BzATP Activates Satellite Glial Cells and Increases the Excitability of Dorsal Root Ganglia Neurons In Vivo

Abstract

The purinergic system plays an important role in pain transmission. Recent studies have suggested that activation of P2-purinergic receptors (P2Rs) may be involved in neuron-satellite glial cell (SGC) interactions in the dorsal root ganglia (DRG), but the details remain unclear. In DRG, P2X7R is selectively expressed in SGCs, which closely surround neurons, and is highly sensitive to 3'-O-(4-Benzoyl) benzoyl-ATP (BzATP). Using calcium imaging in intact mice to survey a large number of DRG neurons and SGCs, we examined how intra-ganglionic purinergic signaling initiated by BzATP affects neuronal activities in vivo. We developed GFAP-GCaMP6s and Pirt-GCaMP6s mice to express the genetically encoded calcium indicator GGCaM6s in SGCs and DRG neurons, respectively. The application of BzATP to the ganglion induced concentration-dependent activation of SGCs in GFAP-GCaMP6s mice. In Pirt-GCaMP6s mice, BzATP initially activated more large-size neurons than small-size ones. Both glial and neuronal responses to BzATP were blocked by A438079, a P2X7R-selective antagonist. Moreover, blockers to pannexin1 channels (probenecid) and P2X3R (A317491) also reduced the actions of BzATP, suggesting that P2X7R stimulation may induce the opening of pannexin1 channels, leading to paracrine ATP release, which could further excite neurons by acting on P2X3Rs. Importantly, BzATP increased the responses of small-size DRG neurons and wide-dynamic range spinal neurons to subsequent peripheral stimuli. Our findings suggest that intra-ganglionic purinergic signaling initiated by P2X7R activation could trigger SGC-neuron interaction in vivo and increase DRG neuron excitability.

Keywords: calcium imaging; dorsal root ganglion; mice; pain; purinergic receptor; satellite glial cell.

Figure 1

Concentration-dependent responses of satellite glial cells (SGCs) to the ganglionic application of BzATP. (A) Representative images illustrate the fluorescence intensity of SGCs at baseline (Frames (F)1–5, 10 s/frame) and 0–20 s (F6–7) after ganglionic application of BzATP (500 µM) in GFAP-GCaMP6s mice. For illustrative purposes, three regions of interest (ROIs) are marked with colored boxes and numbered. (B) Fluorescence intensity traces (F/F₀) of the ROIs shown in (A) before and after the BzATP (500 µM) treatment. Fluorescence intensity was measured for each frame (10 s/frame) and plotted against time or frame number. (C) SGC responses in GFAP-GCaMP6s mice were quantified by measuring the averaged fluorescence intensity in the whole imaging field before (F₀) and after (F) drug treatment. Fluorescence intensity was measured for each frame and plotted against time after ganglionic application of BzATP (5, 50, 500 µM, n = 6 mice/dose). The red arrow indicates the time of drug application. Data are expressed as mean ± SEM. *** p < 0.001 versus vehicle (0 µM); ### p < 0.001 versus indicated group. Two-way repeated measures ANOVA.

Figure 2

BzATP activates more large than small dorsal root ganglion (DRG) neurons in phase I. (A) Upper: Representative images showing changes in fluorescence intensity of L4 DRG neurons after ganglionic administration of BzATP (500 µM) in Pirt-GCaMP6s mice during in vivo calcium imaging in anesthetized mice. Baseline (Frames (F)1–5, 10 s/frame), phase I (F6–7), and phase II post-drug (F8–26). Examples of activated neurons are marked by colored circles and arrows. Lower: The red-outlined areas in phase I and phase II image are shown at higher magnification. DRG neurons were categorized according to the somal area as <450 μm² (small, S), 450–700 μm² (medium, M), and >700 μm² (large, L). (B) Fluorescence intensity traces (F/F₀) of selected neurons activated by BzATP in (A). Red arrow indicates the time of drug application. (C) Number of neurons in each subpopulation that were activated by BzATP (5, 50, 500 µM, n = 6 mice) in phase I (F6–7) and phase II (F8–26). Data are expressed as mean ± SEM. # p < 0.05, ## p < 0.01 versus indicated group. Two-way mixed-model ANOVA with Dunnett’s multiple comparisons test.

