Expression of Shal potassium channel subunits in the adult and developing cochlear nucleus of the mouse
Introduction
The mammalian cochlear nucleus (CN) is the first central nervous system structure in the auditory pathway. CN neurons process information arriving from the inner ear, and form an obligatory relay between auditory nerve fibers and cells of the superior olivary complex and inferior colliculus. Although some CN neurons have response properties similar to those of auditory nerve fibers, most CN neurons have unique structural and functional characteristics that are thought to contribute to their role in processing acoustic stimuli (for review, see Cant and Morest, 1984, Young, 1984). Analysis of the voltage-dependent currents in these cells has revealed that some of the differences among physiological subtypes of CN neurons are due to differences in the biophysical properties of their outward currents (Manis, 1990, Manis and Marx, 1991, Oertel, 1991, Kanold and Manis, 1999).
Voltage-gated K+ channels have been shown to function in the determination of the resting membrane potential, the shaping of the action potential, the rate of spike production, the modulation of transmitter release and the generation of rhythmic firing patterns in other systems (reviewed by Rudy, 1988; see also Storm, 1990, Rehm and Tempel, 1991, Baldwin et al., 1992, Chandy and Gutman, 1995). Molecular biological studies have identified a large number of mammalian genes responsible for the production of voltage-gated K+ channel proteins. These genes have been divided into four subfamilies based on their amino acid sequence (Kv1, Kv2, Kv3 and Kv4; Wei et al., 1990, Chandy et al., 1991, Chandy and Gutman, 1995). In CN, the patterns of expression of three particular subunits from two different subfamilies (Kv1 and Kv3) have been well documented.
Two subunits of the Shaker family of potassium channels, Kv1.1 and Kv1.2, have been localized within the CN (Wang et al., 1994, Grigg et al., 2000). Kv1.1 and Kv1.2 are found in many cells throughout the CN, with particularly dense labeling in the bushy cell region of the anteroventral cochlear nucleus (AVCN) and the octopus cell region of the posteroventral cochlear nucleus (PVCN). A slightly lower level of expression of Kv1.1 in the dorsal cochlear nucleus (DCN) is the only significant regional difference noted. In in vitro systems, expression of Kv1.1 channels results in the production of slowly, partially inactivating (delayed rectifier) currents that have a very low activation voltage (Bosma et al., 1993). Kv1.2 channels have a higher activation threshold and exhibit slightly more inactivation (Werkman et al., 1992, Grissmer et al., 1994). Both Kv1.1 and Kv1.2 channels are sensitive to dendrotoxin. The physiological properties of Kv1.1 and Kv1.2 have led to the hypothesis (Grigg et al., 2000) that these channels are responsible for the low voltage-activated potassium conductance found in CN bushy cells (Manis and Marx, 1991, Rothman et al., 1993).
Kv3.1 potassium channels have been found to be highly expressed in neurons that fire at high frequencies, including the bushy cells in the ventral cochlear nucleus (VCN) and giant cells in the DCN (Perney et al., 1992, Perney and Kaczmarek, 1997). Kv3.1 is present in very low levels in the octopus cell area of the PVCN (Grigg et al., 2000). Expression of splice variants of Kv3.1 is developmentally regulated, as Kv3.1b transcripts increase dramatically during the second week of postnatal life in the rat, while Kv3.1a mRNA levels increase gradually (Perney et al., 1992). Kv3.1 channels have been shown to produce noninactivating (delayed rectifier) currents when expressed in various in vitro systems (Yokoyama et al., 1989, Luneau et al., 1991, Critz et al., 1993), and in the auditory system are thought to improve the ability of neurons to follow high-frequency input (Weiser et al., 1995, Wang et al., 1998).
K+ channels whose openings produce an ‘A-current’ (a transient outward current that exhibits rapid activation and inactivation) are of particular interest with respect to determination of action potential shape and the rate of spike production, and are potentially of significant importance with respect to CN physiology. For example, it has been hypothesized that the ‘pause’ response recorded from fusiform cells in the DCN is due to the presence of a transient potassium conductance (Manis, 1990, Kanold and Manis, 1999). Members of the Shal (Kv4) subfamily of voltage-gated potassium channels are thought to be the primary components of A-type channels in the central nervous system (Pak et al., 1991, Roberds and Tamkun, 1991, Serodio et al., 1994, Serodio et al., 1996, Tsunoda and Salkoff, 1995, Baro et al., 1997, Serodio and Rudy, 1998, Song et al., 1998), and are therefore candidates for the mediators of transient currents in the CN. In contrast to what is known about the Kv1 and Kv3 delayed rectifier channels, very little is known about the expression of Kv4 subunits in the CN, and nothing is known about developmental changes in the expression of these subunits. In their survey of the distribution of Kv4 subunits throughout the adult brain, Serodio and Rudy (1998) indicated that Kv4.2 was very highly expressed in the DCN, with Kv4.3 present at low levels, and Kv4.1 transcripts not being detectable. These authors did not make any mention of the VCN, nor did they describe the specific CN cell types that expressed Kv4.2 and Kv4.3 subunits. The purpose of the experiments described in this paper was to describe the pattern of expression of the three Shal channels, termed Kv4.1, Kv4.2 and Kv4.3, in the developing and mature CN, with the specific objective of identifying the types of projection neurons that express Kv4 mRNA.
Section snippets
Materials and methods
Postnatal CBA/J mice were used to investigate the localization of Kv4 mRNA in CN. Six ages were tested: postnatal day 2 (P2), P6–P7, P10, P14, P18 and adult (P22 and older). All experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, and were designed to minimize animal suffering (Protocol 97034, ‘Potassium Channel Expression in Cochlear Nucleus’, as approved by the IACUC Committee of the University of Minnesota).
Kv4 mRNA detected using RT-PCR
RT-PCR was used to confirm the presence of the Kv4 subunit mRNA in various brain structures, and was not performed in a quantitative manner. The PCR product associated with Kv4.1 mRNA was present in the cerebellum but not in the CN or cortex of adult animals (Fig. 1A). In contrast to the results observed with Kv4.1, Kv4.2 mRNA was found in all three brain regions in the adult (data not shown). Although it is impossible to quantitatively compare expression levels using the PCR methods employed
Differential localization of Kv4 subunits
In their landmark study of the distribution of Kv4 subunits throughout the adult brain, Serodio and Rudy (1998) indicated in their summary table that Kv4.2 was very highly expressed in the DCN, with Kv4.3 present at low levels, and Kv4.1 transcripts not being detectable. The results presented in this study are consistent with this description, but additionally extend these observations to the ventral subdivisions of CN. In addition, this study provides a description of the specific CN cell
Acknowledgements
The authors would like to thank Peter Aas for his assistance in the development of the double-labeling technique and Noelle Bach and Govinda Budrow for their technical support with the Kv4.3 experiments. These experiments were supported by NIDCD #DC03737-01 awarded to J.L.F. and University of Minnesota UROP awards to K.S. and J.R.
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