Abstract
The development of two-photon microscopy has revolutionized our understanding of how synapses are formed and how they transform synaptic inputs in dendritic spines—tiny protrusions that cover the dendrites of pyramidal neurons that receive most excitatory synaptic information in the brain. These discoveries have led us to better comprehend the neuronal computations that take place at the level of dendritic spines as well as within neuronal circuits with unprecedented resolution. Here, we describe a method that uses a two-photon (2P) microscope and 2P uncaging of caged neurotransmitters for the activation of single and multiple spines in the dendrites of cortical pyramidal neurons. In addition, we propose a cost-effective description of the components necessary for the construction of a one laser source-2P microscope capable of nearly simultaneous 2P uncaging of neurotransmitters and 2P calcium imaging of the activated spines and nearby dendrites. We provide a brief overview on how the use of these techniques have helped researchers in the last 15 years unravel the function of spines in: (a) information processing; (b) storage; and (c) integration of excitatory synaptic inputs.
Highlights
A major goal in neuroscience is to understand how neurons integrate and store information they receive from their synaptic inputs and, in turn, transmit signals to their postsynaptic targets
2003; Helmchen and Denk, 2005), and of 2P uncaging microscopy (Matsuzaki et al, 2001; Araya et al, 2006a)—where caged-neurotransmitters can be released from its cage only in the focal plane to mimic presynaptic release at single synapses—have allowed us to image and directly probe dendritic spine function in excitatory and inhibitory synaptic input transformations in pyramidal neurons (Matsuzaki et al, 2001; Araya et al, 2006a,b, 2007, 2014; Bloodgood and Sabatini, 2007; Tanaka et al, 2008; Harnett et al, 2012; Chiu et al, 2013; Oh et al, 2016)
We describe a cost-effective description of the components necessary for the construction of a one laser source-2P microscope capable of nearly simultaneous 2P uncaging of neurotransmitters and 2P calcium imaging of the activated spines and nearby dendrites using a single wavelength with low-laser power for calcium imaging and short high-laser power pulses to uncage caged glutamate
Summary
A major goal in neuroscience is to understand how neurons integrate and store information they receive from their synaptic inputs and, in turn, transmit signals to their postsynaptic targets. 2003; Helmchen and Denk, 2005), and of 2P uncaging microscopy (Matsuzaki et al, 2001; Araya et al, 2006a)—where caged-neurotransmitters can be released from its cage only in the focal plane to mimic presynaptic release at single synapses—have allowed us to image and directly probe dendritic spine function in excitatory and inhibitory synaptic input transformations in pyramidal neurons (Matsuzaki et al, 2001; Araya et al, 2006a,b, 2007, 2014; Bloodgood and Sabatini, 2007; Tanaka et al, 2008; Harnett et al, 2012; Chiu et al, 2013; Oh et al, 2016). Probing the function of individual spines was not previously possible using standard electrophysiological techniques
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