Cannabinoid CB1 receptor facilitation of substance P release in the rat spinal cord, measured as neurokinin 1 receptor internalization

Abstract

The contribution of CB1 receptors in the spinal cord to cannabinoid analgesia is still unclear. The objective of this study was to investigate the effect of CB1 receptors on substance P release from primary afferent terminals in the spinal cord. Substance P release was measured as neurokinin 1 (NK1) receptor internalization in lamina I neurons. It was induced in spinal cord slices by dorsal root stimulation and in live rats by a noxious stimulus. In spinal cord slices, the CB1 receptor antagonists AM251, AM281 and rimonabant partially but potently inhibited NK1 receptor internalization induced by electrical stimulation of the dorsal root. This was due to an inhibition of substance P release and not of NK1 receptor internalization itself, because AM251 and AM281 did not inhibit NK1 receptor internalization induced by exogenous substance P. The CB1 receptor agonist ACEA increased NK1 receptor internalization evoked by dorsal root stimulation. The effects of AM251 and ACEA cancelled each other. In vivo, AM251 injected intrathecally decreased NK1 receptor internalization in spinal segments L5 and L6 induced by noxious hind paw clamp. Intrathecal AM251 also produced analgesia to radiant heat stimulation of the paw. The inhibition by AM251 of NK1 receptor internalization was reversed by antagonists of mu-opioid and GABA(B) receptors. This indicates that CB1 receptors facilitate substance P release by inhibiting the release of GABA and opioids next to primary afferent terminals, producing disinhibition. This results in a pronociceptive effect of CB1 receptors in the spinal cord.

Fig. 1. Images of NK1R neurons in lamina I after dorsal root stimulation

Spinal cord slices were stimulated at the dorsal root at 100 Hz while they were superfused with aCSF alone (A, B), 100 nM AM281 (C), or 100 nM AM251 plus 10 μM CTAP (D). Images in panels A and B were taken from the same histological section and correspond to the dorsal horns contralateral (contra, A) and ipsilateral (ipsi, B) to the stimulated root. C and D are from the ipsilateral dorsal horn. Main panels: images taken with a 20x objective, with a voxel size of 488 × 488 × 1180 nm and 5 confocal planes. Insets: images of lamina I neurons taken with a 100x objective, with a voxel size of 98 × 98 × 285 nm and 3 confocal planes. Scale bars are 50 μm for the main panels and 5 μm for the insets. Neurons with NK1R internalization are indicated with “*” and neurons without internalization by “o”.

Fig. 2. Effect of CB1 receptor agonists and antagonists on NK1R internalization evoked by dorsal root stimulation

Spinal cord slices were stimulated at the dorsal root with 1000 pulses (20 V, 0.4 ms) delivered at 1 Hz (A) or 100 Hz (B) while they were superfused with the indicated compounds (all at 100 nM). Control was aCSF alone. AM251, AM281 and rimonabant are CB1 receptor antagonists, ACEA is a CB1 receptor agonist and O-2640 is a GPR55 agonist. Numbers inside the bars indicate the number of slices used for each set of data (N). Two-way ANOVA yielded p<0.0001 overall for the two variables (drugs and stimulus). Bonferroni’s post-hoc tests: *** p<0.001; ** p<0.01; * p<0.05 compared to control; ††† p<0.001, †† p<0.01, as indicated.

Fig. 3. NK1R internalization induced by exogenous substance P was not affected by CB1 antagonists

Slices were incubated at 35 °C for 10 min with 1 μM substance P alone (control) or with the compounds indicated (all 100 nM). One-way ANOVA: p =0.27.

Fig. 4. Concentration-responses of the CB1 antagonists AM251 and AM281

Slices were superfused with the CB1 antagonists AM251 (A) or the CB1 inverse agonist AM281 (B) while the dorsal root was stimulated at 100 Hz. Data are the mean ± SEM of 3 slices (control, 9 slices). NK1R internalization contralateral to the root (filled symbols) was negligible and unaffected by AM251 or AM281. NK1R internalization ipsilateral to the root (empty symbols) was inhibited in a dose-dependent way by both drugs. Curves represent fitting by non-linear regression to a dose-response function: AM251, IC₅₀ = 13 nM (95% CI, 2-72 nM), ‘bottom’ = 21 ± 5%; AM281, IC₅₀ = 6 nM (95% CI, 2-16 nM), ‘bottom’ = 27 ± 3%. The outlier at 1 μM AM281 was excluded from the fitting. Two-way ANOVA revealed significant effects of the drugs and the stimulus (p<0.001). Bonferroni’s post-hoc tests: * p<0.05, ** p<0.01, *** p<0.001.

