Onset of cholinergic efferent synaptic function in sensory hair cells of the rat cochlea

Abstract

In the developing mammalian cochlea, the sensory hair cells receive efferent innervation originating in the superior olivary complex. This input is mediated by α9/α10 nicotinic acetylcholine receptors (nAChRs) and is inhibitory due to the subsequent activation of calcium-dependent SK2 potassium channels. We examined the acquisition of this cholinergic efferent input using whole-cell voltage-clamp recordings from inner hair cells (IHCs) in acutely excised apical turns of the rat cochlea from embryonic day 21 to postnatal day 8 (P8). Responses to 1 mm acetylcholine (ACh) were detected from P0 on in almost every IHC. The ACh-activated current amplitude increased with age and demonstrated the same pharmacology as α9-containing nAChRs. Interestingly, at P0, the ACh response was not coupled to SK2 channels, so that the initial cholinergic response was excitatory and could trigger action potentials in IHCs. Coupling to SK current was detected earliest at P1 in a subset of IHCs and by P3 in every IHC studied. Clustered nAChRs and SK2 channels were found on IHCs from P1 on using Alexa Fluor 488 conjugated α-bungarotoxin and SK2 immunohistochemistry. The number of nAChRs clusters increased with age to 16 per IHC at P8. Cholinergic efferent synaptic currents first appeared in a subset of IHCs at P1 and by P3 in every IHC studied, contemporaneously with ACh-evoked SK currents, suggesting that SK2 channels may be necessary at onset of synaptic function. An analogous pattern of development was observed for the efferent synapses that form later (P6-P8) on outer hair cells in the basal cochlea.

Figure 1.

nAChR currents in rat apical IHCs are detected first at P0 and are mainly mediated by α9-containing nAChRs. A, Sample traces of ACh-activated currents in IHCs at different ages; holding potential: − 94 mV. B, Comparison of ACh (1 mm)-activated current amplitudes at different ages. ACh current amplitudes were normalized to the membrane capacitance (C_m) of individual IHCs. In parentheses, the number of IHCs in which ACh induced a current per number of IHCs tested is shown. The average amplitudes (±SD) were calculated only for responding IHCs. The average value (±SD) of the IHC Cm varied from 6.7 ± 0.8 pF at P0, 7.2 ± 0.8 pF at P1, 8.1 ± 1.0 pF at P2, 9.0 ± 0.5 at P3 to 10.3 ± 0.6 at P8, and 10.6 ± 1.2 at P21. *p < 0.005 (ANOVA followed by Bonferroni's post hoc test). C, 10 μm strychnine or 10 μm (+)-tubocurarine reversibly block the ACh response to 1 mm ACh, at a holding potential of −94 mV, shown here at P2. D, 300 nm α-conotoxin RgIA (α-RgIA), a subtype-specific blocker of α9/α10 nAChRs, partially and reversibly blocks the ACh response to 1 mm ACh, shown here at P2.

Figure 2.

Development of the coupling of nAChRs and SK channels in apical IHCs. A, B, Current voltage relationships of ACh-activated currents in IHCs at P0 (A) and P2 (B). Holding potentials (V_h) indicated at the right margin of each recording. Every 3 min, 1 mm ACh was applied for 30 s. At P0, ACh-activated currents were inward at −94 mV to −34 mV, whereas at P2, ACh-activated currents were inward at −94 mV, multiphasic at −64 mV, and outward at −34 mV. C, I–V relations of the two IHCs shown in A and B show different reversal potentials at ∼0 mV (P0) and ∼−60 mV (P2). For the IHC at P2, the current amplitude was measured at the time indicated by an arrowhead in B. D, At −34 mV, 1 mm ACh activates an outward current (top trace). In the presence of 300 mm apamin, this outward current is blocked (the SK current) and an inward current is unmasked (the nAChR current) (bottom trace). Representative recording here obtained at P1. E, Percentage of IHCs with an outward current at −34 mV (indicating coupling of SK channels to the ACh response) in response to application of 1 mm ACh at different ages. In parenthesis, number of IHCs studied. F, At P0, application of 1 mm ACh induces depolarization of the IHC membrane potential and in this example, firing of Ca²⁺ action potentials.

