Calcium Imaging Techniques In Vitro to Explore the Role of Dendrites in Signaling Physiological Action Potential Patterns

Neurons generate cell-type-specific action potential (AP) patterns as a result of the integration of synaptic inputs received from many other neurons. In neocortical pyramidal neurons, this AP output is not only transmitted to many other postsynaptic neurons, but also back-propagates into the dendritic arbor, thus fulfilling a number of important functions. Back-propagating APs provide sufficient depolarization to activate voltage-gated Ca²⁺ channels at least in the proximal dendrites, thereby generating Ca²⁺ influx into the dendrites and spines of these dendrites. This Ca²⁺ influx, in turn, triggers a variety of signaling cascades that are involved in the regulation of neuronal signaling and plasticity. To better understand the role of Ca²⁺ influx in the regulation of these different neuronal functions, it is important to quantify the dendritic Ca²⁺ dynamics with a high spatial and temporal resolution. Here, we describe techniques that have been optimized to measure [Ca²⁺ ] dynamics in neocortical dendrites in response to physiological patterns of APs using two-photon imaging. These physiological AP patterns were previously recorded in vivo in response to a sensory stimulus and then replayed in the same neuron type in vitro using properly timed current injections.