Journal: Frontiers in Cellular Neuroscience

Article Title: Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo

doi: 10.3389/fncel.2022.825056

Figure Lengend Snippet: List of custom AAV vectors used for anatomical and functional characterization of IO by “single-AAV” and “double-AAV” approaches.

Article Snippet: The GCaMP6s reporter plasmid was prepared from an AAV plasmid with GCaMP6s (Addgene #50942).

Techniques: Functional Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo

doi: 10.3389/fncel.2022.825056

Figure Lengend Snippet: Comparison of tissue-fixed and live-imaging GCaMP6s fluorescence intensities in-vitro . (A1–A5) Example confocal images (5x) demonstrating wide-area GCaMP6s fluorescence obtained with 5 different constructs (as indicated above each) after 2 weeks of expression. Images are acquired from 300 μm brainstem slices, immersion-fixed after live in-vitro imaging experiments. (B) Comparison of the constructs' baseline (F 0 ) whole-field live fluorescence measurements for individual neurons (B1) and confocal imaging (B2) before (B1) and after (B2) immersion-fixing. (B3) shows the high correlation between the live imaging and confocal imaging results. Colored dots are average values for each construct, where 10 to 20 neurons per slice for the live, and fixed condition in the same slices. (n = 3 slices per animal, min. 2 animals per construct). (C) Distribution of raw live-imaging fluorescence intensities varies between constructs (C1) , but the relative ranges are nearly identical when normalized to maximal intensity in each slice (C2) . Dashed line in (C2) indicates relative intensity of 0.3. For (B,C) , data from different constructs are labeled with colors as in (A) .

Article Snippet: The GCaMP6s reporter plasmid was prepared from an AAV plasmid with GCaMP6s (Addgene #50942).

Techniques: Comparison, Imaging, Fluorescence, In Vitro, Construct, Expressing, Labeling

Journal: Frontiers in Cellular Neuroscience

Article Title: Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo

doi: 10.3389/fncel.2022.825056

Figure Lengend Snippet: Effects of GCaMP6s expression level on the action-potential-related fluorescence signal. (A,B) Comparison between calcium events in units of power (fW) obtained with the 5 different constructs as indicated by the color labels, 2 weeks after injection. The schematic examples on top of the figure (in a dashed-line box; note that the image brightness of the standard deviation time series projection is adjusted for viewing and should not be considered as representative of the dynamic range during live imaging) describe extraction of the calcium fluorescence event parameters in acute IO slices. (A1) Averaged event waveforms aligned on initiation point (black dot). The shaded areas denote ± SEM. (A2) Average calcium event peaks; (A3) their cumulative distributions, with arrow and horizontal line segment highlighting the convergence of maximal values of eCa peaks between high-expressing constructs; (A4) relation between average event peaks and baseline fluorescence in each cell. (B) Same data as in (A) but normalized to F 0 for a baseline-normalized DFF values, similarly represented in boxplot (B2) , distribution plot (B3) and construct-averages represented against their respective F 0 s (B4) . (C) Same data as in (B) but peak-amplitude-normalized to compare event kinetics. (C1) averaged event waveforms; (C2) comparison of average event rise times; (C3) cumulative distributions of event rise times; (C4) individual event rise times and baseline-normalized event amplitudes show linear relation. The colored dots in (C4) represent averages for each construct data, gray dots are individual events. Colored bars in (A2,B2,C2) denote average values. Horizontal and vertical lines in (A4,B4) denote ± SEM values in each dimension. Statistics for eCa F-peak on (A2) (1-way ANOVA, [AAV9-Htr5b(3.7)-tTA/TRE vs. AAV.PHP.eB-Htr5b(3.7)-tTA/TRE] f = 1.56, p = 0.1; [AAV9-Htr5b(3.7)-tTA/TRE vs. AAV.PHP.S-Htr5b(3.7)-tTA/TRE] f = 1.9, p = 0.17).

Article Snippet: The GCaMP6s reporter plasmid was prepared from an AAV plasmid with GCaMP6s (Addgene #50942).

