. 1997). Hensen’s cells create a flying buttress at the outer edge of the reticular lamina, and, thus, are in a position to influence the tilt of the OHC hair bundles. Indeed, in an in vitro study of the guinea pig apical turn, overstimulation-induced collapse and reformation of the Hensen-cell arch correlated with attenuation and recovery, respectively, of hair cell receptor potentials (Flock et al. 1999).
There is no post-natal animal model of selective type-II loss; thus, we do not know if normal cochlear sensitivity requires this local neural circuitry under the OHCs. If type-II reciprocal circuits include a GABAergic component, as in the olfactory bulb (Chen et al. 2000), the OHC-based threshold shifts observed in mice lacking GABAA receptor subunits (Maison et al. 2006) could arise from a such a putative role of type-II circuits in maintaining cochlear sensitivity. The distortion component otoacoustic emission (DPOAE) at f2–f1 is particularly sensitive to the static position of the organ of Corti (Sirjani et al. 2004), and the f2–f1 DPOAE shows a slow amplitude change during continuous two-tone stimulation, which can be blocked by bicucculine, a GABAA receptor antagonist (Kirk and Johnstone 1993). Previous studies have considered only the OC system as a source of GABAergic innervation of the OHCs; the present study suggests that the type-II system must be considered as well.
Research supported by grants from the NIDCD (RO1 DC 00188 and P30 DC05209 to MCL), from the National Organization for Hearing Research and the Hugh Hampton Young Memorial Fund (to FAT), and by a training fund for the Program in Speech and Hearing Science and Technology established by the Amelia Peabody Charitable Fund. The technical assistance of Barbara Burgess is gratefully acknowledged.