Synchronized cluster firing, a distinct form of sensory neuron activation, drives spontaneous pain

Abstract

Spontaneous pain refers to pain occurring without external stimuli. It is a primary complaint in chronic pain conditions and remains difficult to treat. Moreover, the mechanisms underlying spontaneous pain remain poorly understood. Here we employed in vivo imaging of dorsal root ganglion (DRG) neurons and discovered a distinct form of abnormal spontaneous activity following peripheral nerve injury: clusters of adjacent DRG neurons firing synchronously and sporadically. The level of cluster firing correlated directly with nerve injury-induced spontaneous pain behaviors. Furthermore, we demonstrated that cluster firing is triggered by activity of sympathetic nerves, which sprout into DRGs after injury, and identified norepinephrine as a key neurotransmitter mediating this unique firing. Chemogenetic and pharmacological manipulations of sympathetic activity and norepinephrine receptors suggest that they are necessary and sufficient for DRG cluster firing and spontaneous pain behavior. Therefore, blocking sympathetically mediated cluster firing may be a new paradigm for treating spontaneous pain.

Keywords: DRG; adrenergic receptors; cluster firing; dorsal root ganglion neuron; neuropathic spontaneous pain; norepinephrine; sympathetic sprouting.

Figure 1.. Spontaneous CFEs revealed by in vivo DRG calcium imaging after peripheral nerve injury.

(A) Upper: Diagram showing the mating strategy. Middle: Experimental flowchart. Lower: Schematic diagram illustrating in vivo calcium imaging. (B) Representative images of one cluster (orange circle) observed during in vivo calcium imaging of L4 DRG (white outline indicates DRG border) in SNI mouse. Image shows neurons in the cluster firing synchronously at different times (B0– B3). (C) Time course of calcium transients in individual neurons from the cluster in (B), in which all neurons are labeled and traced. (D) Time course of calcium transients in 15 individual singly firing neurons scattered throughout the DRG in (B), labels not shown. (E) Pairwise correlations of 30 neurons in (C) and (D). Left: all pairs’ correlations; color bar indicates the correlations of cluster firing neurons (neuron ID 1 to 15) and singly firing neurons (neuron ID 16-30) that form the pairs. Right: pairs of cluster firing neurons show significantly stronger synchrony than pairs of singly firing neurons. The synchrony index was calculated as the average correlation. There are 15 neurons in each group, so there are 105 correlation values in each group. (F) The distribution of diameters of clustered firing neurons, summarized from 18 mice with cluster firing data. (G) Spontaneous pain scores in naïve mice and SNI mice (Day 21) with and without cluster firing. (H) Pearson correlation between the frequencies of cluster firing recorded during in vivo imaging and spontaneous pain scores (from SNI mice in (G) that showed cluster firing). (I-J) Mechanical pain induced by low force (0.07g) and high force (0.4g) von Frey filaments in SNI mice without and with cluster firing. (K) Diagram for locations of clusters within the whole DRG summarizing 58 clusters identified in 22 SNI mice with cluster firing. Scale bar, 100 μm. Each open circle in (G) to (J) represents an individual mouse. Data are represented as mean ± SEM. **p < 0.01; ****p < 0.0001; n.s., not significant; (E) by unpaired two-tailed t test; (G) by One-way ANOVA with Tukey’s posttest; (I) and (J) by two-way repeated measures ANOVA with Sidak’s posttest.

Figure 2.. Sympathetic nerve fibers sprout into DRGs after peripheral nerve injury

(A) Diagram showing the mating strategy. GFP is expressed in DRG neurons, tdT is expressed in the sympathetic axons. (B–D) Whole-mount cleared L4 DRGs of mice with and without SNI (day 21). (B0–B2) Naive mouse. (B3–B5) SNI mouse. (C) Magnification of the boxed region in (B2), showing no tdT-positive fibers sprouting into the DRG. (D) Magnification of the boxed region in (B5), showing tdT-positive fibers sprouting into the DRG. (E) Schematic of retrograde CTB tracing in a sympathetic ganglion. The red needle indicates the position of CTB555 injection. (F) The experimental procedure: SNI surgery on day 0, injection of CTB555 into L4 DRGs on day 13, and dissection of L3 sympathetic ganglia on day 21. (G) Representative images of L3 sympathetic ganglia of naive and SNI mice. (H) Summary data of the number of CTB-positive neurons in the L3 sympathetic ganglion. Scale bars, 100 um. Each open circle represents an individual mouse. Data are presented as mean ± SEM. *p < 0.05; (H) by two-tailed unpaired t test.

