Spinal endocannabinoids and CB1 receptors mediate C-fiber-induced heterosynaptic pain sensitization

Abstract

Diminished synaptic inhibition in the spinal dorsal horn is a major contributor to chronic pain. Pathways that reduce synaptic inhibition in inflammatory and neuropathic pain states have been identified, but central hyperalgesia and diminished dorsal horn synaptic inhibition also occur in the absence of inflammation or neuropathy, solely triggered by intense nociceptive (C-fiber) input to the spinal dorsal horn. We found that endocannabinoids, produced upon strong nociceptive stimulation, activated type 1 cannabinoid (CB1) receptors on inhibitory dorsal horn neurons to reduce the synaptic release of gamma-aminobutyric acid and glycine and thus rendered nociceptive neurons excitable by nonpainful stimuli. Our results suggest that spinal endocannabinoids and CB1 receptors on inhibitory dorsal horn interneurons act as mediators of heterosynaptic pain sensitization and play an unexpected role in dorsal horn pain-controlling circuits.

Fig. 1

Synaptic effects of CB₁ receptor activation in dorsal horn neuronal circuits. (A–C) Effects of the mixed CB₁/CB₂ receptor agonist WIN 55,212-2 (3 µM) on glycinergic IPSCs (A), GABAergic IPSCs (B) and AMPA-EPSCs (C). Left: current traces averaged from 10 consecutive stimulations under control conditions, after addition of WIN 55,212-2 and after the additional application of AM 251 (5 µM). Right: time course. Mean ± sem, n = 7 – 13 neurons. (D) Inhibition of glycinergic IPSCs in non-glycinergic (EGFP-negative) neurons (n = 8) by the mGluR1/5 agonist DHPG (10 µM) and its reversal by AM 251 (5 µM). Only a minor inhibition was observed in glycinergic (EGFP-positive) neurons (n = 8). (E) DSI (1 s depolarization of the postsynaptic neuron to 0 mV) in non-glycinergic neurons (6 out of 8 neurons) and its prevention by AM 251 (5 µM). No DSI occurred in glycinergic neurons (n = 5). Glycinergic IPSCs were evoked at a frequency of 0.2 Hz.

Fig. 2

Inhibition of glycinergic and GABAergic synaptic transmission via presynaptic CB₁ receptors. (A) Paired pulse experiments. Current traces of two consecutive glycinergic IPSCs (P1 and P2) under control conditions (black) and in the presence of 3 µM WIN 55,212-2 (red). (B) Variation analysis. Top: individual traces of glycinergic IPSCs recorded under control conditions and in the presence of WIN 55,212-2 (3 µM). Bottom: changes in the coefficient of variation in 13 cells are plotted versus changes in the mean amplitude induced by WIN 55,212-2. (C–F) Electron microscopic analysis (a–c and a–b, serial sections) of CB₁ receptor localization in the superficial spinal dorsal horn. Arrowheads, symmetrical synapses. Arrows, immunogold labeling. (Ca–Cc) CB₁-immunostaining coupled to immunoperoxidase reaction (DAB). CB₁ receptors are present in an axon terminal (t) forming a symmetric (inhibitory) synapse on an immuno-negative dendritic shaft (d) in lamina II. Asterisk labels a CB₁-negative bouton of another symmetric synapse on the same dendrite. (Da–Db) High-resolution pre-embedding immunogold staining for CB₁. CB₁ receptor located presynaptically on the plasma membrane of an inhibitory axon terminal (t). (Ea–Eb) DAB staining for vesicular inhibitory aminoacid transporter (VIAAT) and pre-embedding immunogold labeling for CB₁. CB₁ cannabinoid receptors (indicated by arrows) on an inhibitory (VIAAT-positive) axon terminal (t). Note that in this reaction, silver intensification results in weaker electron density of the DAB precipitate. (Fa–Fb) Immunoperoxidase staining for CB₁ combined with pre-embedding immunogold labeling for VIAAT demonstrates co-localization of the two proteins. Similar results were obtained in 4 animals. Scale bar 0.1 µm.

Fig. 3

Extracellular single unit recordings from deep dorsal horn neurons in intact rats. Frequency histograms of action potentials evoked by mechanical stimulation within the receptive field of one hindlimb with brush (br), pressure (pr) and pinch (pi) but outside the capsaicin-injected area. (A) responses (spikes / s) of a representative neuron. (B) statistical analysis of background-corrected action potential activity (mean ± sem). (C) dose response analyses. Repeated measures ANOVA followed by Newman-Keuls multiple comparison post-hoc tests. n = 5 – 6 neurons per group. *, P ≤ 0.05; **, P < 0.01, ***, P < 0.001, against control (pre-capsaicin). ⁺, P ≤0.05; ⁺⁺, P < 0.01, ⁺⁺⁺, P < 0.001, against capsaicin.

Fig. 4

Effects of pharmacological and genetic manipulations of the endocannabinoid system on capsaicin-induced mechanical hyperalgesia in mice. (A) Mechanical paw withdrawal thresholds (mean ± sem) were determined with electronically controlled von-Frey filaments at 20 min intervals for 2 hours following capsaicin injection into the left hindpaw and for another 2 hours after i.t. injections (vehicle [10% DMSO], AM 251 [0.5 nmol / mouse], URB 597 [1.0 nmol], UCM 707 [1.0 nmol], LY 367,385 [1.0 nmol], MPEP [150 nmol]). Left: time course (mean ± sem). Right: treatment-induced changes in hyperalgesia, areas under the curve (AUC) integrated over 2 – 4 h after capsaicin injection. Note that the time course of sensitization in wild-type mice treated with i.t. vehicle is the same as in wild-type mice, which did not receive i.t. injections (B). n = 5 – 6 mice per group, for statistical analyses three groups of vehicle-injected mice were pooled. One-way ANOVA followed by Dunnett’s post-hoc test F (11,74) = 21.18 *, P ≤ 0.05 **, P < 0.01 ***, P < 0.001. (B) Capsaicin-induced secondary hyperalgesia in wild-type mice versus CB₁^−/− mice (n = 9 mice / group), and in ptf1a-CB₁^−/− (n = 7 and 11 mice / group) and sns-CB₁^−/− mice versus mice carrying a CB₁ receptor gene flanked by two loxP sites (CB₁^fl/fl mice) (n = 5 mice / group). Left: time course. Right: AUC (0 – 4 hours after capsaicin injection). ***, P < 0.001 unpaired Student t-test.