Fig. 5. Concentration-response of the CB1 agonist ACEA

Slices were superfused with the CB1 receptor agonist ACEA while the dorsal root was stimulated at 1 Hz. Data are the mean ± SEM of 3-5 slices. NK1R internalization contralateral to the root (filled symbols) was negligible and unaffected by ACEA (filled symbols). NK1R internalization ipsilateral to the root (empty symbols) was increased by ACEA. The curve represents a tentative fitting of the points (excluding the outlier at 300 nM ACEA) to a dose-response function, with the maximum effect (‘top’) fixed at 100%. EC₅₀ = 175 nM (95% CI, 2 nM-17 μM). Two-way ANOVA revealed significant effects of ACEA (p=0.0008) and the stimulus (p<0.0001). Bonferroni’s post-hoc tests: *** p<0.001.

Fig. 6. Effect of AM251 on capsaicin-evoked NK1R internalization

A. The dorsal root was immersed in 1 μM capsaicin for 10 min in a compartment separated from the slice, while the slice was superfused with aCSF alone (control) or 1 μM AM251. Two-way ANOVA yielded p<0.0001 for the two variables (AM251 and capsaicin). Bonferroni’s post-hoc test: *** p<0.001. B. Slices were incubated for 10 min at 35 °C with 0.3 μM capsaicin alone (control) or with 1 μM AM251. Numbers inside the bars indicate the number of slices used for each set of data.

Fig. 7. AM251 inhibits NK1R internalization induced by noxious stimulation

Rats (N =3 per group) were injected intrathecally with 10 μl AM251 (10 nmol) or vehicle (10% DMSO, 1% Tocrisolve in saline; control). Substance P release was induced by clamping of the hind paw with a hemostat for 30 s, 10 min after the injection. After 10 min more the rats were euthanized and fixed. Two-way ANOVA yielded p=0.0014 for AM251 and p<0.0001 for spinal region. Bonferroni’s post-hoc test: ** p<0.01.

Fig. 8. Analgesia produced by AM251

Analgesia was measured as increases in latency in paw withdrawal responses to radiant heat. Baseline latencies were measured at 5 min intervals three times. Immediately after baseline determination, rats received intrathecal injections of 1 nmol AM251 (N =5) dissolved in 1% DMSO or 10 nmol AM251 (N =5) dissolved in 10% DMSO, 1% Tocrisolve. Control rats (N =7) received vehicle: 1% DMSO (4 rats) or 10% DMSO, 1% Tocrisolve (3 rats). Control values with the two vehicles were essentially the same and were pooled in the figure. Ten minutes after the injection, paw withdrawal latencies were measured at 5 min intervals. Two-way ANOVA revealed a significant effect of AM251 (p<0.0001) but not of time (p=0.19) or the interaction of the two variables (p=0.63). Bonferroni’s post-hoc test: * p<0.05, ** p<0.01, *** p<0.001.

Fig. 9. Reversal by MOR or GABA_B antagonists of the inhibition by AM251

Spinal cord slices were stimulated at the dorsal root with 1000 pulses delivered at 100 Hz (A) or 1 Hz (B) while they were superfused AM251 (100 nM), CGP55845 (100 nM) and CTAP (10 μM), alone or combined as indicated. Control was aCSF alone. Numbers inside the bars indicate the number of slices used (N). Two-way ANOVAs: A (100 Hz), p<0.0001 for the variables ‘drugs’, ‘stimulus’ and their interaction; B (1 Hz), p<0.0001 for ‘stimulus’, p=0.041 for ‘drugs’, p=0.012 for their interaction. Bonferroni’s post-hoc tests: *** p<0.001; ** p<0.01, compared to control; ††† p<0.001, †† p<0.01, as indicated.

Fig. 10. Diagram showing the proposed disinhibition mechanism for the facilitation of substance P release by CB1 receptors

Dorsal horn interneurons release GABA or opioids (opi) next to substance P-containing primary afferent terminals. MORs or GABA_B receptors (GABA_BR) coupling to α_o G proteins (G_o) inhibit substance P release. CB1 receptors (CB1R) in the GABAergic and opioidergic terminals inhibit the release of GABA and opioids, preventing the effect of the MORs and GABA_B receptors.