A-type potassium currents dominate repolarisation of neonatal rat primary auditory neurones in situ

doi:10.1016/S0306-4522(01)00454-7

Neuroscience

Volume 109, Issue 1, 18 January 2002, Pages 169-182

https://doi.org/10.1016/S0306-4522(01)00454-7 Get rights and content

Abstract

Spiral ganglion neurones provide the afferent innervation to cochlear hair cells. Little is known of the molecular physiological processes associated with the differentiation of these neurones, which occurs up to and beyond hearing onset. We have identified novel A-type (inactivating) potassium currents in neonatal rat spiral ganglion neurones in situ, which have not previously been reported from the mammalian cochlea, presumably as a consequence of altered protein expression associated with other preparations. Under whole-cell voltage clamp, voltage steps activated both A-type and non-inactivating outward currents from around −55 mV. The amplitude of the A-type currents was dependent on the holding potential, with steady-state inactivation relieved at hyperpolarised potentials. At −60 mV (close to the resting potential in situ) the currents were approximately 30% enabled. The inactivation kinetics and the degree of inactivation varied between cells, suggesting heterogeneous expression of multiple inactivating currents. A-type currents provided around 60% of total conductance activated by depolarising voltage steps from the resting potential, and were very sensitive to bath-applied 4-aminopyridine (0.01–1 mM). Tetraethylammonium (0.1–30 mM) also blocked the majority of the A-type currents, and the non-inactivating outward current, but left residual fast inactivating A-type current. Under current clamp, neurones fired single tetrodotoxin-sensitive action potentials. 4-Aminopyridine relieved the A-type current mediated stabilisation of membrane potential, resulting in periodic small amplitude action potentials.

This study provides the first electrophysiological evidence for A-type potassium currents in neonatal spiral ganglion neurones and shows that these currents play an integral role in primary auditory neurone firing.

Section snippets

Cochlear slice preparation

The preparation of thick transverse slices of the neonatal rat cochlea has been described elsewhere (Jagger et al., 2000). This technique employs the intra-cochlear infusion of cooled Pluronic F127 NF (BASF, Parsippany, USA), a block copolymer that solidifies to a gel (25% solution in artificial perilymph – see below) at room temperature. The gel supports the cochlear partition during microtome slicing. Cochlear slices were prepared from 2–6-day-old (P2–P6) Wistar rats. Following

Identification of inactivating A-type currents in neonatal rat SGN in situ

Whole-cell voltage clamp recordings were made from 74 SG neurones in cochlear slices from 2–6-day-old rats (P2–P6). Cells had membrane capacitance of 7.8±0.3 pF, uncompensated series resistance 6.0±0.2 MΩ, and an input resistance of 499.4±33.7 MΩ, measured from short 10-mV hyperpolarising pulses (see Experimental procedures). Zero-current potential (V_z) was −58.5±1.0 mV, measured from steady-state current values plotted in current–voltage (I–V) relationships. In all neurones tested,

I_As are present in neonatal SGN in a slice preparation, but are absent in age-matched dissociated neurones

Our experiments indicate that neonatal rat primary auditory neurones express ion channels that mediate transient, or A-type potassium currents (I_A; Connor and Stevens, 1971, Rogawski, 1985). The voltage dependence of inactivation of these currents suggests that at the resting potential, around 30% of the total A-type conductance could be activated. At these potentials the A-type currents contributed around 60% of the total current activated by depolarisation. There was considerable variation in

Acknowledgements

This study was supported by the Marsden Fund (Royal Society of New Zealand), the Health Research Council of New Zealand, the Deafness Research Foundation of New Zealand Inc, and the Maurice and Phyllis Paykel Trust.

