RESUMEN
Endogenous glucocorticoids (GC) are known to modulate basic elements of cochlear physiology. These include both noise-induced injury and circadian rhythms. While GC signaling in the cochlea can directly influence auditory transduction via actions on hair cells and spiral ganglion neurons, evidence also indicates that GC signaling exerts effects via tissue homeostatic processes that can include effects on cochlear immunomodulation. GCs act at both the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). Most cell types in the cochlea express both receptors sensitive to GCs. The GR is associated with acquired sensorineural hearing loss (SNHL) through its effects on both gene expression and immunomodulatory programs. The MR has been associated with age-related hearing loss through dysfunction of ionic homeostatic balance. Cochlear supporting cells maintain local homeostatic requirements, are sensitive to perturbation, and participate in inflammatory signaling. Here, we have used conditional gene manipulation techniques to target Nr3c1 (GR) or Nr3c2 (MR) for tamoxifen-induced gene ablation in Sox9-expressing cochlear supporting cells of adult mice to investigate whether either of the receptors sensitive to GCs plays a role in protecting against (or exacerbating) noise-induced cochlear damage. We have selected mild intensity noise exposure to examine the role of these receptors related to more commonly experienced noise levels. Our results reveal distinct roles of these GC receptors for both basal auditory thresholds prior to noise exposure and during recovery from mild noise exposure. Prior to noise exposure, auditory brainstem responses (ABRs) were measured in mice carrying the floxed allele of interest and the Cre recombinase transgene, but not receiving tamoxifen injections (defined as control (no tamoxifen treatment), versus conditional knockout (cKO) mice, defined as mice having received tamoxifen injections. Results revealed hypersensitive thresholds to mid- to low-frequencies after tamoxifen-induced GR ablation from Sox9-expressing cochlear supporting cells compared to control (no tamoxifen) mice. GR ablation from Sox9-expressing cochlear supporting cells resulted in a permanent threshold shift in mid-basal cochlear frequency regions after mild noise exposure that produced only a temporary threshold shift in both control (no tamoxifen) f/fGR:Sox9iCre+ and heterozygous f/+GR:Sox9iCre+ tamoxifen-treated mice. A similar comparison of basal ABRs measured in control (no tamoxifen) and tamoxifen-treated, floxed MR mice prior to noise exposure indicated no difference in baseline thresholds. After mild noise exposure, MR ablation was initially associated with a complete threshold recovery at 22.6 kHz by 3 days post-noise. Threshold continued to shift to higher sensitivity over time such that by 30 days post-noise exposure the 22.6 kHz ABR threshold was 10 dB more sensitive than baseline. Further, MR ablation produced a temporary reduction in peak 1 neural amplitude one day post-noise. While supporting cell GR ablation trended towards reducing numbers of ribbon synapses, MR ablation reduced ribbon synapse counts but did not exacerbate noise-induced damage including synapse loss at the experimental endpoint. GR ablation from the targeted supporting cells increased the basal resting number of Iba1-positive (innate) immune cells (no noise exposure) and decreased the number of Iba1-positive cells seven days following noise exposure. MR ablation did not alter innate immune cell numbers at seven days post-noise exposure. Taken together, these findings support differential roles of cochlear supporting cell MR and GR expression at basal, resting conditions and especially during recovery from noise exposure.
Asunto(s)
Pérdida Auditiva Provocada por Ruido , Ratones , Animales , Pérdida Auditiva Provocada por Ruido/metabolismo , Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Cóclea/metabolismo , Audición , Umbral Auditivo/fisiología , Receptores de Glucocorticoides/metabolismoRESUMEN
Opioids are first-line drugs for moderate to severe acute pain and cancer pain. However, these medications are associated with severe side effects, and whether they are efficacious in treatment of chronic nonmalignant pain remains controversial. Medications that act through alternative molecular mechanisms are critically needed. Antagonists of α9α10 nicotinic acetylcholine receptors (nAChRs) have been proposed as an important nonopioid mechanism based on studies demonstrating prevention of neuropathology after trauma-induced nerve injury. However, the key α9α10 ligands characterized to date are at least two orders of magnitude less potent on human vs. rodent nAChRs, limiting their translational application. Furthermore, an alternative proposal that these ligands achieve their beneficial effects by acting as agonists of GABAB receptors has caused confusion over whether blockade of α9α10 nAChRs is the fundamental underlying mechanism. To address these issues definitively, we developed RgIA4, a peptide that exhibits high potency for both human and rodent α9α10 nAChRs, and was at least 1,000-fold more selective for α9α10 nAChRs vs. all other molecular targets tested, including opioid and GABAB receptors. A daily s.c. dose of RgIA4 prevented chemotherapy-induced neuropathic pain in rats. In wild-type mice, oxaliplatin treatment produced cold allodynia that could be prevented by RgIA4. Additionally, in α9 KO mice, chemotherapy-induced development of cold allodynia was attenuated and the milder, temporary cold allodynia was not relieved by RgIA4. These findings establish blockade of α9-containing nAChRs as the basis for the efficacy of RgIA4, and that α9-containing nAChRs are a critical target for prevention of chronic cancer chemotherapy-induced neuropathic pain.
