Proof of concept of intracochlear drug administration by laser-assisted bioprinting in mice.

Transtympanic administration is used clinically for the injection of gentamicin and/or corticosteroids. This atraumatic route is based on passive diffusion through the round window membrane (RWM). The main limitation of this method is related to the clearance through the Eustachian tube, making the concentration of the therapeutic agent at the intracochlear level uncertain and limited. Moreover, this technique remains unsuitable for molecules of high molecular weight or in the case of gene therapies. The purpose was to study a new technique of intracochlear administration in an atraumatic, direct and controlled manner by laser-assisted bioprinting (LAB). LAB was used to deliver dexamethasone phosphate with thermosensitive hydrogel on the mouse RWM. After validation of the regularity and homogeneity of the pattern, the diffusion in vivo of the dexamethasone into the perilymph after LAB has been confirmed by ELISA. Auditory function measurements showed no hearing impairment suggesting that bioprinting does not induce significant cochlear damage. Hence, the present proof of concept study introduces a promising approach for inner ear drug delivery.

Hyperacusis in the Adult Fmr1-KO Mouse Model of Fragile X Syndrome: The Therapeutic Relevance of Cochlear Alterations and BKCa Channels.

Hyperacusis, i.e., an increased sensitivity to sounds, is described in several neurodevelopmental disorders (NDDs), including Fragile X Syndrome (FXS). The mechanisms underlying hyperacusis in FXS are still largely unknown and effective therapies are lacking. Big conductance calcium-activated potassium (BKCa) channels were proposed as a therapeutic target to treat several behavioral disturbances in FXS preclinical models, but their role in mediating their auditory alterations was not specifically addressed. Furthermore, studies on the acoustic phenotypes of FXS animal models mostly focused on central rather than peripheral auditory pathways. Here, we provided an extensive characterization of the peripheral auditory phenotype of the Fmr1-knockout (KO) mouse model of FXS at adulthood. We also assessed whether the acute administration of Chlorzoxazone, a BKCa agonist, could rescue the auditory abnormalities of adult mutant mice. Fmr1-KO mice both at 3 and 6 months showed a hyperacusis-like startle phenotype with paradoxically reduced auditory brainstem responses associated with a loss of ribbon synapses in the inner hair cells (IHCs) compared to their wild-type (WT) littermates. BKCa expression was markedly reduced in the IHCs of KOs compared to WT mice, but only at 6 months, when Chlorzoxazone rescued mutant auditory dysfunction. Our findings highlight the age-dependent and progressive contribution of peripheral mechanisms and BKCa channels to adult hyperacusis in FXS, suggesting a novel therapeutic target to treat auditory dysfunction in NDDs.

Synaptic Release Potentiation at Aging Auditory Ribbon Synapses.

Age-related hidden hearing loss is often described as a cochlear synaptopathy that results from a progressive degeneration of the inner hair cell (IHC) ribbon synapses. The functional changes occurring at these synapses during aging are not fully understood. Here, we characterized this aging process in IHCs of C57BL/6J mice, a strain which is known to carry a cadherin-23 mutation and experiences early hearing loss with age. These mice, while displaying a large increase in auditory brainstem thresholds due to 50% loss of IHC synaptic ribbons at middle age (postnatal day 365), paradoxically showed enhanced acoustic startle reflex suggesting a hyperacusis-like response. The auditory defect was associated with a large shrinkage of the IHCs' cell body and a drastic enlargement of their remaining presynaptic ribbons which were facing enlarged postsynaptic AMPAR clusters. Presynaptic Ca2+ microdomains and the capacity of IHCs to sustain high rates of exocytosis were largely increased, while on the contrary the expression of the fast-repolarizing BK channels, known to negatively control transmitter release, was decreased. This age-related synaptic plasticity in IHCs suggested a functional potentiation of synaptic transmission at the surviving synapses, a process that could partially compensate the decrease in synapse number and underlie hyperacusis.

Viral Transfer of Mini-Otoferlins Partially Restores the Fast Component of Exocytosis and Uncovers Ultrafast Endocytosis in Auditory Hair Cells of Otoferlin Knock-Out Mice.