Figure 3.. Pharmacological inhibition of sympathetic nerve activity in the DRG and mSYMPX reduce incidence of CFEs.

(A) Diagram showing the mating strategy. Pirt-Cre; GCaMP6s mice were used. (B) Experimental schematic. We first imaged the mice with SNI at POD 21-28 for 2 hours. If CFEs were observed, 0.5 mM 6-OHDA solution was then applied topically onto the DRG and allowed to incubate for 30 minutes, followed by an additional 2 hours of recording with about 5 to 10 μL 6-OHDA solution covering the DRG. (C) An example of cluster firing that decreased after 6-OHDA application. Left is before using 6-OHDA, and there are 2 clusters (orange and purple circle). (D) Representative traces of individual neurons in the orange cluster which are numbered in (C) before and after addition of 6-OHDA. (E-G) The total number of clusters (E), frequency of cluster firing (F), and total number of cluster firing neurons (G) significantly decreased after addition of 6-OHDA to DRG but not after addition of saline. (H) An example of a cluster firing event in the L4 DRG, 4 weeks after SNI. Before (upper) and after (lower) localized microsympathectomy (mSYMPX). (I) Representative traces of individual neurons in (H) before mSYMPX. (J) The number of CFEs was reduced by mSYMPX, whether performed acutely during the recording, at the time of SNI, or 7 days prior to recording after the SNI model had been established for 4 weeks. Scale bar, 100 μm (C); 250 μm (H). Each pair of open circles (before and after) represents an individual mouse. Data are represented as mean ± SEM. **p < 0.01; ****p < 0.0001; n.s., not significant; (E) to (G) by two-way repeated measures ANOVA with Sidak’s posttest, (J) by oneway ANOVA with Dunnett’s posttest.

Figure 4.. Chemogenetic silencing of sympathetic nerve activity in the DRG diminishes the incidence of CFEs and relieves spontaneous pain.

(A) Diagram showing the mating strategy and intrathecal injection with AAV9.CAG.GCaMP6s in Phox2bCre; GiDREADD mice. (B) Diagram showing the experimental procedure. On POD 21, in vivo imaging of L4 DRG was first recorded for 2 hours followed by topical application of 10 μM CNO or vehicle (0.1%DMSO) on the surface of the DRG to locally activate the DREADD receptors in sympathetic nerves. In vivo imaging was then resumed for an additional 2 hours. (C) An example of decreased cluster firing after local addition of CNO on the DRG. Left is quiescent (i.e. period with no cluster firing); Middle is an example of cluster firing before CNO addition. There are 3 clusters (orange, purple and red circles). Right is after CNO addition, all 3 clusters were diminished. (D) Representative traces of neurons in the orange cluster which are numbered in (C) before and after administration of CNO. (E-G) The total number of clusters (E), frequency of cluster firing (F), and total number of cluster firing neurons (G) is significantly inhibited after addition of CNO to DRG, but not with addition of vehicle (0.1% DMSO in saline). (H) Spontaneous pain scores significantly decreased after i.t. administration of CNO in SNI mice (Day 20) with cluster firing. No change was observed with administration of vehicle. Scale bar, 100 μm. Each pair of open circles (before and after) represents an individual mouse. Data are represented as mean ± SEM. ***p < 0.001; ****p < 0.0001; n.s., not significant; (E) to (H) by two-way repeated measures ANOVA with Sidak’s posttest.

Figure 5.. Chemogenetic activation of sympathetic nerve activity in the DRG enhances the incidence of CFEs and spontaneous Pain.

