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: FIGURE 1 | Screening 10 viral constructs for efficient and selective transgene expression in the inferior olive. (A) Confocal images showing EGFP (left) fluorescence driven with three example promoters (Htr5b(3.7), Susd4(3.7), and CAG in (A1,A2,A3), respectively). Middle shows the tdTomato fluorescence driven with the reference construct (AAV.PHP.S-CAG) that was used to normalize the intensity measurements in each sample; the merged fluorescence images are shown on right. White arrows in merged images indicate the sites of injection on one side of IO, showing that the injections included a purposeful “mistargeting” for more fair assessment of labeling specificity. The plasmid used for each construct is schematically depicted above the confocal images. (B) Schematic describing the definition of IO-specific and non-specific measurements. (C) Summary of all the 10 constructs’ normalized expression strengths inside and outside of the IO (vertical and horizontal axes, respectively). Only constructs based on clones of the serotonin receptor 5b (Htr5b) show similar or better performance in the IO than the reference construct. The results from the three example constructs shown in (A) are indicated by white markers. The error bars denote population SEMs.

Article Snippet: Plasmids with tTA (tetracycline-controlled transactivator) and TRE (Tet Response Element) were prepared from AAV plasmids Addgene #104109 and #104110, respectively (Table 3).

Techniques: Construct, Expressing, Injection, Labeling, Plasmid Preparation, Clone 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: FIGURE 3 | AAV capsid type influences transgene expression with both single and dual-AAV approach. (A) Example confocal images of the IO acquired from an animal where three different fluorophores (EGFP, tdTomato and mTagBFP2) were expressed under the control of Htr5b(3.7) promoter but with different AAV capsids (AAV9, AAV.PHP.S, and AAV.PHP.eB in (A1,A2,A3), respectively) after 4 weeks of transfection. Dashed lines indicate the contours of the IO. A schematic labeled alpha on top of the figure depicts the design of the experiment. (B) Example confocal images acquired from the IO of three different animals, in each of which a dual-AAV mixture utilizing different viral capsids (AAV9, AAV.PHP.S, and AAV.PHP.eB in (B1,B2,B3), respectively) were used to deliver the sequences for expressing Htr5b(3.7)-tTA/TRE-controlled GFP. A schematic labeled beta on top of the figure depicts the experiment design. Note that the color scales in each example confocal image are adjusted for visual clarity, and quantitative assessments are done based on the numerical comparisons as shown in other. (C) Distributions of single-soma fluorescence intensities from n = 548 cells from 20 slices in 10 animals, normalized to maximal values for the three different viral capsids. Dashed lines denote data obtained with single virus experiments, solid color bars are from dual-AAV experiments. (D) Comparison of the mean fluorescence intensities between samples obtained with the three capsids. Mean values, indicated by colored bars, are normalized to the mean value of AAV9 intensities (dashed line). (E1,E2) Comparison of the shortest distances between neighboring labeled somata with the three capsids. (1-way ANOVA, [AAV9 vs. AAV.PHP.S] f = 14.5, p<0.001; [AAV9 vs. AAV.PHP.eB] f = 0.3, p = 0.86; [AAV.PHP.S vs. AAV.PHP.eB] f = 13.9, p<0.001).

Article Snippet: Plasmids with tTA (tetracycline-controlled transactivator) and TRE (Tet Response Element) were prepared from AAV plasmids Addgene #104109 and #104110, respectively (Table 3).

Techniques: Expressing, Control, Transfection, Labeling, Virus, 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: FIGURE 12 | 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: Plasmids with tTA (tetracycline-controlled transactivator) and TRE (Tet Response Element) were prepared from AAV plasmids Addgene #104109 and #104110, respectively (Table 3).

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