Transmitter release at auditory inner hair cell (IHC) ribbon synapses involves exocytosis of glutamatergic vesicles during voltage activation of L-type Cav1.3 calcium channels. At these synapses, the fast and indefatigable release of synaptic vesicles by IHCs is controlled by otoferlin, a six-C2-domain (C2-ABCDEF) protein that functions as a high-affinity Ca2+ sensor. The molecular events by which each otoferlin C2 domain contributes to the regulation of the synaptic vesicle cycle in IHCs are still incompletely understood. Here, we investigate their role using a cochlear viral cDNA transfer approach in vivo, where IHCs of mouse lacking otoferlin (Otof-/- mice of both sexes) were virally transduced with cDNAs of various mini-otoferlins. Using patch-clamp recordings and membrane capacitance measurements, we show that the viral transfer of mini-otoferlin containing C2-ACEF, C2-EF, or C2-DEF partially restores the fast exocytotic component in Otof-/- mouse IHCs. The restoration was much less efficient with C2-ACDF, underlining the importance of the C2-EF domain. None of the mini-otoferlins tested restored the sustained component of vesicle release, explaining the absence of hearing recovery. The restoration of the fast exocytotic component in the transduced Otof-/- IHCs was also associated with a recovery of Ca2+ currents with normal amplitude and fast time inactivation, confirming that the C-terminal C2 domains of otoferlin are essential for normal gating of Cav1.3 channels. Finally, the reintroduction of the mini-otoferlins C2-EF, C2-DEF, or C2-ACEF allowed us to uncover and characterize for the first time a dynamin-dependent ultrafast endocytosis in IHCs.SIGNIFICANCE STATEMENT Otoferlin, a large six-C2-domain protein, is essential for synaptic vesicle exocytosis at auditory hair cell ribbon synapses. Here, we show that the viral expression of truncated forms of otoferlin (C2-EF, C2-DEF, and C2-ACEF) can partially rescue the fast and transient release component of exocytosis in mouse hair cells lacking otoferlin, yet cannot sustain exocytosis after long repeated stimulation. Remarkably, these hair cells also display a dynamin-dependent ultrafast endocytosis. Overall, our study uncovers the pleiotropic role of otoferlin in the hair cell synaptic vesicle cycle, notably in triggering both ultrafast exocytosis and endocytosis and recruiting synaptic vesicles to the active zone.

Clustered Ca2+ Channels Are Blocked by Synaptic Vesicle Proton Release at Mammalian Auditory Ribbon Synapses.

A Ca2+ current transient block (ICaTB) by protons occurs at some ribbon-type synapses after exocytosis, but this has not been observed at mammalian hair cells. Here we show that a robust ICaTB occurs at post-hearing mouse and gerbil inner hair cell (IHC) synapses, but not in immature IHC synapses, which contain non-compact active zones, where Ca2+ channels are loosely coupled to the release sites. Unlike ICaTB at other ribbon synapses, ICaTB in mammalian IHCs displays a surprising multi-peak structure that mirrors the EPSCs seen in paired recordings. Desynchronizing vesicular release with intracellular BAPTA or by deleting otoferlin, the Ca2+ sensor for exocytosis, greatly reduces ICaTB, whereas enhancing release synchronization by raising Ca2+ influx or temperature increases ICaTB. This suggests that ICaTB is produced by fast multivesicular proton-release events. We propose that ICaTB may function as a submillisecond feedback mechanism contributing to the auditory nerve's fast spike adaptation during sound stimulation.

Clarin-1 gene transfer rescues auditory synaptopathy in model of Usher syndrome.

Clarin-1, a tetraspan-like membrane protein defective in Usher syndrome type IIIA (USH3A), is essential for hair bundle morphogenesis in auditory hair cells. We report a new synaptic role for clarin-1 in mouse auditory hair cells elucidated by characterization of Clrn1 total (Clrn1ex4-/-) and postnatal hair cell-specific conditional (Clrn1ex4fl/fl Myo15-Cre+/-) knockout mice. Clrn1ex4-/- mice were profoundly deaf, whereas Clrn1ex4fl/fl Myo15-Cre+/- mice displayed progressive increases in hearing thresholds, with, initially, normal otoacoustic emissions and hair bundle morphology. Inner hair cell (IHC) patch-clamp recordings for the 2 mutant mice revealed defective exocytosis and a disorganization of synaptic F-actin and CaV1.3 Ca2+ channels, indicative of a synaptopathy. Postsynaptic defects were also observed, with an abnormally broad distribution of AMPA receptors associated with a loss of afferent dendrites and defective electrically evoked auditory brainstem responses. Protein-protein interaction assays revealed interactions between clarin-1 and the synaptic CaV1.3 Ca2+ channel complex via the Cavß2 auxiliary subunit and the PDZ domain-containing protein harmonin (defective in Usher syndrome type IC). Cochlear gene therapy in vivo, through adeno-associated virus-mediated Clrn1 transfer into hair cells, prevented the synaptic defects and durably improved hearing in Clrn1ex4fl/fl Myo15-Cre+/- mice. Our results identify clarin-1 as a key organizer of IHC ribbon synapses, and suggest new treatment possibilities for USH3A patients.

