The postsynaptic function of type II cochlear afferents

Abstract

The mammalian cochlea is innervated by two classes of sensory neurons. Type I neurons make up 90-95% of the cochlear nerve and contact single inner hair cells to provide acoustic analysis as we know it. In contrast, the far less numerous type II neurons arborize extensively among outer hair cells (OHCs) and supporting cells. Their scarcity and smaller calibre axons have made them the subject of much speculation, but little experimental progress for the past 50 years. Here we record from type II fibres near their terminal arbors under OHCs to show that they receive excitatory glutamatergic synaptic input. The type II peripheral arbor conducts action potentials, but the small and infrequent glutamatergic excitation indicates a requirement for strong acoustic stimulation. Furthermore, we show that type II neurons are excited by ATP. Exogenous ATP depolarized type II neurons, both directly and by evoking glutamatergic synaptic input. These results prove that type II neurons function as cochlear afferents, and can be modulated by ATP. The lesser magnitude of synaptic drive dictates a fundamentally different role in auditory signalling from that of type I afferents.

Figure 1

Recording from Type II terminal arbors. a. OHC cilia (white arrows). b.-c. Pipette attached to Type II fiber below OHCs. d. Confocal projection of dye-filled fiber (green). OHC nuclei (blue, DAPI) visible in rows (1-3), arrows (red) indicate fiber branches toward OHCs. Recording site (white arrowhead) near dye artifact ‘cloud’. e. Drawings of fill from (d) (P6, OHC row 2) and another fiber (P5, OHC row 1). f. Currents evoked by 10 mV steps from −80 mV. Inset: Selected inward currents, expanded. g. Current-clamp evoked action potentials (threshold: −32.1 ± 10.0 mV). Resting potential −56.9 ± 10.2 mV (n=10). Spontaneous action potential (inset) and small EPSPs (arrowhead). P5-9 rats.

Figure 2

Excitatory postsynaptic currents (EPSCs) in Type II fibers. a. Elevated extracellular potassium evoked EPSCs. Inset: EPSC waveform. b. Representative EPSC amplitude distribution (scaled noise in grey). c. Average EPSCs and d., resulting I-V relation. e. EPSC diary plot showing reversible block by NBQX (10 μM). f. EPSC diary plot showing reversible block by nifedipine (50 μM). g.-h. Amplitude versus decay time constant for EPSCs from two fibers. i. Mean decay time constant versus mean EPSC amplitude from 30 fibers. Linear regression fit (F_1,29=16.43, p=0.004; r²=0.37) j. Exemplar EPSC waveforms (fiber in ‘h’). P5-P9 rats.

Figure 3

ATP stimulates Type II fibers. a. ATP-evoked inward currents in postnatal (P5-9) fibers (142.7 ± 73.6 pA, n=6) and increased EPSC frequency. Inset: ATP (1 μM) induced EPSCs in another cell. b. PPADS reversibly blocked ATP-induced repetitive action potentials (loose-patch extracellular record). c. ATP induced inward current (29.9 ± 17.4 pA, n=5) and EPSCs in P12-13 fibers. d. ATP depolarized P12-13 fibers (12.6 ± 4.8 mV, n=4). e. 40 mM extracellular potassium induced EPSCs in a P18 fiber. Inset: expanded waveforms, same cell. f. ATP evoked inward currents in three of six P17-19 fibers (10.1 ± 3.6 pA, n=3). g. ATP depolarized P17-19 fibers (3.1 ± 0.2 mV, n=2).