Elsevier

Hearing Research

Volume 222, Issues 1–2, December 2006, Pages 89-99
Hearing Research

Research paper
Voltage-gated and two-pore-domain potassium channels in murine spiral ganglion neurons

https://doi.org/10.1016/j.heares.2006.09.002Get rights and content

Abstract

The systematically varied firing features of spiral ganglion neurons provide an excellent model system for the exploration of how graded ion channel distributions can be used to organize neuronal firing across a population of neurons. Elucidating the underlying mechanisms that determine neuronal response properties requires a complete understanding of the combination of ion channels, auxiliary proteins, modulators, and second messengers that form this highly organized system in the auditory periphery. Toward this goal, we built upon previous studies of voltage-gated K+-selective ion channels (Kv), and expanded our analysis to K+-selective leak channels (KCNK), which can play a major role in setting the basic firing characteristics of spiral ganglion neurons.

To begin a more comprehensive analysis of Kv and KCNK channels, a screening approach was employed. RT-PCR was utilized to examine gene expression, the major results of which were confirmed with immunocytochemistry. Initial studies validated this approach by accurately detecting voltage-dependent K+ channels that were documented previously in the spiral ganglion. Furthermore, an additional channel type within the Kv3 family, Kv3.3, was identified and further characterized. The major focus of the study, however, was to systematically examine gene expression levels of the KCNK family of K+-selective leak channels. These channel types determine the resting membrane potential which has a major impact on setting the level of neuronal excitation. TWIK-1, TASK-3, TASK-1, and TREK-1 were expressed in the spiral ganglion; TWIK-1 was specifically localized with immunocytochemistry to the neuronal somata and initial processes of spiral ganglion neurons in vitro.

Introduction

Understanding the coding capabilities of a neuron requires the identification of its electrical signature, which is controlled by endogenous ion channels. As with the sensory receptor hair cells in the peripheral auditory end-organ of non-mammalian species (Fettiplace and Fuchs, 1999), the distribution of channels within the murine spiral ganglion display a non-uniform distribution along the tonotopic axis of the cochlea. Previous studies have shown that the basic firing patterns of individual neurons are heterogeneous (Mo and Davis, 1997a), yet organized according to their peripheral innervation patterns (Adamson et al., 2002b). For example, neurons isolated from basal cochlear regions possess fast firing features, displaying abbreviated response latencies to current injections, narrow action potentials, rapid accommodation, and fast onset time courses. In contrast, neurons isolated from apical cochlear regions generally showed longer latencies and onset time courses, broader action potentials, and slow accommodation (Adamson et al., 2002b).

This pattern is shaped in part by the density of a specific sub-set of voltage-gated K+ (Kv) channels that have been characterized electrophysiologically, pharmacologically, and immunocytochemically. Channel types that contribute to fast firing features, such as Kv1.1, Kv1.2, Kv3.1, and KCa, were found to be enriched in the high frequency basal spiral ganglion neurons. A channel type that shows prominent inactivation that could lengthen the latency and spacing of action potentials, Kv4.2, had a higher distribution in the more apical, lower frequency spiral ganglion neurons. The apparent density of these channels is regulated by two different neurotrophins that have been localized to the cochlea: brain-derived neurotrophic factor and neurotrophin-3 (Adamson et al., 2002b, Zhou et al., 2005). From these types of studies it is clear that the electrophysiological properties of the spiral ganglion neurons are established and/or maintained by highly-regulated mechanisms.

In addition to voltage-gated channels, it is likely that K+-selective leak channels (KCNK) also contribute to the neurons heterogeneous firing patterns. KCNK channels help to establish the resting potential, a feature that could have a profound impact upon signaling, since it establishes a fundamental level of neuronal excitability (Plant et al., 2005). Therefore, the present study was designed to broaden our view of the endogenous K+ channels that control the firing patterns of these peripheral auditory neurons by screening with gene expression techniques and confirming our findings with immunocytochemistry.

Section snippets

Tissue preparation

Procedures performed on CBA/CaJ mice were approved by The Rutgers University Institutional Review Board for the Use and Care of Animals (IRB-UCA), protocol 90-073. In order to relate our findings to previous studies of spiral ganglion neurons in vitro (Adamson et al., 2002a, Adamson et al., 2002b), spiral ganglia were isolated from postnatal day 6–7 (P6–P7) mice. From the same animals we extracted the frontal cortex, which was utilized as a reference tissue because it has been shown to express

Results

Neuronal firing patterns are, in large part, controlled by K+ channels, which can profoundly affect neuronal excitability. The sophistication of this process resides within the time course and voltage-dependence of ion channel activation and inactivation, as well as differential modulation by auxiliary proteins and second messengers. We had previously hypothesized that the complex firing patterns displayed by spiral ganglion neurons resulted from differential ion channel distribution, and we

Discussion

In the present study, we identified two additional classes of K+ α-subunits that have the potential to be instrumental in regulating spiral ganglion neuron firing patterns. Initial evidence for a voltage-activated K+ channel, Kv3.3, and a K+-selective leak channel, TWIK-1, was found with RT-PCR analysis designed to screen for genes of high abundance in the spiral ganglion relative to a generic cortical region. Because mRNA levels do not necessarily reflect protein levels due to myriad

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