Dopaminergic modulation of the voltage-gated sodium current in the cochlear afferent neurons of the rat

Abstract

The cochlear inner hair cells synapse onto type I afferent terminal dendrites, constituting the main afferent pathway for auditory information flow. This pathway receives central control input from the lateral olivocochlear efferent neurons that release various neurotransmitters, among which dopamine (DA) plays a salient role. DA receptors activation exert a protective role in the over activation of the afferent glutamatergic synapses, which occurs when an animal is exposed to intense sound stimuli or during hypoxic events. However, the mechanism of action of DA at the cellular level is still not completely understood. In this work, we studied the actions of DA and its receptor agonists and antagonists on the voltage-gated sodium current (INa) in isolated cochlear afferent neurons of the rat to define the mechanisms of dopaminergic control of the afferent input in the cochlear pathway. Experiments were performed using the voltage and current clamp techniques in the whole-cell configuration in primary cultures of cochlear spiral ganglion neurons (SGNs). Recordings of the INa showed that DA receptor activation induced a significant inhibition of the peak current amplitude, leading to a significant decrease in cell excitability. Inhibition of the INa was produced by a phosphorylation of the sodium channels as shown by the use of phosphatase inhibitor that produced an inhibition analogous to that caused by DA receptor activation. Use of specific agonists and antagonists showed that inhibitory action of DA was mediated both by activation of D1- and D2-like DA receptors. The action of the D1- and D2-like receptors was shown to be mediated by a Gαs/AC/cAMP/PKA and Gαq/PLC/PKC pathways respectively. These results showed that DA receptor activation constitutes a significant modulatory input to SGNs, effectively modulating their excitability and information flow in the auditory pathway.

Fig 1. Effects of TTX, nickel and nifedipine on the I_Na.

A) I_Na produced by voltage pulses from −110 mV to 50 mV in control conditions, during 100 nM TTX perfusion and 2 min after washout. In this and the following figures, only representative traces are shown. B) Current-voltage relationship in control conditions (black) and with TTX perfusion (blue). C) Current recording in control conditions and after coapplication of 10 μM nifedipine (Nfd) and 100 μM nickel. D) Current-voltage relationship in control conditions (black) and with Ni²⁺ plus nifedipine perfusion (red). In all traces, the dotted line indicates zero current.

Fig 2. Effects of DA on the I_Na.

A) Representative experiment showing the effect of 10 μM DA after control perfusion. B) Temporal course of the inhibitory action of DA 100 μM on the I_Na amplitude. Bar indicates the DA perfusion. C) Current-voltage relationship in control and with 10 μM DA. DA decreased the I_Na by 43 ± 8% at −20 mV (P = 0.001; n = 9). D) Activation and inactivation curves in control and after DA application. DA caused a hyperpolarizing shift of the V_½ of the inactivation curve of 8 mV at 10 μM (P = 0.8). In this and following activation and inactivation curves the data were fitted with a Boltzmann function (solid lines). E) Concentration-response relationship of the effect of DA (1 nM to 100 μM), with at least n = 6 for each point. The data were fitted with a concentration response curve (solid line) with an IC₅₀ of 2.5 x 10^–6 M and a Hill coefficient of 1. F) Bar graph shows that a mixture of D1 and D2 antagonists (100 μM SCH-23390 + 1 μM eticlopride) completely blocks DA action (P = 0.006). Inset show a representative recording of the SCH-23390 (SCH) and eticlopride (E) actions. Calibration bars 0.2 nA and 1 ms. Asterisks denote a significant effect P < 0.05.

Fig 3. Intracellular mechanism inherent to D1- and D2-like receptors.

Bars indicate the percent I_Na inhibition produced by 100 μM DA and compared with its effect when 500 μM GDP-β-S (P < 0.001) were added intracellularly. The perfusion of 100 nM okadaic acid decreased the I_Na amplitude in a similar percentage as 100 μM DA (P = 0.3 unpaired t-test). While 100 nM okadaic acid added intracellularly occluded the DA action (P = 0.01). Insets above bars show representative recordings in control conditions and after drug application. Asterisks denote a significant difference (P < 0.05 unpaired t-test). Calibration bars are 1 ms and 0.5 nA for all recordings.

Fig 4. Effects of D1 related drugs on the I_Na.