Figure 3.

nAChRs and SK2 clusters appear in apical IHCs at P1 and increase in number during the first postnatal week. A, Three-dimensional reconstruction after confocal analysis of ex vivo cochlear preparations labeled with Alexa Fluor 488 conjugated α-Btx (green), to detect the nAChRs, and FM4-64FX, a fluorescent lipophilic stryryl dye, to visualize the IHCs (red). B, In parallel, SK2 immunoreactivity (red) was studied in the fixed contralateral ear. Myosin VI immunoreactivity (green) was used to label the IHCs. A, B, Representative examples obtained from P0, P1, P3, and P8 rats. From P1 on, discrete clusters of α-Btx and SK2 labeling were detectable, mostly distributed in the basal region of the IHC at the level of the nucleus and below (arrowheads). A few clusters were noticeable above the nucleus of the IHCs (arrows). Scale bars, 5 μm. C, D, For each labeling, the percentage of IHCs with such clusters was quantified (C), as well as the number of clusters per IHC for the IHCs with clusters (D) at the different ages. In parentheses, the number of IHCs analyzed is shown.

Figure 4.

Efferent synaptic currents in apical IHCs appear at P1 and are coupled to SK. A, IHC currents evoked by application of an external solution containing 80 mm K⁺; holding potential −94 mV. No synaptic current could be elicited at P0; only a steady inward current was noticeable (in response to the change of the potassium equilibrium potential from −81 mV to −16 mV). From P1 on, application of 80 mm K⁺ evoked synaptic currents (asterisk) in a subset of IHCs. These currents were completely and reversibly blocked by 1 μm strychnine. B, Percentage of IHCs with synaptic events activated during a 4 min superfusion with external solution containing 80 mm K⁺, studied at different ages. In parentheses, the number of IHCs studied is shown. For comparison, the percentage of IHCs with an SK component in the ACh response is shown (same data as in Fig. 2E). C, Spontaneous synaptic currents recorded in an IHC at different holding potentials. Currents are inward at −94 mV, biphasic at −64 mV, and outward at −34 mV, indicating an SK component. D, Correlation of the presence of SK currents and efferent synaptic activity in IHCs. Individual IHCs were tested for an SK component in the ACh response (at −34 mV holding potential) and for the presence of synaptic currents during a 4 min application of external solution including 80 mm K⁺. Experiments were performed at P1 and P2, the postnatal days during which both these features appear. The occurrence of synaptic events was significantly associated with the presence of an SK component in the ACh response (Fisher's exact test; p < 0.0005).

Figure 5.

The onset of efferent synaptic function in OHCs follows a similar sequence of events as in IHCs. A, B, The percentage of basal OHCs with a response to ACh increased from P6 to P10. OHCs were voltage-clamped at −81 mV and ACh was locally puff-applied for 100–500 ms. B, The current voltage relationships of the ACh response reversed either at ∼0 mV (n = 5) or at ∼−60 mV (n = 6) in different OHCs. In OHCs reversing at −60 mV, 300 nm apamin blocked the outward currents between −60 and 0 mV, suggesting that an SK current had been present (n = 6). Average values are shown. C, Fraction of OHCs with an outward current at −34 mV (indicating coupling of SK to the ACh response) in response to 1 mm ACh, compared with the fraction of OHCs with synaptic currents in response to application of extracellular solution with 40 mm K⁺ (performed in separate experiments). In parentheses, the number of OHCs studied is shown. D, Sample traces of synaptic currents activated by application of extracellular solution with 40 mm potassium. At P8, in an OHC with the ACh response reversing at ∼0 mV, no synaptic current was found. At the same age, in an OHC with the ACh response reversing at ∼−60 mV, synaptic activity appeared at a low rate. At P10, synaptic events occurred at a higher rate. E, Correlation of the presence of SK currents and efferent synaptic activity in OHCs. Individual OHCs were tested for an SK component in the ACh response (at −34 mV holding potential) and for the presence of synaptic currents during application of external solution including 40 mm K⁺. Experiments were performed at P8 and P9, the postnatal days during which both these features appear. The occurrence of synaptic events was significantly associated with the presence of a SK component in the ACh response (Fisher's exact test; p < 0.05).