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In vitro cultures provide a valuable tool in studies examining the survival, morphology and function of cells in the auditory system. Primary cultures of primary auditory neurons have most notably provided critical insights into the role of neurotrophins in cell survival and morphology. Functional studies have also utilized in vitro models to study neuronal physiology and the ion channels that dictate these patterns of activity. Here we examine what influence time-in-culture has on the activity of primary auditory neurons, and how this affects our interpretation of neurotrophin and antibiotic-mediated effects in this population. Using dissociated cell culture we analyzed whole-cell patch-clamp recordings of spiral ganglion neurons grown in the presence or absence of neurotrophins and/or penicillin and streptomycin for 1–3 days in vitro. Firing threshold decreased, and both action potential number and latency increased over time regardless of treatment, whilst input resistance was lowest where neurotrophins were present. Differences in firing properties were seen with neurotrophin concentration but were not consistently maintained over the 3 days in vitro. The exclusion of antibiotics from culture media influenced most firing properties at 1 day in vitro in both untreated and neurotrophin-treated conditions. The only difference still present at 3 days was an increase in input resistance in neurotrophin-treated neurons. These results highlight the potential of neurotrophins and antibiotics to influence neural firing patterns in vitro in a time-dependent manner, and advise the careful consideration of their impact on SGN function in future studies.
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Therefore, in order to maintain the functional status quo of SGNs in a clinical setting, a combined application of BDNF and NT3 might be preferred. The predominance of the rapidly-adapting neurons across our population of SGNs is also an outcome that is consistent with earlier reports of SGN activity in the rat (Jagger and Housley, 2002), guinea-pig (Chen, 1997; Szabo et al., 2002), gerbil (Lin, 1997) and mouse (Sun and Salvi, 2009), in which SGNs that are acutely isolated, in cochlear slices or cultured for only several days typically exhibit a rapidly-adapting profile. Alternatively, studies using cochlear explants cultured for 5–7 days report a higher proportion of slowly-adapting neurons (Adamson et al., 2002b; Liu and Davis, 2007; Mo and Davis, 1997a, 2002).
Neurotrophins provide an effective tool for the rescue and regeneration of spiral ganglion neurons (SGNs) following sensorineural hearing loss. However, these nerve growth factors are also potent modulators of ion channel activity and expression, and in the peripheral auditory system brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) have previously been shown to alter the firing properties of auditory neurons and differentially regulate the expression of some potassium channels in vitro. In this study we examined the activity of the hyperpolarization-mediated mixed-cation current (I_h) in early post-natal cultured rat SGNs following exposure to combined BDNF and NT3. Whole-cell patch-clamp recordings made after 1 or 2 days in vitro revealed no change in the firing adaptation of neurons in the presence of BDNF and NT3. Resting membrane potentials were also maintained, but spike latency and firing threshold was subject to regulation by both neurotrophins and time in vitro. Current clamp recordings revealed an activity profile consistent with activation of the hyperpolarization-activated current. Rapid membrane hyperpolarization was followed by a voltage- and time-dependent depolarizing voltage sag. In voltage clamp, membrane hyperpolarization evoked a slowly-activating inward current that was reversibly blocked with cesium and inhibited by ZD7288. The amplitude and current density of I_h was significantly larger in BDNF and NT3 supplemented cultures, but this did not translate to a significant alteration in voltage sag magnitude. Neurotrophins provided at 50 ng/ml produced a hyperpolarizing shift in the voltage-dependence and slower time course of I_h activation compared to SGNs in control groups or cultured with 10 ng/ml BDNF and NT3. Our results indicate that combined BDNF and NT3 increase the activity of hyperpolarization-activated currents and that the voltage-dependence and activation kinetics of I_h in SGNs are sensitive to changes in neurotrophin concentration. In addition, BDNF and NT3 applied together induce a decrease in firing threshold, but does not generate a shift in firing adaptation.
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This evolution in our view of sensory processing has occurred for the auditory system as well. Before we and others began the examination of spiral ganglion neurons with intracellular recordings in order to characterize their intrinsic electrophysiological features (Chen, 1997; Chen and Davis, 2006; Davis, 1996; Garcia-Diaz, 1999; Hisashi et al., 1995; Jagger and Housley, 2002; Jimenez et al., 1997; Lin, 1997; Lin and Chen, 2000; Mo et al., 2002; Mo and Davis, 1997a; Moore et al., 1996; Santos-Sacchi, 1993; Sheppard et al., 1992; Szabo et al., 2002; Yamaguchi and Ohmori, 1990), the general view of the field was that all type I neurons that composed the ganglion were identical. This was based upon the wealth of in vivo recordings revealing that neurons fire in a similar fashion to pure tone stimuli (Kiang et al., 1965, 1984; Sachs et al., 1974).
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The spiral ganglion accommodates the cell bodies of the acoustic nerve fibres connecting the hair cells to the central nervous system. As the ionic channels containing various voltage-gated K⁺ channel (Kv) subunits play pivotal roles in determining the functional properties and firing behaviour of the spiral ganglion cells (SGCs), every piece of information concerning the Kv expression of the SGCs is valuable. In the present work a comprehensive immunohistochemical analysis was performed to describe the expression of 9 Kv subunits in the guinea pig cochlea on traditional wax-embedded sections as well as employing a newly developed preparation that allowed confocal analysis, reconstruction of the three-dimensional appearance and precise morphological characterisation of the SGCs. Besides determining their Kv expression patterns, differences between type I and type II SGCs were sought. SGCs showed positivity for 8 out of the 9 Kv subunit-specific antibodies with varying intensity and proportion of the immunopositive cells; whereas no obvious Kv3.2 positivity could be noted. Type I and type II cells demonstrated similar expression patterns for all subunits tested, with the exception of Kv1.2, whose presence was confirmed in only 50% of the type II cells. Although the present findings suggest that type I and type II cells do not differ fundamentally in the Kv subunits they possess; they also imply that SGCs may not form a homogeneous cell population, and might provide explanation of the previously noted heterogeneity of the membrane properties of the SGCs.

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A-type potassium currents dominate repolarisation of neonatal rat primary auditory neurones in situ

Abstract

Section snippets

Cochlear slice preparation

Identification of inactivating A-type currents in neonatal rat SGN in situ

IAs are present in neonatal SGN in a slice preparation, but are absent in age-matched dissociated neurones

Acknowledgements

Hear. Res.

Neuron

Neuroscience

Trends Neurosci.

Hear. Res.

J. Neurosci. Methods

Neuroscience

Hear. Res.

Dev. Brain Res.

Hear. Res.

Trends Neurosci.

Hear. Res.

Brain Res.

FEBS Lett.

Neuroscience

Hear. Res.

Neuroscience

Early development and maturation of the spiral ganglion

Acta Otolaryngol.

Three types of depolarisation-activated potassium currents in acutely isolated mouse vestibular neurons

J. Neurophysiol.

Extracellular ATP-induced Ca2+ mobilization of type I spiral ganglion cells from the guinea pig cochlea

Acta Otolaryngol.

Molecular diversity of K+ channels

Ann. N. Y. Acad. Sci.

Voltage clamp studies of a transient outward membrane current in gastropod neural somata

J. Physiol.

Changes in the electrical properties of chick ciliary ganglion neurones during embryonic development

J. Physiol.

Regulation of A-currents by cell–cell interactions and neurotrophic factors in developing chick parasympathetic neurones

J. Physiol.

Development of ganglion cell topography in the postnatal cochlea

J. Comp. Neurol.

I_As are present in neonatal SGN in a slice preparation, but are absent in age-matched dissociated neurones

Extracellular ATP-induced Ca²⁺ mobilization of type I spiral ganglion cells from the guinea pig cochlea

Molecular diversity of K⁺ channels