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. 2023 Dec 23;13(1):38.
doi: 10.3390/cells13010038.

Super-Resolution Analysis of the Origins of the Elementary Events of ER Calcium Release in Dorsal Root Ganglion Neurons

Miriam E Hurley  1 , Shihab S Shah  1 , Thomas M D Sheard  2 , Hannah M Kirton  1 , Derek S Steele  1 , Nikita Gamper  1 , Izzy Jayasinghe  2   3
Affiliations

Super-Resolution Analysis of the Origins of the Elementary Events of ER Calcium Release in Dorsal Root Ganglion Neurons

Miriam E Hurley et al. Cells. .

Abstract

Coordinated events of calcium (Ca2+) released from the endoplasmic reticulum (ER) are key second messengers in excitable cells. In pain-sensing dorsal root ganglion (DRG) neurons, these events can be observed as Ca2+ sparks, produced by a combination of ryanodine receptors (RyR) and inositol 1,4,5-triphosphate receptors (IP3R1). These microscopic signals offer the neuronal cells with a possible means of modulating the subplasmalemmal Ca2+ handling, initiating vesicular exocytosis. With super-resolution dSTORM and expansion microscopies, we visualised the nanoscale distributions of both RyR and IP3R1 that featured loosely organised clusters in the subplasmalemmal regions of cultured rat DRG somata. We adapted a novel correlative microscopy protocol to examine the nanoscale patterns of RyR and IP3R1 in the locality of each Ca2+ spark. We found that most subplasmalemmal sparks correlated with relatively small groups of RyR whilst larger sparks were often associated with larger groups of IP3R1. These data also showed spontaneous Ca2+ sparks in <30% of the subplasmalemmal cell area but consisted of both these channel species at a 3.8-5 times higher density than in nonactive regions of the cell. Taken together, these observations reveal distinct patterns and length scales of RyR and IP3R1 co-clustering at contact sites between the ER and the surface plasmalemma that encode the positions and the quantity of Ca2+ released at each Ca2+ spark.

