Various strategies have been developed recently for imparting light sensitivity onto normally insensitive cells. These include expression of natural photosensitive proteins, photolysis of caged agonists of native cell surface receptors and photoswitching of isomerizable tethered ligands that act on specially engineered ion channels and receptor targets. The development of chemical tools for optically stimulating or inhibiting signaling proteins has particular relevance for the nervous system, where precise, noninvasive control is an experimental and medical necessity.
"For example, phosphorylation of Tyr in tyrosine kinases activates proteins for signal transduction ; 4-hydroxylation of Pro in collagen is essential for the formation of collagen triple helix ; and palmitoylation of Cys localizes proteins on the plasma membrane . Therefore, the post-translational addition of new functionalities to proteins can help to investigate cellular events and regulate cell functions   . Various synthesized molecules that are selectively conjugated with proteins of interest have been developed , in addition to classical reagents that are non-specifically conjugated with functional moieties (-NH2, -COOH, -SH) of proteins. "
"In parallel to the generation of optogenetic tools derived by intrinsically light sensitive proteins, an alternative approach to achieve optical control of neuronal activity has been developed. This approach lies in genetically engineering existing target proteins, channels or receptors, and binding them in vivo to an exogenous chemical photoswitch (Kramer et al., 2005; Gorostiza and Isacoff, 2007; Fortin et al., 2008; Gorostiza and Isacoff, 2008a; Isacoff and Smith, 2009; Kramer et al., 2009). The core of the used phostoswitch consists of an azobenzene functional group that isomerizes when illuminated with UV and green light. "
[Show abstract][Hide abstract] ABSTRACT: Zebrafish became a model of choice for neurobiology because of the transparency of its brain and because of its amenability to genetic manipulation. In particular, at early stages of development the intact larva is an ideal system to apply optical techniques for deep imaging in the nervous system, as well as genetically encoded tools for targeting subsets of neurons and monitoring and manipulating their activity. For these applications,new genetically encoded optical tools, fluorescent sensors, and light-gated channels have been generated,creating the field of \optogenetics." It is now possible to monitor and control neuronal activity with minimal perturbation and unprecedented spatio-temporal resolution.We describe here the main achievements that have occurred in the last decade in imaging and manipulating neuronal activity in intact zebrafish larvae. We provide also examples of functional dissection of neuronal circuits achieved with the applications of these techniques in the visual and locomotor systems.
"First, the caged neurotransmitter must be continually applied, a task difficult to do outside of a specimen chamber. A recent alternative, single neurons expressing genetically encoded light-sensitive ion channels—for example, channelrhodopsin—might extend the ability of AOD-based multisite scanning to investigate this facet of neurophysiology in vivo     . Somewhat implicit in our discussion, we have focused on applying photolytic stimulation techniques to studying the physiology of a single neuron. "
[Show abstract][Hide abstract] ABSTRACT: To study the complex synaptic interactions underpinning dendritic information processing in single neurons, experimenters require methods to mimic presynaptic neurotransmitter release at multiple sites with no physiological damage. We show that laser scanning systems built around large-aperture acousto-optic deflectors and high numerical aperture objective lenses provide the sub-millisecond, sub-micron precision necessary to achieve physiological, exogenous synaptic stimulation. Our laser scanning systems can produce the sophisticated spatio-temporal patterns of synaptic input that are necessary to investigate single-neuron dendritic physiology.
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