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Biology Articles » Biophysics » Medical Biophysics » Flashy Science: Controlling Neural Function with Light » Figures

Figures
- Flashy Science: Controlling Neural Function with Light

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Figure 1. Potentiation of exogenous glutamate responses at single dendritic spines. A, Diagram of the experimental apparatus. UV light is launched into a quartz multimode fiber and focused in a conjugate focal plane using a lens assembly. The intensity of the light is controlled by a neutral density filter wheel. A dichroic mirror (DM) allows the UV light to be combined with wide-field illumination from a conventional mercury arc (HBO) lamp, after passing through an excitation filter (EX). The normal filter cube contains a second dichroic mirror and the desired emission filter (EM). Mechanical shutters gate the UV and visible light pulses. A CCD camera captures images of the cells. Inset, With this apparatus, UV light for microphotolysis can be directed to a spot (gray circle) near the head of a single dendritic spine, visualized with fluorescent dye loaded into the cell from the whole-cell patch pipette. These spots have a full-width at half-maximal height of B, Pooled data showing the mean ± SEM potentiation of photolytic EPSCs in 13 cells. Averaged photolytic EPSCs 3 min before and 20 min after the pairing are shown above and were elicited from the spine illustrated in A. Modified from Bagal et al. (2005Go).

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Figure 2. Glutamate uncaging at the cerebellar mossy fiber-granule cell synapse using diffraction-limited UV spot. A1, Paired-pulse illumination protocol (each 20 µs duration, insets) achieved by shuttering the UV light from an I328 Coherent laser (Coherent, Santa Clara, CA) with an AOTF. The intensity of light, and thus the amount of glutamate uncaged, was varied on pulse 1 by altering the voltage applied to the AOTF. For each trace, the second pulse amplitude was set to match the largest of pulse 1. A2, AMPA receptor-mediated currents (averages of 3 trials) recorded under voltage clamp from the granule cell (GC) soma evoked by uncaging locally perfused MNI-glutamate (10 mM) using a diffraction-limited UV spot using the protocol in A1. The colors of the current traces correspond to those for pulse 1 (A1 inset). B, Simulation of the concentration of MNI-glutamate and free glutamate within the diffraction-limited illumination volume for pulse 1 as in A on a linear (left) and logarithmic (right) timescale. The concentrations were calculated using a three-dimensional diffusion model of the mossy fiber-granule cells synapse (Nielsen etal., 2004Go) and the measured UV point-spread function. The MNI-glutamate concentration recovered to 95% in the 10 ms interpulse interval (D. Digregorio, T. Nielsen, J. Rothman, and R. A. Silver, unpublished observation).

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Figure 3. A system for patterned uncaging and two-photon imaging. A, An ultraviolet uncaging laser beam is projected into the focal plane of a two-photon microscope. IR, Infrared; AOD, acousto-optical deflector; ND, neutral density. B, Patterned activation of cells synaptically connected to a cerebellar Purkinje neuron filled via patch-clamp recording electrode with fluorescent dye. The squares indicate the positions of 20 uncaging locations in the presynaptic granule cell layer. Scale bar, 25 µm. C, Postsynaptic voltage-clamp responses to photolysis of caged glutamate at the colored locations shown in B. Vertical marks indicate times of uncaging. Shown in gray at each location are responses after addition of APV (400 µM) and lidocaine (500 µM). Also shown (colored traces) are single example traces. D, The same uncaging locations as C, activated in current clamp with interlocation intervals of 50 µs. Modified from Shoham et al. (2005Go).

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Figure 4. Dynamics of spontaneous IPSC suppression by photorelease of AEA in cultured hippocampal slices. Left, Chemical scheme showing photorelease of caged AEA. Center, Photorelease of caged AEA, present at 200 µM, by a 100 ms UV laser flash (jagged arrow) transiently suppresses spontaneous IPSCs (downward deflections) recorded under whole-cell voltage clamp from a pyramidal cell at room temperature with ionotropic glutamate receptors blocked. Right, Grouped data showing time course of suppression of inhibition induced by AEA photorelease (17 cells). Modified from Heinbockel et al. (2005Go).

