F-actin coats individual fused granules.

<p>Pancreatic tissue fragments were bathed in paraformaldehyde-fixable fluorescein extracellular dye and then stimulated with 1 µM acetylcholine. Each fused granule is then identified by the uptake of fluorescent dye. After fixation, co-staining with phalloidin Alexa-633, shows that each fused granule is coated with F-actin. The upper panel shows a low magnification image, where-as the lower panel shows high magnification images that are enlargements of the boxed region.</p

Latrunculin treatment abolishes F-actin coating of fused granules.

<p>(<b>A</b>) Low magnification images show complex lumens, identified by SRB (red) and Lifeact-EGFP fluorescence (green), lying between the cells within a pancreatic fragment. (<b>Ai</b>) is an image taken before the appearance of exocytic events, (<b>Av</b>) is taken after, at the time points “i” and “v” as indicated on the graph in panel (<b>C</b>). (<b>B</b>) Shows an image sequence from an enlarged region (box shown in A) of Lifeact-EGFP and SRB and the overlay, for a single exocytic event. The images were taken at the time points i, ii, iii, iv, and v as indicated on the graph in panel (<b>C</b>). (<b>C</b>) Is a graph of fluorescence changes over time taken from a region of interest placed over the exocytic event (indicated by an arrow). SRB fluorescence is plotted normalized to the first, rapid peak and shows a rapid rise followed by a slower increase. The simultaneously recorded Lifeact-EGFP signal, plotted as arbitrary fluorescence units, shows only very small changes over time.</p

Lifeact-EGFP transgenic mice show similar acetylcholine-induced exocytic responses to wild type.

<p>(<b>A</b>) The images were taken before (0 s) and 300 s after exocytic fusion induced by 1 µM acetylcholine stimulation of mouse pancreatic fragments in wild type (upper images) and Lifeact-EGFP transgenic animals (lower images). The tissue fragments were bathed in extracellular fluorescent dye (SRB) which labels the lumens (bright fluorescence between the cells in control) and enters and labels each individual granule (bright fluorescent spots along the lumen after 300 s). (<b>B</b>) We identify the time point of appearance of each fusing granule (cumulative histogram, aligned to the first exocytic event) which shows both wild type and transgenic animals have a similar time-course and extent of exocytic response. (C) Further, we compare the fluorescence profile, over time, of SRB dye entry into each individual granule and observe no differences between wild type and transgenic animals.</p

F-actin coating is initiated simultaneously across the whole of the granule.

<p>(A) a high magnification image sequence (i, ii, iii, iv, v with times shown on graph in <b>B</b>) shows SRB entry and Lifeact-EGFP tracking of F-actin coating of an fusing individual granule. (<b>B</b>) Regions of interest (boxes a, b, c shown in <b>A</b>) placed around single fusing granules showed only small temporal differences in the time-course of F-actin coating. When fitted to a single exponential the τ values were 15.5, 13.3 and 15.5 seconds (for regions a, b, c respectively). (<b>C</b>) shows the graph obtained from the means of 10 events. The mean SRB signal from each granule is shown for reference (black plot). For each event regions a and b were placed at the luminal-granule interface and c was placed at the top of the granule. The extreme minima (labelled min) and maxima (labelled max) of the SEM from all 3 regions of interest are shown as dotted grey lines. The average Lifeact-EGFP change within each region was fitted to a single exponential and these fitted curves are shown, colour-coded, on the graph.</p

Real-time imaging of exocytic fusion events and F-actin coating.

