Fluorescence-based synapse detection enables dense, high-throughput and multichannel analysis of synapse properties and connections in brain tissue. Using fluorogen activating proteins (FAPs) or YFP coupled to a neuroligin tether, we have developed genetically-encoded reagents for fluorescence-labeling of post-synaptic sites. Sparse viral expression of YFP-post or FAP-post in mouse somatosensory (barrel) cortex enables compartment-specific quantitation of synapses across the surface of an individual neuron, as well as cell-type specific presynaptic input assignment. High-resolution, 3D confocal stacks were used for semi-automated, high-throughput assignment of YFP-post and FAP-post synaptic puncta across specific neuron cell-types. Using transgenic mice where specific subtypes of presynaptic inhibitory neurons were fluorescently-labeled with YFP, far red-fluorescence of FAP-post synaptic puncta and dTomato-filled postsynaptic pyramidal cells could be aligned to specific presynaptic partners using tricolor colocalization. Fluorescent post-synaptic puncta properties were evaluated to generate metrics reflecting synapse location, size, shape, and fluorescence intensity that could be used to differentiate synapses from different inhibitory sources. We used these quantitative metrics to evaluate changes in inhibitory inputs after sensory association training for pyramidal neurons in barrel cortex. Genetically-encoded fluorescence-based synaptic labeling reagents provide a powerful approach to enable high-throughput and automated analysis of synapse organization in brain tissue across development, learning, and disease states.
JavaScript jest wyłączony w Twojej przeglądarce internetowej. Włącz go, a następnie odśwież stronę, aby móc w pełni z niej korzystać.