RESUMEN
Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration.
Asunto(s)
Encéfalo/metabolismo , Neuronas/metabolismo , Neurotransmisores/química , Sinaptotagmina I/genética , Sinaptotagminas/genética , Animales , Calcio/química , Calcio/metabolismo , Vesículas de Núcleo Denso/genética , Vesículas de Núcleo Denso/metabolismo , Regulación de la Expresión Génica/genética , Hipocampo/metabolismo , Humanos , Ratones , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/metabolismo , Neuronas/patología , Neuropéptidos/química , Neuropéptidos/metabolismo , Neurotransmisores/metabolismoRESUMEN
Neuropeptides are essential signaling molecules secreted by dense-core vesicles (DCVs). They contribute to information processing in the brain, controlling a variety of physiological conditions. Defective neuropeptide signaling is implicated in several psychiatric disorders. Here, we provide a protocol for the quantitative analysis of DCV fusion events in rodent neurons using pH-sensitive DCV fusion probes and custom-written analysis algorithms. This method can be used to study DCV fusion mechanisms and is easily adapted to investigate fusion principles of other secretory organelles. For complete details on the use and execution of this protocol, please refer to Persoon et al. (2019).