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: For both approaches, when fluorescent cell bodies or axonal branches were in focus, 20–30 fps image series (2.96 pixel per μm) were acquired using the miniscope software (Inscopix data acquisition software, nVoke acquisition system, Inscopix, CA).
Techniques: In Vivo, Expressing, Construct, Imaging, Labeling, Standard Deviation, Fluorescence
![Imaging IO spikes and STOs in vivo . (A) in-vivo calcium spike recording from ventral IO using a GRIN-lens-based <t>miniscope.</t> The schematic on top of the figure describes the approach. (A1) Example image (standard deviation projection of the time series) from a living IO with several IO somata indicated by dashed-line ROIs and numbered labels. Note that the image brightness is adjusted for viewing and should not be considered as representative of the dynamic range during live imaging. The calcium traces extracted from the labeled ROIs are shown below the image, with detected spikes indicated by asterisks. (A2) Detected spikes from pooled N = 4 animals, 22 cells, aligned on the initiation point. Dark green trace is the averaged waveform. (A3-5) Comparison of the calcium event waveforms between in-vivo (dark green) and in-vitro (bright green) recordings. The average amplitudes are not different (A4) but the shortest calcium events are absent in-vivo recordings (A5) . Gray shaded area represents the range of calcium event rise times measured in-vitro corresponding to “short-IO-spikes” (sIO-s; cluster 2 in ). For reference, the vertical dashed line in A5 indicates duration of a single frame in miniscope recording. (B1) Example of oscillating IO neurons recorded in-vivo . Eight active cells could be detected (ROIs indicated with dashed white lines; standard deviation projection of the time series) and traces from the labeled cells are displayed on the right. Welch spectra of the 8 ROI are shown superposed in (B2) , with one example cell [labeled 5 in (B1) ] is highlighted in dark green. (B3) ; Population data of oscillating neurons found in N = 3 animals. Each circle represents a single IO cell.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3741/pmc09093741/pmc09093741__fncel-16-825056-g0011.jpg)
![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.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3741/pmc09093741/pmc09093741__fncel-16-825056-g0012.jpg)