Z-stacks of two single neurons.

<p>(A) Low magnification structural overview (152×152 µm field, M3R, 200 µm depth). (B) Higher magnification image (75×75 µm). (C) Structural images collected at 12 different depths (2 µm depth intervals). (D) Low magnification structural overview (200×200 µm field, M4R, 240 µm depth). (E) Higher magnification image (60×60 µm). (F) Same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013829#pone-0013829-g004" target="_blank">Figure 4C</a>. (A–F) YFP images averaged over 10 frames at 512×512 pixels resolution.</p

Expression of memTNXL in monkey visual cortex over time.

<p>Various time points show different sites with expression of the fluorescent sensor. (A) 148 days post injection (190 µm depth). (B) 154 days post injection (300 µm depth). (C) 297 days post injection (110 µm depth). Labeled neurons vary in size (from 10 to 20 µm). (A–C) YFP images averaged over 10 frames at 512×512 pixels resolution.</p

Directional responses of four selected neurons collected simultaneously (<i>Neurons 4–7</i>, 250 µm depth).

<p>(A) Low magnification structural image (200×200 µm) with the ROIs (white rectangles). (B) High magnification structural image (75×75 µm) of the same site. (A,B) YFP structural images (10 frames averaged). (C) Circular tuning curves (mean and standard error) for the two significant neurons. (D) Circular tuning curves (mean and standard error) for the two non-significant neurons.</p

Two-photon scanning microscope (TPSM) setup for the behaving monkey.

<p>(A) Overview front of TPSM with primate chair in place. Main parts of TPSM are circled in green with magenta arrows indicating path of laser beam to be directed into monkey's chamber (white ellipse on schematic of monkey head). (B) Detail of TPSM from lateral side with motors moving medio-lateral (x-axis, blue arrow) and antero-posterior (y-axis, blue arrow). Scanning mirrors on top of microscope are indicated by green arrow. (C) Detail of TPSM from medial side with micromanipulator allowing precise vertical (z-axis, blue arrow) movement with micrometer precision.</p

Optical chamber with artificial dura for two-photon scanning.

<p>The injections sites are indicated by black dots in V1/V2 for M3R (A) and M4R (B). Olympus Microprobe objective retracted after scanning in M3R (C). Optical chamber is filled with 2% agarose for stability.</p

Functional TPSM of single <i>Neuron 1</i> (M3R, 311 days post injection, 260 µm depth).

<p>(A) Low magnification structural image (200×200 µm) with ROI (white rectangle) submitted to further analysis. (B) High magnification structural image (50×50 µm) with brightly labeled neuron in center. (A,B) YFP images (10 frames averaged). (C) FRET signal plotted as a function of time for all 8 directions (blue and red curves). First panel also shows blank signal (gray curve), onset of fixation (dashed line), and 500 ms baseline signal (gray shaded rectangle) used for normalization (stimulus onset was at 0 ms). (D) Circular tuning curve (mean and standard error).</p

Functional TPSM of single <i>Neuron 3</i> (M4R, 14 days post injection, 300 µm depth).

<p>(A) Low magnification structural image (200×200 µm) with the ROI (white rectangle). (B) High magnification structural image (100×100 µm) with labeled neuron in center. (A,B) YFP structural images (10 frames averaged). (C) FRET signal plotted as a function of time for all 8 directions (blue and red curves). Conventions otherwise as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013829#pone-0013829-g006" target="_blank">Figure 6A–6C</a>. (D) Circular tuning curve (mean and standard error).</p

A Red-Shifted, Fast-Relaxing Azobenzene Photoswitch for Visible Light Control of an Ionotropic Glutamate Receptor

The use of azobenzene photoswitches has become a dependable method for rapid and exact modulation of biological processes and material science systems. The requirement of ultraviolet light for azobenzene isomerization is not ideal for biological systems due to poor tissue penetration and potentially damaging effects. While modified azobenzene cores with a red-shifted <i>cis</i>-to-<i>trans</i> isomerization have been previously described, they have not yet been incorporated into a powerful method to control protein function: the photoswitchable tethered ligand (PTL) approach. We report the synthesis and characterization of a red-shifted PTL, L-MAG0₄₆₀, for the light-gated ionotropic glutamate receptor LiGluR. In cultured mammalian cells, the LiGluR+L-MAG0₄₆₀ system is activated rapidly by illumination with 400–520 nm light to generate a large ionic current. The current rapidly turns off in the dark as the PTL relaxes thermally back to the <i>trans</i> configuration. The visible light excitation and single-wavelength behavior considerably simplify use and should improve utilization in tissue

Long-term integration of the transplanted neurons.

<p>Confocal microscopy images extracted from xyz-tile acquisitions showing GFP+ neuron implantation throughout the hippocampus 24 weeks post-transplantation. <b>a</b>) shows beads at the injection site carrying GFP+ neurons which are projecting their processes in the host hippocampus, <b>b</b>) shows neurons in Or -oriens layer of the hippocampus sending out processes through the radiatum layer, and <b>c</b>) shows cells in the stratum lucidum of the CA3. Brain slices were stained with CD11b a marker for microglia cells (<b>d</b>), and CD68 a marker for macrophages (<b>e</b>). Confocal microscopy images 4 xy frames extracted from xyz-tile acquisitions showing glass bead cluster were projected in z. Increase in microglia cells and macrophages was associated with the presence of GFP+ cells without processes (arrows). Beads without cells were free of microglia and macrophages, suggesting that these cells were there to clear non-integrated GFP+ neurons. All scale bars  =  100 µm.</p

Influence of the injection position on the distribution of the implanted GFP-neurons.

<p><b>a)</b> Schematic representation of the different hippocampus sub-regions. CA1-field SLu- stratum lucidum, Rad- radiatum layer of the hippocampus, PoDG- polymorph layer of the dentate gyrus, GrDG- granular layer of the dentate gyrus, MoDG- molecular layer of the dentate gyrus, LMol- lacunosum moleculare layer of the hippocampus, Py - pyramidal cell layer of the hippocampus, and Or -oriens layer of the hippocampus. Confocal microscopy images extracted from xyz-tile acquisitions showing GFP+ neuron implantation through out the hippocampus: <b>b</b>) shows the radiatum layer, <b>c</b>) the stratum lucidum of the CA3, <b>d</b>) part of the dentate gyrus. <b>f</b>) Fraction of the total GFP+ cells found in each region for injections in the CA3 (blue) and in the DG (red). Error bars represent the standard deviations for series of 10 animals. Scale bars  =  100 µm.</p