Peripheral inflammation affects modulation of nociceptive synaptic transmission in the spinal cord induced by N-arachidonoylphosphatidylethanolamine

Abstract

Background and purpose: Endocannabinoids play an important role in modulating spinal nociceptive signalling, crucial for the development of pain. The cannabinoid CB₁ receptor and the TRPV1 cation channel are both activated by the endocannabinoid anandamide, a product of biosynthesis from the endogenous lipid precursor N-arachidonoylphosphatidylethanolamine (20:4-NAPE). Here, we report CB₁ receptor- and TRPV1-mediated effects of 20:4-NAPE on spinal synaptic transmission in control and inflammatory conditions.

Experimental approach: Spontaneous (sEPSCs) and dorsal root stimulation-evoked (eEPSCs) excitatory postsynaptic currents from superficial dorsal horn neurons in rat spinal cord slices were assessed. Peripheral inflammation was induced by carrageenan. Anandamide concentration was assessed by mass spectrometry.

Key results: Application of 20:4-NAPE increased anandamide concentration in vitro. 20:4-NAPE (20 μM) decreased sEPSCs frequency and eEPSCs amplitude in control and inflammatory conditions. The inhibitory effect of 20:4-NAPE was sensitive to CB₁ receptor antagonist PF514273 (0.2 μM) in both conditions, but to the TRPV1 antagonist SB366791 (10 μM) only after inflammation. After inflammation, 20:4-NAPE increased sEPSCs frequency in the presence of PF514273 and this increase was blocked by SB366791.

Conclusions and implications: While 20:4-NAPE treatment inhibited the excitatory synaptic transmission in both naive and inflammatory conditions, peripheral inflammation altered the underlying mechanisms. Our data indicate that 20:4-NAPE application induced mainly CB₁ receptor-mediated inhibitory effects in naive animals while TRPV1-mediated mechanisms were also involved after inflammation. Increasing anandamide levels for analgesic purposes by applying substrate for its local synthesis may be more effective than systemic anandamide application or inhibition of its degradation.

Linked articles: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Figure 1

Anandamide concentration after 20:4‐NAPE application to spinal cord slices. Three different concentrations of 20:4‐NAPE (20, 100 and 200 μM) were applied to spinal cord slices. Increasing content of anandamide was detected in the extracellular solution after 20:4‐NAPE application, in a concentration dependent manner. *P < 0.05, significantly different from control, ^# P < 0.05, significantly different from 20 and 100 μM 20:4‐NAPE; repeated measures ANOVA on ranks followed by Student–Newman–Keuls test; n = 5).

Figure 2

Inhibitory effect of 20:4‐NAPE application on excitatory postsynaptic currents in spinal cord slices from naive animals. (A) An example of native recording of spontaneous EPSCs from one superficial dorsal horn neuron before (CTRL) and during 20:4‐NAPE (20 μM) application. (B) Application of 20:4‐NAPE (20 μM) robustly decreased the average frequency of sEPSCs. *P < 0.05, significantly different from control; Wilcoxon signed‐rank test; n = 13. (C) This was also evident using cumulative histogram analysis. *P < 0.05, significantly different from control; Kolmogorov–Smirnov test. (D) Decrease of sEPSC amplitude was not significant using cumulative amplitude analysis. (E) Recording of dorsal root stimulation‐evoked EPSC from one neuron before and during 20:4‐NAPE (20 μM) application. (F) Acute application of 20:4‐NAPE (20 μM) significantly decreased the mean amplitude of eEPSCs. *P < 0.05, significantly different from control; Wilcoxon signed‐rank test; n = 15.

Figure 3

The effect of CB₁ receptor and TRPV1 channel antagonists on the 20:4‐NAPE‐induced inhibition of sEPSC frequency in naive slices. (A, C) The application of PF514273 (0.2 μM) alone did not change the frequency of sEPSCs (n = 11). The presence of PF514273 (0.2 μM) prevented 20:4‐NAPE (20 μM) from inhibiting the sEPSCs frequency (B, D) SB366791 (10 μM, n = 10) alone, did not change sEPSCs frequency. The presence of SB366791 (10 μM) did not prevent 20:4‐NAPE (20 μM) from decreasing sEPSCs frequency. *P < 0.05, significantly different from control, ^# P < 0.05, significantly different from SB366791 alone repeated measures ANOVA on ranks followed by Student–Newman–Keuls test. (E) The application of both antagonists, PF514273 (0.2 μM) with SB366791 (10 μM), did not change the frequency of sEPSCs (n = 8). Subsequent co‐application of PF514273 (0.2 μM), SB366791 (10 μM) with 20:4‐NAPE (20 μM) prevented the 20:4‐NAPE induced inhibition. (F) The same data are expressed as a percentage of previous recording conditions. For 20:4‐NAPE alone; *P < 0.05, significantly different from basal frequency of sEPSCs; Wilcoxon signed‐rank test; n = 13. For SB366791 + 20:4‐NAPE ;, *P < 0.05, significantly different from SB366791 pretreatment; Wilcoxon signed‐rank test; n = 10. ^# P < 0.05, significant difference between 20:4‐NAPE alone and PF514273 + 20:4‐NAPE; one‐way ANOVA followed by Student–Newman–Keuls test; n = 11.

