RESUMEN
Simultaneous imaging and manipulation of a genetically defined neuronal population can provide a causal link between its activity and function. Here, we designed a miniaturized microscope (or 'miniscope') that allows fluorescence imaging and optogenetic manipulation at the cellular level in freely behaving animals. This miniscope has an integrated optical connector that accepts any combination of external light sources, allowing flexibility in the choice of sensors and manipulators. Moreover, due to its simple structure and use of open source software, the miniscope is easy to build and modify. Using this miniscope, we demonstrate the optogenetic silencing of hippocampal CA1 neurons using two laser light sources-one stimulating a calcium sensor (i.e., jGCaAMP7c) and the other serving as an optogenetic silencer (i.e., Jaws). This new miniscope can contribute to efforts to determine causal relationships between neuronal network dynamics and animal behavior.
Asunto(s)
Región CA1 Hipocampal/metabolismo , Microscopía/instrumentación , Red Nerviosa/metabolismo , Neuroimagen/métodos , Neuronas/metabolismo , Optogenética/métodos , Animales , Conducta Animal/fisiología , Región CA1 Hipocampal/ultraestructura , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Expresión Génica , Genes Reporteros , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inyecciones Intraventriculares , Luz , Ratones , Microscopía/métodos , Red Nerviosa/ultraestructura , Neuroimagen/instrumentación , Neuronas/ultraestructura , Imagen Óptica/instrumentación , Imagen Óptica/métodos , Optogenética/instrumentación , Rodopsina/genética , Rodopsina/metabolismoRESUMEN
We developed Carignan, a real-time calcium imaging software that can automatically detect activity patterns of neurons. Carignan can activate an external device when synchronized neural activity is detected in calcium imaging obtained by a one-photon (1p) miniscope. Combined with optogenetics, our software enables closed-loop experiments for investigating functions of specific types of neurons in the brain. In addition to making existing pattern detection algorithms run in real-time seamlessly, we developed a new classification module that distinguishes neurons from false-positives using deep learning. We used a combination of convolutional and recurrent neural networks to incorporate both spatial and temporal features in activity patterns. Our method performed better than existing neuron detection methods for false-positive neuron detection in terms of the F1 score. Using Carignan, experimenters can activate or suppress a group of neurons when specific neural activity is observed. Because the system uses a 1p miniscope, it can be used on the brain of a freely-moving animal, making it applicable to a wide range of experimental paradigms.