Expression And Localization Of Atrial Natriuretic Peptide And Its Receptors In The Spiral Ganglion Of Rat Cochlea

The spiral ganglion (SG) is a peripheral cluster of both neurons and glial cellslocalized in Rosenthal’s canal, which coils around the cochlear modiolus. Spiral ganglionneurons (SGNs) are the primary afferent neurons in the spiral ganglion and play a criticalrole in hearing, transmitting primary acoustic information from the mechanosensory haircells in the organ of Corti to the higher auditory centers of the central nervous system(CNS). SGNs are divided into two subpopulations, type I and type II, according to theirdifferent morphology, synaptic connections and functions. The large, bipolar andmyelinated type I neurons innervate inner hair cells with their peripheral processes toprincipally encode the auditory signals and represent approximately90~95%of theafferent auditory neurons. The smaller, pseudomonopolar and non-myelinated type IIneurons innervate the outer hair cells and some of the supporting cells of the cochlea toprovide sensory feedback, controlling the sensitivity of the auditory epithelia to specific sound stimuli. Degeneration of SGNs, primarily resulting from noise trauma, ototoxicdrugs, infection, aging and genetic mutations, or secondarily occurring as a result of haircells loss, ultimately leads to permanent sensorineural hearing loss. Understanding theexpression, function and signaling interactions of the neurotransmitters, neuromodulatorsand other regulatory substances which affect neuronal physiology and neurotransmissionin primary auditory neurons, may offer insights into the mechanisms underlying normaland pathological states of hearing, and provide important clues for effective prophylacticand therapeutic treatment for hearing impairment.The satellite glial cells (SGCs) and Schwann cells are the primary glial cells in thespiral ganglion. Schwann cells are found in close proximity to the neuronal processes oraxons where they from myelin sheaths around type I neuronal axons. Perineuronal SGCs,whose roles are still relatively poorly understood, envelop the somata of SGNs, andtogether they constitute functional units. Additionally, SGCs form loose myelin around thesomatic membrane of type I neurons. SGCs in the peripheral sensory ganglion share manyproperties with astrocytes in the CNS, as they not only express various ion channels,neurotransmitter receptors and trophic factors which modulate neuronal activity insynaptic transmission, but also form a partial diffusion barrier which regulates theneuronal environment, resembling the blood-brain barrier in the CNS. Consequently, theinteraction between neurons and SGCs (Neuron–SGC communication) can modulateneurotransmission, neuroprotection and neuronal homeostasis. Therefore, new strategiesaimed at manipulating communication between SGNs and SGCs within the spiralganglion have the potential to become promising pharmacological targets that facilitatenew and effective therapies for hearing impairment.Atrial natriuretic peptide (ANP) is a28amino acid peptide with potent natriuretic,diuretic, and vasorelaxant activity which is predominantly synthesized and secreted by thecardiac atria, and is the first member of the natriuretic peptide family, which also includesbrain natriuretic peptide and C-type natriuretic peptide. ANP interacts with two specific,high affinity receptors, NPR-A and NPR-C, on the plasma membrane of target cells tomediate its physiological effects on the regulation of cardiovascular homeostasis,maintaining blood pressure and extracellular fluid volume. The natriuretic peptidereceptor-A (NPR-A) is coupled to the particulate guanylyl cyclase (GC) which catalyzesthe synthesis of the second messenger cyclic guanosine monophosphate (cGMP). cGMPmodulates the activity of specific effector molecules including cGMP-regulated isoforms of phosphodiesterases, cyclic-nucleotide-gated ion channels, and cGMP-dependent proteinkinases G, which in turn regulate diverse biological responses associated with blood vesseltone, neuronal excitability, transepithelial ion transportation, and the sensory transductionpathways underlying olfaction and vision. The natriuretic peptide receptor-C (NPR-C),which lacks the GC domain, contributes to the clearance of ANP and other natriureticpeptides from the circulation through receptor-mediated internalization and degradation. Anumber of studies have shown that NPR-C can also affect second messenger systems in avariety of tissues by inhibiting cyclic AMP (cAMP) production via a pertussistoxin-sensitive inhibitory guanine nucleotide regulatory protein (Gi), and by activatingphospholipase-C without affecting cGMP levels.In addition to the cardiovascular system, the tissue-specific distribution and functionof ANP, NPR-A and NPR-C has been established in several tissues including kidney,adrenal, lung, adipose tissue and retina. Furthermore, ANP and its receptors have beenfound in the CNS, leading us to speculate that ANP may function as a neuromodulator orneuropeptide involved in neuronal and glial functions. Importantly, their presence in thesecretory and sensory compartments of the rodent inner ear is well-documented,suggesting ANP may act as a local hormone regulating the fluid and electrolyte balance inthe inner ear. ANP receptors have been localized to the cochlear modiolus of the guineapig and rat spiral ganglion, the neuronal region of the cochlea. However, little is knownregarding the localization and function of ANP and its receptors within the spiral ganglion.In our current study, we investigated the expression and localization of ANP and itsreceptors in the cochlear spiral ganglion of the postnatal rat by immunohistochemistry,reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, withthe aim of identifying their cellular