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. 2010 Aug 11;30(32):10860-71.
doi: 10.1523/JNEUROSCI.1980-10.2010.

Small RNAs Control Sodium Channel Expression, Nociceptor Excitability, and Pain Thresholds

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Free PMC article

Small RNAs Control Sodium Channel Expression, Nociceptor Excitability, and Pain Thresholds

Jing Zhao et al. J Neurosci. .
Free PMC article

Abstract

To examine the role of small RNAs in peripheral pain pathways, we deleted the enzyme Dicer in mouse postmitotic damage-sensing neurons. We used a Nav1.8-Cre mouse to target those nociceptors important for inflammatory pain. The conditional null mice were healthy with a normal number of sensory neurons and normal acute pain thresholds. Behavioral studies showed that inflammatory pain was attenuated or abolished. Inflammatory mediators failed to enhance excitability of Nav1.8+ sensory neurons from null mutant mice. Acute noxious input into the dorsal horn of the spinal cord was apparently normal, but the increased input associated with inflammatory pain measured using c-Fos staining was diminished. Microarray and quantitative real-time reverse-transcription PCR (qRT-PCR) analysis showed that Dicer deletion lead to the upregulation of many broadly expressed mRNA transcripts in dorsal root ganglia. By contrast, nociceptor-associated mRNA transcripts (e.g., Nav1.8, P2xr3, and Runx-1) were downregulated, resulting in lower levels of protein and functional expression. qRT-PCR analysis also showed lowered levels of expression of nociceptor-specific pre-mRNA transcripts. MicroRNA microarray and deep sequencing identified known and novel nociceptor microRNAs in mouse Nav1.8+ sensory neurons that may regulate nociceptor gene expression.