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Dolor en Cáncer/tratamiento farmacológico , Hiperalgesia/tratamiento farmacológico , Péptidos/administración & dosificación , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Analgésicos Opioides/efectos adversos , Animales , Dolor en Cáncer/inducido químicamente , Dolor en Cáncer/genética , Dolor en Cáncer/patología , Humanos , Hiperalgesia/inducido químicamente , Hiperalgesia/genética , Hiperalgesia/patología , Ligandos , Ratones , Ratones Noqueados , Neuralgia/inducido químicamente , Neuralgia/tratamiento farmacológico , Neuralgia/genética , Neuralgia/patología , Antagonistas Nicotínicos/administración & dosificación , Compuestos Organoplatinos/efectos adversos , Oxaliplatino , Receptores de GABA-B/genéticaRESUMEN
Although the major emphasis of Enrico Mugnaini's research has been on investigations of the cerebellum, a significant amount of work over a relatively short span of time was also done in his lab on a number of other brain systems. These centered on sensory systems. One of these extra-cerebellar systems that he embraced was the auditory system. Portions of the cochlear nucleus, the first synaptic relay station along the central auditory pathways, possess a cerebellar-like circuitry and neurochemistry, and this no doubt lured Enrico into the auditory field. As new tools became available to pursue neuroanatomical research in general, which included a novel antibody to glutamic acid decarboxylase (GAD), Enrico's lab soon branched out into investigating many other brain structures beyond the cerebellum, with an overall goal of producing a map illustrating GAD expression in the brain. In collaboration with long-term colleagues, one of these many non-cerebellar regions he took an interest in was an efferent pathway originating in the superior olive and projecting to the cochlea, the peripheral end organ for hearing. There was a need for a more complete neurochemical map of this olivocochlear efferent system, and armed with new antibodies and well-established tract tracing tools, together we set out to further explore this system. This short review describes the work done with Enrico on the olivocochlear system of rodents, and also continues the story beyond Enrico's lab to reveal how the work done in his lab fits into the larger scheme of current, ongoing research into the olivocochlear system.
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Cerebelo/fisiología , Mamíferos/anatomía & histología , Núcleo Olivar/anatomía & histología , Núcleo Olivar/fisiología , Animales , Vías Auditivas/fisiología , Historia del Siglo XX , Humanos , Neuroanatomía/historia , Neuroquímica/historiaRESUMEN
Urocortin is a member of the corticotropin-releasing hormone peptide family and is found in many discrete brain regions. The distinct expression pattern of urocortin suggests that it influences such behaviors as feeding, anxiety and auditory processing. To better define the physiological roles of urocortin, we have generated mice carrying a null mutation of the urocortin gene. Urocortin-deficient mice have normal basal feeding behavior and stress responses, but show heightened anxiety-like behaviors in the elevated plus maze and open-field tests. In addition, hearing is impaired in the mutant mice at the level of the inner ear, suggesting that urocortin is involved in the normal development of cochlear sensory-cell function. These results provide the first example of a function for any peptidergic system in hearing.
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Ansiedad/genética , Conducta Animal/fisiología , Hormona Liberadora de Corticotropina/genética , Trastornos de la Audición/genética , Estimulación Acústica , Animales , Cóclea/fisiopatología , Hormona Liberadora de Corticotropina/metabolismo , Ingestión de Alimentos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Estrés Fisiológico , UrocortinasRESUMEN
Immunohistochemistry is a valuable tool for probing not only scientific questions but also clinical diagnoses. It provides power from localization of a protein within the milieu of a tissue section that may reflect positioning within or beyond the boundaries of a cell that is representative of the tissue at a discrete moment in time. The method can be applied broadly, including to tissues under normal, developmental, chemically, or genetically altered conditions and disease states.Disease manifesting from West Nile virus infection ranges from acute, systemic febrile symptoms to compromise of central nervous system function. Immunohistochemistry has been used to assess WNV infection in the nervous system in postmortem and experimental conditions, despite the lack of understanding of the precise route of viral entry. In addition to imprecise knowledge of initial viral entry into cells and whether entry is even the same between cell types, the fact that spontaneous viral mutations and environmental pressures from climate change may alter the prevalence of the disease state across geographical and climatological boundaries highlights the need for continued assessment of infection. Immunohistochemistry is a useful way to assess these aspects of WNV infection with the aim being to better understand the organs and cell types that are compromised by WNV infection. This chapter outlines how this can be carried out on brain tissue, but the procedures discussed can also be applied more broadly on tissue outside of the central nervous system.
