Uncaging and Photochemistry

Photolabile “caged” compounds are biological signaling inactive molecules with a photoactivatable group. When these compounds absorb photon(s), the caged group can be cleaved and the active biological signaling molecule is released at the site of action. The photochemical reaction can be very fast, with release of the active species often complete within less than a millisecond.

Caged substances range from ions, second messengers and amino acids to fluorescent dyes. A wide range of bioactive molecules such as second messengers or neurotransmitters are now available with conjugated caging groups. These caging groups render the molecule inert until the cage is opened by photolysis. Using this technique it is possible to precisely control in space and time the application of an experimentally applied signal molecule. This technique is providing new avenues of understanding into neurological disorders and drug delivery methods.

Functional mapping of neurotransmitter receptors requires rapid and localized application of transmitter. The usefulness of caged glutamate for this purpose has been limited, because photolysis by unfocused light above and below the target cell limits depth resolution. This problem is eliminated by using a double-caged glutamate that requires absorption of two photons for conversion to active glutamate, resulting in a substantial improvement in spatial resolution over conventional caged glutamate. Thus, the two-photon uncaging offers a general, clean and economical way for spatially localized photolysis of caged compounds.

With caged compounds it is for example possible to investigate second messengers independent of preceding signal transduction chain elements. Two-photon uncaging has also many potential applications to the cardiovascular system. The temporal and three-dimensional spatial resolutions of two-photon uncaging are adequate for mediating a wide range of intra- and intercellular events.

The availability of caged second messengers (e.g. IP₃, cGMP, etc.) and neurotransmitters (e.g. glutamate) can be exploited toward a better understanding of functional compartmentalization as well as distribution and sensitivity of agonist-activated ion channels in the cardiovascular system, respectively. Toward this end, new cages with larger two-photon absorption action cross sections and with a greater variety of active species need to be developed to further enhance this unique and powerful tool.

Calcium Uncaging

The most important chemical messenger that cells use for signaling is ionized calcium (Ca²⁺)¹. Caged calcium molecules are well-defined 3-dimensional structures, as a result of high affinity coordination of Ca²⁺ by photolabile chelators. Chromophore excitation leads to photolysis of a covalent bond, liberating the caged chemical messenger². Highly localized, fast Ca²⁺ transients have been shown to play a critical role in control of a variety of cells or cellular functions, including muscle contraction, secretion of neurotransmitters and hormones, gene transcription, synaptic plasticity, fertilization, movement of cells (nonmuscle motility) and wound healing, cell death, gating of ion channels, and the activity of kinases and phosphatases².

Although the lateral dimension of a tightly focused UV-laser beam in the plane of focus can be in the submicron range, the axial resolution is comparatively poor. One way to perform photolysis on a much smaller spatial scale and at the same time maintain the high temporal resolution of conventional photolysis is two-photon excitation-uncaging. The latter technique is useful for modern neurobiology, as the IR light (700-1000 nm) used for 2-photon imaging is scattered less by brain tissue than the visible light used for confocal microscopy. The second significant advantage of 2-photon imaging over confocal microscopy is that the excited singlet state is only created in a small volume due to the nonlinear nature of the two-photon absorption process. The minute size of the excitation volume eliminates the need for the pinhole that is required for confocal microscopy.

At the time of writing, three selective calcium cages have been reported: azid-1, DMNPE-4, and NDBF. azid-1 is a photosensitive derivative of fura-2 designed initially for photocross-linking the Ca²⁺ dye to cellular proteins. Illumination of this probe preserves the BAPTA coordination sphere but not the fluorescence properties of the fura dye, as an amidoxime photoproduct is generated³. This new electron-withdrawing substituent reduces the molecule’s affinity for Ca²⁺ more efficiently than any of the nitr Ca²⁺ cages. Combined with its superior photochemical properties², this makes azid-1 more (photo)chemically efficient than any nitr cage at releasing Ca²⁺.

DMNPE-4 was the second two-photon calcium cage made⁴. This molecule attempted to combine the choicest properties of DM-nitrophen and NP-EGTA in one photosensitive chelator. This ideal was not completely achievable, as EGTA has a lower affinity for Ca²⁺ than EDTA2.

