An adenylyl cyclase signaling pathway predicts direct dopaminergic input to vestibular hair cells

Abstract

Adenylyl cyclase signaling pathways have been identified in a model hair cell preparation from the trout saccule, for which the hair cell is the only intact cell type. The use of degenerate primers targeting cDNA sequence conserved across adenylyl cyclase (AC) isoforms, and RT-PCR, coupled with cloning of amplification products, indicated expression of AC9, AC7 and AC5/6, with cloning efficiencies of 11:5:2. AC9 and AC5/6 are inhibited by Ca2+, the former in conjunction with calcineurin, and message for calcineurin has also been identified in the trout saccular hair cell layer. AC7 is independent of Ca2+. Given the lack of detection of calcium/calmodulin-activated isoforms previously suggested to mediate adenylyl cyclase activation in the absence of Gαs in mammalian cochlear hair cells, the issue of hair-cell Gαs mRNA expression was re-examined in the teleost vestibular hair cell model. Two full-length coding sequences were obtained for Gαs/olf in the vestibular type II-like hair cells of the trout saccule. Two messages for Gαi have also been detected in the hair cell layer, one with homology to Gαi1 and the second with homology to Gαi3 of higher vertebrates. Both Gαs/olf protein and Gαi1/Gαi3 protein were immunolocalized to stereocilia and to the base of the hair cell, the latter consistent with sites of efferent input. While a signaling event coupling to Gαs/olf and Gαi1/Gαi3 in the stereocilia is currently unknown, signaling with Gαs/olf, Gαi3, and AC5/6 at the base of the hair cell would be consistent with transduction pathways activated by dopaminergic efferent input. mRNA for dopamine receptors D1A4 and five forms of dopamine D2 were found to be expressed in the teleost saccular hair cell layer, representing information on vestibular hair cell expression not directly available for higher vertebrates. Dopamine D1A receptor would couple to Gαolf and activation of AC5/6. Co-expression with dopamine D2 receptor, which itself couples to Gαi3 and AC5/6, will down-modulate levels of cAMP, thus fine-tuning and gradating the hair-cell response to dopamine D1A. As predicted by the trout saccular hair cell model, evidence has been obtained for the first time that hair cells of mammalian otolithic vestibular end organs (rat/mouse saccule/utricle) express dopamine D1A and D2L receptors, and each receptor co-localizes with AC5/6, with a marked presence of all three proteins in subcuticular regions of type I vestibular hair cells. A putative efferent, presynaptic source of dopamine was identified in tyrosine hydroxylase-positive nerve fibers which passed from underlying connective tissue to the sensory epithelia, ending on type I and type II vestibular hair cells and on afferent calyces.

Hyperpolarization-Activated Current (I h) in Vestibular Calyx Terminals: Characterization and Role in Shaping Postsynaptic Events

“…Our data show that cGMP is a potent modulator of I h , and in vivo cGMP levels could be modulated through nitric oxide activation of guanylyl cyclase (Garthwaite 2008). Dopamine is found in auditory efferents (Gáborján et al 1999), and immunoreactivity for dopamine receptor subtypes D1A and D2L was reported on both inner and outer faces of calyces from the rat saccule and mouse utricle (Drescher et al 2010). It is tempting to speculate that elevating levels of intracellular cyclic nucleotides could enhance firing in calyx afferents through I h ; however, further experiments are required to provide a possible direct link between I h modulation and efferent input to the calyx.…”

“…Electrical stimulation of the efferent system leads to an increase in firing in mammalian vestibular afferents and calyx units show the largest responses to efferent stimulation (reviewed in Holt et al 2011). ACh is thought to be a major efferent neurotransmitter, but other candidate transmitter molecules include ATP, calcitonin gene-related peptide, opioid peptides, γ-aminobutyric acid, nitric oxide, and dopamine (Drescher et al 2010;Holt et al 2011). Efferent transmitters could potentially influence I h gating by modulating intracellular levels of cyclic nucleotides.…”

Connectivity and ultrastructure of dopaminergic innervation of the inner ear and auditory efferent system of a vocal fish

“…In contrast, DA increases the excitability of hair cells in the zebrafish lateral line through a D1‐like receptor mechanism (Toro et al, ). Isolated hair cells from rainbow trout express both D1‐like and D2‐like receptor mRNA (Drescher et al, ), as do homogenates of larval zebrafish utricular and saccular maculae (Toro et al, ). To our knowledge, there is no evidence for a direct DA modulation of cholinergic efferents in the peripheral auditory system, but dopaminergic and cholinergic systems have well characterized interactions in the central nervous system (Acquas & Chiara, ; Moore, Fadel, Sarter, & Bruno, ).…”

Catecholaminergic connectivity to the inner ear, central auditory, and vocal motor circuitry in the plainfin midshipman fish porichthys notatus

“…This antibody recognizes ã 60kDa band from mouse brain lysate on Western blot (manufacturer's data) and labels known catecholaminergic neuronal populations in rodent brain (e.g., Kaufling et al, 2009). Importantly, in midshipman, AB1542 shows the same pattern of labeling on adjacent sections in the saccule as the monoclonal anti-TH (MAB318, above) and a similar pattern of innervation in mammalian saccule (Drescher et al, 2010) and cochlear sensory epithelium (Darrow et al, 2006a;Niu and Canlon, 2006; see Discussion) as other polyclonal TH antibodies. Adjacent sections from positively labeled sections incubated without primary or secondary antisera showed a complete absence of labeling.…”

“…Furthermore, dopamine receptor mRNA expression was recently found in hair cell preparations of the trout saccule (Drescher et al, 2010). Thus, there is compelling evidence that dopamine action on the inner ear may be a common feature across unrelated species of teleost fishes, and perhaps vertebrates in general.…”