Techniques: Expressing, Fluorescence, Comparison, Construct, Injection, Standard Deviation, Imaging, Extraction

Journal: Frontiers in Cellular Neuroscience

Article Title: Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo

doi: 10.3389/fncel.2022.825056

Figure Lengend Snippet: Calcium event waveforms do not differ between patched and intact neurons. (A) Example of a combined in vitro patch-imaging experiment. (A1) shows an infrared-contrast image overlaid with standard deviation projection of fluorescence recording time series, and 5 somata identified in the field of view are indicated with dashed lines (ROIs). Cell labeled 1 was recorded with a patch-clamp pipette. (A2) shows the GCaMP6s signals obtained from all of the 5 cells in the field of view (green traces, ROIs indicated with numbers), and the time-aligned electrical recording from cell 1 (black trace). (B) Comparison of calcium event waveforms in patched (black) cells and intact (green cells). (B1) Time-aligned action potential waveforms from all 14 patched cells, aligned at calcium-event onsets. Thick black trace is the average of all spikes. B2, averaged, onset-aligned and normalized calcium event waveforms peak with shaded areas denoting ± SEM. (B2) Comparison between rise times (left) and peak amplitudes (right) of calcium events recorded in patched and intact cells. Green and black bars in box plots represent averages; circles represent outliers. Statistics for eCa event Rise-time between patched and non-patched neurons on (B3) (1-way ANOVA, f = 0.085, p = 0.77). Statistics for eCa event peak amplitude between patched (n = 46 events on 13 neurons) and non-patched neurons on (B3) (1-way ANOVA, f = 11.09, p = 0.0.0012).

Article Snippet: The GCaMP6s reporter plasmid was prepared from an AAV plasmid with GCaMP6s (Addgene #50942).

Techniques: In Vitro, Imaging, Standard Deviation, Fluorescence, Labeling, Patch Clamp, Transferring, Comparison

Journal: Frontiers in Cellular Neuroscience

Article Title: Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo

doi: 10.3389/fncel.2022.825056

Figure Lengend Snippet: Somatic calcium events are only generated by action potentials occurring in the recorded cells regardless of oscillatory status. (A) Simultaneous current-clamp and GCaMP6s imaging shows that a spike in a neighboring IO neuron (A1) is reflected as an electrophysiological “spikelet” in the oscillating cell labeled with an asterisk. The image in (A1) is a standard deviation time series projection of fluorescence time-series recording. Note that the image brightness is adjusted for viewing and should not be considered as representative of the dynamic range during live imaging. Only cells that spiked during the recording are visible. (A2) shows time-aligned fluorescence (top) and V m (bottom) traces, and the neighbor-spike-related spikelet in patched cell is indicated with an asterisk in the inset. (B) Another example of a simultaneous GCaMP6s and current-clamp recording from 3 IO neurons demonstrating presence of electrophysiological “spikelets” in a patched cell [labeled with “*” in (B1) ] linked with neighboring cell calcium events. There are three spikelets in the recording, indicated by thin vertical lines, and two of them are enlarged in insets. Current-injection-evoked spiking in the patched cell results in calcium spikes in the neighbor cells, indicative of gap junction coupling.

Article Snippet: The GCaMP6s reporter plasmid was prepared from an AAV plasmid with GCaMP6s (Addgene #50942).

Techniques: Generated, Imaging, Labeling, Standard Deviation, Fluorescence, Injection

