Embryonic assembly of auditory circuits: spiral ganglion and brainstem

Abstract

During early development, peripheral sensory systems generate physiological activity prior to exposure to normal environmental stimuli. This activity is thought to facilitate maturation of these neurons and their connections, perhaps even promoting efficacy or modifying downstream circuitry. In the mammalian auditory system, initial connections form at embryonic ages, but the functional characteristics of these early neural connections have not been assayed. We investigated processes of embryonic auditory development using a whole-head slice preparation that preserved connectivity between peripheral and brainstem stations of the auditory pathway. Transgenic mice expressing fluorescent protein provided observation of spiral ganglion and cochlear nucleus neurons to facilitate targeted electrophysiological recording. Here we demonstrate an apparent peripheral-to-central order for circuit maturation. Spiral ganglion cells acquire action potential-generating capacity at embryonic day 14 (E14), the earliest age tested, and action potential waveforms begin to mature in advance of comparable states for neurons of the ventral cochlear nucleus (VCN) and medial nucleus of the trapezoid body (MNTB). In accordance, auditory nerve synapses in the VCN are functional at E15, prior to VCN connectivity with the MNTB, which occurs at least 1 day later. Spiral ganglion neurons exhibit spontaneous activity at least by E14 and are able to drive third-order auditory brainstem neurons by E17. This activity precedes cochlear-generated wave activity by 4 days and ear canal opening by at least 2 weeks. Together, these findings reveal a previously unknown initial developmental phase for auditory maturation, and further implicate the spiral ganglion as a potential controlling centre in this process.

Figure 1. Whole-head mouse slices preserve peripheral and central brainstem auditory stations

Selective expression of fluorescent reporter protein in an embryonic mouse whole-head slice preparation highlights auditory and vestibular system structures. A, coronal tissue slice, using transmitted light, of entire mouse head at E17-containing cochleae (dashed red outlines), auditory nerves (red arrowheads), peripheral vestibular system (VS), brainstem region (BS), superior olivary complex (SOC), midbrain (M), cerebellum (Cblm) and oral cavity region (OC). B, same tissue from panel A visualized with DIC optics to better reveal brain fibre tracts; arrowheads identify 7th cranial nerve roots. C, confocal image stack projection shows distribution of td-Tomato expression under control of parvalbumin promoter, overlayed on same tissue slice imaged with DIC optics. At embryonic ages, fluorescence is restricted to spiral ganglion (SG), auditory nerve (AN), vestibular ganglion (VG), ventral cochlear nucleus (VCN) neurons, vestibular nuclei (VsN) and axons of the ventral acoustric stria (VAS). Bipolar stimulating electrodes (black) are positioned on the AN, and recording electrode (arrow) is positioned in the ventral region of the VCN near auditory nerve entry zone.

Figure 6. Spiral ganglion neurons generate action potentials by E14

A, wide field view of an E18 cochlea (left) in Pv-tdTomato mice shows fluorescent inner and outer hair cells (HC), spiral ganglion (SG) and auditory nerve (AN) fibres. By E14, SG cell bodies and proximal neurites can be observed with fluorescence imaging (right), facilitating their identification within the cochlea for electrophysiological recording. B, representative AP waveforms of developing SGCs evoked by depolarizing current injection at SMP. C, spontaneous APs occur in SGCs at E14 (upper left) and older ages (not shown). Voltage responses to serial injection of current steps (5 pA increments) are illustrated by representative cases at E14, E15, E17 and P0. AP response is phasic at E14, but can be tonic (subscript 1) or phasic (subscript 2) in older SG cells. Hyperpolarizing current can convert tonic to phasic AP pattern (E15 subscripts 1 and 2 are the same neuron). Red trace is response to largest depolarizing current. D–F, APs become faster and larger, and voltage threshold becomes hyperpolarized during this developmental time frame. G, resting membrane potential becomes more hyperpolarized with age. H, input resistance at RMP (filled blue circles) does not change dramatically across this age range and is similar for SMP (blue bars). In panels D–G, average values for VCN neurons are depicted for comparison (red bars). I, as a population, SGCs acquire AP capability prior to VCN neurons, and VCN neurons prior to MNTB neurons (latter data re-plotted from Hoffpauir et al. 2010, asterisk). In panels D–H, open black circles indicate values for individual neurons, blue denotes SGC values, red denotes VCN values, bars indicate averaged SMP values, filled circles indicate averaged RMP values and black error bars show SD.

