Limits...
Functional contributions of HCN channels in the primary auditory neurons of the mouse inner ear.

Kim YH, Holt JR - J. Gen. Physiol. (2013)

Bottom Line: We found that HCN1 is the most prominent subunit contributing to Ih in SGNs.Deletion of Hcn1 resulted in reduced conductance (Gh), slower activation kinetics (τfast), and hyperpolarized half-activation (V1/2) potentials.Together, our data indicate that Ih contributes to SGN membrane properties and plays a role in temporal aspects of signal transmission between the cochlea and the brain, which are critical for normal auditory function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neuroscience Graduate Program, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

ABSTRACT
The hyperpolarization-activated current, Ih, is carried by members of the Hcn channel family and contributes to resting potential and firing properties in excitable cells of various systems, including the auditory system. Ih has been identified in spiral ganglion neurons (SGNs); however, its molecular correlates and their functional contributions have not been well characterized. To investigate the molecular composition of the channels that carry Ih in SGNs, we examined Hcn mRNA harvested from spiral ganglia of neonatal and adult mice using quantitative RT-PCR. The data indicate expression of Hcn1, Hcn2, and Hcn4 subunits in SGNs, with Hcn1 being the most highly expressed at both stages. To investigate the functional contributions of HCN subunits, we used the whole-cell, tight-seal technique to record from wild-type SGNs and those deficient in Hcn1, Hcn2, or both. We found that HCN1 is the most prominent subunit contributing to Ih in SGNs. Deletion of Hcn1 resulted in reduced conductance (Gh), slower activation kinetics (τfast), and hyperpolarized half-activation (V1/2) potentials. We demonstrate that Ih contributes to SGN function with depolarized resting potentials, depolarized sag and rebound potentials, accelerated rebound spikes after hyperpolarization, and minimized jitter in spike latency for small depolarizing stimuli. Auditory brainstem responses of Hcn1-deficient mice showed longer latencies, suggesting that HCN1-mediated Ih is critical for synchronized spike timing in SGNs. Together, our data indicate that Ih contributes to SGN membrane properties and plays a role in temporal aspects of signal transmission between the cochlea and the brain, which are critical for normal auditory function.

Show MeSH

Related in: MedlinePlus

Ih in SGNs is carried by HCN channels. (A and B) Representative currents recorded from a WT, P2, basal SGN in response to hyperpolarizing voltage steps before (A) and after (B) 100 µM ZD7288 bath application. Voltage-clamp protocol is shown below. (C) Subtracted currents from datasets A and B show characteristics of Ih. The scale bar applies to all current families.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3753603&req=5

fig3: Ih in SGNs is carried by HCN channels. (A and B) Representative currents recorded from a WT, P2, basal SGN in response to hyperpolarizing voltage steps before (A) and after (B) 100 µM ZD7288 bath application. Voltage-clamp protocol is shown below. (C) Subtracted currents from datasets A and B show characteristics of Ih. The scale bar applies to all current families.

Mentions: To verify that the hyperpolarization-activated inward currents in WT SGN cell bodies were carried by HCN channels, we applied the HCN blocker ZD7288 during voltage-clamp recordings and measured the change in Ih. We applied either 10 or 100 µM ZD7288 in the external bath and found that 10 µM blocked 47 ± 16% (n = 4) of the current at −124 mV, whereas 100 µM blocked 92 ± 4% (n = 6; Fig. 3). Complete Ih block was achieved 15 min after 100 µM ZD7288 treatment and the effect was irreversible. To confirm that the ZD7288 effect was selective for Ih, we subtracted a family of currents recorded in the presence of 100 µM ZD7288 (Fig. 3 B) from control currents (Fig. 3 A) recorded before application of the drug. The subtracted currents (Fig. 3 C) revealed that the ZD7288-sensitive currents were nearly identical in amplitude and activation kinetics to the control currents. Importantly, the subtracted currents did not include properties of any other current, suggesting that 100 µM ZD7288 is specific for Ih in SGNs. Although we have shown ZD7288 to be an Ih-specific antagonist (BoSmith et al., 1993; Harris and Constanti, 1995), it does not distinguish among the four HCN subunits. Thus, we wondered which HCN subunits contribute to the biophysical properties of Ih in WT SGNs. A previous study indicated localization of HCN1 and HCN4 subunits in rat SGN cell bodies at P9–10 (Yi et al., 2010); however, functional evidence implicating specific HCN subunits was not reported. Another study reported the presence of all four HCN subunits in adult guinea pig SGN cell bodies (Bakondi et al., 2009), raising the possibility that all four HCN subunits may be present in WT mouse SGNs.