NDBF (nitrodibenzofuran) is a generic caging chromophore for two-photon photolysis introduced in 2006. The Ca²⁺ cage made with this chromophore, NDBF-EGTA, is almost as photosensitive as azid-1, but is much more chemically efficient at releasing the caged Ca²⁺, due to the large change in affinity upon irradiation². Graham Ellis-Davies’ lab has found that two-photon photolysis of DMNPE-4, loaded into astrocytes in a living mouse using the AM ester technique, can produce cell-wide Ca²⁺ signals, as reported by fluo-4 fluorescence². Uncaging was accomplished using a mode-lock Ti:sapphire laser (at 720 nm), and imaging used a second laser (at 860 nm). The lasers were independently controlled by two sets of x/y galvanometers using a Prairie Technologies Ultima scan head.

Development of other compounds for two-photon uncaging will advance analyses of molecular processes in neurons at the single synapse level.

Glutamate Uncaging

Localized glutamate release can be imitated in vitro by using caged glutamate, an inert derivative of glutamate that can uncage glutamate when it is optically activated by absorption of one or two photons. Suitable compounds are rare. However, it has been shown that MNI-caged glutamate provides a sufficiently large two-photon cross-section (0.06 GM) so that glutamate can be photoreleased in synaptic clefts to mimic unitary synaptic currents^5,6,7. Stimuli can be repeated sufficiently often to allow fluctuation analysis of glutamate receptors at single synapses. MNI-caged glutamate has facilitated new types of experiments. The number and properties of glutamate receptors in single postsynaptic densities have been measured ^5,6,7.

Two-photon excitation is the technique to be used in studying the role of specific components involved in synaptic signaling, such as NMDA (Nmethyl-d-aspartate) receptors. Presynaptic neurons transform their electrical activity into the release of packets of neurotransmitter molecules which will target cells across the synapse. NMDA receptors are protein complexes located on the postsynaptic side of a synapse that transduces pulses of the neurotransmitter glutamate into excitatory electrical potentials in the postsynaptic cell. They are thought to be critical for neuronal plasticity because of their additional dependence of the postsynaptic voltage and their large permeability to calcium, which leads to an increase in intracellular calcium in the post-synaptic cell when NMDA receptors are activated.

The marriage of two-photon excitation glutamate uncaging with two-photon excitation calcium imaging has allowed the measurement of calcium influx through NMDA receptors and calcium-permeable AMPA receptors in single spines ^5,6,7 and also it allowed researchers to understand the role of calcium signaling during NMDA receptor activation in hipppocampal slices⁸.

Two-photon excitation glutamate uncaging has also been used to induce synaptic plasticity in individual spines⁹. In addition, stimulation of multiple spines can be used to study the mechanisms of synaptic integration in dendrites¹⁰. Jai-Yoon Sul et al.¹¹ showed that glutamate released from astrocytes does not mediate astrocyte connectivity in the hippocampus.

References

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2. Graham C. R. Ellis-Davies, Neurobiology with Caged Calcium, Chem. Rev. 2008, 108, 1603–1613.
3. Adams, S. R.; Lev-Ram, V.; Tsien, R. Y. Chem. Biol. 1997, 4, 867.
4. Ellis-Davies, G. C. R. Tetrahedron Lett. 1998, 39, 4.
5. Carter, A.G., and Sabatini, B.L. (2004). State-dependent calcium signaling in dendritic spines of striatal medium spiny neurons. Neuron 44, 483–493.
6. Matsuzaki, M., Ellis-Davies, G.C., Nemoto, T., Miyashita, Y., Iino, M., and Kasai, H. (2001). Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat. Neurosci. 4, 1086–1092.
7. Sobczyk, A., Scheuss, V., and Svoboda, K. (2005). NMDA receptor subunit-dependent [Ca2+] signaling in individual hippocampal dendritic spines. J. Neurosci. 25, 6037–6046.
8. Noguchi, J., Matsuzaki, M., Ellis-Davies, G.C.R. and Kasai, H. (2005) Spine-neck geometry determines NMDA receptor-dependent Ca2+ signaling in dendrites. Neuron 46, 609-622.
9. Matsuzaki, M., Honkura, N., Ellis-Davies, G.C., and Kasai, H. (2004). Structural basis of long-term potentiation in single dendritic spines. Nature 429, 761–766.
10. Gasparini, S., and Magee, J.C. (2006). State-dependent dendritic computation in hippocampal CA1 pyramidal neurons. J. Neurosci. 26, 2088–2100.
11. Jai-Yoon Sul, George Orosz, Richard S. Givens, and Philip G. Haydon, Astrocytic Connectivity in the Hippocampus, Neuron Glia Biol. 2004 February ; 1(1): 3–11.