(A) Diagram showing the mating strategy and intrathecal injection with AAV9.CAG.GCaMP6s in Phox2bCre; GqDREADD mice. (B) Diagram showing the experimental procedure. The details are the same as described in Figure 4B. (C) An example of increased cluster firing after local addition of CNO on DRG. Left is quiescent; Middle is an example of cluster firing before addition of CNO. There are 2 clusters (orange and purple circles); Right is after addition of CNO, and there are 3 clusters (orange, purple and red circles). (D) Representative traces of neurons in the orange cluster which are numbered in (c) before and after administration of CNO. (E-G) The total number of clusters (E), frequency of cluster firing (F), and total number of cluster firing neurons (G) significantly increased after addition of CNO but not vehicle to the DRG. (H) Spontaneous pain scores significantly increased after i.t. administration of CNO to SNI mice (Day 20) with cluster firing, but not after administration of vehicle. Scale bar, 100 μm. Each pair of open circles (before and after) represents an individual mouse. Data are represented as mean ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001; n.s., not significant;(E) to (H) by two-way repeated measures ANOVA with Sidak’s posttest.

Figure 6.. Endogenous NE release in DRGs after SNI.

(A) Diagram showing the i.t. injection with AAV9.CAG.GRABNE in WT mice. (B) Diagram showing the experimental procedure. (C) Representative images of NE sensor signals of naive and SNI mice. Naive mice had no endogenous NE released. Mice with SNI show endogenous NE released sporadically (white rectangle). At the end of the recording session exogenous NE is dropped onto DRG (both naive and SNI mice showed NE sensor activation). (D-E) Enlarged image of rectangle area from SNI image in (C). Red arrowheads indicate 12 neurons with spontaneous NE signals (cluster activation) from quiescent (D) and release periods (E). (F-G) Averaged NE signal traces with ±95% confidence intervals for the 12 neurons indicated by arrowheads in SNI mouse (F) and 12 randomly selected neurons from naive mouse (G). (H) Summary of NE sensor imaging from SNI and Naive mice. DRG of SNI mice exhibit a significantly higher number of neurons responding to endogenous NE release. Scale bar, 100 μm. Each open circle represents an individual mouse. Data are represented as mean ± SEM. **p < 0.01; (H) by two-tailed unpaired t test.

Figure 7.. Block of adrenergic receptors in DRG reduces incidence of CFEs and relieves spontaneous pain.

(A) Diagram showing the mating strategy. (B) Diagram showing the experimental procedure. We imaged the Pirt-GCaMP6 mice with SNI at POD 21-28 for 2 hours. If CFEs were observed, a sympathetic blocker, either phentolamine (5 μM), or propranolol (1 μM) or vehicle (saline) solution was then applied topically onto the DRG and allowed to incubate for 15 minutes, followed by an additional 2 hours of recording with about 5 to 10 μL solution covering the DRG. (C) An example of cluster firing that decreased after using phentolamine (antagonist of α-receptor). There are 3 clusters (orange, purple and red circles) before addition of phentolamine. All clusters are inhibited after. (D) Representative traces of neurons in the orange cluster which are numbered in (C) before and after addition of phentolamine. (E) An example of cluster firing that decreased after using propranolol (antagonist of β-receptor). There are 2 clusters (orange and purple circles) before addition of propranolol. Both clusters are inhibited after. (F) Representative traces of neurons in the orange cluster which are numbered in (E) before and after addition of propranolol. (G-I) The total number of clusters (G), frequency of cluster firing (H), and total number of cluster firing neurons (I) significantly decreased after addition of antagonists of adrenergic receptors on DRG, but not with addition of saline. The data of the saline group are the same as in Fig. 2. (J) Spontaneous pain scores significantly decreased after i.t. administration of either 5μL, 10μM phentolamine or 5μL, 10μM propranolol, or 5μL vehicle alone to SNI mice, but not after administration of vehicle. Scale bar, 100 μm. Each pair of open circles (before and after) represents an individual mouse. Data are represented as mean ± SEM. **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s., not significant; (G) to (J) by two-way repeated measures ANOVA with Sidak’s posttest.