Keywords: calcium signalling; correlative microscopy; dSTORM; dorsal root ganglion neurons; expansion microscopy; inositol 1,4,5-trisphosphate receptor; ryanodine receptor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Observing Ca2+ sparks, RyR3 and IP3R in dorsal root ganglion neurons. (A) A TIRF image of a cultured DRG neuronal soma loaded with Fluo-4AM (resolution ~250 nm). (B) Magnified view of the dashed box region in panel A where spontaneous Ca2+ sparks were recorded in the absence of either caffeine or bradykinin under camera integration time of 50 ms. (C) Overlaid percentage histograms show an increase in the integrated spark mass (shown in arbitrary units) of spontaneous Ca2+ sparks in both cells treated with bradykinin (red) and cells exposed to caffeine (green) compared with controls (in the absence of caffeine or bradykinin), respectively. The equivalent percentage histogram for FWHM of the sparks (inset) shows that it remains unaltered in the presence of bradykinin and caffeine. Spark data pooled from 5 cells in each condition, cells sourced from 5 animals, with the same cell being exposed to each treatment as a repeated measure. Histogram bin sizes: 0.1 for spark mass, 0.2 μm for FWHM. (D) The cartoon illustrates the extensive ER in the DRG soma which occupy the cytoplasm. N notates the nucleus. (E) It is hypothesised that the Ca2+ sparks which are observed frequently at the subplasmalemmal regions (shown in zoomed view of the cartoon in the dashed box in (D)) may arise from either RyR3 (red) and/or IP3R (purple). (F) IP3R1 immunofluorescence near the bottom surface of the cell visualised with 10× ExM combined with confocal microscopy (with a 20× 0.9 NA objective), at an applied resolution of ~39 nm. (G) The region indicated by the region in dashed box in (F) is shown, imaged with a 40× 1.3 NA lens (resolution ~15 nm) illustrating a highly nonuniform distribution of punctate labelling densities. (H) This morphology was qualitatively reproduced with dSTORM images of equivalent regions (resolution ~30 nm). (I) The RyR3 immunofluorescence imaged with 10× ExM, combined with confocal microscopy (with a 0.8 NA lens) at a resolution of ~39 nm. (J) A similar cell from the same sample imaged in Airyscan mode with a 1.3 NA objective (effective resolution ~15 nm) shows a heterogeneous and punctate labelling morphology. Observed were loose clustering patterns at distinct sub-micron-scale domains of the membrane (dashed circles). (K) An equivalent dSTORM image reproduced the same morphologies, including the loose clusters. Scale bars: (A) 5 μm, (B) 3 μm, (F,I) 1 μm, (G,H,J,K) 500 nm.
Figure 2
Figure 2
The experimental pipeline of correlative TIRF Ca2+ spark and dSTORM imaging. (A) The key steps include extracting the DRG neurons from the neonatal rats, dissecting, isolating and coculturing of DRG and glial cells within the gridded dishes, AM-loading of the Ca2+ indicator dye and de-esterification, recording Ca2+-sensitive indicator dye fluorescence from DRG neuronal cells and the specific grid coordinates noted, fixation and immunofluorescence labelling and returning to the coordinates of a cell whose Ca2+ signals were recorded previously to acquire dSTORM data of RyR3 or IP3R. (B) The post-acquisition alignment of the averaged and scale-matched Ca2+ frame TIRF image (green) to the rendered dSTORM image (IP3R image; purple) through a semiautomated programme chiefly relying on the cell outline (indicated with white dashed lines). (C) Shown is an overlay of the two images in their respective colour tables. (D,E) Overlays of the centroids of spontaneous Ca2+ sparks (green) recorded in the absence of either caffeine or bradykinin during a 10 s time window and dSTORM images of RyR3 (red) and IP3R1 (magenta), respectively. Asterisk in panel E denotes regions with a glancing view of the cell surface with lower density of calcium sparks observed. Scale bars: (B,C) 5 μm, (D,E) 1 μm.
Figure 3
Figure 3
Local correlation of spontaneous Ca2+ sparks and localisation patterns of RyR3 and IP3R1. (A) Shown are the TIRF frame (grey colour table) of spontaneous Ca2+ sparks in a DRG neuron in the absence of caffeine or bradykinin and the local RyR3 labelling pattern in the same cell (red). The yellow box shows the magnified view of the sparks and the punctate RyR3 pattern locally. (B) In the local correlation analysis, the FWHM (green dashed line in upper panel) of the localised Ca2+ spark provided a window of confidence (green dashed lines in lower panel) to examine the dSTORM RyR3 puncta in the region of origin for the Ca2+ spark. (C) Scatterplot between the integral of the Ca2+ released (estimated based on ‘spark mass’, shown in arbitrary units) and the number of RyR3 puncta within the interrogation window shows a majority of sparks coinciding with small groups of RyR3 puncta (<10 puncta). (D) Scatterplot between the spark mass and the local IP3R puncta count shows sparks with larger integrals can coincide with a broader range of total IP3R puncta. (E) Dot plot (mean marked with solid line) showing that the densities of both RyR3 and IP3R puncta (per μm2 of 2D image area) were higher in spark sites (51.1 μm−2 and 48.8 μm−2) compared with overall footprint of the cell (13.4 μm−2 and 9.64 μm−2). ** Tukey test results for RyR3 and IP3R comparisons indicated the following: qs = 6.89 and 6.65, respectively; p = 0.0004 and 0.0006, respectively; df = 22 and 22. (F) Overlaid percentage histograms of the nearest neighbour distances of RyR3 (red) or IP3R (purple) puncta within the spark sites. The inset shows the equivalent histograms of RyR3 and IP3R puncta in the cell area overall. Scale bars: (A) left panels: 1 μm, (A) right panels: 250 nm.
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