Figure 4

Effect of CB₁ receptor and TRPV1 channel antagonists on 20:4‐NAPE‐induced inhibition of eEPSC amplitude in naive slices. (A, C) Pretreatment with PF514273 (0.2 μM, n = 13) did not change the amplitude of the recorded eEPSC in spinal cord slices prepared from naive animals. In the presence of PF514273 (0.2 μM), 20:4‐NAPE (20 μM) also did not change the amplitude of eEPSC (n = 13). (B, D) Pretreatment with SB366791 (10 μM) did not change the control eEPSC amplitude. In the presence of SB366791 (10 μM), 20:4‐NAPE (20 μM) decreased the eEPSC amplitude. *P < 0.05, significantly different from control, ^# P < 0.05, significantly different from SB366791 pretreatment; repeated measures ANOVA on ranks followed by Student–Newman–Keuls test; n = 10. (E) Data are shown as a percentage of previous condition to eliminate the effect of antagonist activity. *P < 0.05, significantly different from eEPSC basal amplitude; Wilcoxon signed‐rank test; n = 15. ^# P < 0.05, significantly different from SB366791 pretreatment; Wilcoxon signed‐rank test; n = 10.

Figure 5

Application of 20:4‐NAPE decreased the frequency of sEPSCs under inflammatory conditions. (A) Native recording from one superficial dorsal horn neuron before and during application of 20:4‐NAPE (20 μM) to spinal cord slice dissected 24 h after the induction of peripheral inflammation. (B) Application of 20:4‐NAPE (20 μM) decreased the frequency of sEPSCs. *P < 0.05, significantly different from control; Wilcoxon signed‐rank test; n = 9.

Figure 6

The effect of CB1 and TRPV1 antagonists on 20:4‐NAPE‐induced inhibition of sEPSC frequency under inflammatory conditions: (A, C) The application of PF514273 (0.2 μM, n = 16) did not change the frequency of sEPSCs. Subsequent co‐application of PF514273 (0.2 μM) and 20:4‐NAPE (20 μM) did not significantly change the frequency of sEPSCs, compared with control. (B, D) The frequency of sEPSCs significantly decreased during application of SB366791 (10 μM). *P < 0.05, significantly different from control; repeated measures (RM) ANOVA on ranks followed by Student–Newman–Keuls test; n = 16, In the presence of SB366791 (10 μM), 20:4‐NAPE (20 μM) induced a stronger decrease of sEPSC frequency. *P < 0.05, significantly different from control; RM ANOVA on ranks followed by Student–Newman–Keuls test. (E) The combined application of PF514273 (0.2 μM) and SB366791 (10 μM) did not change the frequency of sEPSCs. In the presence of both antagonists, 20:4‐NAPE (20 μM) significantly decreased the frequency of sEPSCs, compared with control. *P < 0.05, significantly different from control; RM ANOVA on ranks followed by Student–Newman–Keuls test; n = 15. (F) The same data are shown as a percentage of previous recording conditions: 20:4‐NAPE (n = 9) versus sEPSC basal frequency, PF514237 + 20:4‐NAPE versus PF514273 (n = 16) pretreatment, SB366791 + 20:4‐NAPE versus SB366791 (n = 16) pretreatment and PF514237 + SB366791 + 20:4‐NAPE versus both antagonists pretreatment (n = 15). *P < 0.05, significantly different from pretreatment, Wilcoxon signed‐rank test. ^# P < 0.05, significantly different from PF514237 + 20:4‐NAPE co‐application, one‐way ANOVA followed by Student–Newman–Keuls test.

Figure 7

Application of 20:4‐NAPE decreased the amplitude of evoked EPSCs in superficial dorsal horn neurons under inflammatory conditions. (A) An example of native recording from one nociceptive neuron before and during application of 20:4‐NAPE (20 μM) to acute spinal cord slice prepared 24 h after intraplantar injection of carrageenan. (B) Application of 20:4‐NAPE (20 μM) decreased the amplitude of eEPSCs. *P < 0.05, significantly different from control; Wilcoxon signed‐rank test; n = 14.

Figure 8

Antagonists of CB₁ receptors and TRPV1 channels blocked the 20:4‐NAPE‐induced decrease of eEPSC amplitude under inflammatory conditions. (A, C) PF514273 alone (0.2 μM, n = 16) did not change the amplitude of eEPSCs. In the presence of PF514273, 20:4‐NAPE (20 μM) did not significantly affect the amplitude of eEPSCs. (B, D) Pretreatment with SB366791 (10 μM, n = 11) did not change the amplitude of eEPSC. In the presence of SB366791 (10 μM), 20:4‐NAPE (20 μM) also did not change the eEPSC amplitude. (E) The same data are shown as a percentage of previous recording conditions: *P < 0.05, significantly different from eEPSC basal amplitude; Wilcoxon signed‐rank test; n = 14.