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Rapid neurotransmitter uncaging in spatially defined patterns

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Abstract

Light-sensitive 'caged' molecules provide a means of rapidly and noninvasively manipulating biochemical signals with submicron spatial resolution. Here we describe a new optical system for rapid uncaging in arbitrary patterns to emulate complex neural activity. This system uses TeO2 acousto-optical deflectors to steer an ultraviolet beam rapidly and can uncage at over 20,000 locations per second. The uncaging beam is projected into the focal plane of a two-photon microscope, allowing us to combine patterned uncaging with imaging and electrophysiology. By photolyzing caged neurotransmitter in brain slices we can generate precise, complex activity patterns for dendritic integration. The method can also be used to activate many presynaptic neurons at once. Patterned uncaging opens new vistas in the study of signal integration and plasticity in neuronal circuits and other biological systems.

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Figure 1: A system for patterned uncaging and two-photon imaging.
Figure 2: Using patterned uncaging to emulate dendritic integration.
Figure 3: Local amplification of glutamate responses in pyramidal neuron dendrites.
Figure 4: Repeatability of responses to patterned activation of multiple dendrites.
Figure 5: Patterned activation of presynaptic cells.

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Change history

  • 28 November 2005

    In the version of this article initially published online, Supplementary Videos 1 and 2 were in AVI format. These videos have been replaced with new versions in MOV format.

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Acknowledgements

This work was supported by grants from the Princeton Center for Photonics and Optoelectronic Materials, the US National Institutes of Health, the National Science Foundation (NSF), and the W.M. Keck Foundation to S.S.-H.W., a Lewis Thomas fellowship to S.S., an NSF Graduate Research Fellowship to D.H.O. and a Burroughs-Wellcome Interfaces of Science fellowship to D.V.S. We thank J. Soos from Brimrose Corp. for designing the AOD device and K. Visscher, M. McDonald, J. Puchalla, G. Wittenberg, G. Major and T. Adelman for advice and discussion.

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Correspondence to Samuel S-H Wang.

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Supplementary information

Supplementary Fig. 1

Axial and lateral resolution depends on depth in brain slices. (PDF 82 kb)

Supplementary Fig. 2

Patterned uncaging and fluorescence measurement of intracellular calcium signals. (PDF 112 kb)

Supplementary Video 1

Current summation in a pyramidal neuron. In the first video segment, uncaging locations are shown in blue on a two-photon image of a dye-filled CA1 pyramidal neuron. Voltage-clamp current responses to a complex uncaging pattern are highly reproducible when the stimulation pattern is repeated (first three current traces, shown in color). In contrast, the current response is different when a new stimulation pattern with the same mean rate is delivered across the same dendritic locations (fourth current trace, shown in black). In the second video segment, a freely-spiking pyramidal neuron shows highly reproducible voltage responses to identical stimuli (first three voltage traces, shown in color). A different stimulus with the same mean rate gives a different voltage response (fourth voltage trace, shown in black). (MOV 1779 kb)

Supplementary Video 2

IP3 uncaging in a Purkinje neuron. Ca2+ release responses are shown after patterned release of caged IP3 at six locations in a rat cerebellar Purkinje neuron. The Purkinje cell was filled through a patch-pipette with 100 μM double-caged IP3 and 300 μM of the Ca2+-sensitive dye fluo-5F, and imaged with a two-photon microscope at a rate of 32 ms per frame. The first segment of the video shows responses to uncaging at 1-second intervals. The second segment shows responses to uncaging at the same locations at 320-millisecond intervals. (MOV 1735 kb)

Supplementary Data 1

Axial resolution in brain slices. (PDF 24 kb)

Supplementary Data 2

Calcium release in response to focal uncaging. (PDF 25 kb)

Supplementary Note

Optical system description and plan. (PDF 269 kb)

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Shoham, S., O'Connor, D., Sarkisov, D. et al. Rapid neurotransmitter uncaging in spatially defined patterns. Nat Methods 2, 837–843 (2005). https://doi.org/10.1038/nmeth793

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