Research reportSubstance P inhibits potassium and calcium currents in inner ear spiral ganglion neurons
Introduction
Substance P (SP), a neuropeptide of the neurokinin family, is widely expressed in the peripheral and central nervous system [3], [13], [16], [34]. SP has been shown to modulate potassium (K+), chloride (Cl−) and calcium (Ca2+) channels and nonselective cation channels in a number of neuronal systems [3], [15], [26], [36]. SP can also bind to G-proteins, which can modulate ionic conductances either directly or through second messenger systems [1], [6], [30]. In addition, SP can also evoke slow excitatory postsynaptic potentials (EPSP) in dorsal horn neurons in the spinal cord [35].
SP has been identified at several locations in the auditory pathway [29], [53], including the inner ear [25], [37], [55]. SP immunolabeling has been observed on the labyrinthine and spiral modiolar arteries, but not in the stria vascularis [25], [47], suggesting that the SP could be involved in regulating cochlear blood flow. SP immunolabeling has also been observed on inner hair cells (IHCs) and outer hair cells (OHCs), on spiral ganglion neurons (SGNs), which synapse on OHCs and IHCs, and on the axons of SGNs, which form the auditory nerve [37], [55]. SP is expressed in most human SGNs and on 50% of rabbit SGNs [2], [55].
The physiological effects of SP on cochlear function have been examined using several different methods. Infusion of SP into the anterior inferior cerebellar artery, which supplies the cochlea, increased cochlear blood flow [28], but did not alter the compound action potential (CAP), generated by SGNs or the cochlear microphonic (CM), generated predominantly by OHCs. These results suggest that circulating SP affects cochlear blood flow, but not neural or sensory cell function. In addition, intracochlear perfusion of substance P methyl ester (SPME), a selective NK1 receptor agonist, had no effect on the endolymphatic potential or the CM. However, SPME produced a slight increase in the amplitude of the negative summating potential, generated predominantly by IHCs, as well as a slight increase in CAP amplitude [31]. Conversely, intracochlear perfusion of an antagonist of NK1 receptor decreased the amplitude and increased the latency of the CAP [21]. These results clearly indicate that SP modulates auditory nerve function. Whether the changes in CAP amplitude are mediated by a direct effect of SP on SGNs or an indirect effect through the IHCs is not clear.
The cellular mechanisms by which SP affects SGN activity were recently investigated in acutely dissociated adult SGNs using calcium imaging and whole-cell patch-clamp recordings [14]. Puffing SP on to SGNs caused a transient increase in intracellular Ca2+. This response persisted in the absence of extracellular Ca2+ and declined with repeated stimulation, suggesting Ca2+ release from intracellular stores. Inhibitors of G-proteins and phospholipase C blocked the SP-induced Ca2+ increase.
The preceding results suggest that SP exerts a direct effect on SGNs. However, we are unaware of any published reports showing that NK1 receptors are present on SGNs. In the present study, we used immunolabeling to identify the presence of NK1 receptors on SGNs. In addition, using whole-cell patch-clamp recordings, we found that SP prolonged the latency and duration of the action potential, made the resting potential (RP) more positive and suppressed voltage-activated potassium currents (IK+) and calcium currents (ICa2+) of SGNs in cochlear organotypic cultures.
Section snippets
Cochlear organotypic cultures
Postnatal (P0–P5) 10J mice were killed by cervical dislocation, the inner ear removed and cochlear organotypic cultures prepared as in our previous reports [44]. The cochlea was dissected out and placed in cold Hank's balanced salt solution (HBSS, GIBCO). The cochlear capsule was opened, the lateral wall of the cochlea and the spiral lamina were removed, and the basilar membrane (BM), containing the organ of Corti and SGNs, was carefully removed as a flat surface preparation. The surface
RT-PCR
Fig. 1 shows the RT-PCR result from cochlear and brain samples taken from four P2 mice using primers designed to detect the mRNA for the NK1 receptor. As a negative control, water was added to the reaction mixture instead of cochlear mRNA. In the cochlear sample, a PCR product of 400 bp, consistent with that previously reported for the NK1 receptor [45], was present; this band was also present in the brain stem consistent with the previous results [27], but was absent in the water control
Discussion
Previous studies have shown that SP is present on the afferent synapses, soma and axons of SGNs in the inner ear [33], [40], [54]; however, we are unaware of any studies showing that the high affinity receptor for SP, NK1, is present on SGNs. Our RT-PCR results show that the mRNA for the NK1 receptor is expressed in the cochlea of postnatal mice. In addition, Western blots indicated that the NK1 receptor protein is present in postnatal cochlea and our immunolabeling studies show that the NK1
Acknowledgements
This work was supported by a grant from National Institutes of Health P01 DC03600-01A1 and an American Tinnitus Association grant. We gratefully acknowledge Te-Chung Lee for the assistance with the Western blots.
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