<p>(<b>A</b>) Low magnification images show a lumen lying diagonally between two acinar cells identified with SRB (red) and Lifeact-EGFP (green) in the sub-apical region. (<b>A, i</b>) is an image taken before the appearance of exocytic events at the time point indicated “i” on the graph of fluorescence intensity over time in panel (<b>C</b>). (<b>A, v</b>) Is an image at a time point after induction of a number of exocytic events which can be seen as bright spots of SRB fluorescence along the lumen; the time point “v” is indicated on the lower graph (<b>C</b>). (<b>B</b>) Shows an image sequence from an enlarged region (box shown in <b>A</b>) of Lifeact-EGFP and SRB and the overlay, for two exocytic events. The images were taken at the time points i, ii, iii, iv, and v as indicated on the graph in panel (<b>C</b>). (<b>C</b>) is a graph of fluorescence changes over time taken from a region of interest placed over the lower exocytic event (indicated by an arrowhead). SRB fluorescence is plotted normalized to the peak and rises rapidly to a peak and then decays to a plateau. The simultaneously recorded Lifeact-EGFP signal, plotted in arbitrary fluorescence units, rises slowly and nearly reaches a maximum by the end of the record. The starting points of the SRB and Lifeact-EGFP signals, as determined by a positive deflection of the signal by more than 5 times the standard deviation of the signal noise, are shown by the colour-coded triangles on the X axis. The black dotted lines were mono-exponential fits to the data with τ values of 6.9 s (SRB) and 29.4 s (Lifeact-EGFP).</p

Reduced relaxin-3 does not alter body weight, food or water intake or blood glucose concentration.

<p>A. Body weight of each treatment group (mean ± SEM) for the study duration. Stereotaxic surgery was completed between days 7 and 14. B. 24 hr food intake for each treatment group (mean ± SEM) following surgery. C. 24 hr water intake for each treatment group (mean ± SEM) following surgery. Saline n = 13, EmGFP n = 14, miRC n = 13, miR499 n = 14. D. Concentration of blood glucose for each treatment group (mean ± SEM) following surgery. At each time point n = 4–7 for each group.</p

Reduced relaxin-3 has no effect on performance in paradigms of spatial memory or anxiety-like behaviour.

<p>A. Spatial learning and memory was assessed in the spontaneous alternation task. Percentage alternation scores for each treatment group are represented as mean ± SEM. Saline n = 13, EmGFP n = 14, miRC n = 13, miR499 n = 14. B. There was no correlation between percentage alternation score and relaxin-3 mRNA levels (n = 24, 5–7 rats per group, r = 0.0814, p = 0.705). C. Anxiety-like behaviour was investigated in the light/dark box. Time spent in the light side for each treatment group is represented as mean ± SEM. Saline n = 13, EmGFP n = 14, miRC n = 13, miR499 n = 14. D. There was no correlation between time and relaxin-3 mRNA levels (n = 24, 5–7 rats per treatment group, r = −0.292, p = 0.166).</p

Hindbrain relaxin-3 mRNA is reduced following rAAV1/2 EmGFP miR499 treatment whereas RXFP3 mRNA remains unchanged.

<p>qRT- PCR was used to determine the levels in a subset of rats (5–7 rats) for each treatment group. Relaxin-3 levels were reduced compared to all control groups following infusion of rAAV1/2 EmGFP miR499 (A). For statistical analysis relaxin-3 expression was log₁₀ transformed (B). *indicates significant difference between the group indicated and all other groups as determine by a one-way ANOVA and Holm-Sidak post-hoc analysis with significance set to p<0.05. There was no significant difference between RXFP3 levels between treatment groups (C).</p

rAAV1/2 EmGFP miR499 reduces relaxin-3-like immunoreactivity from one week post infusion.

<p>EmGFP (green) is expressed in cells transduced by rAAV1/2 EmGFP miR499. Relaxin-3-like immunoreactivity (red) in the nucleus incertus decreases as transgene expression increases from 1 week (A), 3 weeks (B), 6 weeks (C) and 9 weeks (D) post infusion. n = 4 rats for each time point. Scale bar indicates 200 µm.</p

rAAV1/2 EmGFP miR499 reduces relaxin-3-like immunoreactivity in nucleus incertus.

<p>EmGFP transgene expression (A, D, G) and relaxin-3-like immunoreactivity (B, E) in no infusion controls (A, B, C) and following bilateral infusion of rAAV1/2 EmGFP miR499 (D, E, F) or rAAV1/2 EmGFP miRC (G, H, I). Scale bar indicates 200 µm (A, B, D, E, G, H) or 50 µm (C, F, I).</p