Most of our knowledge is based on painstaking patch-clamp recordings from individual dendrites or simulating synaptic activity by local glutamate uncaging ( Antic et al., 2010 Major et al., 2013).
As the dendritic arbor of even a single cortical neuron spans several square millimeters and dendritic calcium spikes are stochastic, capturing these rare events with laser scanning microscopy is technically challenging. These non-linear processes may endow dendrites with the capability to serve as computational subunits ( Polsky et al., 2004) and gate the output of cortical neurons during perception of sensory stimuli ( Takahashi et al., 2020). In addition to NMDA receptors, voltage-gated calcium channels also contribute to local dendritic depolarization and calcium spikes ( Losonczy and Magee, 2006 Remy and Spruston, 2007 Takahashi et al., 2020). When a cluster of excitatory synapses is simultaneously activated on a basal dendrite of a pyramidal neuron, the combined depolarization triggers a local NMDA spike ( Schiller et al., 2000). As the read-out of fluorescence can be performed several hours after photoconversion, TubuTag will help investigating dendritic signal integration and calcium homeostasis in large populations of neurons. Photoconversion was strongest in the most distal parts of the apical dendrite, suggesting a gradient in the amplitude of dendritic calcium signals. We used a custom two-photon microscope with a large field of view to image pyramidal neurons in CA1 at subcellular resolution. TubuTag integrates cytoplasmic calcium signals by irreversible photoconversion from green to red fluorescence when illuminated with violet light. To investigate calcium signaling in dendrites, we introduce TubuTag, a genetically encoded ratiometric calcium sensor anchored to the cytoskeleton. Under specific conditions, large neurons produce calcium spikes that are locally restricted to a dendritic section. In addition, they are capable of releasing calcium from intracellular stores. Dendrites contain a rich diversity of ligand- and voltage-activated ion channels as well as metabotropic receptors. The extensive dendritic arbor of neurons is thought to be actively involved in the processing of information. 2Rapp OptoElectronic GmbH, Wedel, Germany.1Institute for Synaptic Physiology, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.Alberto Perez-Alvarez 1,2*, Florian Huhn 2, Céline D.
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