RESUMO
Dynamic regulation of the expression of surface AMPA receptors (AMPARs) is a key mechanism to modulate synaptic strength and efficacy in the CNS and also to regulate auditory sensitivity. Here we address the role of surface AMPAR expression in excitotoxicity by blocking clathrin-mediated AMPAR endocytosis in auditory neurons. We used a membrane-permeable, dynamin-derived, myristoylated peptide (myr-Dyn) to inhibit surface AMPAR endocytosis induced by glutamate receptor agonists in culture and by noise exposure in vivo. Myr-Dyn infused into the mouse cochlea induced excitotoxic responses to acoustic stimuli that were normally not excitotoxic. These included vacuolization in the nerve terminals and spiral ganglion as well as irreversible auditory brain stem response threshold shifts. In cultured spiral ganglion neuronal cells, blockade of the reduction of surface AMPARs exacerbated neuronal death by incubation with N-methyl-d-aspartate and AMPA. This excitotoxic neuronal death could be prevented by calpeptin, a calpain-specific inhibitor. These results suggest that the reduction of surface AMPAR by endocytosis during excitatory stimulation plays an important role in limiting the excitotoxic damage to the neuron.
Assuntos
Vias Auditivas/fisiologia , Membrana Celular/fisiologia , Receptores de AMPA/metabolismo , Células Receptoras Sensoriais/fisiologia , Estimulação Acústica , Animais , Limiar Auditivo/fisiologia , Tronco Encefálico/fisiologia , Morte Celular/fisiologia , Células Cultivadas , Cóclea/fisiologia , Exocitose/fisiologia , Camundongos , Camundongos Endogâmicos CBA , N-Metilaspartato/toxicidade , Neurotoxinas/toxicidade , Distribuição Aleatória , Receptores de Glutamato/metabolismo , Gânglio Espiral da Cóclea/fisiologia , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/toxicidadeRESUMO
We report a robust regulation of surface AMPA receptors in mouse auditory neurons, both with application of glutamate receptor agonists in cultured neurons and in response to acoustic stimulation in vivo. The reversible reduction of surface AMPA receptors following acoustic stimulation correlated with changes in acoustic sensitivity. Thus we show that AMPA receptor cycling is important for optimizing synaptic transfer at one of the most exacting synapses in the body.
Assuntos
Limiar Auditivo/fisiologia , Regulação da Expressão Gênica/fisiologia , Neurônios Aferentes/metabolismo , Receptores de AMPA/metabolismo , Estimulação Acústica/métodos , Animais , Animais Recém-Nascidos , Limiar Auditivo/efeitos dos fármacos , Relação Dose-Resposta à Radiação , Potenciais Evocados Auditivos/efeitos dos fármacos , Potenciais Evocados Auditivos/efeitos da radiação , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Neurônios Aferentes/efeitos dos fármacos , Gânglio Espiral da Cóclea/citologia , Fatores de TempoRESUMO
We have developed an automated, high-throughput behavioral screening method for detecting hearing defects in zebrafish. Our assay monitors a rapid escape reflex in response to a loud sound. With this approach, 36 adult zebrafish, restrained in visually isolated compartments, can be simultaneously assessed for responsiveness to near-field 400 Hz sinusoidal tone bursts. Automated, objective determinations of responses are achieved with a computer program that obtains images at precise times relative to the acoustic stimulus. Images taken with a CCD video camera before and after stimulus presentation are subtracted to reveal a response to the sound. Up to 108 fish can be screened per hour. Over 6500 fish were tested to validate the reliability of the assay. We found that 1% of these animals displayed hearing deficits. The phenotypes of non-responders were further assessed with radiological analysis for defects in the gross morphology of the auditory system. Nearly all of those showed abnormalities in conductive elements of the auditory system: the swim bladder or Weberian ossicles.