Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase

Benjamin F. Cravatt, Kristin Demarest, Matthew P. Patricelli, Michael H. Bracey, Dan K. Giang, Billy R. Martin, and Aron H. Lichtman
PNAS July 31, 2001 98 (16) 9371-9376; https://doi.org/10.1073/pnas.161191698

  1. Edited by L. L. Iversen, University of Oxford, Oxford, United Kingdom, and approved June 11, 2001 (received for review April 18, 2001)

Article Figures & SI

Figures

  • Figure 1
    Figure 1

    Generation and biochemical characterization of FAAH−/− mice. (A) The genomic structure surrounding the deleted FAAH exon 1 (E1). Only relevant restriction sites are designated. The deleted E1 exon encodes amino acids 1–65 of the FAAH protein. (B) Southern blot analysis of EcoRV-digested genomic DNA by using the indicated probe (External probe in A), where 4.5- and 14-kb bands correspond to FAAH−/− and FAAH+/+ genotypes, respectively. (C) Western blot analysis of tissues from FAAH+/+, FAAH+/−, and FAAH−/− mice demonstrating the selective absence of FAAH protein in FAAH−/− animals. (D) Confocal microscopy immunofluorescence images of cerebellar sections of FAAH+/+ (Left) and FAAH−/− (Right) mice. Green signal, anti-FAAH; red signal, propidium iodide (stains nuclei). Arrowheads highlight intense FAAH immunoreactivity in the cell bodies of Purkinje neurons (Left).

  • Figure 2
    Figure 2

    Pharmacological activity of anandamide in FAAH+/+ and FAAH−/− mice. Anandamide elicited dose-dependent pharmacological effects in FAAH−/− mice (■), but failed to produce any significant effects in FAAH+/+ mice (○). (A) Hypomotility (locomotor activity), ED50 < 6.25 mg/kg; (B) antinociception (tail-immersion), ED50 [95% confidence limits (CL)] = 13 (5–30) mg/kg; (C) catalepsy, ED50 (95% CL) = 20 (11–35) mg/kg; (D) hypothermia (rectal temperature), ED50 (95% CL) = 11 (6–19) mg/kg. ★, P < 0.05; ★★, P < 0.01; and ★★★, P < 0.001, for FAAH−/− versus FAAH+/+ mice receiving the same treatment (planned comparison). *, P < 0.05 and **, P < 0.01 for anandamide-treated versus vehicle-treated FAAH−/− mice (Dunnett's test). The results are presented as means ± SE. n = 6–8 mice per group.

  • Figure 3
    Figure 3

    Time course of the hypothermia (A) and catalepsy (B) in mice treated with either vehicle (FAAH+/+, open circles; FAAH−/−, filled circles) or 50 mg/kg anandamide (□, FAAH+/+; ■ FAAH−/−). ***, P < 0.001 for anandamide-treated FAAH−/− mice versus the other three test groups (Scheffé test). The results are presented as means ± SE. n = 6–8 mice per group.

  • Figure 4
    Figure 4

    The behavioral effects of anandamide in FAAH−/− mice are mediated by the CB1 cannabinoid receptor. The effect of vehicle (open bars) or SR141716A (filled bars) administered 10 min before treatment with anandamide in FAAH−/− mice. SR141716A (10 mg/kg) completely blocked the hypomotility (A), antinociception (B), catalepsy (C), and hypothermia (D) induced by anandamide (50 mg/kg), as SR141716A-pretreated, anandamide-treated FAAH−/− mice were indistinguishable in all behavioral assays from FAAH−/− mice treated with vehicle alone (0 mg/kg anandamide data in Fig. 2 A–D). **, P < 0.01 and ***, P < 0.001 for SR141716A-treated versus vehicle-treated FAAH−/− mice (planned comparison). The results are presented as means ± SE. n = 6–8 mice per group.

  • Figure 5
    Figure 5

    Altered pain responses in FAAH−/− mice. (A) FAAH−/− mice (filled columns) exhibited prolonged response latencies in both the tail-immersion (n = 48, 50, and 62 for FAAH+/+, FAAH+/−, and FAAH−/− genotypes, respectively) and hot plate (n = 12–15 mice per group) tests for thermal pain sensation relative to FAAH+/+ (open columns) and FAAH+/− mice (hatched bars; for hot plate data with FAAH+/− mice, see Fig. 6B Left). (B) Duration of licking during the early phase of the formalin test was reduced in FAAH−/− mice (filled columns) relative to FAAH+/− (hatched columns) and FAAH+/+ (open columns) mice; n = 8–10 mice per group. **, P < 0.01 and ***, P < 0.001 for FAAH−/− versus FAAH+/− or FAAH+/+ mice (planned comparisons). The results are presented as means ± SE.

  • Figure 6
    Figure 6

    Enhanced endogenous cannabinoid levels and activity in FAAH−/− mice. (A) FAAH−/− mice (filled column) possessed greatly increased endogenous brain levels of anandamide relative to FAAH+/+ mice (open bar); FAAH+/+, 50 ± 10 pmol/g of tissue; FAAH−/−, 775 ± 113 pmol/g of tissue; ***, P < 0.001 for FAAH−/− versus FAAH+/+ mice (planned comparison); n = 7–8 mice per group. (B) The prolonged response latency of FAAH−/− mice in the hot plate assay (Left, filled bar) was reversed by treatment with SR141716A (Right, filled bar). Vehicle administration failed to significantly affect the response latencies of FAAH−/−, FAAH+/−, and FAAH+/+ mice (Right, open bars), and SR141716A failed to significantly affect the response latencies of FAAH+/− and FAAH+/+ mice (Right, filled bars). n = 8–11 mice per group, assayed 30 min posttreatment. **, P ≤ 0.01 for SR141716A-treated FAAH−/− mice versus either vehicle-treated FAAH−/− mice or baseline latencies (planned comparison); ***, P < 0.005, for FAAH−/− versus FAAH+/− or FAAH+/+ mice (planned comparison). The results are presented as means ± SE.

Tables

  • Table 1

    Anandamide and oleamide hydrolytic activities in FAAH+/+, FAAH+/−, and FAAH−/− mice (nmol/min/mg)

    Anandamide hydrolysisOleamide hydrolysis
    Brain+/+0.33  ± 0.040.24  ± 0.03
    +/−0.20  ± 0.030.15  ± 0.01
    −/−0.003  ± 0.0020.004  ± 0.002
    Liver+/+0.53  ± 0.060.54  ± 0.01
    +/−0.29  ± 0.030.29  ± 0.07
    −/−0.01  ± 0.010.03  ± 0.01

    • Fatty acid amide hydrolytic activities were measured as described in Materials and Methods. 

  • Table 2

    ED50 values for the behavioral effects of THC in FAAH+/+ and FAAH−/− mice [mg/kg (95% confidence limits)]

    HypomotilityAntinociceptionHypothermia
    FAAH+/+13 (10–17)13 (7–26)12 (9–17)
    FAAH−/−13 (9–18)12 (7–20)15 (10–24)

    • FAAH+/+ and FAAH−/− mice were injected with 5, 10, 20, or 40 mg/kg THC (i.p.) and tested as described in Materials and Methods. The highest dose of THC failed to elicit more than 50% catalepsy in either genotype. n = 6–8 mice per group. 

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Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase
Benjamin F. Cravatt, Kristin Demarest, Matthew P. Patricelli, Michael H. Bracey, Dan K. Giang, Billy R. Martin, Aron H. Lichtman
Proceedings of the National Academy of Sciences Jul 2001, 98 (16) 9371-9376; DOI: 10.1073/pnas.161191698
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