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Fiebre del Nilo Occidental , Virus del Nilo Occidental , Humanos , Fiebre del Nilo Occidental/diagnóstico , Proteínas del Envoltorio Viral , Encéfalo , InmunohistoquímicaRESUMEN
Cells of the inner ear face constant metabolic and structural stress. Exposure to intense sound or certain drugs destroys cochlea hair cells, which in mammals do not regenerate. Thus, an endogenous stress response system may exist within the cochlea to protect it from everyday stressors. We recently described the existence of corticotropin-releasing factor (CRF) in the mouse cochlea. The CRF receptor type 1 (CRFR1) is considered the primary and canonical target of CRF signaling, and systemically it plays an essential role in coordinating the body-wide stress response via activation of the hypothalamic-pituitary-adrenal (HPA) axis. Here, we describe an essential role for CRFR1 in auditory system development and function, and offer the first description of a complete HPA equivalent signaling system resident within the cochlea. To reveal the role of CRFR1 activation in the cochlea, we have used mice carrying a null ablation of the CRFR1 gene. CRFR1(-/-) mice exhibited elevated auditory thresholds at all frequencies tested, indicating reduced sensitivity. Furthermore, our results suggest that CRFR1 has a developmental role affecting inner hair cell morphology and afferent and efferent synapse distribution. Given the role of HPA signaling in maintaining local homeostasis in other tissues, the presence of a cochlear HPA signaling system suggests important roles for CRFR1 activity in setting cochlear sensitivity, perhaps both neural and non-neural mechanisms. These data highlight the complex pleiotropic mechanisms modulated by CRFR1 signaling in the cochlea.
Asunto(s)
Cóclea/fisiología , Células Ciliadas Auditivas/fisiología , Sistema Hipotálamo-Hipofisario/fisiología , Sistema Hipófiso-Suprarrenal/fisiología , Receptores de Hormona Liberadora de Corticotropina/fisiología , Hormona Adrenocorticotrópica/biosíntesis , Animales , Umbral Auditivo , Cóclea/inervación , Corticosterona/farmacología , Hormona Liberadora de Corticotropina/biosíntesis , Ratones , Ratones Noqueados , Vías Nerviosas , Comunicación Paracrina , Proopiomelanocortina/biosíntesis , Receptor de Melanocortina Tipo 2/biosíntesis , Receptores de Hormona Liberadora de Corticotropina/biosíntesis , Receptores de Hormona Liberadora de Corticotropina/genética , Transducción de SeñalRESUMEN
Suppression of ipsilateral distortion product otoacoustic emissions (DPOAEs) by contralateral noise is used in humans and animals to assay the strength of sound-evoked negative feedback from the medial olivocochlear (MOC) efferent pathway. However, depending on species and anesthesia, contributions of other feedback systems to the middle or inner ear can cloud the interpretation. Here, contributions of MOC and middle-ear muscle reflexes, as well as autonomic feedback, to contra-noise suppression in anesthetized mice are dissected by selectively eliminating each pathway by surgical transection, pharmacological blockade, or targeted gene deletion. When ipsilateral DPOAEs were evoked by low-level primaries, contra-noise suppression was typically ~1 dB with contra-noise levels around 95 dB SPL, and it always disappeared upon contralateral cochlear destruction. Lack of middle-ear muscle contribution was suggested by persistence of contra-noise suppression after paralysis with curare, tensor tympani cauterization, or section of the facial nerve. Contribution of cochlear sympathetics was ruled out by studying mutant mice lacking adrenergic signaling (dopamine ß-hydroxylase knockouts). Surprisingly, contra-noise effects on low-level DPOAEs were also not diminished by eliminating the MOC system pharmacologically (strychnine), surgically, or by deletion of relevant cholinergic receptors (α9/α10). In contrast, when ipsilateral DPOAEs were evoked by high-level primaries, the contra-noise suppression, although comparable in magnitude, was largely eliminated by MOC blockade or section. Possible alternate pathways are discussed for the source of contra-noise-evoked effects at low ipsilateral levels.
