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Science 325 (5941): 760-764

Copyright © 2009 by the American Association for the Advancement of Science

Spinal Endocannabinoids and CB1 Receptors Mediate C-Fiber–Induced Heterosynaptic Pain Sensitization

Alejandro J. Pernía-Andrade1,*,{dagger}, Ako Kato1,9,*, Robert Witschi1,9,*, Rita Nyilas2, István Katona2, Tamás F. Freund2, Masahiko Watanabe3, Jörg Filitz4, Wolfgang Koppert4,{ddagger}, Jürgen Schüttler4, Guangchen Ji5, Volker Neugebauer5, Giovanni Marsicano6, Beat Lutz7, Horacio Vanegas8, and Hanns Ulrich Zeilhofer1,9,§

1 Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
2 Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1083 Budapest, Hungary.
3 Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan.
4 Department of Anesthesiology, University of Erlangen-Nürnberg, Krankenhausstrasse 12, D-91054 Erlangen, Germany.
5 Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555–1069, USA.
6 U862 Centre de Recherche INSERM François Magendie, 33077 Bordeaux, France.
7 Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg-University Mainz, D-55099 Mainz, Germany.
8 Instituto Venezolano de Investigaciones Cientificas, Apartado 20632, Caracas 1020A, Venezuela.
9 Institute of Pharmaceutical Sciences, ETH Zurich, Wolfgang Pauli Strasse 10, CH-8093 Zurich, Switzerland.

Figure 1
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Fig. 1. Synaptic effects of CB1 receptor activation in dorsal horn neuronal circuits. (A to 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 panels: 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 panels: Time course. Mean ± SEM, n = 7 to 13 neurons. (D) Inhibition of glycinergic IPSCs in nonglycinergic (EGFP-negative) neurons (n = 8 neurons) 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 neurons). (E) DSI (1-s depolarization of the postsynaptic neuron to 0 mV) in nonglycinergic neurons (six out of eight neurons) and its prevention by AM 251 (5 µM). No DSI occurred in glycinergic neurons (n = 5 neurons). Glycinergic IPSCs were evoked at a frequency of 0.2 Hz.


Figure 2
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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) are shown. (B) Variation analysis. Top panel: Individual traces of glycinergic IPSCs recorded under control conditions and in the presence of WIN 55,212-2 (3 µM). Bottom panel: Changes in the CV in 13 cells are plotted versus changes in the mean amplitude induced by WIN 55,212-2. (C to F) Electron microscopic analysis (a to c and a and b are serial sections) of CB1 receptor localization in the superficial spinal dorsal horn. Arrowheads indicate symmetric synapses; arrows indicate immunogold labeling. (Ca to Cc) CB1 immunostaining coupled to immunoperoxidase reaction [3,3'-diaminobenzidine (DAB)]. CB1 receptors are present in an axon terminal (t) forming a symmetric (inhibitory) synapse on an immunonegative dendritic shaft (d) in lamina II. The asterisk labels a CB1-negative bouton of another symmetric synapse on the same dendrite. (Da and Db) High-resolution pre-embedding immunogold staining for CB1. The CB1 receptor is located presynaptically on the plasma membrane of an inhibitory axon terminal (t). (Ea and Eb) DAB staining for VIAAT and pre-embedding immunogold labeling for CB1. CB1 cannabinoid receptors (indicated by arrows) are on an inhibitory (VIAAT-positive) axon terminal (t). In this reaction, silver intensification results in weaker electron density of the DAB precipitate. (Fa and Fb) Immunoperoxidase staining for CB1 combined with pre-embedding immunogold labeling for VIAAT demonstrates colocalization of the two proteins. Similar results were obtained in four animals. Scale bar, 0.1 µm.


Figure 3
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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 on one hindlimb with brush (br), pressure (pr), and pinch (pi) but outside the capsaicin-injected area are shown. (A) Responses (spikes/s) of three representative neurons. (B) Statistical analysis of background-corrected action potential activity (mean ± SEM). (C) Dose-response analyses by repeated-measures analysis of variance (ANOVA), followed by Newman-Keuls multiple comparison post-hoc tests; n = 5 or 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.


Figure 4
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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 dynamic von Frey filaments at 20-min intervals for 2 hours after capsaicin injection into the left hindpaw and for another 2 hours after intrathecal injections of vehicle (10% dimethyl sulfoxide), AM 251 (0.5 nmol per mouse), URB 597 (1.0 nmol), UCM 707 (1.0 nmol), LY 367,385 (1.0 nmol), or MPEP (150 nmol). Left panel: Time course (mean ± SEM). Right panel: Treatment-induced changes in hyperalgesia. Areas under the curve (AUC) were integrated over time from 2 to 4 hours after capsaicin injection. The time course of sensitization in wild-type mice treated with intrathecal vehicle is the same as in wild-type mice that did not receive intrathecal injections (B). n = 5 or 6 mice per group; for statistical analyses, three groups of vehicle-injected mice were pooled. Analyses were by 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 per group) and in ptf1a-CB1–/– mice (n = 7 and 11 mice per group) and sns-CB1–/– mice versus mice carrying a CB1 receptor gene flanked by two loxP sites (CB1fl/fl mice) (n = 5 mice per group). Left: Time course. Right: AUC (0 to 4 hours after capsaicin injection). ***P < 0.001, unpaired Student’s t test.


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