Spatiotemporal Analysis of Cochlear Nucleus Innervation by Spiral Ganglion Neurons that Serve Distinct Regions of the Cochlea

Highlights

• Functionally distinct cochlear neurons use different schemes to innervate targets.

• Axons of cochlear neurons show a gradient of development based on tonotopy.

• Low-frequency auditory nerve fibers form smaller endbulb synaptic endings.

Abstract

Cochlear neurons innervate the brainstem cochlear nucleus in a tonotopic fashion according to their sensitivity to different sound frequencies (known as the neuron’s characteristic frequency). It is unclear whether these neurons with distinct characteristic frequencies use different strategies to innervate the cochlear nucleus. Here, we use genetic approaches to differentially label spiral ganglion neurons (SGNs) and their auditory nerve fibers (ANFs) that relay different characteristic frequencies in mice. We found that SGN populations that supply distinct regions of the cochlea employ different cellular strategies to target and innervate neurons in the cochlear nucleus during tonotopic map formation. ANFs that will exhibit high-characteristic frequencies initially overshoot and sample a large area of targets before refining their connections to correct targets, while fibers that will exhibit low-characteristic frequencies are more accurate in initial targeting and undergo minimal target sampling. Moreover, similar to their peripheral projections, the central projections of ANFs show a gradient of development along the tonotopic axis, with outgrowth and branching of prospective high-frequency ANFs initiated about two days earlier than those of prospective low-frequency ANFs. The processes of synaptogenesis are similar between high- and low-frequency ANFs, but a higher proportion of low-frequency ANFs form smaller endbulb synaptic endings. These observations reveal the diversity of cellular mechanisms that auditory neurons that will become functionally distinct use to innervate their targets during tonotopic map formation.

Introduction

The sense of hearing allows humans and animals to distinguish different sound stimuli they are exposed to, not only in the strength and pitch of the sound, but also in the direction and duration. To accomplish this complicated task, the auditory system is organized with precisely wired circuits and specialized synaptic structures (Yu and Goodrich, 2014). The auditory circuit first arises from spiral ganglion neurons (SGNs) in the cochlea extending the peripheral processes of their auditory nerve fibers (ANFs) to receive inputs from hair cells (Rubel and Fritzsch, 2002, Appler and Goodrich, 2011). SGNs then transmit sound information to the auditory brainstem through the central projections of ANFs. Upon entering the brainstem, each individual ANF bifurcates and innervates the three subdivisions of the cochlear nucleus (CN) (Fig. 1A) (Fekete et al., 1984). The ascending branch projects toward the anteroventral cochlear nucleus (AVCN) and elaborates a large synaptic ending, known as the endbulb of Held, on the bushy cell (Ryugo and Fekete, 1982). By comparison, the descending branch travels through the posteroventral cochlear nucleus (PVCN), terminates in the dorsal cochlear nucleus (DCN), and innervates a variety of target neurons along the way with conventional bouton-type synapses (Rouiller et al., 1986).

In each of the three subdivisions of the CN, the innervation by ANFs forms tonotopic maps where neuronal connectivity is organized in an orderly arrangement according to frequency responses (Fekete et al., 1984, Ryugo and May, 1993, Ryugo and Parks, 2003, Kandler et al., 2009, Muniak et al., 2013). Each SGN is most sensitive to a particular sound frequency, which is known as the neuron’s characteristic frequency (Kiang and Moxon, 1972). SGNs with high-characteristic frequencies in the base of the cochlea send their ANFs to dorsal regions of the CN subdivision, while SGNs having low-characteristic frequencies at the apical end of the cochlea project their ANFs to ventral portions of the CN subdivision (Fekete et al., 1984). The axon terminal arbors of ANFs with similar characteristic frequencies then form isofrequency bands to activate nearby target CN neurons (Young and Rubel, 1983). This tonotopic arrangement allows animals to separate a complex sound into its frequency components, which forms the basis of sound discrimination. Despite the importance of tonotopy in auditory functions, how auditory circuits assemble to form tonotopic maps remains largely unknown.

While the gross organization of the tonotopic projections has been examined (Fekete et al., 1984, Leake et al., 2002, Molea and Rubel, 2003, Koundakjian et al., 2007), the cellular events (e.g., initial mapping precision/targeting/pruning) of how ANFs with different characteristic frequencies innervate the CN have not yet been determined. It is also unclear whether SGN populations that serve distinct regions of the cochlea use different cellular strategies to assemble the circuit during tonotopic map formation. One obstacle to address these questions is the lack of a reliable way to differentially label these populations of SGNs. Although traditional histology approaches using dye labeling in anatomic tracing studies have provided valuable insight on how cochlear ganglion neurons innervate the CN during tonotopic map formation (Snyder and Leake, 1997, Leake et al., 2002), they have several limitations. As a surgical intervention is required to inject the dye into the cochlea, it is technically challenging to perform this procedure in embryonic stages. Consequently, these studies only assessed postnatal development, long after the initial establishment of the circuit. Additionally, since dye injections are made through the round window in the inner ear, only a limited subset of SGNs from the relatively high-frequency region at the base of the cochlea can be labeled. Therefore, it is difficult to use this approach to compare the cellular strategy used by SGNs that supply high- versus low-frequency regions of the cochlea.

SGNs originate from a neurogenic domain of the otic vesicle by transiently expressing the transcription factor Neurogenin1 (Ngn1) in a basal to apical progression along the length of the cochlea between E9.5 and E12.5 in mice (Ma et al., 1998, Koundakjian et al., 2007). A small subset of SGNs can be genetically labeled using the Ngn1-creER^T2 mouse line and a Cre-dependent reporter upon induction of Cre recombination by a single low-dose tamoxifen administration (Koundakjian et al., 2007). Using this approach, Koundakjian et al. were able to reproducibly label SGNs and their ANFs that ultimately respond to different sound frequencies by providing tamoxifen at a specific time point between E9.5 (start of neurogenesis) and E12.5 (end of neurogenesis). For simplicity, although characteristic frequencies of SGNs develop after innervation of the CN, we will refer to SGNs that will ultimately have high- or low-characteristic frequencies as “prospective high- or low-frequency (pHF or pLF) SGNs” and refer to them as “functionally distinct.” In this study, we employed the same genetic strategy to respectively label pHF and pLF SGNs and their ANFs and investigate how distinct populations of SGNs explore and innervate the CN. We first used the Ngn1-creER^T2 line and the R26iAP Cre reporter to label pHF and pLF ANFs and compared their overall innervation patterns in the CN at different stages. We then used the Ngn1-creER^T2 line and the Ai14-tdTomato Cre reporter to trace individual ANFs at single-synapse resolution throughout development to determine if pHF and pLF ANFs synapse differently on CN neurons. We found that functionally distinct SGN populations employ different cellular mechanisms to target and innervate CN neurons during tonotopic map formation.