Asunto(s)
Anestesia General , Biorretroalimentación Psicológica/fisiología , Cóclea/fisiología , Músculo Esquelético/fisiología , Núcleo Olivar/fisiología , Emisiones Otoacústicas Espontáneas/fisiología , Vías Aferentes/fisiología , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Relación Señal-RuidoRESUMEN
The transduction of sound in the auditory periphery, the cochlea, is inhibited by efferent cholinergic neurons projecting from the brainstem and synapsing directly on mechanosensory hair cells. One fundamental question in auditory neuroscience is what role(s) this feedback plays in our ability to hear. In the present study, we have engineered a genetically modified mouse model in which the magnitude and duration of efferent cholinergic effects are increased, and we assess the consequences of this manipulation on cochlear function. We generated the Chrna9L9'T line of knockin mice with a threonine for leucine change (L9'T) at position 9' of the second transmembrane domain of the alpha9 nicotinic cholinergic subunit, rendering alpha9-containing receptors that were hypersensitive to acetylcholine and had slower desensitization kinetics. The Chrna9L9'T allele produced a 3-fold prolongation of efferent synaptic currents in vitro. In vivo, Chrna9L9'T mice had baseline elevation of cochlear thresholds and efferent-mediated inhibition of cochlear responses was dramatically enhanced and lengthened: both effects were reversed by strychnine blockade of the alpha9alpha10 hair cell nicotinic receptor. Importantly, relative to their wild-type littermates, Chrna9(L9'T/L9'T) mice showed less permanent hearing loss following exposure to intense noise. Thus, a point mutation designed to alter alpha9alpha10 receptor gating has provided an animal model in which not only is efferent inhibition more powerful, but also one in which sound-induced hearing loss can be restrained, indicating the ability of efferent feedback to ameliorate sound trauma.
Asunto(s)
Acetilcolina/metabolismo , Colinérgicos/metabolismo , Células Ciliadas Auditivas/fisiología , Neuronas Eferentes/fisiología , Mutación Puntual , Receptores Nicotínicos/genética , Animales , Vías Auditivas/fisiología , Umbral Auditivo/fisiología , Cóclea/metabolismo , Modelos Animales de Enfermedad , Retroalimentación Fisiológica/fisiología , Pérdida Auditiva Sensorineural/prevención & control , Ratones , Ratones Mutantes , Canales de Potasio/fisiología , Receptores Nicotínicos/fisiología , Transducción de Señal/fisiología , Sinapsis/fisiologíaRESUMEN
Acetylcholine is the major neurotransmitter of the olivocochlear efferent system, which provides feedback to cochlear hair cells and sensory neurons. To study the role of cochlear muscarinic receptors, we studied receptor localization with immunohistochemistry and reverse transcription-PCR and measured olivocochlear function, cochlear responses, and histopathology in mice with targeted deletion of each of the five receptor subtypes. M2, M4, and M5 were detected in microdissected immature (postnatal days 10-13) inner hair cells and spiral ganglion cells but not outer hair cells. In the adult (6 weeks), the same transcripts were found in microdissected organ of Corti and spiral ganglion samples. M2 protein was found, by immunohistochemistry, in olivocochlear fibers in both outer and inner hair cell areas. M3 mRNA was amplified only from whole cochleas, and M1 message was never seen in wild-type ears. Auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) were unaffected by loss of Gq-coupled receptors (M1, M3, or M5), as were shock-evoked olivocochlear effects and vulnerability to acoustic injury. In contrast, loss of Gi-coupled receptors (M2 and/or M4) decreased neural responses without affecting DPOAEs (at low frequencies). This phenotype and the expression pattern are consistent with excitatory muscarinic signaling in cochlear sensory neurons. At high frequencies, both ABRs and DPOAEs were attenuated by loss of M2 and/or M4, and the vulnerability to acoustic injury was dramatically decreased. This aspect of the phenotype and the expression pattern are consistent with a presynaptic role for muscarinic autoreceptors in decreasing ACh release from olivocochlear terminals during high-level acoustic stimulation and suggest that muscarinic antagonists could enhance the resistance of the inner ear to noise-induced hearing loss.
Asunto(s)
Cóclea/fisiología , Neuronas Aferentes/fisiología , Neuronas Eferentes/fisiología , Terminales Presinápticos/fisiología , Receptores Muscarínicos/metabolismo , Animales , Umbral Auditivo/fisiología , Cóclea/crecimiento & desarrollo , Potenciales Evocados Auditivos del Tronco Encefálico , Retroalimentación Fisiológica , Femenino , Células Ciliadas Auditivas Internas/fisiología , Células Ciliadas Auditivas Externas/fisiología , Pérdida Auditiva Provocada por Ruido/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Emisiones Otoacústicas Espontáneas , ARN Mensajero/metabolismo , Receptores Muscarínicos/genética , Ganglio Espiral de la Cóclea/crecimiento & desarrollo , Ganglio Espiral de la Cóclea/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
HYPOTHESIS: Localized cooling of the external ear has a protective effect on the susceptibility to cisplatin-induced hearing loss. BACKGROUND: We previously demonstrated significant protection from cisplatin-induced hearing loss using cool water ear canal irrigation. However, the study was limited to a single bolus injection of cisplatin and an acute time period. Here, we examined the application of localized cooling of the ear canal with repeated doses of cisplatin, over an expanded period of time, and using two methods of cooling. METHODS: Twenty-four guinea pigs (12 male and 12 female) underwent auditory physiological testing (auditory brainstem response and distortion product otoacoustic emissions at 8-32âkHz) and pre/postadministration of cisplatin. Cisplatin (4âmg/kg i.p.) was administered in 3 weekly single injections for a total of 12âmg/kg. While anesthetized, the left ears of the guinea pigs were exposed to either cool water (22°C; ICS Water Caloric Irrigator), a cool ear bar (15°C, cooled by a Peltier device; TNM, Scion NeuroStim), or left uncooled as a sham control. The animals were tested 3 days post each dosage and 1 month post the final dose. At the end of the experiment the animals were euthanized for histological evaluation. RESULTS: We found that hearing loss was significantly reduced, and hair cell survival greatly improved, in animals that received cooling treatments compared to cisplatin-only control animals. No significant difference was observed between the two methods of cooling. CONCLUSION: Localized cooling of the ear canal during administration of cisplatin mitigated loss of auditory function and loss of hair cells.
