Rapid neurotransmitter uncaging in spatially defined patterns

Shy Shoham, Daniel H O'Connor, Dmitry V Sarkisov & Samuel S-H Wang

Supplementary Fig. 1 (pdf 84K)
Axial and lateral resolution depends on depth in brain slices.

Supplementary Fig. 2 (pdf 116K)
Patterned uncaging and fluorescence measurement of intracellular calcium signals.

Supplementary Video 1 (mov 1.8M)
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).

Supplementary Video 2 (mov 1.7M)
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 M double-caged IP₃ and 300 M 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.

Supplementary Data 1 (pdf 28K)
Axial resolution in brain slices.

Supplementary Data 2 (pdf 28K)
Calcium release in response to focal uncaging.

Supplementary Note (pdf 272K)
Optical system description and plan.

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