Different CaV1.3 Channel Isoforms Control Distinct Components of the Synaptic Vesicle Cycle in Auditory Inner Hair Cells.

The mechanisms orchestrating transient and sustained exocytosis in auditory inner hair cells (IHCs) remain largely unknown. These exocytotic responses are believed to mobilize sequentially a readily releasable pool of vesicles (RRP) underneath the synaptic ribbons and a slowly releasable pool of vesicles (SRP) at farther distance from them. They are both governed by Cav1.3 channels and require otoferlin as Ca2+ sensor, but whether they use the same Cav1.3 isoforms is still unknown. Using whole-cell patch-clamp recordings in posthearing mice, we show that only a proportion (∼25%) of the total Ca2+ current in IHCs displaying fast inactivation and resistance to 20 µm nifedipine, a l-type Ca2+ channel blocker, is sufficient to trigger RRP but not SRP exocytosis. This Ca2+ current is likely conducted by short C-terminal isoforms of Cav1.3 channels, notably Cav1.342A and Cav1.343S, because their mRNA is highly expressed in wild-type IHCs but poorly expressed in Otof-/- IHCs, the latter having Ca2+ currents with considerably reduced inactivation. Nifedipine-resistant RRP exocytosis was poorly affected by 5 mm intracellular EGTA, suggesting that the Cav1.3 short isoforms are closely associated with the release site at the synaptic ribbons. Conversely, our results suggest that Cav1.3 long isoforms, which carry ∼75% of the total IHC Ca2+ current with slow inactivation and confer high sensitivity to nifedipine and to internal EGTA, are essentially involved in recruiting SRP vesicles. Intracellular Ca2+ imaging showed that Cav1.3 long isoforms support a deep intracellular diffusion of Ca2+SIGNIFICANCE STATEMENT Auditory inner hair cells (IHCs) encode sounds into nerve impulses through fast and indefatigable Ca2+-dependent exocytosis at their ribbon synapses. We show that this synaptic process involves long and short C-terminal isoforms of the Cav1.3 Ca2+ channel that differ in the kinetics of their Ca2+-dependent inactivation and their relative sensitivity to the l-type Ca2+ channel blocker nifedipine. The short C-terminal isoforms, having fast inactivation and low sensitivity to nifedipine, mainly control the fast fusion of the readily releasable pool (RRP); that is, they encode the phasic exocytotic component. The long isoforms, with slow inactivation and great sensitivity to nifedipine, mainly regulate the vesicular replenishment of the RRP; that is, the sustained or tonic exocytosis.

A synaptic F-actin network controls otoferlin-dependent exocytosis in auditory inner hair cells.

We show that a cage-shaped F-actin network is essential for maintaining a tight spatial organization of Cav1.3 Ca(2+) channels at the synaptic ribbons of auditory inner hair cells. This F-actin network is also found to provide mechanosensitivity to the Cav1.3 channels when varying intracellular hydrostatic pressure. Furthermore, this F-actin mesh network attached to the synaptic ribbons directly influences the efficiency of otoferlin-dependent exocytosis and its sensitivity to intracellular hydrostatic pressure, independently of its action on the Cav1.3 channels. We propose a new mechanistic model for vesicle exocytosis in auditory hair cells where the rate of vesicle recruitment to the ribbons is directly controlled by a synaptic F-actin network and changes in intracellular hydrostatic pressure.

Exocytotic machineries of vestibular type I and cochlear ribbon synapses display similar intrinsic otoferlin-dependent Ca2+ sensitivity but a different coupling to Ca2+ channels.