Asunto(s)
Antineoplásicos , Pérdida Auditiva , Animales , Antineoplásicos/efectos adversos , Cisplatino/toxicidad , Potenciales Evocados Auditivos del Tronco Encefálico , Femenino , Cobayas , Células Ciliadas Auditivas , Audición , Pérdida Auditiva/inducido químicamente , Pérdida Auditiva/tratamiento farmacológico , Pérdida Auditiva/prevención & control , Masculino , Emisiones Otoacústicas EspontáneasRESUMEN
Noise-induced hearing loss is a highly prevalent occupational injury, yet little is known concerning the signals controlling normal cochlear sensitivity and susceptibility to noise-induced trauma. While the corticotropin-releasing factor (CRF) system is involved in activation of the classic hypothalamic-pituitary-adrenal axis, it is also involved in local physiological responses to stress in many tissues, and is expressed in the inner ear. We demonstrate that mice lacking the CRF receptor CRFR2 exhibit a significantly lower auditory threshold than wild type mice, but this gain of function comes at the price of increased susceptibility to acoustic trauma. We further demonstrate that glutamatergic transmission, purinergic signaling, and activation of Akt (PKB) pathways within the cochlea are misregulated, which may underlie the enhanced sensitivity and trauma susceptibility observed in CRFR2(-/-) mice. Our data suggest that CRFR2 constitutively modulates hearing sensitivity under normal conditions, and thereby provides protection against noise-induced hearing loss.
Asunto(s)
Umbral Auditivo/fisiología , Cóclea/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Pérdida Auditiva Provocada por Ruido/genética , Audición/fisiología , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Estimulación Acústica , Animales , Audiometría , Vías Auditivas/metabolismo , Vías Auditivas/fisiopatología , Western Blotting , Ácido Glutámico/metabolismo , Pérdida Auditiva Provocada por Ruido/metabolismo , Pérdida Auditiva Provocada por Ruido/fisiopatología , Inmunoprecipitación , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-akt/metabolismo , Purinas/metabolismo , Receptores de Hormona Liberadora de Corticotropina/genética , Receptores de Glutamato/metabolismo , Receptores Purinérgicos/metabolismo , Transducción de Señal/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
Generation of reactive oxygen species (ROS) is a common denominator in many conditions leading to cell death in the cochlea, yet little is known of the cochlea's endogenous mechanisms involved in preventing oxidative stress and its consequences in the cochlea. We have recently described a corticotropin-releasing factor (CRF) signaling system in the inner ear involved in susceptibility to noise-induced hearing loss. We use biochemical and proteomics assays to define further the role of CRF signaling in the response of cochlear cells to aminoglycoside exposure. We demonstrate that activity via the CRF(2) class of receptors protects against aminoglycoside-induced ROS production and activation of cell death pathways. This study suggests for the first time a role for CRF signaling in protecting the cochlea against oxidative stress, and our proteomics data suggest novel mechanisms beyond induction of free radical scavengers that are involved in its protective mechanisms.
Asunto(s)
Hormona Liberadora de Corticotropina/metabolismo , Gentamicinas/toxicidad , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/toxicidad , Animales , Caspasa 3/metabolismo , Muerte Celular , Línea Celular Transformada , Oído Interno/citología , Cromatografía de Gases y Espectrometría de Masas/métodos , Proteoma/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Superóxido Dismutasa/metabolismoRESUMEN
Cochlear hair cells use SK2 currents to shape responses to cholinergic efferent feedback from the brain. Using SK2(-/-) mice, we demonstrate that, in addition to their previously defined role in modulating hair cell membrane potentials, SK2 channels are necessary for long-term survival of olivocochlear fibers and synapses. Loss of the SK2 gene also results in loss of electrically driven olivocochlear effects in vivo, and down regulation of ryanodine receptors involved in calcium-induced calcium release, the main inducer of nAChR evoked SK2 activity. Generation of double-null mice lacking both the alpha10 nAChR gene, loss of which results in hypertrophied olivocochlear terminals, and the SK2 gene, recapitulates the SK2(-/-) synaptic phenotype and gene expression, and also leads to down regulation of alpha9 nAChR gene expression. The data suggest a hierarchy of activity necessary to maintain early olivocochlear synapses at their targets, with SK2 serving an epistatic, upstream, role to the nAChRs.