localizations, expression levels and potential functions.We showed here that ANP and its receptors were colocalized in both subtypes of SGNsand perineuronal SGCs within the rat spiral ganglion. Additionally, we analyzed thedifferential expression levels of both mRNA and protein of ANP and its receptors withinthe rat spiral ganglion during postnatal development. Collectively, our data provide directevidence for the presence and synthesis of ANP as well as its receptors in both neuronaland nonneuronal cells within the cochlear spiral ganglion, suggesting possible roles forANP in modulating neuronal and glial functions as well as neuron–SGC communicationwithin the spiral ganglion during auditory neurotransmission and development. Part one: Expression and localization of atrialnatriuretic peptide and its receptors in the spiralganglion of the rat cochlea[Objectives] To investigate the expression and cellular localization of ANP and itsreceptors in the spiral ganglion of the rat cochlea.[Methods] All cochleae used in this investigation were obtained from postnatalSprague Dawley rats (postnatal day7, P7). To reveal the cellular localization of ANP andits receptors in the rat spiral ganglion, both the cochlear cryosections and ultrathin sectionswere subjected to immunohistochemistry, and then examined under a scanning laserconfocal microscope or a transmission electron microscope, respectively. To investigatethe expression of ANP and its receptors in the rat spiral ganglion, both mRNA and proteinproducts of ANP and its receptors were detected in the cochlear modiolus tissues byRT-PCR and Western blot analysis.[Results] The co-localization of ANP and its receptors were found in both subtypes ofSGNs and in perineuronal SGCs by immunohistochemistry, while the heterogeneity ofimmunoreactivity of ANP and its receptors were less co-localizated in a sub-population ofSGNs. Specifically, ANP was predominantly immunoreactive in the perikaryal cytoplasm ofboth type I and type II SGNs and in the perinuclear cytoplasm of SGCs. The distribution ofNPR-A and NPR-C was rather similar, and they were predominantly immunoreactive in theplasma membrane and adjacent cytoplasm of both type I and type II SGNs as well assurrounding SGCs. Subsequently, the transcripts of ANP and its receptor were detected inthe cochlear modiolus tissues by RT-PCR using specific primer pairs for ANP, NPR-A andNPR-C. The protein products were also present in the modiolus tissues by Western blottingusing specific antibodies against ANP, NPR-A and NPR-C.[Conclusions] We have demonstrated that ANP and its receptors colocalize in bothsubtypes of SGNs and perineuronal SGCs of the rat spiral ganglion. Furthermore, we haveconfirmed the expressions of ANP and its receptors in the rat spiral ganglion at both theprotein and mRNA level. Collectively, our research provides a direct evidence for thepresence and synthesis of ANP and its receptors in both neuronal and non-neuronal cells within the spiral ganglion of the rat cochlea, suggesting possible roles for ANP inmodulating neuronal and glial functions, as well as neuron–SGC communication withinthe spiral ganglion during auditory neurotransmission via its receptors.Part two: Expression patterns of atrial natriureticpeptide and its receptors within the spiral ganglion ofthe rat cochlea during postnatal development[Objectives] To investigate the expression profiles of ANP and its receptors withinthe spiral ganglion of the rat cochlea during postnatal development.[Methods] All cochleae used in this investigation were obtained from postnatalSprague Dawley rats at five different ages (from postnatal day0to day28, P0-P28). Toreveal the cellular localization of ANP and its receptors in the rat spiral ganglion duringpostnatal development, the cochlear cryosections were subjected to immunohistochemistryand examined under a scanning laser confocal microscope. To investigate the expressionlevels of ANP and its receptors in the rat spiral ganglion, both mRNA and proteinproducts of ANP and its receptors were detected and analyzed in the cochlear modiolustissues by RT-PCR and Western blot analysis.[Results] The co-localization of ANP and its receptors were confirmed within thespiral ganglion of the rat cochlea at various stages of postnatal development byimmunohistochemistry. Subsequently, the differential expressions of ANP and itsreceptors at both the protein and mRNA level were detected in the cochlear modiolustissues by RT-PCR and Western blotting. Specifically, ANP mRNA was expressed at thelowest level after birth but strongly increased to a maximum level at P28. NPR-A mRNAwas expressed at a maximum level after birth but gradually decreased to a minimum levelat P28. The expression of NPR-C mRNA was decreased at early postnatal developmentalstages but slightly increased in adulthood. In contrast to the qRT-PCR data, ANP proteinwas expressed at a maximum level at P7but sharply decreased to the lowest level at P28.NPR-A and NPR-C proteins were expressed at a maximum level after birth but graduallydeclined during postnatal development.[Conclusions] We have demonstrated that ANP and its receptors colocalize in the spiral ganglion of the rat cochlea during postnatal development. Furthermore, we haveanalyzed the differential expression levels of both mRNA and protein of ANP and itsreceptors within the rat spiral ganglion during postnatal development. Collectively, ourresearch provides a direct evidence for the presence and synthesis of ANP and itsreceptors in the spiral ganglion of the rat cochlea during postnatal development. Theprotein expression levels of ANP and its receptors decreased with auditory development,suggesting possible roles for ANP in modulating neuronal and glial development, as wellas establishment of the neuron–SGC communication via its receptors.