Journal: PLoS ONE

Article Title: An Improved Genetically Encoded Red Fluorescent Ca 2+ Indicator for Detecting Optically Evoked Action Potentials

doi: 10.1371/journal.pone.0039933

Figure Lengend Snippet: A , A projection image of a CA3 pyramidal cell expressing R-CaMP1.07 and ChR2. The cell was whole-cell recorded to measure the number of APs. B , Imaging of ChR2-triggered-APs by changes in the R-CaMP1.07 fluorescence (top). APs were evoked by a pulse of 470-nm light with a duration of 300 to 3000 ms (blue region indicates time of photostimulation). Putative Ca 2+ increases during the photostimulation are represented by broken lines (Bottom). The number of APs was recorded using the current-clamp mode. C , Δ F / F amplitude of the R-CaMP1.07 responses as a function of the number of APs induced by photostimulation. The peak Δ F / F amplitudes were calculated from the first frames after termination of the photostimulation. Individual data are plotted as black dots and their averages are shown in red. Error bars, s.e.m. ( n = 4 cells).

Article Snippet: The cDNAs encoding R-GECO1 and ChR2 were chemically synthesized (GenScript).

Techniques: Expressing, Imaging, Fluorescence

Journal: eLife

Article Title: Gamma rhythms and visual information in mouse V1 specifically modulated by somatostatin + neurons in reticular thalamus

doi: 10.7554/eLife.61437

Figure Lengend Snippet: ( A ) Neural activity was recorded from V1 in freely moving mice. Mice were presented with a gray blank screen while a blue light (473 nm, ~63 mW/mm 2 ) was delivered to Channelrhodopsin-2 (ChR2)-expressing SST cells in nRT using an optical fiber implanted above the nRT. Mouse movement was tracked over the course of the experiment. ( B ) Representative extracellular raw voltage trace is shown along with its power spectrum. Blue shading areas indicate optogenetic activation. Mouse movement speed is shown at the bottom. Note that optogenetic activation of SST cells in nRT reduced the power of local field potentials (LFPs) regardless of the locomotion state of the animal. ( C ) Optogenetic stimulations at baseline: across-trial average power of all channels in the absence (black) and presence (blue) of optogenetic activation in one representative mouse shows significant decreases across theta (4–8 Hz), beta (15–30 Hz), and gamma bands (30–80 Hz) (see for statistics). ( D ) Effect of locomotion without optogenetic stimulations: across-trial average power of all channels in one representative mouse indicates that locomotion has no significant effect on theta power, significantly reduces beta power, while causing a strong enhancement in gamma power . ( E ) Effect of optogenetic stimulations in still and running conditions: average power across all channels in four mice shows that optogenetic activation of SST nRT cells selectively reduces gamma power both in the still (black vs. blue marks) and running conditions (red vs. blue marks) , and has no significant effect on theta and beta powers from four mice. ( F ) Visual responses were recorded while mice were presented with moving gratings (eight directions, each moving in one of two possible directions; 2 s duration; randomly interleaved with optogenetic stimulation) in the visual field contralateral to the recording site. ( G ) Firing rate (averaged over all eight drifting directions) of an example cell during the course of the experiment. Black marks: visual responses when the laser is off; blue marks: visual responses when visual stimuli and optogenetic activation of SST nRT cells are coupled. Red shadings: locomotion state. Error bars: SEM. ( H ) Effect of visual stimulation with and without optogenetic manipulation: stimulus-evoked (average over all 20 trials) minus ongoing power of all channels when the laser is off (black circles) versus the laser-on condition (blue circles) in one representative mouse indicates a significant shift across all frequencies . ( I ) Same as in ( E ) in the presence of visual stimulus . Note that optogenetic activation of SST nRT cells selectively reduces gamma power in both still and running conditions, without significant effects on the other frequency bands. ( J ) Using three parameters calculated from average waveforms, cells were classified into narrow- (NS, cyan) or broad- (BS, magenta) spiking (height of the positive peak relative to the negative trough: −0.20 ± 0.01, –0.34 ± 0.02 [p=1.02e-9, Wilcoxon rank-sum test]; the time from the negative trough to the peak: 0.73 ± 0.02, 0.32 ± 0.02 ms [p=3.9e-33], slope of the waveform 0.5 ms after the negative trough: 0.01 ± 0.00, –0.01 ± 0.00 [p=5.94e-35], BS [n = 169], and NS [n = 73] cells, respectively). Subplot: average spike waveforms for all units, aligned to minimum, demonstrating BS (magenta) and NS (cyan) cells. ( K ) Optogenetic light significantly reduced firing rate of BS (magenta) and NS (cyan) cells during visual stimulus in both still and running conditions . ( L ) Percentage change in visually evoked firing rate of both cell types versus percentage change of power in channels that each cell is recorded from for still and running states , Spearman’s rho and p: 0.05, 0.58 BS and still [n = 100 BS in four mice]; −0.12, 0.22 BS and running; 0.19, 0.29 NS and still [n = 32 NS in four mice]; 0.05, 0.77 NS and running). Figure 2—source data 1. Results of significance testing accross different conditions.