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Supervivencia Celular/fisiología , Cóclea/citología , Cóclea/inervación , Vías Eferentes/anatomía & histología , Células Ciliadas Auditivas Externas/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Sinapsis/metabolismo , Animales , Cóclea/fisiología , Vías Eferentes/fisiología , Células Ciliadas Auditivas Externas/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Núcleo Olivar/anatomía & histología , Núcleo Olivar/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Sinaptofisina/metabolismoRESUMEN
Although homomeric channels assembled from the alpha9 nicotinic acetylcholine receptor (nAChR) subunit are functional in vitro, electrophysiological, anatomical, and molecular data suggest that native cholinergic olivocochlear function is mediated via heteromeric nAChRs composed of both alpha9 and alpha10 subunits. To gain insight into alpha10 subunit function in vivo, we examined olivo cochlear innervation and function in alpha10 null-mutant mice. Electrophysiological recordings from postnatal (P) days P8-9 inner hair cells revealed ACh-gated currents in alpha10(+/+) and alpha10(+/-) mice, with no detectable responses to ACh in alpha10(-/-) mice. In contrast, a proportion of alpha10(-/-) outer hair cells showed small ACh-evoked currents. In alpha10(-/-) mutant mice, olivocochlear fiber stimulation failed to suppress distortion products, suggesting that the residual alpha9 homomeric nAChRs expressed by outer hair cells are unable to transduce efferent signals in vivo. Finally, alpha10(-/-) mice exhibit both an abnormal olivocochlear morphology and innervation to outer hair cells and a highly disorganized efferent innervation to the inner hair cell region. Our results demonstrate that alpha9(-/-) and alpha10(-/-) mice have overlapping but nonidentical phenotypes. Moreover, alpha10 nAChR subunits are required for normal olivocochlear activity because alpha9 homomeric nAChRs do not support maintenance of normal olivocochlear innervation or function in alpha10(-/-) mutant mice.
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Cóclea/fisiología , Células Ciliadas Auditivas/fisiología , Núcleo Olivar/fisiología , Receptores Nicotínicos/fisiología , Sinapsis/fisiología , Animales , Cóclea/citología , Estimulación Eléctrica , Electrofisiología , Ratones , Ratones Noqueados , Receptores Nicotínicos/genéticaRESUMEN
Zika virus (ZIKV) has been recently recognized as a causative agent of newborn microcephaly, as well as other neurological consequences. A less well recognized comorbidity of prenatal ZIKV infection is hearing loss, but cases of hearing impairment following adult ZIKV infection have also been recognized. Diminished hearing following prenatal ZIKV infection in a mouse model has been reported, but no cellular consequences were observed. We examined the effects of ZIKV infection on inner ear cellular integrity and expression levels of various proteins important for cochlear function in type I interferon receptor null (Ifnar1-/-) mice following infection at 5-6 weeks of age. We show that ZIKV antigens are present in cells within the cochlear epithelium, lateral wall, spiral limbus and spiral ganglion. Here we show that ZIKV infection alters cochlear expression of genes that signal cell damage (S100B), transport fluids (AQP1), are gaseous transmitters (eNOs) and modulate immune response (F4/80). Morphological analyses shows that not only are cochlear structures compromised by ZIKV infection, but damage also occurs in vestibular end organs. ZIKV produces a graded distribution of cellular damage in the cochlea, with greatest damage in the apex similar to that reported for cytomegalovirus (CMV) infection. The graded distribution of damage may indicate a differential susceptibility to ZIKV along the cochlear tonotopic axis. Collectively, these data are the first to show the molecular and morphological damage to the inner ear induced by ZIKV infection in adults and suggests multiple mechanisms contributing to the hearing loss reported in the human population.