A) Recordings of the I_Na in control condition and after perfusion of A-68930 (300 nM). B) Current-voltage relationship of the I_Na under control conditions and after 300 nM A-68930. The maximum decrease of the current was 29 ± 4% at −10 mV. C) Steady state inactivation of the I_Na in control conditions and after 300 nM A-68930, which caused a leftward shift of the inactivation. D). Bar graph comparing the inhibitory effect of 100 nM dihidrexidine with A-68930 effect. Non-significant difference was found (P = 0.445; unpaired t-test). E) Current-voltage relationship of the I_Na in control conditions (with 100 μM SCH-23390), and after the co-application of 300 nM A-68930. F) Steady state inactivation of the I_Na in control (with 100 μM SCH-23390) and after A-68930 + SCH-23390, which caused non-significant changes. G) Typical recordings of the I_Na showing that SCH-23390 significantly reduced the inhibitory action of 300 nM A-68930.

Fig 5. Transducer mechanisms activated by D1-like receptors.

Bars show the effect of A-68930 in control condition in comparison with its action while other drugs were used. The use of H89 in the intracellular solution decreased the inhibitory effect of A-68930 from 29 ± 4% to 16 ± 5% at −10 mV (P = 0.08). When cells were preincubated with 50 μM Rp-cAMP, A-68930 effect was completely blocked (P < 0.001). Coapplication of 8-Br-cAMP and IBMX mixture caused a decrease in the I_Na current which mimicked the effect of A-68930 (P = 0.114). Forskolin also mimicked the effect of D1-like agonist decreasing the I_Na 18 ± 11% (P = 0.27). The NPC-15437 in the intracellular solution did not significantly modify the A-68930 effect (P = 0.92). Insets above bars show typical recordings of the I_Na under control conditions and after drug application. Calibration bars are 2 ms and 0.5 nA for all recordings.

Fig 6. Effects of quinpirole on the I_Na.

A) Typical recordings of the I_Na under control conditions and after 1 μM quinpirole perfusion. B) Current- voltage relationship of the I_Na in control conditions and after 1 μM quinpirole. The maximum inhibition was 28 ± 6% at −10 mV. C) Conductance activation and steady state inactivation curves of the I_Na in control conditions and after 1 μM quinpirole perfusion. Quinpirole caused a 5 mV hyperpolarizing shift in the V_½ of the inactivation curve. D) Current-voltage relationship of the I_Na in control condition (with 1 μM eticlopride) and after coapplication of 1 μM quinpirole. E) Bar graph of the percent inhibition of the I_Na by quinpirole in control, with NPC-15437 in the intracellular solution (P = 0.004) and when the cells were preincubated with U-73122 (P = 0.02) or PTx (P = 0.82). Insets above bars show typical recordings of the I_Na in control and after drug application. Calibration bars 0.5 nA, 1 ms for all recordings.

Fig 7. Current clamp response to sinusoidal stimulation and DA receptor activation.

A) The use of 100 nM dihydrexidine (blue traces) shifted the maximum repolarization rate and increased the action potential duration. B) Phase plane plot of action potentials under control conditions and after dihydrexidine application. C) Action potentials produced by a current pulse injection of 150 pA were reduced from 13 under control conditions to 4 after dihydrexidine (100 nM). D) Typical response to sinusoidal current injection (10 Hz, 150 pA). Before the stimuli, the cells were held at −80 mV. E) In a cell discharging in a 2:1 phase lock to sinusoidal stimuli, the use of DA reduced action potential discharges per cycle to a phase lock of 1:1 F) Bar graph showing the percent change in the number of action potentials in the control, after 100 μM DA application and after washout of the drug.

Fig 8. Scheme of the signaling pathways activated by D1- and D2-like receptors in the spiral ganglion neurons.

Receptors are shown as homodimers. Phosphorylation and glycosylation sites are indicated (P and Y). The drugs used in this work are shown in red. Lines with transverse-endings indicate blockade and those with circle-endings stimulation. D1-like receptors are coupled to a G_αs protein leading to AC stimulation, thus increasing cAMP levels and subsequent PKA activation. PKA phosphorylates the Na⁺ channels thus decreasing the I_Na. D2-like receptors are coupled to a G_αq protein whose activation stimulates the PLC, which cleaves PIP₂ into IP₃ and DAG, the IP₃ increases the Ca²⁺ concentration, and both Ca²⁺ and DAG activates PKC leading also to a Na⁺ channel phosphorylation thus decreasing the I_Na. In both cases, phosphorylation was prevented by okadaic acid.