Article Snippet: Equipment , Blue laser for ChR2 , OPTO ENGINE LLC , Cat #: MDL-III-450 , .

Techniques: Activity Assay, Expressing, Activation Assay

Journal: eLife

Article Title: Gamma rhythms and visual information in mouse V1 specifically modulated by somatostatin + neurons in reticular thalamus

doi: 10.7554/eLife.61437

Figure Lengend Snippet: ( A ) Representative composite images (individual images, 10×) of coronal brain sections from Sst-Cre mice labeled with parvalbumin (PV) antibody (red). The sections span anterior, middle, and posterior portions of the nucleus reticularis thalami (nRT) and were obtained 4–6 weeks after an intra-nRT injection of an AAV virus containing ChR2-enhanced yellow fluorescent protein (eYFP) targeting Sst-Cre cells in nRT. ( B ) Representative composite images (individual images, 10×) of coronal brain sections from Sst-Cre mice labeled with PV antibody (red). The sections were obtained 4–6 weeks after an intra-nRT injection of an AAV virus containing ChR2-eYFP targeting Sst-Cre cells in nRT. Note dense axonal projections from PV nRT cells to the somatosensory ventrobasal thalamus (VB), but not to dorsal lateral geniculate nucleus (dLGN), and the opposite for somatostatin (SST) nRT cells. ( C ) A high magnification image showing that both PV (red) and SST (green) neurons are present in the visual sector of the nRT. The results are representative of six mice out of six from each group. ( D ) 63× confocal images showing the entire field of view (FOV) and a zoomed cropped region (‘High mag’) to show details of axonal boutons and nRT somata. Each channel is obtained from the maximal intensity projection of a 30 μm z-stack (5 μm intervals). . The expression of the viral constructs in different brain regions was confirmed using the mouse brain atlas . ( E ) Same as ( A ) but for Pvalb-Cre mice. ( F ) Same as ( D ) but for Pvalb-Cre mice. Arrows indicate co-localization of the PV antibody staining (red) and opsin expression (green). nRT confocal image is presented as a single z-stack for clarity.

Article Snippet: Equipment , Blue laser for ChR2 , OPTO ENGINE LLC , Cat #: MDL-III-450 , .

Techniques: Labeling, Injection, Virus, Expressing, Construct, Staining

Journal: eLife

Article Title: Gamma rhythms and visual information in mouse V1 specifically modulated by somatostatin + neurons in reticular thalamus

doi: 10.7554/eLife.61437

Figure Lengend Snippet: ( A ) Changes in the dLGN ongoing power due to optogenetic activation (blue) or inhibition (green) of SST nRT neurons across the three frequency bands (Channelrhodopsin-2 [ChR2] vs. enhanced Natronomonas halorhodopsin [eNpHR]; theta, still: −77.6e3 vs. 53.4e3, p=0.06, nested t-test, mean difference −132.0e3 with a 95% CI of [−105.1e3, 163.0e3], run: 8.2e3 vs. 74.1e3, p=0.09, nested t-test, mean difference −65.9e3 with a 95% CI of [−75.1e3, −51.8e3]; beta, still: −141.3e3 vs. 6.7e3, p=0.012, mean difference −134.6e3 with a 95% CI of [−178.0e3, −87.2e3], run: −134.0e3 vs. 28.5e3, p=0.009, mean difference −162.5e3 with a 95% CI of [−184.0e3, −123.6e3]; gamma, still: −137.0e3 vs. 109.3e3, p=3e-5, mean difference −246.3e3 with a 95% CI of [−289.5e3, −191.7e3], run: −65.0e3 vs. 118.9e3, p=6e-3, mean difference −183.9e3 with a 95% CI of [−202.0e3, −58.8e3]; all in three mice, all powers in uV2/Hz). *p<0.016, **p<0.0016, ***p<0.00016. ( B ) Effects of optogenetic activation or inhibition of SST neurons on the ratio of laser-on to laser-off firing rates of dLGN cells across locomotion conditions (ChR2 vs. eNpHR; multi-unit [MU], still: 0.65 vs. 1.28, p=5e-22, run: 0.66 vs. 1.65, p=5e-11, n: 158 vs. 185 cells; single-unit [SU], still: 1.13 vs. 1.30, p=7e-6, run: 1.34 vs. 1.23, p=6e-8, n: 35 vs. 49; all in three mice). ***p<2.5e-4.

Article Snippet: Equipment , Blue laser for ChR2 , OPTO ENGINE LLC , Cat #: MDL-III-450 , .

Techniques: Activation Assay, Inhibition

Journal: eLife

Article Title: Gamma rhythms and visual information in mouse V1 specifically modulated by somatostatin + neurons in reticular thalamus

doi: 10.7554/eLife.61437

Figure Lengend Snippet:

Article Snippet: Equipment , Blue laser for ChR2 , OPTO ENGINE LLC , Cat #: MDL-III-450 , .

Techniques: Sequencing, Plasmid Preparation, Immunohistochemistry, Recombinant, Software