The hair cell ribbon synapses of the mammalian auditory and vestibular systems differ greatly in their anatomical organization and firing properties. Notably, vestibular Type I hair cells (VHC-I) are surrounded by a single calyx-type afferent terminal that receives input from several ribbons, whereas cochlear inner hair cells (IHCs) are contacted by several individual afferent boutons, each facing a single ribbon. The specificity of the presynaptic molecular mechanisms regulating transmitter release at these different sensory ribbon synapses is not well understood. Here, we found that exocytosis during voltage activation of Ca(2+) channels displayed higher Ca(2+) sensitivity, 10 mV more negative half-maximum activation, and a smaller dynamic range in VHC-I than in IHCs. VHC-I had a larger number of Ca(2+) channels per ribbon (158 vs 110 in IHCs), but their Ca(2+) current density was twofold smaller because of a smaller open probability and unitary conductance. Using confocal and stimulated emission depletion immunofluorescence microscopy, we showed that VHC-I had fewer synaptic ribbons (7 vs 17 in IHCs) to which Cav1.3 channels are more tightly organized than in IHCs. Gradual intracellular Ca(2+) uncaging experiments revealed that exocytosis had a similar intrinsic Ca(2+) sensitivity in both VHC-I and IHCs (KD of 3.3 ± 0.6 µM and 4.0 ± 0.7 µM, respectively). In otoferlin-deficient mice, exocytosis was largely reduced in VHC-I and IHCs. We conclude that VHC-I and IHCs use a similar micromolar-sensitive otoferlin Ca(2+) sensor and that their sensory encoding specificity is essentially determined by a different functional organization of Ca(2+) channels at their synaptic ribbons.

Developmental acquisition of a rapid calcium-regulated vesicle supply allows sustained high rates of exocytosis in auditory hair cells.

Auditory hair cells (HCs) have the remarkable property to indefinitely sustain high rates of synaptic vesicle release during ongoing sound stimulation. The mechanisms of vesicle supply that allow such indefatigable exocytosis at the ribbon active zone remain largely unknown. To address this issue, we characterized the kinetics of vesicle recruitment and release in developing chick auditory HCs. Experiments were done using the intact chick basilar papilla from E10 (embryonic day 10) to P2 (two days post-hatch) by monitoring changes in membrane capacitance and Ca(2+) currents during various voltage stimulations. Compared to immature pre-hearing HCs (E10-E12), mature post-hearing HCs (E18-P2) can steadily mobilize a larger readily releasable pool (RRP) of vesicles with faster kinetics and higher Ca(2+) efficiency. As assessed by varying the inter-pulse interval of a 100 ms paired-pulse depolarization protocol, the kinetics of RRP replenishment were found much faster in mature HCs. Unlike mature HCs, exocytosis in immature HCs showed large depression during repetitive stimulations. Remarkably, when the intracellular concentration of EGTA was raised from 0.5 to 2 mM, the paired-pulse depression level remained unchanged in immature HCs but was drastically increased in mature HCs, indicating that the Ca(2+) sensitivity of the vesicle replenishment process increases during maturation. Concomitantly, the immunoreactivity of the calcium sensor otoferlin and the number of ribbons at the HC plasma membrane largely increased, reaching a maximum level at E18-P2. Our results suggest that the efficient Ca(2+)-dependent vesicle release and supply in mature HCs essentially rely on the concomitant engagement of synaptic ribbons and otoferlin at the plasma membrane.

Control of exocytosis by synaptotagmins and otoferlin in auditory hair cells.

In pre-hearing mice, vesicle exocytosis at cochlear inner hair cell (IHC) ribbon synapses is triggered by spontaneous Ca(2+) spikes. At the onset of hearing, IHC exocytosis is then exclusively driven by graded potentials, and is characterized by higher Ca(2+) efficiency and improved synchronization of vesicular release. The molecular players involved in this transition are still unknown. Here we addressed the involvement of synaptotagmins and otoferlin as putative Ca(2+) sensors in IHC exocytosis during postnatal maturation of the cochlea. Using cell capacitance measurements, we showed that Ca(2+)-evoked exocytosis in mouse IHCs switches from an otoferlin-independent to an otoferlin-dependent mechanism at postnatal day 4. During this early exocytotic period, several synaptotagmins (Syts), including Syt1, Syt2 and Syt7, were detected in IHCs. The exocytotic response as well as the release of the readily releasable vesicle pool (RRP) was, however, unchanged in newborn mutant mice lacking Syt1, Syt2 or Syt7 (Syt1(-/-), Syt2(-/-) and Syt7(-/-) mice). We only found a defect in RRP recovery in Syt1(-/-) mice which was apparent as a strongly reduced response to repetitive stimulations. In post-hearing Syt2(-/-) and Syt7(-/-) mutant mice, IHC synaptic exocytosis was unaffected. The transient expression of Syt1 and Syt2, which were no longer detected in IHCs after the onset of hearing, indicates that these two most common Ca(2+)-sensors in CNS synapses are not involved in mature IHCs. We suggest that otoferlin underlies highly efficient Ca(2+)-dependent membrane-membrane fusion, a process likely essential to increase the probability and synchrony of vesicle fusion events at the mature IHC ribbon synapse.