Asunto(s)
Oído Interno , Infección por el Virus Zika , Virus Zika , Animales , Infecciones por Citomegalovirus , Modelos Animales de Enfermedad , Femenino , Pérdida Auditiva , Ratones , Embarazo , Infección por el Virus Zika/complicacionesRESUMEN
The development of knockin mice with Cre recombinase expressed under the control of the promoter for choline acetyltransferase (ChAT) has allowed experimental manipulation of cholinergic circuits. However, currently available ChATCre mouse lines are on the C57BL/6J strain background, which shows early onset age-related hearing loss attributed to the Cdh23753A mutation (a.k.a., the ahl mutation). To develop ChATCre mice without accelerated hearing loss, we backcrossed ChATIRES-Cre mice with CBA/CaJ mice that have normal hearing. We used genotyping to obtain mice homozygous for ChATIRES-Cre and the wild-type allele at the Cdh23 locus (ChATCre,Cdh23WT). In the new line, auditory brainstem response thresholds were â¼20 dB lower than those in 9 month old ChATIRES-Cre mice at all frequencies tested (4-31.5 kHz). These thresholds were stable throughout the period of testing (3-12 months of age). We then bred ChATCre,Cdh23WT animals with Ai14 reporter mice to confirm the expression pattern of ChATCre. In these mice, tdTomato-labeled cells were observed in all brainstem regions known to contain cholinergic cells. We then stained the tissue with a neuron-specific marker, NeuN, to determine whether Cre expression was limited to neurons. Across several brainstem nuclei (pontomesencephalic tegmentum, motor trigeminal and facial nuclei), 100% of the tdTomato-labeled cells were double-labeled with anti-NeuN (n = 1896 cells), indicating Cre-recombinase was limited to neurons. Almost all of these cells (1867/1896 = 98.5%) also stained with antibodies against ChAT, indicating that reporter label was expressed almost exclusively in cholinergic neurons. Finally, an average 88.7% of the ChAT+ cells in these nuclei were labeled with tdTomato, indicating that the Cre is expressed in a large proportion of the cholinergic cells in these nuclei. We conclude that the backcrossed ChATCre,Cdh23WT mouse line has normal hearing and expresses Cre recombinase almost exclusively in cholinergic neurons. This ChATCre,Cdh23WT mouse line may provide an opportunity to manipulate cholinergic circuits without the confound of accelerated hearing loss associated with the C57BL/6J background. Furthermore, comparison with lines that do show early hearing loss may provide insight into possible cholinergic roles in age-related hearing loss.
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Tronco Encefálico/enzimología , Colina O-Acetiltransferasa/metabolismo , Fibras Colinérgicas/enzimología , Pérdida Auditiva/prevención & control , Audición , Integrasas/metabolismo , Animales , Umbral Auditivo , Tronco Encefálico/fisiopatología , Cadherinas/genética , Colina O-Acetiltransferasa/genética , Cruzamientos Genéticos , Proteínas de Unión al ADN/metabolismo , Potenciales Evocados Auditivos del Tronco Encefálico , Femenino , Técnicas de Sustitución del Gen , Pérdida Auditiva/enzimología , Pérdida Auditiva/genética , Pérdida Auditiva/fisiopatología , Integrasas/genética , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas , Especificidad de la EspecieRESUMEN
Several ganglionic nicotinic acetylcholine receptor (nAChR) types are abundantly expressed in nonneuronal locations, but their functions remain unknown. We found that keratinocyte alpha7 nAChR controls homeostasis and terminal differentiation of epidermal keratinocytes required for formation of the skin barrier. The effects of functional inactivation of alpha7 nAChR on keratinocyte cell cycle progression, differentiation, and apoptosis were studied in cell monolayers treated with alpha-bungarotoxin or antisense oligonucleotides and in the skin of Acra7 homozygous mice lacking alpha7 nAChR channels. Elimination of the alpha7 signaling pathway blocked nicotine-induced influx of 45Ca2+ and also inhibited terminal differentiation of these cells at the transcriptional and/or translational level. On the other hand, inhibition of the alpha7 nAChR pathway favored cell cycle progression. In the epidermis of alpha7-/- mice, the abnormalities in keratinocyte gene expression were associated with phenotypic changes characteristic of delayed epidermal turnover. The lack of alpha7 was associated with up-regulated expression of the alpha3 containing nAChR channels that lack alpha5 subunit, and both homomeric alpha9- and heteromeric alpha9alpha10-made nAChRs. Thus, this study demonstrates that ACh signaling through alpha7 nAChR channels controls late stages of keratinocyte development in the epidermis by regulating expression of the cell cycle progression, apoptosis, and terminal differentiation genes and that these effects are mediated, at least in part, by alterations in transmembrane Ca2+ influx.
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Células Epidérmicas , Queratinocitos/citología , Queratinocitos/fisiología , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Animales , Bungarotoxinas/farmacología , Calcio/metabolismo , Ciclo Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Antagonistas de Aminoácidos Excitadores/farmacología , Expresión Génica/fisiología , Humanos , Ratones , Ratones Noqueados , Oligodesoxirribonucleótidos/farmacología , Transducción de Señal/fisiología , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Modern biologists have at their disposal a large array of techniques used to assess the existence and relative or absolute quantity of any molecule of interest in a sample. However, implementing most of these procedures can be a daunting task for the first time, even in a lab with experienced researchers. Just choosing a protocol to follow can take weeks while all of the nuances are examined and it is determined whether a protocol will (a) give the desired results, (b) result in interpretable and unbiased data, and (c) be amenable to the sample of interest. We detail here a robust procedure for labeling proteins in a complex lysate for the ultimate differential quantification of protein abundance following experimental manipulations. Following a successful outcome of the labeling procedure, the sample is submitted for mass spectrometric analysis, resulting in peptide quantification and protein identification. While we will concentrate on cells in culture, we will point out procedures that can be used for labeling lysates generated from other tissues, along with any minor modifications required for such samples. We will also outline, but not fully document, other strategies used in our lab to label proteins prior to mass spectrometric analysis, and describe under which conditions each procedure may be desirable. What is not covered in this chapter is anything but the most brief introduction to mass spectrometry (instrumentation, theory, etc.), nor do we attempt to cover much in the way of software used for post hoc analysis. These two topics are dependant upon one's resources, and where applicable, one's collaborators. We strongly encourage the reader to seek out expert advice on topics not covered here.
