Journal: Nature Communications

Article Title: Improved methods for marking active neuron populations

doi: 10.1038/s41467-018-06935-2

Figure Lengend Snippet: In vitro characterization of CaMPARI2. a Primary (bottom) and tertiary (top) structures of CaMPARI2. Mutations relative to CaMPARI1_W391F-V398L are shown in red. Two orthogonal views of the same CaMPARI crystal structure (PDB ID 4OY4 [10.2210/pdb4OY4/pdb] are shown. b Absorption (left) and fluorescence (right) excitation (full line) and emission (dotted line) spectra of CaMPARI2. Green and magenta spectra represent the green and red forms of CaMPARI; bright and dark lines represent the calcium-free and calcium-saturated states. c Photoconversion timecourse showing the red fluorescence of CaMPARI1 (black) and CaMPARI2 (red) as a function of exposure to 405 nm light. Left: photoconversion of purified CaMPARI protein in the presence (solid lines) or absence (dashed lines) of calcium. Right: photoconversion of primary rat hippocampal neurons with (solid lines) or without (dashed lines) 80 Hz stimulation. d Fold increase of the red-to-green ratio of CaMPARI variant-expressing neurons after 2 s of photoconversion during different electrical stimulation frequencies relative to no stimulation. Error bars are standard deviation, n = 3, asterisks denote values significantly differing from the corresponding CaMPARI1 value ( p < 0.001, Dunnett’s multiple comparisons test)

Article Snippet: CaMPARI1 (Addgene #604021) or CaMPARI2 (without C-terminal epitope tags) were subcloned into a pAAV vector under the control of a human synapsin1 promoter.

Techniques: In Vitro, Fluorescence, Purification, Variant Assay, Expressing, Standard Deviation

Journal: Nature Communications

Article Title: Improved methods for marking active neuron populations

doi: 10.1038/s41467-018-06935-2

Figure Lengend Snippet: Photophysical properties of CaMPARI1, CaMPARI2, CaMPARI2_F391W, and CaMPARI2_L398T

Article Snippet: CaMPARI1 (Addgene #604021) or CaMPARI2 (without C-terminal epitope tags) were subcloned into a pAAV vector under the control of a human synapsin1 promoter.

Techniques:

Journal: Nature Communications

Article Title: Improved methods for marking active neuron populations

doi: 10.1038/s41467-018-06935-2

Figure Lengend Snippet: Brightness and photoconversion of CaMPARI1 and CaMPARI2. Two-photon images of CaMPARI1 and CaMPARI2_F391W-L398V (no epitope tags) expressed in CA1 neurons of rat hippocampal slice cultures before and after pairing of stimulation (100 bAPs at 100 Hz) with UV light (2 s, 16 mW mm −2 ). Two lasers were simultaneously employed to acquire images of the green (980 nm) and red species (1040 nm) of the CaMPARI variants. A single cell, denoted by a pipette drawing, is patched and stimulated during the UV illumination. Green and red fluorescence is quantified along with the fold R/G in both stimulated (CaMPARI1 n = 5; CaMPARI2 n = 6) and unstimulated neighboring neurons (CaMPARI1 n = 5; CaMPARI2 n = 14) as a function of the number of pairings. Error bars are SEM. Note that fluorescence intensity is normalized to the laser power (see Methods), showing the increased brightness of CaMPARI2. Scale bars are 25 µm

Article Snippet: CaMPARI1 (Addgene #604021) or CaMPARI2 (without C-terminal epitope tags) were subcloned into a pAAV vector under the control of a human synapsin1 promoter.

Techniques: Transferring, Fluorescence