Is otospiralin inner ear specific? Evidence for its expression in mouse brain.

The small protein otospiralin has initially been identified as an inner ear specific molecule. However, compelling evidence from high throughput sequencing projects suggested that otospiralin is likely expressed in the central nervous system. Here, we tested this hypothesis using a combination of molecular biology, immunological, and histological techniques, and found that otospiralin is expressed in numerous regions of the central nervous system in mouse. In situ hybridization and immunohistochemistry revealed that otospiralin is widely expressed in neuronal cell bodies and glia. Ultrastructural observations in the cerebral cortex located the small protein in close proximity to membranous organelles in perikarya, the inner face of post-synaptic neuronal membranes, and in astrocytic processes. These results are in agreement with the predicted structure of the protein which revealed a single N-terminal transmembrane helix domain followed by a C-terminus cytosolic tail. Interestingly, 2 weeks after a mechanical trauma in the cerebral cortex, otospiralin expression increased in reactive astrocytes located within the vicinity of the site of injury, but not in neurons. Collectively, our observations suggest that otospiralin is possibly involved in signaling pathways, and could play a role in repair mechanisms subsequent to an injury in the central nervous system.

Calcium- and otoferlin-dependent exocytosis by immature outer hair cells.

Immature cochlear outer hair cells (OHCs) make transient synaptic contacts (ribbon synapses) with type I afferent nerve fibers, but direct evidence of synaptic vesicle exocytosis is still missing. We thus investigated calcium-dependent exocytosis in murine OHCs at postnatal day 2 (P2)-P3, a developmental stage when calcium current maximum amplitude was the highest. By using time-resolved patch-clamp capacitance measurements, we show that voltage step activation of L-type calcium channels triggers fast membrane capacitance increase. Capacitance increase displayed two kinetic components, which are likely to reflect two functionally distinct pools of synaptic vesicles, a readily releasable pool (RRP; tau = 79 ms) and a slowly releasable pool (tau = 870 ms). The RRP size and maximal release rate were estimated at approximately 1200 vesicles and approximately 15,000 vesicles/s, respectively. In addition, we found a linear relationship between capacitance increase and calcium influx, like in mature inner hair cells (IHCs). These results give strong support to the existence of efficient calcium-dependent neurotransmitter release in immature OHCs. Moreover, we show that immature OHCs, just like immature IHCs, are able to produce regenerative calcium-dependent action potentials that could trigger synaptic exocytosis in vivo. Finally, the evoked membrane capacitance increases were abolished in P2-P3 OHCs from mutant Otof-/- mice defective for otoferlin, despite normal calcium currents. We conclude that otoferlin, the putative major calcium sensor at IHC ribbon synapses, is essential to synaptic exocytosis in immature OHCs too.

Identification of differentially expressed genes in the developing mouse inferior colliculus.

Although injured neurons of inferior colliculus (IC) display a robust axonal outgrowth through a lesion site at postnatal day six (P6) in vitro, and are capable to re-innervate their target cells, injured neurons from P10 IC are unable to regenerate their axons across the lesion site. This axonal regenerative failure has been attributed to an increase of expression of inhibitory molecules in endogenous tissue, during development. As a first step to identify such inhibitory molecules, the present study reports the isolation of molecules differentially expressed in the IC during development. A two-directional (forward and backward) suppression subtractive hybridization (SSH) was performed on IC tissue between P6 and P10 stages. One hundred cDNAs from P6 (P6-P10) and 200 cDNAs from P10 (P10-P6)-subtracted libraries were randomly sequenced. A dot-blot screening of sequenced cDNAs revealed the differential expression for the majority of these cDNAs at their respective developmental stage. Then, the analysis of sequenced clones showed that P6 library was highly enriched in molecules expressed early in the development, such as GAP43 or vimentin proteins. By contrast, the P10 library contained mostly molecules expressed at later stages of development in the central nervous system, such as myelin-related proteins. Our results show that SSH is a suitable method for identifying differentially expressed genes in the developing IC. In addition, these results provide a foundation for further studies dealing with molecules involved in the IC development before and at the onset of hearing, some of which being probably involved in the axonal outgrowth mechanism.

