Abstract
Determining how neurons transform synaptic input and encode information in action potential (AP) firing output is required for understanding dendritic integration, neural transforms and encoding. Limitations in the speed of imaging 3D volumes of brain encompassing complex dendritic arbors in vivo using conventional galvanometer mirror-based laser-scanning microscopy has hampered fully capturing fluorescent sensors of activity throughout an individual neuron's entire complement of synaptic inputs and somatic APs. To address this problem, we have developed a two-photon microscope that achieves high-speed scanning by employing inertia-free acousto-optic deflectors (AODs) for laser beam positioning, enabling random-access sampling of hundreds to thousands of points-of-interest restricted to a predetermined neuronal structure, avoiding wasted scanning of surrounding extracellular tissue. This system is capable of comprehensive imaging of the activity of single neurons within the intact and awake vertebrate brain. Here, we demonstrate imaging of tectal neurons within the brains of albino Xenopus laevis tadpoles labeled using single-cell electroporation for expression of a red space-filling fluorophore to determine dendritic arbor morphology, and either the calcium sensor jGCaMP7s or the glutamate sensor iGluSnFR as indicators of neural activity. Using discrete, point-of-interest scanning we achieve sampling rates of 3 Hz for saturation sampling of entire arbors at 2 μm resolution, 6 Hz for sequentially sampling 3 volumes encompassing the dendritic arbor and soma, and 200–250 Hz for scanning individual planes through the dendritic arbor. This system allows investigations of sensory-evoked information input-output relationships of neurons within the intact and awake brain.
Highlights
Neurons within brain circuits receive inputs from hundreds to thousands of upstream neurons at synaptic contacts distributed across their elaborate dendritic arbors
Information received in synaptic currents is integrated within dendrites and transformed into neural output encoded in action potential (AP) firing at the soma, which is transmitted via the axon to downstream targets
Calcium transients can be imaged as a proxy for neural activity using calcium-sensitive fluorescent dyes or genetically-encoded calcium indicators (GECIs) (Nakai et al, 2001; Rose et al, 2014), such as the family of engineered GCaMPs (Chen et al, 2013)
Summary
Neurons within brain circuits receive inputs from hundreds to thousands of upstream neurons at synaptic contacts distributed across their elaborate dendritic arbors. Our understanding of neural information processing and encoding is limited due to technical challenges in simultaneously tracking activity throughout complex three dimensional (3D) dendritic arbor structures and the soma, which is needed to establish full input-output relationships. Approaches to this problem have been aided by the development of fluorescent sensors of neural activity, those capable of detecting rapid changes in intracellular free calcium. The genetically encoded protein iGluSnFR can be expressed on the surface of neurons and increases fluorescence upon binding to pre-synaptically-released glutamate (Marvin et al, 2013, 2018)
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