Anandamide [arachidonylethanolamide (AEA)] appears to be an endogenous agonist of brain cannabinoid receptors (CB₁), yet some of the neurobehavioral effects of this compound in mice are unaffected by a selective CB₁ antagonist. We studied the levels, pharmacological actions, and degradation of AEA in transgenic mice lacking the CB₁ gene. We quantified AEA and the other endocannabinoid, 2‐arachidonoyl glycerol, in six brain regions and the spinal cord by isotope‐dilution liquid chromatography‐mass spectrometry. The distribution of endocannabinoids and their inactivating enzyme, fatty acid amide hydrolase, were found to overlap with CB₁ distribution only in part. In CB₁ knockout homozygotes (CB₁‐/‐), the hippocampus and, to a lesser extent, the striatum exhibited lower AEA levels as compared with wild‐type (CB₁+/+) controls. These data suggest a ligand/receptor relationship between AEA and CB₁ in these two brain regions, where tonic activation of the receptor may tightly regulate the biosynthesis of its endogenous ligand. 2‐Arachidonoyl glycerol levels and fatty acid amide hydrolase activity were unchanged in CB₁‐/‐ with respect to CB₁+/+ mice in all regions. AEA and Δ⁹‐tetrahydrocannabinol (THC) were tested in CB₁‐/‐ mice for their capability of inducing analgesia and catalepsy and decreasing spontaneous activity. The effects of AEA, unlike THC, were not decreased in CB₁‐/‐ mice. AEA, but not THC, stimulated GTPγS binding in brain membranes from CB₁‐/‐ mice, and this stimulation was insensitive to CB₁ and CB₂ antagonists. We suggest that non‐CB₁, non‐CB₂ G protein‐coupled receptors might mediate in mice some of the neuro‐behavioral actions of AEA.

Acid-sensing ion channel 3 (ASIC3), a proton-gated ion channel of the degenerins/epithelial sodium channel (DEG/ENaC) receptor family is expressed predominantly in sensory neurons including nociceptive neurons responding to protons. To study the role of ASIC3 in pain signaling, we generated ASIC3 knockout mice. Mutant animals were healthy and responded normally to most sensory stimuli. However, in behavioral assays for pain responses, ASIC3 null mutant mice displayed a reduced latency to the onset of pain responses, or more pain-related behaviors, when stimuli of moderate to high intensity were used. This unexpected effect seemed independent of the modality of the stimulus and was observed in the acetic acid-induced writhing test (0.6 vs. 0.1–0.5%), in the hot-plate test (52.5 and 55 vs. 50°C), and in tests for mechanically induced pain (tail-pinch vs. von Frey filaments). We postulate that ASIC3 is involved in modulating moderate- to high-intensity pain sensation.

Neuropathic pain is a clinical manifestation of nerve injury difficult to treat even with potent analgesic compounds. Here, we used different lines of genetically modified mice to clarify the role played by CB₂cannabinoid receptors in the regulation of the central immune responses leading to the development of neuropathic pain. CB₂knock-out mice and wild-type littermates were exposed to sciatic nerve injury, and both genotypes developed a similar hyperalgesia and allodynia in the ipsilateral paw. Most strikingly, knock-outs also developed a contralateral mirror image pain, associated with an enhanced microglial and astrocytic expression in the contralateral spinal horn. In agreement, hyperalgesia, allodynia, and microglial and astrocytic activation induced by sciatic nerve injury were attenuated in transgenic mice overexpressing CB₂receptors. These results demonstrate the crucial role of CB₂cannabinoid receptor in modulating glial activation in response to nerve injury. The enhanced manifestations of neuropathic pain were replicated in irradiated wild-type mice reconstituted with bone marrow cells from CB₂knock-outs, thus demonstrating the implication of the CB₂receptor expressed in hematopoietic cells in the development of neuropathic pain at the spinal cord.

Osteoporosis is one of the most common degenerative diseases. It is characterized by reduced bone mineral density (BMD) with an increased risk for bone fractures. There is a substantial genetic contribution to BMD, although the genetic factors involved in the pathogenesis of human osteoporosis are largely unknown. Mice with a targeted deletion of either the cannabinoid receptor type 1 (Cnr1) or type 2 (Cnr2) gene show an alteration of bone mass, and pharmacological modification of both receptors can regulate osteoclast activity and BMD. We therefore analyzed both genes in a systematic genetic association study in a human sample of postmenopausal osteoporosis patients and matched female controls. We found a significant association of single polymorphisms (P=0.0014) and haplotypes (P=0.0001) encompassing the CNR2 gene on human chromosome 1p36, whereas we found no convincing association for CNR1. These results demonstrate a role for the peripherally expressed CB2 receptor in the etiology of osteoporosis and provide an interesting novel therapeutical target for this severe and common disease.

Caffeine, an adenosine A₁, A_2A, and A_2B receptor antagonist, is frequently used as an adjuvant analgesic in combination with nonsteroidal anti-inflammatory drugs or opioids. In this study, we have examined the effects of novel specific adenosine receptor antagonists in an acute animal model of nociception. Several A_2B-selective compounds showed antinociceptive effects in the hot-plate test. In contrast, A₁- and A_2A-selective compounds did not alter pain thresholds, and an A₃ adenosine receptor antagonist produced thermal hyperalgesia. Evaluation of psychostimulant effects of these compounds in the open field showed only small effects of some antagonists at high doses. Coadministration of low, subeffective doses of A_2B-selective antagonists with a low dose of morphine enhanced the efficacy of morphine. Our results indicate that analgesic effects of caffeine are mediated, at least in part, by A_2B adenosine receptors.