Figure 2. VCN neurons can be activated by auditory nerve stimulation at E15

A, E15 whole-head slice preparation has gross structure similar to E17, with AN (arrowheads) extending from cochlea to VCN (bracket). Bipolar stimulating electrodes (black) are placed within cochlea, and typical recording location in VCN at the level of auditory nerve entry is indicated by asterisk. B, DIC image of VCN field defines cellular boundaries for Fura-2 imaging of Ca²⁺ levels. C, ratiometric Ca²⁺ signals (340 nm/380 nm) induced by AN stimulation for numbered cells shown in D–G. Cells 1–3, but not 4, exhibit stimulus-locked Ca²⁺elevation. Temporal sequence of Fura-2 response maps (D–G) is depicted with lower arrows. D–G, pseudocolour maps of Ca²⁺ signal intensity for field shown in B. Ca²⁺ intensity depicted with thermal scale (D), with lowest intensity blue and highest red. Several cells show acute stimulus-evoked Ca²⁺ increase (E, 12 V and G, 16 V stimuli) in comparison with baseline (D) preceding stimulation. Neurons in F have returned to values near baseline about 3 s after 12 V stimulus. Scale in B applies to D–G.

Figure 3. Synaptic currents in VCN neurons are evoked at E15 and grow rapidly after E16

A, AN stimulation (time indicated by arrow) evoked synaptic currents in VCN neurons as early as E15. Representative traces shown for ages E15–P0. B, maximal evoked current amplitude shown for each responsive neuron (open black circles) across age (n = 8 per age for E15–18, n = 7 for P0). Average peak current amplitude (filled red circles) is initially very low, but increased rapidly after E16 to almost 1.5 nA by P0. C, minimum response latency from stimulus to current onset (left axis values, open black circles, n = 8 per age for E15–18, n = 7 for P0) decreased with age. Estimated conduction velocity (right axis) increased across developmental age. Holding potential was –73 mV for all recordings. Open black circles indicate values for individual neurons, filled red circles indicate average values, blue triangles depict average conduction velocity measures, and error bars show SD.

Figure 4. Action potentials are evoked in VCN neurons at embryonic ages

A, APs are first reliably elicited by AN stimulation at E16. For comparison, representative waveforms for ages E15–P0 are aligned (0 mV) at resting membrane potential, with latency relative to AN stimulus (0 ms, arrow). B, AP half-width decreased with age. C, latency from AN stimulation to AP peak decreased with age (n = 6–9 for all ages in panels B and C). Open black circles indicate values for individual neurons, filled red circles indicate average values and error bars show SD.

Figure 5. Electrophysiological properties of developing VCN neurons

A, representative voltage responses of VCN neurons to serial injection of current steps (5 pA increments, 200 ms duration). Range of plotted current step values are reported for each series; some traces at larger positive values removed to clarify presentation. Voltage response to largest plotted depolarizing current step shown in red. Small AP responses followed by a steady depolarization are present in some cells at E14 and E15. Later ages show progressive appearance of tonic AP firing patterns during current step and sag (arrows) in hyperpolarizing voltage. B, AP amplitude, measured as difference between peak and inflection point, increased with VCN neuron maturation from 21 mV at E14 (n = 7) to over 40 mV by P0 (n = 11–14, each remaining age). C, resting membrane potential (RMP) for VCN neurons declines from –32 mV to about –60 mV by P0 (n = 15–19, all ages). D, input resistance at RMP (filled red circles) declines from very large values (average 2.7 GΩ) after E15 (n = 15–19, all ages). Red bars indicate average values with cells at standardized membrane potential (SMP, –73 mV), open black circles indicate values for individual neurons and black error bars show SD.

Figure 7. Peripheral auditory system can drive MNTB activity by E17

A, whole-head mouse slice at E17 depicting basic connectivity for MNTB input from peripheral SGCs, with electrode stimulation of AN at base of cochlea. We investigated ability to activate ipsilateral and contralateral MNTB from AN stimulation site. B, responses of E17 MNTB principal neuron to series of current steps. C, AN stimulus-locked Ca²⁺ increases, measured using Fura-2, occur in contralateral, but not ipsilateral, E17 MNTB neurons. D, whole-cell recordings of a single E17 MNTB principal neuron showing evoked synaptic currents and postsynaptic potentials with AP following AN stimulation. E, comparison of latencies for postsynaptic currents (onset and peak values) and APs (inflection point and peak) at E17 in VCN and MNTB following AN stimulation. One VCN cell had very long latency (asterisks), and was excluded from mean calculations (filled circles). Range of responses depicted with bars.

Figure 8. Timeline for assembly of auditory circuits

Upper timeline, birthdates of constituent cell groups indicated by coloured ovals. Age of initial connectivity between auditory stations is depicted by lines with open symbols for anatomical proximity (axons have entered postsynaptic cell group), and lines with filled symbols for initial functional communication. Earliest age at which neurons and hair cells generate spontaneous APs is indicated with lightning bolt symbol. Left-facing arrows with question marks denote events that may occur at earlier ages. Lower timeline, three phases of cochlea-generated activity are proposed: phase I from time at which SGCs generate spontaneous activity to time at which hair cells can drive SGC activity, phase II from hair cell-driven SGC activity to onset of ATP generated Ca²⁺ waves, phase III from onset of ATP Ca²⁺ waves to onset of sensitivity to airborne sound. Also depicted are temporal sequences comparing auditory stations across measures of AP competence, input resistance, AP breadth and AP height. In general, SGCs develop ahead of VCN and MNTB neurons.