Figures

Figure 1.
Figure 1.
Neural marker expression in DRGs of Dicer conditional null mice. A, DRG sections of Dicer conditional null mice and Dicer floxed littermate controls were labeled with anti-neurofilament (N200 and N52), anti-peripherin, anti-CGRP, anti-IB4, and anti-c-RET antibodies. The panels in the columns marked “Merged” are merged images of the panels in the two columns to the left. B, Cross sections of lumbar spinal cord were stained with anti-CGRP and IB4. In lamina I-II, CGRP-positive terminals were identified in both the Dicer knock-out mice and floxed littermate controls, whereas all detectable IB4-positive terminals were deleted compared with littermate controls. C, The proportions of neurofilament (N200) and peripherin-expressing neurons were normal in Dicer conditional null animals compared with floxed littermate controls. WT, White symbols; KO, black symbols. Scale bars, 50 μm.
Figure 2.
Figure 2.
Acute pain behavior and in vivo electrophysiology of wide dynamic range (WDR) in Dicer mutant mice. A, Rotarod studies showed no motor deficits in conditional Dicer-null animals (n = 12, WT; n = 20, KO). B, Acute mechanical pressure applied with the Randall–Selitto apparatus demonstrated identical behavior in KO and WT mice (n = 12, WT; n = 20, KO). C, Responses to low-threshold mechanical stimulation by Von Frey filaments are normal in the null mutants (n = 8, WT; n = 15, KO). D, Hargreaves' apparatus demonstrates identical latencies of response to thermal stimulation (n = 8, WT: n = 16, KO). E, Supra-spinal reflexes to heat (50 and 55°C) using the hot plate apparatus demonstrates identical latencies in KO and WT mice (n = 9, WT; n = 14, KO). F, Cold behavior (number of liftings) is reduced in the Dicer-null mutant mice (0°C) using the cold plate apparatus (n = 16, WT; n = 22, KO). G, A-fiber and C-fiber firing thresholds of WDR neurons in control Nav1.8Cre+/− mice (black) (n = 14) and Dicer KO mice (white) (n = 16). H, Responses to transcutaneous electrical stimulation of the receptive field (a train of 16 2-ms-wide electrical pulses at 0.5 Hz at 3× C-fiber threshold). Spikes evoked between 0 and 50 ms were classified as evoked by A-fiber input, and those evoked between 50 and 250 ms were classified as evoked by C-fiber input. Postdischarge (PD) describes the spikes that are evoked between 250 and 800 ms and correlate with C-fiber activity. Input is determined by the first stimulus of the train × 16 and reflects nonpotentiated C-fiber-evoked WDR neuron responses. Wind-up (WU) is determined by calculating additional spikes above the predicted constant baseline response and describes the increased neuronal excitability evoked by C-fibers to repeated constant stimulation. C-fiber activity, wind-up, and postdischarge were all significantly decreased in diphtheria toxin A chain (DTA) mice. I, Evoked responses from innocuous dynamic brush, noxious pinch, and a cold (1°C) water jet stimulus. J, Evoked responses from Nav1.8 Cre+/− mice (solid line) and Dicer−/− mice (dashed line) to graded von Frey filaments. K, Thermally evoked responses using water jets at graded temperatures (Temp). There was no significant difference between the two groups.
Figure 3.
Figure 3.
Inflammatory, neuropathic pain behavior, c-Fos expression in spinal cord, and excitability of DRG neurons. A, Time course of pain behavior in Dicer conditional null mutants after intraplantar injection of formalin. Time spent licking/biting the injected hindpaw was recorded (n = 8, WT; n = 10, KO). B, Two phases (0–10 min, 10–60 min) of licking behavior after formalin injection were analyzed, the second phase of which is attenuated in the Dicer-null mice (p < 0.01, t test). C, Intraplantar injection of carrageenan (n = 9, WT; n = 11, KO) caused a long-term thermal hyperalgesia that is attenuated in Dicer conditional mutant mice (p < 0.05, two-way repeated measured ANOVA). D, CFA-induced mechanical hyperalgesia was measured using von Frey filaments (n = 5, WT; n = 7, KO). A strong difference was observed. (p < 0.01, two-way repeated measured ANOVA). E, CFA-induced thermal hyperalgesia was measured using the Hargreaves instrument (n = 5, WT; n = 7, KO). A significant difference was observed (p < 0.05, two-way repeated measured ANOVA). F, Behavioral signs of spontaneous pain behavior were tested on CFA-treated mice. The number of spontaneous foot liftings (SFLs) was measured on days 1, 2, and 4 after intradermal injections of CFA in the right hind-paw and right knee (n = 11, WT; n = 10, KO). SFL on day 1 was much less in Dicer-null mutant mice than in floxed control mice (p < 0.05, t test). G, Sciatic nerve ligation following the Seltzer procedure caused a long-lasting mechanical hyperalgesia. Both Dicer conditional null mutants and floxed Dicer littermate controls showed an identical time course and level of allodynia after this injury. All data presented as means ± SEM; *p < 0.05, **p < 0.01. WT, White symbols, KO, black symbols.
Figure 4.
Figure 4.
c-Fos response in the spinal cord and sensitization of nociceptors by inflammatory mediator(s). A, Representative immunostaining for c-Fos in superficial laminae I and II of spinal cord. Contra, Contralateral side to formalin injection; Ispi, ipsilateral side to formalin injection. Scale bar, 100 μm. B, Quantification of c-Fos-positive neurons. The number of c-Fos-positive nuclei in superficial (laminae I, II) cord in L4 to L5 ipsilateral to the injection of formalin was counted (n = 4, WT; n = 5, KO). The number of c-Fos-positive neurons is reduced in the ipsilateral side in Dicer-null mutants (p < 0.05, t test). C, Sensitization of WT and KO nociceptors by inflammatory mediators. Top, Representative traces showing action potential firing produced by a 100 pA depolarizing current in control conditions (left) and in the presence of inflammatory soup (IS, right) in a WT DRG neuron. Bottom, Same as top, but in a Dicer KO DRG neuron.
Figure 5.
Figure 5.
Microarray analysis. A, Summary of changed transcripts in Dicer conditional null mutant. After isolation of total RNA from DRGs of Dicer conditional null mice (n = 6, males) and floxed littermate control (n = 6, males), the expression of mRNA was detected with GeneChip (mouse genome 430 2.0 array). Four thousand six hundred and eighteen (4618) transcripts (p < 0.05) (left) and 2015 transcripts (p < 0.01) (right), changed from 43,086 transcripts in the DRGs of Dicer conditional null mice compared with floxed littermate controls. B, The number of transcripts with fold change is ≥4 and ≥2, respectively, in Dicer conditional null mutant versus floxed littermate controls (p < 0.01). C, The fold change of representative genes in Dicer conditional null mutant, p < 0.0001.
Figure 6.
Figure 6.
Representative qRT-PCR analysis of selected transcripts and correlation between fold decrease in Nav1.8+ neuron-depleted mice [diphtheria toxin A chain (DTA)] (Abrahamsen et al., 2008) and fold decrease in Dicer conditional null mutants. A, After isolation of DRG, total RNA was extracted from DRG. Representative mRNA and pre-mRNAs were subjected to analysis by qRT-PCR. Some of the DRG-specific gene (Edg7, TRPC6, Nav1.9, Nav1.8, Nav1.7, Runx1, CaMKIIa, and Mrgpra3) transcripts were significantly downregulated in Dicer knock-out mice. B, Representative DRG nonspecific gene (Mtap2, Gbp1, ApoD, and Rgs5) transcripts show significant upregulation in Dicer knock-out mice. C, The pre-mRNAs of DRG-specific genes (Nav1.8, Nav1.9) and Edg7 and CaMKIIa were downregulated in Dicer knock-out mice. No alteration of pre-mRNA of ApoD was detected in Dicer knock-out mice. The data are normalized with β-actin. Each bar represents n = 3 mice. All data are presented as means ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, using t test. WT, White symbols, KO, black symbols. D, The Pearson product-moment correlation (r) measures the strength of the association between Log2 fold change of gene expression in DTA mice and Log2 fold change of gene expression in Dicer KO. The correlation coefficient is r = 0.937. If there was a perfect correlation (r = 1), all points would fall along the straight diagonal line.
Figure 7.
Figure 7.
Expression and function of representative genes in DRGs of Dicer conditional null mice. A, Protein was extracted from DRGs and CaMKIIa and ApoD were analyzed with Western blots. B, Western blot relative density. The experiments were repeated at least three separate times. The data are normalized with β-actin. Each bar represents n = 3 mice. C, DRG sections were labeled with anti-CaMKIIa antibody and anti-neurofilament (N200) antibody. CaMKIIa is mainly expressed in small diameter DRG neurons of floxed dicer littermate controls, but not in Dicer conditional null mice. D, TTX-resistant sodium currents were measured in small diameter (≤25 μm) DRG neurons (n = 23) and presented as maximum current density. E, Effect of cycloheximide on expression of Nav1.8. The DRGs from Dicer conditional null mice and floxed littermate controls were cultured in DMEM at 37°C, 5% CO2. Three hours after treatment with or without 10 μg/ml cycloheximide, mRNA and pre-mRNA of Nav1.8 from DRG were subjected to analysis by qRT-PCR. The data are normalized with β-actin. Each bar represents n = 3 mice. F, Effect of cycloheximide on expression of ApoD. The data are normalized with β-actin. Each bar represents n = 3 mice. All data presented as means ± SEM; *p < 0.05, **p < 0.01, using t test. WT, White symbols, KO, black symbols. Scale bars, 50 μm.

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