Journal: Frontiers in Cellular Neuroscience

Article Title: Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo

doi: 10.3389/fncel.2022.825056

Figure Lengend Snippet: Electrophysiological features of IO action potentials are reflected in the GCaMP6s event waveform. (A) Principal component analysis of IO action potential waveforms. (A1) 60 voltage spike events detected and aligned on initiation point. (A2) Relative contributions of the principal components to the waveform variability. The 3 first PCAs explain 95%. (A3) Time-varying contributions of the 3 first PCAs, corresponding to the “calcium shoulder” (solid line), after-hyperpolarizarion (dashed line), and the sodium spike (dotted line). (B) Clustering IO calcium events (eCAs) based on the PCA analysis in (A) . (B1) k -means clustering (indicated by colored markers) of the IO action potentials shown in (A1) . (B2) Averaged electrophysiological waveforms from the clusters identified in (B1) . (B3) The average calcium event waveforms linked to the electrophysiological clusters in (B1,B2) . Note that no noticeable calcium transients are seen linked with cluster 1 that corresponds to “spikelet” events (see ). Also, the calcium events belonging liked to cluster 6 events lacking a full sodium spike are very small in amplitude. Spikes from cluster 6 are excluded from rest of analysis, and only the 46 spikes [corresponding to clusters 1–5] recorded in 13 healthy IO neurons are depicted in following. (C) Calcium event peak amplitudes (C1) and rise times (C2) strongly correlate with the electrophysiological spike widths (n = 31 events, clusters 2–5). Note that the eCa amplitude-to-spike width relation saturates with longest spikes, while rise time to spike width relation is more robust. (C3) Relation between calcium event rise time and peak amplitude. Note slight non-linearity with largest events. The colored points and black circles represent average values for respective clusters and individual observations respectively. Events from clusters 1 and 6 are not shown in (C1,C2) as they correspond to spikelets and unhealthy neuron spikes. (C3) consists of data from clusters 1–5. (D) Spike cluster diversity in different recordings. (D1) Number of spike cluster observations for each cell. Note that the “incomplete spikes” (cluster 6) and “short IO spikes” (cluster 2) are only seen in single cells each while spikes from other clusters are seen in multiple cells. (D2) Box-plot including data from clusters 2–5 providing suggestive translation scale between observed calcium event rise time (bottom scale) and the corresponding electrophysiological spike width (top scale). Calcium events with rise times longer than 150 ms correspond to “normal” IO spikes (IO-S). The “short IO spikes” (sIO-s) do not present a clear calcium shoulder and may be caused by unusual physiological state of the IO neuron, possibly due to major damage to dendrites caused by slice preparation.

Article Snippet: The GCaMP6s reporter plasmid was prepared from an AAV plasmid with GCaMP6s (Addgene #50942).

Techniques: Slice Preparation

Journal: Frontiers in Cellular Neuroscience

Article Title: Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo

doi: 10.3389/fncel.2022.825056

Figure Lengend Snippet: In-vitro calcium imaging of IO subthreshold oscillations. (A,B) Example in vitro calcium imaging recordings from dense (A) and sparse (B) transfection, using the constructs as indicated above the panels. Identified IO neuron somata are indicated with dotted ROIs in time-series projection images in (A1,B1) ; note that the image brightness is adjusted for viewing and should not be considered as representative of the dynamic range during live imaging. The GCaMP6s traces corresponding to labeled ROIs are shown in (A2,B2) . In the example shown in (B) , only one cell is labeled, and 4 neighboring regions indicated by dotted squares in (B1) , are shown as ROIs representing signal from non-transfected cells. Asterisks in (A2) indicate detected calcium spikes. Note the different vertical scales in (A2,B2) , reflecting lower GCaMP6s concentration in (B) . (A3,B3) : Welch spectra for the example recordings show a sharply defined frequency peak for the oscillating cells (left). Right show the peak frequency of the example recordings (colored arrows) with respect to the entire population (gray bars) (C) The power of observed oscillations in calcium traces is stronger with higher baseline fluorescence level resulting from higher concentration of GCaMP6s. Colored lines [in C1,C3 ] and markers (C2,C4) represent data obtained with the 5 different constructs as indicated at the bottom right corner of the figure. (C1,C2) display the ranges of oscillation in terms of STO-bandwith power (C1) and oscillation peak-to-peak amplitude (C2) . Note that the oscillation amplitude increase saturates below 1% (C3) , while all of the constructs were shown to report spike amplitudes with several-fold higher . (C2,C4) display the relation between single-cell baseline fluorescence intensity (F 0 , x -axis) and oscillation power (C2) and peak-to-peak amplitude (C4) . Dotted horizontal line in (C4) represents the threshold of 0.12 % DFF for classification of the signal as “oscillating” as determined in . (D) Summary of imaging capabilities of the different constructs for reporters of IO action potentials (vertical axis) and STOs (horizontal axis). The constructs with intermediate expression levels (orange and green data points; see ) provide better resolution of spike waveforms. The constructs with highest expression (blue data) under-perform with spike amplitudes but provide high resolution of the subthreshold oscillations.

Article Snippet: The GCaMP6s reporter plasmid was prepared from an AAV plasmid with GCaMP6s (Addgene #50942).

