ReviewStimulating neurons with light
Introduction
Experimental investigations into neuronal function require the ability to manipulate neuronal activity. In most cases, this has been accomplished by electrically stimulating neurons, using electrodes inserted into the tissue of interest. This method has a long tradition going back to Galvani's electrical stimulation of frog nerves. Although very useful, using electrodes to stimulate is cumbersome, provides only crude spatial resolution, requires mechanical stability of the electrodes, does not allow sequential stimulation at multiple closely spaced sites, and can result in substantial mechanical damage to the tissue. In addition, electrical stimulation activates not only neurons with somata at the stimulation site, but also axons of passage; stimulation at multiple sites requires implantation of multiple electrodes, which induces extensive tissue damage.
All of these problems can be solved by stimulating neurons with light. Light can be easily focused with fine resolution, it can be delivered quickly, and it is relatively harmless. It is not surprising then, that neuroscientists have devoted considerable effort to the development of optical stimulation methods and, in particular, to the use of optical methods to ‘uncage’ glutamate, because this neurotransmitter is able to excite most mammalian neurons. The basic approach is to convert inactive ‘caged’ glutamate to active glutamate with ultraviolet light. This method has been used to mimic synaptic input and to map the glutamate sensitivity of the dendritic arbors of single cells (Fig. 1b). Glutamate uncaging can also be used to generate action potentials with fine spatial resolution in small populations of neurons in brain slices; by combining this method with intracellular recording, it is possible to map the locations of neurons connected to a single cell (Fig. 1a). Connectivity can also be investigated by direct photoactivation of action potentials in neurons. Finally, neurons can be made sensitive to light by using genetic methods to express photosensitive proteins. Here, we review the use of both single-photon and two-photon optical methods to stimulate neurons in order to unravel neural circuits, mimic synaptic connections, and investigate glutamate sensitivity.
Section snippets
Early development of optical stimulation methods
The invention of lasers enabled the development of the first optical stimulation methods. In 1971, Fork [1] focused laser light on Aplysia abdominal ganglion neurons and reported that the cells were depolarized to action potential threshold by the illumination. Although the mechanism of the depolarization was not characterized, this effect was reversible and the neuron returned to a resting membrane potential. In this pioneering study, Fork proposed the systematic use of optical methods to
Excitation with caged glutamate — single-photon
During the last decade, investigators have developed light-sensitive bioactive compounds, typically referred to as caged compounds [3] (see also [4]). These compounds are molecules that are rendered inactive by the addition of chemical groups, typically nitrobenzyl groups (Fig. 1a), which are broken up by the absorption of light. Because virtually every neuron in the mammalian central nervous system can be stimulated by glutamate, caged glutamate is as an ideal compound for conferring light
Two-photon excitation
A problem with optical stimulation methods that use traditional (one-photon) light sources is that the light is scattered by living tissue and the distribution of energy, and therefore the uncaging region, is not very precise. For example, the light from a tightly focused, diffraction limited (0.2 μm) spot at the surface of a brain slice diffuses in the slice such that the lateral spread will become comparable to the depth beneath the surface (D Kleinfeld, personal communication). Even though
Genetic methods for photostimulation
Finally, another novel approach for increasing the sensitivity of neurons to light is to express a genetically engineered photoactivatable sensor in them [33••]. Zemelman et al. [33••] coexpressed arrestin, rhodopsin and a subunit of a G-protein in cultured hippocampal neurons, to artificially recreate the Drosophila phototransduction cascade and sensitize neurons to light. The depolarization of the cells occurs, presumably, through the opening of cation channels by a poorly understood
Conclusions and future directions
Overall, we are very optimistic about the future use of optical stimulation methods. In the last few years, the relatively few studies performed with caged glutamate have already demonstrated that these can be powerful methods to probe dendritic function and to map circuits. In addition, we feel that the ‘optical revolution’ is only starting and novel optical methods using either novel caging groups, genetically based probes, or two-photon excitation, as well as the combination of optical
Acknowledgements
We thank the National Institutes of Health for the support of both laboratories and C Ellis Davies for comments.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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