Functional contributions of HCN channels in the primary auditory neurons of the mouse inner ear.

Kim YH, Holt JR - J. Gen. Physiol. (2013)

Ih in SGNs is carried by HCN channels. (A and B) Representative currents recorded from a WT, P2, basal SGN in response to hyperpolarizing voltage steps before (A) and after (B) 100 µM ZD7288 bath application. Voltage-clamp protocol is shown below. (C) Subtracted currents from datasets A and B show characteristics of Ih. The scale bar applies to all current families.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3753603&req=5

fig3: Ih in SGNs is carried by HCN channels. (A and B) Representative currents recorded from a WT, P2, basal SGN in response to hyperpolarizing voltage steps before (A) and after (B) 100 µM ZD7288 bath application. Voltage-clamp protocol is shown below. (C) Subtracted currents from datasets A and B show characteristics of Ih. The scale bar applies to all current families.
Mentions: To verify that the hyperpolarization-activated inward currents in WT SGN cell bodies were carried by HCN channels, we applied the HCN blocker ZD7288 during voltage-clamp recordings and measured the change in Ih. We applied either 10 or 100 µM ZD7288 in the external bath and found that 10 µM blocked 47 ± 16% (n = 4) of the current at −124 mV, whereas 100 µM blocked 92 ± 4% (n = 6; Fig. 3). Complete Ih block was achieved 15 min after 100 µM ZD7288 treatment and the effect was irreversible. To confirm that the ZD7288 effect was selective for Ih, we subtracted a family of currents recorded in the presence of 100 µM ZD7288 (Fig. 3 B) from control currents (Fig. 3 A) recorded before application of the drug. The subtracted currents (Fig. 3 C) revealed that the ZD7288-sensitive currents were nearly identical in amplitude and activation kinetics to the control currents. Importantly, the subtracted currents did not include properties of any other current, suggesting that 100 µM ZD7288 is specific for Ih in SGNs. Although we have shown ZD7288 to be an Ih-specific antagonist (BoSmith et al., 1993; Harris and Constanti, 1995), it does not distinguish among the four HCN subunits. Thus, we wondered which HCN subunits contribute to the biophysical properties of Ih in WT SGNs. A previous study indicated localization of HCN1 and HCN4 subunits in rat SGN cell bodies at P9–10 (Yi et al., 2010); however, functional evidence implicating specific HCN subunits was not reported. Another study reported the presence of all four HCN subunits in adult guinea pig SGN cell bodies (Bakondi et al., 2009), raising the possibility that all four HCN subunits may be present in WT mouse SGNs.

Bottom Line: We found that HCN1 is the most prominent subunit contributing to Ih in SGNs.Deletion of Hcn1 resulted in reduced conductance (Gh), slower activation kinetics (τfast), and hyperpolarized half-activation (V1/2) potentials.Together, our data indicate that Ih contributes to SGN membrane properties and plays a role in temporal aspects of signal transmission between the cochlea and the brain, which are critical for normal auditory function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neuroscience Graduate Program, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

ABSTRACT
The hyperpolarization-activated current, Ih, is carried by members of the Hcn channel family and contributes to resting potential and firing properties in excitable cells of various systems, including the auditory system. Ih has been identified in spiral ganglion neurons (SGNs); however, its molecular correlates and their functional contributions have not been well characterized. To investigate the molecular composition of the channels that carry Ih in SGNs, we examined Hcn mRNA harvested from spiral ganglia of neonatal and adult mice using quantitative RT-PCR. The data indicate expression of Hcn1, Hcn2, and Hcn4 subunits in SGNs, with Hcn1 being the most highly expressed at both stages. To investigate the functional contributions of HCN subunits, we used the whole-cell, tight-seal technique to record from wild-type SGNs and those deficient in Hcn1, Hcn2, or both. We found that HCN1 is the most prominent subunit contributing to Ih in SGNs. Deletion of Hcn1 resulted in reduced conductance (Gh), slower activation kinetics (τfast), and hyperpolarized half-activation (V1/2) potentials. We demonstrate that Ih contributes to SGN function with depolarized resting potentials, depolarized sag and rebound potentials, accelerated rebound spikes after hyperpolarization, and minimized jitter in spike latency for small depolarizing stimuli. Auditory brainstem responses of Hcn1-deficient mice showed longer latencies, suggesting that HCN1-mediated Ih is critical for synchronized spike timing in SGNs. Together, our data indicate that Ih contributes to SGN membrane properties and plays a role in temporal aspects of signal transmission between the cochlea and the brain, which are critical for normal auditory function.

Show MeSH
Related in: MedlinePlus