Techniques: In Vitro, Imaging, Transfection, Construct, Labeling, Concentration Assay, Fluorescence, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo

doi: 10.3389/fncel.2022.825056

Figure Lengend Snippet: GCaMP6s-STOs are reporting electrophysiological STOs in vitro . (A) Example of a simultaneous patch clamp and calcium imaging recording of an IO cell in-vitro . (A1) shows the standard deviation projection image of the recorded time series, with the patched cell indicated with a dashed line. Note that the image brightness is adjusted for viewing and should not be considered as representative of the dynamic range during live imaging. (A2) time-aligned GCaMP6s trace (top) and (V m ) trace (middle). The section of traces indicated by dotted rectangle is shown overlaid in bottom. The autocorrelation of the calcium trace (A3) as well as comparison of its Welch spectrum with that of the electrophysiological recording (A4) show stable oscillatory behavior in this cell. (B) The frequency of GCaMP6s STOs closely matches that of electrophysiological recordings. (B1) Frequency ratios of voltage ( y -axis) and fluorescence ( x -axis) signals is flat over large range of oscillation power. Dotted line indicates a threshold of 1 mV 2 RMS used to classify cells as “oscillating” or “non-oscillating.” (B2) Relation between electrophysiological STO power and GCaMP6s STO amplitude. Dotted line indicates the 1 mV 2 RMS threshold value as in (B1) . (B3) Comparison of STO power of the GCaMP6s signal obtained from cells classified as oscillating (green) or non-oscillating (black) based on the 1 mV 2 RMS threshold (Welch t-test, t = -4.99, p < 0.05).

Article Snippet: The GCaMP6s reporter plasmid was prepared from an AAV plasmid with GCaMP6s (Addgene #50942).

Techniques: In Vitro, Patch Clamp, Imaging, Standard Deviation, Comparison, Fluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo

doi: 10.3389/fncel.2022.825056

Figure Lengend Snippet: Recording of climbing fiber calcium events in vivo. (A1–A3) Expression of GCaMP6s in cerebellar climbing fibers with AAV9, AAV.PHP.S, AAV.PHP.eB-Htr5b(3.7)-tTA/TRE constructs, respectively. (A1-A3) are tiled and Z-projected 40x confocal images of sagittal cerebellar sections showing expression of GCaMP6s in axons in the cerebellar cortex with samples prepared for anatomical study. Expression of GCaMP6s in CF is sparse in when the viral serotype is AAV.PHP.S, while dense and widespread with AAV.PHP.eB serotype. The inset [in (B1) ] indicates the region that was imaged in with the miniscope (see schematic on top of the figure). (B) Example recording from CFs in a living mouse. (B1) Is a sagittal slice from tissue on the same brain where in-vivo calcium imaging was done. A schematic labeled beta describes the experiment procedure. (B2) , z -projection image (standard deviation) of miniscope calcium recording time series (20 second recordings per zone) as seen from the dorsal surface of the cerebellar cortex. Anterio-posterior (AP) and medio-lateral (ML) directions are indicated with white arrows. The dashed lines indicate manually-drawn ROIs for CFs, and the respective calcium traces are shown in (B3) . Parts of two of the traces [2 and 5, indicated by dashed rectangles in (B4) ] are shown with expanded time scale at the bottom of the panel. Detected events are indicated by asterisks. (C) Comparing in vivo calcium events recorded in IO axons (CFs; blue) with the somata (green). n = 70 events in 2 animals for axon recordings, 22 events in 4 animals for somata. (C1) Average in-vivo GCaMP6s transients from CFs and IO somata, aligned at initiation point. Shaded regions represent ± SEM. (C2) Comparison of calcium event amplitudes in IO somas and axons when normalized to basal fluorescence (1-way ANOVA, f = 8.19, p < 0.001). (C3) Comparison of event rise-times between IO soma and climbing fibers (1-way ANOVA, f = 5.3, p < 0.001). (C4) Comparison of the instantaneous frequency of events (inverse of inter-event interval) in the axon vs. the soma (1-way ANOVA, f = 5.35, p < 0.023). Note the slower spike rate in somatic recordings, possibly due to tissue cooling during the ventral surgery needed for somatic recordings.

Article Snippet: The GCaMP6s reporter plasmid was prepared from an AAV plasmid with GCaMP6s (Addgene #50942).

Techniques: In Vivo, Expressing, Construct, Imaging, Labeling, Standard Deviation, Comparison, Fluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo

doi: 10.3389/fncel.2022.825056

Figure Lengend Snippet: In-vitro properties of GCaMP6s signals found in inferior olive.

Article Snippet: The GCaMP6s reporter plasmid was prepared from an AAV plasmid with GCaMP6s (Addgene #50942).

Techniques: Fluorescence