Expression analysis of neuroleukin, calmodulin, cortactin, and Rho7/Rnd2 in the intact and injured mouse brain.

Subtracted cDNA libraries from the mouse developing inferior colliculus were previously constructed between postnatal day (P) 6 and 10. In the P10-P6 subtracted library, neuroleukin, calmodulin I, cortactin, and Rho7 were identified. The goal of the present study was to analyze their distribution, at the mRNA and protein levels, in both the adult and the developing mouse brain. The four molecules showed a wide expression throughout the brain, with a neuronal-enriched localization in structures such as the cortex, the hippocampus, the cerebellum, and the inferior colliculus. The level of expression of their corresponding mRNAs increased during brain postnatal development. The expression of these molecules was also investigated 2 weeks after a mechanical lesion in the adult cerebral cortex. Neuroleukin and cortactin were found to be expressed by reactive astrocytes, while there were no changes in the expression of calmodulin and Rho7. The expression of neuroleukin, calmodulin, cortactin, and Rho7 is discussed in the context of their putative role in the maturation of the brain and in the axonal regeneration process.

Substance P mobilizes intracellular calcium and activates a nonselective cation conductance in rat spiral ganglion neurons.

We demonstrate the expression of functional tachykinin receptors in rat spiral ganglion neurons (SGNs) using calcium signal measurement and whole-cell patch clamp recording. Substance P (SP; 10 microm, 1 s application) induced a transient increase in intracellular calcium. The SP dose-response study showed an EC50 of 18.8 microm and a Hill slope of 0.77. Comparison between specific agonists for the three tachykinin receptor (NKR) types showed the potency NKR3 > NKR1 > NKR2 at 10 microm. The Ca2+ response could be evoked in Ca2+-free medium and was blocked by N-ethylmaleimide and U-73122, indicating that Ca2+ was released from intracellular stores via a G-protein and phospholipase C pathway. Under whole-cell voltage clamp recording at a holding potential of -50 mV, SP (10 microm, 1 s) evoked a slowly developing transient inward current. The current reversed near to 0 mV and ionic permeability experiments revealed a cation nonselective conductance also permeable to large organic cations such as N-methyl-D-glucamine and tetraethylammonium. Neither removing extracellular calcium nor chelating intracellular calcium with 10 mm BAPTA could block the SP-evoked current. This conductance appeared coupled to G-protein activation as intracellular GDP-betaS blocked the SP-evoked current. Mutual desensitization and occlusion studies with acetylcholine and ATP showed that the SP-evoked conductance share effector channels and/or intracellular processes with the purinergic/cholinergic conductance. In SGNs, SP could have both a trophic action, via a calcium response, and a neuromodulatory role, by a depolarizing action through the activation of nonselective cation channels.

Comparative distribution of NK1, NK2, and NK3 receptors in the rat brainstem auditory nuclei.

While the distribution of substance P in the auditory system is well illustrated, the localization of its receptors has not yet been documented. The goal of our study was to characterize the distribution of the tachykinin receptors NK1-R, NK2-R and NK3-R in the brainstem auditory nuclei of the adult rat using immunohistochemical techniques. The immunoreactivity of the neurokinin receptors was found to be widely distributed in most neurons of the cochlear nucleus (CN), the lateral superior olive (LSO), the medial nucleus of the trapezoid body (MNTB) and in the inferior colliculus (IC). Immunoreactivity was generally confined to post-synaptic targets (neuronal cell body and proximal or primary dendrites) in all auditory nuclei. However, unlike brainstem nuclei, the IC showed, in addition to neuronal cell body staining, a positive axonal immunolabeling (axons and pre-synaptic terminals) with the anti-NK1-R antibody. This axonal staining, revealing a pre-synaptic expression of NK1-R, is in good agreement with the known presence of substance P in the IC neurons. The absence of axonal staining in the superior olivary complex nuclei which projects afferent to the IC indicated that the NK1-R labeled axons are rather intrinsic IC fibers or descending thalamic projections to the IC. Overall, the wide distribution of the three types of tachykinin receptors observed in the present study argues for an important role of tachykinin neuropeptides in the central auditory system.