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Figure 5. Stimulation of corneal nerve terminals by two-photon photorelease of vanilloid agonist. Rat cornea was loaded in vivo with the fluorescent Ca2+ indicator Oregon Green 488 BAPTA-1 dextran (10 kDa), as described previously (Gover et al., 2003Go). Confocal fluorescence images of the dissected cornea were acquired with 488 nm excitation on a confocal microscope (LSM-510 NLO; Zeiss, Oberkochen, Germany) fitted with a titanium:sapphire laser (Mira 900-F; Coherent-AMT). The cornea was superfused with 5 µM Nv-VNA in Locke's solution. Three corneal nerve terminals are discernible in the field of view. For two-photon photolysis, the 720 nm output of the titanium:sapphire laser was scanned seven times over a ~5 µm2 area centered at the site marked by the yellow arrow (pixel dwell time, 1.6 µs). Changes in Ca2+ -sensitive fluorescence from the two terminals marked by the red and green arrows are shown in the correspondingly colored traces at right; data are represented as fluorescence change relative to baseline intensity ({Delta}F/F0). The images shown were acquired immediately before (Pre) and 5.05 s after (Post) two-photon excitation and correspond to the points marked by filled symbols in the {Delta}F/F0 traces. The nerve terminal stimulated by vanilloid photorelease showed a marked increase in [Ca2+]i, whereas the adjacent, unstimulated terminal showed no change. Scale bar, 5 µm. (T. D. Gover, J. P.Y. Kao, and D. Weinreich, unpublished observation.)

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Figure 6. Light-activated gene expression. A549 cells were infected with adenoviruses Ad-VgRXR (bearing constructs for the ecdysone and RXR coreceptors) and AdEGIKir2.1 [bearing genes for enhanced GFP (EGFP) and Kir2.1 K+ channel with an intervening internal ribosomal entry site, under ecdysone promoter control]. Twenty hours after infection, cultures were subjected to NSE uncaging, and fluorescence imaging and electrophysiological measurements were undertaken 24 h later. Cells stimulated by NSE photorelease expressed EGFP reporter abundantly (top right image), as evidenced by the bright green fluorescence. Expression of the exogenous K+ channel was also high, as shown by the massive outward currents measured under whole-cell voltage clamp. In contrast, control cells expressed neither EGFP reporter (top left image) nor exogenous K+ channels. A light image (bottom left) and merged light and EGFP image (bottom right) are also shown. From a holding potential of -45 mV, outward currents were evoked by a series of voltage steps to -15 to +25 mV in 5 mV increments (D. A. Freilich, J. Ni, D. C. Johns, T. H. V. Moreira, A. Garzino-Demo, and J. P. Y. Kao, unpublished observation).

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Figure 7. Photoinactivation of Syt I impairs endocytosis after normal vesicle fusion at the Drosophila neuromuscular junction. A, Schematic of experimental protocol and results. Nerve stimulation causes exocytosis, but no endocytosis, at the shibirets restrictive temperature in both conditions. Synaptic vesicle proteins and membrane are therefore trapped at the plasma membrane. When returned to the permissive temperature, control synapses endocytose synaptic vesicles, as indicated by synapto-pHluorin fluorescence decay (Control). At synapses in which Syt I has been photoinactivated (FlAsH), there is no synapto-pHluorin fluorescence decay, indicating that endocytosis is blocked. B, Images demonstrating synapto-pHluorin intensity changes at the neuromuscular junction after the shift from the restrictive to the permissive temperature. Time indicates minutes at the permissive temperature. Modified from Poskanzer et al. (2003Go).

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Figure 8. Design and use of light-activated SPARK channels. A, The channels contain a synthetic photoswitch molecule (MAL-AZO-QA; top) that changes length from 17Å (trans) to 10Å (cis) during exposure to light. Covalent attachment of the molecule to the outside of a Shaker K+ channel (bottom) allows the pore-blocking QA group (blue ball) to be retracted or advanced into the pore with different wavelengths of light. B, Rapid control of SPARK channels with light. Activation of the channels with 380 nm light (violet bars) and deactivation with 500 nm light (green bars). Data are from a membrane patch excised from a Xenopus oocyte expressing the H-SPARK channel. C, Expression of the H-SPARK channel in cultured hippocampal pyramidal neurons allows 380 nm light (violet bars) to suppress spontaneous action potential firing and 500 nm light (green bars) to restore activity. Modified from Banghart et al. (2004Go).

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