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Espectrometría de Masas/métodos , Proteínas/análisis , Proteómica/métodos , Animales , Línea Celular , Cóclea/citología , Cóclea/metabolismo , Oído Interno/citología , Oído Interno/metabolismo , Humanos , Marcaje Isotópico/métodos , Espectrometría de Masas/instrumentación , Proteómica/instrumentaciónRESUMEN
Alterations in bone strength and structure were found in knockout (KO) mouse strains with deletion of several acetylcholine receptors. Interestingly, the expression of the nicotinic acetylcholine receptors (nAChR) subunit α10 was down-regulated in osteogenic differentiated mesenchymal stem cells of patients with osteoporosis whereas the expression of subunit α9 was not altered. Since nAChR subunits α9 and α10 are often combined in a functional receptor, we analyzed here the bone of adult female KO mice with single deletion of either nAChR alpha9 (α9KO) or alpha10 (α10KO). Biomechanical testing showed a significant decrease of bending stiffness and maximal breaking force in α9KO compared to their corresponding wild type mice. Furthermore, an increase in trabecular pattern factor (Tb.Pf) and structure model index (SMI) was detected by µCT in α9KO indicating reduced bone mass. On the mRNA level a decrease of Collagen 1α1 and Connexin-43 was measured by real-time RT-PCR in α9KO while no alteration of osteoclast markers was detected in either mouse strain. Using electron microcopy we observed an increase in the number of osteocytes that showed signs of degeneration and cell death in the α9KO compared to their wild type mice, while α10KO showed no differences. In conclusion, we demonstrate alterations in bone strength, structure and bio-marker expression in α9KO mice which imply the induction of osteocyte degeneration. Thus, our data suggest that nAChR containing the α9 subunit might be involved in the homeostasis of osteocytes and therefore in bone mass regulation.
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Huesos/anatomía & histología , Eliminación de Gen , Receptores Nicotínicos/genética , Fosfatasa Alcalina/metabolismo , Animales , Animales Recién Nacidos , Huesos/fisiología , Hueso Esponjoso/anatomía & histología , Hueso Cortical/anatomía & histología , Femenino , Fémur/anatomía & histología , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteocitos/metabolismo , Osteocitos/ultraestructura , Receptores Nicotínicos/deficienciaRESUMEN
The α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits are likely to be the evolutionary precursors to the entire cys-loop superfamily of ligand-gated ion channels, which includes acetylcholine, GABA, glycine and serotonin ionotropic receptors. nAChRs containing α9 and α10 subunits are found in the inner ear, dorsal root ganglia and many non-excitable tissues, but their expression in the central nervous system has not been definitely demonstrated. Here we show the presence of both α9 and α10 nAChR subunits in the mouse brain by RT-PCR and immunochemical approaches with a range of nAChR subunit-selective antibodies, which selectivity was demonstrated in the brain preparations of α7-/-, α9-/- and α10-/- mice. The α9 and α10 RNA transcripts were found in medulla oblongata (MO), cerebellum, midbrain (MB), thalamus and putamen (TP), somatosensory cortex (SC), frontal cortex (FC) and hippocampus. High α9-selective signal in ELISA was observed in the FC, SC, MO, TP and hippocampus and α10-selective signal was the highest in MO and FC. The α9 and α10 proteins were found in the brain mitochondria, while their presence on the plasma membrane has not been definitely confirmed The α7-, α9- and α10-selective antibodies stained mainly neurons and hypertrophied astrocytes, but not microglia. The α9- and α10-positive cells formed ordered structures or zones in cerebellum and superior olive (SO) and were randomly distributed among α7-positive cells in the FC; they were found in CA1, CA3 and CA4, but not in CA2 region of the hippocampus. The α9 and α10 subunits were up-regulated in α7-/- mice and both α7 and α9 subunits were down-regulated in α10-/- mice. We conclude that α9 and α10 nAChR subunits are expressed in distinct neurons of the mouse brain and in the brain mitochondria and are compensatory up-regulated in the absence of α7 subunits.