Figure 3

Pretreatment with A317491, A438079, and probenecid inhibited the activation of neurons and satellite glial cells (SGCs) by BzATP. (A) Upper: Representative images showing DRG neurons and SGCs after ganglionic administration of BzATP (100 µM) in Pirt:GFAP-GCaMP6s mice. Baseline (Frames (F)1–5, 10 s/frame), phase I (F6–7), and phase II post-drug (F8–26). Lower: The high-magnification images of the boxes outlined in the upper panel. (B) Upper: Examples of activated neurons (#1–4) and SGCs (#5–6) are marked with colored circles and arrows. DRG neurons were categorized by somal area as <450 μm² (small, S), 450–700 μm² (medium, M), and >700 μm² (large, L). Lower: Fluorescence-intensity traces (F/F₀) of DRG neurons and SGCs activated by BzATP. (C) Number of neurons in each subpopulation activated by BzATP (5, 50, 500 µM, n = 6 mice) in phase I and phase II. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 versus vehicle (0 µM); # p < 0.05, ## p < 0.01, ### p < 0.001 versus indicated group. Two-way mixed-model ANOVA with Dunnett’s multiple comparisons test. (D) Number of neurons in each subpopulation that were activated by BzATP (100 µM) after pretreatment with vehicle (ACSF), the P2X3R antagonist A317491 (100 µM), the P2X7R antagonist A438079 (100 µM), or the Panx1 antagonist probenecid (1 mM; n = 6 mice/group). Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 versus pre-drug; # p < 0.05, ## p < 0.01 versus vehicle + BzATP in the same phase. Two-way mixed-model ANOVA with Tukey’s multiple comparisons test. (E–G) Average SGC fluorescence intensity produced by BzATP (100 µM) application after pretreatment with A438079 (100 µM, n = 6 mice, E), A317491 (100 µM, n = 5 mice, F), probenecid (1 mM, n = 6 mice, G), or vehicle (ACSF, n = 6 mice). Fluorescence was measured for each frame and then plotted against time. Red arrow indicates the time of drug application. The curves representing the changes in fluorescence intensity after different drug treatments were compared between groups. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01 versus antagonist + BzATP; ### p < 0.001. Two-way mixed-model ANOVA with Tukey’s multiple comparisons test.

Figure 4

Changes in the percentage of DRG neurons that respond to brush and heat stimulation after ganglionic application of BzATP. (A) Upper: Representative images show L4 DRG neuronal fluorescence in response to brush stimulation (~1 Hz for 10 s) at the hind paw before and 5 min after BzATP (100 µM) application in Pirt-GCaMP6s mice. Fluorescence-intensity traces (F/F₀) of the activated neurons marked with colored arrows. Lower: Percentage of neurons in each size of population that responded to brush stimulation before and after BzATP treatment (n = 5 mice). DRG neurons were categorized according to somal area as <450 μm² (small), 450–700 μm² (medium), and >700 μm² (large). (B) Upper: Representative images show neuronal fluorescence in response to non-noxious heat stimulation of the hind paw (41 °C water bath, 10 s) before and 10 min after BzATP (100 µM) application. Lower: Percentage of neurons in each subpopulation that responded to heat stimulation (n = 5 mice). Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01 versus pre-drug. Paired t-test.

Figure 5

Effects of ganglionic BzATP application on spinal wide-dynamic-range (WDR) neuronal responses to electrical stimulation. (A) WDR neuron responses to supra-threshold electrical stimulation (3.0 mA, 2 ms) at the sciatic nerve were separated into A- and C-fiber components (number of action potentials) based on latency. Quantification of A-component and (B) C-component response to electrical stimulation before and 5 min after ganglionic application of BzATP (100 µM) with and without the P2X7R antagonist A438079 (100 µM, n = 5 mice/group; 1 mM, 200 µL), which was applied to a bath of ~2 mL ACSF 2 min before the agonist was added. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01 versus pre-drug. Paired t-test.

Figure 6

Expression of P2X3 protein in the dorsal root ganglion (DRG) of Pirt-GCaMP6s mice. (A) The P2X3 receptor immunoreactivity was detected in neurons, but not satellite glial cells (SGCs), in the DRG of Pirt-GCaMP6s mice. The distribution of P2X3 (red), GFP (green, which labels GCaMP6s-expressing neurons), and GFAP (blue, which labels SGCs) fluorescence signals in the DRG. * indicates P2X3 and GFP co-labeled neurons. (a,b,c) Single-label images of P2X3, GFP and GFAP. (d) An overlaid image of (a–c). (e,f,g) Double-label images. (h) A high-magnification image of the box outlined in (d). (B) Quantification of the percentage of P2X3–GFP co-labeled neurons in total GFP-positive neurons (n = 3 mice). (C) Distribution of somata diameters (5 µm bins) of P2X3-positive (+) neurons from lumbar DRGs of Pirt-GCaMP6s mouse (n = 200 neurons from 3 mice).

Figure 7

Hypothetical model illustrating the activation of DRG neurons and SGCs by BzATP. (A) 1. In phase I, BzATP strongly activates P2X7Rs, which are abundant in SGCs. To a smaller degree, BzATP may also activate other P2XR (e.g., P2X4R), which may be expressed on SGCs and large DRG neurons, as suggested by previous findings, and P2YR on SGCs. 2. In phase 2, the activation of SGCs and large neurons leads to Panx1 opening and release of ATP. 3. ATP further activates both small and large neurons and SGCs. 4. The activation of small neurons also leads to Panx1 opening and release of ATP to the extracellular space. 5. ATP, in turn, activates more SGCs and neurons. This process ends rapidly as ATP is degraded or cleared from the extracellular space. Neurons and SGCs return to the resting state, but neurons may become sensitized to subsequent stimulation. (B) The diagram illustrates the sequential activation of large neurons, SGCs, and small neurons after BzATP treatment.